WO2010012687A2

WO2010012687A2 - Preparations based on artemisia annua, the conversion thereof into microparticles and nanoparticles, and use of same

Info

Publication number: WO2010012687A2
Application number: PCT/EP2009/059671
Authority: WO
Inventors: Wolf-Dieter Juelich; Hans-Peter Welzel; Ulrike Lindequist; Gerold Lukowski; Ottmar Geiger; Sabine Mundt
Original assignee: Hc Berlin Pharma Ag; Ernst-Moritz-Arndt-Universität Greifswald
Priority date: 2008-07-26
Filing date: 2009-07-27
Publication date: 2010-02-04
Also published as: EP2320867A2; WO2010012687A3; DE102008035442A1

Abstract

The invention relates to preparations based on Artemisia annua, and to a plurality of methods for producing microparticles and nanoparticles from the annual mugwort (Artemisia annua). Ultrafine particles with improved properties are obtained as a result, that can be used as food supplements and feed supplements, in cosmetics and for medical purposes.

Description

Preparations from Artemisia Annua, their transformation into micro- and nanoparticles and their use

The subject of the invention are preparations of Artemisia annua and several processes for the production of micro and nanoparticles from the annual mugwort (Artemisia annua). As a result, ultrafine particles with improved properties can be obtained, which can be used as food and feed supplements, for use in cosmetics as well as for medical applications.

State of the art

1. Preparation of nanoparticles and microparticles

For the production of nanoparticles and microparticles different manufacturing methods based on lipids or polymers are already known. Thus, processes for the preparation of lipid microparticles based on phospholipids have already been described, which have antifungal properties and can be used in the pharmaceutical or cosmetic sector (DE 69 00 2905 T2). Other methods describe lipid nanoparticles based on extracted mono-, di- and triglycerides, oils or waxes. With the help of these lipids obtained also pharmaceutical active ingredients are encapsulated (WO 94/20072 Al). Other lipid nanoparticles also describe substances based on lipids for parenteral administration (WO 98/56362 A1). Special techniques for the preparation of lipid nanoparticles are also presented (eg EP 0 526 666 A). So far, however, no micro- and nanoparticles based on Artemisia Annua and their preparation have been described in the literature.

The production of micro- and nanoparticles using species-specific lipids is already known (WO2004 / 075907 A2 and WO2003 / 72118 Al).

The use of the plant Artemisia annua has so far focused on the use of artemisinin as an antimalarial agent. This requires extraction with apolar solvents such as n-hexane. It inevitably removes the lipids. A direct use of the teaching of WO2004 / 075907 A2 and WO2003 / 72118 Al for the conversion of the remaining biomass of Artemisia annua, which contains even more valuable ingredients in micro- and nanoparticles is therefore not possible because of the separated lipids. The remaining biomass and thus also the active substances contained therein remain largely unnoticed in the previous utilization concepts.

2. Previous use of Artemisia annua

The annual mugwort (Artemisia annua) has been used for millennia as a medicinal plant. The plant is native to Europe and Asia. It grows 2 to 3 m high and has fresh green, heavily divided leaves and tiny cream-colored flower heads. For medical purposes, the aerial parts of the plant are used. The preparation and use of extracts from Artemisia and their use in the fields of nutrition, cosmetics and medicine is known from the literature (eg Applied Biochemistry and Biotechnology, 1990, VoI 24/25, pp 213-222).

Part of the ingredients of the annual mugwort (Artemisia annua) has a high activity against the malaria parasite. In contrast to other malaria drugs, the drug has few side effects and also a very favorable resistance, whereby it can be combined with other substances.

The actual active ingredient is artemisinin (artemesin, cotexin), a secondary plant substance (sesquiterpene with peroxide group), which occurs in the leaves and flowers of annual mugwort (Artemisia annua). Like all phytochemicals, these are associated in the biomass with other similar substances, since the biochemical pathways are not single-tracked. Thus, 9 other peroxide-containing substances, which also act against the malaria pathogen, contained in the plants. Over 200 ingredients could be detected in the plant parts in total.

The mode of action of artemisinin discussed in the literature is closely related to the chemical structure. The peroxide group contained in the molecule is unstable in the presence of high concentrations of iron ions and forms free radicals. Such high levels are found in red blood cells and also in malaria pathogens that accumulate iron. If artemisinin gets into erythrocytes, free radicals are formed and the parasite may be killed. There is also evidence that artemisinin derivatives inhibit certain enzymes. From the artemisinin or related plant substances, several semi-synthetic derivatives have been prepared, but all must contain the Peroxidgruppierung to the effectiveness.

Further applications of the peroxide-containing structures are described in US2007269537, for example, for local wound treatment.

It is expected that the general demand for artemisinin preparations will increase to several hundred million treatments in the next few years. To produce this volume, enormous amounts of the herbal active ingredient are needed. It would therefore be of great advantage if there were the highest possible utilization of the resulting in large excess biomass.

From one hectare of plants can harvest one to two tons of leaves from which about two to three kilograms of artemisinin can be obtained. Extraction from vegetable raw materials is complicated by the fact that the artemisinin content of the plants is very low and has considerable fluctuations. It is usually between 0.1 and 0.4%, based on the dry weight. Ideally, one to two tons of leaves can be harvested from one hectare of plant material, from which approximately 2 to 3 kg of artemisinin can be obtained by extraction with nonpolar solvents such as hexane and subsequent treatment with petroleum ether. The result is an oily-yellow extract of the plant, which is refined to gel and then to white, crystalline powder containing artemisinin as a so-called Blutschizontozid.

The remaining biomass remains largely ignored in the previous utilization concepts, since the use of the plant so far focused on the use of artemisinin as an antimalarial agent. To solve the task, suitable combination partners must be found.

3. Marine organisms as potential combination partners

For the task suitable natural products have been found for example in marine microalgae (Lukowski G, Lindequist U, Mundt S, Jülich WD: Pharmaceutically or cosmetically active agents from lipid-containing marine organisms. (2003) WO2003 / 72118 Al). Because marine organisms have very special lipid compositions In each case, it is possible to adapt formulations prepared therefrom by selection of the marine organisms to a particular end use (Lindequist U, Schweder T: Marine Biotechno Io gy. In: Rehm HJ: Biotechno Io gy. 2nd ed., Wiley-VCH Weinheim 2001: 442-473).

In addition, cyanobacteria have remarkable biosynthetic potencies. For example, Haematococcus pluvialis is a single-celled green alga that can produce up to 5% of its biomass, the astaxanthin keto-carotenoid. Astaxanthin is a valuable cosmetic raw material as a natural antioxidant. While cyanobacteria are known to be highly productive producers of secondary metabolites with a variety of biological effects, the eukaryotic microalgae are known in particular as producers of primary substances such as fatty acids, pigments and the like. estimated.

Compounds that are biogenetically attributable to the polyketides have been found less frequently in microalgae (Falch, B. Phaarmacy in Our Time 1996, 25, 311-312; Burja, AM; Banaigs, B. Abou-Mansour, E .; Burgess, JG; Wright, PC Tetrahedron 2001, 57, 9347-9377). The first paracyclophanes, chloride-containing secondary metabolites of polyketide metabolism, were isolated from Nostoc linkia and Cylindrospermum licheniforme in the early 1990s and demonstrated an inhibitory effect on KB and LoVo cell line proliferation (Moore, BS; Patterson GML, Moore, RE Brinen, L .; KatoY; Clardy, JJ Am. Chem. Soc. 1990, 112, 4061-4063; Chen, JL; Moore, RE; Patterson, GMLJ Org. Chem. 1991, 56, 4360-4364; Moore Chen, JL; Patterson, GML; Moore RE Tetrahedron 1992, 48, 3001-3006). From a naturally occurring Nostoc sp. Bloom was another cyclophane characterized by a triple bond and has antibacterial and antialgal effects isolated (Ploutno, A., Carmeli, S.J. Nat, Prod., 2000, 63, 1524-1526).

4. Medicinal mushrooms as potential combination partners

The lipid content of fungi can vary from less than 1% up to 15-20% of the dry weight. On average, can be expected with about 2 to 8%. Fungus fat contains all types of lipids such as free fatty acids (predominantly unsaturated fatty acids), mono-, di- and triglycerides, sterols (especially ergosterol), sterol esters and phospholipids (Chang, S. Miles, PG: The nutritional attributes and medicinal value of edible Mushrooms, Edible mushrooms and their cultivation, 27-40, CRC Press, BocaRaton (1989); Garcha, HS, Khanna, PK, Soni, GL: Nutritional importance of mushrooms. Mushroom Biology and Mushroom Products, Ed. Chang, ST, Buswell, JA, Chiu, SW: The Chinese University Press, JonKong, 227-236 (1993); Breene, WM: Nutritional and medicinal value of specialty mushrooms, Journal of Food Protection 53, 883-894 (1990); Boetticher, W .: Technology of mushroom utilization, publishing house Eugen Ulmer, Stuttgart 21-55, (1974). The Lackporlinge (Ganoderma spec) are wood inhabitants (Saprobionten and parasites) on coniferous and deciduous trees, which are characterized, inter alia, by a high Triterpern content. Biologically active compounds from fungi of the genus Ganoderma have long been described (U. Lindequist: Ganoderma In: Hager Handbook of Pharmaceutical Practice / Ed. F. Bruchhausen, 5th completely revised edition, Springer- Verlag Berlin- Heidelberg New York 1998, follow-up tape 2 Drugs AK (Ed. W. Blaschek), pp. 750-761.

5. Previously realized combinations with Artemisia annua

In contrast to the use of artemisinin as malaria drug relatively few other applications of plants of the genus Artemisia have been described. Thus, in KR20030051517 a dietary supplement is described which, in addition to Hovenia dulcis Thunberg and Semisulcospira libertine contains as main constituents an addition of 8% Artemisia luayomogi.

In KR20030005127 a dietary supplement is described, which in addition to Hoving Dulis Thunb and Alnus rubra Hovenia dulcis contains as main ingredients an addition of 10% Artemisia capillaris. Artemisia capillaris is described in CN1615927 among other things as an anti-hypertensive agent, the prior art is a mixture with various Chinese plants. CN 1969679 describes the combined use of Artemisia capillaris and G. lucidum in the form of a tea. JP2000143437 describes a cosmetic containing at least two of the following plant extracts: Veronica undulata Wall, Ottelia alismoides Pers., Artemisia apiacea Hance, Artemisia annua L., Andrographis paniculata Nees, Dichroa febrifuga Lour., Eclipta prostrata L., Dipsacus asper Wall, Dipsacus japonicus Miq., Boehmeria nivea Gaud., Polygonum aviculare L., Sterculia lychnophera Hance, Carpesium abrotanoides L. and Polyporus mylittae Cook. A combination of sugar alcohols, such as xylitol or erythritol, and plant extracts, which may also contain, among others, Artemisia capillaries, is described in JP2001081008 for external application to the skin. A dietary supplement containing an Artemisia capillaris Thunb. Extract is described in KR20050024920. This nutritional supplement is said to counteract skin aging. Also Artemisia annua mainly used in combinations. The health-promoting ingredients of the genus Ganoderma are often with extracts of Artemisia spec. combined. Thus, the combination described in KR20020078314 contains, inter alia, 3% Artemisia capillaris Thunb. and 2% G. lucidum. Also in JP2006143711 a combination of extracts of plants and fungi is described, including the Artemisia argyi Levl. et Vant and G. lucidum. The food described in KR20040032288 contains Artemisia capillaris Thunb in combination with brewer's yeast and G. lucidum.

KR20050001730 describes a dietary supplement with immunoactivating and antitumor activity, which i.a. G. lucidum (FR) Karst and Artemisia Messerschmidtiana Besser contains.

CN1351886 describes an agent of 20 components of Chinese medicine which i.a. G. lucidum and Artemisia capillaris and which is to be used in liver diseases. Also, CN 1092295 teaches the use of G. lucidum and Artemisia capillaris in a combination of many herbal extracts to combat various

Diseases.

JP2048517 describes a hair care composition, which i.a. Artemisia apiacea Hance and

G. lucidum contains.

As can be seen from this list, although different species of the genus Artemisia are used in the described combinations of extracts of the genera Artemisia and Ganoderma, while the use of Ganoderma species has been largely limited to the use of the species G. lucidum , A combination of Artemisia annua with Ganoderma pfeifferi is not yet known, although extracts from G. pfeifferi from the German patent application DE 10 2005 031 363 Al are known.

6. State of the art in intended applications

6.1. Anti-Aging

Skin aging is the complex biological process of alteration of the skin associated with aging. While the intrinsic aging, ie the genetically controlled diminished reactivity of skin cells can not be influenced, extrinsic factors (environmental factors such as ultraviolet light, chemical light) can be influenced by extrinsic factors

Reagents, mechanical stress, stress, heat and cold) caused cell aging Anti-aging preparations are reduced. In particular, under the influence of free radicals, there is an exhaustion of the cell processes, cell division, increased permeability of the cell membranes and an insufficient supply of the cells.

As a visible sign of this environmental cell aging, the skin gets deep wrinkles and wrinkles, their dry surface tends to tears and pseudo colors, the epidermis is thinner. It is produced less fat, the skin loses its elasticity and is no longer so regenerative. Because UVA radiation penetrates deep into the skin, it produces singlet oxygen in the dermis. This causes the production of enzymes that damage the collagen fibers and thus reduce the firmness of the skin. At the same time, elastic fibers swell, resulting in loss of elasticity of the skin.

However, the problem of all cosmetic preparations in connection with anti-aging is that so far mainly the visible external signs of aging are assessed. Prior art creams can only reduce these visible signs as long as the cause - environmental cell aging - is not eliminated. Existing wrinkles can not be made to disappear but essentially moisturise and moisturize the skin so that it temporarily appears smoother.

Since the influence on the environmental aging of the cells could not be detected experimentally, the aging process of the skin cells by the prior art corresponding means is practically not affected.

6.2. sun protection

A major cause of premature skin aging is UV radiation. In conventional sunscreens based on titanium dioxide (TiO ₂ ), the reflection and adsorption by microfine particles of titanium dioxide or zinc oxide, which reflect the incident UV light, exploited to avoid erythema (sunburn). Act as inorganic UV filters. In modern preparations, the pigment particles are reduced to about 200 nanometers.

When exposed to ultraviolet (UV) light, photocatalytic substances such as titanium dioxide (TiO ₂ ;) highly absorb UV radiation. In the reaction of titanium dioxide with UV radiation, however, form in the presence of water and oxygen free radicals. It has been proven that the photocatalytic effect of titanium dioxide with the use of titanium dioxide in UV protectants causes not only premature cell aging, but also free radical damage to DNA, which is not prevented by the protective agent but is even enhanced by the photocatalytic reaction.

Virtually no sunscreen therefore dispensed with the addition of radical scavengers today. This makes sense because reactive oxygen species are involved in all inflammatory processes and, above all, attack the unsaturated compounds (amino acids, proteins, lipids) that make up the cell walls and DNA structures of the cell nuclei. Free radicals and reactive oxygen species also play a role in polymorphic photodermatosis - often referred to by the layman as a sun allergy. In sunscreen products there are therefore a large number of substances which are intended to neutralize free radicals: vitamin E, vitamin C, glucosylrutin, follicular glucitol, ginkgo extract, thermal water, silymarin, superoxide dismutase, green tea extract, bacterial lysates, organic melanin, ferulic acid or carboxymethyl glucan. For some substances, however, it is questionable whether they also act as free-radical scavengers when applied topically. A potent scavenger is the flavonoid glucosylrutin, which is applied in combination with vitamin E as a Pre Sun cream for the prevention of polymorphic light dermatosis days before staying in the sun (Hadschiew 1997). The same principle follows the recommendation to saturate the skin early with a highly concentrated vitamin E cream (example Optolind E) (Heinrich 1994).

Almost every sunscreen contains vitamin E (tocopherol). Pure vitamin E creams achieve a sun protection factor of about 3. The peroral intake in the epidermis can not achieve sufficiently high tocopherol concentrations. In addition, studies have shown that exposure to sunlight can reduce skin vitamin E levels by up to fifty percent (Thiele 1998). Therefore, a local application is required. As an acetate, vitamin E penetrates well into the epidermis, where it is cleaved by esterases into free vitamin E. Due to its structure, it can be stored well in the cell wall and protects it from the attack of radicals. To characterize the quality of a sunscreen protection against sunburn in the future will no longer be in the foreground. Although the SPF helps to prevent erythema when used properly, it does not provide guidance on how the consumer can avoid immunosuppression or irreparable nuclear damage. This requires new measurement criteria. The photocatalytic reaction produces, inter alia, very reactive free OH radicals which have a strong antimicrobial action (A. Heller: Chemistry and Applications of Photocatalytic Oxidation of Thin Organic Films, Acc. Chem. Res., Vol 12 (1995) 503 / D. Bahnemann: Photocatalytic Detoxification of Polluted Waters.The Handbook of Environmental Chemistry, Springer Verlag 1999, Volume 2, Part L, 285-351. It becomes a very strong reduction under photocatalytic activation by UV light The initial germ content of E. faecium is 0.01%, that of S. aureus is 0.001% and that of E. coli is 0.00002%, and this strong biocidal activity is undesirable in skin applications.

6.3. Anti-tumor effect

There are initial indications of an effect of Artemisia annua ingredients on various human cancers.

According to results of the German Cancer Research Center Heidelberg also this effect is based on the formation of free radicals by the peroxide grouping in the presence of iron ions.

The examination of artemisinin as a potential anticancer drug is still at an early stage.

Triterpenes isolated from Ganoderma concinna can induce apoptosis in HL-60 cells (Gonzalez AG, Leon F, Rivera A, Padron JI, Gonzalez-Plata J, Zuluaga JC et al., New Lanostanoids from the Fungus Ganoderma concinna J Nat Prod 2002; 65: 417-21). Triterpenes, available from Ganoderma applanatum, are effective against murine skin tumors (Chairul, Tokuyama T, Hayashi Y, Nishizawa M, Tokuda H, Chairul SM, Hayashi Y. Applanoxidic acids A, B, C and D, biologically active tetracyclic triterpenes from Ganoderma applanatum, Phytochemistry 1991; 30: 4105-9; Chairul, Chairul SM, Hayashi Y. Lanostanoid triterpenes from Ganoderma applanatum, Phytochemistry 1994; 35: 1305-8). A derivative of illudin was effective in clinical studies (Murgo A, Cannon DJ, Blatner G, Cheson BD, Clinical Trials of MGI-114, Oncology 1999, 13: 233-8).

The addition of lentinan in addition to chemotherapy in patients with gastric cancer, colon cancer and other tumors has proved to be successful (Hazama S, Oka M, Yoshino S, Iizuka N, Wadamori K, Yamamoto et al., Clinical effects and immunological analysis of intraabdominal and intrapleural injection of lentinan for malignant ascites and pleural effusion of gastric carcinoma. Cancer & Chemotherapy 1995; 22: 1595-7).

According to the P53 model (Nature 415, 26-27 (2002)), there are close relationships between low tumor morbidity and anti-aging effects.

6.4. Reduction of the contamination of implants

The infection rates in permanent implants are usually between 0.5 and 6% Prevention strategies are urgently needed. Together with Candida, staphylococci are the most common causes of catheter-associated sepsis, of which about 50% are fatal. In an organic film forming on the plastic surface, the staphylococci are partially protected from attack by antibiotics and very often develop multiple resistance.

Each implant-associated infection is associated with a sharp increase in the cost of treatment [Kann & Jansen, 2001]. , In Germany one assumes from 3000 to 4000 catheter-associated deaths per year.

Antiseptic-treated implants may e.g. in the gastrointestinal and genitourinary area (Brauers et al., Biocompatibility, Cell Adhesion and Degradation of Surface Modified Biodegradable Polymers designed for the Upper Unrinary Tract. Tech. Urol. (1998) 4, 214-220, Multanen et al. Bacterial adherence to ofloxacin-hides polyacetone-coated self-reinforced 1-lactide acid polymer uroological stents BJU Intern (2000), 900, 44-56; Schierholz, JM, H.-M. Wenchel, D. König, J. Beuth and G. Pulverer: The Importance of Slow-Release Antimicrobial Systems for the Prevention of Catheter-Associated Infections Hyg. Med. 23 (1998), 548-556.

The adhesion of microorganisms and cells to an implant is the first important step in the pathogenesis of nosocomial foreign body infections on plastic surfaces. PCT / EP 2008/056 730 of 31.5.08 describes methods of coating surfaces with micropores and microplates Nanoparticles described by means of plasma process, which can be used to prevent adhesion by germs. Work on the coating of medical devices with lipid nanoparticles made from Artemisia annua are not available according to our research.

7. Disadvantages of the prior art All combinations described in the prior art use extracts from the whole plant Artemisia annua.

The use of the plant Artemisia annua has so far focused on the use of artemisinin as an antimalarial agent. This requires extraction with apolar solvents such as n-hexane. It inevitably removes the lipids. Thus, the conversion of biomass into micro- and nanoparticles using the species-specific lipids described in the prior art is no longer possible.

The remaining biomass and its ingredients remain largely unnoticed in the previous utilization concepts.

Object of the present invention

An object to be solved by the invention was to provide from the biomass of Artemisia annua means that can be used after appropriate clinical testing for tumor treatment.

An additional object was to provide lipid-containing micro- and nanoparticles for the coating of instruments and / or implants.

An urgent object of the present invention was to find new applications for the remaining after separation of Arteminsin from Artemisia annua residues and to provide new formulations for it.

Presentation of the essence of the invention

The objects were achieved according to the features of the claims, on the one hand by preparations of biomasses of the plant Artemisia annua and on the other hand by biomass lipid-containing algae or fungi, and by the conversion of this preparation into micro- and nanoparticles.

According to the invention novel particles having a mean diameter, depending on the production between 10 nm and 10 microns, obtained, which integrate the ingredients of Artemisia annua and the respective combination partners. Depending on whether artemisin-containing or artemisin-free biomasses from Artemisia annua are used, micro- and nanoparticles are obtained which can be used for very different tasks.

Micro- and nanoparticles, characterized in that biomass of the plant Artemisia annua are used for their production, which contain all ingredients including artemisinin (sesquiterpene peroxides), on the one hand provide new means that are used after appropriate clinical examination for tumor treatment On the other hand, agents suitable for coating surfaces and their antimicrobial finish are also obtained.

In this application, the strong radical formation upon decomposition of the peroxide group, especially in the presence of iron ions, is exploited for cytotoxic and antimicrobial effects.

To protect itself from the radical action Artemisia annua contains radical scavenging agents. Micro- and nanoparticles, characterized in that biomass of the plant Artemisia annua are used for their production, whose content of artemisinin (sesquiterpene peroxides) has been removed before the conversion into micro- and nanoparticles, use these radical scavenging properties. The microparticles and nanoparticles prepared by separating off the peroxide structures are therefore suitable for anti-aging preparations, skin care products and sun protection products.

Irrespective of whether artemisin-containing or artemisin-free biomasses are used, the conversion into microparticles and nanoparticles required for the solution of the problem can be carried out optionally with the homogenization process, the solvent homogenization process or the solvent emulsion process.

1. Homogenization process

The comminuted biomass of Artemisia annua or the residues after separation of the sesquiterpene peroxides are first heated. This fraction is suspended, dispersed or adsorbed in the fatty acids of other lipid-containing fungi or cyanobacteria) or of the entire lipid-containing biomass. In parallel, a surfactant-water mixture is produced. In this biomass, one or more active ingredients (solid or liquid) can be added. This surfactant-water mixture is heated to a temperature above the Melting temperature of the fatty acids heated. The two phases are combined at the selected temperature. Subsequently, a pre-suspension is produced with the aid of a stirrer (rotor-stator principle) or with the aid of ultrasound. The pre-suspension is then homogenized using a high-pressure homogenizer, the number of homogenization cycles and the working pressure being chosen according to the desired particle size and stability of the preparation. Make sure that the production temperature is set again and again between the individual cycles. The surfactant serves to stabilize the suspension.

If there are problems with the level of the temperature (eg sensitive active ingredients) during production, it is possible to carry out the entire process even at room temperature. In this case, the method is carried out in the same manner as described above, wherein the active ingredient is adsorbed on the lipid-containing organisms or dispersed with the addition of a small amount of water.

2. Solvent Homogenization Process

The crushed biomass of Artemisia Annua or extraction residues after separation of the sesquiterpene peroxides and lipid-containing biomasses of a second species and optionally selected active ingredients preferably vitamin mixtures are suspended in a vaporizable organic solvent. Thereafter, this mixture is predispersed (stator-rotor principle or ultrasound), homogenized

(High-pressure homogenizer) and then spray-dried or freeze-dried (scheme 2). When freeze-drying, it should be noted that suitable cryoprotectants are used. There is also the possibility of removing the organic solvent by means of suitable evaporators (eg rotary evaporators). Subsequently, the particles can be redispersed in suitable aqueous surfactant solutions. Thereafter, a new dispersion (stator-rotor principle or ultrasound) and homogenization (Hochdruckhomogenisator) is necessary.

3. Solvent emulsion process

This method is based on the preparation of an emulsion of water and a solution of Artemisia annua or the extraction residue of Artemisia annua after separation of the sesquiterpene peroxides in a suitable organic solvent. For this purpose, an emulsifier for dispersing the biomass active ingredient is used. Emulsifier and

Biomass are dissolved in a suitable organic solvent. To this solution For example, an aqueous phase containing a water-soluble cosurfactant is added and mixed with the lipid-containing biomass of a second type. Thereafter, this mixture is vorisp ergiert (stator-rotor principle or ultrasound). After a homogenization step with the aid of a high-pressure homogenizer, the organic solvent is removed by evaporation, wherein the active substance-containing biomass precipitates in the form of solid particles.

The biologically active ingredients from Artemisia annua or from the combination partner can, depending on the charge and size distribution of the ingredients and the production conditions in the solid matrix (nanopellet) and / or in the lipidhaltigen shell (nanocapsule), enclosed in the shell, and / or distributed in the dissolved or highly dispersed state in the formulation and / or adsorbed or adhered to the edge zones of the nanopellets. By converting the combination of Artemisia annua and the lipid-containing biomass of a second species into micro- and nanoparticles, ingredients of both components are released in a controlled manner.

In the preparation of the micro- and nanoparticles, both artemisin-containing and artemisin-free biomasses with lipid-containing algae can preferably be combined with cyanobacteria having a lipid content of at least 10% and / or with the unicellular green alga Haematococcus pluvialis.

In a preferred embodiment of the invention, artemisin-containing biomasses are combined with microalgae, which additionally have a natural content of carbamidocyclophanes. Both carbamidocyclophane-containing biomasses of cynanobacteria and the peroxide-containing structures of Artemisia annua exert a strong cytotoxic effect on tumor cells, but have different mechanisms of action, so that there is a synergistic effect. Particularly suitable for this are microalgae, the Carbamidocyclophane according to the formula image 1, characterized by a symmetrical carbon skeleton with carbamido structures in the side chain and by a different substitution pattern of the Carbamidostrukturen opposite side chains, wherein Ri to R _{7 is} both halogen, hydroxyl, carbonyl - as well as alkyl groups may be included. The carbamidocyclophanes according to formula 1 and preparations prepared therefrom inhibit the growth of various tumor cell lines (MCF-7 cells breast carcinoma, 5637 cells Bladder carcinoma) particularly strong. Particularly preferred are micro- and nanoparticles containing the active ingredient of formula image 1 in a concentration of 0.01 to 3%.

The inventive conversion into micro- and nanoparticles on the one hand against a synergistic cytotoxic effect of the combination of active ingredients according to formula 1 and the peroxide-containing structures from Artemisia annua

Tumor cells.

Formula 1: carbamidocyclophanes

In order to increase the cytotoxic activity, the proportion of active compounds according to formula 1 or the sesquiterpene peroxides in the inventive preparations can be increased by adding the pure active ingredient.

On the other hand, if artemisin-free biomasses and carbamidocyclophane-free microalgae are used for the production of the micro- and nanoparticles, particularly favorable skincare products are obtained. For this task are suitable as lipid-containing biomasses of a second species in particular microalgae, which are characterized by a special fatty acid pattern in the lipid content. By providing care with formulations containing the micro- and nanoparticles prepared according to the invention from Artemisia annua and microalgae, protective lipids are added to the skin. In addition, some microalgae have an extremely high water retention capacity that can be used in these formulations. This counteracts the transepidermal water loss with the formulations according to the invention. With the micro- and nanoparticles according to the invention, the skin is supplied with moisture and fat in a particularly favorable form, so that it appears smoother.

In a particularly favorable embodiment, the single-celled green alga Haematococcus pluvialis is used as a combination partner of Artemisia annua. This green alga contains up to 5% of its biomass the astaxanthin ketocarotenoid. Due to the microencapsulation according to the invention, astaxanthin is protected and released in a controlled manner. The radical scavenging properties of the microparticles and nanoparticles according to the invention, with regular use, prevent the premature aging of the skin caused by environmental factors such as UV light, chemical reagents, mechanical stress, stress, heat and cold.

In another embodiment, both artemisin-containing and artemisin-free biomasses can be combined with lipid-containing fungi in the production of the microparticles and nanoparticles. Particularly preferred are the species Ganoderma lucidum and / or Ganoderma pfeifferi and / or Ganoderma applanatum and / or Ganoderma concinna and / or Ganoderma tsugae and / or Auricularia auricula-judae and / or Grifola frondosa and / or Hericium erinaceus and / or Lentinula edodes and / or Pleurotus ostreatus and / or Pleurotus eryngii.

If artemisin-containing biomasses or combinations of artemisin-containing biomasses and carbamidocyclophane-containing microalgae are combined with fungi, in particular of the genus Ganoderma, a synergistic effect occurs.

Both carbamidocyclophanes of the microalgae and the peroxide-containing structures of Artemisia annua exert a direct cytotoxic effect on tumor cells. In contrast, the biomass of the genus Ganoderma causes an induction of apoptosis and an immune stimulation in particular by the glucan content of the Ganoderma species. Ingredients of Ganoderma species cause activation of phagocytosis by macrophages. The efficacy may be enhanced when combinations with triterpenes from Ganoderma concinna and / or from Ganoderma applanatum and / or from Ganoderma lucidum, Ganoderma tsugae and / or from Ganoderma pfeifferi are used. Advantageously, triterpenes from these five species of Ganoderma can be combined for this therapeutic use.

The interaction of cytotoxic action, induction of apoptosis and macrophage activation is surprising and allows new ways in the prevention and control of tumors. The micro- and nanoparticles according to the invention may contain cytostatic agents, apoptosis-inducing agents and immune immunulants as combinations of two or three.

On the other hand, if artemisin-free biomasses or combinations of artemisin-free biomasses and carbamidocyclophane-free microalgae are combined with fungi which are have been proven in health promotion and prophylaxis, preparations with special care properties are obtained. The micro- and nanoparticles have pronounced scavenging properties, which is advantageous for many applications. Also, by the different fatty acid patterns of the fungi and / or the cyanobacteria, the properties of the resulting products can be adapted to the requirements.

In particular Ganoderma pfeifferi suitable as a combination partner for these applications. The metabolism of human cells (FL cells) micro- and nanoparticles, produced from G. pfeifferi, is very strongly stimulated. Surprisingly, it has been found that with microparticles and nanoparticles prepared from a combination of Artemisia annua and Ganoderma pfeifferi, similarly strong stimulating effects on cell metabolism are achieved, even if Ganoderma pfeifferi is greatly reduced in concentration and the Artemisia annua component in the large excess is present.

The ingredients of G. pfeifferi reduce oxidative stress and enhance endogenous radical defense. The ingredients ganomycin A and B, which are only found in G. pfeifferi, inhibit luminol-induced and spontaneous oxygen radical release at a concentration of 10 μg / ml.

The controlled release of ingredients increases the proliferation of human keratinocytes. Under the influence of the combination, the glucose consumption of human cells increases, indicating a stimulation of the metabolism. It has been found that respiration is enhanced, protein production is promoted and cell aging is slowed down. Under the same experimental conditions, extracts of G. lucidum do not show any slowing of the aging process.

The increased cell permeability under UV influence - measurable by determining the LDH activity in the medium - is reduced. The regeneration capacity of the cells after UV damage is significantly increased. The micro- and nanoparticles also have the advantage of having pronounced radical scavenging properties, which is advantageous for many applications. From the micro- and nanoparticles, the radical-scavenging ingredients are released in a controlled manner by both Artemisia annua and G. pfeifferi.

In addition, it is not insignificant that the production of special Ganoderma species, which are not yet used commercially on a large scale, is very complicated due to the slow fungus growth. The combination partner from Artemisia annua In contrast, in the production of the active ingredient against malaria as a by-product. With the erfϊndungsgemäßen combination of Ganoderma species and Artemisia annua residues is therefore a cost effective way to produce an anti-aging drug with the cell metabolism stimulating properties provided.

Particularly advantageous application properties are obtained when the residues of Artemisia annua remaining after separation of the sesquiterpene peroxides are combined with biomass from both microalgae and fungal species. The micro- and nanoparticles resulting from this triple combination are preferably suitable for use in sunscreen preparations.

This effect can be enhanced when vitamins are integrated into the micro- and nanoparticles. Vitamins can be added both in the homogenization process, in the solvent homogenization process or in the solvent emulsion process during the production of the micro- and nanoparticles, whereby they are integrated into the micro- and nanoparticles. In a particularly preferred embodiment, vitamin E is used as the acetate. The vitamins are released from the micro- and nanoparticles in a controlled manner to the skin. As an acetate, vitamin E penetrates well into the epidermis, where it is cleaved by esterases into free vitamin E. Due to its structure, it can be stored well in the cell wall and protects it from the attack of radicals. Since damage by free radicals play a special role in the formation of skin tumors, the risk of tumor development is reduced with regular prophylactic use of sunscreens which contain the micro- and nanoparticles according to the invention. Cells which nonetheless degenerate under the influence of UV radiation are stimulated to apoptose by the triterpenes from Ganoderma species contained in the preparations, in particular from Ganoderma concinna.

The object of providing lipid-containing microparticles and nanoparticles for the coating of surfaces, for example of instruments and / or implants, has been achieved by producing micro- and nanoparticles from which the biomass of Artemisia annua is separated without separating the peroxides. This results in micro- and nanoparticles with strong antimicrobial activity. These particles can be used to reduce the adhesion of proteins, cells and germs. In the patent application PCT / EP2008 / 056730 of 31.05.2008 unpublished at the time of application becomes Process for coating surfaces with micro- and nanoparticles by means of plasma method described, which can be used to prevent adhesion by germs. The micro- and nanoparticles according to the invention can be used for an extension of this method.

The features of the invention will become apparent from the claims, but also from the description, wherein the individual features each represent alone or to several advantageous combinations in the form of combinations for which protection is sought with this document.

The invention will be explained by means of embodiments, without being limited to these examples.

embodiment

Example 1: Fatty acid composition of the microalgae suitable for the preparation of micro- and nanoparticles from a combination of Artemisia annua with marine organisms

methodology

Per order, 5-6 strains were examined in each case with a double determination.

Results

Table 1 shows the fluctuation range of the contents found in this case.

In the case of the Chroococcales, the tetradecanoic acid and the tetradecenoic acid are found at a constant high content. Also strikingly high is the content of either n-hexadec-cis-9-enoic acid or 11-hexadecenoic acid. Eight other fatty acids are found, each with a low content, usually <1%.

In the Oscillatoriales, the tetradecenoic acid was not found at all, the saturated C-14 acid was detected only in individual strains. n-Hexadecanoic acid was detected at a level between 10 and 14% and n-octadeca-cis-9, cis 12, ice 15-trienoic acid at a level between 20 and 30% in all strains. Also dominant was the octadecadienoic acid, the position of the double bond in the individual strains varied.

In nostocales, n-hexadecanoic acid, which was detected between 26 and 72%, dominates, while the corresponding undersaturated acid was found only in a small proportion.

On the other hand, in the case of fatty acids of higher chain length, the polyunsaturated forms, in particular the n-octadeca-cis-9, cis 12, ice 15-trienoic acid or octadecatetraenoic acid predominated. Since polyunsaturated fatty acids with a large chain length are of particular influence on the growth of S. aureus, this finding is particularly pronounced remarkable. In the further investigations, the strain Bio 33 from the family of the Nostocaceae was found to be particularly suitable, whose fatty acid composition is shown in Tab. 2 in comparison to other strains of this order.

Table 2: Fatty acid composition in strains of the order Nostocales

Combination partners of different orders with sufficiently high lipid content are available. The properties of the micro- and nanoparticles can be varied by selecting the appropriate lipids for each application.

Example 2 Antioxidant Properties Methodology

The determination of the antioxidative properties was carried out by thin-layer chromatography (SIEVERS A et al., (2002) Simple thin-layer chromatography test for antioxidative compounds using DPPH assay, CBS Camag Bibliography Service 88: 14-15).

Result

FIG. 1 shows the qualitative DPPH test of the Artemsia annua / Ganoderma pfeifferi microparticles (left) in comparison to ascorbic acid (right).

As can be seen from the size of the decolorizing zone of the Artemsia annua / Ganoderma pfeifferi microparticles, there are strong antioxidant properties of the microparticles investigated.

Example 3 Influence of the microparticles according to the invention on the cell metabolism

methodology

A test arrangement was used which was described in DP 19709649.2 (Jülich, W.-D., Woedtke, Th. Von, Abel, P .: Measuring arrangement for the detection of toxic, subtoxic, chronic toxic or stimulatory effects of active and Pollutants using perfusion cell cultures) has been described. Confluent FL cells were treated with mitomycin C to inhibit growth.

Results

Micro- and nanoparticles, made from G. pfeifferi, strongly stimulate the metabolism of human cells (FL cells). With a combination of Artemisia annua and Ganoderma pfeifferi, produced according to the invention by conversion into micro- and nanoparticles using the species-specific lipids of G. pfeifferi, these effects could be achieved, even if Ganoderma pfeifferi is greatly reduced in its concentration and Artemisia αnnwα Component is present in large excess.

Example 4 Cytostatic Effect Methodology

The cytotoxicity of the carbamidocyclophanes and of the carbamidocyclophan-containing combinations is carried out on in vitro cultured MCF-7 cells, 5637 cells and on Fl cells in 96-well microtiter plates. The MCF-7 cells and the 5637 cells are pre-cultured for 48 h in IMDM (Invitrogen) with 10% fetal bovine serum or the Fl cells in Eagle-MEM + 8% fetal bovine serum (37 ° C, 5% CO2). The carbamidocyclophane-containing extracts of the aquatic organisms or the isolated carbamidocyclophanes are then added in concentrations of from 100 μg / ml to 0.0000 μg / ml and the cells are incubated for a further 48 hours under the same conditions. The cells are then fixed and stained with 0.02% crystal violet for 30 min. After washing and drying, the cell-bound dye is dissolved with 70% ethanol and the optical density at λ = 550 nm is measured with an Anthos ht II plate reader (Anthos, Salzburg, Austria) (Bracht, K. Boubakari, Grünert, R .; Bednarski, PJ, Anti-Cancer Drugs 2006, 17, 41-51) The inhibition of cell proliferation is calculated from the optical density measured in the batches incubated with the carbamidocyclophanes or the carbamidocyclophan-containing extracts of the aquatic organisms in comparison to the cell control (cells incubated with medium only) and for the isolated carbamidocyclophanes the IC50 values are calculated.

[Olli] For the two tumor cell lines, the isolated carbamidocyclophanes have IC50 values between 0.7 μg / mL and 1.7 μg / mL, for the Fl cells, which are to be regarded as non-transformed cells, IC50 values between 2.5 μg / mL, 3.1 μg / mL and 3.5 μg / mL. With microparticles and nanoparticles containing combinations of sesquiterpene peroxides and carbamidocyclophanes, IC50 values of less than 0.3 μg / ml are determined.

Example 5 Preparation of Micro- and Nanoparticles Table 3: Formulation of the nano- and microparticles

The biomass are combined and heated to a temperature of 80 ⁰ C. Separately, an aqueous emulsifier solution is heated to the appropriate temperature (80 ⁰ C). Then both phases are combined and processed using an Ultra Turrax T25 Fa. Janke and Kunkel GmbH & Co KG (Staufen, Germany) in an emulsification process at 8000 revolutions per minute and a duration of 30 seconds. The suspension is then reacted with a piston-gap high-pressure homogenizer Micron Lab 40 (APV-Gaulin, Lubeck) at a pressure of 500 bar and a temperature of 80 ⁰ C homogenised four times.

Example 6 Determination of the minimum effective dose

Methodology according to Example 3

6.1. Limitation of the application period

The addition of G. pfeifferi to the medium as a 2% suspension was limited in contrast to Example 3 to 12 h (from H 148 to h 160).

Result:

Already after this brief exposure time, a promoting effect can be detected, which is detectable for at least 3 days (FIG. 3). An extension of the exposure time of the 2% suspension to 72 h increases the effect (FIG. 4), the promoting effect is detectable over a longer period of time.

6.2. Limitation of daily feed In order to determine the minimum effective concentration, the daily intake was limited to 0.5 ml of the 2% suspension given within 111 min, ie thereafter the cells received a drug-free medium over 1329 min. The daily delivery of the drug was reduced to 0.012% of the cell culture medium with this assay.

Even at this low concentration, a promoting effect can be seen (FIG. 5). When the concentration is doubled (delivery 2 times a day for 111 min), the effect is more pronounced (FIG. 6).

Claims

claims

1. Micro- and nanoparticles from biomasses of the plant Artemisia annua on the one hand and biomasses of lipid-containing algae or fungi on the other hand.

2. micro- and nanoparticles according to claim 1, characterized in that it is such biomass of the plant Artemisia annua, containing all ingredients including artemisinin (sesquiterpene peroxides).

3. micro- and nanoparticles according to claim 1, characterized in that it is such biomass of the plant Artemisia annua, whose content of artemisinin (sesquiterpene peroxides) was removed before the conversion into micro- and nanoparticles.

4. micro- and nanoparticles according to claim 1 to 3, characterized in that it is used in the production of lipid-containing algae to cyanobacteria having a lipid content of at least 10% and / or cyanobacteria having a lipid content of at least 10% and / or is the unicellular green alga Haematococcus pluvialis.

5. micro- and nanoparticles according to one of claims 1 to 4, characterized in that it is the selected lipid-containing cyanobacteria are strains with a natural content of carbamidocyclophanes

6. micro- and nanoparticles according to one of claims 1 to 4, characterized in that the selected carbamidocyclophane-containing strains active compounds of the formula. 1

Preferably in a concentration of 0.01% to 3%, where R can stand to R ₇ are both halogen, hydroxyl, carbonyl and alkyl groups, or that these agents of formula 1 are added to the microparticles and nanoparticles in the preparation until a concentration of 0.01 to 3% is reached.

7. micro- and nanoparticles, characterized in that active ingredients of formula 1 and sesquiterpene peroxides from Artemisia annua are combined.

8. micro- and nanoparticles according to claim 1 to 7, characterized in that it is the mushroom species used in the production of lipid-containing fungi

• Ganoderma lucidum and / or

• Ganoderma pfeifferi and / or

• Ganoderma applanatum and / or

• Ganoderma concinna and / or

• Ganoderma tsugae and / or

Auricularia auricula-judae and / or

• Grifola frondosa and / or

• Hericium erinaceus and / or

• Lentinula edodes and / or

• Pleurotus ostreatus and / or

• Pleurotus eryngii acts.

9. micro- and nanoparticles, characterized in that active ingredients according to formula 1 and biomass from Ganoderma species are combined.

10. Micro- and nanoparticles, characterized in that sesquiterpene peroxides from Artemisia annua and biomasses from Ganoderma species are combined.

11. micro- and nanoparticles, characterized in that active ingredients according to formula 1, sesquiterpene peroxides from Artemisia annua and biomass from Ganoderma species are combined.

12. micro- and nanoparticles according to one of claims 1 to 11, characterized in that they additionally contain at least one of the vitamins A, C or E, wherein vitamin E is preferably used as acetate.

13. A process for the preparation of the micro- and nanoparticles according to one of claims 1 to 12, characterized in that either comminuted biomass from Artemisia annua, which contains all active substances, or otherwise biomass from Artemisia annua, whose content of artemisinin (sesquiterpene peroxides) was removed, mixed with the algae or fungi according to claim 1 and / or additives and converted by known methods into micro- and nanoparticles.

14. Use of the micro- and nanoparticles according to one of claims 1 to 13 for cosmetic and / or pharmaceutical preparations and / or as dietary supplements.

15. Use of compositions according to claim 3 as a) agents for slowing cell aging and / or b) sunscreens and / or c) agents with anti-aging effect. d) means for the prophylaxis of skin tumors e) means for inducing apoptosis in tumor cells

16. Use of compositions according to any one of claims 2, 9, 10, 11, 13 as a) anti-tumor agents b) agents for the treatment of tumors c) agents for inducing apoptosis in tumor cells

17. Use of the microparticles and nanoparticles according to one of claims 1 to 3 for coating surfaces.