In this study, naringenin in Dendrobium officinale Kimura et Migo was sucessfully extracted by supercritical carbon dioxide by a pilot supercritical fluid extraction (SFE) unit at 35 MPa and 50 ℃.
Supercritical carbon dioxide with ethanol is employed as the desorbent for a three-section simulated moving bed with open-loop design, called SF-SMB, to separate the naringenin from Dendrobium officinale crude extract, and the experimental validation of the separation is accomplished in this work.
Silica was used as the stationary phase, and the crude extract of Dendrobium officinale extracted by supercritical carbon dioxide containing mainly the naringenin was used as the feedstock for the SF-SMB. From single column chromatography, the operating conditions for series experiments with 18 wt% of ethanol were conducted.
The results were compared to that predicted by the Triangle theory to determine the separable operating conditions and the dead volume of the SF-SMB unit.
This study provides effects of operating conditions on the extraction of Dendrobium officinale, which are useful for effectively operating a full scale unit and future study on economical feasibility. From this study, the SF-SMB is demonstrated as a useful technology to the separation of natural products and provides greener potential alternative for developing botanical drugs.
Dendrobium officinale Kimura et Migo is the dried stem of Dendrobium officinale in the genus Dendrobium.
The extraction of Dendrobium candidum
At present, the extraction of Dendrobium candidum mainly adopts the traditional cold immersion water / alcohol extraction, or the reflux extraction of organic solvents. These methods have the disadvantages of large solvent consumption, time-consuming and low dissolution components.
In the process of separation and purification, the column chromatography is usually repeated many times to purify the target. This commonly used adsorption and chromatography technique often leads to serious product dilution, high solvent consumption, low efficiency of the adsorbent, and high operating costs.
Not suitable for industrial production.
Supercritical fluid extraction
At present, supercritical fluid extraction technology (SFE), as a new type of extraction and separation technology, is widely used in the extraction and separation of active ingredients of medicinal plants.
In particular, supercritical carbon dioxide has the advantages of gas diffusibility and liquid solubility, low critical point (7.2 MPa, 32 ℃), easy operation, and fluidization to reduce gasification and extract separation after decompression, and can effectively reduce the extraction The temperature and the amount of organic solvent can achieve the purpose of keeping activity and reducing energy consumption.
Simulated Moving Bed
In addition, the Simulated Moving Bed (SMB) is an effective continuous adsorption chromatography method, which is suitable for industrial separation of natural products.
In view of this, this paper expects to extract and separate the effective components of Dendrobium candidum by supercritical carbon dioxide extraction combined with supercritical fluid simulated moving bed technology (SF-SMB), which will provide a reliable basis for the industrial production of Dendrobium candidum natural products.
Supercritical CO2 extraction of Dendrobium candidum
Take fresh Dendrobium officinale to dry and crush it to an appropriate particle size, take about 180 g in the extraction tank, the temperature during extraction is set to 50 ℃, the extraction tank pressure is set to 35 MPa, and the carbon dioxide flow rate is set to 60 g / min;
After the extraction tank pressure and temperature reach the set conditions, use the infusion pump to inject the auxiliary solvent (ethanol) into the system, and pump 7.42 wt% (weight percent) of ethanol and carbon dioxide simultaneously for a total of 11 hr.
The pressure is reduced in the gas-liquid separation tank, and the ethanol dissolved in the crude extract of Dendrobium candidum is separated from the carbon dioxide gas-liquid.
During the extraction process, the extracts dissolved in ethanol were collected at the separation tank every 30 min and given the numbers TPSH001-TPSH022. Take 6 mL of crude extract from each numbered sample and vacuum dry (40 ° C) until the weight is no longer reduced, calculate the weight of the crude extract. Based on the residual weight obtained above, the weight of the extraction product collected within 30 min was calculated inversely.
If the accumulated weight of the extracted product and the weight of the supercritical fluid passed are divided by the weight of the loaded raw material, it can be drawn to draw an extraction curve (referred to as qE diagram), where q refers to the total weight of carbon dioxide flowing through The weight ratio of the dry weight of Dendrobium candidum, E represents the weight ratio of the extract to the dry weight of Dendrobium candidum, that is, the extraction yield of the supercritical fluid extraction product of Dendrobium candidum.
Screening auxiliary solvents with SFC
Since the crude extract obtained by extracting Dendrobium candidum differs greatly in polarity, and the solubility of pure carbon dioxide is extremely low, the solubility of polar substances is poor, so when using supercritical carbon dioxide as the mobile phase for separation, it is necessary to add polar organic Solvent (called auxiliary solvent) to increase the solubility of the mobile phase.
At the same time, Dendrobium is used as a Chinese medicinal material, and its products are generally taken orally to improve the immunity of the body.
To ensure the safety and non-toxicity of Dendrobium products, ethanol was used as a co-solvent in this study. In order to select the appropriate ratio of co-solvent to improve the solubility of carbon dioxide, before simulating the moving bed separation experiment, first use SFC (Supercritical Fluid Chromatography) to investigate the effect of the co-solvent ratio on the separation effect, and screen out the optimal co-solvent ratio to obtain iron skin The best separation effect of Dendrobium extract.
The conditions set by the SFC experiment: the pressure is 20 MPa, the temperature is 40 ℃, the CO2 flow rate is 3.625 g / min, the column size used is 250 mm * 10 mm ID, the packing is LiChroprep Si 60 (40 ~ 63 μm), UV-visible light is selected as the detector, and the detection wavelength is 230 nm. To explore the effect of the proportion of cosolvent ethanol at 12 wt%, 15 wt%, 18 wt% and 20 wt% on the resolution of the sample, according to the results obtained by SFC, the theoretical operating conditions of SF-SMB were calculated.
SF-SMB Separation of Dendrobium officinale crude extract
Simulated moving bed chromatography technology is to periodically change the position of the material and eluent inlet and the outlet of each product to simulate the countercurrent circulation movement of the stationary phase relative to the mobile phase, while Supercritical Fluid Simulated Moving Bed (SF-SMB) and SMB are operating The biggest difference is that the mobile phase is replaced with supercritical carbon dioxide.
The SMB in the SF-SMB used in this study is designed as three sections, with a total of two inlets, namely feed and mobile phase, and two outlets, namely Extract, Raffinate consists of 6 columns.
The extracts of Dendrobium officinale (that is, the samples numbered TPSH001-TPSH022) obtained by SFE extraction were all mixed and used as SF-SMB feed (Feed). The amount of crude extract in the feed was about 1600 mg / L.
The conditions set when conducting the SF-SMB experiment: the pressure is 1800 psi, the temperature is 40 ℃, the mass flow rates of carbon dioxide at the Extract end, Raffinate end and Feed end are 4 g / min, 5.6 g / min, 0.6 g / min,
The volume flow rates at the feed end and the Desrobent end are 0.164 mL / min and 2.455 mL / min, respectively. The flow rate setting of the SMB part is shown in Figure 2. After setting all the conditions, fix the flow rate of each inlet and outlet, change the switching time of the valve, and then observe the composition changes of the two outlets of SF-SMB to discuss the effect of different switching times on the separation results.
Results and discussion
The crude extract of Dendrobium candidum extracted by SFE is mixed and analyzed according to the analysis method in 2.4.
It can be found that the SFE crude extract has various components. According to related calculations, SFE technology can obtain crude extract 46 g / kg and naringenin 30 mg / kg from Dendrobium candidum. It can be found that most of the soluble substances can be extracted 2 hr before extraction.
After 2 hr, the extraction rate gradually decreases, but there are still substances with lower solubility that are continuously extracted.
The cumulative yield of naringenin shows that the extraction trend of naringenin and the total product of Dendrobium officinale is basically the same, and the extraction rate rises sharply within 2 hr before extraction, indicating that naringenin can be extracted together with soluble substances. After 9 hr, the extraction rate gradually leveled off, indicating that most of the naringenin had been basically extracted.
Co-solvent proportion screening results
At 20 MPa, 40 ℃ and CO2 flow rate of 3.625 g / min, the proportion of ethanol is 12 wt%,
In addition, the standard naringenin and the crude extract of Dendrobium officinale are mixed in a ratio of 2: 1, and then analyzed by SFC.
It can be seen that when ethanol is 12 wt%, most of the substances in Dendrobium extract can not be washed out due to insufficient solvent power of the mobile phase.
When the percentage of ethanol is increased to 15 wt%, although there is a better Separation effect, but its residence time is longer; if the proportion of co-solvent is increased to 20 wt%, it will lead to poor relative selectivity.
Only when ethanol is 18 wt%, not only is the selectivity good, but the residence time is more appropriate, and the mobile phase has better dissolving power. It was found that naringenin appeared at about 8.8 minutes, while strongly adsorbed substances appeared at about 11.9 minutes.
Based on the above results, the final proportion of the auxiliary solvent ethanol was 18 wt%.
Supercritical co2 extraction process
Pulverization degree:40 mesh
Extraction pressure: 35 MPa
Extraction temperature: 50°C
Separation kettle Ⅰ pressure: 0.5 MPa
Cosolvent: Ethanol； Concentration ：18 wt%
Extraction time: 9HR