Int J App Pharm, Vol 14, Issue 6, 2022, 120-126Original Article

APPLICATION OF MATHEMATICAL MODELING AND PHYSICO-CHEMICAL ANALYSIS METHODS IN THE PREDICTION OF BIOLOGICAL ACTIVITY AND QUALITY CONTROL OF GOSSYPOL DERIVATIVES

ELENA V. USPENSKAYA*, POLYNA А. ZABORKINA, EVGENIYA A. RYNDINA, TATYANA V. PLETENEVA, MARIYA A. MOROZOVA, ILAHA V. KAZIMOVA, ANTON V. SYROESHKIN

Department of Pharmaceutical and Toxicological Chemistry, Peoples Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St, Moscow, 117198, Russian Federation
*Email: [email protected]

Received: 02 Aug 2022, Revised and Accepted: 09 Sep 2022


ABSTRACT

Objective: The purpose of this work was to evaluate in silico biological activity profiles of real and virtual molecular structures of gossypol derivatives and to develop methods of Physico-chemical analysis to control their quality.

Methods: Substance of gossypol-acetic acid (GAA) and 14 virtual derivatives; PASS and ChemicDescript QSAR methods; low angle and dynamic laser light scattering (LALLS, DLS) methods; IR Spectroscopy–Cary 630 Fourier Transform IR Spectrometer, UV spectrometry–Cary-60 spectrophotometer, Optical microscopy (Altami BIO 2 microscope); Spirotox method for a sample’s biological activity.

Results: A distance-based topological Balaban index (J) was successfully selected by ChemicDescript analysis; the Pa meaning by PASS Online program showed maximum (from 0.8 to 0.9) variations of antitumor and antiandrogenic and minimum of antiviral activities of GAA derivatives (Pa<0.5) despite the existing literature data. Microscopy and DLS methods demonstrated the values of high powder dispersion d=0.8 nm and weak stability of colloidal particles =-0.9 mV. According to UV data =42.4±0.8 (100 ml·g-1·cm-1) at λmax=380 nm. The LALLS method determined the GAA dissolution rate constant in ethanol: k=0.041±0.004 s-1. The calculated activation energy values of cell biosensor death process in 1 mmol solution of GAA in N,N-DMF: °bsEa=174.36±0.45 kJ·mol-1 in comparison with the solvent medium: °bsEa=213±1.55 kJ·mol-1

Conclusion: The developed approach of chemometric, laser and biotesting methods can be used for the identification of biologically active properties, as well as for qualitative analysis within the development of the standard for the pharmaceutical substance of natural polyphenols.

Keywords: Gossypol-acetic acid, In silico methods, Pa/Pi ratios, Quality control, Analysis of physical and chemical properties


INTRODUCTION

The natural polyphenol gossypol contained in cotton (genus Gossypium, family Malvaceae) is of great interest in pharmacy due to the high biological activity detected in this natural compound [1, 2]. Gossypol was discovered by Longmore and Marchlewski at the turn of the 19th and 20th centuries, having purified it by precipitation from an essential solution using acetic acid [3]. Adams et al. elucidated and reported the gossypol-acetic acid structure as a 1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-5,5′-disopropyl-(2,2′-binaphthalene)-8,8′-dicarboxaldehyde (С32Н34О10, Mr=578.6). Gossypol, characterized by a rich yellow color due to a long conjugation chain, was positioned as an industrial dye; however, it demonstrated significant instability in the light. Later it was found that its instability is not due to molecular decomposition but to the existence of three tautomeric keto-enol forms in equilibrium (fig. 1).

Fig. 1: The keto-enol tautomerization equilibrium of gossypol [4]

As can be seen in fig. 1 gossypol is an asymmetrical molecule and has restricted rotation around the binaphthyl bond. Therefore, enantiomers with different biological activity exist [5]. 

As a sesquiterpenoid and polyphenol, gossypol exhibits the properties of a polybasic-OH acid (pKa1 = 6.73; pKa2 = 7.38; pKa3 = 11.82; pKa 4 = 12.68; pKa5 = 13.89) and is capable of forming azomethines (Schiff bases), oxidation, methylation products, etc. with different biological activity [6]. Numerous biological effects of gossypol on cell lines, animals and humans have been claimed: hypolipidemic, antibacterial, antiparasitic, antiviral, antitumor action [7-9]. The authors [10] characterize gossypol as a promising substance in the development of drugs for diseases such as resistant tumors, HIV, malaria, and psoriasis. However, its use in medicine is limited due to toxicity [11], insufficient accumulated data on the mechanisms of action, as well as the lack of existing techniques to assess authenticity, purity and content [12].

The present work is aimed on in silico methods application to predict the biological activity of gossypol derivatives and the development of Physico-chemical analysis techniques for gossypol-acetic acid (GAA) quality control.

MATERIALS AND METHODS

Powder substance

In this work, gossypol-acetic acid (GAA) produced by the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry (the content of API ≥97, 5%) is presented in a real (fig. 2) and virtual molecular structures (RMS, VMS, respectively) of gossypol derivatives [13].

Sample preparation

The solvents–N, N-dimethylformamide anhydrous, 99.8% (DMF), ethanol 96% (Sigma-Aldrich, USA); deionized high resistance water (18.2 MΩ•cm at 25 °C, Milli-Q system); chloroform (anhydrous, ≥99%), acetone (reagent, ≥99.5%)–were used to evaluate the solubility of GAA under equilibrium and kinetic conditions.

Fig. 2: Structure of gossypol-acetic acid (GAA) [14], real molecular structure of GAA: 7-(8-formyl-1,6,7-trihydroxy-3-methyl-5-propan-2-ylnaphthalen-2-yl)-2,3,8trihydroxy-6-methyl-4-propan-2-ylnaphthalene-1-carbaldehyde

Methods of research

To select "candidate substances", we used QSAR (Quantitative Structure-Activity Relationships) modeling-a procedure for constructing models of structures to predict their properties. We used ChemicPen software to construct and then save VMS in. chp and. mol formats and ChemDesc to work with Wiener, Rouvray, Balaban, bypass and electropy topological indices [15-18].

In silico modelling: PASS (Prediction of Activity Spectra for Substances) Online [19], was used to predict the biological activity of loaded VMS using the criteria of Pa (probability to be active) and Pi (probability to be inactive) [16]. The ratio Pa/Pi>1 was taken as the maximum value of activity; while the quantitative value of the property, close to zero (Pa/Pi→0), was considered as "not active".

The GAA analysis in the mid-infrared region was carried out using an Agilent Cary 630 FTIR spectrophotometer with a diamond ATR accessory (Agilent Technologies, USA). The spectral range is 4000–750 cm-1. The resolution is less than 2 cm-1, the correctness of the wave number is 0.05 cm-1, the reproducibility of the wave number is 0.005 cm-1. The thickness of the absorbing layer is 1.5 nm (the clamping device guarantees the setting of an optimal and reproducible pressure).

The optical density of GAA solutions in N, N-DMF in the UV region was measured on a Cary-60 spectrophotometer (Agilent Technologies, USA).

The GAA particles size and shape were determined by the optical microscopy method (microscope Altami BIO 2, Russia) with a 10-fold increase.

Kinetic dissolution of the GAA in ethanol was carried out by the method of low-angle laser light scattering (LALLS) [20] on Mastersizer 3600 optical set-up (Malvern Panalytical, United Kingdom) (fig. 3).

A 30-second background measurement was made prior to adding the examined disperse system to account for electrical background and scattering from the optics and ‘clean’ dispersant. The measurement of Laser Obscuration Time (LOT) was started while adding water to the cell and continued with 10 s intervals until complete dissolution of the sample with no more change of the examined parameter.

Fig. 3: Optical set-up of kinetic dissolution test. 1-He-Ne laser 632.8 nm; 2-capacity cell with examined disperse system; 3–diffraction pattern; 4–detector [21]

Spirotox method

Research of GAA biological activity was carried out with the use of test culture Spirostomum ambiguum, characterized by statistically reliable sensitivity to toxicants. The kinetic scheme of ligand-induced death of S. ambiguum is well described in [22]. The dependence of test culture lifetime (tL,s) on temperature (T,K) was linearized in "Arrenius coordinates" [23, 24]:

Statistics

All obtained results were processed by statistical methods using software packages of OriginPro 2017. Each value in the fig. represents "mean±SD"; significant differences were considered when p<0.05.

RESULTS AND DISCUSSION 

In silico research

The fourteen structures of VMS of GAA constructed using ChemicDescript software are presented in table 1 [25].

Table 1: Gossypol derivatives-virtual related chemical structures

RMS of gossypol (fig. 2)
VMS of gossypol derivatives (by chemic descript)
Molecular structures Molecular structures
Name Structure Name Structure
1 Gihsypolic acid 8 Methanol derivative
2 Imine-the Schiff base 9 Hexamethyl ether
3 Secondary amine 10 Dimethyl ether
4 Gossiden 11 Tetramethyl ether
5 Gossypol-o-binaftoketon 12 Apogossypol
6 Gossypolom 13 Hexamethyl ether of apogossypol
7 Methyl-derivative 14 Hexamethyl ether of diisopropyl apogossypol

Fig. 4: The structure-activity ratio between balaban index and predicted properties of activity for gossypol derivatives, expressed in Pa: red-antitumor; blue-antiandrogenic; green-antiviral. The fig. in the diagram correspond to the GAA VMS from table 1

QSAR screening of VMS made it possible to choose one of five topological indiсes-distance-based Balaban index (J), reflecting the most significant differences for this type of compound with a certain activity, topology and geometry [26]. With the help of PASS Online product, we analyzed the quantitative values of the activity expressed in Pa probabilities. Fig. 4 shows a two-dimensional diagram of "structure-property" correlations for the most significantly manifested indicators of biological activity: antitumor (antagonism of the apoptosis regulator Bcl2), antiandrogenic (induction of azoospermia) and antiviral.

Analysis of the obtained diagrams showed that for most VMS, as well as for RMS of GAA, in fig. 4, the values of antitumor activity are as close as possible to “1” (Pa from 0.8 to 0.9), which may indicate a high probability of a positive effect when using GAA derivatives as part of antitumor agents [27]. A review of scientific publications and the results of experiments conducted in China [28] indicate the ability of drugs containing Gossypol to suppress spermatogenesis, causing azoospermia. Our QSAR study confirmed the literature data: the probability to be active (Pa) for the majority (the exception are VMS 2 and 9) of gossypol derivatives was in the range from 0.6 to 0.8 (blue circles in see fig. 4). According to the analysis of the literature data [29], Gossypol has antiviral properties against HIV, influenza and other diseases. The results of our studies demonstrate the mutual correspondence of the Balaban index and the Pa indicator in fig. 4 with the absence of significant activity of this property in 15 representatives of VMS–values of Pa ≤0.5.

Physico-chemical research

Description of GAA

The GAA substance solubility was evaluated in different solvents (table 2), which made it possible to determine the purity and consequently prognose the rate of release of the active pharmaceutical ingredient (API) from the finished dosage form, its absorption into the bloodstream and the achievement of a therapeutic effect [30].

Table 2: Solubility of GAA substance in various solvents

Solvent Approximate volume of solvent in millilitres per gram of solute Descriptive term
Acetone from 1 to 10 Freely soluble
Chloroform from 30 to100 Sparingly soluble
Ethanol from 100 tо 1000 Slightly soluble
Water more than 10 000 Slightly soluble

The observed differences in the solvent solubility indicate the high lipophilicity of GAA pharmaceutical substance (fig. 2).

The shape of substance particles plays a significant role for the proper quality of manufactured dosage forms, including such parameter as the release of API (fig. 5).

Microscopy results showed that GAA particles are irregularly shaped, with sharp corners and smooth surface, and mostly highly dispersed. The predominance of fine particles facilitates the release of API from the dosage form, and, taking into account the lipophilicity of gossypol log P=8.2 also increases bioavailability [31].

The high dispersion of the GAA powder and the low colloidal stability were confirmed by the DLS method according to the parameters: size distribution, polydispersity index (Pdl), average scattering light intensity (in kilo counts per second, kcps) and ζ-potential (table 3).

Fig. 5: Visualization of particles (size and shape) and granulometric composition (on the insert) of GAA by optical microscopy

Table 3: Characterization of nanoparticles in polyelectrolyte dispersions

Sample C, g ml-1 Size±SD, nm PDl±SD Count rate kcps, ±SD, mV
Gossypol-acetic acid in ethanol 3.0·10-4 0.8±0.2 0.36±0.01 271 -0.9±8

mean±SD, n=5, P=0.95

Fig. 6: IR spectra of GAA substance

Fourier transform infrared (FT-IR) spectroscopy

The vibrational-rotational spectrum of the GAA substance obtained by the ATR technique is shown in fig. 6.

The analysis of the IR spectrum showed the presence of characteristic light transmission bands corresponding to vibrations of bonds between atoms in the structural fragments of alcohol and phenolic hydroxyls, carbonyls, and numerous methylene groups (table 4).

Table 4: The main transmittance bands in the IR spectrum of GAA substance

Frequency range, sm-1 Group Compound class Appearance/Comments
3550-3200 O-H stretching alcohol strong, broad/
3100-3000 C-H stretching alkene medium
1690-1640 C=O stretching conjugated ketone strong
1650-1566 C=C stretching cyclic alkene medium
1450 C-H bending alkane methyl group
1390-1310 O-H bending phenol medium
1085-1050 C-O stretching primary alcohol strong

Thus, the results of FT-IR spectroscopy analysis allowed to propose this method as the first identification technique to characterize the investigated compound.

Ultraviolet-visible spectroscopy

Electronic spectra of GAA solutions in N,N-DMF exhibited characteristic absorption maximum at 380 nm, rising with concentration change from 2.5·10-6 tо 2.5·10-5 mmol · L-1. This allowed not only identify the samples but also made the calibration curve for API assay using linear regression analysis (fig. 7).

The results of the UV method allowed us to calculate the specific absorption coefficient for a series of GAA solutions and use it to determine the identity of the GAA substance. The specific absorption coefficient =42.4±0.8 (100 ml·g-1·cm-1) was also calculated at λmax=380 nm), and mean error =0.02% (n=5, P=0.95) (table 5).

A B

Fig. 7: UV spectra of GAA solutions in N,N-DMF in the concentration range from 2.5·10-6 tо 2.5·10-5 mmol · L-1(A); calibration line (B)

Table 5: The specific absorption coefficient of GAA in N,N-DMF

С ·10-5, mol · L-1 (at λmax=380 nm) (100 ml·g-1·cm-1)
2,5 0.060 41.4
5 0.126 43.5
9 0.226 43.3
15 0.374 43.0
25 0.590 40.7

n=5, P=0.95

A B

Fig. 8: Determination the dissolution rate of GAA: dependence of LO values on time in dispersed GAA ethanol solution depicted in linear (А) and semi-log coordinates (B), (n=5; P=0.95)

Dissolution kinetics

The kinetic of GAA dissolution based on light scattering indicatrix registered in time was used to determine the Laser Obscuration (LO) values: LO= 1 ─ I/I0 ∙100%. Fig. 8 depicts the dependence of OL from the time during which GAA substance was dissolved in ethanol.

The kinetics of API’s dissolution in ethanol turned out to be a two-step process: a sharp decrease in the recorded LO parameter from the onset of dissolution (first stage) was replaced by gradual decrease in the LO value until the plateau (second stage). We fixed it as complete dissolution of the substance. The first stage was the speed‒determining. We calculated the values of the constant dissolution rate for log-linearization as k =–tgα=0,041 s-1. The calculated dissolution rate constant can be used to characterize the identity of GAA in quality control and standardization process [32].

Spirotox

To estimate the biological activity of GAA substance, the temperature dependence of S. ambiguum death in the range of 295-303 K in increments of 2 K was analyzed (fig. 9).

A B

Fig. 9: Dependence of life span of S. ambiguum on temperature in the control N,N-DMF: water 1:30 solution (A) and in 1 mmol solution of GAA in N,N-DMF: water 1:30 (B) in linear and Arrhenius (on inserts) coordinates (n=5, P=0.90)

Table 6: The calculated °bsEa values of ligand-induced S. ambiguum death process in control N,N-DMF: water 1:30 solution and 1 mmol solution of GAA in N,N-DMF: water 1:30

Sample °bsEa±SD, kJ·mol-1
N,N-DMF: water solution (1:30)–control solution 213.4±1.5
1 mmol N,N-DMF: water solution (1:30)–test solution 174.2±0.5

mean±SD, n=5, Р=0.90

The observed activation energy (°bsEa) values for test and control solutions were found using Arrhenius coordinates (table 6).

The value of °bsEa reflects the ligand-receptor interaction accompanied by cell death. The lower activation energy, as well as the reduction of S. ambiguum's lifetime by about 1.2 times at the same temperature for control N,N-DMF: water solution compared to 1 mmol GAA in N,N-DMF, indicates a higher biological activity/toxicity of the nature polyphenol compound [33].

CONCLUSION

An integrated approach to the study of the structure and properties of natural polyphenol originating from the cotton plant (Gossypium), based on an in silico approach and analytical techniques, allowed not only give the substance a complete characteristic for the purpose of standardization but also to predict its biological activity for the development of ready-made targeted drugs.

FUNDING

This paper has been supported by the RUDN University Strategic Academic Leadership Program.

AUTHORS CONTRIBUTIONS

All the authors have contributed equally

CONFLICT OF INTERESTS

The authors declare no other conflict of interest.

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