Part Ⅰ:Cistanche:Highly Efficient Adsorption Of Phenylethanoid Glycosides On Mesoporous Carbon
Mar 04, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Helin Xu, Wenjing Pei, Xueqin Li, and Jinli Zhang
Phenylethanoid glycosides are the major active compounds of Cistanche tubulosa（Click for products）, and it is extremely desirable for obtaining high purification of Phenylethanoid glycosides by adsorption from their extracts. To explore the highly efficient adsorption of Phenylethanoid glycosides, a novel adsorption material for the efficient separation and purification of phenylethanoid glycosides (Phenylethanoid glycosides) from Cistanche tubulosa was explored. The three mesoporous carbons of ordered mesoporous carbon (CMK-3), disordered mesoporous carbon (DMC), and three-dimensional cubic mesoporous carbon (CMK-8) were compared for adsorption of Phenylethanoid glycosides. Meanwhile, adsorption isotherms, adsorption kinetics, and the optimization of adsorption conditions were investigated. The results indicated that CMK-3 showed the highest adsorption capacity of 358.09 ± 4.13 mg/g due to its high specific surface area, large pore volume, and oxygen-containing functional groups. The experimental data can be accurately described using the Langmuir model and pseudo-second-order model. The intra-particle diffusion model suggested that the rate-limiting steps of adsorption were intra-particle diffusion.
INTRODUCTION
Cistanche tubulosa was an Orobanchaceae parasitic plant (Li et al., 2016; Wang X. et al., 2017), and it mainly grew on the roots of Tamarix plants and Calotropis species (Zhang W. et al., 2016; Yan et al., 2017). Cistanche tubulosa was originally recorded in Shen Nong’s Chinese Materia Medica in ca. 100 B.C. The growth and cultivation of Cistanche tubulosa required severe environmental conditions, and it was widely planted in arid lands and deserts of the northern hemisphere, such as the provinces of Xinjiang, Inner Mongolia, Gansu, Qinghai, and the Ningxia Autonomous Region in China (You et al., 2016). Cistanche tubulosa was a precious Chinese tonic herb that had the functions of nourishing the kidney, anti-aging, boosting the essence of blood, and moistening the large intestines to free stool (Gu et al., 2016; Shimada et al., 2017; Cui et al., 2018), and it has been reputation as “Ginseng of the Deserts” (Song et al., 2016; Wang et al., 2018). Cistanche tubulosa was officially recorded in the Chinese Pharmacopeia as the authentic source of Cistanches Herba (Chinese name: Rou cong rong) from the 2005 edition (Wang T. et al., 2016; Pei et al., 2019).
The previous study had revealed several main chemical constituents of Cistanche tubulosa, including PhGs, iridoids, and polysaccharides (Li et al., 2018a). The structures of PhGs were mainly composed of cinnamic acid and polysaccharides alcohol that was attached to a β-glucopyranose through ester and glycosidic linkages (Luo et al., 2010), and phenylethanoid glycoside has been regarded as the major active components of Cistanche tubulosa possessing various pharmacological activities (Liao et al., 2018). The study showed that PhGs had a variety of medicinal properties, such as neuroprotection, immune regulation, anti-inflammatory, liver protection, and antioxidant (Aiello et al., 2015; Shiao et al., 2017; Wu et al., 2018, 2019). According to phytochemical evaluations, PhGs such as echinacoside, acteoside were considered to be the main active components and markers of Cistanche tubulosa (Li et al., 2017b), which were usually chosen as marker compounds for the quality evaluation of Cistanche tubulosa and the species of Cistanche were distinguished through these compounds. PhGs were naturally occurring water-soluble compounds because they had many hydroxyl groups and phenolic hydroxyl groups in the molecule. Thus, the phenylethanoid can be separated from Cistanche tubulosa in an aqueous solution.
Many methods for the separation and purification of natural products have been developed including adsorption (Liu et al., 2016), membrane separation (Zhang et al., 2018b; Li et al., 2019) and solvent extraction, and so on (Li et al., 2015a,b; Wang S. et al., 2016; Zhang H. et al., 2016). However, membrane separation and solvent extraction were not suitable for large-scale preparation and they were difficult to achieve high recovery of the products (Zhang et al., 2018a). Adsorption was one of the most widely adopted methods for the separation of natural products (Wang S. et al., 2016; Konggidinata et al., 2017). Owing to its unique and tunable pore structures, high surface areas, and mechanical stability, mesoporous carbons (pore size between 2 and 50 nm) have been proven to be a kind of efficient adsorbents for adsorptive natural products. The study has shown that mesoporous carbons were more suitable for adsorbing macromolecules, such as mesoporous carbons have been used by Qin et al. to the enrichment of chlorogenic acid from eucommia ulmoides leaves (Qin et al., 2018). Li et al. synthesized two mesoporous carbons via a hydrothermal treatment approach and evaluated the adsorption performance of two mesoporous carbons for berberine hydrochloride and matrine from water (Li et al., 2018b). It was considered to be a kind of promising material as a highly efficient adsorbent (Zhang et al., 2013; Tian et al., 2015; Zhou et al., 2016). Additionally, mesoporous carbons also have been applied on adsorptive removal of aromatic compounds, dyes, and heavy metals from wastewater (Kong et al., 2016). In previously published works, Liu et al. used a macroporous resin to adsorb PhGs from Cistanche tubulosa, and the purity of the PhGs increased but the adsorption capacity and desorption rate were low. Compared with macroporous resin, mesoporous carbons had the characteristics of large specific surface area, suitable pore size, and a high pore volume. Therefore, mesoporous carbon was considered to be a highly efficient adsorbent for PhGs. In this study, the three kinds of mesoporous carbon were selected as adsorbents for the separation and purification of phenylethanoid glycosides from Cistanche tubulosa.
This work's main objective was to explore the adsorption performance of CMK-3 for the separation and purification of phenylethanoid glycoside from Cistanche tubulosa. The effects of different concentrations, pH, and temperature on the adsorption performance of CMK- 3 were investigated and optimal adsorption conditions of PhGs were screened out. Mesoporous carbons were characterized by FT-IR, BET, TEM, and TGA, adsorption isotherms, and kinetics were performed and analyzed in detail.
Cistanche tubulosa
EXPERIMENTS
Materials and Reagents
Cistanche tubulosa stem was purchased from Congrongtang Biological Technology Co., Ltd. (Xinjiang). The standards of echinacoside (purities ≥ 98%) and acteoside (purities ≥ 98%) were purchased from Sunny Biotech Co., Ltd. (Shanghai). Acetonitrile, methanol, and acetic acid of HLPC were purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai). The ethanol of analytical grade was purchased from Yongsheng Fine Chemical Co., Ltd. (Tianjin). Ordered mesoporous carbon (CMK-3), disordered mesoporous carbon (DMC), and three-dimensional cubic ordered mesoporous carbon (CMK-8) were purchased from Xianfeng Nano Material Technology Co., Ltd. (Nanjing).
Characterization
The morphology and microstructures of the prepared samples were investigated using Transmission electron microscopy (TEM, Tecnai G2 F20) operated at 200 kV. The TEM samples were prepared under ambient conditions by depositing droplets of the ethanol solution with the mesoporous materials onto carbon films supported by Cu grids. Generally, a light source with a shorter wavelength was selected to increase the resolution of the microscope, and the structure of the mesoporous carbons can be clearly observed. The surface functional groups were qualitatively measured by Fourier transform infrared spectroscopy (FT-IR, AVATAR360) using the interaction between infrared radiation and matter molecules. FT-IR use attenuated total reflection method test, the conditions were step size of 2 cm−1 and scanning range was 4,000–400 cm−1. The physical structure data such as the specific surface area, pore size, and pore volume of the mesoporous carbons were calculated by Brunauer-Emmett-Teller (BET, ASAP 2460). The procedure for the adsorbent was as follows: mesoporous carbons were degassed at 60◦C for 12 h, and the N2 adsorption-desorption curves were tested at −196◦C to calculate the specific surface area, pore size, and pore volume of the mesoporous carbon. Thermogravimetric analyzer (TGA, STA 449 F3) is an instrument that uses the thermogravimetric to detect the temperature-mass relationship of a substance, and TGA measures the mass of a substance as a function of temperature under program temperature control. TGA data were obtained using a TGA in the temperature ranging from 30 to 800◦C at a heating rate of 10◦C/min under an air atmosphere.
HPLC Analysis
The content of echinacoside and acteoside was detected by high-performance liquid chromatography (HPLC, Waters Co., USA). The system included an autosampler, high-pressure pump, and ultraviolet (UV) detector. The analysis was conducted on the asymmetry C18 column (100Å, 5µm, 4.6 × 250 mm). HPLC used the gradient elution method to separate and detect samples. The volume of the injection loop was 10µm, the column temperature was 30◦C, detection wavelength of the UV spectrophotometer was 330 nm, the flow rate was 1 ml/min and the mobile phase was (A) acetonitrile and (B) acetic acid/water (1:44, v/v).
Adsorption Equilibrium
The optimization experiment of adsorption condition for CMK- 3 has been carried out using a mixture of acteoside and echinacoside and under the optimal conditions, the crude extract of Cistanche tubulosa was carried out on adsorption cycle experiment and all adsorption experiments were repeatedly carried out at least 3. In the same batch of experiments, mesoporous carbons of CMK-8 and DMC were run in parallel with the CMK-3. The three kinds of mesoporous carbon (CMK- 3, DMC, and CMK-8) each 10 mg were added to the three bottles, respectively. Then 15 mL sample solution with the initial concentration of C0 (mg/mL) was added to the bottle. The bottle was placed in a constant temperature shaker of 30◦C for 24 h until the adsorption equilibrium was reached. Then 1 ml of adsorption solution was filtered through a 0.22µm filter and the equilibrium concentration Ce (mg/mL) of the sample solution was determined by HPLC.
Desorption Experiment
Then the desorption experiment of mesoporous carbon was carried out. The adsorbed mesoporous carbon under 15 mL of methanol/acetic acid (9:1, v/v) mixed solution, which was placed in the water bath of ultrasonic for 1 h at 30◦C. The obtained desorption solution was filtered by a 0.22 filter before analysis by HPLC. The adsorption capacity QE (mg/ml) was evaluated as follows:
QE = (C0 − Ce) · v/w (1)
(1)where V is the volume of the solution (mL) and W is the weight of the mesoporous carbons (g)
RESULTS AND DISCUSSION
Characterization
Figure 1 showed a TEM of the three kinds of mesoporous carbons. DMC was a disordered porous network, CMK-8 was a network structure of three-dimensional porous, and CMK-3 was a clearly striped structure with ordered one-dimensional pore, which was similar to the reported results (Wang et al., 2006; Luo et al., 2010).
Figure 2 showed the FT-IR spectrum of the mesoporous carbons (CMK-3, DMC, and CMK-8) and the FT-IR spectrum before and after CMK-3 adsorption. It can be seen from Figure 2A that the functional groups on the surfaces of the mesoporous carbons were mainly oxygen-containing groups. The overall shapes of the spectra for the three kinds of mesoporous carbons were similar. The mesoporous carbons showed a peak band at 3,423 cm−1 referring to the stretching vibration band of O-H. The bands in the region of 1,580 and 1,629 cm−1 correspond to stretching vibrations of the carbonyl and carboxyl C=O. Additionally, the peak occurring at 1,384 cm−1 was found to be stretching vibrations of alcoholic C-O and the tensile vibration at 2,922 and 2,852 cm−1 corresponds to the C-H on methylene and methyl groups, respectively. This indicated that the oxygen-containing groups existing on the surfaces of the mesoporous carbons might lead to a weak chemical interaction between PhGs molecules and the mesoporous carbons.
Figure 2B shows the FT-IR spectra of CMK-3 before and after adsorption, acteoside, and echinacoside. The characteristic peak at 1,697 cm−1 derived from the C=C of olefin in acteoside and echinacoside, while the bands in the region of 1,519–1,423 cm−1 corresponded to stretching vibration peak of the aromatic ring C=C in acteoside and echinacoside. The tensile vibration at the 1,604 cm−1 was the C=O bond and the peak at 1,157 cm−1 was caused by the stretching vibration of the ether bond in acteoside and echinacoside. Compared with the FT-IR spectrum of CMK-3 before adsorption, the FT-IR spectrum of CMK-3 after adsorption appeared the new peaks, which belonged to the characteristic peak of acteoside and echinacoside.
The N2 adsorption-desorption isotherms were an important parameter for the adsorption of PhGs on CMK-3 and the comparison of adsorbent structure. Figure 3 showed the N2 adsorption-desorption isotherms of CMK-3, CMK-8, DMC, and CMK-3 after PhGs adsorption, respectively. As can be seen from Figure 3, the isotherm of the mesoporous carbons was similar to type-IV isotherm in that this type of isotherm was predominantly mesoporous, in which the range of pore size was between 2 and 50 nm (Sanz Pérez et al., 2019). The gap between adsorption and desorption isotherm was referred to as hysteresis loop caused by capillary condensation reaction. For capillary condensation reactions, capillary condensation occurs first in the smallest pores (Barsotti et al., 2016). This shows that CMK-3 had a smaller mesopore than does DMC and CMK-8, which was consistent with the results of Table 1. The isotherm of CMK-3 exhibits an H1 hysteresis loop that was indicative of the rapid pore filling associated with capillary condensation and the pore structure of CMK-3 was reasonably orderly. The isotherm of DMC exhibits an H3 hysteresis loop, this type of hysteresis had disordered pores due to a network of pores that caused an undefined structure of porous adsorbent. CMK-8 isotherms exhibit an H2 hysteresis loop, indicating that pore structure was complicated and pore size distribution was uneven.
The N2 adsorption-desorption isotherms of CMK-3 were compared before and after the adsorption of PhGs. The isotherm of the CMK-3 after adsorption was also similar to type-IV isotherm in Figure 3B. It indicated that the CMK-3 maintained its mesoporous structure after the adsorption. As can be seen from Table 1, the specific surface and pore volume of CMK-3 after adsorption exhibited a marked decrease, the specific surface area of CMK-3 before and after adsorption decreased from 1,098.02 to 227.75 m2 /g, and the pore volume that reduced from 1.32 to 0.42 cm3 /g. It indicated that PhGs molecules were adsorbed on CMK-3
Table 1 summarized the BET-specific surface area, pore-volume, and pore size of the four samples. The BET surface areas of CMK-3, DMC, and CMK-8 were 1,098.02, 430.42, and 596.00 m2 /g, and the pores volume were 1.32, 0.70, and 0.85 m3 /g, respectively. The pore size of CMK-3 was 4.31 nm, lower than that of CMK-8 (9.58 nm) and DMC (5.18 nm). It can be seen that the pore volume and specific surface area follow the order: CMK-3 >CMK-8 >DMC, while pore size follows the order: DMC >CMK-8 >CMK-3.
Figure 4 shows the TGA curves of the three kinds of mesoporous carbons (CMK-3, CMK-8, and DMC). As can be seen from Figure 4, the three kinds of mesoporous carbons all have two distinct stages of mass loss: the first stage of mass loss was due to the evaporation of moisture in the mesoporous carbons before 100◦C, the second mass loss stage of CMK- 3, DMC and CMK-8 approximately occurs at 660, 427, and 615◦C, respectively, which corresponds to the oxidative thermal decomposition of mesoporous carbons materials. It can be seen that the thermal decomposition temperature of CMK-3 was higher than CMK-3 and CMK-8, the thermal stability of CMK-3 was better than that of CMK-8 and DMC.
