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Title:
EXTRACT OF PARTS OF SAMBUCUS FOR USE IN THE TREATMENT OF INFLAMMATORY PROCESSES, INTIMAL HYPERPLASIA AND VEIN GRAFT DISEASE
Document Type and Number:
WIPO Patent Application WO/2011/144639
Kind Code:
A1
Abstract:
The present invention relates to an extract of parts of Sambucus for the treatment of inflammatory processes and disorders, intimal hyperplasia, vein graft disease, a method for producing such an extract, and a preparation and/or formulation comprising the extract for the treatment of inflammatory diseases, intimal hyperplasia and vein graft disease.

Inventors:
BERNHARD, David (Augasse 27A TOP 26, Wörgl, A-6300, AT)
SCHWAIGER, Stefan (Höttinger Au 38, Innsbruck, A-6020, AT)
STUPPNER, Hermann (Steinangerl 7a, Götzens, A-6091, AT)
Application Number:
EP2011/058005
Publication Date:
November 24, 2011
Filing Date:
May 17, 2011
Export Citation:
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Assignee:
BIONORICA SE (92318 Neumarkt, DE)
BERNHARD, David (Augasse 27A TOP 26, Wörgl, A-6300, AT)
SCHWAIGER, Stefan (Höttinger Au 38, Innsbruck, A-6020, AT)
STUPPNER, Hermann (Steinangerl 7a, Götzens, A-6091, AT)
International Classes:
A61K36/185; A61P29/00
Attorney, Agent or Firm:
SIMANDI, Claus (Höhenstrasse 26, Hennef, 53773, DE)
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Claims:
Claims

1 . Extract of parts of Sambucus for treatment of inflammatory processes and disorders, intimal hyperplasia, vein graft disease, wherein the extract comprises ursolic acid.

2. Extract according to claim 1 , wherein Sambucus comprises Sambucus ebulus,

Sambucus nigra, Sambucus racemosa, or other species of Sambucus.

3. Extract according to claim 1 , wherein the extract comprises ursolic acid, chlorophyll and/or accompanying compounds that reduce the toxicity of ursolic acid.

4. Extract according to claim 1 , wherein the extract comprises ursolic acid and one or more compound selected form the group Isoquercitrin, Luteolin-7-O-beta-D- glucoside, lsorhamnetin-3-O-beta-D-glucoside, Oleanolic acid, Lutein, Pheophorbide, Hydroxypheophorbide, Hydroxypheophorbide ethylester, Pheophorbide,

Pheophorbide ethylester, Hydroxypurpurin-7-lacton-ethyl-methyldiester and (-)- Loliolide, preferably Isoquercitrin, lsorhamnetin-3-O-beta-D-glucoside, (-)-Loliolide.

5. Extract according to claim 4, wherein one or more compounds are enriched in the extract.

6. Method for the production of an extract according to claim 1 , wherein the parts of Sambucus are extracted by maceration, percolation, and C02-extraction.

7. Method according to claim 6 comprising the following steps:

- evaporating the obtained solution,

- separation by means of liquid-liquid extraction, wherein the solvent is preferably non polar,

- separation by column chromatography and optionally further separation steps are carried out.

8. Method according to claim 7, wherein the solvent is selected from the group diethyl ether and / or petroleum ether.

9. Method according to claim 7, wherein a silica gel column chromatography is used.

10. A preparation and/or formulation for the treatment of inflammatory processes and disorders, intimal hyperplasia, vein graft disease comprising the extract according to claim 1 .

1 1 . A preparation and/or formulation according to claim 10, comprising the extract in the form of an alcoholic or hydroalcoholic extract.

12. A preparation and/or formulation according to claim 1 1 and 1 1 , for oral

administration.

13. A preparation and/or formulation according to claim 12, in the form of tablets,

dragees, capsules or tinctures.

14. A preparation and/or formulation according to claim 10, wherein the extract is in admixture with a pharmaceutically acceptable carrier.

15. A preparation and/or formulation according to claim 10 to 14, wherein the extract is administered in combination with another therapeutic agent.

Description:
Extract of parts of Sambucus for use in the treatment of inflammatory processes, intimal hyperplasia and vein graft disease

Description:

The present invention relates to an extract of parts of Sambucus for the treatment of inflammatory processes and disorders, intimal hyperplasia, vein graft disease, a method for producing such an extract, and a preparation and/or formulation comprising the extract for the treatment of inflammatory diseases, intimal hyperplasia and vein graft disease.

Extracts of roots and leaves of Sambucus ebulus have been used in traditional medicine to treat health problems associated with inflammation such as inflammatory joint diseases, rheumatic pain and sore throat (Hiermann, 2007). Several studies have confirmed the antiinflammatory potential of dwarf elder extracts, but the nature of the active principle remained unsolved. Leave extracts showed an impact on the concentration of cytokines (interleukin- 1 a, interleukin-1 β, TNFa) when mixed with whole blood of healthy volunteers (Yesilada et al., 1997). Despite the traditional use of the leaves of S. ebulus especially in Mediterranean countries, up to now only one attempt was performed to identify the active principle (Yesilada, 1997). An extract of the aerial plant parts was subjected to activity guided isolation using different animal models (carrageenan or serotonin induced in mice paw edema assays and others) ending up with identification of chlorogenic acid as active principle. However, the whole work was published without any experimental results concerning the pharmacological part.

Ursolic acid is a widespread pentacyclic tripernoid and has previously been isolated from a number of different plants species e.g. rosemary and basil. Recent reports have ascribed to ursolic acid chemo-preventive properties (Lu et al., 2007; Martin-Aragon et al., 2001 ), cell death-inducing (Achiwa et al., 2005; Choi et al., 2000), anti-mutagenic (Ramos et al., 2008) and anti-viral activities (Serra et al., 1994), direct (Ramachandran and Prasad, 2008) and indirect (Martin-Aragon et al., 2001 ) anti-oxidative properties, anti-inflammatory as well as pro-inflammatory properties (Ikeda et al., 2008), pro-angiogenic and antiangiogenic properties (Cardenas et al., 2004; Sohn et al., 1995), ability to induce differentiation (Lee et al., 1994), and anti-invasive activities (Cha et al., 1996).

In addition, ursolic acid is generally considered to act as an anti-inflammatory agent, e.g. by inhibition of COX-2 transcription (Subbaramaiah et al., 2000), release of macrophage migration inhibitory factor (Ikeda et al., 2005), inhibition of IL-8 secretion (Thuong et al., 2005), and downregulation of enzymes required for permeation of lymphocytes/monocytes through basal lamina such as MMP-9 and elastase (Cha et al., 1996; Ying et al., 1991 ). In general, a number of effects of ursolic acid on cells in culture were ascribed to an interaction with cellular signalling at or around NFKB activation and/or activity. However, up to now only one report suggested a precise site of interaction of ursolic acid with NFKB, namely inhibition of ΙκΒα-kinase and phosphorylation of p65 (Shishodia et al., 2003).

Pozo et al. (Atherosclerosis 184, 53-62; 2006) have investigated the "in vitro" activity of ursolic acid regarding the vascular smooth muscle cell (VSMC) behaviour and the "in vivo" effects of ursolic acid on an experimental model of vascular damage.

The exposure of VSMC to ursolic resulted in a chemotaxis inhibition, in a reduction of the expression of proliferating cell nuclear antigen (PCNA) and in a disorganization of β-tubulin and vimentin cytoskeletal proteins. Pozo et al. demonstrated that ursolic acid inhibits the migratory response of VSMC toward the growth factor PDGF-BB.

Pozo et al suggest that ursolic acid may be a promising therapeutical tool in the prevention and attenuation of vascular diseases and may be used as a possible treatment strategy for the prevention of the progression of atherosclerosis.

As described above, it is known that ursolic acid has an anti-inflammatory effect. However, if ursolic acid is administered as a pure compound, it causes toxic effects. There is no teaching in the state of the art dealing with the problem how the toxic effects of ursolic acid can be avoided or reduced.

It is an object of the present invention to provide ursolic acid in a suitable non-toxic form for use in the treatment of inflammatory processes and disorders, intimal hyperplasia, vein graft disease, which has less side-effects than the pure compound and is well tolerated. It is a further object to provide a method for the production of such an extract.

The present invention provides an extract of parts of Sambucus for the treatment of inflammatory processes and disorders, intimal hyperplasia, vein graft disease, wherein the extract comprises ursolic acid.

The term "inflammatory processes and disorders" relates to any kind of inflammation, which underlie a vast variety of human diseases, in particular non-immune diseases with etiological origins in inflammatory processes are thought to include cancer, atherosclerosis, and ischaemic heart disease, in particular autoimmune diseases, degenerative joint diseases, diseases of the rheumatic type with cartilage degradation, all the progressive forms of arthritis, especially rheumatoid and chronic polyarthritis, joint trauma, diseases with disrupted leucocyte adhesion, diseases as a result of increases TNF-alpha concentrations, cachexia.

The term "Intimal hyperplasia" is directed to the thickening of the Tunica intima of a blood vessel as a complication of a reconstruction procedure or endarterectomy. Intimal hyperplasia is the universal response of a vessel to injury and is an important reason of late bypass graft failure, particularly in vein and synthetic vascular grafts.

The term "vein graft disease" is directed to a generic reference to the pregressive degradation and build up atheroma and clots within the ever thickening wall of veins which are used as arteries during surgical bypass operations.

To identify an anti-inflammatory compound a general model of inhibition of the TNFa induced expression of vascular cell adhesion molecule 1 (VCAM-1 ) on the surface of human umbilical vein endothelial cells (HUVECs) can be used. Increased expression of VCAM-1 is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention.

The extract according to the invention reduces TNFa induced VCAM-1 expression of HUVECs in a dose dependent manner. The reduction of VCAM-1 corroborates the use of extracts of parts of Sambucus for the therapy of (chronically) inflammatory processes (see Figure 1 ).

In addition, extracts of parts of Sambucus may be used for the treatment of intimal hyperplasia and vein graft disease. The extracts according to the invention were used in an autologous rat bypass model to test their in vivo applicability. It could be shown in this in vivo model, that the application of the extracts according to the invention to vein grafts potently inhibits intimal hyperplasia.

Surprisingly, the inventors have found that toxic effects of ursolic acid can be drastically reduced by using an enriched extract with ursolic acid instead of the pure compound. It could be shown that the toxicity of ursolic acid as constituent of an extract is diminished when comparing the extract according to the invention with the pure compound ursolic acid (see Figure 1 ). The extract according to the invention is made of Sambucus, in particular Sambucus ebulus, Sambucus nigra, Sambucus racemosa, or other species of Sambucus. "Parts of Sambucus" shall mean preferably the leaves, flower or roots or any other suitable part of Sambucus.

In a further embodiment of the invention, the extract of the invention comprises chlorophyll and/or accompanying compounds that reduce the toxicity of ursolic acid apart from ursolic acid. Such preferred accompanying compounds are selected from the group Isoquercitrin, Luteolin-7-O-beta-D-glucoside, lsorhamnetin-3-O-beta-D-glucoside, Oleanolic acid, Lutein, Pheophorbide, Hydroxypheophorbide, Hydroxypheophorbide ethylester, Pheophorbide, Pheophorbide ethylester, Hydroxypurpurin-7-lacton-ethyl-methyldiester and (-)-Loliolide.

Such "detoxification compounds" can be enriched in an extract in accordance with the invention by using the herein described methods.

In a preferred embodiment the said extract may contain compounds with a detoxifying effect selected from the group Isoquercitrin, Luteolin-7-O-beta-D-glucoside, lsorhamnetin-3-O- beta-D-glucoside, Lutein, (-)-Loliolide, most preferably Isoquercitrin, lsorhamnetin-3-O-beta- D-glucoside, (-)-Loliolide.

Hence, the invention is directed to such enriched and optimized extracts having ursolic acid and containing Isoquercitrin, Luteolin-7-O-beta-D-glucoside, lsorhamnetin-3-O-beta-D- glucoside, Oleanolic acid, Lutein, Pheophorbide, Hydroxypheophorbide, Hydroxypheophorbide ethylester, Pheophorbide, Pheophorbide ethylester, Hydroxypurpurin- 7-lacton-ethyl-methyldiester and (-)-Loliolide, preferably Isoquercitrin, lsorhamnetin-3-O- beta-D-glucoside, (-)-Loliolide.

One reason for the observed alleviation of side effects regarding the extract of the invention might be related to an induction of phase II detoxification enzymes. Since also detoxification compounds, which are present in the extract of the invention, are able to induce phase II enzymes like heme oxygenase-1 (= HO-1 ) and quinone oxidoreductase-1 (= NQ01 ) (Zhang et al. 2008) it is assumed that the observed effect is a result of an interaction between ursolic acid and present accompanying compounds like the above mentioned.

In addition, the invention relates to a method for the production of the extract according to claim 1 , wherein the parts of Sambucus are extracted for example by maceration.

The extraction is carried out with e.g. milled air-dried leaves, which are macerated with ethanol for preferably 24 h at room temperature. However, the preparation of the basic extract of Sambucus may comprise mechanical pulping, sonication, use of mortars and pestles, freeze- thawing cycles, use of blenders (like Waring-Blenders, Polytron), liquid homogenization and maceration, or e.g. Dounce homogenization, French Press etc. However, the extracts may be obtained by disrupting the cells and cells from Sambucus species by any mechanical/physical or chemical means, like by use of detergents.

Mechanical methods rely on the use of rotating blades to grind and disperse large amounts of complex tissue, such as plant leaves, flowers, seeds and roots. The Waring blender and the Polytron are commonly used for this purpose, Unlike the Waring blender, which is similar to a standard household blender, the Polytron draws tissue into a long shaft containing rotating blades.

Liquid-based homogenization is the most widely used cell disruption technique for cultured cells. Cells are lyzed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. Three different types of homogenizers are in common use. A Bounce homogenizer consists of a round glass pestle that is manually driven into a glass tube. A Potter-Elvehjem homogenizer consists of a manually or mechanically driven Teflon pestle shaped to fit a rounded or conical vessel. The number of strokes and the speed at which the strokes are administered influences the effectiveness of Dounce and Potter-Elvehjem homogenization methods. Both homogenizers can be obtained in a variety of sizes to accommodate a range of volumes. A French press consists of a piston that is used to apply high pressure to a sample volume of 40 to 250 ml, forcing it through a tiny hole in the press. Only two passes are required for efficient lysis due to the high pressures used with this process. It is of note that in more industrial applications also other, larger devices may be employed to prepare the extracts from Sambucus.

Sonication is also a physical disruption commonly used to break open cells. The method uses pulsed, high frequency sound waves to agitate and lyse cells and finely diced tissue. To prevent excessive heating, ultrasonic treatment may be applied in multiple short bursts to a sample immersed in an ice bath. Sonication is best suited for volumes <100 ml.

Cells, organisms as well as tissue might be treated with various agents to aid the disruption process. Chemical substances, such as hexane, petroleum benzene, chloroform, dichloromethane, acetone, ethyl acetate, diethyl ether, ethanol and mixtures of water and alcohol or mixtures of different solvents may be added during or before mechanical disruption. Lysis can also be promoted by suspending cells in a hypotonic buffer, which cause them to swell and burst more readily under physical shearing. Processing can be expedited by treating cells with glass beads in order to facilitate the crushing of cell walls. Less preferred, however envisaged, is the use of detergents in the preparation of the extracts to be treated in accordance with the present invention. Detergents are a class of molecules whose unique properties enable manipulation (disruption or formation) of hydrophobic-hydrophilic interactions among molecules in biological samples. Such detergents may be used to lyse cells, solubilize membrane proteins and lipids. Generally, moderate concentrations of mild (i.e., nonionic) detergents compromise the integrity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form. Using other conditions, detergents effectively penetrate between the membrane bilayers at concentrations sufficient to form mixed micelles with isolated phospholipids. Detergents may be, e.g. Triton; CHAPS, Tween-20, Tween-40, Tween-80, SDS and the like. However, it may be useful to stabilize the extract by certain chemical means.

The cells and plants to be employed in order to obtain the basic extract may be cells of natural origin as well as cultured cells or plants. It is preferred herein that the cells or plants and in particular leaves of the plants are dried before mechanical disruption/maceration. The cells or plants may be air dried, lyophilized (freeze-dried) or, though less preferred, dried in an oven. It is preferred herein that the "cell(s)"' and "'plant(s)" to be used as a basic material are fresh, i.e. harvested shortly before the extract is prepared. Nonetheless, it is possible to store the basic material before its use in the preparation of the extract. For example, the basic material may be lyophilized (freeze-dried) or simply frozen and stored at low temperatures, e.g. at about -20 to -300C or as low as -800C.

In context of the present invention, the term "cell" and "plant" to be used as basic material for preparing the extract to be treated by the method of the present invention also comprises the use of "tissues". Such tissues may be leaves, sprouts, or reproductive organs e.g. flowers. Preferably, the tissues are leaves.

Methods for preparing the extract are known in the art and also described herein. Preferably, the extract is further processed shortly after its preparation (e.g. the extract is used in the preparation of a herein disclosed pharmaceutical composition); however, it is also possible to store the extract for some time before they are used in accordance with the present invention. The extracts may, for example, be stored in lyophilized form or in form of dried extracts. However, each storage form known in the art is be employed, as long as the storage has the effect that the extract (and its components) remain efficacious over a long time period, i.e. the stored extract has, preferably, substantially the same efficacy as the fresh extract. Dried extracts can be routinely prepared by methods known in the art. For example, following mechanical disruption of the basic (plant) material by e.g. maceration or percolation, the material can be extracted using (a) solvent(s) or mixtures thereof as described herein. The fluid extract (i.e. the fluid phase of the obtained extract) may be concentrated taking advantage of routine techniques, some of which are exemplarily described herein below. Such concentration techniques include, but are not limited to fluidised-bed drying, concentration to a syrup or concentrated fluid extract, spray drying, freeze drying or the use of a vacuum dryer, a drying tunnel, vacuum band dryer or a drying hurdle. Often organic-hydrous fluid extracts (such as the fluid extract obtained herein using an organic solvent) are concentrated by nucleate boiling or surface evaporation.

Routine drying techniques employed in the pharmaceutical field comprise distillation and drying under normal conditions (i.e. room temperature) also methods which take advantage of variations in pressure and temperature in order to obtain the dried extracts. One well known method for preparing a dried extract is as follows; First, a fluid extract or tincture is prepared; after subsequent distillation of the solvent a viscous extract is obtained, to which often adjuvants and/or excipients (e.g. lactose, polyvinylpyrrolidone, sucrose, silicon dioxide and the like are added. This moist mass is then dried in suitable driers. Also employed in this context is the use of a vacuum band dryer (Mitchell Dryers Ltd) , wherein a dried extract is obtained from the viscous extract after a pre-drying step using downdraft vaporizers.

Also envisaged herein is the use of commercially available extracts, in particular dried extracts, obtained from (a) plant(s) belonging to the genus Sambucus.

After mechanical disruption of the cell(s), tissue(s) or whole plant(s) the plant material may be further macerated and/or dissolved/suspended in an organic solvent, such as hexane, petroleum benzene, chloroform, dichloromethane, acetone, ethyl acetate, diethyl ether, liquid carbon dioxide, ethanol and mixtures of water and alcohol with any of the solvents separately or subsequently with a second solvent or mixtures of different solvents.

Preferably, diethyl ether and petroleum ether are used as extraction solvents.

Hence, in a further preferred embodiment of the invention the method comprises further the following steps:

The mixture is sonicated and filtered. The obtained solution is evaporated to dryness using e.g. a rotavapor at 25 'Ό. This procedure is repeated several times with fresh solvent each time yielding crude extract. The initial separation is performed by means of liquid-liquid extraction. The crude extract is suspended in water and transferred into the separation funnel. The suspension is extracted several times with solvents of different polarity. The obtained fractions are evaporated to dryness and are subjected to gel column chromatography. For further enrichment, the fractions may divided in a dichloromethane (= DCM)-soluble and a DCM-insoluble part. For this purpose, the fractions are suspended in DCM and sonicated. The mixture is filtered over a sintered plate funnel. The same procedure is repeated several times yielding two fractions: DCM-soluble and DCM-insoluble. A further separation can be performed for example by means of liquid-liquid extraction, percolation, C0 2 -extraction or by other means.

The solvent used for the method according to the invention is non-polar, preferably diethyl ether or petroleum ether.

In a further embodiment of the invention a chromatography, in particular silica gel column chromatography, is used for separation. Silica gel column chromatography is well known in the art and described in detail in standard textbooks, such as "Preparative Chromatography Techniques" by Hostettmann, K. Marston, Andrew Hostettmann, Maryse, Springer-Verlag GmbH, 2007, 260 p.

The invention relates further to a preparation / medicinal drug and/or formulation / pharmaceutical composition for the treatment of inflammatory diseases, intimal hyperplasia, vein graft disease comprising the extract according to claim 1 . In a preferred embodiment, the preparation and/or formulation contains the extract in the form of an alcoholic or hydroalcoholic extract. Preferably, the preparation and/or formulation is suitable for oral administration, in particular in the form of tablets, dragees, capsules or tinctures.

In addition, the preparation and/or formulation according to the invention is preferably in admixture with a pharmaceutically acceptable carrier and it is preferably administered in combination with another therapeutic agent.

Preferably, at least one extract is formulated as a pharmaceutical composition comprising in addition one or more pharmaceutically acceptable carriers or diluents.

The medicinal drugs / preparation that are manufactured with extracts in accordance with the invention can be administered orally, intramuscularly, peri-articularly, intra-articularly, intravenously, intraperotoneally, subcutaneously, or rectally. The invention pertains to processes for the manufacture of medicinal drugs that are characterized by the feature that at least one extract according to the invention is brought into a suitable form of agent for administration together with a pharmaceutically suitable and physiologically tolerated vehicle and, optionally, further suitable active substances, additives, or ancillary substances. Suitable solid or liquid galenic forms of preparation or formulations are, for example, granulated materials, powders, sugar-coated pills, tablets, (micro)capsules, suppositories, syrups, juices, suspensions, emulsions, drops, or injectable solutions as well as preparations with a protracted release of the active substance, whereby use is made in their preparation of conventional ancillary substances, such as vehicle substances, agents that lead to the disintegration of the preparation, binders, coating agents, swelling agents, slippage promoting agents or lubricants, taste improving agents, sweeteners, and solubilizers. Mention may be made of the following as ancillary substances: magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talcum, milk protein, gelatine, starch, cellulose and its derivatives, animal and vegetable oils such as cod-liver oil, sun flower oil, groundnut [oil] or sesame oil, poly(ethylene glycols), and solvents such as, for example, sterile water and monohydric or polyhydric alcohols, e.g. glycerine.

The medicinal drugs are preferably manufactured and administered in dosage units, whereby each unit contains, as the active component, a defined dose of extracts according to the invention. In the case of solid dosage units, such as tablets, capsules, sugar-coated pills or suppositories, this dose can amount to 1 to 1000 mg and preferably 50 to 300 mg, and in the case of injection solutions in ampoule form, this dose can amount to 0.3 to 300 mg and preferably 10 to 100 mg.

Daily doses of 20 to 1000 mg of active substance, and preferably 100 to 500 mg of active substance, are indicated for the treatment of an adult patient weighing 50 to 100 kg, e.g. 70 kg. However, higher or lower daily doses can also be applied under certain circumstances. The administration of the daily dose can take place via an administration on one single occasion in the form of an individual dosage unit or several smaller dosage units, or via the multiple administration of subdivided doses at defined intervals.

In the following, the present invention is described in more detail by way of examples. However, these examples are not intended to limit the scope of protection of the present invention in any way.

The examples also refer to several figures, the legends of which are given below:

Examples

Extracts and fractions of the leaves of Sambucus ebulus (dwarf elder) are able to inhibit TNFa-stimulated expression of VCAM-1 and ICAM-1 in HUVECs. The responsible principal for this effect was identified as ursolic acid. Extracts and preparations of S. ebulus, rich in ursolic acid, are suitable for the treatment of chronically inflammatory processes.

Interestingly, as constituent of an extract the toxicity of ursolic acid seems to be diminished. This was quite evident when comparing the dwarf elder diethyl ether fraction (DEE-fr. containing 14.79 ± 0.03 % (w/w) ursolic acid) with the pure compound ursolic acid: despite its slightly lower VCAM-1 expression-repressing activity (Figure 1 ; IC50 of 20 μΜ compared to 5-10 μΜ ursolic acid) it is significantly less toxic as suggested by the content of ursolic acid in the extract demonstrated a clearly more pronounced reduction of toxicity (IC50 > 108 μΜ compared to 10 - 20 μΜ ursolic acid).

Activity-guided Isolation

Results of the activity-guided fractionation are summarized in Fig. 2. Activity was evaluated monitoring the reduction of TNFa induced VCAM-1 expression of HUVECs. A crude ethanol leave extract which reduced VCAM-1 expression by 74.5% (c = 33 g ml) compared to control was used as starting material.

First, this extract was divided in five fractions by liquid-liquid extraction. Activity of the petroleum ether fraction was comparable to that of the starting ethanol extract, whereas the diethyl ether fraction caused an even more pronounced reduction of VCAM-1 expression (90.7%). The more polar fractions had little effects or were almost inactive (ethyl acetate: 39.4%, n-butanol: 15.2%, water: 13.9%). Silica gel column chromatography of the diethyl ether fraction yielded eight fractions. Fraction 4 (SED4) which showed potent activity, was separated into two fractions according to their solubility in DCM. The active DCM insoluble part was subjected to liquid/liquid partitioning followed by HSCCC. Of the resulting four fractions fraction 4 (SHE RS) turned out to contain the active principle. Final crystallization from CH3CN/THF yielded ursolic acid as major constituent. Its structure (Fig. 3) was confirmed by mass spectrometry and 1 - and 2D-NMR spectroscopy.

Biological characteristics of the isolate and comparison with commercially available ursolic acid

Ursolic acid isolated from the leaves of S. ebulus (purity 85%) reduced TNFa induced VCAM-1 expression of HUVECs in a dose dependent manner with an IC50 value 12.5 μΜ. In comparison commercial ursolic acid (purity >98.5%) showed an IC50 value of 6.25 μΜ Besides inhibition of VCAM-1 expression purified and commercial ursolic acid significantly reduced viability of both endothelial and smooth muscle cells (SMCs). IC50 values of purified and commercial ursolic acid determined by the XTT assay (Fig. 4) were 22 μΜ and 1 1 .5 μΜ, respectively. Viability data for SMCs were almost identical with those for ECs and are therefore not shown. Reason for the differences between ursolic acid isolated from plant material and the commercially available compound may be the different grade of purity (85% compared to >98.5%).

Ursolic acid inhibits TN Fa-mediated ICAM-1 surface expression

Since exposure of HUVECs to TNFa is known to cause not only expression of VCAM but also expression of other adhesion molecules, it was tested whether ursolic acid is antagonizing also ICAM-1 expression. Fig. 5 shows the outcomes: ursolic acid inhibits TNFa stimulated surface expression of ICAM-1 on endothelial cells in a dose dependent manner. In the absence of TNFa ursolic acid does not have any effect on ICAM-1 expression.

Preparation of an extract

The leaves of Sambucus ebulus were collected close to Schonberg/Stubaital; Tirol, Austria in July 2009. A voucher specimen (VP-07200901 ) is deposit in the Herbarium of the Institute of Pharmacy/Pharmacognosy University of Innsbruck. The collected plant material was dried for 10 days in a shaded room at room temperature. The extract was prepared with ethanol (96 vol%) using a accelerated solvent extractor device (ASE 100, Dionex). The milled plant material (5.00 g) was mixed with diatomaceous earth and filled in a 34 ml cartridge. The extraction parameters were set to: temperature. l OO 'C, static time: 5 min; flush volume: 130%, purge time: 70 sec, number of static cycles: 5; the total procedure was repeated three times to afford 1 .6761 g dry extract. For further enrichment 1 .00 g of the obtained extract was suspended in 20.0 ml water and extracted three times with 15.0 ml n-hexane followed by three times 20.0 ml diethyl ether. The obtained organic solvents were combined an evaporated to dryness to afford 168.36 mg of the desired diethyl ether fraction.

The obtained extract was analyzed by HPLC-ESI-MS (see figure 6) and HPLC-APCI-MS: HP 1 100 system (Agilent, Waldbronn, Germany) equipped with autosampler, DAD and column thermostat; Esquire 3000plus ion trap (Bruker Daltonics, Bremen, Germany); stationary phase: Phenomenex Luna 3μ C8 100A (150 mm χ 2.0 mm) with Synergy Fusion guard column; mobile phase: solvent A: H20; solvent B: acetonitrile; DAD: 205 and 425 nm, temp.: 35.0°C; injection volume: 10 μΙ_; flow: 0.2 ml/min; composition during run: start: 75% A; 15 min: 60% A; 20 min: 30% A; 25 min: 30% A; 55 min: 0% A; 65 min: stop; post time: 15 min. MS conditions: ESI alternating mode: scan from 100 m/z to 1500 m/z; nebulizer 40.00 psi; dry gas: 10.00 l/min; HV capillary: 4.5 kV; HV end plate offset: -0.5 kV; dry temp.: 350°C; APCI alternating mode: scan from 100 m/z to 1500 m/z; nebulizer 50.00 psi; dry gas: 8.00 l/min; capillary -2.0 kV; end plate offset: -0.5 kV; dry temp.: 250 < C; vaporizer temp.: 300 °C. Preparation of an extract

Leaves of Sambucus ebulus were collected in Magdalensberg (Austria) in September 2003. A voucher specimen (CS-09200301 ) is deposit in the Herbarium of the Institute of Pharmacy/Pharmacognosy University of Innsbruck. The collected plant material was dried for 10 days in a shaded room at room temperature. 570 g of dried leaves were milled and macerated in 2.5 L EtOH (96 vol%) for 24 h at room temperature. The mixture was then sonicated for 10 minutes and filtered. The obtained solution was then evaporated to dryness using a Heidolph Laborota 4000 Rotavapor (25 'C). This procedure was repeated eight times, using fresh solvent each time. The extraction yielded 1 16.25 g of extract.

95.23 g of crude extract were further enriched by means of liquid/liquid extraction. Therefore, the extract was suspended in 1 .0 I water and extracted with 500 ml Petroleum ether (PE). The procedure was repeated 5 times with 500 ml of fresh PE. The remaining aqueous solution was extracted analogous six times with portions of 500 ml diethyl ether. The combined organic layer were evaporated to dryness using a Heidolph Laborota 4000 Rotavapor (25 °C) to afford 20.45 g of the desired diethyl ether fraction.

The obtained extract was analyzed by HPLC-ESI-MS (see figure 7) and HPLC-APCI-MS: HP 1 100 system (Agilent, Waldbronn, Germany) equipped with autosampler, DAD and column thermostat; Esquire 3000plus ion trap (Bruker Daltonics, Bremen, Germany); stationary phase: Phenomenex Luna 3μ C8 100A (150 mm χ 2.0 mm) with Synergy Fusion guard column; mobile phase: solvent A: H20; solvent B: acetonitrile; DAD: 205 and 435 nm, temp.: 35.0°C; injection volume: 10 μί; flow: 0.2 ml/min; composition during run: start: 75% A; 15 min: 60% A; 20 min: 30% A; 25 min: 30% A; 55 min: 0% A; 65 min: stop; post time: 15 min. MS conditions: ESI alternating mode: scan from 100 m/z to 1500 m/z; nebulizer 40.00 psi; dry gas: 10.00 l/min; HV capillary 4.5 kV; HV end plate offset: -0.5 kV; dry temp.: 350 < C; APCI alternating mode: scan from 100 m/z to 1500 m/z; nebulizer 50.00 psi; dry gas: 8.00 l/min; capillary: -2.0 kV; end plate offset: -0.5 kV; dry temp.: 250 °C; vaporizer temp.: 300 < C.

200.12 mg of the fraction DCM insoluble part were suspended in 2.0 ml MeCN+H20 (1 +1 ; v/v) and filtered over cotton wool. The obtained yellow solution was applied on the top of a Strata C18-E SPE column (1 g), preconditioned with 6 ml MeCN and 6 ml MeCN+H20 (1 +1 ; v/v). The sample was eluted with 5 ml MeCN+H20 (1 +1 ; v/v); 5 ml MeCN+H20 (7.5+2.5; v/v), 5 ml MeCN and 10 ml tetrahydrofurane. The obtained fractions were evaporated to dryness and analyzed by TLC (figure 3). Compound X was located in the fraction eluted with 50% MeCN. In order to increase the amount of the fraction containing compound X, the whole procedure was repeated with further 195.53 mg of the DCM insoluble fraction to afford 15.58 mg of the desired fraction (50% MeCN). The procedure was repeated a third time but as starting material the obtained residues of the filtration of both previous separations (total of 335.13 mg) were used. This third separation afforded 22.83 mg of the desired fraction (50% MeCN).

For further enrichment of compound X the obtained fractions (50% MeCN) were combined (54.18 mg) and separated by Sephadex CC (35cm x 2.5 cm bed size) using a mixture of water and methanol (1 +1 ; v/v) as mobile phase with a flow rate of 1 ml/min. The eluate was collected in portions of 3.0 ml and analyzed by TLC. Compound X was eluted in fraction 42- 45, which were combined and evaporated to dryness to afford 9.23 mg. The compound was analyzed by LC-ESI-MS to afford m/z of 197 ([M+1 ]+) and 393 ([2M+1 ]+) in positive mode and 195 ([M-1 ]-) and 391 ([2M-1 ]-) in negative mode. The further structure elucidation was carried out by means of NMR-spectroscopy. Therefore, the compound was dissolved in 0.6 ml MeOH-d4 containing 0.03 % TMS and measured at a Bruker avance II 600 (Bruker Biospin Rheinstetten, Germany) at 600 MHz (1 H) and 150.9 MHz (13C) at 300 °K.

The proton NMR of compound X showed four singlets which were assigned to three methyl groups ( H 1 .27; 1 .46; 1 .76 ppm) and one low field shifted methine group ( H 5.74 ppm). The remaining signals were identified as two methylene groups ( H Ha 1 .97, Hb 1 .53; Ha 2.42, Hb 1 .74 ppm) and a muliplet of a methine group ( H 4.21 ppm). The 13C spectrum reveled signals for 1 1 carbon atoms. The connectivity of the obtained signals was deduced from the recorded COSY, HMBC and HSQC spectra and enabled the identification of compound X as (-)-Loliolide (Structure Fig. 3), a C1 1 -terpenelactone (in agreement with Ki Eui Park et al., Journal of the Korean Chemical Society, 2004, 48(4): 394-8).

Material and methods

General

All reagents used were of purissimum or analytical grade and were purchased from Sigma Aldrich (Sigma-Aldrich, Vienna, Austria) unless specified otherwise. HPLC solvents were of gradient grade and purchased from Sigma-Aldrich as well. Water was produced by reverse osmosis followed by distillation. Pure grade solvents were distilled prior to use. Ursolic acid purchased from Sigma was of 98.5 % purity.

Cell isolation and culture Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords (kindly donated by the Gynaecology and Obstetrics Department, Innsbruck Medical University) by enzymatic detachment using collagenase, as previously described (Bernhard et al., 2003). Cells were routinely passaged in 0.2% gelatine-coated (Sigma, Steinheim, Germany) polysterene culture flasks (Becton Dickinson, Meylan Cedex, France) in Endothelial Cell Basal Medium (CC-3121 , BioWhittaker, Inc., Walkersville, MD, USA) supplemented with EGM SingleQuots Supplements and growth factors (CC-4133, BioWhittaker, Inc.) in a humidified atmosphere containing 5% C0 2 . Fetal bovine serum (FBS; 2%) was routinely used for cell culturing and experiments. The isolation and analysis of human umbilical cord ECs was approved by the Ethics Committee of the Innsbruck Medical University.

Quantification of endothelial surface vascular cell adhesion molecule 1 (VCAM-1 ) expression

To analyze and quantify TNFa-induced VCAM-1 expression on the surface of HUVECs, 10,000 HUVECs were seeded into each well of a 96-well plate and allowed to adhere overnight. Following replacement of medium with 200 μΙ of fresh medium, different dilutions of plant extracts or solvents (as controls) were added to the wells. After an incubation period of 30 min, human recombinant TNFa (Peprotech (Eubio), 300-01 A) was added to the wells at a final concentration of 100 ng/mL. After incubation for 15 h, supernatants were discarded, cells were washed 5 times in ice-cold PBS, and the 96-well plates were put on ice. Following blocking of non-specific bindings sites with 1 % BSA in PBS for 60 min, mouse anti-human CD106/VCAM-1 antibody (Neomarkers, MS-1 101 -P, final concentration 0.2 g/ml in PBS, 1 % BSA) was added to the wells or isotype control (Mouse lgG1 , Dako, X0931 , final concentration 0.2 μg/ml in PBS, 1 % BSA) and incubated for 30 min on ice on a horizontal shaker. Following washing of cells 5 times in ice cold PBS, cells were incubated with HRP labelled secondary antibodies (goat anti-mouse HRP, Dako, P0447, final concentration 1 μg/ml in PBS, 1 % BSA) for 30 min on ice. After another washing step (5 times with ice cold PBS), 0.32 mg/ml of ABTS (Roche) in ABTS buffer (512 mg NaB0 3 -trihydrate, 8.799 g citric acid 1 -hydrate, and Di-NaHP0 4 -dihydrate 1 1 .214 g per liter (pH = 4.4)) was added to the wells and allowed to develop for 45 min in the dark. OD was determined at 405 nm. At least three dilutions per extract/fraction were tested in four parallels, and each experiment was repeated three to five times. All data were verified by FACS-based quantification of VCAM-1 (data not shown).

Quantification of endothelial surface inter cellular adhesion molecule 1 (ICAM-1) expression The protocol for the quantification of surface ICAM-1 on endothelial cells was identical to the protocol for VCAM-1 detection (see above). Antibodies used were: anti-mouse anti-human CD54/ICAM-1 antibody (Neomarkers, MS-1094-S, 1 :15 in PBS, 1 % BSA), isotype control (Mouse lgG1 , Dako, X0931 , final concentration 0.2 pg/ml in PBS, 1 % BSA), HRP-labelled secondary antibodies (Goat anti-Mouse HRP, Dako, P0447, final concentration 1 g/ml in PBS, 1 % BSA).

Analysis of the number of viable cells

The number of viable cells in the cultures was determined by the XTT assay (Biomol, Hamburg, Germany) according to the manufacturer's instructions.

Plant material

Leaves of Sambucus ebulus were collected in Gaaden (Austria) in September 2003. A herbarium specimen is stored at the Institute of Pharmacy/Pharmacognosy, University of Innsbruck.

Chromatography and NMR analyses

High-speed counter-current chromatography (= HSCCC): P.C. Inc. (Potomac; MD, USA; model: HSCCC multilayer (triple) coil, ser. 690) HSCCC instrument with Gilson 302/803 C pump system Model 302 (Villiers-la-Bel, France). LC for quantification: LC-parameter: HP 1050 system (Agilent, Waldbronn, Germany) equipped with auto sampler, DAD and column thermostat; stationary phase: Phenomenex Luna 3μ C8 100A (150 x 3.0 mm) with Synergy Polar RP 80A guard column; mobile phase: solvent A: H 2 0 with 0.02 trifluor acetic acid, (v/v); solvent B: acetonitrile; DAD: 205 nm, temp.: 35.0 ^; injection volume: 5 μί; flow: 0.2 mL/min; composition during run: start: 60% A; 10 min: 300% A; 40 min: 2% A; 55 min: stop; post time: 15 min. LC-MS: LC-parameter: HP 1 100 system (Agilent, Waldbronn, Germany) equipped with auto sampler, DAD and column thermostat; stationary phase: Phenomenex Synergy Polar RP 80A (150 x 4.6 mm) with identical guard column; mobile phase: solvent A: H 2 0 with 0.9 % formic acid, 0.1 % acetic acid, (v/v); solvent B: acetonitrile + methanol (v/v, 1 +1 ); DAD: 250 nm, temp.: 40 ^; injection volume: 10 μί; flow: 1 .0 mL/min; composition during run: start: 50% A; 20 min: 42% A; 21 min: 28% A; 40 min: 4% A; 45 min: stop; post time: 10 min. MS-parameters: Esquire 3000 plus (Bruker Daltonics, Bremen, Germany); split: 1 :5; ESI, alternating mode; spray voltage: -4.5 kV, 350°C; dry gas: 10.00 L/min; nebulizer 40 psi; full scan mode: m/z 100-1500. TLC: silica gel 60 F 254 plates (VWR Darmstadt, Germany) mobile phase: toluol + ethyl acetate (1 +1 ; v/v), chemical derivatization with vanillin-sulfuric acid reagent. NMR: 1 D- and 2D-experiments were measured on a Bruker DRX 300 (Bruker Biospin Rheinstetten, Germany) operating at 300.13 MHz ( H) and 75.47 MHz ( 3 C) at 300 K (chemical shifts δ in ppm, coupling constants J in Hz); NMR solvent: DMSO-d 6 with 0.03 % TMS (Eurisotop Gif-Sur-Yvette, France), which was used as internal standard. Melting point (m.p.): Kofler hot-stage microscope; uncorr. Optical rotation: Perkin-Elmer 341 polarimeter (Wellesley, MA, USA) at 25°C.

Extraction and separation

Extraction was carried out with 570 g of the milled air-dried leaves, which were macerated with 2.5 L EtOH 96 % for 24 h at room temperature. The mixture was sonicated for 10 min and filtered. The obtained solution was evaporated to dryness using a rotavapor at 25 °C. This procedure was repeated eight times with fresh solvent each time yielding 1 16.25 g crude extract. The initial separation was performed by means of liquid-liquid extraction; 95.23 g of the crude extract were suspended in 1 .0 L water and transferred into the separation funnel. The suspension was extracted six times with 500 ml of solvents of different polarity starting with petroleum ether, diethyl ether, ethyl acetate and finally water saturated n-butanol. The obtained fractions were evaporated to dryness yielding 18.94 g petroleum ether, 20.45 g diethyl ether, 7.69 g ethyl acetate, 18.24 g n-butanol and 35.10 g water fraction. Since the activity was concentrated in the obtained diethyl ether fraction, 19.0 g of the fraction were subjected to silica gel column chromatography using 230 g Merck silica gel 60, (0.040- 0.063 mm, 230^100 mesh) as stationary phase and a gradient of petroleum ether and increasing amounts of ethyl acetate as mobile phase. The eluate was monitored by TLC and combined to eight fractions (SE-D 1 to SE-D 8). The activity was located in fractions SED 4, 5 and 6. Due to the higher amount and purity of fraction SE-D 4 (2.55 g) compared to the total amount of SE-D 5 and 6 (1 .50 g), the further separation steps were carried out with fraction SE-D 4. For further enrichment, SE-D 4 was divided in a dichloromethane (= DCM)- soluble and a DCM-insoluble part. For this purpose, 2.20 g of SE-D 4 were suspended in 10.0 ml_ DCM and sonicated for 5 min. The mixture was filtered over a sintered plate funnel (porosity nr. 4). The same procedure was repeated four times yielding two fractions: SE-Ak- f-DCM-soluble (0.48 g) and SE-Ak-f-DCM-insoluble (1 .71 g). The further separation of the active fraction SE-Ak-f-DCM-insoluble was performed by means of liquid-liquid extraction using 1 .64 g of SE-Ak-f-DCM-insoluble and 900 ml_ of a solvent mixture of petroleum ether, ethyl acetate, acetonitrile and tertiary butyl-methyl ether (10+1 +5+2, all v/v). The evaporated upper layer yielded 1 .10 g, the lower layer afforded yielded 0.54 g (SE-Ak-g). The residue of the lower layer was further purified by means of HSCCC using a solvent mixture of petroleum ether, ethyl acetate, acetonitrile and tertiary butyl-methyl ether (10+1 +5+2, all v/v) using the upper layer as mobile phase. The sample (458.12 mg of SE-Ak-g) was suspended in 10.0 ml_ of the lower phase and 5.0 ml_ of the upper phase and filtered before injection yielding an insoluble part of 129.80 mg. The used coil had a volume of 325 ml and was used in the tail-to-head-modus with a rotation of 800 rpm. The used flow rate was 1 .00 mL/min, fractions were collected in portions of 5 mL. Although the separation technique enabled a good separation of the compounds, which were combined to three fractions (SEAk-h 1 to 3), the activity remained in the insoluble part (SE-Ak-h RS). Since the residue (SE-Ak-h RS) formed pale yellow crystals, the insoluble part was subjected to crystallization with a mixture of acetonitrile and tetrahydrofuran (v/v; 1 +1 ). The first crystallization afforded 67.7 mg of off- white platelets, which were crystallized a second time from the same solvent mixture yielding 47.3 mg of white platelets.

Structure elucidation of the active principle

LC-MS analysis of the crystallized residue afforded in positive mode m/z of 439.3; 457.3 and 479.4 corresponding to the pseudo-molecular ions of [M-H 2 0] + , [M+1 ] + and [M+Na] + as well as a m/z of 455.2 in negative mode corresponding to [M-1 ] " suggesting a molecular weight of 456 g/mol. The 1 D- and 2D-NMR-analysis of 20.0 mg of the obtained compound enabled the identification of the isolated compound as ursolic acid with 85% purity with following NMR data (solvent DMSO-d 6 ; 300.13 MHz ( H) and 75.47 MHz ( 3 C); data sorted after position, chemical shifts δ 0 Ιδ Η in ppm, multiplicity, number of protons and coupling constants (J in Hz): 1 : 38.3/H a 1 .52 m, H b 0.90 m\ 2: 27.4 /H a 1 .82 m, H b 0.98 m\ 3: 76.7/3.00 dd 1 H (9.1 ; 6.3); 4:38.2/-; 5: 54.7/0.68 m 1 H; 6: 19.1/1 .30 m 2H; 7: 32.6/ H a 1 .43 m, H b 1 .25 m\ 8: 39.1 /-; 9: 46.9/1 .46 m 1 H; 10: 36.5/-; 11 : 23.0/ 1 .57 m 2H; 12: 124.4/5.12 br s 1 H; 13: 138.2/-; 14: 41 .5/-; 15: 27.5/0.98 m 2H; 16: 23.7/H a 1 .90 m, H b 1 .52 m; 17: 46.7/-; 18: 52.3/2.1 1 cM H (1 1 .3); 19: 38.4/1 .30 m 1 H; 20: 38.4/ 1 .30 m 1 H; 21 : 30.1/H a 1 .41 m, H b 1 .25 m; 22: 36.3/1 .54 m 2H; 23: 28.1/0.90 s 3H; 24: 15.9/0.68 s 3H; 25: 15.2/0.86 m 3H; 26: 16.8/0.75 s 3H; 27: 23.2/1 .04 s 3H; 28: 178.6/-; 29: 16.9/0.81 d 3H (6.3); 30: 21 .0/0.90 d 3H (4.7). m.p.: 283 °C (293 °C lit.).

Determination of ursolic acid concentration in the diethyl ether fraction

Quantification of the ursolic acid concentration was carried out by a HPLC-DAD method using the method of external standard with a calibration curve of y = 6446x + 98.509 (R 2 = 0.9999). For this purpose, the commercial ursolic acid was dissolved in three concentrations (5 mM, 0.5 mM and 0.25 mM) in DMSO and analyzed by HPLC in triplicate. Determination of the ursolic acid concentration in DMSO solution in the obtained diethyl ether fraction (10 mg/ml) afforded a content of 14.79 ± 0.03 % (w/w).

Brief description of figures Figure 1 shows the effect of diethyl ether fraction adjusted to the

corresponding ursolic acid concentration on VCAM-1 expression (light

bars) and cell viability (dark bars). Shown are representative experiments

performed in quadruplicates + S.D.

Figure 2 shows an activity guided isolation schema. The ethanolic leaf

extract of S. ebulus was subjected to an activity-guided fractionation.

Activity was tested by monitoring the reduction of TNFa induced VCAM-1

expression of HUVECs. PE = petroleum ether, DEE = diethyl ether,

EtOAc = ethyl acetate.

Figure 3 shows the chemical structure of several compounds.

Figure 4 shows a comparison of the activity of isolate and commercially available ursolic acid (UA). The upper diagram shows a comparison of the isolated vs. the commercial product on the basis of inhibition of VCMA-1 expression. To confirm specificity an isotype control was included in the experiments. Mean values of a representative experiment performed in quadruplicate +/- S.D. are shown. The lower diagram shows an analysis of cytotoxicity/cell viability by the XTT assay. Data represent mean values of a representative experiment performed in triplicate +/- S.D. Cells were exposed to the compounds for 24 h (compounds were added 30 min prior to TNFa addition).

Figure 5 shows that ursolic acid inhibits TNFa-mediated ICAM-1 expression of endothelial cells. To confirm specificity an isotype control was included in the experiments. Mean values of a representative experiment performed in quadruplicate +/- S.D. are shown.

Figure 6: Chromatogram of the HPLC-ESI-MS analysis of the obtained diethyl ether fraction (c = 5.49 mg/ml tetrahydrofurane). A: total ion chromatogram negative mode; B: total ion chromatogram positive mode; C: UV-Chromatogram at 205 nm; D: UV- Chromatogram at 425 nm. Assigned compounds: 1 . Isoquercitrin, 2...Luteolin-7-0- beta-D-glucoside, 3... lsorhamnetin-3-0-beta-D-glucoside, 4...Ursolic acid; 5...0leanolic acid; D1 ...Pheophorbide b; D2...Pheophorbide a; X...(-)-Loliolide.

Figure 7: Chromatogram of the HPLC-ESI-MS analysis of the obtained diethyl ether fraction (c = 5.25 mg/ml tetrahydrofurane). A: total ion chromatogram negative mode; B: total ion chromatogram positive mode; C: UV-Chromatogram at 205 nm; D: UV- Chromatogram at 435 nm. Assigned compounds: 1 ... Isoquercitrin, 2... lsorhamnetin-3- O-beta-D-glucoside, 3...Ursolic acid; 4...0leanolic acid; 5...Lutein; D1/D1 a....Hydroxypheophorbide b ethylester; D2/2a... Pheophorbide b ethylester; D3...152-Methoxylacetone pheophorbide a methylester or 151 -Hydroxypurpurin-7- lacton-ethyl-methyldiester; X...(-)-Loliolide.

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