WANG Renmin (王仁敏), LIN Chan (林嬋), LIU Jingliang (劉晶靚), YU Fang (余方), GAO Jianpei (高建培) and PAN Xuejun (潘學(xué)軍)**

Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming 650093, China

1 INTRODUCTION

Licorice, derived from the dried roots and rhizomes of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L., has been widely used in both Eastern and Western medicines [1, 2].Licorice is recommended for the treatments of spleen deficiency, sore throat, cough, injury or swelling and used as demulcent, expectorant and antidote [2]. As mentioned in Chinese medical classic, Shang Han Lun,more than 60% of the Chinese traditional prescriptions include licorice [3]. Licorice shows a variety of pharmacological activities, including antiulcer, antiinflammatory, antispasmodic, antioxidative, anti-allergic,antiviral, anti-diabetic, anticancer, antidepressive and memory enhancing activities [1, 4], especially the anti-HIV activity. Now, it is used extensively in the tobacco, food, confectionary and pharmaceutical industry throughout the world. Glycyrrhetinic acid (GRA) and glycyrrhizic acid (GA) are the main active constituents of licorice. Orally administered GA is almost completely hydrolyzed by intestinal bacteria and reaches the systematic circulation as GRA [5]. When the two glucuronic acid moieties are removed from the GA molecule by hydrolysis, the aglycon glycyrrhetinic acid remains [6],which is considered to be ultimately responsible for the pharmacological effects of licorice. Besides, GRA has more potential in vitro anti-platelet aggregation activity than GA. GRA also shows the most potential cytotoxicity against tumor cell lines and the potent inhibitory activity on rotavirus infection as well as growth of helicobacter pylori [7]. Both GA and GRA have the activities of inhibiting the proliferation of hepatoma carcinoma cells and inducing their differentiation.Furthermore, the dose of GRA is only 2.5% of GA to obtain the equal effect [8]. The direct application of GRA in the treatment of diseases can strike a balance between the dose and therapeutic effectiveness of licorice-related drugs in order to reduce the pharmacological side effect [9].

One of the traditional methods employed for preparation of glycyrrhetinic acid is hydrolyzing glycyrrhizic acid in acidic solution (mostly sulfuric acid solution), which is a hydrolysis process that is carried out with appropriate heating and pressure. It usually takes more than 10 hours to reach high yield of GRA.There are also some reports [7, 10, 11] about hydrolyzing glycyrrhizic acid by biocatalysts. The results showed that some special germs should be found or cultivated as the catalysts, indicating the biohydrolysis was also a complex and time-consuming process. To overcome the disadvantages of the conventional methods, especially long hydrolization time, a new hydrolization method should be developed.

The microwave-assisted technique, which was firstly performed for the extraction of organic substances by Ganzler et al. [12] in 1986, has been adapted for scientific applications, including microwave-assisted extraction, dehydration, synthesis, as well as hydrolysis process. Without the usual limitations of heat conduction and convection, the microwave energy conversion mode allows the selection of target-specific molecules and deposition of the energy in the whole volume of the sample. It is considered to be fast, clean, simple and often more efficient than conventional heating methods.Pan et al. [13] dehydrated penicillin G sulfoxide under microwave irradiation. Saponites were successfully synthesized by using the microwave hydrothermal method [14]. Aluminium polycations were rapidly synthesized by microwave assisted hydrolysis of aluminium[15]. Qu et al. [16] used high pressure microwaveassisted hydrolysis to hydrolyze the proteins in the root ofpanax quinquefolius L. Halaszet al. [17] found that hydrolytic degradation of nitroglycerin using microwave heating was much higher than its degradation using conventional heating.

However, there is no report on preparation of GRA from crude GA by pressured microwave-assisted hydrolysis (PMAH) technique. In order to reduce the hydrolysis time and increase the hydrolysis efficiency,a novel pressured microwave-assisted hydrolysis technique is developed in this study for efficient preparation of glycyrrhetinic acid from crude glycyrrhizic acid. Various hydrolysis approaches are compared.Pressured microwave-assisted hydrolysis (PMAH) is identified as the simplest and most efficient method,and its process parameters are optimized.

2 MATERIALS AND METHODS

2.1 Materials

Licorice root (from Inner Mongolia, China) was purchased from Fulintang Medicine Co., Kunming,Yunnan Province, China. The licorice root was large pieces (diameter about 5-10 mm and thickness about 3-5 mm).

2.2 Reagents

Ethanol, glacial acetic acid, ammonium acetate,sulfuric acid and chloroform are analytical reagents.Ammonia solution (25% NH3) is chemical reagent.Methanol is reagent for high performance liquid chromatography. Ammonium glycyrrhizinate (purity≥95%) and glycyrrhetinic acid (purity≥98%) were purchased from Institute of Medicine and Bio-product of China, Beijing.

2.3 Preparation of crude glycyrrhizic acid

Licorice root (total 200 g) was mixed with an appropriate solvent [2000 ml, 70% (by volume) ethanol/2% (by volume) ammonia/water]. After heating for 4 h at 70 oC in a stainless steel pot, the licorice leaching solution was filtered and concentrated to 400 ml in a rotary evaporator (Buchi Rotavapor RII, Switzerland). Glycyrrhizic acid was deposited in the bottom when pH of the solution was adjusted between 2 to 3 with H2SO4(98%). After treatment in centrifuge and freeze drier separately, the crude glycyrrhizic acid samples were obtained. The purity of GA in the crude GA sample was 13.67% (by mass). This crude GA sample was used in the following experiments.

2.4 Hydrolysis of glycyrrhizic acid to produce glycyrrhetinic acid

2.4.1Conventional hydrolysis method (heat reflux hydrolysis)

Heat reflux hydrolysis was performed with 5 g crude GA and 100 ml solvent (5% H2SOs) in a flask(250 ml) with water condenser for 14 h with saline water-bath. Mechanical stirrer (300 r·min-1) was used during the hydrolysis.

2.4.2Microwave-assisted hydrolysis (atmospheric pressure)

A household microwave oven (Midea, China, full power 700 W) was modified in our laboratory with addition of a mechanical stirrer (300 r·min-1), water condenser, temperature measurement and time controlling [18]. 5 g crude GA was mixed with 100 ml 5%(by mass) H2SO4. The suspension was irradiated under microwave in pre-setting procedures [15 s power on,15 s power off for three times to the desired temperature (about 95-100 oC) and then 3 s power on for heating and 15 s power off for cooling], but not allowed to super-boil.

2.4.3Pressured microwave-assisted hydrolysis

The hydrolysis was carried out in the ETHOS 1 advanced microwave digestion system (Milestone,Italy) equipped with a 12-sample tray. Airtight and pressure-resisting vessels were employed to hold the samples. The volume of the teflon lined vessel was 100 ml. There was no pressure indicator in the microwave unit, but the inner temperature of the sample vessel can be indicated with a special sensor. The conditions for microwave-assisted hydrolysis of GA can be optimized by adjusting the magnetron power output, the heating time and the temperature. Crude GA sample was accurately weighed and transferred into the vessel. Sulfuric acid solution with different volume and concentration were used as the solvent.The vessels were symmetrically placed on the microwave turntable. PMAH was carried out under the pre-setting procedures (see Table 1).

Table 1 The procedures for pressured microwave-assisted hydrolysis

In the present work, hydrolysis efficiency was defined as follows:2.4.4High performance liquid chromatographic analysis

GA and GRA were quantified at the same run by HPLC (Agilent Technologies 1200) equipped with a reversed-phase C8 column (5 μm, 4.6×150 mm) and UV detector at UV 254 nm. The column temperature was maintained at 45 oC. Gradient elution (See Table 2)was used in HPLC runs. The mobile phase A was 60∶40∶1 (by volume) of methanol, ammonium acetate solution (0.2 mol·L-1) and glacial acetic acid;the mobile phase B was 90∶10∶1 (by volume) of methanol, ammonium acetate solution (0.2 mol·L-1)and glacial acetic acid. The flow rate was 0.8 ml·min-1.All the products prepared by the three hydrolysis techniques were treated by chloroform firstly, then after vacuum filtration, rotary evaporator and nitrogenblowing, refined GRA were obtained. Refined GRA and crude GA were dissolved in ethanol and mobile phase B separately, and filtered with micro porous(0.45 μm) membranes. The treated solutions were analyzed. Retention time of GA was about 5.3 min and that of GRA was about 11.4 min. This method was sensitive and accurate with good reproducibility. The relative standard deviations (RSDs) of GA and GRA were 1.96% (n=5) and 1.90% (n=5) separately. A good linearity was ranged from 6 to 600 μg·ml-1for ammonium glycyrrhizinate (Y=12397X-43.7,R2=0.999) and from 3 to 300 μg·ml-1for glycyrrhetinic acid (Y=36011X-59.2,R2=0.999), respectively. The analytical operation was completed in 16 min. As shown in Fig. 1, this reversed-phase HPLC method was validated by good specificity for the analysis of ammonium glycyrrhizinate and glycyrrhetinic acid.Fig. 2 showed that there was no GRA detected in crude GA. Moreover, there was no GA detected in GRA samples since the GRA samples were treated by chloroform. (See Fig. 3).

Table 2 The elution gradients for HPLC separation

Figure 1 HPLC chromatogram of ammonium glycyrrhizinate and glycyrrhetinic acid standards at UV 254 nm (Peak 1: ammonium glycyrrhizinate; Peak 2: glycyrrhetinic acid)

3 RESULTS AND DISCUSSION

3.1 Mechanism of hydrolyzing GA

Figure 2 HPLC chromatogram of crude glycyrrhizic acid at UV 254 nm (Peak 1: glycyrrhizic acid)

Figure 3 HPLC chromatogram of refined glycyrrhetinic acid at UV 254 nm (Peak 2: glycyrrhetinic acid)

The relative molecular mass of GA is 822.93,which is relatively large, so acid, alkali or bioactive enzyme should be employed as catalysts to promote hydrolysis. In this paper, sulfuric acid was selected.The reaction mechanism of hydrolyzing GA to produce GRA was shown in Fig. 4. With the help of acid,the glucosidic bond in GA was broken and GA was decomposed into a molecule of GRA and two molecules of glucuronic acid. During this process, there were several factors affecting the yield of GRA, including liquid-solid ratio, hydrolysis time, sulfuric acid concentration, and the most important one, temperature.

3.2 Optimization of pressured microwave-assisted hydrolysis method

3.2.1The effect of liquid-solid ratio on yield of GRA

The results (see Fig. 5) indicated that the yield of GRA increased with the increase of liquid-solid ratio,but when the ratio increased to 30∶1, the yield of GRA did not increased any more. High liquid-solid ratio was favorable for hydrolysis process because of more efficient mass transfer between liquid and solid phases, but excessive high liquid-solid ratio requires more energy and time for the post-treatment of the leaching solution. Thus, a suitable intermediate liquid-solid ratio of 25∶1 (ml·g-1) was chosen.

3.2.2The effect of hydrolysis time on yield of GRA

The results in Fig. 6 indicated that the yield of GRA increased with the increase of hydrolysis time(the sum of heating-up time 15 min and temperature-holding time) before 21 min and declined with the prolongation of hydrolysis time after 23 min. This was probably due to that hydrolysis of GA was almost done before 23 min. With longer temperature-holding time, there were some undesirable reactions occurring,such as carbonization, thus lowering the yield of GRA.To compromise between improving product yield and energy saving, 21 min (heating-up time: 15 min; temperature-holding time: 6 min) was thought suitable for the total hydrolysis. PMAH reached high productivity only taking 17-23 min, which showed the significant advantage over the conventional techniques (about 14 h needed).

Figure 4 Reaction mechanism of hydrolyzing GA to produce GRA

Figure 5 The effect of liquid-solid ratio on yield of GRA[heating-up time: 15 min; temperature-holding time: 30min;temperature: 150 oC; 25 ml 5% (by mass) H2SO4]

Figure 6 The effect of hydrolysis time on yield of GRA[L-S ratio: 25∶1; crude GA: 1 g; heating-up time: 15 min;temperature: 150 oC; 5% (by mass) H2SO4]

3.2.3The effect of sulfuric acid concentration on yield of GRA

Figure 7 The effect of sulfuric acid concentration on yield of GRA (L-S ratio: 25∶1; crude GA: 1 g; heating-up time:15 min; temperature-holding time: 6 min; temperature: 150 oC)

The effect of sulfuric acid concentration on yield of GRA was shown in Fig. 7. At low sulfuric acid concentration, the yield of GRA increased with the increase of sulfuric acid concentration because more hydrogen ions were favorable for hydrolysis reaction.But the yield of GRA decreased obviously in sulfuric acid of high concentration owing that carbonization could easily occur under high temperature. Thus, the best product yield of GRA was obtained at 3%-5%(by mass) sulfuric acid concentration.

3.2.4The effect of temperature on yield of GRA

The effect of temperature on yield of GRA was shown in Fig. 8. The yield of GRA increased sharply with the increase of hydrolysis temperature below 130 oC, and increased slightly above 130 oC. As is known, temperature is one principal factor to chemical reactions, directly influencing equilibrium and reaction rate constants. In this study, the yield of GRA decreased when the temperature increased above 150 oC probably due to carbonization, as evidenced by a little black color of the hydrolysis solution and emission of scorched flavor. Consequently, 150 oC was the optimal temperature for the pressured microwave-assisted hydrolysis of GA.

Figure 8 The effect of hydrolysis temperature on yield of GRA [L-S ratio: 25∶1; crude GA: 1 g; heating-up time: 15 min; temperature-holding time: 6 min; 5% (by mass) H2SO4]

Table 3 Comparison of three different hydrolyzation methods

3.3 Comparison of PMAH with other techniques

Microwave energy is a non-ionizing radiation(0.3-300 GHz) that causes molecular motion by migration of ions and rotation of dipoles, but does not cause changes in molecular structure. In heat transfer,energy is transferred due to thermal gradients, but microwave heating is by conversion of electromagnetic energy to thermal energy, rather than heat transfer.Microwave energy can penetrate materials, and heat the whole sample simultaneously. The application of microwave energy for sample preparation can be performed either in closed vessels with pressure and temperature control (PMAH) or in open vessels at atmospheric pressure (MAH) [19-21]. Limited by the boiling point at atmospheric pressure, even when solution approaching its boiling, the yield of GRA was not satisfactory by using neither microwave-assisted atmospheric hydrolysis (MAH) nor heat reflux hydrolysis. Under the same boiling point at atmospheric pressure and other hydrolysis conditions, the yield of GRA was largely dependent on the leaching time.Thus, GRA yield achieved by heat reflux hydrolysis taking 14 h was higher than that by MAH taking 3 h(see Table 3 for detail). Only in PMAH, when the boiling point was elevated by enhancing the pressure,the solution can reach higher temperature to supply a desirable temperature for quick hydrolysis of GA.This result was consistent with Ref. [22] that the hydrolysis process was hardly completed at atmospheric pressure. It is worth mentioning that without sulfuric acid as catalyst, PMAH can still obtain a GRA yield around 70% (see Fig. 7).

4 CONCLUSIONS

PMAH was proved to be an efficient method for hydrolyzing GA to produce GRA. Compared with the conventional techniques, PMAH provided higher hydrolysis efficiencies (up to 90%), as well as considerable time-saving (21 min to 14 h). Optimal conditions for the PMAH of GA to produce GRA were as follows:PMAH of crude GA for 21 min (15 min to reach 150 oC,and holding for 6 min) at 150 oC (at a radiation power of 450 W) in 3%-5% sulfuric acid solution with the liquid-solid ratio of 25∶1. This novel method was suitable for fast preparation of glycyrrhetinic acid from crude glycyrrhizic acid and its prospect will be very well.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the support of Analysis and Testing Foundation of Kunming University of Science and Technology.The authors also wish to thank Professor Bo PAN for providing HPLC equipment.

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