Industrial Processing of Natural Products from Medicinal Plants

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extraction. It consists in practice of a battery of countercurrent percolators, in which each compartment of the extractor represents a percolator. The percolator is ...
Industrial Processing of Natural Products from Medicinal Plants Ezio Bombardelli and Antonella Riva Indena SpA, Viale Ortles 12 Milan Italy Keywords: good manufacturing practices (GMP), good agricultural practices (GAP), standardization Abstract A presentation with advice on preparing and characterizing traditional pharmaceutical drugs with the objective of producing plant materials and extracts with attributes that make them acceptable as modern drugs. INTRODUCTION Natural products in various forms have been used since immemorial time for the treatment of pathological conditions or for health benefits all over the world. The use of plant or animal ingredients has been reported in several ancient books dating back thousands of years in Chinese, Indian, Egyptian, Greek and Latin cultures and many preparations are still used in several countries. In all continents, even when there are not ancient written memories, the use of plants considered useful are traditionally used and they are still the basis of therapy in developing countries. In western countries, where an allopathic system of medicine substituted almost completely the use of crude extracts, plant derived compounds constitute 25% of the existing drugs. In developing countries the situation is exactly the opposed. From many plants described or traditionally used today, the active principles have been isolated, biologically characterized and used in pure form or in form of suitable derivatives to ameliorate their original profile. For the pure active natural principles, the regulatory requirements both for the preparation and final use are the same as those used for synthetic drugs; as far as the extracts are concerned which are still today very popular in several industrialized countries and predominant in developing countries the regulatory aspects are contradictory and need an harmonization. The target of my presentation today is to present some advice to prepare and characterize this important class of traditional pharmaceutical drugs, in order to produce reproducible items with the dignity of modern drugs. DISCUSSION Today, many extracts apart from the biological and clinical documentation which is normally insufficient are also poorly chemically characterized, the active constituents are not identified, the constancy and stability are erratic and so on. For these reasons many extracts with totally different composition are claiming the same therapeutic properties. It is clear that to obtain biological reproducible data in terms of safety and efficacy the active ingredients must be the same over the time, must be stable and devoid of unpredictable toxicity or side effects. The question is to prepare standardized extracts, identifying the active constituents when it is possible but controlling also the unknown substances in terms of stability and constancy. Standardization Standardization is a difficult task which starts from the bio-mass obtained, possibly cultivating the plant according to Good Agricultural Practice, to the chemical isolation and characterization of active reference substances, to the establishment of validated analytical methods and finally to GMP production of the final ingredient. For many plants traditionally used in the countries of origin, the scenario in the last 15 years changed drastically due to internationalisation and strong demand in the Proc. WOCMAP III, Vol. 5: Quality, Efficacy, Safety, Processing & Trade in MAPs Eds. E. Brovelli, S. Chansakaow, D. Farias, T. Hongratanaworakit, M. Botero Omary, S. Vejabhikul, L.E. Craker and Z.E. Gardner Acta Hort. 679, ISHS 2005

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industrialised countries. For many plants, wild harvest is currently not sufficient and cultivation is requested for the following reasons: - Natural sources of plants traditionally used are not enough for the world market - Environmental and ecological problems after the Rio de Janeiro convention are strictly ruling the harvest of many plants - The standardization of the finished product does not allow the use of any kind of wild biomass available, so restricting their quantity. The cultivation as discussed later presents many advantages but also a few disadvantages: Advantages include: - The standardization of the biomass is usually excellent - The reproducibility and quality of the obtainable extracts is upstanding - The requested quantity of the biomass is unlimited Disadvantages include: - The cultivation of a medicinal plant takes years of agronomic research and technical and financial organization - The content in active substances even in the cultivated plants is very often changeable for perennial plants - The cost of the biomass harvested according to GAP is usually higher Starting from wild or cultivated crops the standardization of extracts for clinical purpose is drastically influenced by several parameters dependent on the quality of the biomass and on the process. The following parameters influence the standardization of botanical extracts: - Use of the correct botanical species - Area of plant origin - Plant harvesting period - Drying conditions - Storage of the plant material - Content of active principles and markers - Ratio among the various active compounds - Qualitative composition of the plant - Extraction process - Contaminants The first point for the identification of an extract useful in therapy concerns the active components able to justify its use with characteristic markers of the plant which are then able to link together at least two classes of substances within narrow limits. Following some criteria discussed later it is possible to obtain a reproducible product. A suitable analytical method must be performed from the beginning in order to check the quali-quantitative composition of the biomass. As far as the biomass is concerned through controlled cultivation, it is possible to avoid a variety of problems that might otherwise derive from incorrect use of different chemical varieties, the presence of contaminants, etc. Most important of all, a cultivated plant can be gathered at the peak of the balsamic period, and desiccating process can be done mechanically and under optimal conditions before enzymatic and microbial degradations which very often occur in the wild due to transportation problems or adverse climatic conditions. As far as the standardization of extracts is concerned, the aim has to be the reproducibility of all the chemical components contained in an extract, including the unknown ones. An extract normally contains several classes of substances, some of which are active principles, whilst others can be their natural vehicles or they are inert substances. To standardize any extract the best procedure to follow is to first fix a quantitative value of the active principle or principles and contemporaneously to fix reciprocal ratios, within very strict limits, between at least two classes of substances present in the extract; by analyzing several lots of biomass before extraction and combining them in a

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mathematical ratio so as to obtain a homogeneous and constant mix it is possible to obtain extracts that are completely reproducible, as can be deduced specific fingerprints and by complementary spectroscopic analyses, such as FT-IR and 13C-NMR which are discussed later in practical applications. Every standardized extract must then have a fingerprint to enable it to be qualitatively evaluated, and validated analytical methods for its quantitative determination. As far as the preparation of the extracts is concerned they must be obtained according to GMP fixing all the parameters usually involved during extraction, concentration, purification and drying process. To summarize the technical requirements for an extract of acceptable quality we can say that the following points are necessary: - Cultivation of the plant according to Good Agricultural Practice - A chromatographic fingerprint has to be established for vegetal material and extract - A quantitative ratio among the selected active principles and markers has to be fixed - A spectroscopic technique (1H-NMR, 13C-NMR, NIR) has to be introduced for evaluation of unknown substances and global stability - Solubility and bio-availability of the active principles - GMP procedure has to be used at production level In conclusion, following the GMP procedures starting from a cultivated plant material to the final product makes possible the preparation of a standardized extract which complies with the safety and efficacy requirements demanded of any product for human use. The parameters influencing the quality of any extract are related to the process and equipment used in their preparation. The scheme of operation for a purified and standardized extract is the following: - Grinding of the plant material - Extraction of plant material - Concentration of the pooled extracts - Counter-current extraction of the concentrates - Drying process of the phase of interest Extraction As far as the extraction of the biomass is concerned, the equipment is very different according to what one would want to obtain. For example, with the grinding process, different equipment adapted to the material to be processed is needed. The procedures for extraction of the ground biomass may be classified into main groups: - Procedures in which it is sufficient to achieve within set limits the equilibrium of concentration between drug and solution (tinctures, decoctions, macerates) - Procedures in which the drug is extracted until exhaustion of the soluble substances in the chosen medium The most important processes today are those described in the second point. The parameters influencing the extraction, operating on large scale, are the swelling of the biomass in contact with the solvent, the particle size, the pH and temperature. The swelling of the biomass is a crucial point and the configuration of the equipment is important for the following reasons: - To avoid sudden swelling of the drug in a closed container, because, if the solvent is aqueous, the drug may swell to two or three times its original volume and so burst the extractor or make percolation impossible - To ensure uniform moistening of the material for extraction and so prevent the formation of preferential channels, increasing the contact and passage of the solvent - To increase the porosity of the cell wall, thus facilitating diffusion of the extractive substances from cell to solvent - To increase the penetration of the solvent in the tissue The value of the first two points is technically self-evident. The third point is of particular importance when, for reasons of selectivity, an extraction has to be performed in a heterogeneous phase, as is the case with the extraction of many alkaloids. Swelling of

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the vegetal material with water in these cases ensures permeation of the solvent, and consequent removal of the substances soluble in it, simply and selectively. Diffusion, pH, particle size, and temperature are each equally important case by case. As far as the equipment is concerned for small to relatively large production and with high versatility the equipment of choice is the classical percolators which can be used alone or in series in order to reduce the volume of solvent needed for the biomass exhaustion. Conceptually, the extraction, as indicated in the second point, is that in countercurrent regulated by the equation: T = 1/KSV2(C1 – C2), where T = time needed to reach desired concentration, K = constant depending on the solid/solvent ratio, S = surface of contact, V = rate of passage of the solvent, and C1 – C2 = solid/liquid concentration difference. It is clearly of interest to increase V in order to decrease the time since V is in the denominator and is squared. For large production or when there is a possibility to perform long campaign the most important equipment for the continuos countercurrent extraction is the screw extractor, carousel extractor, and for larger quantities there is the belt extractor. Screw extractors operate on the absolute countercurrent principle, according to which drug and solvent move in opposite directions. The most common screw extractor is Niro extractor and consists of a long pressure-tight steel cylinder, which can be heated and cooled in sections, equipped inside with a system of screw feeders. The drug reaches the extractor through a loading device at one end of the cylinder and is driven by the screw to the opposite end. The solvent enters the extractor at the opposite end to the drug entry point. Mixing of drug and solvent is a very vigorous process, the intensity of which can be regulated by the number of revolutions of the screw, the slope of the cylinder, the temperature, and the pressure. Through modification of these parameters, when a drug has been through the length of the extractor, continually meeting fresh solvent, it is practically exhausted. The carousel extractor operates on the principle of relative countercurrent extraction. It consists in practice of a battery of countercurrent percolators, in which each compartment of the extractor represents a percolator. The percolator is usually made up of 10 to 20 segments, on one or two planes. The plane rotates and during the movement each chamber containing the biomass is washed by a rain of solvent, which comes from the underlying part and has a different concentration in each point. The exhausted drug at the end of the run is conveyed to the desolventizer. One needs to know all the “ins and outs” of this apparatus to make it work properly. As a rule, the drug has to be pretreated and particle size has to be right to get a constant speed of percolation. Grinding is best done with knife mills to prevent the formation of too fine particles, which should not exceed 10%. The carousel extractor is popular among pharmaceutical manufacturers for the extraction of numerous active principles. Like the carousel, the belt moving tank extractor operates on the principle of relative continuous extraction. This extractor likewise contains the drug in a fixed compartment, through which the solvent passes in the opposite direction. This equipment is also used for the production of oils in the food industry. Other extractors operating in a similar manner are the Hildebrandt extractor and modification of moving tank extractors. Isolation and Concentration After extraction of a drug, the resulting solutions containing the soluble fraction as a rule undergo a process of concentration or of liquid/liquid extraction, if the aim is to select a particular class of components. These extracts may be: - Concentrated partially or totally according to whether they are to be used for preparing total fluid soft or dry extracts - Concentrated partially or counter-extracted with compatible solvents for conversion into purified extracts - Concentrated partially and counter-extracted with selective solvents for the isolation of

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a given active principle - Counter-extracted right away, without concentration, for the isolation of particular products Apart from the last case, in which concentration takes place at a later stage, extracts must be concentrated. Concentration is a tricky stage in the process in which many chemically labile products may undergo degradation, mainly due to the temperature. For this reason several types of high performance concentrators have been invented for operating at reasonably low temperatures (25 to 30°C) or at high temperatures for very short periods. The parameters that govern the choice of an evaporator are the quantity of solution to be concentrated per unit and the properties of the solution. Stable solutions, which do not tend to encrust, may be concentrated in simple apparatus, like ordinary reactors, in which the solutions can be evaporated at ordinary pressure or under vacuum. Industrially, for large quantities of solution one of the most widely used concentrators of the old type is the Robert concentrator consisting of a bundle of concentrator tubes arrayed around a central groove. The tubes are steam heated from the outside and the solution to be concentrated passes through them and on course through a chamber, from which it then passes to the condenser. 1. Concentrators. The most up-to-date types of concentrator in the field of natural products are the descending film, thin layer or plate concentrators. The thin layer evaporator, of which there are many versions, both in design and in capacity, consists basically of a hollow cylindrical body, heated externally, in which a rotor rotates. The rotor design governs the evaporation capacity, which can be adapted to the solution to be evaporated. The rotor spreads the liquid to be concentrated over the hot wall and the evaporation is very rapid. In film evaporators, the phase change occurs in a very thin layer of liquid. The volume of solution involved is very small and so the time spent in the apparatus is very short. By operating under vacuum and hence at a fairly low evaporation temperature, it is possible to ensure gentle treatment of thermo-labile products. After concentration very often the extracts need purification particularly when they have to be counter-extracted. In this context this purification of extracts means the treatment of extraction liquids to remove drug residues or undesirable matter formed in the process. The clarification phase is practically indispensable, whether one intends to obtain the finished product by simple concentration or whether the solution must be purified. In the extraction of a drug a small portion of very fine vegetal material usually manages to pass through the filtering system of the extractor. Inert substances extracted, especially in hot maceration processes, often give rise in the subsequent cooling to flocculates or precipitates that render the solution cloudy and inhomogeneous. The presence of solid particles or thick sediment makes it impossible to carry out the normal continuous countercurrent extractions involving the use of liquid/liquid centrifugal separators, which would thus get blocked, or even the use of extractions in separators, because of the emulsions or formation of additional phases that such material generates. Since products intended for pharmaceutical use must not contain solid particles extraneous to the extract, clarification is obviously essential. Another aspect of the purification of extracts is the reduction of the bacterial content which is not dealt with here as it cannot be dealt with in general terms. Sterile methods of filtration, such as sterilizing membrane filtration, are not suitable for extracts, especially when they contain mucilaginous or polymeric substances which hinder filtration. These processes therefore need to be devised case by case. 2. Dryers. The last phase in extract preparation is the drying process. For the drying process there are today several versions of dryers including lyophilizers, spray-dryers. Lyophilization is the most expensive process and has to be applied to very labile compounds, Spray-dryers represent a good alternative and this technique allows the obtainment of very homogeneous and reproducible powder.

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CONCLUDING EXAMPLE After examination of the basic principles of the extract preparation, it is convenient to consider in practice the obtainment of a standardized extract containing different classes of active principles along with toxic compounds. As a practical example of the application of theories above reported, I would like to consider the preparation of the Ginkgo biloba extract which is a very popular product, having been commercialized in several countries where it is both a prescription drug and/or a health food product. The extract is produced mainly in western countries and in China. The quality of the drug substance is often very different because it is based only on the evaluation of single markets and not the global profile. In the last five years several copies of the leading compound have been realized modifying the composition of documented extracts simply in order to bypass the existing patents and regulations, but the main problem remains the standardization criteria. Ginkgo biloba leaves contain several classes of substances; the derived extracts are controlled for the quali-quantitative presence of five of them. In this presentation I would like to examine the various phases of the process. The plant for the production of the extract is cultivated and the leaves harvested in July/August and mechanically dried within 12 hours. The constituents in the biologically and clinically controlled extracts must be present in this ratio. For the determination of all these components it is necessary to combine different techniques like HPLC, chemical reactions and spectroscopy. For the evaluation of procyanidines, which are prodelphinidenes, it is necessary to perform the Bathe-Smith oxidative hydrolysis and to determine delphinidine by HPLC. The determination of Ginkgolic acids, which are the allergenic substances is also carried out by HPLC. What is important is the strict correlation among the different classes of compounds, each harmonized with the others for the final therapeutic goal. This extract indicates a perfect balance of the different fractions and any modification of this balance, e.g., any attempt to increase the content of any active principle which when tested separately, has displayed a marked specific activity, has turned out to be disadvantageous. On the basis of specific determination this extract is completely known for about 40%; the remaining 60% is only roughly known. To ensure the uniformity of the different batches, due to the complexity of the composition, we have introduced the 13C-NMR determination in the quality control of the extracts. Using this new approach it is also possible to verify the quality of the other extract present on the market. This example can illustrate better than words the difference existing between these two products. Comparing in expansion scale our extract with another, one as you can see there are macroscopic differences which of course are due mainly to the unknown substances, considering that the flavonoids and terpenes are very similar. These substances can influence the bioavailability of the active principles through inhibition of cellular pumps or other mechanisms. There are many other examples of extracts where a proper technology would have an important impact on their safety and efficacy. Respecting certain logic criteria it is possible to prepare at an industrial level valid items derived from an important heritage of human kind; to get reproducible biological results the products must be chemically reproducible. Starting from plants cultivated in proper way and combining suitable analytical techniques as mentioned above, it is really possible both to produce highly standardized extracts, which in terms of reproducibility can compete with pure pharmaceutical compounds, and to check their stability. A biological evaluation in my opinion is necessary when important variations in process are performed in the preparation of extracts already used. Most importantly: is to have the will to solve the problem in respect of the common sense of the quality.

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