INTRODUCTION:

Most organic elements in soil, peat, lignites, brown coals, sewage, natural waterways, and their sediments are humic compounds¹. These natural complexing substances come from a variety of places, including the final products of biosynthetic pathways in microbes, degradation and transformation products in plants, synthetic oxidation products of phenolic compounds, and polymers formed during roasting processes, such as coffee roasting². Shilajit, a powerful rejuvenator and anti-aging chemical used in eastern medicine, is found in Himalayan regions bordering India, China, Tibet, and portions of Central Asia in the form of rock³. It's also mentioned in the Ayurvedic texts Charaka Samhita and Susruta Samhita as a rejuvenator and treatment for all illnesses⁴. Fulvic acids, humic acids (HAs), and humin are the three components of humic substances. The most essential component of humic compounds is hydroxyapatite (HA).

Many researchers from many fields have been drawn to it because of its amazing characteristics. Intensive study over the years has revealed fresh information on the structure and physicochemical characteristics of this intriguing natural molecule, pointing to its potential in a variety of practical applications in agriculture, industry, the environment, and medical ¹ . In recent years, this amazing biopolymer has also become a useful tool in the pharmaceutical industry.

HA is mostly acidic, with a wide range of functional groups such as carboxylic, hydroxylic, phenolic, carbonylic, and aminic groups (Figure 1a), all of which are amorphous or colloidal in form and have a high molecular weight. The chemical flexibility of HA, as well as its lack of toxicity, makes it a good candidate for pharmacological research. There are just a handful similar Indian patents, according to³. Saluja and Agarwal's patent application number 814/Del/2001 asserts that when piroxicam is complexed with HA, it has a higher bioavailability. Another patent investigates the components of Shilajit's bio-enhancement potential in depth. The separation and characterisation of HA and fulvic acid from Shilajit is detailed in patent application number 531/Del/2005⁴. Recently, HA has been investigated as a solubilizing agent for medicines that are weakly water soluble (BCS II and IV, respectively). The hydrophobic core of HA macromolecules is big, whereas the hydrophilic outside is little. These may entrap and form inclusion complexes with improved solubility, wettability, dissolution, and permeability properties ³ combined carbamazepine, an anticonvulsant, with HA to create complexes. They discovered that complexation enhanced carbamazepine's absorption and pharmacokinetic characteristics ⁴. looked at several ratios of b-carotene–HA complexes and found that the complexation not only boosted water solubility but also improved photostability of b-carotene, a potential dietary antioxidant ⁵ described the usage of HAs derived from biomass as natural surfactants in industrial settings. Furosemide complexed with HA improved solubility and diuresis, according to ⁶.

The ability of HA with excellent solubilization capability to increase the anticancer effect of paclitaxel (PTX) by enhancing its solubility was examined in this work. Paclitaxel (PTX), also known by the trade names Taxol and Taxol, is a chemotherapy drug used to treat a variety of cancers. Ovarian cancer, oesophageal cancer, breast cancer, lung cancer, Kaposi's sarcoma, cervical cancer, and pancreatic cancer are among the cancers that fall under this category. It is administered by a venous injection. A formulation that is albumin-bound is also available.

Hair loss, bone marrow suppression, numbness, allergic responses, muscular aches, and diarrhoea are all common adverse effects. Heart issues, a higher risk of infection, and lung inflammation are among the more significant adverse effects. There are worries that using it while pregnant might result in congenital abnormalities.

MATERIALS AND METHODS:

Synthesis of PTX–HA complexes:

Solvent evaporation was used to make PTX-HA complexes in the weight ratios of 1:1, 1:2, and 2:1 (HA: PTX). In a nutshell, PTX was dissolved in methanol and then added to a solution of HA. They were well combined and evaporated under vacuum for 20 minutes at 700C using a rotary evaporator (EV11.JH.084 Equitron, Maharashtra, India) (time required till dryness). The resulting complex was kept in sealed containers for further research.

Solubility study:

By vortexing for 10 minutes, known concentrations of HA–PTX complexes were dissolved in water and phosphate buffer pH 6.8. A 0.45 mm membrane filter was used to filter the suspension (Whatman Ltd., Piscataway, NJ). UV spectroscopy at 226 nm was used to determine the concentration of PTX. All of the results were made in triplicate.

Differential scanning calorimetry (DSC):

DSC (Mettler-Toledo DSC 823e, Labcompare, South San Francisco, CA) was used to investigate the thermal behaviour of PTX, HA, and HA–PTX at a temperature range of 40–3200C at a scan rate of 5 C/min. Aluminium pans were used to conduct the experiment in a nitrogen atmosphere.

Fourier transform infrared spectroscopy (FT-IR):

Separately, appropriate quantities of PTX, HA, and the HA–PTX complex were combined with KBr. On a Jasco FT/IR-4100 (Jasco, Tokyo, Japan) equipped with Spectra manager version 2, the IR spectra of the resulting mixture were recorded. Each measurement was based on an average of 45 scans with a 1 cm-1 resolution.

X-ray diffraction study (XRD):

Using an X-ray diffractometer, the crystalline characteristics of pure PTX, HA, and the HA–PTX complex were evaluated (PW 1729, Philips, Eindhoven, The Netherlands). The samples were exposed to monochromatized Cu Ka radiation (1.542 A) and evaluated at 2 between 5 and 50 degrees Celsius.30 kV and 30 mA were utilised as the voltage and current, respectively. The chart speed and range were 2 103 CPS and 10 mm/ 2 correspondingly.

Dissolution experiment:

An in vitro dissolving experiment was conducted using a paddle type dissolution equipment (Biomedica, BMI-599, Curno Bergamo, Italy). The dissolution research was carried out in 900 mL of phosphate buffer pH 6.8 for four hours at 37 0.5 C. At specified intervals, 5 mL aliquots were extracted and filtered using Whatman filter paper. To maintain sink conditions, the volume removed was replaced with an equivalent volume of new dissolving media. At 226 nm, the filtered samples were spectrophotometrically examined. A calibration curve was used to determine the amount of medication released (PCP Disso Software IIPC, Pune, India). Dissolution tests were carried out in triplicate for each formulation.

Scanning electron microscopy (SEM):

The technique is used for examining objects using electrons. Scanning electron microscope (Stereoscan S120, Cambridge Instruments, Cambridge, UK) with 10 kV acceleration voltage was used to characterize the exterior topography of the PTX, HA, and HA–PTX complex. Surface topography was studied at various magnifications after samples were placed on double-faced sticky tape and coated with a thin gold–palladium layer (20 mm) using a sputter-coated machine (VGMicrotech, Uckfield, UK).

RESULT:

The goal of this work was to find HA–PTX complexes in order to improve the phytopharmaceutical's solubility. Water solubility of PTX has been reported to be poor. Due to complexation with HA, its solubility was greatly enhanced (60.2 mg/ml). This might be because HA polymers have a hydrophilic surface and a big hydrophobic core, which can entrap poorly soluble medicines and form inclusion complexes, resulting in increased solubility. The increase in solubility in buffer pH 6.8 was greater than in water. The 2:1 ratio showed the greatest increase in PTX aqueous solubility and was employed in physicochemical characterization procedures.

DSC investigated the thermal analysis of PTX and its complex (2:1). Figure 1 shows the thermograms of PTX, HA, and the HA–PTX complex. The thermogram (A) of PTX in this figure shows an endothermic peak at 234.7 C, which corresponds to its melting point⁷. Water desorption and structural alterations were shown by the wide endothermic curve (B) of HA. At around 60 degrees Celsius, reversible water loss occurs. Beyond 70 degrees Celsius, irreversible water loss begins; above 100 degrees Celsius, HA breakdown occurs, followed by the emission of carbon dioxide, and above 140 degrees Celsius, carbon monoxide is produced. According to Laplante, the skeleton of HAs is not destroyed, and the functional groups are not removed (save maybe for a small quantity of carboxyl groups) ⁸. The thermogram (C) of the complex revealed an endothermic peak at 110.4 C; this change in the endotherm suggested that PTX and HA could interact. The complex exhibits an exothermic activity at much higher temperatures, which differs from that found in HA curves. All of the evidence points to a possible connection between HA and PTX.

FT-IR analysis of the drug (Figure 2) confirmed characteristic peaks at wave numbers (cm1) corresponding to 3400.85 (–OH stretching), 2845.35, 2932.23 (CH stretching), 1733.6 (C1 4O stretching due to a, b unsaturation), 1457.92 (C1 4C stretching), 1363.4 (C–H deformation), 1223.6 (C–O–C stretch of lactone ring), 972.3, 1024. The FT-IR absorption bands of HA matched those previously described in the literature⁹. The FT-IR absorption bands of HA matched those described in the literature as well. The spectra displayed typical HA features (e.g., OH stretching at 3450 cm1, methylene bands C–H at 2920 and 2850 cm1, C14O stretching vibrations owing to carboxylic groups at 1720 cm1, and C–C bonds conjugated with C–O and COO groups at 1620 cm1). However, the complex's FT-IR spectra revealed a change in the C1 4O absorption band towards a lower wave number at 1546 cm1. These findings also showed that PTX and HA could interact.

PTX's X-ray diffraction spectra (Figure 3) revealed that the medication was crystalline, as shown by several peaks at diffraction angles of 10.1, 29, and 35.3. HA, on the other hand, did not display any diffraction peaks, indicating that it is amorphous¹⁰. The XRD pattern of the 2:1 complex showed a substantial reduction in the strength of PTX's main characteristic peak, indicating partial crystallinity loss. The increase in solubility of PTX in the complexed form may have been aided by the reduction in crystallinity.

Figure 5 shows the results of the dissolving research. In four hours, the pure medication released a maximum of 30% of its content. The complexes, on the other hand, showed a distinct pattern of drug release. The results demonstrated that PTX release was fast for all complexes, with total drug release (about 100 percent) within four hours. The 2:1 complex, on the other hand, obtained full release the quickest (after 40 minutes). The 1:2 complex had a somewhat sluggish drug release, with total release taking 60 minutes. The dissolution curves in Figure 4 show that drug release of 1:1 and 2:1 complex follows a similar pattern. The commonly used ‘‘f2” or ‘‘similarity factor” was computed to determine this similarity, and it was found to be 62.55, indicating that these two dissolution profiles were not substantially different. As a result, the 1:1 complex, which uses a less quantity of HA, was chosen for future research.

Figure 5 shows a scanning electron micrograph of PTX, HA, and the HA–PTX (2:1) complex. PTX was made up of a variety of huge cylindrical crystals. The crystal structure of PTX was shown by the broad, sharp edges of the particles, whereas HA looked randomly shaped with soft edges^11-14,
16. The complex was made up of smaller aggregates with a structure that was completely different from that of its members. It had a very airy texture at higher magnifications. It also had smaller particles, which might give more surface area for better dissolving. The permeation research was carried out to see how PTX affected intestinal permeability. The penetration of the 1:1 complex over the everted rat intestinal sac was investigated for this purpose. The complex was found to penetrate the body more quickly than the pure drug. Approximately 70% of the complexed drug penetrated within 300 minutes, whereas only about 34% of the drug permeated during the same time frame. The surfactant property of HA is responsible for the enhanced permeability^11,12,15-17. HA is known to produce micelles, which aid in breakdown, while the surfactant feature aids in intestinal penetration ^{12,15,18-21,23}.

Table 1. Solubility determination (mg/ml) of PTX (Results are expressed as mean SD of three determinations)

Solvent	(1:1) Complex (HA:PTX)	(1:2) Complex (HA:PTX)	(2:1) Complex (HA:PTX)	Pure Drug PTX
Water	95.30 ± 1.01	76.25 ± 1.55	100.50 ± 2.05	6.00 ± 0.20
Phosphate Buffer 6.8	170.50 ± 2.41	169.00 ± 3.21	185.00 ±3.78	26.00 ±0.66

Figure 1. DSC thermogram of PTX, HA, and their complex.

Figure 2. FT-IR spectra of (A) PTX, (B) Humic acid, and (C) complex.

Figure 3. X-ray diffraction pattern of humic acid, PTX, and their complex

Figure 4. In vitro release profile of PTX complexes and PTX

Figure 5. SEM micrographs of HA, PTX, and their complex ²².

CONCLUSION:

With its diverse uses in various fields, HA has shown to be a gift from nature. The current research adds to the few existing pharmacological uses that are relevant to increasing solubility. PTX's solubility and hepatoprotective efficacy skyrocketed when it was complexed with HA. Poorly soluble medicines are a major source of worry for formulators all over the world since they have issues with bioavailability. The biomaterial HA, which was investigated in this study, can be used to alleviate this problem.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 26.08.2021 Modified on 06.10.2021

Research J. Pharm. and Tech 2022; 15(11):5089-5093.

DOI: 10.52711/0974-360X.2022.00855