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19
Section 3. Biotechnology
https://doi.org/10.29013/AJT-20-9.10-11-22
Lolakhon Ettibaeva,
Senior Teacher of the Department of Chemistry at Gulistan State University
E-mail: [email protected] Abdurakhmanova Ugiloy Head of the Department of Chemistry at Gulistan State University
E-mail: [email protected] Boburjon Yusupov, Master of chemistry department Makhammadiev Sharoffiddin, Master of chemistry department Faculty of Natural Science, Gulistan State University, Uzbekistan
ACQUISITION OF SUPRAMOLECULAR COMPLEX OF GLITSIRRIZIN ACID WITH MENTHOL AND THEIR CHEMICAL IDENTIFICATION
Abstract. In this study, the supramolecular complexes were obtained from glycyrrhizic acid (GA) and Menthol (1R, 2S, 5R) -2-isopropyl-5-methylcyclogeksanol) with various ratios. It has been shown the highest yield of the supramolecular complex with GA and Menthol in a 4:1 (GA: Menthol) ratio. This combination has been chemically identified by comparing the IR-Fure spectra of the initial agents. Similarity coefficients of the initial agents for the supramolecular complex GA: Menthol (4:1) showed as a 0.84 and 0.70 coefficient with known functional groups of GA.
Keywords: Glycyrrhiza glabra L., glycyrrhizic acid, menthol, supramolecular complex, IR spec-troscopy.
Introduction
Glycyrrhizic acid (GA) (20 ^-carboxy-11-oxo-30-norolean- 12-en-3 j3-il-2-O-^-D-glucopyranuronosyl-a-D-glucopyranose-duronic acid) is a valuable raw material, which is important in the food industry, cosmetology, and other industries. Based on the chemical structure of glycyrrhizic acid, it is a gly-coside, which is formed by glucuronic acid residue with triterpene-glycyrrhizic acid [1-2] (Figure 1 A). Many studies on the physical-chemical features of GA
showed the molecule contains hydrophobic (triter-pene fragment) and hydrophilic (two glucuronide residues) parts that are assumed to make a mycelium in the complexes [3-4].
The GA is extracted from the root of licorice (GlycyrrhizaglabraL. and Glycyrrhiza uralicaL.) [5]. Several compounds of GA were obtained and used for various purposes. The GA can be easily formed with other molecules to make a supramolecular compound called a "guest-host" type [6]. Other studies
were also confirmed that GA provides a "host-guest" type of auto association [7-9].
The supramolecular complex of GA is characterized as a ring dimer structure with hydrophobic cavities due to intermolecular hydrogen bonds. This cavity can provide a "guest-host" type formation [10].
The GA reacts with many other molecules to yield a supramolecular complex [11-15]. Particularly, increasing GA concentration stimulated the auto association of the "guest-host" type of supramolecular complex with streptomycin under the various ratio of GA: streptomycin (1:1, 2:1, 3:1, 3:2) [16].
At present, a lot of supramolecular complexes based GA with various agents were chemically synthesized and used in various fields. For instance, before sowing wheat seed treated GA and its denatured salt combined with "Tebukonazol" enhanced the resistance to various pathogenic infections at the early stage of plant organogenesis and increased yield productivity [9]. It is assumed that GA molecules (~60-100) form vesicles/mycelium and provide increased transmembrane penetration with the main drug molecules involved [9].
The therapeutic dosage of some medicines can often cause adverse effects [17-18]. It is worth noting that positive results are obtained based on the use ofsu-pramolecular compounds ofGA. For example, a supramolecular complex of GA and streptomycin can reduce the dose of the drug used [19-20]. Supramolecular complexes are used in practice as an effective method of increasing the water solubility level ofpharmacological preparations based on mechanical and chemical mechanisms. In particular, studies have shown a significant increase in the solubility levels of drugs such as Diazepam (Sibazon), "Nifedipine", "Ibuprofen". It is assumed that this is due to the physicochemical properties ofthe micelles that form the GA molecule [9].
Menthol (C10H20O) is a terpenoid that found in the essential oils of the mint family (Mentha sp.). It is a white crystalline solid that can dissolve well at room temperature or partially at high temperatures. There are several isomers of menthol including isomenthol, neomenthol, neo-isomenthol with peppermint odor. Among them, (-) menthol (1R, 2S, 5R)-2-isopropyl-5-methylcyclohexaneanol) is one of the strong aromatic molecules in nature (Figure 1 B).
Figure 1. Molecular structures of glycyrrhizic acid (GA) and Menthol. (A) Glycyrrhizic acid (Empirical formula - C42H62O16; 20P -carboxy-11-oxo-30-norolean-12-en-3 P-il-2-O- P -D-glucopyranuronosyl-a-D-glucopyranose-duronic acid) [2]; (B) Menthol (C10H20O);
The (-) Menthol is a known with strong cooling and refreshing agent and four times higher efficacy than (+) menthol isomers [21]. Therefore, some of its derivatives are widely used in medicine, pharmaceutics, perfumery and food industries. Menthol cools the skin when it applied to the skin; therefore it is used as a medicine during the headache. Moreover, (-) menthol can also use as antiseptic properties to nose and throat mucus [22]. Nowadays, one of the crucial tasks in agriculture involves the enhancing of plant productivity with sustainable chemicals. In the present research, the GA was extracted from the licorice root and synthesized it's a supramolecular complex with menthol.
Main Part
Materials and methods
The spectrophotometric methods for quantitative/qualitative chemical identification of GA in biomaterial content are used effectively [23-26]. Some researchers have noted the efficacy of Glycyrrhiza glabra L. root mass extraction in high-temperature aqueous environments, in the next step, the formation of low concentrations in a vacuum apparatus, HNO3 solution (3%) and extraction of GA based on acetone extracting [27]. Another study used an NH4OH (1%) for the extraction of GA from the root of the licorice. In case, the acid was removed by H2SO4 solution [28].
Extraction of GA. Some methods are available to extraction and chemical identification for the GA from licorice (Glycyrrhiza glabra L.) root [29]. We have used a local licorice root from a Sirdarya region, Uzbekistan. Approximately 100mg which is 92% technical GA dissolved with 3% Sulphate acid and boiled in a water bath until to get a white powder. After then this product was cooled at room temperature overnight. The contents then were filtered with filter paper and washed with cold distilled water. Final extract dried and stored in a dark condition for further experiment. Purity: 92%, Tliq = 210-213 °C / a / D25 = =+48 (dis.water. Ethanol 1: 1); Rf = 0.83 (1), Yield: 98%. IR spectra: 1041 cm-1 (-O ); 1656 cm-1 (CO); 2873 cm-1 (CH3); 3247 cm-1 (OH).
Identification of GA. Chemical identification of GA was performed by using standard methods which are mentioned in some reference [30-31]. In our experiments, root extract was analyzed by using a Perkin Elmer Spectrum IR (Germany; version 10.6.1) IR-Fure spectrometer in the range of4000--500 cm - 1. KBr tablet was used as a standard GA for the preparation of test samples. The IR spectra of GA and ^-indolyl-3-acetic acid were compared with the spectra of the "Biochemica" Biblioteca.
Obtaining a supramolecular complex.
Supramolecular compounds were obtained by the following steps. Firstly, 1.68 g (0.002 M) of GAMAT dissolved 25 ml distilled water and 25 ml ethanol. This solution widely mixed with a magnetic stirrer at 50-60 °C. Then we added 0.156 g (0.001 M) Menthol and continued the mixing process for 5-6 hours. After then the reaction mixture was filtered. The rest ofthe ethanol was removed in a vacuum, and the aqueous part was dried in a lyophilic manner. Final product was a yellow powder: liquid=205-210 °C Rf = = 0.9 (System 2) Units: 85% IR spectrum:1042 (-O-) cm1; 1655 (CO) cm1 2948 (CH3) cm1; 3600-3200 (OH) cm-1. Other supramolecular complexes of GA with Mt were also obtained by this synthesis method:
1. GA: MT (2: 1). Liquid = 218-220 ° C Rf= 0.8 (System 2) Productivity 95%
2. GA: MT (4: 1). Liquid = 220-225 °C, Rf(1) Productivity: 90%.
Reagents and equipment. In our experiment, we have used ethanol, benzene, acetone acid solutions, as well as alkaline solutions as organic solvents for dissolving. The Chromatography (TLC) was used for thick layer:
1) benzine: acetone 5:3,
2) acetonitrile: water 1:2,
3) benzine -ether 15:3;
4) benzine: acetone 5:1
5) acetone: Alcohol is treated with 1: 1.
10% alcohol solution of sulfuric acid (H2SO4) and iodine chamber for chromatography staining were monitored.
Continuously stirring was performed by using a magnetic mixer MM-5 TU25-11834-80. The organic solvent was evaporated from the system on an IR-1M2 rotor evaporator. For drying we have used the Automatic FREEZE-Dryer10-010, a lyophilic device and a PTP TU25-11-1144 unit were used to measure the fluid temperature of the substances. The structure of supramolecular complexes was performed on the IR spectrometer FT-IR System-2000. The Silufol (Czech Republic) plates were used for thin-layer chromatography.
Preparation of a working standard solution for glycyrrhizic acid: 0.10 precise drawer was weighed on analytical weight and placed in a 100ml measuring tube. 10 ml 96% ethanol was added, diluted thoroughly and filled with distilled water to the measuring line (Solution A). After thoroughly shaking the tube, 1 ml of aliquots were taken and diluted with 9 ml. fluid system (Solution B, working standard solution), 0.1 mg/ml. The standard working solution was placed on a chromatograph for analysis.
Preparation of the solvent system: 14 ml of acetonitrile and 0.5 ml of acetic acid and water-filled till 50 ml tube. This solution (pH 3.0-3-5) always used in our work.
Results and discussion
Currently, modern physic-chemical methods are available for GA extraction including boiling of root tissue at high-temperature water [27; 32], NaOH
Table 1. - Quantification of GA from root tissue
Our next experiment was root biomass (5 ± ± 0.5 g) of licorice (Glycyrrhiza glabra L.) was mixed with dilution (distilled water+ammonia solution (3%); +150 °C; 5 MPa) at a ratio of1:10. The prepared extagent in the tube for 120 min continued boil and cooled and filtered. The method was
aqueous solution [33], methanol [34], ethanol [35-36], and the use of ammonia solutions. As well as, the vacuum-pulse methods [33], ultrasound [37] also used to increase the efficiency of extraction.
In our experiment, local licorice (Glycyrrhiza glabra L.) roots were washed with distilled water and dried. Then the roots were mechanically milled to < 0.5 - 1 mm size. Extra agents were selected according to the literature data [38]. Firstly, pieces of the roots were cracked in 70% ethanol at 1:5 ratios and kept in dark for 5 days that stirring constantly. In the next step, the extract was filtered and the ethanol was evaporated under + 75 °C [39]. The mass column (h = 20 cm; 0 = 2.5 cm) was divided into fractions (3) by using a chromatography. The elution was carried out in 1% ethanol (75%) of the ammonia solution. The fractions were analyzed by using high-performance liquid chromatography 1200 series DAD detector with Agilent Technologies (USA), high-performance liquid chromatography (150 x 4.6 mm); 5 ^m) at +20 °C. The flow rate value of the moving phase shows a 0.5 ml/min. The methanol and an acidic acid solution (0.05%) were used as the active phase. Detection was carried out in the250 nm, 275 nm, and 350 nm full-length sectors. As a standard sample, we have used ammonium salts of GA (Sigma-Aldrich, Germany). In results, the higher concentration of the GA can observe at a second fraction (Table 1).
extract of the licorice (Glycyrrhiza glabra L.)
used to determine the amount of GA in the extract. Acetonitrile+methanol+distilled water+acetic acid (35 : 20 : 45 : 1) was used as an active phase (flow rate 0.5 cm/min). Detection was performed at a wavelength of256 nm (Table 2).
Fractions Concentration (in accordance with dry specimen, mg/g) Weight share (%)
1 2.4 0.24
2 5.8 0.65
3 3.6 0.38
It reported that the GA content from aqueous extract is a 13.6% [32] compared to the dry plant mass, a 7.3% [38] in some data, and 0.88% in etha-nol extract [35], and also when used ultra-sound, it was found to be 3.64% [37].
Synthesis and chemical identification of su-pramolecular complex of glycyrrhizic acid with menthol.
The supramolecular complex of GA with menthol was obtained according to (Scheme 1).
Table 2.- GA content in Glycyrrhiza glabra L.root extract (n = 3 - 5)
Conditions of extradiction Identification through gravimetric method Identification through HPLS
Quantity in water extract (%) Quantity in filtered content (%) Quantity in accordance to dry plant mass (%) Concentration in extract (mg/mO Share in extract content (%)
Distilled water + ammonia solution (3%); + 150 °C; 5 MPa 0.074 ± 0.005 0.083 ± 0.04 14.3 ± 0.28 7.75 ± 0.3 0.9 ± 0.04
Scheme 1. Reaction of the supramolecular complex between GA and Menthol
No. Substances T °c Rf (system) Solubility IR spectrum, cm 1
1 GA: Menthol 2:1 218-220 °C 0.8 Water alcohol 3400-3500(0H); 2924(CH3) 2857-(CH2); 1637-1725-(CO) 1085-(-0 )
2 GA: Menthol 4:1 220-225 °C 0.9 Water alcohol 3400-3500(0H); 2924(CH3) 2857-(CH2); 1637-1725-(CO) 1085-(-0 )
3 GA: Menthol 9:1 228-230 °C 0.8 Water alcohol 3400-3500(0H); 2924(CH3) 2857-(CH2); 1637-1725-(CO) 1085-(-0 )
Supramolecular complexes with molar ratio 2:1; Some physical-chemical properties of the result-
4:1; 9:1 of glycyrrhizic acid with menthol were ob- ing supramolecular compounds were studied and tained in water: acetone system. their chemical structures were investigated by IR
spectroscopy (Table 3).
Table 3.- Some physical-chemical descriptions of supramolecular complex of GA with menthol
As shown in (Table 3), all obtained complex compounds are well soluble in water. It can be seen that the liquid temperature is between 200 and 230 °C.
System: benzine: chloroform 3:1
The structure of the supramolecular complex was studied by physical methods based on the interaction of organic molecules with electromagnetic radiation, in particular their IR spectrum (the frequency of vibration of atoms in the molecule, X= 10-4 -10-2 cm-1).
The valence vibrations of hydroxyl groups in the resulting complex compounds are observed in the 3500-3400 cm-1 area, and the valence oscillations of the carboxyl groups in the GA are observed in the field 1725-1690 cm-1 (Table 3). In the IR spectrum of the supramolecular complex GA formed with menthol, asymmetric valence oscillations of the -CH3 group were observed in 2924-2927 cm-1. At the wavelength of 2857-2860 cm 1, it is noted that the symmetric valence oscillations of the -CH2 group are weak, and in the area 1085 cm-1 there are valence oscillations of the -O- group.
Due to the absence of chromophore groups in the L-(-)- menthol molecule, no apparent absorption was observed in their UV spectra. Therefore, the IR spectra of menthol and its complex with GA were studied.
In the formation of a supramolecular compound, it was found that the host molecule contains several active bonds that form several bonds. It was noted that the host and guest geometric structure, that is, the diameter of the gap in the receptor molecule, corresponds to the radius of the substrate molecule. The complementarity feature allows the host molecule to select guests in a well-defined structure.
It is worth noting that the GA molecule has a "guest-host" formation of clathrate compounds, its complexity with a number of medications used in medicine, to increase their activity and increase the treatment index by the effect of molecular capsules [40-41].
In the picture below, the supramolecular complex of 4:1 GA with mentolol was recorded by using "Shimadzu" IR-Fure spectrophotometer device (Japan) and "Perkin-Elmer Spectrum IR" - 10.6.1 in the absorption range of 3400-3500 cm-1 (Figure 3; Figure 4).
In this case, the shape of the oscillation is explained by the amplitude of all the atoms vibrating at the same frequency and, in turn, the change in the length of the chemical bond and the interconnection angle at normal vibrations.
If the angles between atoms change in the course of vibration, this type of oscillation is called deformation vibration. However, pure valence or pure deformation oscillations occur only in linear molecules or in highly symmetric molecules and ions (octahedrons, tetrahedrons, squares, etc.). In most multi-atom molecules and ions, mixed valence-deformation oscillations occur together, and the angles between them change as the valence bond distances change.
One of the most widely used methods for the identification of menthol with GA derived supra-molecular compounds has been the use ofhigh-per-formance liquid chromatography. Chromatography isoconate flow rate 1ml/min and acetylitrile: acetate buffer system was used as an eulent.
The following are the chromatographic conditions:
- chromatograph- Agilent Technologies - 1200
- column - Exlipce XDB - C18, 5mkm, 4.8 x 150 mm
- eluent-acetoneitrile: acetone buffer (21:89)
- detector - UV (254nm)
- regime-isocratic
- Temperature - 25 ° C
- vcol-5mkl
The results showed that the amount of glycyrrhi-zic acid in the supramolecular complex compounds was theoretically calculated and the error rate was ± 1-1.5%.
LTTI-I
Figure 3. IR-Fure spectra of the supramolecular complex GA generated by menthol. The IR-Fure spectra were recorded using an IR-Fure spectrophotometer device (Perkin-Elmer Spectrum IR - 10.6.1 ; USA) in the absorption range of 3400-3500 cm-1. The spectra were recorded at a resolution of >4 cm-1. The vacuum conditions (0.1-0.05 mm s.u) were pressed in the form of a spectral pure KBr ("Merck", Germany) for adsorption of moisture in the test samples
Figure 4. IR spectrum of menthol. The IR-Fure spectra were recorded using an IR-Fure spectrophotometer device (Perkin-Elmer Spectrum IR - 10.6.1 ; USA) in the absorption range of 3400-3500 cm1. The spectra were determined at a resolution value of > 4 cm1. The vacuum conditions (0.1 -0.05 mm s.u) were pressed in the form of a spectral pure KBr ("Merck", Germany) for adsorption of moisture in the test samples
Quantitative determination was performed with respect to standard GA solution. Because the menthol molecule does not contain chromophore groups, it cannot be detected by simple spectroscopic methods. Therefore, in our experiments, we used
quantitative GA to qualitatively and quantitatively determination. Quantitative computations were compared relative to the peak area at the time of the standard solution retention (Table 4).
Table 4.- Results of the HPLS-based study of supramolecular complex of GA with menthol
Complexes Time for holding complexes in the column, min. Quantity calculated from the theoretical point. mg/100ml Result obtained practically mg/100ml
GA 6.967 100.0 100.0
2:1 7.118 100.0 98.7
4:1 7.129 100.0 99.1
9:1 7.159 100.0 98.6
Initial agents for the IR-Fure spectra GA: Menthol (4:1) supramolecular complex recorded in the experiments by using an IR-Fure spectrophotometer device (Perkin-Elmer Spectrum IR-10.6.1; USA). The correlation coefficients of functional groups in the molecules GA (С42Н62О16; "Biochemica", Ger-many)and ISK (C12H9NO2; Biochemica, Germany) were 0.84 and 0.70, respectively.
It was noted that the resulting GA: Menthol (4:1) supramolecular complex is 99.1%.
As can be seen from the values presented in (Table 2), the quantities oftheoretically obtained complexes are consistent with the results obtained in practice, and their difference does not exceed 1.0-1.5%. This proves that the method of HPLS can be used to standardize the obtained supramolecular complex compounds.
Qualitatively standard of GA hold time in the column was set as (6.5-7.0 min).
Chemical synthesis the supramolecular complexes of GA with physiologically active compounds
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Conclusion
In the experiments, we have obtained the supramolecular complexes of glycyrrhizic acid and menthol. The resulting supramolecular complexes have the highest yields in the ratio of 4:1, and the resulting GA: Menthol (4:1) complex has been chemically identified by comparing the IR-Fure spectra of the primary agents.
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