Article pubs.acs.org/IECR
Conceptual Process Synthesis for Recovery of Natural Products from Plants: A Case Study of Artemisinin from Artemisia annua Chandrakant R. Malwade, Haiyan Qu, Ben-Guang Rong,* and Lars P. Christensen Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Niels Bohrs Allé 1, DK-5230, Odense M, Denmark ABSTRACT: A systematic method of conceptual process synthesis for recovery of natural products from their biological sources is presented. This methodology divides the task into two major subtasks namely, isolation of target compound from a chemically complex solid matrix of biological source (crude extract) and purification of target compound(s) from the crude extract. Process analytical technology (PAT) is used in each step to understand the nature of material systems and separation characteristics of each separation method. In the present work, this methodology is applied to generate process flow sheet for recovery of artemisinin from the plant Artemisia annua (A. annua). The process flow sheet is evaluated on the basis of yield and purity of artemisinin obtained in bench scale experiments. Yields of artemisinin obtained in individual unit operations of maceration, flash column chromatography, and crystallization are 90.0%, 87.1% and 47.6%, respectively. Results showed that the crystallization step is dominant to the overall yield of the process which was 37.3%.
1. INTRODUCTION Natural products form a vast resource of compounds with a huge chemical and functional diversity that may serve as a major resource for drug discovery.1 Traditional medicinal systems of China and India have been using natural products in their crude form in various medicinal preparations to treat different ailments for thousands of years.2,3 However, in order for natural products to be able to enter today’s highly regulated healthcare market they need to be standardized in medicinal preparations. It is almost impossible to standardize natural products in their crude form for example medicinal plant preparations as the chemical composition depends on the origin of the plant material such as geographical location, cultivar, cultivation practice, etc. In addition, natural products in their pure form may offer enhanced activity with reduced side effects.4 Therefore, recovery of natural products from their biological sources in pure form has significant importance. However, the development of processes for the recovery of target compound(s) from their biological sources represents one of the most challenging tasks due to the presence of many other compounds in the biomass and also due to lack of basic process information about the impurities. Very often, the development and design of a production process are problemspecific and rely on the trial-and-error method, especially for the two major steps of isolation and purification, which are commonly involved in recovery of natural products from plants. On the other hand, the current chemical and biochemical process industries face challenges like quicker development of new products and processes, making existing processes more efficient by reducing capital and operating costs, and improving safety and environmental performance. Taking the characteristics of the problem into account, it is worthwhile to investigate systematic process syntheses, which are well-used in bulk chemicals and petrochemicals process synthesis,5,6 for generation of process alternatives for recovery of natural products from plants. © 2013 American Chemical Society
However, there are features of process synthesis for natural products which are distinct from those of conventional process synthesis. First, the concentration of desired natural products is usually very low which makes the problem much more difficult than conventional chemical separation problems. Second, the material systems very often contain hundreds of compounds, and little or no information is available concerning the thermodynamics and kinetics of the isolation and purification separation processes. Third, associating compounds with similar chemical and physical properties as that of the target compound significantly influence the performance of a stand alone separation technique. As a consequence, the conventional process synthesis methods are either not sufficient or not suitable for the conceptual process synthesis of natural products from plants. For example, Douglas’s hierarchical procedure7 which is a heuristic-based methodology and which is widely used for conventional process synthesis is not sufficient. On the one hand, very few heuristics are available for conceptual process synthesis of natural products. On the other hand, even if heuristics can preliminarily help to select the possible separation methods, there are neither shortcut nor rigorous methods to predict process stream information. Similarly, due to the complexity of the process, the mathematical programming methodology like a mixed integer nonlinear programming (MINLP) based optimization method8 is not practical because of a lack of suitable equations for the process. Previous studies by Harjo et al.9 and Ndocko et al.10 describe manufacturing process design for phytochemicals with emphasis on heuristics and guidelines for selection of individual unit operations to generate process flow sheets. However, the Special Issue: PSE-2012 Received: Revised: Accepted: Published: 7157
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Figure 1. Basic process structure for recovery of natural products from plant.
described methodologies do not take into account the molecular level understanding of process streams to aid rational decision making. Also, the general heuristics mentioned are not sufficient to support the decisions as there is less experience to summarize the heuristics. For example, according to one of the heuristics provided by Harjo et al.9 compounds that contain C atoms fewer than four times the combined number of O and N atoms are poorly soluble in nonpolar solvents, but artemisinin (C15H22O5) is freely soluble in dichloromethane and slightly soluble in n-hexane.11 For conceptual process synthesis of natural products from plants, the suitable separation methods together with the suitable separating-agent (e.g., solvents) for isolation and purification of the target compound from the biological source must be selected, which can partly be based on the heuristic support. However, understanding the characteristics of the selected separation methods and their performance requires molecular level information of the target compound and the impurities, for example, target compound distribution and process stream information. Due to hundreds of compounds being present in the mixtures, there are no thermodynamic models to predict the process stream information. Needless to say, for such natural products process synthesis, process analytical technology (PAT) is indispensable in order to get the molecular-level information for all the major separation steps whenever specific samples are processed with defined separation methods and separating agents. The objective of this work is to investigate the PAT-support methodology for the conceptual process synthesis of natural products from plants. The proposed methodology emphasizes the combination of heuristics-based approach and PAT methods for the selection of separation methods in the isolation and purification steps, as well as the generation of the conceptual process alternatives. The proposed methodology is applied for isolation and purification of artemisinin from the plant A. annua. A process flow sheet is generated and evaluated on the basis of yield and purity of artemisinin recovered from the dried leaves of A. annua. Molecular level process data obtained by PAT tools at different process steps are used for understanding the characteristic behaviors of the individual separation methods
as well as their interactive effect regarding the target compound distribution and the impurities.
2. METHODOLOGY 2.1. Basic Process Structure. The basic process structure for recovery of natural products from plants is shown in Figure 1. The task is divided into two subtasks of isolating the target compound(s) from its source and then purification from a complex crude extract. Depending upon the complexity of the crude extract, the purification task is further done in different steps. The problem decomposition is aimed at dealing with the complexity of the problem in a stepwise and the most economical way. Initial selection of individual unit operations and operating conditions can be achieved with the help of available heuristics and bench scale tests to generate a process flow sheet followed by experimental evaluation on the basis of yield and purity of target compound(s). PAT tools such as different analytical techniques and chemometrics are emphasized in the process development for recovery of natural products. Analytical results collected during each step combined with heuristics can improve the rational decision making to a great extent and also can make preliminary evaluation of the feasibility and performance of the processing operations selected. Process development for recovery of natural products from plants is always preceded by the botanical, phytochemical, and pharmacognostical studies of the plant under investigation.12−14 Such studies provide to some extent basic information (Table 1) required in the process development as a starting point. However, very often there is little information available about the target compound(s) physicochemical properties. In the case of availability of target compound(s), these properties can be measured by performing bench scale tests. Otherwise, property prediction tools such as quantitative structure−property relationship (QSPR),15 group contribution method,16 and computational chemistry 17 based tools can also be used to predict physicochemical properties of target compound(s). 2.1.1. Isolation. Most of the natural products are embedded into chemically complex soft tissue matrix of the biological source. In order to recover natural products from such biological sources, first they need to be isolated from the 7158
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such as enantioseparation where initial enantiomeric enrichment is obtained by using chiral chromatography followed by final purification with the help of cost-effective techniques like crystallization.22 In practice, high purity natural products can be obtained by highly efficient stand-alone separation techniques like preparative high performance liquid chromatography (HPLC), but the cost of such a process is always high. Table 4 presents the widely used separation techniques for purification of natural products along with their advantages and disadvantages.18 The present methodology suggests the use of hybrid separation in such a way that preparative separation methods with less operating costs are first used to partially purify target compound(s), then cost-effective techniques like crystallization are used to reach the final purification. The present methodology strongly recommends use of chromatography for partial purification coupled with cost-effective techniques such as crystallization for final purification of crystalline natural products from plants. However, the use of the relatively simple and cost-effective technique of liquid− liquid extraction can also be employed either in combination with chromatography or alone for partial purification of a crude extract. Chromatography and crystallization being powerful separation techniques should accommodate most of the natural product purification problems.23,24 The key to the successful use of such hybrid separation techniques could be finding the synergy between these two operations which in turn largely depends upon the chemical constituents present in the crude extract. Table 5 shows some heuristics for selection of suitable methods and their operating conditions for purification of a crude extract. 2.2. Process Analytical Technology (PAT). PAT is being widely accepted as an important process systems engineering tool used for better understanding and control of the manufacturing processes. It has already been a well-established practice in petrochemical and biotechnology industry.28 Application of PAT as a process system engineering tool in the development of pharmaceutical manufacturing processes is described by Gernaey et al.29 However, application of PAT in petrochemical and biotechnology industry has been introduced mostly at the manufacturing plant level and intended at controlling the processes through continuous measurement of critical process parameters. Recently, the US Food and Drug Administration (FDA) released draft guidance for industry,30 which recommends extensive use of PAT and also suggests its use right from the early stage of process design for better process understanding. It has also outlined various tools such as advanced process analyzers and multivariate data analysis methods encompassing PAT framework. Successful application of multivariate data analysis methods such as PARAFAC and PCA to identify natural compounds present in ripened apples from a complex GC-MS data is described by Amigo et al.31 For grassroots design of a separation process for recovery of natural products, there is always the bottleneck of dearth of basic information and most of the impurities are unknown. Recent advances in the analytical technologies in detecting and identifying chemical compounds rapidly even at very low concentrations can be harnessed successfully in designing separation processes for recovery of natural products. Therefore, in our present methodology, we emphasize application of PAT to get the basic information during the conceptual design, which is to build up the foundation in the design of the separation process for recovery of natural products. This foundation of process information could include molecular
Table 1. Basic Information about the Natural Products and the Raw Materials Required for Separation Process Design9 information about target compound(s) • required purity • form (solid or liquid) • structure (only for solid phase compounds, including amorphous/crystalline, polymorphism, solvation states) • type (single or multiple compounds) • chemistry (molecular structure, chirality, class of phytochemicals, etc.) • intended use (pharmaceutical, food, neutraceutical, etc) • performance (thermal stability, stability toward light, pH stability, stability in solvents, microbial degradation) • physical properties (solubility, melting point, boiling point, etc) information about the harvested plant • plant species • growth conditions, cultivation, and harvest time • location of target compound(s) in the plants (leaves, flowers, barks, roots, stems, etc.) • physical characteristics (soft, hard, fibrous, woody, etc.) information about the compounds contained in the plant • chemical composition • chemistry (molecular structure, class of phytochemicals) • pure component properties (molecular weight, melting point, boiling point, ΔHfus, ΔHvap, partition coefficient, aqueous solubility, diffusivity, vapor pressure, heat capacity, etc.) • other properties (heat stability, photostability, chemical reactivity/stability, etc.)
source. Unit operation 1 (Figure 1) is designated for this purpose in the basic process structure. Unit operation 1 can be represented by one of many industrial applied extraction methods shown in Table 2, each having their own advantages and disadvantages.18 A comprehensive review of the separation techniques, including extraction methods, used to isolate natural products from biological sources and examples is given by Sarkar et al.19 and Sticher.20 The decision to pick up the best suitable separation method and selection of optimal operating conditions is problem specific and depends upon many factors like concentration of target compound in the biomass, distribution of target compound(s) in the source, final product specifications, and also the physicochemical properties of the target compound(s). Heuristics are frequently employed combined with the basic information available about the raw material and target compound(s) to aid selection of suitable separation technique and operating conditions. Table 3 presents the heuristics for the selection of extraction methods and solvents. In addition, the results obtained from the use of PAT during the bench scale tests is also given due consideration. Selection of suitable separation technique and solvent in the isolation step is very important as it not only determines the recovery of the target compound from the source but also has direct impact on the chemical composition of crude extract and thereby downstream processing of the crude extract. 2.1.2. Partial and Final Purification. The process of isolation of natural products from plants often yields chemically complex crude extract containing many unknown impurities. This makes the purification task of target compound(s) challenging, hence difficult to achieve the goal by using a single separation technique. Therefore, it would be beneficial to take advantage of different separation techniques to form an efficient hybrid separation process for purification of natural products. Use of hybrid separation processes for purification of complex mixtures has been applied successfully in many fields 7159
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supercritical fluid extraction ultrasound assisted extraction
steam distillation
pressurised liquid extraction solid phase extraction soxhlet extraction
selective for volatiles, e.g. essential oils, very simple and cheap technique, no organic solvent required, very clean extracts, high enrichment factor, suitable for preparative scale mild technique, no organic solvents needed (environmentally friendly), short extraction times, automation possible, with 100% CO2 no solvent removal step, solvating power variable via pressure short extraction times, automation possible, reduced solvent consumption, relatively simple and cheap
short extraction times, high efficiency, selective, automation possible, reduced solvent consumption, relatively simple and cheap continuous extraction possible, low solvent consumption
short extraction times, high efficiency, automation possible, reduced solvent consumption
mild technique, exhaustive extraction possible, no heat required
simple and cheap process, does not require special equipment, limited use of solvent, can give good and selective extraction short extraction times, high efficiency, automation possible, reduced solvent consumption
maceration
microwave assisted extraction percolation
no solvent and heating required, relatively simple equipment
advantages
cold pressing
isolation method
Table 2. Commonly Used Extraction Methods for Isolation of Natural Products from Plants
special equipment required, high energy consumption, harsh technique, thermal decomposition, only pure solvents or solvent mixtures forming azeotropes can be used only suitable for nonpolar volatiles, possible decomposition due to water and high temperatures, long extraction times complex apparatus required, less suitable for very polar products, scale up is difficult, fresh plant materials are difficult to extract, involves high pressures heating effect, thermal decomposition, limited to small scale volumes
require target compound(s) in solution, selectivity can be a problem
selectivity can be a problem, difficulty in recovering residual solvent, can be slow, large amounts of solvent required high operating pressures and temperatures, thermal decomposition, harsh technique
need special equipment, thermal decomposition, solvents transparent to microwaves are needed, raw material needs to contain high moisture, limited to small scale volumes
applicable only to raw materials having high content of target compound, e.g., oil seeds, often additional solvent extraction required to recover residual content long extraction times, problem of recovering residual solvent from biomass
disadvantages
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membrane based techniques (reverse osmosis; ultrafiltration; nanofiltration; microfiltration; membrane extraction dialysis; pervaporation; membrane distillation; electrodialysis)
liquid−liquid extraction
most effective and cheap method for final purification of solid natural products, final product quality e.g. crystal size, polymorphs, purity can be manipulated as per the requirement of further formulation process, chiral separations possible no special apparatus needed, high capacity, easy scale up, solvents can be adjusted according to compound, no irreversible adsorption high selectivity can be obtained, mild technique
advantages
powerful separation technique, easy scale up, availability of different separation mechanisms therefore applicable to wide range of samples, continuous operation possible
3. CASE STUDY OF ARTEMISININ FROM A. ANNUA (SWEET WORMWOOD) Artemisinin is an important natural product recommended by World Health Organization (WHO)32 for use in combination
separation methods
level identification of different components during separating crude extract at different process steps and relating their properties to the separation process characteristics and operating conditions. Different PAT tools that can be used in obtaining specific characteristics of process streams at various process steps of the methodology are shown in Figure 2. It is also shown that the generated information can be used as input combined with heuristics for rational decision making at various process steps. Thus, the knowledge base provided by the PAT approach can be valuable in three main ways: it is a foundation for robust process and product design, it supports and justifies flexible regulatory paths for innovative new approaches, and finally it facilitates the improvement of the process flow sheet in case of unacceptable quality attributes of the product such as purity and yield, which are obtained during the evaluation of the process flow sheet. As shown in Figure 2, different process stream characteristics will be defined with the help of PAT tools during the process flow sheet evaluation, which can be effectively used to optimize the process flow sheet in terms of sequencing of unit operations and selection of optimal operating conditions if poor performance of the flow sheet is observed.
chromatography (countercurrent chromatography; liquid−liquid chromatography; thin layer chromatography (TLC); liquid−solid chromatography; ion-exchange chromatography; size exclusion chromatography; affinity chromatography) crystallization (antisolvent crystallization; evaporative crystallization; cooling crystallization; reactive crystallization; precipitation)
Table 4. Widely Used Separation Techniques for Purification of Natural Products
disadvantages
pretreatment of raw material • consider pretreatment of raw material, e.g., grinding, enzymatic treatment, freezing, thawing, cooking, etc., when active components are present inside the cellular matrix21 • materials destined for oil production containing large amounts of proteins (e.g., cotton seeds, soya beans, flax seeds, sesame seeds, peanuts) must be cooked in order to coagulate the proteins before oil extraction21 • consider drying of raw material destined for long-term storage to avoid microbial contamination • portion of fine particles with sizes
