Modern Extraction and Isolation Methods Faculty of Pharmacy MU Department of Natural Drugs prof. PharmDr. Karel Šmejkal, Ph.D. Introduction to Extraction and Isolation Ústav přírodních léčiv2 Why to extract? ̶ Content of active compounds in drug low ̶ Content of active compounds variable ̶ Presence of unwanted compounds ̶ Drug not acceptable because of bad organoleptic properties ̶ The amount for direct preparation of application form/administration too big ̶ Better possibility of dosage ̶ Better compliance of patient Ústav přírodních léčiv3 Content compounds ̶ Main active compounds ̶ Supporting content compounds ̶ Ballast compounds ̶ The aim of extraction: ̶ Remove ballast substances, to maintain main and supporting content compounds, the obtain extract rich in target substance Ústav přírodních léčiv4 Drug Fresh plant or its part Medicinal wines Pressed juices Oil concentrates Cold macerations Water extracts Infusions Decoctions Instant products External preparations (ointments, balms, compresses) Herbal (medicinal) teas Essential oils Cosmetic preparations Aromatic alcohols Pulverized drug Tinctures Extracts Liquid Thick Dry Lipophilic extracts EtOHWater Traditional processing of medicinal plants, their parts and drugs Ústav přírodních léčiv5 Methods of standardization Analytical techniques for determination of markers and standardization Determination of class of compounds Total phenolics, total flavonoids, total alkaloids… Amount of volatile substances (essential oil quantification) Common utilization of color reactions Fast, simple, cheap Possible false-positive results, possible falsification Single compounds as markers • HPLC-DAD, HPLC-MS, GC-MS • Quantification of one or more compounds • Precise, more expensive, low chance of falsifying Requirements for marker: 1) Bioactivity 2) Sufficient content 3) Physico-chemical stability Combination of analytical technique and biological activity Ústav přírodních léčiv6 Extraction - Fick diffusion law Δn/ Δ t = - (DA/h) × (c0 - c) ̶ Δn/Δt – velocity of diffusion ̶ D – diffusion coefficient based on temperature and diameter of diffunding particles ̶ A – diffusion space (surface) ̶ h – diffusion layer ̶ (c0-c) – concentration gradient Ústav přírodních léčiv7 Improved effectiveness of extraction ̶ Maceration (one batch method – periodic) ̶ Decantation, centrifugation, filtration Improved methods (semicontinual) – Percolation – Digestion – Variations on Soxhlet extractor Matrix effects Desintegration of material Meating Stirring Repetition of process Solvent selection Sonication Ústav přírodních léčiv8 MUNI Biopharma Hub9 Solid-liquid extraction techniques ̶ Conventional extraction techniques ̶ maceration, percolation, squeezing, counter-current extraction, extraction through Soxhlet, distillation, etc. ̶ high quantities of expensive and pure solvents, a low selectivity of extraction; a high solvent evaporation rate during the process; long extraction times, thermal decomposition of thermolabile compounds ̶ Unconventional (innovative) extraction techniques ̶ ultrasound-assisted extraction (UAE), supercritical fluid extraction (SFE), microwaveassisted extraction (MAE), extraction with accelerated solvent, solid phase microextraction, enzyme-assisted extraction, and rapid solid-liquid extraction dynamic (RSLDE) via the Naviglio extractor Ústav přírodních léčiv10 Water steam destillation Ústav přírodních léčiv11 Ústav přírodních léčiv12 Dalton´s law Steam distillation Suitable for water-insoluble substances, only volatile compounds Selective Simple Cheap Ústav přírodních léčiv13 Ústav přírodních léčiv14 Products of steam distillation ̶ Essential oils ̶ Eucalyptus, tea-tree oil, camphora ̶ Hydrolates ̶ Rose water ̶ Orange blossom water ̶ Aromatic spirits MUNI Biopharma Hub15 Equipment MUNI Biopharma Hub16 Equipment MUNI Biopharma Hub17 Superkritical fluid extraction ̶ Extraction with utilization of supercritical fluids ̶ Supercritical fluid Pressure and temperature over critical values Physical properties form a transition between the properties of gases and liquids Density close to liquids good dissolving ability Diffusion constant close to gases rapid mass transfer Viscosity lower than liquid advantage of better flow properties Low surface tension easy material penetration Ústav přírodních léčiv18 MUNI Biopharma Hub19 Supercritical fluid extraction Ústav přírodních léčiv20 Advantages of SFE: Gentle technique. Ideally, no organic solvents are needed. Ecologically harmless. Cheap. Fast. Possibility of automation. Changes of solvation strength by changes of pressure. Disadvantages of SFE: Less suitable for polar compounds. More demanding instrumentation. Requires the use of high pressures. Less suitable for leaf extraction. Extraction tuning issues. Difficult extraction of fresh material (water content). CO2 – non-flammable, non-explosive, easily available, cheap, environmentally friendly, advantageous supercritical region (T=31.1 °C; P=7.28 MPa), suitable for the extraction of low polar substances (essential oils, oils, waxes, carotenoids) ̶ Utilization: Hop extraction. Decaffeination of coffee. Extraction of taxol from Taxus brevifolia. Extraction of essential oils and spices. Nonpharmaceutical purposesÚstav přírodních léčiv21 SFE Ústav přírodních léčiv22 https://www.youtube.com/watch?v=DunzmKCDk0c Ústav přírodních léčiv23 Accelerated solvent extraction (ASE) ̶ Increased extraction yields and reduced time ̶ An increased diffusion ̶ Liquids operating above their boiling temperature while being maintained in a liquid state by the increase in pressure ̶ A cylindrical steel container, the extracting solvent is introduced ̶ The temperature of the system is raised above the boiling point of the solvent, which is maintained in the liquid state thanks to a simultaneous increase in pressure (the vial is sealed to resist high pressure values: 100–200 bar) ̶ Not suitable for thermally labile substances Ústav přírodních léčiv24 Accelerated solvent extraction (ASE) https://www.thermofisher.com/cz/en/home/industrial/chromatography/chromatography -sample-preparation/automated-sample-preparation/accelerated-solvent-extraction- ase.html https://www.thermofisher.com/order/catalog/product/083114 MUNI Biopharma Hub25 Subcritical water extraction (SBWE) MUNI Biopharma Hub26 ̶ 1) reversed-phase liquid chromatography using subcritical water as the sole mobile phase-subcritical water chromatography ̶ 2) extraction of environmental samples ̶ 3) hydrolysis, degradation, polymerization, and synthesis reactions using subcritical water as both a solvent or a reactant ̶ 4) environmental remediation such as cleaning contaminated sewages and soils, decomposing pollutants (pesticides, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls) and explosives ̶ 5) extraction of active ingredients from medicinal and seasoning herbs, vegetables, fruits, and other plant related matrices Subcritical water extraction (SBWE) MUNI Biopharma Hub27 Subcritical water extraction (SBWE) ̶ High-temperature and high-pressure water ̶ Temperature and pressure below its critical point (Tc = 374.15 °C, Pc = 22.1 MPa). ̶ Its polarity can be dramatically decreased with increasing temperature ̶ Dielectric constant, viscosity, and surface tension all decrease steadily ̶ Its diffusion coefficient is improved with increasing water temperature ̶ SW can behave similar to methanol or ethanol ̶ Co-solvents include ethanol, methanol, salts, and ionic liquids ̶ Support by microwave and sonication Static model Static × dynamic MUNI Biopharma Hub28 The SWE process can be proposed to have six sequential steps: (1) rapid fluid entry; (2) desorption of solutes from matrix active sites; (3) diffusion of solutes through organic materials; (4) diffusion of solutes through static fluid in porous materials; (5) diffusion of solutes through layer of stagnant fluid outside particles; and (6) elution of solutes by the flowing bulk of fluid Subcritical water extraction (SBWE) MUNI Biopharma Hub29 Subcritical water extraction (SBWE) Offline coupling of SBWE with SBWC MUNI Biopharma Hub30 Subcritical water extraction (SBWE) MUNI Biopharma Hub31 ̶ The major advantage of SBWE ̶ Water is nontoxic → suitable for the extraction of herbs, vegetables, and fruits ̶ No liquid waste disposal ̶ Ecology, economy, and safety ̶ Density, ion product, and dielectric constant can be adjusted by temperature ̶ Optimal temperature 130 to 240 °C ̶ Flavonoids, organic acids, glycosides, carbohydrates, polyphenols, essential oils, alkaloids, lignans, steroids Subcritical water extraction (SBWE) MUNI Biopharma Hub32 Subcritical water extraction (SBWE) Ústav přírodních léčiv33 Ultrasound assisted extraction doi: 10.3390/ijerph18179153 ̶ An innovative technique, used in different settings ̶ “Clean technology” ̶ Use of low solvent volumes ̶ Short Ets ̶ Few instrumental requirements ̶ Low economic and environmental impact ̶ Technique employs ultrasonic waves frequencies between 20 kHz and 10 MHz ̶ power ultrasound (20–100 kHz), characterized by a high intensity, used for extraction and processing applications ̶ signal or diagnostic ultrasound (100 kHz– 10 MHz), employed as a clinical diagnostic technique, and for control and quality assessment ̶ Acoustic cavitation (AC) Ústav přírodních léčiv34 ̶ UAE-Associated Mechanisms • Fragmentation → the reduction of matrix particle size guided by the ultrasonic action → increases the solid surface area to develop mass transfer, driving to better extraction yields • Erosion → the release of solid structures from the matrix into the extractive solvent, caused by the collapse of cavitation bubbles • Sonocapillary → an enhanced penetration of solvent into the canals and pores of the matrix • Detexturation → the solid matrix destruction • Sonoporation → an increase in cell membranes permeability, forming of membrane pores • Local shear stress → generation of shear forces onto the matrix surface, causing the later rupture of its structures and the extraction of inner compounds in the solvent ̶ Relevant Parameters Associated with UAE • Physical parameters - power, frequency, and ultrasound intensity, related with the ultrasound equipment – ET, shape and size of ultrasonic reactors • Medium parameters - the solvent nature and its properties (polarity,, viscosity, surface tension, solvent vapor pressure), extraction time, the presence of gases, new green solvents for lipophilic extraction → ionic liquids and deep eutectic solvents • Matrix parameters • type of matrix, structure, pre-treatment, particle size, or solid-liquid ratio Ultrasound assisted extraction (UAE) Ústav přírodních léčiv35 Ultrasound assisted extraction Advantages ̶ Similar results as extraction by pressing (squeezing) ̶ High speed ̶ Economic advantage ̶ Relatively low-cost technology involved Disadvantages ̶ The system heats up due to the prolonged treatment ̶ The solid matrix is completely crushed → difficult to separate the mass from extract ̶ The use of ultrasound energy of more than 20 kHz may influence the active phytochemicals through the formation of free radicals. Ústav přírodních léčiv36 Microwave assisted extraction Ústav přírodních léčiv37 Microwave assisted extraction ̶ Microwaves at 2.45 GHz ̶ Electric field causes heating ̶ Dipolar rotation Molecules with dipol moment (permanent or induced), both in solvent and solid Oscillation caused collisions and interactions with surrounding molecules → deliberation of thermal energy Larger dielectric constant of solvent – greater heating ̶ Ionic conduction Ion currents formation – resistence induces heat ̶ Extraction ̶ Disruption of weak hydrogen bonding ̶ Viscosity decreases effect ̶ Migration of ions increases solvent penetration into matrix ̶ In some cases, the matrix itself interacts with microwaves while the surrounding solvent possesses a low dielectric constant and thus remains cold Advantageous in the case of thermosensitive compounds - the extraction of essential oils, microwaves interact selectively with the polar molecules present in glands, trichomes or vascular tissues. Localized heating leads to the expansion and rupture of cell walls and is followed by the liberation of essential oils into the solvent Ústav přírodních léčiv38 Microwave assisted extraction ̶ Closed and open vessels Ústav přírodních léčiv39 Microwave assisted extraction ̶ Combination of MAE with different methods ̶ Maceration ̶ Percolation ̶ Distillation Ústav přírodních léčiv40 Rapid Solid-Liquid Dynamic Extraction (RSLDE) ̶ a negative gradient of pressure between the inner material and the outside of the solid matrix (high pressure inside and low pressure outside; Naviglio’s principle). ̶ when the gradient of pressure is removed, the liquid flows out of the solid in a very fast manner and carries out all substances not chemically bonded to the main structure of the solid. an “active” process → the gradient of pressure forces out the molecules techniques based on diffusion and osmosis → “passive” processes, molecules are not forced out of the matrix https://doi.org/10.3390/foods8070245 Ústav přírodních léčiv41 Rapid Solid-Liquid Dynamic Extraction (RSLDE) Figure 1. Schematic representation of the Naviglio extractor consisting of two extraction chambers connected via a conduit: the first two images show the dynamic phase, while the third image the static phase. ̶ An extractive cycle consists of both static and dynamic phases ̶ The static phase ̶ → the liquid is maintained under pressure at about 10 bar on the solid to be extracted ̶ left long enough to let the liquid penetrate inside the solid and to balance the pressure between the inside and the outside of the solid (about 1–3 min) ̶ The dynamic phase ̶ the pressure immediately drops to atmospheric pressure ̶ a rapid flowing of liquid from the inside to the outlet of the solid matrix ̶ a suction effect of the liquid from the inside towards the outside of the solid. ̶ Extractable material transported by solvent ̶ The cycles can be repeated until the solid runs out, about 30 extractive cycles (two-minute static phase; two-minute dynamic phase) Ústav přírodních léčiv42 Comparison of different extraction techniques LUPULI FLOS – Hop flowers (ČL 2017) Humulus lupulus L. – Hop, Chmel otáčivý (Cannabaceae) • a dioecious, right-handed winding climbing plant • cultivated in US, Europe and northern Asia • only female plants are grown, they reproduce vegetatively https://doi.org/10.1016/j.tifs.2019.08.018. Ústav přírodních léčiv43 Hop processing Hop material (raw, granulate, pelets)Phenolics Essential oils Total resins Soft resins (hexan soluble) α-bitter acids β-bitter acids unspecified soft resins (resupones) Hard resins (hexan unsoluble) γ-hard resins δ-hard resins Hot water extraction Steam distillation Methanol, ethanol, diethylether extraction Hexane extraction Ústav přírodních léčiv44 MUNI Biopharma Hub45 Hop products and extracts Products ̶ Mechanical products – compressed hop, granulates Extracts ̶ Ethanol (MW assisted, Rapid Solid-Liquid Dynamic Extraction (RSLDE) – Naviglio´s principles) ̶ Extract containg besides α-acids, β-acids and hop essential oils also phenolics and tannins ̶ 90% ethanol, possible fractionation to get hop tannins (water soluble) ̶ CO2 ̶ Supercritical or subcritical – effectivity/yield/conditions ̶ pure hop resin extract containing α-acids, β-acids and hop essential oils. ̶ Steam distillation (MW assisted) ̶ pure extract hop essential oils ̶ Vaccum distillation and chromatographic purification of CO2 extracts MUNI Biopharma Hub46 Extraction of hop components conventionally or assisted by intensified technologies, a) solvent extraction optionally assisted by microwave (Jeliazkova et al., 2018; Tyśkiewicz et al., 2018), b) extraction assisted by pulsed electric field (Held & Stanis, 2018), and c) pressurized extraction https://doi.org/10.1016/j.tifs.2019.08.018. 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