*Corresponding Author:
X. Zhu
Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, No.18 Chaowang Road, Hangzhou 310014, China
Date of Submission 30 March 2017
Date of Revision 04 May 2018
Date of Acceptance 11 January 2019
Indian J Pharm Sci 2019;81(2):211-218  

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Candesartan cilexetil was complexed with glycyrrhizic acid to enhance its aqueous solubility. Solid dispersion of candesartan cilexetil and glycyrrhizic acid were prepared by mechanochemical technique of milling at 120 rpm for 8 h. This solid dispersion could significantly improve the solubility of candesartan cilexetil in water. In vitro drug release showed a significant increase in drug dissolution rate after formation of a solid dispersion with glycyrrhizic acid. It was also found that the chemical stability of the solid dispersions was enhanced compared to pure candesartan cilexetil in a stability study involving heat, light, and long-term storage. The results of differential scanning calorimetry, X-ray powder diffraction shown that the drug existed in the amorphous state. Fourier-transform infrared spectra demonstrated that hydrogen bonding occurred between candesartan cilexetil and glycyrrhizic acid with mechanical and chemical treatment.


Candesartan cilexetil, glycyrrhizic acid, mechanochemical technique, solubility, stability

Poorly water-soluble drugs often require higher dosage in order to reach the therapeutic plasma concentrations after oral administration. Improvement in the extent and rate of dissolution is highly desirable for such compounds, as this can lead to an increased and more reproducible oral bioavailability and subsequently to clinically relevant dose reduction and more reliable therapy [1]. A solid dispersion approach could increase the dissolution rate of poorly water-soluble drugs thereby improve their bioavailability [2,3]. Usually, solid dispersions are two-component systems in which the drug is incorporated in the hydrophilic carrier. The drug within the hydrophilic carrier may be dispersed molecularly or occur as amorphous components [4].

Candesartan cilexetil (Figure 1A), an inactive ester prodrug, is widely used to treat essential hypertension and heart failure [5]. During absorption from the gastrointestinal tract, it is rapidly and completely hydrolysed into candesartan, which is a highly potent and selective non-peptide angiotensin-II type-1 (AT1) receptor antagonist [6]. Candesartan cilexetil, as a new antagonist of AT1, has attracted greater attention of the antihypertensive drugs market [7]. However, the oral bioavailability of candesartan cilexetil (~15 %) is not as good as expected, mainly because of its poor water-solubility, efflux by drug resistance pumps in intestinal epithelial cells (P-glycoprotein), and first pass metabolism by cytochrome P450 3A4 [8,9]. Thus, several attempts were made to improve the dissolution rate and bioavailability of candesartan cilexetil, such as preparation of solid dispersions [10-12], cyclodextrins inclusion complexes [13], self-emulsifying drug delivery system [14], nanoparticles [15], or preparation of micelles [16]. Although much progress has been achieved through previous research, many formulations were normally prepared as liquids that is considered unfriendly to the environment and some formulations used large quantity of surfactants, which can induce gastrointestinal irritation.


Figure 1: Chemical structures
A. Candesartan cilexetil and B. glycyrrhizic acid

In this study, solid dispersion of candesartan cilexetil were prepared employing the solid-state mechanochemistry, which has been found to be green, environment protecting, fast and an efficient process technology for pharmaceutical solid dispersion preparation [17,18]. Moreover, high fever, which could result by the use of a partial decomposed drug could be avoided [19]. Mechanochemical technology was widely used in terms of improving the water solubility of poorly soluble drugs, such as the preparation of solid dispersions, cyclodextrins inclusion complexes, eutectic and so on [20]. Recently, the use of mechanochemistry has become very attractive in the field of pharmaceutical industry to enhance the dissolution rate of poorly water-soluble drugs [21]. In fact, compared with traditional “liquid phase” method, mechanochemical process offers significant advantages such as one-stage process, absence of solvents or melts and respective additional procedures, high strength of formed complexes and low operating costs [22,23]. Recent publications confirmed the prospectivity of mechanochemical approach on the examples of α-lipoic acid, simvastatin and other drugs [24-27].

Glycyrrhizic acid (GA, Figure 1B) is a good soluble natural saponin, extracted from liquorice roots, and widely applied as flavouring and sweetener. GA is a conjugate of two glucuronic acid molecules and a GA molecule and is capable of formation large micellelike aggregates in aqueous and water-alcohol solutions due to its amphiphilic properties [28]. Poorly watersoluble drugs can form solid dispersion with GA, which leads to increased solubility and therapeutic effect and, consequently, reduces the therapeutic doses of these drugs [29-31]. In addition, GA can increase the permeability of cell membranes for small molecules [32]. In the present investigation, a solid dispersion of candesartan cilexetil with GA was synthetized via ball milling. The complex formed was evaluated with X-ray diffraction (XRD), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and its dissolution behaviour. In addition, the stability of ball milling products regarding illumination and hyperthermia behaviour was investigated.

Materials and Methods

Candesartan cilexetil (purity ≥99 %) was obtained by Aladdin Industrial Corporation, China; GA was supplied by TCI Chemical Industry Co., Ltd, Shanghai, hina; PM-200 planetary ball mill (Retsch, Germany); HTY-EU802 rotating paddle apparatus (Tailin Bioengineering Equipments Co., Ltd., Hangzhou, China); Waters 2695 Liquid chromatograph (Waters Corporation, USA).

Preparation of samples:

Candesartan cilexetil/GA solid dispersion was prepared in a planetary micro mill with stainless steel jars, which possessed 50 ml volume. Rotational speed of stainless steel jars is 120 rpm. Steel balls (diameter 12 mm) were used as grinding bodies. The duration of mechanical processing was from 0.5 to 16 h (0.5, 2, 4, 8, and 16 h). To prepare the solid dispersion, candesartan cilexetil/ GA mass ratio 1/5, 1/10, 1/20 and 1/50 was used. All the products should over 100 mesh sieve and preserved in a driers with a low temperature after sealed. Physical mixtures of candesartan cilexetil and GA were prepared by blending them in a small bottle.

Solubility study:

Solubility of samples was determined in water using a standardized shake bath method. The samples contained 30 mg candesartan cilexetil were placed in a HY-4 shaker-incubator (+37°, 200 rpm, Hangzhou Dewei Instrument Technology Co., Ltd. Hangzhou, China) for 48 h. The samples were then filtered through a 0.45 μm membrane filter and the filtrate was assayed per high performance liquid chromatography (HPLC). The test was parallel to the determination of three times.

HPLC analysis:

Waters 2695 HPLC system was used to determine the concentration of candesartan cilexetil. The HPLC system equipped with an UV detector set at 254 nm. Chromatographic analysis was performed on a reverse phase column (5 μm, 4.6×50 mm, Zorbax Eclipse XDB-C18) at ambient temperature. The mobile phase consisted of potassium dihydrogen phosphate buffer solution (pH 4.0) and acetonitrile (20:80, v/v). The flow rate was 1.0 ml/min and the injection volume was 5 μl for each sample. The calibration curve was rectilinear in the concentration range of 10-100 μg/ml with a correlation coefficient of 0.9999. The inter/ intraday accuracy and precision was within a relative standard deviation of ≤5 %.

Content test for candesartan cilexetil and its solid dispersions:

The co-milled products (containing 0.6 mg candesartan cilexetil) were weighed accurately by a BSA124S analytical balance (Sedorius, Germany). Then put them into different volumetric flasks with a capacity of 50 ml, respectively, and then added the ethanol into the volumetric flask as a solution until all components of complexes were completely dissolved. Added in ethanol up to the scale mark. The samples were suitably diluted and assayed by HPLC. The test replicates three times and calculates the percentage of practical and theoretical drug content in the grinding product (Table 1).

Sample Drug content, % from initial
Candesartan Cilexetil/GA (1:5, 8 H) 98.8±1.07
Candesartan Cilexetil/GA (1:10, 8 H) 97.4±1.22
Candesartan Cilexetil/GA (1:20, 8 H) 99.5±0.98
Candesartan Cilexetil/GA (1:50, 8 H) 97.9±1.14

Table 1: Content Homogeneity

Same quality (~0.2 g) of candesartan cilexetil and the co-milled binary systems complexes (w/w=1:5,1:10 and 1:20 milling time 8 h) were divided into 3 equal portions. One was stored in a stability test chamber (DHG-9240A, Yiheng Scientific Instrument Co., LTD., Shanghai) under dry conditions at 60±0.5° to test the thermal stability of samples. The samples were taken out on day 5 and day 10, dissolved in ethanol and assayed using HPLC to calculate the contents of candesartan cilexetil.

Second portion was subjected to light stability test. The samples were kept in a photostability test chamber, whose light intensity was set at 4500±500 lux [33]. Samples were taken out and dissolved in ethanol, assayed using HPLC to calculate the contents of candesartan cilexetil at 5th and 10th d. The third portion was stored for 6 mo under room temperature in a sealed dryer, samples were taken out, dissolved in ethanol and assayed using HPLC to calculate the contents of candesartan cilexetil after 30, 90, 120 and 180 d.

In vitro dissolution test:

Determination of the dissolution rate in this study was according to method of the FDA [34]. The amount of milled products equivalent to 16 mg candesartan cilexetil and pure candesartan cilexetil were determined using a HTY-EU802 rotating paddle apparatus. Phosphate buffer of pH 6.7 was used as the dissolution medium (37±0.5°, 900 ml), and the paddle speed was set at 50 rpm. Five millilitre aliquots were withdrawn at 5, 10, 15, 30, 45 and 60 min, each time replacing the volume of sample with equal volume of preheated dissolution medium to maintain sink condition. The samples were filtered through 0.45 μm membrane filter and assayed for drug concentration using a HPLC method that was described earlier. Each dissolution rate test was repeated three times.

DSC analysis:

DSC thermograms were obtained under argon gas flow of 10 ml/min. Calibration of the DSC instrument (Instrument Scientific Specialists Inc., Omaha, NE, USA) was carried out using indium as a standard. Sample powders (5 to 7 mg) were crimped in an aluminium pan and heated at a rate of 10 K/min from 20° up to 250°.

XRD analysis:

Powder XRD was used to assess the degree of crystallinity of cogrinding products. XRD analysis was performed using an X'Pert PRO X-ray diffractometer (PA Analytical, Holland) with a CuKα radiation source at 40 kV voltage, 30 mA current and a scanning speed of 2 degrees/min from 0 to 60°.

FTIR analysis:

Infrared spectra of the samples were obtained using a Niclolet Avatar 370 instrument (Thermo Niclolet Corporation, USA). All samples were mixed with KBr for compressing into a thin tablet. The wave number of FTIR spectra ranged from 400 to 4000 cm−1 with a resolution of 2 cm−1.

SEM analysis:

Electron microscopic images were acquired using a Hitachi TM-1000 microscope (Tokyo, Japan). Samples were coated with gold using a Jeol JFC-1600 auto fine coater (Tokyo, Japan). The coating parameters were as follows: sputtering time 30 s, amperage 30 mA, and film thickness 15 nm.

Results and Discussion

Thermal behaviours of candesartan cilexetil, a physical mixture of 1:20 candesartan cilexetil and GA, and the ball milled 1:20 solid complexes were shown in figure 2A. DSC thermogram of candesartan cilexetil showed a sharp endothermic peak at about 170°, which was the result of the melting for candesartan cilexetil [13]. The thermogram of GA showed a very broad endothermic effect in the range from 70 to 100° due to release of water molecules. The characteristic melting endotherm peak appeared in the physical mixture of candesartan cilexetil and GA, but the complete disappearance of the candesartan cilexetil endothermic peak suggested that no crystal of candesartan cilexetil existed in GA solid dispersion.


Figure 2: Spectral characterization of candesartan cilex etil, physical mixture and solid dispersion.
A. Differential scanning spectra B. powder X-ray diffraction and C. FTIR spectra of a. candesartan cilexetil, b. physical mixture, c. solid dispersion (1:20, 8 h) and d. glycyrrhizic acid

The physical status of candesartan cilexetil in solid dispersion was further confirmed by XRD analysis (Figure 2B). Candesartan cilexetil showed the typical peaks at 2 θ values of 9.9, 17.26, 18.7, 19.62, 20.52, 23.14, and 25.16 degrees, indicating that candesartan cilexetil existed as crystalline form [13]. The characteristic peaks also existed in the physical mixture, which indicated that there was no interaction between drug and GA by the way of direct superposition. On the contrary, a broad peak without any drug crystalline peak was detected in the diffraction pattern of the mechanical activation complexes. The XRD data indicated that candesartan cilexetil was in the amorphous or molecular state in the mechanical activation solid dispersion, which accorded with the DSC measurements.

FTIR spectroscopy was used to investigate possible chemical reactions or H-bonding formation in the binary systems of candesartan cilexetil with GA. Pure candesartan cilexetil (Figure 2C) showed characteristic peaks ideal for candesartan cilexetil at 2942.83 cm−1 due to aromatic (−C-H) stretching, 2865.16 cm−1 for (O-H) stretching, 1751.99 cm−1 and 1716.92 cm−1 for ester (−C=O) stretching vibration, 1270.42 cm−1 and 1316.28 cm−1 due to (−C-O) stretching of carbonyl group of aromatic ester. It was found that the overall peaks were similar to the free GA in physical mixture. However, the C=O stretching vibration peak of candesartan cilexetil was still observed in the physical mixture indicating that there was no interaction between drug and GA. On the contrary, the C=O stretching vibration peak of candesartan cilexetil was completely disappeared in the milled complex of candesartan cilexetil/GA, probably indicating that the carbonyl group in candesartan cilexetil interacted with the hydroxyl group of GA.

SEM images of the pure candesartan cilexetil, GA and corresponding solid dispersion in 1:20 mass ratio were shown in figure 3. Candesartan cilexetil demonstrated a slightly bacilliform crystallites within an irregular shaped particle. The morphological feature of GA was in the shape of spherical particle with different size. Emergence of the new morphological shape in solid dispersion compared to the pure drug and GA was indicative of the efficient formation of solid dispersion system. Moreover, according to SEM image of the solid dispersion, there were no observable drug particles on the surface of prepared solid dispersion, representing homogeneous distribution of the drug molecules in the solid dispersion system.


Figure 3: Scanning electron microscopy images
A. Unprocessed candesartan cilexetil, B. glycyrrhizic acid, C. solid dispersion (1:20, 8 h)

The solubility of candesartan cilexetil was enhanced with varied extent in formulation of mechanical-treated solid dispersions (Figure 4). In addition, the contents of candesartan cilexetil in solid dispersion were all nearly 100 % from theoretically calculated. It indicated that during mechanical treatment, there was no significant destruction and loss of candesartan cilexetil in solid dispersions. The increase of GA amount increased candesartan cilexetil solubility. As an amphiphilic carrier, GA can promote drug micellar solubilisation. The micelle formation occurs when the GA concentration is above its critical micellar concentration. Additionally, the solubility of candesartan cilexetil basically had no change when milled over 8 h. Therefore, 8 h was the best milling time for this system to prepare solid dispersions with good solubility of drug.


Figure 4: Solubility of milled compounds in different proportions image

The dissolution of coground mixtures and candesartan cilexetil were shown in figure 5. Dissolution from unprocessed candesartan cilexetil order kinetics with a very slow dissolution rate (≈1 % in 60 min). Compared to pure drug, the dissolution rate of 1:5 and 1:10 (w/w) compounds has increased significantly, reached to 50.2 and 75.4 % in 60 min, respectively. Moreover, the candesartan cilexetil approximation acquired a complete release in 60 min when the ratio of candesartan cilexetil and GA has reached to 1:20. The mechanochemically obtained compositions have the most improved dissolution profile may be attributed to the decrease in the drug crystallinity during the preparation process. The improved dissolution rate of candesartan cilexetil in the physical mixture might be attributed to the hydrophilic nature of GA, which can increase the dissolution rate by reduction in the interfacial tension between the poorly-soluble drug and the release medium.


Figure 5: Dissolution profiles of candesartan cilexetil from different formulations
image 1:20, milling 8 h; image 1:10, milling 8 h; image 1:20 physical mixture, image 1:5, milling 8 h; image candesartan cilexetil

The stability tests of pure candesartan cilexetil and its mechanochemically obtained complexes were carried out at high temperature, light irradiation and long-time storage, and results of drug content were represented in figure 6. It was found that the content of candesartan cilexetil in all samples decreased with the extended storage time. For mechanochemically obtained solid dispersions, the drug content was higher than pure candesartan cilexetil, which suggested the stability of solid dispersion has an enhanced stability. In addition, the proportion of candesartan cilexetil and GA has a great effect for the stability of the compounds.


Figure 6: Stability Studies of candesartan cilexetil and co-milled compounds
A. Thermal stability, B. light stability and C. long-term storage of candesartan cilexetil and co-milled compounds. A and B. image 1:20, milling 8 h; image 1:10, milling 8 h; image 1:5, milling 8 h; image candesartan cilexetil; C. image 1:20, milling 8 h; image candesartan cilexetil

In this study, mechanochemical milling process that allows getting final product in one technological step without using of any organic solvents has been applied to prepare solid dispersions of candesartan cilexetil with GA. Formation of the solid dispersions was proved by XRD, FTIR and DSC analysis. In vitro drug release showed a significant increase in drug’s dissolution rate after formation a solid dispersion with GA. On the other hand, the stability assay showed that the chemical stability of solid dispersions had an enhanced compared with pure candesartan cilexetil. In conclusion, solid state mechanochemical milling process could be a most useful method for the preparation of complexes of candesartan cilexetil with GA, which are promising to improve the oral bioavailability of candesartan cilexetil and might be considered for development of the lowdose drug market. This study still has some limitation. In stability study, we only calculate the drug content, without using other determination methods. In next studies, it is better to use drug content determination combined with FTIR, DSC and NMR to investigate the stability, which makes the results more convincing.

Conflict of interest

The authors report no conflicts of interest.