Optimization of ultrasonic frequency for the improvement of extraction yields of bufadienolides from the Chinese medicine ChanSu by using a novel ultrasonic system

Abstract

Optimization of the ultrasonic frequency for the improvement of extraction yields of bufadienolides from ChanSu was evaluated successfully using an online extraction system, which can convert the ultrasonic frequency information to intuitive waveforms that are easily identified by the naked eye. This can provide the information that can quickly select the optimum ultrasonic frequency to obtain the maximum extraction yields. This work has systematically studied the influence of ultrasonic frequency of 20–84 kHz on the bufadienolides yield, whereas existing ultrasonic-assisted extraction (UAE) uses only one or very few frequency points. The higher bufadienolides yield was achieved at the ultrasonic frequency of 54–55 kHz, which was found to be weakly affected by other extraction parameters such as the type of solvent, solvent to solid ratio, ultrasound power, extraction temperature and particle size. The extraction results, which were converted to the intuitive waveforms by the laboratory virtual instrument engineering workbench (LabVIEW) on a computer, could be easily observed by the naked eye by the real-time monitoring of the product concentration in the treatment tank. Hence, it would be favorable to minimize the time-consuming treatments of samples and the required laborious manual manipulations. Comparative studies between existing methods and the proposed method have been carried out. Results indicate that this method can achieve a higher extraction yield compared with the existing extraction technologies.

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1 Introduction

ChanSu (also called toad venom) is one of the important Chinese traditional medicines. Many researchers have reported that the major active constituents of ChanSu are bufadienolides. ChanSu is widely used as a local anesthetic agent and cardiotonic in China and other countries and it is responsible for the characteristic structural feature of the bufadienolides, which is a doubly unsaturated six membered lactone ring on position 17 of C-24 steroids.^1–4 In recent years, many different methods have been employed for the extraction of bufadienolides from animal and natural sources, such as toad skin,^5,6 the Chinese traditional medicine of ChanSu,^7,8 Kalanchoe pinnata,⁹ and Helleborus thibetanus.¹⁰ Among these techniques, UAE was proven to significantly shorten the extraction time, lower the consumption of solvent and enhance extraction yields in many natural products.^11–15

Although these studies on UAE mainly focused on the optimization of extraction conditions, the effect of ultrasonic frequency on the extraction efficiency of bufadienolides from ChanSu has not been systematically investigated. Existing extraction techniques are well proven, widely accepted and available for obtaining higher extraction yields,^16,17 but they are often viewed as laborious and complex, requiring time-consuming treatment of the samples.^18–20 The analysis process usually has to be performed in a specialized laboratory by skilled personnel. Moreover, these extraction processes may result in less satisfactory results due to taking roughly only one or very few frequency points.^21,22 Therefore, to develop an extraction technology with higher efficiency by seeking the optimum ultrasonic frequency is extremely urgent. However, very few studies regarding the automation technology have been found to determine the optimum ultrasonic frequency of bufadienolides.

The objective of this work is to assess the optimization of ultrasonic frequency in a range of 20–84 kHz with regards to the bufadienolides yield using an online extraction system by a two-step procedure, which can improve the extraction yield and minimize the required manual manipulations. Moreover, we have deepened our investigation by looking at several processing conditions that may influence recovery yields such as the particle size, solvent type, solvent to solid ratio and ultrasound power.

2 Experimental

2.1 Extraction and detection system

The experimental setup for the system is shown in Fig. 1 in detail. The whole system consists of eight groups undergoing the same treatment with different ultrasonic frequency bands. There are essentially two subsystems within this system: a concentration detection subsystem, which performs the functions of product concentration detecting and signal processing, and an ultrasonic processing subsystem, which is used to extract the sample in the tanks. The two subsystems are controlled by a microprocessor that directs them to perform the sequential steps in the analysis.

Fig. 1 Experimental setup for the system: (a) block diagram of the whole system; (b) schematic diagram of one of the treatment groups.

The ultrasonic processing subsystem is composed of a rectangular stainless steel treatment tank (300 × 250 × 200 mm) and a thermostatic bath to maintain the treatment tank at the desired temperature. As shown in Fig. 1(b), the treatment fluids are continuously stirred by a small pump (30 W) to avoid precipitation and to assure the homogeneity in composition and temperature; an ultrasound power supply and an ultrasonic generator were used to generate power waveforms to drive twelve piezoelectric transducers (>1 W cm⁻²), which are mounted on a rectangular aluminum ultrasonic vibrating plate (300 × 250 × 30 mm³). To improve the accuracy of determining the optimum frequency, the resonance frequency of each transducer is maintained at less than 1 kHz. Therefore, even if there is a frequency drift during processing, one or more transducer can work at the sound resonant condition.

In the initial development of the detection subsystem, an ultraviolet light emitting diode (LED) and a transparent glass tube was encapsulated into a pair of UV transceivers, which includes a UV emitter (which is used as the ultraviolet light source) and a UV receiver (which is used as the ultraviolet light receiver). As shown in Fig. 1(b), a compromise was found concerning the distance between the UV emitter and the UV receiver. The distance between them has to be, on the one hand, not too far due to ultraviolet attenuation, and on the other hand, not too close because of insufficient ultraviolet absorption. Many preliminary tests have been performed to determine the most suitable distance. Finally, it was found that the optimum distance between them is 6 cm. Moreover, they should be mounted at the same height level. Note that the ultraviolet LED with a peak absorbance wavelength of 296 nm was selected to overlap with the maximum absorption of bufadienolides.²³

In recent years, as an innovative technology, UAE has shown great expectations in the natural products extraction field with promising results. Povey²⁴ examined that ultrasonic frequencies used for the extraction of plant products are mainly concentrated using low frequency (20–100 kHz). In a similar study, Mizrach²⁵ also reported that ultrasonic frequencies less than 100 kHz yield the best results when applied to natural materials. In addition, many studies on the extraction of natural products have been reported using low ultrasonic frequencies, which have achieved higher extraction yields. Hence, in our investigation, the ultrasonic frequency band of the extraction system was initially set in a range of 20–84 kHz to achieve more extraction yield and efficiency.

In this study, the automatic work process of the system is summarized in the following steps. Initially, the ultrasonic frequency bands of the eight treatment groups were set at 20–28 kHz, 28–36 kHz,…, and 76–84 kHz, respectively. Then, the operating parameters in all treatment groups were initialized. Subsequently, each treatment group began to run, in which the frequencies were sequentially sought by the microprocessor from the upper frequency to the lower one. Moreover, the bufadienolides concentration was proportionally converted to a voltage signal by the concentration detection subsystem. When the maximum yield was found in a treatment group, the operating parameters would be automatically saved in the memory. However, if the maximum yield appeared at the upper frequency or the lower frequency (meaning that the obtained maximum yield may not be the real maximum value, the real maximum yield may present at out of 84 kHz or less than 20 kHz), in order to obtain the real maximum yield, the operating parameters in each treatment group should be reset, and then the previous steps should be repeated. Finally, according to the obtained optimum frequency band (for example: 52–60 kHz), it was further divided into eight smaller frequency bands of 1 kHz. The extraction experiments were restarted by substituting treatment liquid in each group, until the presence of the maximum yield and the optimum ultrasonic frequency was finally determined.

2.2 Materials and methods

Dried ChanSu crude drug was purchased from a local market in Bing Hu district, Wuxi, Jiangshu province, China. The dried crude drug was pulverized into powder form with a blade mixer and sieved with stainless steel sieves to classify the particle size. The ground samples were stored in plastic inside desiccators prior to use. All the reagents used in the experiments were purchased from the Tianjin Chemical Factory, Tianjin, China. All the solvents used in the extraction, including methanol, acetonitrile, and ethanol, were of analytical reagent grade. Acetonitrile used in the analysis of high performance liquid chromatography (HPLC) was purchased from TEDIA Company. Water used was redistilled water, and was passed through a 0.45 μm filter before use in all the studies.

2.2.1 Extraction of the present method. For the extraction of the present system, 5 g drug powders of ChanSu (each group) were loaded into eight rectangular containers in our extraction system. The selected solvents were added. The samples were immersed into the ultrasonic tanks for irradiation under different extraction conditions, including solvents (70% acetonitrile, 70% ethanol and 70% methanol), solvent to solid ratio (5, 25 and 50 ml g⁻¹), ultrasound power (50, 100 and 200 W), particle size (40–60, 60–80, 80–100 mesh), temperature (15, 20 and 40 °C) and extraction time of 15 min. Finally, the solutions were filtered through a Millipore filter (0.45 μm) and the filtrates were analyzed by HPLC. All the samples were prepared and analyzed in triplicate.

2.2.2 HPLC analysis. HPLC analysis was carried out on a Hypersil C₁₈ column (250 × 4.6 mm², 5 μm) from Dalian Elite Analytical Instruments Co. Ltd. The mobile phase: CH₃CN–H₂O (85 : 15, v/v); the flow rate: 1 ml min⁻¹; the sample volume of injection: 20 μl; the wavelength of detection: 300 nm and the column temperature: 20 °C. The extraction yield of bufadienolides was calculated based on the integration of the chromatographic peak areas.

The extraction yields of bufadienolides from ChanSu by our extraction system and existing UAE methods were calculated using the following equation:

Yields (mg g⁻¹) = weight of purified sample (mg)/weight of dried sample (g)

2.2.3 Statistical analysis. Three triplicates for each treatment were performed. The significance of the differences of bufadienolides was calculated through a one-way ANOVA procedure. The results of HPLC analysis were expressed as the mean value ± the standard deviation. Duncan's multiple range tests were used to determine significant differences among treatments at p-values < 0.05.

3 Results and discussion

3.1 Mechanism of action

Comprehending the role of ultrasonic frequency in the reported enhanced extraction of objective components is of utmost significance. For sonication, not all bubbles are capable of producing significant cavitation effects. The greatest coupling of the ultrasonic energy will occur when the natural resonance frequency of a bubble is equal to the ultrasonic frequency.^26,27 Based on suitable approximations, it can be assumed that the bubble on expansion (or contraction) increases (or decreases) its radius by an amount r, such that R, the bubble radius at any time, is given by R = R_e + r. R_e is the bubble radius at equilibrium. The relationship between ultrasonic frequency and the resonance frequency of a bubble in liquid was given by Huang et al.²⁸ as follows:where ω_r is the resonance circular frequency of the bubble (=2πf_r), ω_a is the applied ultrasonic circular frequency (=2πf_a), P_a is the amplitude of sound pressure, and ρ is the density of the surrounding medium. It is important to recognize that r (or the bubble radius R) increases with the reduction of the difference between ω_r and ω_a. When the resonance frequency of the bubble is equal to ultrasonic frequency (ω_r = ω_a), r will tend to infinity (or R), meaning that the bubbles are more likely to resonate, and undergo more intense acoustic cavitation and collapse. Thus, more objective constituents can be removed quickly from the samples, and therefore with higher extraction yields, which suggested that the extraction yield in liquid can achieve a maximum value at an optimum ultrasonic frequency that helps to promote bubble collapse by driving the bubbles into resonance. The objective of the developed system was to seek the resonance frequency of bubble to improve the extraction yield.

3.2 System testing

Testing experiments were done on the developed system to verify its effectiveness. To evaluate each treatment group, experiments for the extraction efficiency of bufadienolides were carried out in eight treatment groups, in which their working frequency bands were set at 20–28 kHz, 28–36 kHz, 36–44 kHz, 44–52 kHz, 52–60 kHz, 60–68 kHz, 68–76 kHz, and 76–84 kHz. Other fixed operating conditions were an ultrasound power of 100 W, 70% aqueous methanol, a temperature of 20 °C and a solvent to solid ratio of 25 ml g⁻¹.

In this study, the automatic work of the system is mainly reflected in the concentration detection, signal processing and display. First, the ultraviolet LED was driven by a constant current. It was modulated by a square wave that was mainly used to remove the interference such as low frequency noise in the amplifier circuit and ambient light. The photoelectric converting unit was highly sensitive and incorporated a transimpedance amplifier and an integrated filter to realize a light-to-voltage function. The output voltage from the photoelectric converting unit was directly proportional to the ultraviolet light intensity and therefore to the bufadienolides concentration. The voltage was then buffered using a high sensitivity instrumentation amplifier, and digitized using a 12 bit analogue to digital converter (A/D) at a rate of six million samples per second. The digitized signal was further filtered using a software low-pass filter to remove other components. Digital signal processing (or a microprocessor) was applied to analyze and calculate the recorded return signal from the A/D converter using the Fourier Transform algorithm software. Finally, LabVIEW was used as a software platform to develop the whole software package. Thus, the waveform of extraction result could be displayed on the computer via USB for manual analysis.

The intuitive waveforms of extraction efficiency are shown in Fig. 2. In the automatic operating mode, the bufadienolides signal was measured only once during a single measurement cycle, once to get a background measurement, and then again at a time when the maximum bufadienolides signal was present. Moreover, the obtained waveform of the 4^th treatment group had the best extraction efficiency, where the bufadienolides signal was monitored continuously every 2–3 s as the system proceeded through its normal sequences. The obtained data were presented as memory units, which were just the readings taken from the computer. They represent the amount of charge generated by the UV emitter in response to a light pulse, and therefore were proportional to the bufadienolides concentration in the active zone, which we have observed to be a linear approximation from 0 to 15 min. Therefore, the extraction system can convert the ultrasonic frequency information to intuitive waveforms that can be easily identified by the naked eye. This can provide the information that can quickly select the optimum ultrasonic frequency to obtain the maximum extraction yields.

Fig. 2 The waveforms of the extraction efficiency of bufadienolides from ChanSu by LabVIEW for eight treatment groups (20–28 kHz, 28–36 kHz, 36–44 kHz, 44–52 kHz, 52–60 kHz, 60–68 kHz, 68–76 kHz, and 76–84 kHz) using 70% aqueous methanol at the solvent to solid ratio of 25 ml g⁻¹, temperature of 20 °C and 100 W sonication power.

3.3 Optimization of the ultrasonic frequency band under the influence of other extraction conditions

3.3.1 Effect of the type of solvent. The effect of the type of solvent on the extraction yield of bufadienolides was determined for three commonly used solvents: 70% acetonitrile, 70% ethanol and 70% methanol. The results are shown in Fig. 3(a). It was found that the three different solvents exhibited different effects on the extraction yield under same extraction conditions. Methanol gave the maximum extraction yield, followed by acetonitrile and ethanol. The different extraction yields might be caused by the polarities of the solvents. Another possible reason might be the fact that the molecular structure of acetonitrile is very different from that of bufadienolides, resulting in the lowest solubility compared with the other solvents. Thus, 70% methanol was chosen as the best solvent in the following extraction experiments. In addition, we can clearly find that the highest extraction yield of bufadienolides appeared at 52–60 kHz. When the ultrasonic frequency was outside of 52–60 kHz, extraction yields obviously decreased. This suggests that the ultrasonic frequency of 52–60 kHz is the optimum value.

Fig. 3 Effect of solvent type (a), solvent to solid ratio (b), ultrasound power (c), temperature (d), and particle size (e) on the yields of bufadienolides from ChanSu.

3.3.2 Effect of solvent to solid ratio. The effect of solvent to solid ratio on the extraction yield of bufadienolides is observed in Fig. 3(b). It could be found that under the fixed conditions of other factors, the bufadienolides yields were greatly affected by the solvent to solid ratio. The extraction yield increased significantly when the solvent to solid ratio was increased from 5 to 25 ml g⁻¹. Subsequently, the rate of increase declined, and little increase between 25 ml g⁻¹ and 50 ml g⁻¹ was observed. This was probably due to increasing the surface area for solute–solvent contact and the larger volume of methanol, which caused effective swelling of the material by water.^29,30 Furthermore, the presence of water reduced the mixture viscosity, which was conducive for improving mass transfer. Thus, the solvent to solid ratios of 25 ml g⁻¹ was chosen as the suitable value.

In addition, it was observed that the higher yield was observed at 52–60 kHz, although the extraction yield at 44–52 kHz was a little more than that observed at 52–60 kHz at the solvent to solid ratios of 5 ml g⁻¹. Therefore, 52–60 kHz was the most optimum ultrasonic frequency for extracting bufadienolides, which was not sensitive to the solvent to solid ratio.

3.3.3 Effect of ultrasound power. The effect of ultrasound power on the extraction yield of bufadienolides from ChanSu was determined for three ultrasound powers (50, 100 and 200 W). The results are shown in Fig. 3(c), which illustrates that the increment of extraction yield is obvious when the ultrasound power was between 50 and 100 W. However, when the ultrasound power increased from 100 to 200 W, the bufadienolides yields were not significantly different. Considering commercial applications, 100 W should be selected as the optimum ultrasound power. The extraction results also showed that the highest extraction yield also appeared at 52–60 kHz.

3.3.4 Effect of temperature. In this study, the samples were extracted at a temperature of 15, 20 and 40 °C. The effect of temperature on the extraction yield of bufadienolides is shown in Fig. 3(d). Note that the extraction yield did not significantly change with the increased extraction temperature. The extraction yield leveled out when the temperature was increased from 20 °C to 40 °C, which is supported by the fact that high temperature is not beneficial for ultrasonic extraction due to evaporation of solvent. Therefore, 20 °C was used as the optimal temperature in the following study. In addition, it was found that all the maximum yields of bufadienolides were observed in treatment group 5 (52–60 kHz) at the extraction temperature of 15, 20 and 40 °C. This indicates that the optimum ultrasonic frequency of 52–60 kHz was weakly affected by the extraction temperature.

3.3.5 Effect of particle size. The bufadienolides yields were dependent on the particle size (Fig. 3(e)). It was observed that the minimum value for yield appeared at the particle size of 80–100 mesh. Then, with the reduction in particle size, the yield significantly increased. When the particle size was decreased to 60–80 mesh, the yield reached the peak value. After that, however, a slight reduction of yield was found at 40–60 mesh. This result demonstrates that the particle size has both positive and negative influences on the extraction yield. In fact, the small particle size was more favorable for the extraction yield than the large one. However, if the particle size was reduced too much, the use of ultrasound would be less effective. This is probably attributed to the reason that the cell walls were already considerably ruptured and there was a greater surface area, and therefore it permitted close contact of the extracting solvent with the matrix,^31–33 which indicates that ultrasound has a less significant effect on the extraction yield as the particle size is reduced to a certain smaller level. Thus, the particle size of 60–80 mesh was selected as the optimum size because of its maximum extraction yield.

Besides the particle size, the ultrasonic frequency was another important variable for the extraction yield. It could be seen clearly from the extraction results that the ultrasonic frequency of the highest extraction yield was between 52 kHz and 60 kHz in each group. This may be partially attributed to the fact that the frequency of 52–60 kHz was closer to the natural resonant frequency of the bubbles, which indicates that the particle size has less pronounced effects on the optimal extraction frequency. Therefore, the ultrasonic frequency band between 52 kHz and 60 kHz was finally determined as the optimal extraction frequency band for bufadienolides from ChanSu.

3.4 Optimization of ultrasonic frequency under optimum extraction variables

According to the abovementioned study, the optimum frequency band (52–60 kHz) and other optimum extraction variables were determined. In this step, we further sought the optimum frequency from 52 to 60 kHz. Other optimized extraction variables were applied in this step. The results obtained by HPLC analysis are presented in Fig. 4.

Fig. 4 Determination of optimal ultrasonic frequency under the optimum experimental conditions: 70% methanol; solvent to solid ratio of 25 ml g⁻¹; ultrasound power of 100 W; particle size of 60–80 mesh; extraction temperature of 20 °C; extraction time of 15 min and ultrasonic frequency of 52–60 kHz.

The extraction yields of bufadienolides from ChanSu increased gradually as the ultrasonic frequency was increased; the higher extraction yield was obtained in the third treatment group (54–55 kHz). Then, the extraction yield declined along with the increase of ultrasonic frequency. It is worth noting that the extraction yield between 54 kHz and 55 kHz in this step was higher than that of the first step. Moreover, the maximum value of extraction yield between 54 kHz and 55 kHz was significantly higher than that of others. The result might demonstrate that the suitable ultrasonic frequency can cause more disintegration of cells and give higher mass transfer, which results in higher extraction yields. Furthermore, the optimum ultrasonic frequency can attract bubble collapse by driving the bubble into resonance because the extraction depends on the degree of cavitation activity. When the ultrasonic frequency is equal to the natural resonance frequency of the bubble, the bubble will collapse violently; therefore, it is favorable to enhance extraction yields. Hence, 54–55 kHz is the optimum ultrasonic frequency for bufadienolides, which can give a maximum yield. Another probable reason was that transducers in the third treatment group (54–55 kHz) might work under a sound condition, in which all transducers could produce maximum output efficiency and lead to more disintegration of cells.

3.5 Comparison of the results between this system and other methods

To understand the advantage of this extraction system, this method was compared with the existing UAE and SE methods.

In this study, Soxhlet extraction (SE) was performed with 0.5 g (60–80 mesh) of powder. 100 ml of methanol was placed into a classical Soxhlet apparatus. Extraction was carried out at a temperature of 90 °C. After 6 h of extraction, the concentration of the sample was determined by HPLC analysis.

For the existing UAE experiments, a rectangular ultrasonic bath (KJ-300, Wuxi Kejie Ultrasonic Electronic Equipment Co. Ltd., inner dimension: (300 × 240 × 150 mm³)) with an ultrasound power of 125 W and ultrasonic frequency of 40 kHz was used as the ultrasound source. The extraction temperature was maintained at 20 °C. The sample beakers were immersed into the ultrasonic bath for irradiation under the extraction conditions, including 70% methanol, particle size of 60–80 mesh, and solvent to solid ratio of 25 ml g⁻¹. When the extraction was finished, the mixtures were filtered through a 0.45 μm membrane filter. Finally, the filtrates were collected for HPLC analyses. All the samples were prepared and analyzed in triplicate.

The comparison of yields is summarized in Table 1. It could be seen that the extraction yield in this system over 15 min was higher than that of the existing 20 min of the UAE method and 3600 min of the SE method. Clearly, by reducing the power and time required for extraction, this extraction system has proven to be a promising method that offers an improved extraction yield. These results showed that this extraction system was very efficient for the extraction of bufadienolides from ChanSu when compared to the existing UAE and SE methods.

Table 1 Comparison of the present system with other extraction methods

Comparison	SE	UAE	This system
Time (min)	3600	20	15
Temperature (°C)	90	20	20
Power (W)	—	125	100
Frequency (kHz)	—	40	54–55
Yield (mg g^−1)	205.53 ± 1.26	226.46 ± 3.12	289.75 ± 3.42

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4 Conclusions

This work has studied systematically the influence of ultrasonic frequency in a continuous range of 20–84 kHz on the extraction yield of bufadienolides from the Chinese medicine ChanSu, in which an on-line extraction system was developed to perform the determination of the optimum ultrasonic frequency. Other extraction parameters, such as the type of solvent, solvent to solid ratio, ultrasound power, extraction temperature and particle size, also have been optimized. A maximum extraction yield was obtained at a frequency of 54–55 kHz, which had a weak effect under other extraction conditions. In comparison with the existing UAE and SE methods, the developed system not only minimized the time-consuming treatments of samples and other required manual manipulations but also improved the extraction yield.

Acknowledgements

The authors acknowledge the support of Natural Science Foundation of Fujian Province of China (no. 2015J01661) and the Program of Motor Design and Control System by the Innovation Team of Ningde Normal University (no. 2013T04).

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