The Effect of pH and Cocrystal Quercetin-Isonicotinamide on Quercetin Solubility and its Thermodynamic

Budipratiwi Wisudyaningsih¹, Solihatus Sallama¹, Siswandono², Dwi Setyawan²*

¹Faculty of Pharmacy, University of Jember, Jember 68121, Indonesia.

²Faculty of Pharmacy, Universitas Airlangga, Surabaya 60115, Indonesia.

*Corresponding Author E-mail: dwisetyawan-90@ff.unair.ac.id

ABSTRACT:

This study aimed to improve the solubility of quercetin by solvent pH control method and crystal modification through co-crystal formation using isonicotinamide as its co-former. Solubility of quercetin was tested at nine pH levels using phosphate buffer solvents. Quercetin-isonicotinamide co-crystal was prepared by a solvent evaporation method. Co-crystal preparation was carried out using two different stoichiometric ratios of quercetin-isonicotinamide (1:1 and 1:3). The co-crystalline solubility test was performed in 50 mL citrate buffer (pH 5.0 ± 0.05) at a temperature of 37 ± 0.5°C. The thermodynamic parameters of quercetin and co-crystal were analyzed to determine the mechanism of the quercetin solubility process. Increasing the pH of solvents has proven to increase the solubility of quercetin. The quercetin oxidation reaction starts at pH level of 7.4. The formation of quercetin-isonicotinamide co-crystal at ratio of 1:1 and 1:3 shows the increase of quercetin solubility by 1.36 and 1.27 times, respectively. The thermodynamic parameters of the quercetin and quercetinco-crystal, which include entropy, enthalpy, and free energy values, can be used to explain the solubility process of quercetin. Quercetin has increased solubility under alkaline pH conditions, but undergoes an oxidation reaction at pH 7.4 and easily oxidized at alkaline pH. Crystal modification of quercetin by the co-crystal formation method has proven to increase the solubility of quercetin so that it can be used for the development of quercetin as a candidate for effective, safe, and acceptable active pharmaceutical ingredient.

KEYWORDS: Quercetin, solubility, pH, co-crystal, isonicotinamide.

INTRODUCTION:

Development of active pharmaceutical ingredient is important for obtaining a stable and acceptable drug with safer and more effective therapeutic effects. Quercetin is one of chemical compounds found in plants¹. This compound is a super-antioxidant compound that has been shown to have various pharmacological activities as anticancer, anti-inflammatory, and can even be used as an anti-aging agent². Previous research has proven that the anticancer pharmacological effects of quercetin are related to its activity as an antioxidant³. Various studies on quercetin pharmacological activities have been carried out, making this compound very potential to be developed as candidates for active pharmaceutical ingredients.

Developing quercetin as an active pharmaceutical ingredient is challenging as it is difficult to dissolve in water and has chemical instability which causes low bioavailability. Therefore, there is no pharmaceutical preparation with quercetin as the active ingredient^4,5,6.

The strategy used to increase the solubility of quercetin is by modifying the solvent pH and quercetin crystals through the co-crystal formation. Quercetin has shown different solubility in different pH conditions, and other studies suggest that quercetin undergoes an oxidation reaction at alkaline pH. This condition makes quercetin a natural compound with high antioxidant potential⁷. Another strategy that was carried out to improve the solubility of quercetin was by the formation of pharmaceutics co-crystal⁸. Previous studies have shown that modification of quercetin crystals through co-crystal formation increases its dissolution 1.08 times than that of quercetin⁹. In this study, quercetin-isonicotinamide co-crystals were prepared at a ratio of 1: 1 and 1:3. The solubility test was done to determine the changes in quercetin solubility characteristic.

This study was conducted to design a product of quercetin which provides better solubility and stability so that it has good bioavailability. Thus, it is expected that quercetin can be developed as an active ingredient in the drug.

MATERIAL AND METHODS:

Chemicals and reagents:

The following materials were used in the study: quercetin and isonicotinamide (Tokyo Chemical Industry, Japan); pro analysis buffer component: H₃PO₄, NaH₂PO₄, Na₂HPO₄, Na₃PO₄, citric acid, ethanol, NaOH, HCl and aquadest.

Quercetin Solubility Test at Different pH Levels:

Quercetin solubility test was carried out in phosphate buffer, pH 1.5; 2.0; 3.0; 6.5; 7.4; 8.0; 11.5; 12.0; and 12.5 using a solubility test chamber with a temperature of 37±0.5°C, stirred at 50rpm for 4 hours. Samples of 5 mL were taken, filtered with 0.45mm Millipore filter paper, and the absorbance was determined at the maximum wavelength using a UV-Vis spectrophotometer (Genesys 10S UV-Vis, USA). The solubility test was replicated three times.

Preparation of Quercetin-Isonicotinamide Co-crystal:

Quercetin-isonicotinamide co-crystals were prepared with a 1:1 and 1:3 stoichiometric ratio by the solvent evaporation method⁹.

Solubility Test:

Determination of solubility was carried out in four sample groups, namely quercetin, physical mixture of quercetin-isonicotinamide, and quercetin-isonicotinamide co-crystal at stoichiometry ratios of 1:1 and 1:3 obtained from the solvent evaporation method using ethanol. Each sample was weighed equivalent to 25mg quercetin. The samples were placed in the solubility chamber filled with 50mL citrate buffer pH 5.0 ±0.05 with a stirring speed of 100 rpm and a temperature of 37 ± 0.5°C. The samples were taken at saturated solubility of 4 hours as much as 5.0mL, then filtered with a 0.45µm cellulose filter membrane. The absorbance was then observed in a UV-Vis spectrophotometer (Genesys 10S UV-Vis, USA) at 369 nm. The analysis was replicated three times.

Thermodynamic Determination of Quercetin and Quercetin-Isonicotinamide Co-crystal:

Quercetin thermodynamic parameters (DG, DH, and DS) were determined by solubility test of quercetin at a temperature of 32, 37, and 42°C. The solubility tests were carried out under saturated solubility of quercetin and co-crystal. The solubility data obtained were then used to determine free energy, enthalpy, and entropy in the solubility process of quercetin.

RESULTS:

Solubility Test of Quercetin at Different pH Levels:

The higher pH value of the solvent has proved to increase the solubility of quercetin. However, at alkali pH, an oxidation reaction occurs. Changes in pH also affect the maximum absorbance wavelength of quercetin in the determination of quercetin absorbance by the UV spectrophotometry method. (Table 1) shows quercetin solubility at various pH levels.

Table 1: Quercetin solubility at different pH levels

pH	l max(nm)	Quercetin Solubility (% w/v 10^-4) ± SD
1.5	366	0.12 ± 0.003
2.0	366	0.12 ± 0.003
3.0	366	0.17 ± 0.007
6.5	369	0.36 ± 0.005
7.4	373	4.07 ± 0.050
8.0	377	13.33 ± 0.010
11.5	391	33.91 ± 0.022
12.0	321	73.60 ± 0.013
12.5	320	71.70 ± 0.126

The quercetin solubility test shows the occurrence of chemical instability at pH 7.4, which is the oxidation reaction. It was shown as yellow quercetin solution and became increasingly yellow to brown-yellow at a higher pH. (Fig. 1) shows the quercetin solubility at different pH levels which does not undergo oxidation reactions.

Solubility Determination of Quercetin-Isonicotinamide Co-crystal:

Quercetin-isonicotinamide co-crystals showed an increase in solubility compared to pure quercetin and a physical mixture of quercetin-isonicotinamide. Co-crystal formed using a 1:1 stoichiometry ratio showed higher solubility compared to the solubility of the co-crystal at a ratio of 1: 3.

Fig.1: The solubility of quercetin at pH not causing an oxidation reaction

Fig. 2: The solubility of quercetin co-crystal, stoichiometric ratio of 1: 1 and 1: 3 in pH 5.0

Thermodynamic Parameter:

Quercetin and co-crystal thermodynamic parameters (DG, DH and DS) were obtained from solubility test data at temperatures of 32, 37, and 42 °C in equilibrium. The thermodynamic parameters of quercetin co-crystal show lower free energy value, enthalpy and entropy compared to quercetin. Thermodynamic parameter data of solubility of quercetin and co-crystal are presented in (Table 2).

DISCUSSION:

Quercetin Solubility at Various pH Levels:

Quercetin has different solubility characteristics at different pH levels. This is because quercetin is ionized under certain pH conditions¹⁰. With the increase of pH in the solvent, the proton is released from the carbonyl group to form an ionized molecule. This ionization causes a shift in the maximum absorbance wavelength at certain pH conditions. At pH 1.5 – 3.0 quercetin is in the form of H₅A. So, it has the same maximum wavelength. However, a bathochromic shift occurs at pH 6.2 - 11.0 and a hypochromic shift at pH 12.0 - 12.5. This is caused by the change in the hydroxyl group which can also function as an auxochrome group in the quercetin structure. Table 3 shows different forms of ionized quercetin at different pH conditions.

In the molecular structure of quercetin, there are 5 hydroxyl groups which cause intra-molecular and inter-molecular interactions, thus making these compounds have low solubility in water¹¹. The increase of pH in the solvent causes the release of protons in the quercetin carbonyl group so that the hydrogen bonds between quercetin molecules will be released. This event caused an increase in the solubility of quercetin.

At pH level 7.4 more protons are released. So, quercetin will undergo an oxidation reaction as indicated by the color change of the solution. The yellowish color that appears at pH 7.4 becomes yellow to brownish yellow with an increase to pH 12.5. This oxidation reaction causes changes in the molecular structure of quercetin which provides high absorbance at the quercetin wavelength at pH 7.4 - 12.5. Thus, the solubility data shown at pH 7.4 – 12.5 cannot be used to determine the solubility of pure quercetin.

Solubility of Quercetin-Isonicotinamide Co-crystal:

Isonicotinamide preference as a co-former is based on the synthon approach by utilizing a molecule fragment of active ingredients and co-formers to form a supramolecular synthons^12,13.

Quercetin-isonicotinamide co-crystal showed higher solubility compared to quercetin and physical mixture of quercetin-isonicotinamide. The increase in solubility of the quercetin-isonicotinamide co-crystal occurs because of hydrogen bonds interactions between quercetin hydroxyl group and the amide group in the structure of isonicotinamide^14,15.

The computational approach is used to see the possibility of interactions between quercetin and isonicotinamide through hydrogen bonds. The computational results using ChemBioDraw Ultra 12.0 are shown in (Fig. 3).

Figure 3. Predicted supramolecular Heterosynthon Quercetin-Isonicotinamide

Based on the computational results, hydrogen bonds interaction occurs between the hydroxyl group on the A ring of quercetin and group C = O amide in isonicotinamide. Another hydrogen bond interaction occurs between carbonyl group in C ring of quercetin and isonicotinamide amide group, with energy minimize value of 50.18kcal/mol (figure 3A). Figure 3B shows the presence of hydrogen bonding between hydroxyl and carbonyl groups in the ring C of quercetin attached with C = O amide group in isonicotinamide molecule with energy minimize value of 39.50kcal/mol. The last interaction predicted in Figure 3C shows hydrogen bond between the hydroxyl groups of the A ring of quercetin and aromatic N group of isonicotinamide with energy minimize value of 52.53kcal/mol.

The stoichiometric ratio difference in the co-crystal system will affect the solubility of the resulting crystalline¹⁶. In this study, quercetin-isonicotinamide co-crystal with a stoichiometric ratio of 1: 1 had better water solubility (0.44 10^-4 % w/v ± 0.012) than the ratio of 1: 3 (0.41 10^-4 % w/v ± 0.018). This is because with the increase of co-former stoichiometry ratio, there will be an increase in the mass of material to get the concentration of certain active ingredients¹⁷.

Thermodynamic Parameters:

The thermodynamic parameters analyzed included free energy, enthalpy and entropy. Thermodynamic parameter data will be used to explain the dissolution mechanism of an active ingredient in a particular solvent¹⁸. The calculation results of thermodynamic parameters show that the solubility process of quercetin and quercetin-isonicotinamide co-crystal takes place with the same mechanism, which is positive free energy and enthalpy, while entropy is negative. The positive value of free energy indicates that the solubility reaction taking place is not spontaneous. Enthalpy with a positive value indicates that heat absorption occurs during the process of dissolving an active ingredient. This heat absorption is the absorption of energy needed in the solubility process. With this thermodynamic data, it can be seen that the solubility process of quercetin and quercetin-isonicotinamide co-crystal is an endothermic reaction. The negative value of entropy shows that quercetin and quercetin-isonicotinamide co-crystal are an order or less disorder system. This order system shows the characteristic of quercetin which is difficult to dissolve in water.

CONCLUSION:

Increasing the pH of solvents can increase the solubility of quercetin. However, at pH 7.4 to pH 12.5, an oxidation reaction occurs due to the release of protons on the molecular structure of quercetin. The formation of quercetin-isonicotinamide co-crystal can increase the solubility of quercetin. Quercetin-isonicotinamide co-crystal with a 1: 1 stoichiometry ratio has a higher solubility compared to that with a 1: 3 ratio. The increases in the solubility of quercetin on the quercetin-isonicotinamide co-crystal with ratio stoichiometry of 1: 1 and 1: 3 were 1.36 and 1.27 times, respectively. The solubility mechanism of quercetin and quercetin-isonicotinamide co-crystal occurs in an order system, takes place non spontaneously, and is an endothermic reaction.

ACKNOWLEDGEMENT:

Thanks to the ministry of technology research and higher education for doctoral scholarships program.

CONFLICT OF INTEREST:

No confict of interest

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Received on 22.04.2020 Modified on 04.08.2020

Research J. Pharm. and Tech. 2021; 14(9):4657-4661.

DOI: 10.52711/0974-360X.2021.00809

Temperature (°C)	Quercetin			Cocrystal
Temperature (°C)	DG (kal/mol)	DH (kal/mol)	DS (kal/mol.der)	DG (kal/mol)	DH (kal/mol)	DS (kal/mol.der)
32	8688.31	16.25	-28.43	8481.96	10.56	-27.78
37	8628.36		-27.83	8438.93		-27.22
42	8457.84		-26.85	8412.21		-26.71

pH	Ionization form	Molecular structure
1.0 – 3.0	H₅A
4.0 – 8.0	H₅A, H₄A^-
9.0 – 12.5	H₄A^-, H₃A^2-, H₂A^3-, HA^4-, A^5-