Extraction Of Phenylethanoid Glycosides From Cistanche Tubulosa By High-Speed Shearing Homogenization Extraction

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Wenjing Pei and Ruili Guo

Background:

Cistanche tubulosa is a well-known traditional Chinese medicine originating in Xinjiang. It is widely distributed in northern Africa, India, etc. Objective: The major bioactive component of C. tubulosa is phenylethanoid glycosides (PhGs). Echinacoside and acteoside are the indicative components for the determination of PhGs and are mainly used for liver protection, immune protection, etc. Therefore, it is very important to extract the PhGs from C. tubulosa. Methods: In this study, the ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and high-speed shearing homogenization extraction (HSHE) methods were compared. Furthermore, the extraction conditions of the HSHE method were optimized. Results: The results showed that the HSHE method was better than both the UAE and MAE methods, and the optimal extraction parameters of HSHE were an ethanol concentration of 50%, an extraction temperature of 70°C, a rotation speed of 16000 rpm, an extraction time of 2 min, a solid-to-liquid ratio of 1:9, and one extraction cycle. The yields of echinacoside and acteoside were 1.366 and 0.519%, respectively, and the transfer rates of echinacoside and acteoside reached 87 and 94%, respectively. Conclusions: It can be concluded that the HSHE method is a simple, rapid, and efficient technique for extracting PhGs from C. tubulosa. Highlights: An efficient and eco-friendly HSHE method has been investigated for the extraction of PhGs from C. tubulosa. The optimum conditions of the HSHE method for the extraction of PhGs from C. tubulosa were obtained. This research provides a new method for the industrial extraction of PhGs from C. tubulosa.

Cistanche tubulosa

Cistanche tubulosa is a common traditional Chinese medicine that originates in Xinjiang. It is widely distributed in northern Africa, the Arabian Peninsula, Pakistan, and India and was officially recorded in the Chinese Pharmacopoeia in 2010. The main bioactive component of C. tubulosa is phenylethanoid glycosides (PhGs), a class of water-soluble natural products including echinacoside, acteoside, isoacteoside, and 2-acetylacteoside (1, 2). The structure of PhGs consists of cinnamic acid and hydroxyphenylethyl moieties, both of which are attached to a β-glucopyranose through ester and glycosidic linkages, respectively (3). Pharmacological studies have shown that PhGs of C. tubulosa have many medicinal functions, such as antifatigue (4), neuroprotective (5–7), liver protective (8–10), and immunoprotective effects (11). Therefore, the effective extraction of PhGs from C. tubulosa is of great significance for the development and utilization of natural products.

The current traditional extraction methods of PhGs are hot-reflux extraction, ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE; 12–16). These methods have different disadvantages, such as low efficiency, long extraction times, and high costs. Compared with the traditional methods, the high-speed shearing homogenization extraction (HSHE) method is an emerging, novel extraction method and has the advantages of faster extraction times, less solvent waste, higher yields, simpler device, and easier operation (17). The working principle of the HSHE method is based on a rotating inner cutter driven by a high-speed motor. A powerful shearing force is produced by the inner and outer cutters to disrupt and mix the samples. Meanwhile, the cutter with high-speed rotation causes pressure differences between the inner and outer cavities of the cutter, resulting in a mass transfer between the samples and solvents. With the combination of high shearing force, collision force, pressure, and other forces, a soluble balance between solid samples and solvents can be rapidly achieved (18). Thus, the extraction process of HSHE can be completed in a few minutes, and even as quickly as dozens of seconds (19–22).

Recently, HSHE has been explored in the extraction of bioactive components from various natural products (23–25). Cheng et al. used HSHE to extract five lignans from the fruit of Schisandra Chinensis (26). The results showed that the optimal conditions were an ethanol concentration of 75%, an extraction time of 1 min, a solid-to-liquid ratio of 1:19, an extraction voltage of 180 V, and a sample particle size of 120 meshes. The total yield of the five lignans was 13.89 ± 0.014 mg/g under the optimized conditions. Tang et al. used HSHE to obtain bioactive oil from perilla seeds (27). The optimal conditions were an extraction time of 2 min, a solid-to-liquid ratio of 1:10, an extraction voltage of 150 V, and two extraction cycles. The yield of perilla seed oil reached 59.3%. The above results indicate that HSHE has the advantages of short extraction times, low costs, and high yields. However, there is a lack of research on the extraction of PhGs by HSHE.

In the present study, HSHE was used to explore its potential application in the extraction of PhGs from C. tubulosa. The yield of PhGs was determined by HPLC. The three extraction methods, HSHE, UAE, and MAE, were compared under the same conditions. The effects of ethanol concentration, extraction temperature, rotation speed, extraction time, solid-to-liquid ratio, and the number of extraction cycles of the yield were investigated. The advantages and disadvantages of this work and the references are compared and discussed.

Materials and Methods

Material and Chemical Reagents

C. tubulosa was purchased from Xinjiang Congrongtang Biological Technology Co., Ltd (Xinjiang, China). The standards of echinacoside (purities ≥98%) and acteoside (purities ≥98%) were purchased from Shanghai Standard Biological Technology Co., Ltd (Shanghai, China). Acetonitrile, methanol, and acetic acid were of HPLC grade (Fisher Scientific, Fair Lawn, NJ). The analytical-grade ethanol was purchased from Tianjin Yongsheng Fine Chemical Co., Ltd (Tianjin, China). Deionized water was used throughout the experiments.

HPLC Analysis

An HPLC analysis was performed on a Waters e2695 liquid chromatography (Waters, Milford, MA) equipped with a 2489 UV/Visible Detector and a Symmetry C18 column (Waters), 250 × 4.6 mm, 5 μm particle size. The concentrations of echinacoside and acteoside were analyzed by the HPLC method. The mobile phase consisted of (1) acetonitrile and (2) a 2% glacial acetic acid aqueous solution. The gradient elution program is presented in Table 1.

The detected wavelength was 330 nm, the flow rate was 1 mL/min, the injection volume was 10 μL, and the oven temperature was set at 30°C. The chromatographic peak of the analyte was confirmed by comparing its retention time with that of the standards. Quantification was carried out by integrating the peak using an external standard method.

The precision test was carried out by injecting the same extraction samples five times. For the stability test, the same samples were analyzed in increments of 0, 1, 2, 4, 12, 24, and 48 h. The recovery tests of echinacoside and acteoside were carried out by adding the standards into the extraction samples. All the peak areas and retention times of echinacoside and acteoside were recorded. The results of the calibration curves for echinacoside and acteoside are listed in Table 2.

Calculation of the Yield

Echinacoside and acteoside were used as the indicative components for the determination of PhGs. The yield of echinacoside or acteoside was calculated using the following equation:

Y=CV/W×100% (1)

where Y = yield of echinacoside or acteoside (%); C = concentrations of echinacoside or acteoside (mg/mL); V = volume of the extraction solution (mL); and W = weight of the C. tubulosa powder (mg).

Extraction Methods of PhGs

The stems of C. tubulosa were first milled by an electric grinder (JC-500g; Yongkang Kaiyuan Industry & Trade Co., Ltd, Yongkang, China) and filtrated by a filter sieve (approximately 380 μm). Then, the C. tubulosa powder was dried in the oven at 70°C until the weight was constant. Five grams C. tubulosa powder was mixed with the extraction solvent and placed in an extraction bottle. After, the liquid mixture was preheated (30–80°C) for 5 min in a constant-temperature water bath (DF-101S; Jintan Analytical Instrument Co., Ltd, Jintan, China).

(a) HSHE method.—HSHE was carried out by an FA25 system (FLUKO Co., Ltd, Shanghai, China), which consisted of the rotating cutter, high-speed motor, and controller. The rotation speed of the inner cutter was regulated in the range of 10000–28000 rpm, and the extraction times were adjusted in the range of 2–15 min.

(b) UAE method.—Five grams dried sample and 300 mL methanol was accurately weighed and added into an extraction bottle. The extraction bottle was immersed into an ultrasonic bath and carried out at an ultrasonic power of 300 W for 10 min. The extraction temperature was set at 30°C.

(c) MAE method.—Five grams dried sample and 300 mL methanol was accurately weighed and added into an extraction bottle. The extraction bottle was put into the microwave reactor and carried out at 180 W for 10 min. The extraction temperature was set at 30°C.

The HSHE method was compared with the UAE and MAE methods with the same extraction solvent, extraction time, extraction temperature, solid-to-liquid ratio, and extraction cycle number. The extraction solution was filtered by a 0.22 μm membrane and analyzed via HPLC to determine the concentrations of echinacoside and acteoside. All the extraction processes were repeated three times

Determination of Transfer Rates

Based on the standard method in the 2015 China Pharmacopoeia, the echinacoside and acteoside contents in C. tubulosa powder were determined (28). One gram C. tubulosa powder and 50 mL 50% methanol were accurately weighed and added into a 50 mL brown volumetric flask. The liquid mixture was weighed and soaked for 30 min. Then, the brown volumetric flask was immersed in an ultrasonic bath and carried out at an ultrasonic power of 250 W and frequency of 35 kHz for 40 min. The liquid mixture was reweighed, and 50% methanol was added to supply for solvent loss. The extraction solution was filtered by a 0.22 μm membrane and was analyzed using HPLC to determine the concentrations of echinacoside and acteoside. The extraction processes were repeated three times.

The transfer rates of echinacoside or acteoside were calculated using the following equation:

X= M1/M2 ×100% (2)

where X = transfer rate of echinacoside or acteoside (%); M1 = quality of echinacoside or acteoside in the extract (mg); and M2 = quality of echinacoside or acteoside in C. tubulosa powder (mg).

Results and Discussion

Validation of the HPLC Method

The HPLC profiles of echinacoside and acteoside from C. tubulosa using HSHE are shown in Figure 1. The RSD of the peak areas of echinacoside and acteoside were 1.89 and 1.40%, respectively. The results showed that the HPLC method has good precision in the quantitative analysis of the echinacoside and acteoside. At various times (0–48 h), the RSDs of the peak areas of echinacoside and acteoside were 1.71 and 1.70%, respectively, indicating that the sample was stable in 48 h. The recovery of echinacoside and acteoside was 99.3 and 99.1%, respectively, and their RSD was 0.70 and 0.67%, respectively, which confirmed the HPLC method’s reliability.

Comparison of the Different Extraction Methods

A comparison of the HSHE method with the MAE and UAE methods regarding the yields of echinacoside and acteoside was conducted, the results of which are shown in Figure 2. It was observed that the yields of echinacoside and acteoside obtained by HSHE were higher than those obtained by both MAE and UAE. Compared with MAE, the yields of echinacoside and acteoside obtained by HSHE were greater by 20.5 and 20.8%, respectively. And compared with UAE, the yields of echinacoside and acteoside obtained by HSHE were greater by 6.6 and 7.4%, respectively. These results indicate that HSHE can significantly increase the yields of PhGs.

Optimization of the HSHE Method

The extraction parameters of PhGs by HSHE include ethanol concentration, extraction temperature, rotation speed, extraction time, solid-to-liquid ratio, and the number of extraction cycles (26, 29).

Effect of Ethanol Concentration on the Extraction of PhGs

Ethanol concentration is an important factor that affects the yields of PhGs. As shown in Figure 3, the yields of echinacoside and acteoside from C. tubulosa under different ethanol concentrations were investigated. It revealed that the yield of echinacoside increased from 0 to 30% and then decreased from 30 to 90% with increasing the ethanol concentrations. The yield of acteoside increased from 0 to 70% and then decreased from 70 to 90% with increasing ethanol concentrations. When the ethanol concentration was 50%, the sum of the echinacoside and acteoside yields was at its maximum. The different ethanol concentrations have different polarities, which leads to different solubilities of echinacoside and acteoside. Thus, the optimal ethanol concentration was 50%.

Effect of Rotation Speed on the Extraction of PhGs

Figure 5 shows the effects of different rotation speeds on the yields of PhGs. The yields of echinacoside and acteoside increased with increasing rotation speeds. The increase of rotation speed can increase the shearing force of the inner and outer cutter, thus increasing the yields of PhGs. The optimal rotation speed was 16000 rpm.

Effect of Extraction Time on the Extraction of PhGs

The yields of echinacoside and acteoside under the different extraction times are shown in Figure 6. The yields of echinacoside and acteoside were stable with increasing extraction times. It can therefore be concluded that PhGs can be completely extracted from C. tubulosa in 2 min, and the optimal extraction time was 2 min.

Effect of Solid-to-Liquid Ratio on the Extraction of PhGs

As shown in Figure 7, the yields of echinacoside and acteoside under the different solid-to-liquid ratios were discussed. The yields of echinacoside and acteoside increased from 1:3 to 1:9 and became stable from 1:9 to 1:24. Because a small amount of extraction solvent cannot completely dissolve echinacoside and acteoside, the mass transfer rates were reduced. The excessive extraction of solvents caused unnecessary waste. Therefore, the optimal solid-to-liquid ratio was 1:9.

Effect of Number of Extraction Cycles on the Extraction of PhGs

Figure 8 shows that the effects of a number of extraction cycles on the yields of PhGs. The yields of echinacoside and acteoside increased with the increasing number of extraction cycles. However, the extraction times remarkably increased with increasing extraction cycles, and more energy was consumed. The optimal number of extraction cycles was one.

Comparing the HSHE Method with Other Extraction Methods for Extracting PhGs

The results of the HSHE method in comparison with other extraction methods for extracting PhGs from C. tubulosa are listed in Table 3. HSHE is simpler and easier than the coupling methods such as the ultrasound-assisted aqueous two-phase extraction and ultrasonic microwave extraction technology methods. In addition, the extraction pressure of HSHE is lower than the pressurized liquid extraction method, and the operation conditions of the former are safer. More importantly, it can be seen from Table 3 that HSHE shows the shortest extraction time and the least solvent consumption among all the extraction methods. It can be concluded that the HSHE method has many advantages such as high efficiency, mild operating conditions, low consumption levels, simple equipment, and easy operation for the extraction of natural products.

Conclusions

In this study, an efficient and ecofriendly HSHE method has been investigated for the extraction of PhGs from C. tubulosa. Compared with the UAE and MAE methods, the novel HSHE method produces a higher yield of PhGs under the same conditions. Compared with the MAE method, the yields of echinacoside and acteoside were increased by 20.5 and 20.8%, respectively. And compared with UAE, the yields of echinacoside and acteoside were increased by 6.6 and 7.4%, respectively. The extraction parameters of HSHE of PhGs from C. tubulosa were optimized. Under the optimized parameters, the yields of echinacoside and acteoside reached 1.366 and 0.519%, respectively, and the transfer rates of echinacoside and acteoside reached 87 and 94%, respectively. The HSHE method has the advantages of high efficiency, mild operating conditions, low consumption levels, simple equipment, easy operation, and low costs. This research provides a new method for the industrial extraction of PhGs from C. tubulosa.