**2.4. Supramolecular structures of glycyrrhizic acid (GA) and poorly soluble drugs water solutions: Synthesis and properties**

582 The Complex World of Polysaccharides

Fibregum

n

Arabinogalacta

Hydroxyethyl starch 200/0,5

Dextran 70

Dextran 40

Dextran 10

single intragastric injection.

[23,39]. Note that AG macromolecules with MM (Molecular Mass) ~9 kDa are likewise the

Furthermore, the analyzed polysaccharides experience almost no mechanical failure on lowrate grinding in a rotary mill (Table 3). Thus, ball rotary milling appears to be most often preferable, as molecular mass changes in technologically produced polymers are commonly unwanted in view of their further use in dietary supplements and drugs, otherwise

kDa

Native 146.6 256.7 1.8 <75.9 <528.2 Planetary mill, 20g, 20min 31.4 55.2 1.8 <16.3 <113.5 Ball mill, 1g, 4hours 120.3 231.6 1.9 <60.3 <478.4

Native 13.9 17.3 1.2 <9.0 <27.9 Planetary mill, 20g, 20min 9.3 11.2 1.2 <6.1 <18.4 Ball mill, 1g, 4hours 13.1 16.3 1.2 <8.1 <26.2

Native 47.9 116.9 2.4 <20.9 <265.3 Planetary mill, 20g, 20min 26.6 55.2 2.1 <12.7 <118.9 Ball mill, 1g, 4hours 45.6 105.5 2.3 <20.0 <237.6

Native 30.9 76.4 2.5 <14.0 <174.7 Planetary mill, 20g, 20min 22.7 54.8 2.4 <10.4 <123.5 Ball mill, 1g, 4hours 29.6 73.5 2.5 <13.4 <169.2

Native 24.6 38.0 1.5 <13.3 <72.3 Planetary mill, 20g, 20min 19.5 31.9 1.6 <10.4 <61.3 Ball mill, 1g, 4hours 24.3 37.4 1.5 <13.0 <71.2

Native 8.3 13.4 1.6 <4.2 <26.4 Planetary mill, 20g, 20min 8.0 12.1 1.5 <41.9 <22.7 Ball mill, 1g, 4hours 8.3 13.4 1.6 <4.2 <26.3

Toxicological tests of the milled polysaccharides show that a single intragastric injection administered at doses from 500 to 6000 mg/kg body weight caused no death in experimental animals. Their appearance, behavior, and state were within the background over the whole dose range; no statistically significant changes in body temperature relative to the control was observed, and body weight growth was uniform in all groups. Injections of the tested polysaccharides neither induced any considerable effect on the central nervous system of the mice. Patomorphological postmortem examination of mice in 14 days after polysaccharide administration revealed no pathology in thoracic and abdominal cavities. The median lethal dose LD50 for all polysaccharides was over 6000 mg/kg body weight on

Mw, kDa

Mw/ Mn

Weight shares of macromolecules, kDa 10% 90%

main product of chemical destruction of Canadian larch AG [42].

additional tests and standardization may be required.

Sample Treatment Mn,

**Table 3.** Molecular mass distribution of polysaccharides

Biosynthetic and natural plant-derived carbohydrate-bearing metabolic agents have been increasingly used for obtaining complexes (clathrates) with drugs in drug delivery research. The mechanism of GA-pharmacon interaction in solutions may consist in involving drug molecules into self-associates (micelles) that exist in a wide range of concentrations in GA solutions. Until recently however, the existence of micelles in GA solutions had no direct proof but was either inferred from measured concentration dependences of solution viscosity [43] or was studied by dynamic NMR spectroscopy in water-methanol solutions [44]; the latter (30% concentration) were at the same time used as solvent for technical reasons. Thus, the molecular mechanism of drug complexing remained unclear, whether it was incorporation into micelles or supramolecular complexes with GA in water solutions, without organic solvents which change notably the GA-pharmacon reactions.

GA water solutions, with and without the presence of poorly soluble drugs, were investigated in [29] using gel permeation chromatography, which allows detecting selfassociates/micelles and estimating their sizes and concentrations. On the other hand, solid GA-drug dispersions were obtained with the mechanochemical approach developed earlier [10, 11]. The binding strengths in GA-pharmacon supramolecular complexes or GA micelles in water solutions were compared using the criterion of solubility increase in poorly soluble drugs [22] and studied in terms of pharmacological activity.

Chromatograms of GA water solutions (Fig. 3) show peaks of high-molecular (~ 46-67 kDa) forms over all studied concentration ranges, while the GA molecular weight is 836.96 Da (Table 4).

The peak areas, being proportional to the solution concentrations and calculated relative to the known amounts of standard dextrans, show that almost all GA is stored in the solution. This, in our view, is evidence for the existence of GA self-associates (micelles). The critical concentration of micelle formation (CMC) was estimated earlier [43] from viscosity change in GA solutions to be 0.004 wt. % (0.05 mM). In our case estimating exact CMC is difficult for the limited sensitivity of refractometric detector and for dilution in the chromatographic column. However, it may be inferred from the time to the chromatographic peak (~0,5 min) and the elution rate. The solution we studied underwent about 10-fold dilution, and the derived CMC is ~< 0.0001 wt.%, (0.001mM), or far less than in water-methanol solution (0.04-0.08 wt.% or 0.5-1.0 mM) [44]. In diluted 0.01-0.001 wt. % solutions, there is only one type of micelles (~ 66 kDa) with a very low Mw/Mn ratio of 1.08-1.06. As the GA solutions reach concentrations of 0.5 wt.%, micelles decrease in weight to form ~ 46 kDa bodies and increase in Mw/Mn ratios. Therefore, almost all glycyrrhizic acid in water solutions from 0.0001 to 0.5 wt.% exists in the self-associated form of micelles, out of which the ones with MM= ~ 66 kDa consisting of about 80 GA molecules are most stable.

**Figure 3.** GPS chromatograms of glycyrrhizic acid water solution. 1 - concentration - 0.001, 2 - 0.01, 3 - 0.1, 4 - 0.5 wt.%.


**Table 4.** Molecular-mass characteristics of micelles in solutions of glycyrrhizic acid and its composition with ibuprofen

#### *2.4.1. GA solid dispersions with poorly water-soluble drugs*

Solid GA dispersions with ibuprofen, phenylbutazone, clozapine, and diazepam were obtained by milling with GA (10:1 mass, or 2.5/1 – 4/1 molar ratios). Their thermal analysis data indicate that the crystalline phase of the drugs becomes disordered, until complete loss of crystallinity. In our view, the molecules of drugs may disperse into the excess solid GA with formation of solid solutions. Other investigated systems behave in a similar way.

As the dispersions dissolve, the drugs become more soluble in water (Table 1), this being evidence of the efficiency of GA as a solubilizing agent and the mechanochemical treatment as a tool for synthesis of water-soluble solid dispersions. GA has a nearly intermediate solubilizing effect higher than HES but lower than AG.

#### *2.4.2. GPC of dissolved GA-drug solid dispersions*

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0.1, 4 - 0.5 wt.%.

COMPOSITION Molecular

GLYCYRRIZIC ACID Mw/Mn

GLYCYRRIZIC ACID/

Treated in planetary

/IBUPROFEN 10/1 w/w

mill 3 min

with ibuprofen

masses

Mw/Mn

*2.4.1. GA solid dispersions with poorly water-soluble drugs* 

solubilizing effect higher than HES but lower than AG.

**Figure 3.** GPS chromatograms of glycyrrhizic acid water solution. 1 - concentration - 0.001, 2 - 0.01, 3 -

**Table 4.** Molecular-mass characteristics of micelles in solutions of glycyrrhizic acid and its composition

Solid GA dispersions with ibuprofen, phenylbutazone, clozapine, and diazepam were obtained by milling with GA (10:1 mass, or 2.5/1 – 4/1 molar ratios). Their thermal analysis data indicate that the crystalline phase of the drugs becomes disordered, until complete loss of crystallinity. In our view, the molecules of drugs may disperse into the excess solid GA with formation of solid solutions. Other investigated systems behave in a similar way.

As the dispersions dissolve, the drugs become more soluble in water (Table 1), this being evidence of the efficiency of GA as a solubilizing agent and the mechanochemical treatment as a tool for synthesis of water-soluble solid dispersions. GA has a nearly intermediate

Solution concentration, wt. %

0.001 0.01 0.1 0.5

kDa 65.93/61.0 66.2/62.5 60.7/57.3 45.3/36.5

kDa 69.0/67.2 69.4/65.3 65.2/61.7 48.6/39.0

The GPC data for GA-ibuprofen dispersions in water are shown in Table 4. Similar results of MM increase in micelles were obtained for the GA-diazepam, GA- phenylbutazone, and GA-clozapine systems. The peak areas, proportional to the concentrations of the analyzed solutions, indicate that they bear almost the entire mass of GA-drug samples. Thus, the dissolved drugs in the GA-drug complexes are likewise self-associated in micelles which are stable in a broad range of concentrations, as well as in the solutions of native GA. Therefore, we suggest that poorly soluble drugs increase their solubility by incorporating into GA micelles/self-associates. GA molecules contain a hydrophilic (two glucuronide residues) and a hydrophobic (triterpene) components (Fig. 4).

**Figure 4.** The structure of glycyrrhizic acid

In micelles, the latter are most likely oriented inward and the former toward the outer surface of the self-associate, while the drug molecules may occur either in the interior hydrophobic part or complex with the exterior hydrophilic part of the micelles. Unfortunately, the experimental evidence is insufficient to judge about these subtle GA-drug interaction mechanisms. Generally, the MM of GA-pharmacon micelles are 5-7% higher than those in GA water solutions, all over the studied range of concentrations. Another possibility is that drug molecules may substitute for some GA molecules while the micelles generally grow in size. As the solution concentration increases, the size difference of micelles grows correspondingly. Note that the region of high GA concentrations is proximal to the conditions in which the solubility of drugs was measured, this being additional evidence for the suggested mechanism of water solubility increase. An explanation for the decreasing MM differences on dilution may be that drug molecules can escape from GA micelles during GPC process to diluted solutions and appear in chromatograms as individual substances eluted at different rates [45]. Anyway, further investigation is needed to gain more insights into the GA-drug interaction.
