Abstract: Objective To purify andrographolide by supercritical CO2 extraction and crystallization. Methods: The effects of supercritical CO2 extraction and crystallization on the morphology, purity and crystallinity of andrographolide were investigated by systematic observation. The results were analyzed by scanning electron microscopy and HPLC . Results The crystal form of andrographolide became thinner and shorter with increasing pressure. With the increase of pressure, the purity of crystal increased and the amount of crystallization increased. Conclusion: Supercritical CO2 can extract and synthesize high-efficiency crystal separation of andrographolide, which provides a shortcut for the development of high-quality active ingredients of traditional Chinese medicine.
Key words: andrographolide; supercritical CO 2 fluid extraction; crystalline andrographis paniculata is the dry aerial part of Andrographis paniculata (Burm.f.) Nees. The main active sites of Andrographis paniculata are diterpene lactone and flavonoids, which have pharmacological effects such as inducing cell differentiation, protecting liver and reducing enzymes, increasing the amount of bile secretion, and changing its physical properties. Andrographolide is clinically used to treat acute gastroenteritis, tonsillitis, hepatitis, and cancer. In order to improve the separation efficiency and product purity of andrographolide, the author used supercritical fluid extraction and crystallization technology to efficiently separate and purify andrographolide. The effect of supercritical CO2 extraction crystallization pressure on the morphology, purity and crystallinity of andrographolide was reported. The effect of extraction crystallization temperature on purity was also investigated.
1 Materials and equipment, Andrographolide extract (andrographolide content of 30%) and andrographolide raw material (andrographolide content of 98%) are provided by Hefei Tuofeng Bioengineering Co., Ltd.; Andrographolide reference substance is purchased from Chinese medicine Biological Products Laboratory; CO2 food grade.
32MPa 1.6 L supercritical CO2 extraction-crystallization device; extraction crystallizer 1.6 L (10:1 aspect ratio); Waters HPLC system: 515 infusion pump, 2487 UV double-beam detector; Hitachi X-650 scanning electron microscope ( Hitachi, Japan).
2 methods and results
2.1 Chromatographic conditions: column: Waters Symmetryshield C18 (150 mm × 3.9 mm, 5 μm); mobile phase: methanol-water (65: 35); flow rate: 1.0 mL / min; detection wavelength: 205 nm.
2.2 Preparation of the standard curve: Weigh accurately 1.82mg of andrographolide reference substance, placed in a 5mL volumetric flask, dissolved in methanol and added to the mark, shaken to obtain the reference stock solution. Pipette 1.0 mL of the solution, transfer to a 5 mL volumetric flask, dilute to volume, and shake well. Inject 2, 6, 10, 14, 18 μL in sequence. The peak area is plotted on the ordinate and the injection volume is plotted on the abscissa. Andrographolide has a good linear relationship with peak area from 0.12 to 1.30 μg. The regression equation is: Y=3470+674X, r=0.9990.
2.3 Preparation of the extract solution for the extract: The andrographis extract was dissolved by heating with ethanol, filtered, and the filtrate was obtained, and the content of andrographolide was determined to be 45 mg/mL.
2.4 Preparation of crystallization test solution: Weigh accurately 2.00mg of crystalline product, placed in a 25mL volumetric flask, dissolved in methanol and added to the mark, shake well, that is.
2.5 Pressure on the crystal morphology: Take the andrographolide on the crystallizer in the extraction kettle. After a period of extraction with supercritical CO 2 , a thin layer of andrographolide crystals appeared on the crystal plate. Scanning electron microscopy of the crystal. It can be seen that the morphology of the andrographolide crystals after supercritical CO2 extraction and crystallization is thinner and shorter as the pressure increases.
2.6 Effect of pressure on andrographolide
2.6.1 Effect of pressure on andrographolide content: Controlled extraction-crystallization kettle temperature was 50 °C, extraction time was 90 min, flow rate was 20 L/min, and pressure was chosen to be 8, 12, 18, 22 MPa, respectively. The crystallizer was equally divided into 3 sections from the bottom from the top and sampled in the middle section. The experimental materials were treated with Andrographis paniculata and pressure-andrographolide content.
2.6.2 Effect of pressure on the crystallizing amount of andrographolide: The temperature of the extraction-crystallization kettle is still controlled at 50 ° C, the extraction time is 90 min, the flow rate is 20 L/min, and the pressure is selected as 8, 12, 15, 18, respectively. 22MPa, still sampling in the middle of the crystallizer, the experimental materials used the andrographolide raw material, pressure-andrographolide mass fraction (mass fraction = local crystallization / total crystallization) curve.
2.7 Effect of temperature on andrographolide content: The experimental materials were treated with Andrographis paniculata extract. The control extraction-crystallization kettle pressure was 18 MPa, the extraction time was 90 min, the flow rate was 20 L/min, and the temperature was selected to be 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, respectively. The crystallizer was equally divided into 3 sections from the bottom from the top and sampled in the middle section. Temperature-andrographolide content curve. It can be seen that under the condition that Other process parameters are unchanged, the content of andrographolide is also positively correlated with temperature, but the degree of influence is small and the curve changes gently.
3 Discussion Andrographolide is a diterpenoid, relative to flavonoids, alkaloids, low polarity, suitable for supercritical CO2 extraction; and ethanol modification, blending CO2 polarity, is more conducive to improving supercritical CO2 extraction A natural active substance with a high polarity and a relatively high molecular weight.
In the supercritical state, the extraction phase equilibrium is affected by the external field, such as crystallizer interference, surface adsorption force, gravity, intermolecular force, etc., crystallization and dissolution form competition, and the simultaneous extraction and crystallization of andrographolide occurs; and the higher the pressure, The greater the solubility of supercritical CO2, the more the number of nucleation per unit time, the shorter the crystal. The higher the extraction pressure, the greater the solubility of andrographolide in supercritical CO2. The stronger the negative effect of overcoming other forces, the higher the growth of andrographolide crystals on the upper end of the crystallizer.
Since the active ingredient in the natural medicine is a crystalline component, when the product is developed by supercritical technology, the phenomenon of crystallization is caused by the separation of the extraction, which causes blockage of the pipeline, which is a great obstacle to the development of the supercritical fluid extraction technology. The simultaneous extraction crystallization technique used in this experiment improves the traditional supercritical fluid extraction and separation process.
References:
E1] Matsuda T, Kuroyanagi M, Della G, et. Cell differentiation—inducing diterpene from Andrographis paniculata Nees [J]. Chem Pharm Bull, 1994, 42(6): 1216-1225.
Key words: andrographolide; supercritical CO 2 fluid extraction; crystalline andrographis paniculata is the dry aerial part of Andrographis paniculata (Burm.f.) Nees. The main active sites of Andrographis paniculata are diterpene lactone and flavonoids, which have pharmacological effects such as inducing cell differentiation, protecting liver and reducing enzymes, increasing the amount of bile secretion, and changing its physical properties. Andrographolide is clinically used to treat acute gastroenteritis, tonsillitis, hepatitis, and cancer. In order to improve the separation efficiency and product purity of andrographolide, the author used supercritical fluid extraction and crystallization technology to efficiently separate and purify andrographolide. The effect of supercritical CO2 extraction crystallization pressure on the morphology, purity and crystallinity of andrographolide was reported. The effect of extraction crystallization temperature on purity was also investigated.
1 Materials and equipment, Andrographolide extract (andrographolide content of 30%) and andrographolide raw material (andrographolide content of 98%) are provided by Hefei Tuofeng Bioengineering Co., Ltd.; Andrographolide reference substance is purchased from Chinese medicine Biological Products Laboratory; CO2 food grade.
32MPa 1.6 L supercritical CO2 extraction-crystallization device; extraction crystallizer 1.6 L (10:1 aspect ratio); Waters HPLC system: 515 infusion pump, 2487 UV double-beam detector; Hitachi X-650 scanning electron microscope ( Hitachi, Japan).
2 methods and results
2.1 Chromatographic conditions: column: Waters Symmetryshield C18 (150 mm × 3.9 mm, 5 μm); mobile phase: methanol-water (65: 35); flow rate: 1.0 mL / min; detection wavelength: 205 nm.
2.2 Preparation of the standard curve: Weigh accurately 1.82mg of andrographolide reference substance, placed in a 5mL volumetric flask, dissolved in methanol and added to the mark, shaken to obtain the reference stock solution. Pipette 1.0 mL of the solution, transfer to a 5 mL volumetric flask, dilute to volume, and shake well. Inject 2, 6, 10, 14, 18 μL in sequence. The peak area is plotted on the ordinate and the injection volume is plotted on the abscissa. Andrographolide has a good linear relationship with peak area from 0.12 to 1.30 μg. The regression equation is: Y=3470+674X, r=0.9990.
2.3 Preparation of the extract solution for the extract: The andrographis extract was dissolved by heating with ethanol, filtered, and the filtrate was obtained, and the content of andrographolide was determined to be 45 mg/mL.
2.4 Preparation of crystallization test solution: Weigh accurately 2.00mg of crystalline product, placed in a 25mL volumetric flask, dissolved in methanol and added to the mark, shake well, that is.
2.5 Pressure on the crystal morphology: Take the andrographolide on the crystallizer in the extraction kettle. After a period of extraction with supercritical CO 2 , a thin layer of andrographolide crystals appeared on the crystal plate. Scanning electron microscopy of the crystal. It can be seen that the morphology of the andrographolide crystals after supercritical CO2 extraction and crystallization is thinner and shorter as the pressure increases.
2.6 Effect of pressure on andrographolide
2.6.1 Effect of pressure on andrographolide content: Controlled extraction-crystallization kettle temperature was 50 °C, extraction time was 90 min, flow rate was 20 L/min, and pressure was chosen to be 8, 12, 18, 22 MPa, respectively. The crystallizer was equally divided into 3 sections from the bottom from the top and sampled in the middle section. The experimental materials were treated with Andrographis paniculata and pressure-andrographolide content.
2.6.2 Effect of pressure on the crystallizing amount of andrographolide: The temperature of the extraction-crystallization kettle is still controlled at 50 ° C, the extraction time is 90 min, the flow rate is 20 L/min, and the pressure is selected as 8, 12, 15, 18, respectively. 22MPa, still sampling in the middle of the crystallizer, the experimental materials used the andrographolide raw material, pressure-andrographolide mass fraction (mass fraction = local crystallization / total crystallization) curve.
2.7 Effect of temperature on andrographolide content: The experimental materials were treated with Andrographis paniculata extract. The control extraction-crystallization kettle pressure was 18 MPa, the extraction time was 90 min, the flow rate was 20 L/min, and the temperature was selected to be 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, respectively. The crystallizer was equally divided into 3 sections from the bottom from the top and sampled in the middle section. Temperature-andrographolide content curve. It can be seen that under the condition that Other process parameters are unchanged, the content of andrographolide is also positively correlated with temperature, but the degree of influence is small and the curve changes gently.
3 Discussion Andrographolide is a diterpenoid, relative to flavonoids, alkaloids, low polarity, suitable for supercritical CO2 extraction; and ethanol modification, blending CO2 polarity, is more conducive to improving supercritical CO2 extraction A natural active substance with a high polarity and a relatively high molecular weight.
In the supercritical state, the extraction phase equilibrium is affected by the external field, such as crystallizer interference, surface adsorption force, gravity, intermolecular force, etc., crystallization and dissolution form competition, and the simultaneous extraction and crystallization of andrographolide occurs; and the higher the pressure, The greater the solubility of supercritical CO2, the more the number of nucleation per unit time, the shorter the crystal. The higher the extraction pressure, the greater the solubility of andrographolide in supercritical CO2. The stronger the negative effect of overcoming other forces, the higher the growth of andrographolide crystals on the upper end of the crystallizer.
Since the active ingredient in the natural medicine is a crystalline component, when the product is developed by supercritical technology, the phenomenon of crystallization is caused by the separation of the extraction, which causes blockage of the pipeline, which is a great obstacle to the development of the supercritical fluid extraction technology. The simultaneous extraction crystallization technique used in this experiment improves the traditional supercritical fluid extraction and separation process.
References:
E1] Matsuda T, Kuroyanagi M, Della G, et. Cell differentiation—inducing diterpene from Andrographis paniculata Nees [J]. Chem Pharm Bull, 1994, 42(6): 1216-1225.
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