Plant hydroethanolic extracts

Abstract

The present disclosure relates to hydroethanolic extracts of the plants Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera. and Baccharis articulata and their therapeutic use, for example in the treatment and prevention of cancer. Specifically, the present disclosure relates to hydroethanolic extracts of the plant Vernonanthura nudiflora.

Description

PLANT HYDROETHANOLIC EXTRACTS FIELD OF THE INVENTION

[0001] The present disclosure relates to hydroethanolic plant extracts and their therapeutic use, for example in the treatment and prevention of cancer. Specifically, the present disclosure relates to hydroethanolic extracts of the plant Vernonanthura nudiflora. Additionally, the present disclosure relates to combinations of isolated plant- derived compounds, for example compounds found in V. nudiflora extracts. BACKGROUND OF THE INVENTION

[0002] Several natural products with anti-cancer activity are currently clinically used, for example the plant-derived paclitaxel, and docetaxel. In addition, several plant species have demonstrated anticancer properties, and are used as herbal medicines in developing countries.

[0003] Vernonanthura nudiflora is a species of perennial plants in the family Asteraceae that includes more than 23,500 species spread over about 1600 genera. (Vernonanthura nudiflora (Less.) H. Rob. | Plants of the World Online | Kew Science". Plants of the World Online. Retrieved 2020-08-10) with distribution in Argentina, Brazil and Uruguay (Vega AJ, Dematteis M.2011. Pollen morphology of some species of Vernonanthura (Asteraceae, Vernonieae) from southern South America. Palynology. 35(1):94–102. 2011). The Vernonanthura (Vernonia) genera includes more than 1000 species (Bremer, K. (1994) Asteraceae: Cladistics and Classification. Timber Press, Portland). The anti-proliferative and antioxidant activities of an organic extract of Vernonanthura nudiflora and of some of the chemical constituents thereof was reported by Ramos, A.V.G. et al. 2019 (Ramos, A.V.G. et al., J. Braz. Chem. Soc. 2019, Vol.30 (8), 1728-1740). In addition, some metabolites isolated from the flowers of Vernonanthura nudiflora showed antimicrobial activities (A. V. G. Ramos et al., 2021, The chemistry of Vernonanthura nudiflora (Less.) H. Rob. flowers and its antimicrobial activities Natural Product Research, DOI: 10.1080 / 14786419.2021.1980790).

[0004] Plants of Baccharis family have found significant use in traditional South- American medicine. Among them, Baccharis trimera (B. trimera) is the most studied one,and has been reported in traditional treatment of gastrointestinal disorders and hepatic diseases (Garcia et al. 2014 A comparative study of two clerodane diterpenes from Baccharis trimera (Less.) DC on the influx and mobilization of intracellular calcium in rat cardiomyocytes. Phytomedicine 21:1021–1025; Losqui YR, Rozete FSS, Almeida MB et al (2009) Activity of Baccharis trimera (Less.) DC. Asteraceae on culture of retinal ganglion cells in vitro. Rev Bras Farmacogn 19:931–936) and several biological activities have been reported for this plant, including antihepatotoxic, antidiabetic, schistosomicidal, antioxidant, antinociceptive, and anti-inflammatory effects that are attributable to flavonoids, diterpenes, triterpenes, saponins, essential oils, and caffeoylquinic acids (Abad MJ, Bermejo P (2007) Baccharis (Compositae): a review update. Arkivoc 7:76–96; Campos FR, Bressan J, Jasinski VC et al (2016) Baccharis (Asteraceae): chemical constituents and biological activities. Chem Biodivers 13: 1–17). A hydroethanolic extract of B. trimera was also reported (Francislaine Aparecida Dos Reis Lívero, Luisa Mota da Silva et al., Hydroethanolic extract of Baccharis trimera promotes gastroprotection and healing of acute and chronic gastric ulcers induced by ethanol and acetic acid Naunyn Schmiedebergs Arch Pharmacol. 2016 Sep;389(9):985- 98). Several Baccharis species are used interchangeably, e.g., B. trimera, B. crispa, and B. articulata, with the two former being even very difficult to distinguish between.

[0005] Furthermore, the plant Plantago major (P. major) commonly known as “great plantain”, is a widespread used medicinal plant from the Plantaginaceae family (Wang H, Zhao C, Huang Y, Wang F, Li Y, Chung HY. Chemical Constituents and Bioactivities of Plantaginis Herba. Hong Kong Med J.2015;22:29–35). Plantago major contains several active compounds, such as flavonoids, polysaccharides, terpenoids, lipids, iridoid glycosides and caffeic acid derivatives (Samuelsen AB. The traditional uses, chemical constituents and biological activities of Plantago major L. A review. J Ethnopharmacol. 2000;71:1–21) and is used in treatment of various diseases such as constipation, coughs, wounds, infection, fever, bleeding and inflammation (Aghili M. Makhzan-O-L Advieh. Tehran: Tehran University of Medical Science Press; 2008. (3)). In addition, water and ethanol extracts of Plantago major leaves show anti- inflammatory activity on oral epithelial cells (Zubair et al. Journal of Traditional and Complementary Medicine 9 (2019) 169e171170).

[0006] Mitochondria are central to basic life functions for the generation of cellular energy and of key components of the biosynthetic pathways and play a central role in apoptosis. During transduction of an apoptotic signal into the cell, an alteration in the mitochondrial membrane permeability occurs, allowing the release of apoptogenic proteins, such as cytochrome c (Cyto c), an apoptosis inducing factor (AIF), and Smac / DIABLO (Kroemer, G. et al., Mitochondrial membrane permeabilization in cell death. Physiol Rev, 2007. 87(1): p. 99-163). When released from the mitochondria, all participate in the complex processes resulting in the activation of proteases and nucleases leading to DNA and protein degradation, and ultimately to apoptotic cell death. One of the mitochondrial proteins serving as a mitochondrial gatekeeper, controlling both cell life and death, is the voltage-dependent anion channel 1 (VDAC1). As previously shown by some of the present inventors, VDAC1 assumes a crucial position in the cell, being located in the outer mitochondrial membrane (OMM), thus forming the main interface between mitochondrial and cellular metabolisms; VDAC1 is a also key protein in the regulation of metabolism controlling the passage of adenine nucleotides and other metabolites and of Ca2+into and out of mitochondria, and a key protein in the regulation of mitochondria-mediated apoptotic cell death and controls other biological and cellular functions, and VDAC1 is overexpressed in various cancer cell lines and in different tumors, indicating its importance for their development and persistence, and also in many diseases other than cancer (Shoshan-Barmatz, V. VDAC1 at the intersection of cell metabolism, apoptosis and diseases, Biomolecules 2020, 26;10(11):1485). The crucial role VDAC1 plays in regulating the metabolic and energetic functions of mitochondria in cancer cells is demonstrated by the findings that down-regulation of VDAC1 expression by specific siRNA leads to a decrease in energy production and cell growth and inhibits tumor growth (Arif, T. et al., Silencing VDAC1 Expression by siRNA Inhibits Cancer Cell Proliferation and Tumor Growth In Vivo. Mol Ther Nucleic Acids, 2014. 3: p. e159). Several mechanisms for the release of the pro-apoptotic proteins have been proposed, including a channel formed by VDAC1 oligomers (Keinan, N. et al., Oligomerization of the mitochondrial protein VDAC is coupled to the induction of apoptosis, Molecular Cell Biol. 30(24), 5698-5709; Weisthal, S., et al., Ca(2+)-mediated regulation of VDAC1 expression levels is associated with cell death induction. Biochim Biophys Acta, 2014. 1843(10): p. 2270-81). Defects in the regulation of apoptosis are often associated with drug resistance and diseases such as cancer, with apoptosis evasion being a cancerhallmark (Hanahan, D. and R.A. Weinberg, Hallmarks of cancer: the next generation. Cell, 2011.144(5): p.646-74).

[0007] All of the mitochondrial apoptotic proteins known to translocate to the cytoplasm following an apoptotic stimulus reside in the mitochondrial intermembrane space (IMS). Thus, only the permeability of the OMM needs to be modified for their release (Halestrap, A.P. et al., The permeability transition pore complex: another view. Biochimie, 2002. 84(2-3): p.153-66). Hence, VDAC1, as an OMM channel could mediate Cyto c release. It has been previously demonstrated that VDAC1 can exist in the form of oligomers that mediate the release of Cyto c, by forming a large channel that enables the release of pro- apoptotic proteins, leading to cell death (Keinan, N. et al., Oligomerization of the mitochondrial protein VDAC is coupled to the induction of apoptosis, Molecular Cell Biol. 30(24), 5698-5709). Moreover, it was shown that apoptosis induction by cisplatin, selenite, H2O2, UV light, and more, lead to apoptosis via inducing VDAC1 overexpression, shifting the equilibrium towards oligomers, followed by the release of pro-apoptotic proteins and apoptosis (Keinan, N. et al., The role of calcium in VDAC1 oligomerization and mitochondria-mediated apoptosis. Biochim Biophys Acta, 2013. 1833(7): p.1745-54).

[0008] There remains an unmet need in the art to identify and harness the natural products, such as the ones produced by V. nudiflora and optionally by other plants, for effective interaction with VDAC1-mediated apoptosis pathway, for the use, inter alia, in treating cancer, either alone or as an adjuvant to apoptosis-inducing chemotherapy, or in reducing the prevalence of cancer recurrence. SUMMARY OF THE INVENTION

[0009] It has now been unexpectedly found that extracts of the plant V. nudiflora, alone or in combination with extracts of other plants that have been used in some complex herbal remedies, e.g., for general wellness improvement, can be used against cancer cells in culture, reducing their cell viability and inducing cell death. In a mouse model of cancer, V. nudiflora extracts were shown to inhibit tumor growth, being more effective at high dilutions. However, unlike in the homeopathic pseudo-theory, and without being bound to a particular narrative it is believed that the anti-apoptotic, pro-survivalcompounds reported to be present in the extract and responsible for its beneficial effects in other areas of human health, such as anti-oxidation and / or anti-ageing, at the concentration present in the extract overcome the pro-apoptotic effectors of the extracts, thereby negating their effect on the malignant cells. Reducing the concentration of the extract by high dilutions reduces the effects of anti-apoptotic / pro-survival components of the extract, which are probably less potent than the anti-cancer compounds present therein. It has been further unexpectedly found that prolonged hydroethanolic extraction of plant substance, as described in greater detail below, provides an extract characterized by a set of compounds which differs from the set of compounds obtained by exhaustive organic ethanolic extraction, the set of compounds having pronounced pro-apoptotic effects, possibly, mediated by VDAC1 pathway. Additionally, as demonstrated in the examples, administering Vernonantura-containing extracts to cancer patients, both alone and as a co-therapy with conventional chemotherapy, has led to either a prolonged remission or a complete response.

[0010] Therefore, in a first aspect thereof the present disclosure provides a composition comprising a hydroethanolic plant extract or any fraction thereof, wherein said plant is selected from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera, and Baccharis articulata, and any combination thereof, e.g., a hydroethanolic plant extract or any fraction thereof of Vernonanthura nudiflora or a combination of Vernonanthura nudiflora and Plantago major. The extract is essentially identical to an extract obtained by a process comprising combining the biomass of at least one of said plant(s) with a hydroethanolic mixture comprising 20 to 50 volume percent of water, and extracting said biomass into said hydroethanolic mixture for a time interval of between about 3 and 35 days, preferably between about 18 and 23 days. In various embodiments the extract is manufactured by a process comprising the steps of combining the biomass of said plant(s) with a hydroethanolic mixture comprising 20 to 50 volume percent of water and extracting said biomass into said hydroethanolic mixture for a time interval of between about 3 and 35 days, for example between about 3 and 28 days, or between 10 and 30 days, preferably between about 18 and 23 days.

[0011] In the second aspect provided herein a method of extracting bioactive compounds from the biomass of the above plants, e.g., from V. nudiflora biomass, by contacting said biomass with hydroethanolic mixture comprising 20 to 50 percent by volume of water in ethanol, and extracting said biomass into said hydroethanolic mixture for a time interval (time period) of between about 3 and 35 days, for example between about 3 and 28 days, or between 10 and 30 days, preferably between about 18 and 23 days. The method of extracting of said plant biomass into said hydroethanolic mixture may be performed at a temperature range of between about 15°C and 40°C, preferably between 20°C and 25°C. The extraction method may further include harvesting the plants’ (e.g., V. nudiflora) areal parts, e.g., stems, branches, leaves, and optionally flowers, and optionally drying them to furnish dried biomass, i.e., drying of said plants prior to extracting. The drying may preferably be performed at ambient to slightly elevated temperatures, e.g., at a temperature of between 15 and 45°C, preferably between about 20°C and 25°C. The drying step may preferably be carried out until a certain mass is lost as water, e.g., until a weight loss of about 40 to 60 % by weight remains of the initial weight of the plant, e.g., the aerial parts thereof. The process may further comprise grinding said dried biomass, to furnish biomass suitable for extracting. The extracting may be carried out in a ratio of between 1:2 to 1:8, e.g., between 1:3 and 1:6 between the biomass being extracted and the hydroethanolic mixture, preferably about 1:4, e.g. about between 13 and 33 weight percent, or between about 15 and 25 weight percent, or about between 18 and 22 weight percent, of biomass to the total volume of the hydroethanolic solution. The process may further comprise separating residues of said plant biomass from said hydroethanolic mixture to furnish said extract, e.g., filtering said biomass to produce crude liquid extract. The process may further comprise clarifying said crude liquid extract, e.g., by centrifugation at a suitable g-force to effect sedimentation of dispersed fine matter. Furthermore, obtained by the presently disclosed method of extraction are a dried hydroethanolic extract or a liquid hydroethanolic extract, in the latter case the concentration of plant-derived matter on dry basis in said liquid hydroethanolic extract is between 0.5% and 15.0%, preferably between 1.0% and 5.0% wt, or the concentration of plant-derived matter on dry basis in said liquid hydroethanolic extract is between 2.5% and 3.7% wt. in particular embodiments, the hydroethanolic extract comprises between 40 and 80% of ethanol, the balance of the solvent consisting essentially of water. In certain embodiments, the method further comprises a sub-extraction of thehydroethanolic plant extract, preferably by butanol, hexane, chloroform, or ethyl acetate extraction, or any combination thereof, thereby obtaining a sub-extraction (also referred to herein as a fraction of said hydroethanolic extract).

[0012] By a further aspect thereof the preset disclosure provides a composition comprising a hydroethanolic plant extract, said extract containing less than 5-20% by weight of the compounds listed in Table 1 below (e.g., less than 5%) by weight or is essentially devoid of these compounds, wherein said plant is from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera, and Baccharis articulata, and any combination thereof, e.g., the plant is Vernonanthura nudiflora or a combination of Vernonanthura nudiflora and Plantago major. Moreover, in a further aspect provided herein an artificial (i.e., unnatural) mixture, also referred to herein as fraction of components that may be present in at least one of the plant(s) from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera, and Baccharis articulata, and any combination thereof (e.g., V. nudiflora) hydroethanolic extract. By way of example, the fraction of components comprises at least two compounds selected from the group consisting of rutin, keampferol, myricetin, stachydrine chloride, cyanidin chloride, glycitein diosmetin, 8-hydroxyquinoline, quercetin-3-beta-glucoside, phytol, myristicin, alpha-lapachone and 1,2:2,4-di-p-methylbenyliedene.

[0013] By a further aspect thereof the present disclosure provides the composition as herein defined for use in treating of a proliferative disorder (e.g., cancer) in a subject in need thereof. The composition as herein defined and the method of treatment comprising the same are particularly suitable for treating a proliferative disorder or cancer which is a primary or metastatic prostate cancer, Langerhans cell histiocytosis, testicular cancer, colon cancer, or breast cancer. In some embodiments, the composition as herein defined is supplied to said subject in amounts of between 0.033 mg / kg and 8.6 mg / kg of extract by dry basis per kilogram weight of said subject, for example, in amounts of between 20 and 500 mg of extract by dry basis per subject. The composition as herein defined may be administered by a route selected from the group consisting of intravenous administration, oral administration, intramuscular administration, and intraperitoneal administration. Furthermore, in certain embodiments the composition as herein definedis sutable for treating a subject that concomitantly receives a treatment with an additional anti-cancer agent, preferably an apoptosis-inducing chemotherapy.

[0014] Still further the composition according to the present disclosure is useful for the manufacture of a medicament for treating a proliferative disorder in a subject in need thereof.

[0015] The present disclosure further provides a method for the treatment of a proliferative disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a composition as herein defined, namely a composition comprising a hydroethanolic plant extract or any fraction thereof, wherein said plant is from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from the group consisting of Baccharis crispa, Baccharis trimera, and Baccharis articulata, and any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0017] Fig. 1A – Fig. 1F. SHSY5Y cancer cells were incubated (24 hours) with the indicated dilutions of the hydroethanolic plant extracts of Vern (Vernonanthura nudiflora), Bac (Baccharis trimera), Pla (Plantago major), or their mixture (40%: 40%: 20%, respectively). Fig. 1A1 and Fig. 1A2 are representative results of FACS analysis of propidium iodine (PI) stained cells showing live and dead cells in the control (Fig. 1A1) and cells incubated for 24 hours with Vern plant extract (Fig.1A2, 1:500). Fig.1B is a bar diagram showing analysis of apoptosis (cell death % based on FACS analysis of propidium iodine (PI) stained cells) in cells incubated with the indicated different dilutions of extracts from Vern plant, Bac, or Pla. The results are the mean ± SEM of three independent experiments. Fig. 1C is a graph showing apoptosis percentage in SHSY5Y cells incubated (24 hours) with the indicated dilution of Vern plant extract, and then analyzed for apoptosis using Annexin V / PI staining and FACS. Fig. 1D – Fig. 1Fare graphs showing SHSY5Y cell viability as revealed by the XTT assay of cells incubated with different dilutions of the indicated plant extract for 24, 48, or 72 hours.

[0018] Fig.2A – Fig.2G F. SHSY5Y and U-87MG cells were incubated 24 hours with the indicated dilution of Vern plant extract, and then subjected to different analyses. Fig. 2A is an immunoblot performed using anti-VDAC1-specific antibodies, showing VDAC1 expression levels in treated cells at the indicated dilution of Vern plant extract. Immunoblotting with actin as a loading control is also shown. The levels of VDAC1 are given below the immunoblot as relative units (RUs), showing increase in VDAC1 levels by the Vern plant extract. Fig. 2B is a graph showing samples of the pant Vern extract treated cells analyzed for apoptosis, inducing over 80% apoptosis. Fig. 2C is an immunoblot of SHSY5Y cells incubated (24 hours) with the indicated dilutions of the plant extracts Vern, Bac and Pla, or their mixture and then analyzed for VDAC1 oligomerization by incubation with the cross-linking reagent EGS (100 ^M), followed by immunoblotting using anti-VDAC1 antibodies. The positions of the VDAC1 monomers, dimers, trimers, tetramers, and higher oligomers are indicated. Fig. 2D is a bar graph showing the level of VDAC1 dimers as analyzed using Image J software and presented relative to its levels in control cells subjected to EGS. The results are the mean ± SEM of three independent experiments. Fig. 2E is an immunoblot showing SHSY5Y cells that were incubated (24 hours) with the indicated dilutions of the plant extracts Vern, Bac and Pla, or their mixture without EGS treatment, and subjected to immunoblotting. The position of the VDAC1 monomers, dimers, trimers, and tetramers is indicated. Fig.2F is a bar graph showing the level of VDAC1 dimers as analyzed using Image J software and presented relative to its levels in control cells.

[0019] Fig.3A – Fig. 3D. Fig.3A is a graph showing MitoSox fluorescence percentage in SHSY5Y cells incubated 24 hours with the indicated dilutions of Vern plant extract and analyzed for reactive oxygen species (ROS) production using MitoSox Red reagent and FACS analysis. Fig. 3B is a graph showing intracellular [Ca2+]i using the calcium indicator Fluo-4 reagent and FACS analysis. Fig. 3C and Fig. 3D, respectively, show representative Operetta imaging visualization of the cells in Fig. 3B and quantification thereof. The results are the mean ± SEM of three independent experiments.

[0020] Fig. 4A – Fig. 4C. Fig. 4A is a TLC separation photograph of hydroethanolic extracts of Vern plant, Bac, and Pla, along with known amounts of phytol and ethyl linoleate, using the solvent mixture of petroleum ether: diethyl ether: acetic acid (85:15:1,V:V:V) and developed by exposure to iodine vapor. Fig. 4B is a bar graph showing a quantification of phytol and ethyl linoleate in plant extracts using the compounds’ calibration curves and Image J software (n=3). Fig. 4C is a graph showing SHSY5Y cells incubated 24 or 48 hours with and without the indicated concentration of phytol or ethyl linoleate, and then evaluated for cell death using PI staining and FACS analysis. The results are the mean ± SEM of three independent experiments.

[0021] Fig. 5A – Fig. 5D. Fig. 5A1 and Fig. 5A2 are immunoblots showing SH-SY5Y cells incubated 24 hours with and without the indicated concentrations of phytol (Fig. 5A1) or ethyl linoleate (Fig. 5A2), and then analyzed for VDAC1 expression levels by immunoblotting. Fig. 5B is a graph showing the level of VDAC1 in the cells detailed in Fig. 5A, as relative units (RUs), quantified using Image J software. Fig. 5C is an immunoblot showing control and phytol- or ethyl linoleate-treated cells that were also analyzed for VDAC1 oligomerization by incubation with the cross-linking reagent EGS (100 ^M), followed by immunoblotting using anti-VDAC1 antibodies. The position of the VDAC1 monomers, dimers, trimers, and tetramers is indicated. Fig. 5D is a graph showing a quantification of VDAC1 dimer level in the experiment detailed in Fig. 5C. Results are the mean ± SEM (n=3).

[0022] Fig.6A – Fig.6H. Fig.6A is a schematic presentation of the experimental design in nude mice inoculated subcutaneously (s.c.) with U-87MG cells (1.8 × 106cells / mouse), in which the tumor volume was monitored (using a digital caliper), and on day 14, when the tumor volume was about 50 mm3, the mice were divided into three groups with a similar average volume calculated per group (5 or 6 mice per group). The three mice groups were subjected to the following treatments: control (ethanol to a final concentration 0.14%) or Vern plant extract to a final dilution of 1:250 or 1:500, and phytol to a final concentration of 75 ^M. The injected volume is calculated according to the tumor volume and the stock solution used. The mice were monitored and sacrificed on day 34 (indicated by 34D) post cells inoculation. Fig.6B is a graph showing the calculated average tumor volumes as a function of time in the participating mice, presented as means± SEM (n=5 or 6 mice). Fig. 6C is a graph showing calculated tumor volume before scarifying the mice (day 34), presented as % of the control. Fig. 6D is a graph showing the calculated average tumor weights presented as means ± SEM. *p < 0.05. Fig. 6E shows confocal images of representative immunofluorescent images of paraffin- embedded sections from U-87MG-derived tumors in mice treated with control, Vern plant extract (1:500) or phytol (75 ^M), the immunofluorescent images were obtained using antibodies against the proliferation marker, Ki-67. A quantification of staining intensity is shown in Fig.6F. Results are the mean ± SEM (n=3), ****p < 0.0001. Fig. 6G shows micrographs of TUNEL staining on paraffin-embedded sections cut from control tumors, Vern plant extract- or phytol-treated tumors. TUNNEL staining of tumor sections was carried out as described in the Examples section. Representative confocal images with red staining indicates PI nuclear staining, and green-stained cells indicates TUNEL staining. Fig. 6H is a graph showing a quantification of TUNEL positive cells. Results are the mean ± SEM (n=3), *p < 0.0; ****p < 0.0001;

[0023] Fig. 7A – Fig. 7B. Fig.7A is a graph showing the average tumor volume (mm3) as a function of the number of treatments in nude mice inoculated s.c. with U-87MG cells (1.8 × 106cells / mouse). Tumor volume was monitored (using a digital caliper) and on day 14, when the tumor volume was between 40 and 60 mm3, the mice were divided into three groups with a similar average volume calculated per group (5 or 6 mice per group). The three mice groups were then subjected to the following treatments: control (ethanol to a final concentration 0.14%) or Vern plant extract to a final dilution of 1:100 or 1:300, calculated according to the tumor volume. The calculated average tumor volumes as a function of time are presented as means ± SEM (n=5 or 6 mice). Fig. 7B is a graph showing the calculated average tumor weights, presented as means ± SEM. *P<0.05.

[0024] Fig. 8A – Fig. 8D. Confocal images of sections from U-87MG-derived tumors, control or treated with Vern plant extract (1:500) or phytol (75 ^M), and immunofluorescent stained for glucose transporter 1 (Glut-1) and glyceraldehyde de hydrogenase (GAPDH) are shown in Fig.8A or stained for VDAC1 and hexokinase (HK- I) are shown in Fig. 8C, using specific antibodies. Staining intensity was quantitative using Image J software as a measure for relative protein level and in shown in Fig. 8Band Fig.8D for the images detailed in Fig. 8A and Fig. 8C, respectively. Results reflect the mean ± SEM (n=3), ****p ≤ 0.0001.

[0025] Fig. 9A – Fig. 9D. Confocal images of sections from U-87MG-derived tumors obtained from mice treated with control, Vern plant extract (1:500), or phytol (75 ^M) that were immunofluorescent stained for CD-31 or ^-SMA are shown in Fig.9A and Fig. 9C, respectively. Quantifications of the above images are presented in the graphs of Fig. 9B (showing the relative level of CD-31) and in Fig.9D (showing the relative level of ^- SMA). Results = means ± SEM (n =3 mice) ****p ≤ 0.0001.

[0026] Fig.10A – Fig.10B. Fig.10A shows representative IF staining of tumor sections from U-87MG-derived tumors, control or treated with Vern plant extract (1:500) or phytol (75 ^M), immunofluorescent stained with specific antibodies against the CSCs markers, Sox2 and Nestin. Fig. 10B is a graph showing a quantitative analysis of the images shown in Fig.10A. Results are means ± SEM (n = 3 tumors), ****p ≤ 0.0001.

[0027] Fig. 11A – Fig. 11I are graphs showing SHSY5Y cells incubated 24 hours with Vern extract at the indicated dilutions (Fig. 11A) or with the compounds Caffeic acid (Fig. 11B), Myricetin (Fig. 11C), Rutin (Fig. 11D), Quercetin (Fig. 11E), Kaempferol (Fig.11F), Alpha-Lapachone (Fig.11G), 1,2:2,4-Di-p-Methylbenyliedene Sorbitol (Fig. 11H) and Myristicin (Fig.11I) at the indicated concentrations and then evaluated for cell death using PI staining and FACS analysis. The results are the mean ± SEM of three independent experiments.

[0028] Fig.12A – Fig. 12G are graphs showing SHSY5Y cells incubated 24 hours with the compounds Quercetin-3-beta-glucoside (Fig. 12A), Diosmetin (Fig. 12B), Stachidrine chloride (Fig. 12C), Glyciteine (Fig. 12D), 8-hydroxyquinoline (Fig. 12E), Di-p-Methlbenyliedene sorbitol (Fig. 12F) and Cyanidin chloride (Fig. 12G) at the indicated concentrations and then evaluated for cell death using PI staining and FACS analysis. The results are the mean ± SEM of three independent experiments.

[0029] Fig.13A – Fig. 13G are graphs showing SHSY5Y cells incubated 24 hours with the compounds Yangonin (Fig. 13A), Chloragenic acid (Fig. 13B), 4-hydroxycoumarin(Fig. 13C), Betaine (Fig. 13D), Rosmarinic acid (Fig. 13E), Nobiletin (Fig. 13F) and Murrangatin (Fig. 13G) at the indicated concentrations and then evaluated for cell death using PI staining and FACS analysis. The results are the mean ± SEM of three independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0030] Vernonanthura nudiflora extracts have been prepared in the past by exhaustive organic ethanolic extraction, followed by fractionation into a variety of organic solvents. Some of the compounds identified in these extracts, for example, as reported in the publication mentioned above by Ramos A.V.G. et al., 2019, namely piptocarphin sesquiterpene lactones, in particular, 8α-tigloyloxy-10α-hydroxy-hirsutinolide, some triterpenes, glycosylated steroids, some flavonoids, including velutin, and chlorogenic acid derivatives, have been also tested for anti-cell proliferation activity in cell lines with a varying degree of success. It has now been unexpectedly found that subjecting V. nudiflora to hydroethanolic extraction, as opposed to organic ethanolic extraction, for prolonged time intervals, furnishes a completely different extract that contains significantly larger number of compounds. As demonstrated in the appended examples, in the hydroethanolic extract according to some embodiments of the present invention, apart from chlorogenic acid, the vast majority of the compounds reported in Ramos A.V.G. et al., 2019 were not identified by gas-chromatography (GC) and liquid chromatography (LC) coupled with mass spectroscopy (MS). The extract prepared according to the present disclosure was able to not only kill cancer cells by leveraging VDAC1 pro-apoptotic pathway, but also demonstrated a pronounced effect in xenograft murine model and remarkably, also showed a beneficial effect in treatment of various cancers in human patients, particularly, when administered as a co-therapy.

[0031] Therefore, by a first aspect thereof, the present disclosure provides a composition comprising a hydroethanolic plant extract or any fraction thereof, wherein said plant is from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from the group consisting of Baccharis crispa, Baccharis trimera, and Baccharis articulata, and any combination thereof (referred to herein: “the Plants”). By way of example, the plant species Vernonanthura nudiflora, Baccharis trimera or Baccharis artuculata, and Plantago major are extracted individually, and the mixture thereof isprepared such that a mixing ratio of 40% Vernonanthura nudiflora, 40% Baccharis articulata / trimera, and 20% Plantago major is obtained. By way of a further example, the plant is Vernonanthura nudiflora or a combination of Vernonanthura nudiflora and Plantago major.

[0032] According to various embodiments, the composition as herein defined comprises a hydroethanolic extract of at least one of Vernonanthura nudiflora, Baccharis articulata / trimera, and Plantago major, said extract having a composition essentially identical to an extract being manufactured (obtained) by a process comprising combining the biomass of at least one of these plants with a hydroethanolic mixture comprising between 20 and 50 volume percent of water in ethanol, and extracting said biomass into said hydroethanolic mixture for a time interval of between about 3 and 35 days, e.g., 3 and 28 days, or between about 10 and 30 days, preferably between about 18 and 23 days. In specific embodiments, the composition as herein defined comprises a hydroethanolic extract of at least one of the Plants manufactured by an extraction process as detailed herein below, namely a process comprising combining biomass of at least one of the Plants with a hydroethanolic mixture comprising between 20 and 50 volume percent of water in ethanol, and extracting said biomass into said hydroethanolic mixture for a time interval of between about 3 and 35 days, e.g., 3 and 28 days, or between about 10 and 30 days, preferably between about 18 and 23 days.

[0033] As detailed above, the inventors have found that prolonged hydroethanolic extraction of plant substance, as described herein below, provides an extract characterized by a set of compounds which differs from the set of compounds obtained by exhaustive organic ethanolic extraction, the set of compounds having pronounced pro-apoptotic effects. In specific embodiments, the hydroethanolic extract of at least one of the Plants manufactured by the extraction process as detailed herein below may be defined as comprising the compounds listed in at least one of Tables 5-11 below, for example, as comprising at least 60% of the compounds in ratios as enumerated therein, preferably at least 65%, or at least 70%, or at least 75%, preferably at least 80%, 85%, or at least 90%, at least 95%, or at least 97%, as readily determined by the skilled artisan, e.g., by comparing output of analytical methods, such as mass-spectrometry-coupled chromatography, e.g., LC-MS / MS, GC-MS / MS. The peak positions, the molecularweight of the compounds, their relative intensities (areas), and / or their fragmentation patterns may be used. In the tables 5-11, enumerated compounds pertain to their respective extracts as follows: A, B, C: compounds identified exclusively in V. nudiflora B. trimera and P. major, respectively, and AB, AC, BC, and ABC enumerate compounds identified in two or more respective plants.

[0034] The compounds identified in Ramos A.V.G. et al 2019 are presented in Table 1 below. By a further aspect thereof the present disclosure provides a composition comprising a hydroethanolic plant extract, said extract containing less than 5-20% by weight of the compounds listed in Table 1 below (e.g., less than 5%), i.e., compounds 1- 26 as disclosed in Ramos A.V.G. et al., 2019 (which is hereby incorporated by reference) or is essentially devoid of these compounds, wherein said plant is selected from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera, and / or Baccharis articulata and any combination thereof. Compounds 1-26 are identified in Ramos A.V.G. et al., 2019, e.g., in Figure 1 (compounds 1-17) and in Figure 3 (compounds 18-26). The names of compounds 1-26 are, respectively, lupeol, ^-amyrin, ^-amyrin, taraxasterol, pseudotaraxasterol, piptocarphin A, piptocarphin B, piptocarphin D, 8 ^-tigloyloxy-10 ^ hydroxy- hirsutinolide, velutin, apigenin, chrysoeriol, β-Sitosterol β-D-glucoside (sitosterol-3-β-O- D‑glicopyranoside), Stigmasterol-3‑β‑O-D‑glicopyranoside, 3-O-caffeoylquinic acid (Chlorogenic acid), rutin, luteolin, (E)-3-(acetoxymethyl)-6-hydroxy-6,10-dimethyl-2- oxo-2,4,5,6,9,10-hexahydro-7,10-epoxycyclodeca[b]furan-4-yl (E)-2-methylbut-2-eno- ate, (E)-3-(acetoxymethyl)-6-hydroxy-6,10-dimethyl-2-oxo-2,4,5,6,9,10-hexahydro-7,- 10-epoxycyclodeca[b]furan-4-yl methacrylate, 3-(acetoxymethyl)-7,11-dihydroxy-6,10- dimethyl-2-oxo-2,4,5,6,7,8,9,10,11,11a-decahydro-7,10-epoxycyclodeca[b]furan-4-yl methacrylate, 3-(acetoxymethyl)-7,11-dihydroxy-6,10-dimethyl-2-oxo-2,4,5,6,7,8,9,10,- 11,11a-decahydro-7,10-epoxycyclodeca[b]furan-4-yl (E)-2-methylbut-2-enoate, 8α-tig- loyloxyhirsutinolide 13-O-acetate, piptocarphin E, diacetyl piptocarphol, 5-acetoxy-8- (acetoxymethyl)-1a,5-dimethyl-4,9-dioxo-1a,2,3,4,5,6,7,9,-10a,10b-decahyd-rooxireno- [2',3':9,10]cyclodeca[1,2-b]furan-7-yl (E)-2-methylbut-2-enoate and glaucolide A.

[0035] As mentioned above, it has now been unexpectedly found that prolonged hydroethanolic extraction of plant substance, as described hereinbelow, provides anextract with beneficial effect for treating subjects afflicted, inter alia, with various cancers. The extract, or a fraction thereof, as herein defined, is obtainable by a process as described in greater detail herein. That is, the extract, or a fraction thereof, may be prepared by the process, or may be prepared by a different process provided that the obtained extract or a fraction thereof is essentially identical to an extract obtained by the process as described herein or to the specific fraction thereof. The essentially identical extract may have at least 60% of the similarity in the composition with the extract prepared by the process as described herein. Preferably, the essentially identical extract has at least 65% similarity, or at least 70% similarity, or 75, 80, 85, 90, or at least 95% similarity, preferably at least 80% similarity and above. The similarity may be conventionally determined as known in the art, e.g., by comparing output of analytical methods, such as mass-spectrometry coupled chromatography, e.g., LC-MS / MS, GC- MS / MS. The peak positions, the molecular weight of the compounds, and / or their fragmentation patterns may be used to assess the similarity. Additionally, suitable statistical and / or chemometric methodology may be employed to determine the similarity and the extent thereof. As demonstrated in the appended examples, fractions of the hydroethanolic extract manufactured as described herein demonstrated anti-cancer properties comparable or even superior to the original extract; therefore, the extract fractions may also be obtainable by the process as described herein, including the sub- extractions, or may be obtained otherwise, e.g., directly by extracting the plant material, V. nudiflora in particular, with a solvent, such as hexane, chloroform, ethyl acetate, or mixtures thereof, or equivalent solvents, provided that obtained extract is essentially identical, in the sense as described above, to the sub-extract manufactured by the process as specified generally herein.

[0036] In addition, since the hydroethanolic extract contains a significantly different composition of compounds, a process for preparation of such hydroethanolic extract embodies a further aspect of the present invention. Thus, provided herein a process of manufacturing of plant extracts of the plants indicated herein, also termed herein an extracting process, said process comprising combining the biomass of at least one of V. nudiflora, P. major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera, and Baccharis articulata with a hydroethanolic mixture comprising between 20 and 50 volume percent of water in ethanol. The extraction, however, is carriedout over very long-time intervals, of between 3 and 35 days, but preferably between 18 and 23 days, e.g., three weeks. Without being bound by a particular theory, it is currently believed that this extraction process is different from exhaustive organic ethanolic extraction, for example since it is performed in the presence of a significant amount of water, which in turn allows for extraction of more of water-soluble compounds, and since some chemical modifications to the compounds that may be sensitive to water and other ambient factors may occur during the prolonged extraction period, rendering the final extract stable for a long shelf life. Thus, preferably, the process comprises combining V. nudiflora biomass with hydroethanolic mixture and extracting said biomass for a time interval of between 3 and 35 days, preferably between 18 and 23 days.

[0037] Preferably, the extraction of plant biomass into the hydroethanolic mixture is performed in a suitable vessel, equipped by agitation means, and is agitated intermittently, with a periodicity of between 24 and 48 hours, for intervals of between 1 and 2 hours. The extracting step may be advantageously performed in an ambient temperature, e.g., between 15 and 25°C. However, the extracting may be performed at a slightly elevated temperatures, to facilitate and / or accelerate the extraction, e.g. at a temperature of between 25 and 45°C. Therefore, the temperature may be adjusted according to the needs of the process, between 15 and 45°C, but preferably the temperature would be between 20 and 25°C. In specific embodiments, extracting plant biomass into the hydroethanolic mixture is performed at a temperature range of between about 15°C and 40°C, preferably between 20°C and 25°C.

[0038] The hydroethanolic mixture comprises ethanol, in a concentration of between 40 and 80 volume percent. The balance of the hydroethanolic mixture consists essentially of water. Preferably, the concentration of ethanol is between 65 and 75 volume percent. Further preferably, the concentration of ethanol is about 70%. Without being bound by a particular theory, it is also currently believed that utilizing 70% ethanol for extraction medium may be particularly advantageous due to the antiseptic properties of the solvent, thereby contributing to the microbial stability of the extracts in their liquid form.No Name IUPAC name CAS No. 1Lupeol (1R,3aR,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a,5a,5b,8,8,11a-545-47-1 hexamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,- 10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysen-9-ol 2^-Amyrin (3S,4aR,6aR,6bS,8aR,11R,12S,12aR,14aR,14bR)-4,4,6a,6b,8a,-638-95-9 11,12,14b-octamethyl-2,3,4a,5,6,7,8,9,10,11,12,12a,14,14a- tetradecahydro-1H-picen-3-ol 3β -Amyrin 3S,4aR,6aR,6bS,8aR,12aR,14aR,14bR)-4,4,6a,6b,8a,11,11,14b-octa-559-70-6 methyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-ol 4Taraxasterol (3S,4aR,6aR,6bR,8aR,12S,12aR,12bR,14aR,14bR)-4,4,6a,6b,8a-1059-14-9 ,12,14b-Heptamethyl-11-methylidenedocosahydropicen-3-ol 5Pseudotaraxa-4,4,6a,6b,8a,11,12,14b-octamethyl-2,3,4a,5,6,6a,7,8,- 464-98-2 sterol 9,12,12a,13,14,14a-tetradecahydro-1H- picen-3-ol 6Piptocarphin A (4S,6R,7S,10R,11E)-5,6,7,8,9,10-Hexahydro-3-(acetoxymethyl)-6,7-76248-63-0 dihydroxy-4-(methacryloyloxy)-6,10-dimethyl-7,10- epoxycyclodeca[b]furan-2(4H)-one 7Piptocarphin B (E)-3-(acetoxymethyl)-6,7-dihydroxy-6,10-dimethyl-2-oxo-76215-49-1 2,4,5,6,7,8,9,10-octahydro-7,10-epoxycyclodeca[b]furan-4-yl (E)-2- methylbut-2-enoate 8Piptocarphin D [(2Z)-8,10,11-trihydroxy-1,10-dimethyl-5-oxo-4,14-76215-51-5 dioxatricyclo[9.2.1.03,7]tetradeca-2,6-dien-6-yl]methyl acetate 98 ^-tigloyloxy-10 ^hydroxy-hirsutinolide: (E)-3-ethyl-6,7-dihydroxy-6,10-dimethyl-2-oxo-2,4,5,6,7,8,9,-10-octahydro-7,10-epoxycyclodeca[b]furan-4-yl (E)-2-methylbut-2-enoate 10Velutin 5-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-4H-1-25739-41-7 benzopyran-4-one 11Apigenin 5,7-Dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one520-36-5 12Chrysoeriol 5,7-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopyran-491-71-4 4-one 13β-Sitosterol β-D-glucoside (sitosterol-3-β-O-D‑glicopyranoside)474-58-8 14Stigmasterol-3‑β‑O-D‑glicopyranoside153-O-caffeoyl-(1S,3R,4R,5R)-3-{[(2E)-3-(3,4-Dihydroxyphenyl)prop-2- 202650-88-2 quinic acid enoyl]oxy}-1,4,5-trihydroxycyclohexane-1-carboxylic acid 16Rutin (42S,43R,44S,45S,46R,72R,73R,74R,75R,76S)-153-18-4 13,14,25,27,43,44,45,73,74,75-Decahydroxy-76-methyl-24H-3,6- dioxa-2(2,3)-[1]benzopyrana-4(2,6),7(2)-bis(oxana)-1(1)- benzenaheptaphane-24-one 17Luteolin 2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one491-70-3 18(E)-3-(acetoxymethyl)-6-hydroxy-6,10-dimethyl-2-oxo-2,4,5,6,9,10-hexahydro-7,10-epoxycyclodeca[b]furan-4-yl (E)-2-methylbut-2-enoate 19(E)-3-(acetoxymethyl)-6-hydroxy-6,10-dimethyl-2-oxo-2,4,5,6,9,10-hexahydro-7,10-epoxycyclodeca[b]furan-4-yl methacrylate 203-(acetoxymethyl)-7,11-dihydroxy-6,10-dimethyl-2-oxo-2,4,5,6,7,8,9,10,11,11a-decahydro-7,10-epoxycyclodeca[b]furan-4-yl methacrylate 213-(acetoxymethyl)-7,11-dihydroxy-6,10-dimethyl-2-oxo-2,4,5,6,7,8,9,10,11,11a-decahydro-7,10-epoxycyclodeca[b]furan-4-yl (E)-2-methylbut-2-enoate 228α-Tigloyloxy-2-Butenoic acid, 2-methyl-, (4S,6R,7S,10R,11E)-3- 83182-58-5 hirsutinolide 13- [(acetyloxy)methyl]-2,4,5,6,7,8,9,10-octahydro-7-hydroxy-6,10- O-acetate dimethyl-2-oxo-7,10-epoxycyclodeca[b]furan-4-yl ester, (2E)- 23Piptocarphin E [(2E)-6-(acetyloxymethyl)-11-ethoxy-10-hydroxy-1,10-dimethyl-5-76215-52-6 oxo-4,14-dioxatricyclo[9.2.1.03,7]tetradeca-2,6-dien-8-yl] 2- methylprop-2-enoate 24Diacetyl(E)-(4-acetoxy-6,7-dihydroxy-6,10-dimethyl-2-oxo-2,4,5,6,7,8,9,10-octahydro-7,10- piptocarphol epoxycyclodeca[b]furan-3-yl)methyl acetate 255-acetoxy-8-(acetoxymethyl)-1a,5-dimethyl-4,9-dioxo-1a,2,3,4,5,6,7,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-7-yl (E)-2-methylbut-2-enoate 26Glaucolide A 5-acetoxy-8-(acetoxymethyl)-1a,5-dimethyl-4,9-dioxo-11091-29-5 1a,2,3,4,5,6,7,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2- b]furan-7-yl methacrylate Table 1 Compounds 1-26 identified in Ramos A.V.G. et al., 2019

[0039] As known in the art, the plant Vernonanthura nudiflora (V. nudiflora) is a species in the genus Vernonanthura in the family Asteraceae, native to Uruguay, Brazil, and Argentina. It is a 50-80 cm high, flowering sub-shrub. The extract of V. nudiflora is referred to herein interchangeably as “plant Vern” and “Vern plant” extract.

[0040] V. nudiflora biomass as well as biomass of the plants Baccharis trimera / articulata and Plantago major (namely plant-based material) may be obtained by harvesting the plants, checking the quality of the aerial parts of the plants, i.e., parts residing above the soil, including the stems, leaves, petioles, flowers, fruit, and seeds, cleaning and / or washing the plant material to remove loose dirt and contaminants, and drying the clean aerial plant parts. In particular, stems, leaves, petioles and seeds aerial parts of the plants are useful for preparing the extract of the present disclosure. In some embodiments, the extraction process as herein defined comprises drying of the plant (e.g., V. nudiflora) prior to extracting. The drying is preferably carried out indoors at ambient or slightly elevated temperature, i.e., between 15 and 45°C, preferably between 20 and 25°C. The drying is usually carried out until at least 40% weight loss of the original plant material mass. The drying may be continued until between 40 and 60 weight percent weight loss is achieved. Such weight loss is usually achieved after 7-8 days at room temperature and after 3-4 days at a temperature of 45°C. The dried biomass may then be ground using a suitable size-reduction equipment, such as a mill, e.g., dried biomass is size reduced using electric a hammer mill. Dried milled biomass obtained presents a particle size of 5.0-10.0 x 1.0 -2.0 x 0.2-1.0 mm. Dried ground plant biomass (e.g., of V. nudiflora) may then be used in extracting steps of the process.

[0041] Unlike exhaustive extractions wherein the extraction mixture is exposed to high volumes, concomitantly or repeatedly in smaller portions, of extraction solvent, the extraction process according to the present disclosure relies on the same amount of extracting solvent. Therefore, the concentration of the botanical biomass can be controlled as required. Usually, the concentration of the plant biomass (e.g., of V. nudiflora biomass) during the extracting step is between 10 and 30% of weight by volume of the extraction solvent, preferably between 15 and 25% of weight by volume of the extraction solvent. Preferably, the concentration of the biomass is between 18 and 22 % weight by volume, e.g., about 20%. In other words, the extracting may be carried out ina ratio of between 1:2 to 1:8, e.g., between 1:3 and 1:6 between the biomass being extracted and the hydroethanolic mixture, preferably about 1:4.

[0042] After the completion of the extraction step, namely the completion of extracting the biomass into the hydroethanolic mixture, the extract may be separated from the depleted biomass (i.e., residues) to furnish said extract. The separating of the extract may be performed by decantation of the extract from the biomass sediment. Alternatively, or additionally, the spent biomass may be separated by filtration via a suitable filter. The filtering may be performed via a cheesecloth, or may be performed through a suitable filter with defined pore size, e.g., cellulotic 15 µm filter. Alternatively, filtration can be performed by gravity, or using electrical devices with the above pore size. The separating may also comprise, additionally or alternatively, centrifugation of the mixture to effect complete sedimentation of the spent biomass and to enable easy decantation of the extract.

[0043] In some embodiments, the resulting extract as herein defined has a plant-derived solid content (dried plant-derived content after evaporating the solvent, interchangeably referred to herein in terms “dry basis”, “dried extract”, and the like) at a weight of between 0.5% and 15.0%, preferably between 1.0% and 5.0%, or preferably between 1.8% and 4.5%, or between 2.5 and 3.7 %, weight by volume of the extract’s initial volume. Exemplary dry basis values characteristic to the hydroethanolic extract may be any one of the following numbers: 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, or 3.7%, preferably 3.1%, 3.2%, 3.3%, or 3.4%. In some cases, it may be advantageous to dilute the extract, either as a solid or as a dissolved solid, with further solvent or other diluting medium (e.g., similar or identical to the extraction hydroethanolic mixture). In various embodiments, the original extract (i.e., comprising dissolved solid) may be further diluted, e.g., by dilution of 1 part of extract with 9 parts of distilled water or 2 parts of extract with 8 parts of distilled water. Another suitable dilution is 1 part of extract to 8 parts of distilled water.

[0044] It is evident that the process furnishes a liquid hydroethanolic plant extract (e.g., of V. nudiflora). However, the extract may be dried using suitable drying means, e.g., under reduced pressure (for example as exemplified below), to furnish dried hydroethanolic extract. Dried ethanolic extract may further be adsorbed onto a suitableinert carrier, to be used as a powder. Alternatively, or additionally, the liquid hydroethanolic extract may be dried in presence of at least a portion of the inert carrier, to furnish adsorbed dried hydroethanolic extract.

[0045] In a further aspect provided herein a hydroethanolic extract manufactured as described herein and, in particular, above.

[0046] As demonstrated in the examples section herein, the hydroethanolic extract exhibits unexpected properties in anti-cancer cell activities in cells in culture and in intra- tumor injection in mouse xenograft model. Surprisingly, the efficacy of the cancer cell eradication increased with increased dilution of the crude extract. As mentioned above, the plant extracts are complex mixtures comprising materials with a variety of possible pharmacological activities. Therefore, in a further aspect thereof the present disclosure provides an artificial mixture (i.e., a mixture not necessarily present in nature, also referred to herein as a “fraction” of the compounds present in the extract) consisting essentially of the compounds present in the hydroethanolic extract as defined herein and containing less than 5%-20% (e.g., less than 5%) by weight of the compounds listed in Table 1 above (i.e., compounds 1-26 as disclosed in Ramos, A.V.G. et al., 2019), or is essentially devoid of these compounds. In various embodiments the fraction of compounds may be based only on compounds present in Vernonanthura nudiflora or on a combination of Vernonanthura nudiflora and Plantago major. In further embodiments the fraction may be a sub-extraction of the hydroethanolic plant extract, preferably by butanol, hexane, chloroform, or ethyl acetate, or any combination thereof.

[0047] However, the artificial mixture may further comprise compounds of botanical origin, that are not necessarily present exclusively in V. nudiflora extract, provided that they exhibit at least certain anti-proliferative activity, e.g., as demonstrated in the appended examples. Thus, in further embodiments the artificial mixture according to the present disclosure comprises at least two compounds selected from the group consisting of Rutin, Keampferol, Myricetin, Stachydrine chloride, Cyanidin chloride, Glycitein, Diosmetin, 8-hydroxyquinoline, Quercetin-3-beta-glucoside, Phytol, Myristicin, Alpha- lapachone and 1,2:2,4-Di-p-methylbenyliedene.

[0048] As shown by the examples below, the hydroethanolic extracts have demonstrated significant pro-apoptotic activity, inducing apoptosis and mitochondrial dysfunction mediated by VDAC1. Furthermore, the appended examples provide evidence for the beneficial therapeutic effect of the composition comprising the extract as herein defined, when administered in combination with standard of care therapy, or even standalone. Therefore, in a further aspect thereof, the present disclosure provides a method of treating a proliferative disorder, e.g., cancer, or for reducing the prevalence of recurrence or occurrence thereof, or for alleviating the symptoms associated therewith in a patient in need thereof, by administering to said patient a therapeutically effective amount of the composition comprising said hydroethanolic extracts as generally described herein or the artificial mixture as herein described.

[0049] As it is appreciated, by the term “treatment”, “treating” or “treat” it is meant to include ameliorating one or more of the clinical manifestations of disease activity in a subject having a proliferative disease, e.g., cancer, or reducing the prevalence of disease or condition, its recurrence or occurrence, or at least alleviating the symptoms associated therewith. As known in the art, “proliferative diseases” are characterized by excessive proliferation of cells and turnover of cellular matrix, for example, cancer. As used herein to describe the present invention, “cancer” and “tumor” equivalently relate to a malignant proliferative disorder of a tissue or organ. As generally used herein, the cancer is preferably a disease or disorder classified in a subclass 02 [Neoplasms] of the 11threvision of World Health Organization International Classification of Diseases (herein: “ICD - 11”).

[0050] In general, the methods and compositions of the present invention are useful in the treatment of solid and / or non-solid and solid tumors. The efficiency of treating of a disease or a disorder may be assessed as known in the art, e.g., with clinical trials. Preferably, the clinical trials are controlled clinical trials. Depending on the purpose of the clinical study and the objectives thereof, the control group to measure an effect of a clinical trial involving cancer patients may be a group receiving a supportive treatment, or a known standard of care, depending on the severity of the condition. The clinical study is usually a randomized clinical study, wherein the control group receives one of the control treatments, and the treatment groups receive varying doses of the compositionscomprising hydroethanolic extracts as described generally herein. The tested doses’ range may be determined in a previous pilot clinical study, based on a pre-clinical data, and otherwise procured as known in the art. The clinical study may have one or more of primary end points, i.e., measurable outcomes, and may further have one or more of secondary end points, i.e., further measurable outcomes. The clinical study may be considered successful if at least one primary and / or at least one secondary outcomes are reached, that is, the measurable outcomes of a treatment group is statistically significantly different and within the predefined range of difference in comparison to the measurable outcome of a control group. The clinical studies may enroll sufficient number of patients, the number being controlled by the outcomes sought and the anticipated differences, to meet the required statistical power. The enrolled patients must meet the inclusion criteria on the disease or disorder, the condition, and / or the symptoms, and must not have any of the exclusion criteria defined to disallow certain patients that might not benefit from the treatment or may be in danger developing a side effect or having otherwise compromised medical safety as the result of the treatment.

[0051] The present disclosure further provides a composition comprising hydroethanolic extracts as herein defined (e.g., in therapeutically effective amount) for use in a method of treating a proliferative disorder, e.g., cancer, or for reducing the prevalence of recurrence or occurrence of a proliferative disorder in said subject.

[0052] By way of example, the proliferative disorder or cancer as herein defined is a primary or metastatic prostate cancer, Langerhans cell histiocytosis, testicular cancer, colon cancer, or breast cancer.

[0053] The present invention relates to the treatment of subjects, also referred to herein as patients, in need thereof. By “patient” or “subject in need” it is meant any organism who may be affected by the above-mentioned conditions, and to whom the treatment methods herein described are desired, such as mammals, e.g., humans. Diagnosing a disease or condition in a subject to whom the compositions and methods described herein are useful is within the skills of a clinician.

[0054] Administering according to the present disclosure is by a route selected from the group consisting of oral administration, intravenous administration, intramuscular administration, and intraperitoneal administration.

[0055] The term “therapeutically effective amount” is intended to mean that amount of the composition as herein defined or any fraction thereof eliciting the desired biological or medical response which may be determined by such means known in the art, for example, by preclinical and clinical experiments, e.g., such as those described herein. Treating of a patient in need thereof as disclosed herein entails administering to the patient an effective amount of the hydroethanolic extract. Dosages (i.e., therapeutically effective amounts) may vary between 0.008 mL / kg (kg-weight of the patient) and 2 mL / kg of the diluted 1:8 hydroethanolic extracts, i.e., between 0.001 mL / kg and 0.25 mL / kg of concentrated mL, which means between 0.033 mg / kg (of dry mass / kg of patient) and 8.6 mg / kg (of dry mass / kg of patient). Preferably, dosages (i.e., therapeutically effective amounts) may vary between 0.048 mL / kg (kg-weight of the patient) and 1 mL / kg of the diluted 1:8 hydroethanolic extracts, i.e., between 0.006 mL / kg and 0.125 mL / kg of concentrated mL, which means between 0.2 mg / kg (of dry mass / kg of patient) and 4.3 mg / kg (of dry mass / kg of patient). As seen in the appended examples, the amount administered perorally could be between 8 and 80 mL of 1:8 diluted extract daily, and the amounts administered intravenously could be 6 mL undiluted extracts once a week. The dose is preferably expressed based on the dry basis of the extract. Thus, in some embodiments, the dose may vary between 25 and 500 mg per day.

[0056] Specific doses for specific diseases or disorders may be adjusted depending on the severity of the condition, the patient’s factors, comorbidities, and the desired treatment outcome.

[0057] The peroral daily doses as described herein may be administered in a variety of regimens, depending on the patient mental status, general health condition, and desired outcome. The regimens include once daily administration, that is, the whole daily dose is administered at once every day; twice daily administration, that is, the total daily dose is divided into two preferably equal doses and administered in a reasonable interval,preferably every 12 hours; three time a day, that is, the total daily dose is divided into three preferably equal doses and administered in a reasonable interval, preferably every 8 hours; or four times a day, that is, the total daily dose is divided into four preferably equal doses and administered in a reasonable interval, preferably every 6 hours. Administered perorally, the hydroethanolic extracts may be administered with or without food, preferably without food.

[0058] In some embodiments, the methods of treatment or the use of the hydroethanolic extracts are in addition to standard of care therapy, e.g., conventional chemotherapy, in particular with cell proliferation inhibition – chemotherapy or in conjunction with apoptosis-inducing chemotherapy. Since the effect of the hydroethanolic extracts as described herein are mediated directly by VDAC1, it is hypothesized without being bound by a theory, that the disclosed hydroethanolic extracts may be effective also in cancers overexpressing anti-apoptotic intracellular factors.

[0059] In some further embodiments, the hydroethanolic extract may be provided in a composition comprising same. For administration by a parenteral route, the extract may be provided as a liquid for injection. Preferably, e.g., for intravenous administration, the liquid comprising the extract is diluted in a large volume, e.g., of a physiological solution as used in medicine. for this purpose, the hydroethanolic extract (or a dried form thereof) may be combined with inert ingredients, e.g., pharmaceutical excipients. The skilled artisan is referred to the Remington’s Pharmaceutical Sciences, 18thedition, 1990, ISBN 9780912734040, or to a comparable textbook, for the principles of formulation of dosage forms.

[0060] The hydroethanolic extract may be formulated in a liquid dosage form. The liquid dosage form may be the original hydroethanolic extract, in which case it will be in a form or a tincture. The concentration of the dried basis of the tincture may be adjusted by dilution, to enable more accurate dosing. The extract may also be in a form of a diluted extract, with the dilution of more than 1:5. The diluted extract may contain the same solvent as the original hydroethanolic extract, or may have a higher amount of water. Thediluted extract may further comprise preservatives, if the concentration of ethanol in the diluted extract is below 20% by volume.

[0061] The hydroethanolic extract may be further formulated into a medicated syrup, e.g., by mixing the original tincture with a predetermined amount of a syrup. To maintain the hydroethanolic composition, the tincture may be further formulated into an elixir, e.g., by mixing the original tincture with a predetermined amount of a syrup diluted with ethanol.

[0062] The hydroethanolic extract, particularly dried or lyophilized hydroethanolic extract, may be formulated into a capsule, e.g., a gelatin capsule, or hypromellose capsule. the liquid hydroethanolic extract may be adsorbed onto a carrier, and then carefully evaporated till dryness, to furnish a powder suitable for filling a capsule. The dried extract or adsorbed extract may further be compressed into a tablet, as known in the art. The tablets or capsules may be further coated, e.g., with a protective coating, and packaged into a suitable container, such as a bottle or a blister-pack.

[0063] Inactive ingredients, suitable for the dosage forms as described herein, may be found in the Handbook of Pharmaceutical Excipients, 9thedition, 2020, ISBN 9780857113757, or an equivalent text as known to the skilled artisan. ***

[0064] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0065] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. As used herein the term "about" refers to ± 10 %.

[0066] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of".

[0067] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. The following examples are representative of techniques employed by the Inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

[0068] It is appreciated that certain features of the invention, which are, for brevity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, combinations of various features of the invention, which are, for clarity and demonstration, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination, or as suitable in any other described embodiment of the invention.

[0069] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0070] Various other features according to the invention as described herein for the aspect of the methods of treating of conditions are applicable mutatis mutandis to compositions and / or dosage forms for use in these methods according to the teachings herein and vice versa. The terms “composition” and the like as used herein interchangeably to hydroethanolic plant extracts as described herein, and to compositions comprising at least a fraction of the hydroethanolic extracts, e.g., compositions ready for administering to a patient in need thereof, or to the dosage forms as described herein.EXAMPLES

[0071] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion. Cell lines and culture

[0072] U-87MG (human glioblastoma) and SH-SY5Y (human neuroblastoma) were maintained at 37°C and 5% CO2 in the recommended culture medium supplemented with 10% FBS, 1 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin. Table 2. Antibodies used in the experiments Antibody Source and Cat. No. WB IF Rabbit polyclonal anti-VDAC1 Abcam, Cambridge, UK, ab158951:5000 1:500Rabbit monoclonal anti-HK1 Abcam, Cambridge, UK, ab150423- 1:750Rabbit monoclonal anti-Lactate dehydrogenase Abcam, Cambridge, UK, ab52488- 1:750Rabbit monoclonal anti-Nestin Abcam, Cambridge, UK, ab105389- 1:750Rabbit monoclonal anti-Glut-1 Abcam, Cambridge, UK, ab115730- 1:750Mouse monoclonal anti-SOX2 Abcam, Cambridge, UK, ab171380- 1:200Anti- ^-SMA Abcam, Cambridge, UK, ab5694- 1:750Anti-CD31 Abcam, Cambridge, UK, ab28364- 1:750Mouse monoclonal anti-GAPDH Abcam, Cambridge, UK, ab9484- 1:750Mouse monoclonal anti-Ki-67 Biolegend, San Diego, California, - 1:750 US, 350502 Donkey anti-mouse-Alexa fluor 488 Abcam, Cambridge, UK, ab150109- 1:750Goat anti-rabbit IgG- Alexa fluor 555 Abcam, Cambridge, UK, ab150086- 1:850Goat anti-rabbit Alexa fluor 488 Abcam, Cambridge, UK, -ab150078- 1:750Goat anti-mouse Alexa fluor 555 Abcam, Cambridge, UK, ab150114- 1:750Goat anti-rabbit HRP Promega, Wisconsin W40181:15,000 -Donkey anti-mouse HRP Abcam, Cambridge, UK, ab987991:10,000 -Mouse monoclonal anti- ^-actin Millipore, Billerica, MA, MAB15011:40,000 -Materials

[0073] 4’,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), propidium iodide (PI), Tris, and trypan blue were purchased from Sigma (St. Louis, MO). The cell transfection agent JetPRIME was from PolyPlus (Illkirch, France). Annexin-V (FITC) was obtained from Alexis Biochemicals (Lausen, Switzerland). Dulbecco’s modified Eagle’s medium (DMEM), BSA 7.5%, MEM non-essential amino acids (NEAA), phosphate-buffered saline (PBS), and Roswell Park Memorial Institute medium (RPMI)were purchased from Gibco (Grand Island, NY). Fluo-4-AM, and Mito-SOX-Red were acquired from Invitrogen (Waltham, MA). TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) kit was obtained from Promega (Madison, WI). XTT cell viability assay kits were obtained from Biological Industries (Beit Haemek, Israel). Table 2 above lists the primary and secondary antibodies used against the indicated proteins, their catalogue number, source, and the dilutions used in immunoblotting (WB), and immunofluorescence (IF) experiments. TLC silica gel 60 F254 plates were obtained from Merck (Darmstadt, Germany). Lyophilization of the aqueous-ethanolic plant extract

[0074] A dry product of the aqueous-ethanolic plant extract produced as detailed above was obtained by lyophilization as follows. A sample of plant extract (1 liter) in a water- ethanol phase was first subjected to removal of the ethanolic phase from the mother liquor. This step was performed by applying a vacuum of 750 mbar at 45°C over a period of 80 hours. Upon completion of this drying step, the volume of the sample was reduced to 450 mL, therefore apparently all ethanol initially present was completely eliminated. A starch-based carrier (N-Zorbit 2144, Ingredion) was then added for homogenization of the final product by stirring for three (3) minutes, at a ratio of 1 gr modified starch for every 5 mL of the original extract (i.e., 200 g per liter of the original extract prior vacuum drying). The sample was then frozen at -80°C for 72 hours (by introducing the sample in an ultra-freezer that reaches the mentioned temperature)l. Once the sample was frozen, it was lyophilized at -50°C and 1 MPa vacuum, for 96 hours. A homogeneous and dry solid was obtained (234 gr). Once collected, the sample was stored at room temperature, preserving it from light. Cell treatment with plant extracts and cell death analysis

[0075] Cells (6×105 / ml at 70–80% confluence) were incubated with ethanol extracts of plant A (V. nudiflora), B (B. trimera), or C (P. major), or their mixture, prepared as described above, at the indicated dilution, in culture plates containing 500 µl culture medium (each) for 24 hours or the indicated time, at 37oC and 5% CO2. The cells were then trypsinized, centrifuged (1500×g, 5 minutes), washed with PBS and analyzed for the desired activity. For cell death analysis, propidium iodide (PI) staining was performed by addition of PI (6.25 µg / ml) to the cells, followed by immediate analysis using the EC800Flow cytometer Analyzer–Eclipse or the fluorescence-activated cell sorter (FACS) caliber (Beck-ton-Dickinson, San Jose, CA) and BD CellQuest Pro software.

[0076] For apoptosis analysis by PI and annexin V-FITC staining, cells (2×105), untreated or treated with the plant extracts, were collected (by centrifugation at 1,500×g for 5 min), washed and re-suspended in binding buffer (200 µl of 10 mM HEPES-NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Annexin V–FITC staining was performed according to the recommended protocol. Cells were then washed once with binding buffer and re-suspended in binding buffer (200 µl), to which PI was added immediately before flow cytometric analysis using the EC800 Flow cytometer Analyzer–Eclipse. At least 10,000 events were collected and recorded on a dot plot. Cell viability assay

[0077] The effect of the various plant extracts on SH-SY5Y cell survival was assayed using an XTT-based kit (Biological Industries, Beit Haemek, Israel) according to the manufacturer’s protocol. Briefly, cells were seeded in a 96-well plate and incubated at 37°C with 5% CO2, and 24 hours later were treated with different concentrations of the extracts for the time indicated in the legends to the Figures. XTT reagent (2,3-bis-(2- methoxy-4-nitro-5sulphenyl)-(2H)-tetrazolium-5-carboxanilide) was then added to each well, and the absorbance was measured at 450 nm and 630 nm (Tecan, Infinite M1000, Mannedorf, Switzerland). The absorbance obtained at 630 nm was subtracted from the absorbance at 450 nm to obtain the specific reduced XTT reaction product. Determination of reactive oxygen species, and intracellular Ca2+levels

[0078] For measuring mitochondrial accumulated reactive oxygen species (ROS), SH- SY5Y cells were seeded in a 6-well plate (1×105 / well). Cells were treated for 24 hours with the indicated plant extract, and then were incubated with MitoSOX-Red (a mitochondrial superoxide indicator for live-cell imaging) for 10 minutes at 37°C. Fluorescence was measured using flow cytometry (iCyt, Sony Biotechnology, San Jose, CA). At least 10,000 events were recorded on the FL2 detector, represented as a histogram, and analyzed with ec800 software (Sony Bio-technology, San Jose, CA). Positive cells showed a shift to an enhanced level of green fluorescence (FL2).

[0079] Cytosolic Ca2+levels [Ca2+]i were analyzed using Fluo-4-AM (Invitrogene, Grand Island, NY) as follows. Cells (1x106cells / ml) were harvested after the appropriate treatment, collected by centrifugation (1,500xg for 10 minutes) and washed with HBSS buffer (5.33 mM KCl, 0.44 mM KH2PO4, 138 mM NaCl, 4 mM NaHCO3, 0.3 mM Na2HPO4, 5.6 mM glucose, 0.03 mM phenol red), supplemented with 1.8 mM CaCl2(HBSS+) and incubated with 2 ^M Fluo-4 in 200 ^l HBSS(+) buffer in the dark for 30 minutes at 37°C. After washing the remaining dye, the cells were incubated with 200 μl HBSS(+) buffer, and changes in [Ca2+]i were measured immediately by FACS and analyzed with an EC800 Flow cytometer Analyzer–Eclipse (Sony Biotechnology). Positive cells showed a shift to an enhanced level of green fluorescence (FL1).

[0080] Changes in cellular Ca2+were monitored in live cells using the high content Operetta screening system (Perkin-Elmer, Hamburg, Germany). In each well, ten fields were imaged using a 20×wide field objective with an excitation filter of 520–550 nm and emission filter of 560–630 nm. Cross-linking experiments

[0081] In order to assess the amount of VDAC1 protein dimers formed, cells were treated with the plant extract for the indicated time and concentration, harvested, washed with PBS, pH 8.3, and incubated for 15 minutes with the cross-linking reagent EGS (ethylene glycol bis(succinimidyl succinate), (Pierce chemicals TX, USA ) at a ratio of 3 mg protein / ml / 300 ^M EGS. Next, protein aliquots (30 µg) were subjected to sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using anti- VDAC1 antibodies. Quantitative analysis of VDAC1 dimers was performed using FUSION-FX (Vilber Lourmat, France). Gel electrophoresis and immunoblotting

[0082] Cells or tumor tissues were lysed using lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, supplemented with a protease inhibitor cocktail (Calbiochem, UK)). The lysates were then centrifuged at 12000xg (10 minutes at 4°C) and protein concentration was determined. Aliquots (10– 20 ^g of protein) were subjected to SDS-PAGE and immunoblotting using various primary antibodies (sources and dilutions are provided in Table 2 above), followed byincubation with appropriate HRP-conjugated secondary antibodies (i.e., anti-mouse, anti- rabbit, or anti-goat). Blots were developed using enhanced chemiluminescence (Biological Industries). Band intensities were analyzed by densitometry using FUSION- FX (Vilber Lourmat, France) software, and values were normalized to the intensities of the appropriate ^−actin signal that served as a loading control. Gas chromatography-mass spectroscopy (GC-MS) analysis

[0083] CG-MS analysis of plant extracts A, B and C was carried out using a 7890B Mass- Detector; 5977A, Agilent Technologies; Column 5MS UI. The compounds were identified using Library Name W 10N 14L (NIST MS Search 2.2). The various names representing each compound, quality of identification (maximum is 100%), and peak area (Ab*s) are given in Table 4 below. Liquid chromatography-mass spectroscopy (LC-MS / MS) analysis

[0084] The plant extract compounds were identified by an ultra-high pressure liquid chromatography (LC) system (ACQUITY UPLC® I Class Waters) coupled to a Q Exactive mass spectrometer equipped with an electrospray ionization (ESI) source in positive and negative mode.

[0085] The Q Exactive is an instrument that has an API source with S-lens ion optics technology, a quadrupole mass filter for precursor ion selection, and a collision cell for performing MS / MS experiments. It is equipped with an Orbitrap analyzer.

[0086] The chromatographic separation of the compounds was carried out using an ACQUITY UPLC®HSS T3 Waters Column (2.1x100mm., particle size 1.8^m μm) (Waters). The column temperature was set at 40°C, and the injection volume of the samples was 5 ^l. The mobile phase consisted of 0.1% formic acid in water (eluent A) and acetonitrile in 0.1% formic acid (eluent B) at a flow rate of 0.40 mL / min. The elution conditions were as follows: 0–1 min, 1% B; 1–11 min, 1–40% B; 11–13 min, 40–70% B; 13–15 min, 70–99% B; 15-16, 99% B, 17–20 min, equilibration to 1% B.

[0087] The ESI source was operated in the positive and negative ion mode with the parameters as follows: the capillary voltage was 3.5 kV, sheath drying and auxiliary gas(N2) flow rates were 60 and 20 L / min, respectively, the capillary temperature was 275°C, and the drying gas temperature was 300°C.

[0088] The collision-induced dissociation was ultra-high purity grade (99.999%) nitrogen with a flow rate of 0.5ml / min.

[0089] The main compounds of the plant extracts were identified by comparing their retention time (RT) and mass spectrometry (Q Exactive; Thermo Scientific) fragmentation patterns with 25 nce (collision energy normalized to m / z 500). The mass spectrometer was operated in positive mode and negative mode, and the fragmentation patterns of the compounds were obtained. The precursor ions were [M+H]+for the positive and [M−H]−for negative modes.

[0090] The data were processed by the related X-Calibur and Compound Discoverer programs to investigate the matching compounds. The MS data, MS / MS fragmentation profiles, and molecular formula were proposed by the predicted compositions*, M / Z Cloud and ChemSpider, and were compared with the literature data and some databases to annotate the compounds in the extracts. The molecular formula proposed by the predicted compositions in the MS experiments was compared with the literature data, and a maximum error of 2 ppm was accepted. Thin Layer Chromatography (TLC) separation

[0091] TLC silica gel 60 F254 plates (Merck, 20x20 cm) were used to separate the A, B and C hydroethanolic plant extracts as well as purified phytol and ethyl linoleate, using a mobile phase mixture of petroleum ether:diethyl ether: acetic acid: (85:15: 1,V / V / V). The plates were air dried and visualized by exposure to iodine vapor. Xenograft mouse model

[0092] U-87MG cells (3×106cells / mouse) were inoculated subcutaneously (s.c.) at day zero (0D) into the hind leg flanks of athymic eight-week-old male nude mice (Envigo). Tumor size was measured daily using a digital caliper, and volume was calculated. When it reached 50 mm3(at day 14, “14D”), mice were randomly divided into several groups (5 mice / group). One group was intra-tumorally injected with HBSS (5.33 mM KCl, 0.44mM KH2PO4, 138 mM NaCl, 4 mM NaHCO3, 0.3 mM Na2HPO4, and 5.6 mM glucose, pH 7.3) containing 0.14% ethanol (control, un-treated), and other groups were treated with V. nudiflora extract to a final dilution of 1:100, 1:250, 1:300, or 1:500 or with phytol to a final concentration of 75 ^M. The xenografts were injected three times a week. The mice were sacrificed 34 days post-cell inoculation, and tumors were excised. The experimental design is schematically presented in Fig. 6A. Tumors were fixed in 4% buffered formaldehyde, paraffin-embedded, and processed for immunofluorescence (IF). These experimental protocols were approved by the Institutional Animal Care and Use Committee of the Ben-Gurion University. Immunofluorescence (IF) of tumor tissue sections

[0093] Formalin-fixed, paraffin-embedded sections (5 μm thick) of U-87MG cell-derived tumors from control and from V. nudiflora extract- or phytol-treated tumors were de- paraffinized by placing the slides at 60°C for 1 hour and using xylene, followed by rehydration with a graded ethanol series (100%–50%). Antigen retrieval was performed in 0.01M citrate buffer (pH 6.0) at 95°C–98°C for 20 min. After washing sections in PBS, pH 7.4, sections were incubated in 10% normal goat serum for 2 hours, followed by overnight incubation at 4°C with primary antibodies (as detailed in Table 2). Sections were washed thoroughly with PBST, incubated with the fluorescently-labeled secondary antibodies (as detailed in Table 2) for 2 hours, washed five times with PBST, and cover slipped with fluoroshield mounting medium (Immunobioscience, Mukilteo, WA). Fluorescent images were viewed with an Olympus IX81 confocal microscope. Quantitation of protein levels, as reflected in the staining intensity, was analyzed in the whole area of the sections using Image J™ software. TUNEL assay

[0094] Paraffin-embedded-fixed tumor sections (5 μm thick) were processed for a Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling TUNEL assay using the Dead End Fluorometric TUNEL system according to the manufacturer’s instructions. Sections were de-paraffinized, equilibrated in PBS, permeabilized with proteinase K (20 μg / ml in PBS), post-fixed in 4% paraformaldehyde, and incubated in TdT reaction mixture for 1 hour at 37°C, in the dark. Slides were then washed in saline- sodium citrate buffer, counter-stained with PI (1 µg / ml), and cover slipped withfluoroshield mounting medium (Immunobioscience, Mukilteo, WA). Fluorescent images of apoptotic cells (green) and cell nuclei (red) were captured using a confocal microscope (Olympus 1X81). Quantification analysis of stained slides was done using an Image J program. Statistics and data analysis

[0095] Means ± SE of results obtained from three independent experiments are presented. Statistical significance is reported at p < 0.05(*), p < 0.01(**), p < 0.001(***), or p < 0.0001(****). EXAMPLE 1 Preparing extracts of Vernonanthura nudiflora, Baccharis trimera and Plantago major

[0096] The following extracting procedure was performed for each one of the indicated plants, separately. Wild-growing plants of each one of the plants Vernonanthura nudiflora, Baccharis trimera and Plantago major species were harvested and selected based on the overall conditions of the botanical material (also referred to herein as “biomass”), e.g., absence of parasites, absence of agrochemicals in the vicinity, etc. Next, botanical material of each one of the plants was carefully cleaned prior to extraction, leaving only the aerial parts (namely the plant parts residing above the soil, including the stems, leaves and petioles). The botanical material was washed with copious amount of water, and then was air-dried indoors, until a 50% reduction in its total mass was obtained, for about 7-8 days at room temperature or for about 3-4 days in a heated chamber (at 45°C). The dried botanical material was then ground by using a mechanical mill to the final size of about 5.0-10.0 mm x 1.0-2.0 mm x 0.2-1.0 mm. The ground botanical biomass was then extracted during a period of 21 days in a reactor containing hydroalcoholic solution (at 70% ethanol in water solution, interchangeably termed herein a hydroethanolic solution), where the concentration of the botanical material in the hydroalcoholic solution was 0.2 g / ml. The solution was periodically agitated for about 1- 2 hours, every 24-48 hours. The obtained extract was then filtered by gravity, through a filter with a pore size of 15 µm, and packaged in opaque and hermetically sealed containers, which were stored at room temperature in a cool and ventilated place, protected from exposure to light. To produce a mixture of three extracts, plant parts ofthe plant species Vernonanthura nudiflora, Baccharis trimera, and Plantago major were extracted individually, and the mixture thereof was prepared such that a mixing ratio of 40% Vernonanthura nudiflora, 40% Baccharis trimera, and 20% Plantago major was obtained. The obtained extracts were stored at 4oC and was active over a year. EXAMPLE 2 Apoptosis induction by hydroethanolic plant extracts

[0097] In order to investigate the effects of hydroethanolic extracts of plants from the Vernonanthura nudiflora, the Baccharis trimera and the Plantago major and their mixture on cancer cell survival and apoptotic cell death, cells of the cancer cell line SHSY5Y were incubated, as detailed above, in the presence of various dilutions of the extracts from Vernonanthura nudiflora, the Baccharis trimera and the Plantago major or their 2:2:1 (V:V;V) mixture.

[0098] First, the activity of the three plant hydroethanolic extracts and their mixture in cell death induction as a function of their amount was analyzed by propidium iodine (PI) staining and flow cytometer analysis. PI is a fluorescent intercalating agent used to stain nucleic acids by binding to DNA and intercalating between the bases with little or no sequence preference and is not membrane-permeable, rendering it useful to differentiate between apoptotic and intact healthy cells based on membrane integrity. Fig. 1A1 and Fig.1A2 shows a representative analysis of cell death induced by control (Fig. 1A1) and plant Vern extract (Fig.1A2).

[0099] The results presented in Fig.1B clearly show that V. nudiflora (Vern plant) extract was the most active in triggering massive cell death, followed by the P. major (Pla plant) extract, with the B. trimera (Bac plant) extract being the least active. It is noteworthy that the extract Vern, at a dilution of 1:500 induced higher cell death than the mixed extracts at 1:100 dilution as shown in Fig. 1B, or the plant Bac extract at a dilution of 1:166, suggesting it is 5- and 3-fold more active, respectively.

[0100] In order to determine whether the induced cell death relates to apoptosis, apoptosis was analyzed by Annexin V / PI and FACS analysis. Annexin V-FITC has a high affinity for the anionic phospholipid phosphatidylserine, which is located on theextracellular side of the plasma membrane in apoptotic cells. The results presented in Fig. 1C clearly show that the Vern extract induced apoptotic cell death.

[0101] Next, the effects of the plant extracts on cell viability was analyzed using an XTT assay, as detailed above. As shown in Fig.1D–F, for experiments conducted with Vern plant, Bac and Pla extracts, respectively. Vern extract highly reduced cell viability following 24, 48, or 72 hours of incubation in a dilution-dependent manner (Fig. 1D), while the Bac extract at a dilution of 1:500 showed some decreased cell viability following incubation of 48 and 72 hours (Fig. 1E) and the Pla plant extract showed no decrease in cell survival at all dilutions tested and incubation times (Fig.1F). Considering the fact that the XTT assay relies on reduction by the NADH produced in the mitochondria, these results suggest that Vern extract, but not the Bac or Pla plant extracts, induced mitochondrial dysfunction. In addition, without wishing to be bound by theory, the results suggest that the cell death induction by the three plant extracts involves different active compounds and modes of action.

[0102] Furthermore, as shown in Table 3 below, Vernonanthura nudiflora plant extract induction of cell death activity remained stable even upon heating of the extract. This was demonstrated by incubating Vernonanthura nudiflora plant extract at dilutions of 1:500, 1:1000 and 1:2000 for 10 minutes at 4°C, 45°C, 60°C and 80°C and then assaying (as described above) the ability of the extract to induce death of SH-SY5Y cells upon incubation (24 hours) of the cells in the presence of the dilutions of the heated extracts. Table 3 Vern plant extract cell death activity is stable after heating the extract Temp,oC Cell death, % V. nudiflora extract 1:2000 1:1000 1:500 dilution 4 37 83 98 45 - 84 98 60 40 91 98 80 57 92 99EXAMPLE 3 The hydroethanolic plant extracts induced VDAC1 overexpression and its oligomerization

[0103] It has been previously shown that apoptosis triggers, such as chemotherapeutic drugs, stress conditions and radiation, induce VDAC1 overexpression, whereby the equilibrium is shifted from monomeric towards VDAC1 oligomers, followed by the release from the mitochondria of pro-apoptotic proteins and apoptosis. Thus, the effects of the plant extracts on VDAC1 expression levels and its oligomerization were next tested in SHSY5Y and U-87MG cells, which were incubated for 24 hours with the plant extracts at various dilutions, and then analyzed for VDAC1 expression levels using immunoblotting, as detailed above.

[0104] Fig. 2A demonstrates that Vern plant extract induces VDAC1 overexpression in the two different cell lines tested, namely the neuroblastoma-derived cell line SHSY5Y and glioblastoma-derived U-87MG cell line. In both cell lines, the Vern extract highly increased the level of expression of VDAC1, up to 3-fold (Fig.2A), which was accompanied by pro-apoptotic activity as shown in Fig.2B, showing the level of cell death as a function of Vern extract dilution (IC50 = 1:800). Furthermore, all three extracts increased, in a concentration-dependent manner, VDAC1 oligomeric forms that were stabilized by chemical cross-linking using EGS and monitored by immunoblotting, as detailed above (Fig. 2C). It is clear that the highest VDAC1 oligomerization levels were induced by the Vern plant extract (Fig. 2C, Fig. 2D), in agreement with its highest cell death-inducing activity.

[0105] Interestingly, the presence of VDAC1 oligomers was also observed without chemical cross-linking, and even after exposing the cells to a high detergent concentration (i.e., 1% SDS) and heating at 70oC for 5 minutes (results are shown in Fig. 2E, Fig. 2F). In this case as well, the level of the oligomeric VDAC1 was highest when the cells were treated with the Vern plant extract, as found for VDAC1 overexpression, oligomerization, and apoptosis induction. This suggests that the VDAC1 oligomers induced by the plant extract are very stable.

[0106] Without being bound by theory, the above results suggest that the active compounds in the Vern plant extract act via enhancing VDAC1 expression levels leading to VDAC1 oligomerization and apoptosis. EXAMPLE 4 Vernonanthura nudiflora extract increased intracellular Ca2+and reactive oxygen species (ROS) production

[0107] Reactive oxygen species (ROS) are known to be associated with induction of apoptosis. Thus, mitochondrial ROS was next measured upon treatment with Vern plant extract, as detailed above.

[0108] As shown in Fig.3A, treatment of cells with the Vern plant extract induced production of ROS. It has been previously shown that an increase in the level of calcium ions (Ca2+) is involved in apoptosis induction and that Ca2+is required for apoptosis- stimuli-induced VDAC1 overexpression and VDAC1 oligomerization. Therefore, the effect of Vern plant extract on cellular Ca2+levels ([Ca2+]) was analyzed using Flu-4 and FACS or by Operetta as detailed above (Fig.3B–Fig 3D). Both assays demonstrated that this extract highly increased cellular [Ca2+] levels. EXAMPLE 5 Analysis of phytol and ethyl linoleate identified in the plant extracts and their effects on cell death and on VDAC1 expression and oligomerization

[0109] In order to identify at least some of the chemical compounds present in the hydroethanolic extracts, they were subjected to gas chromatography–mass spectroscopy (GC-MS) analysis, as detailed above. A plurality of compounds have been identified in these extracts as listed in Table 4. The cell death induction activity of two of abundant compounds identified in the extracts, namely phytol and ethyl linoleate (linoleic acid ethyl ester), was tested. First, the relative amounts of phytol and ethyl linoleate in the plant extracts were determined using known amounts of the two compounds and TLC as detailed above (Fig. 4A). The amounts of phytol in the three plant extracts were about 1800, 800, and 1200 nmol / ml (or ^M) in extract of Vern (Vernonanthura nudiflora), Bac(Baccharis trimera) and Pla (Plantago major), respectively (Fig. 4B, also showing the concentrations of ethyl linoleate in these extracts). Vernonantura plant extract Baccharis plant extract Plantago plant extract 1) Hexadecanoic acid ethyl ester 1) Hexadecanoic acid, ethyl ester 1) Hexadecanoic acid, ethyl ester (Peak 1) Area (Ab*s)=33,110,310 (Peak 26) Area (Ab*s)= (Peak 7) Area (Ab*s)=Quality=9910,220,250 50,748,144 Quality=99 Quality=99 2) Phytol (Peak 2) Area 2) Phytol (Peak 29) Area 2) Phytol (Peak 8) Area (Ab*s)= (Ab*s)=13,850,839 Quality=99 (Ab*s)=3,989,191 Quality=93 18,787,619 Quality=96 3) Linoleic acid ethyl ester (Peak 3) Linoleic acid ethyl ester 9,12- 3) Linoleic acid ethyl ester (Peak 3) |ETHYL (9z,12z)- 9,12- Other names: Octadecadienoic 11) Area (Ab*s)= 30,507,168 Octadecadienoate acid, ethyl ester (Peak 30) Area Quality=99 (Ab*s)=20,555,645 Quality=99 (Ab*s)= 5,514,452 Quality=99 4) 9,12,15-Octadecatrienoic acid 4) 9,12,15-Octadecatrienoic acid, 4) 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- (Peak 4) ethyl ester, (Z,Z,Z)- ethyl ester, (Z,Z,Z)- (Peak 12) (Ab*s)=23,074,287 Quality=99 (Peak 31) Area (Ab*s)= 5,710,334 Area (Ab*s)= 63,412,281 Quality=99 Quality=99 5) Octadecanoic acid ethyl ester 5) Benzene, 1-methoxy-2- 5) Octadecanoic acid, ethyl ester (Peak 5) (Ab*s)=3,107,973 (1-methylethenyl)- (Peak 13) Area (Ab*s)= Quality=99 4,630,724,Quality=99 6) 1,2- Benzenedicarboxylic acid, 6) 4H -Pyran-4-one, 2,3-dihydro- 6) 4H-Pyran-4-one, 2,3- bit (2-ethylhexyl) ester (Peak 6) 3,5-dihydroxy-6-methyl- (Peak dihydro-3,5-dihydroxy-6- 3) ) Area (Ab*s)= 24,981,669 methyl- (Peak 2) ) Area Quality=96 (Ab*s)= 27,382,476 Quality=95 7) 2,5 Dimethyldiphenylsulfone 7) 2-Methoxy- 4-vinylphenol 7) 9,12,15-Octadecatrienoic (Peak 10) acid, (Z,Z,Z)- (Peak 10) Area Area (Ab*s)= 7,305,730 (Ab*s)= 47,918,691 Quality=97 Quality=99 8) Stigmasta-5,22-dien-3-ol (Peak 8) 4-Methyleneisophorone 8) 2-Methoxy-4- vinylphenol 11) Area (Ab*s)= 11,567,27423. (peak 6) Area (Ab*s)= (Peak 3) ) Area (Ab*s)= 21,458,921 Quality=97 3,985,200 Quality=94 9)OLEAN-12-EN-3-OL(Peak 8) 9)1H-Cycloprop[e]azulen-7-ol, 9) 9,12-Octadecadienoic acid (Ab*s)= 143,738,307 decahydro-1,1,7-trimethyl-4- (Z,Z)- (Peak 9) Area Quality=93 methylene-, [1ar (1a.alpha., (Ab*s)=8,205,041 4a.alpha.,7.beta.,7a.beta.,7b.alp Quality=99 ha.)]-(Peak 18) Area (Ab*s)= 13,447,105 Quality=99 10) URS-20(30) –EN- 3-OL 10)1,1,4,7 tetramethyldecahydro- 10) n-Hexadecanoic acid 1h-cyclopropa[e]azulen-4-OL, (Peak 6) Area (Ab*s) = (Peak 19) Area (Ab*s)= 25,158,853 Quality=93 60,683,772 Quality=97 11) (3.beta.21.beta.) –A’- 11) Ledol 11) Hexadecanoic acid, 2- neogammacer-22(29)- en-3-ol hydroxy-1 –(hydroxymethyl) (Peak 10) Area (Ab*s)= ethyl ester 59,020,551 Quality=93Vernonantura plant extract Baccharis plant extract Plantago plant extract 12) Pentacyclic Triterpene – 12) 4aH-Cycloprop[e]azulen-4a- 12)1,3,12- Nonadecatriene Alcohol (Peak 11) Area (Ab*s)= ol, decahydro-1,1,4,7-tetramethyl-, 22,398,502 Quality=93 [1aR (1a.alpha., 4.beta.,4a.beta., 7.alpha.,7a.beta.,7b.alpha.)]- (Peak 17) Area (Ab*s)= 30,609,427, Quality=99 13) 2(10)-Pinene (Peak 2) ) 13) gamma. – Sitosterol (Peak Area (Ab*s)= 5,807,189 22) Area (Ab*s)= 35,635,132 Quality=95 14) Trans-Sinapyl alcohol (Peak 27) Area (Ab*s)= 5,523,775 Quality=96 15) 2-Naphthalenemethanol, decahydro-.alpha,.alpha.,4a- trimethyl-8-methylene-, [2R- (2.alpha.,4a.alpha.,8a.beta.)]- (Peak 23) Area (Ab*s)= 26,189,433 Quality=97 16) 4-oxo-adamantane-1- carboxylic acid methyl ester Pyrene, 1,2,3,6,7,8-hexahydro- (Peak 28) Area (Ab*s)= 28,617,055 Quality=90 17) 2-Hydroxy-3,5,5-trimethyl- cyclohex-2-enone, (peak 4) Area (Ab*s)= 10,619,964 Quality=96 18) 3-Cyclohexen-1-ol,5- methylene-6-(1-methylethenyl)- (peak 5) ) Area (Ab*s)= 34,153,434 Quality=91 19) (4H)1-Benzopyran-4-one, 3,5,6,7-tetrahydroxy-8-methyl- 2-phenyl- 20) Valerena-4,7(11)-diene (peak 15 Area (Ab*s)= 6,007,4363 Quality=96 Table 4. The compounds identified in plant extracts – Vern, Bac, Pla by GC-MS

[0110] CG-MS analysis was carried out using: a 7890B Mass-Detector; 5977A, Agilent Technologies; Column 5MS UI. The compounds were identified using Library Name W 10N 14L (NIST MS Search 2.2). The various names presenting each compound, the quality of identification (maximum is 100%), and the peak area (Ab*s) are given.

[0111] Next, the activity of phytol and ethyl linoleate in inducing cell death was analyzed by incubating SHSY5Y cells with different concentrations (50–200 ^M) of each compound for 24 or 48 hours. As shown in Fig. 4C, phytol induced cell death, with maximal cell death of 100% and with half maximal cell death (IC50) obtained at 70 ^M. Ethyl linoleate showed a weak cell death activity, increasing from about 15% in non- treated cells to about 40% at 200 ^M of ethyl linoleate (Fig.4C).

[0112] Next, VDAC1 expression levels and oligomerization thereof was tested in cells incubated with phytol or ethyl linoleate. As shown in Fig.5A1, phytol but not ethyl linoleate (Fig. 5A2), in particular at the high concentrations used, induced VDAC1 overexpression (Fig.5B) and VDAC1 oligomerization (Fig. 5C, Fig.5D). As graphically shown in Fig. 5B and Fig. 5D, phytol induced VDAC1 overexpression and oligomerization in a concentration-dependent manner, and in correlation with the higher activity shown therefore in cell death induction.

[0113] Notably, Vern plant extract induced cell death at a dilution of 500–1,000, with phytol concentration in these dilutions being is in the range of 2.5 to 5 ^M. When used per se, phytol demonstrated cell death at a concentration of over 50 ^M, suggesting, without wishing to be bound by theory that extract components other than phytol are involved in cell death induction. Without being bound by a specific theory it is therefore believed that neither phytol nor ethyl linoleate are responsible for the activity seen with the extract A of V. nudiflora. EXAMPLE 6 Anti-tumor activity of Vern plant extract and phytol in a murine xenograft model

[0114] Next, the effect of Vern plant extract at two dilutions as well as the effect of phytol were tested on tumor growth, in a xenograft GBM mouse model as detailed above and as schematically presented in Fig. 6A. Briefly, U-87MG malignant glioma cells were inoculated subcutaneously (s.c.) into the hind leg flanks of 7-week-old male athymic nude mice. When the tumor volume was around 50 mm3, the mice were divided into four groups, with a similar average volume, and treated with Vern plant extract (at two different final concentrations, of 1:500 and 1:250) or phytol (at a concentration of 75 ^M). Control tumors were injected with PBS containing 5% ethanol (final concentrationin the tumor 0.14%). Treatment was given three times a week, and tumor growth was monitored three times a week. All mice were sacrificed 34 days post-cell inoculation, tumors were excised, weighed, and fixed, and sections were immunofluorescent stained for selected proteins.

[0115] The results shown in Fig. 6B demonstrate that the tumors in the control grew exponentially with time, and in a similar way when the tumors were injected with Vern plant extract to a final diluted of 1:250. However, tumors treated with a higher dilution of Vern plant extract, namely of 1:500, showed about a 70% decrease in tumor volume and weight, as shown in Fig. 6B – Fig. 6D. The results indicate that Vern plant extract at higher concentration (1:250) is less effective than at the dilution of 1:500. Similar results with substantially no effect on tumor growth were obtained using 1:100 and 1:300 dilutions of Vern plant extract, as shown in Fig. 7A and Fig. 7B presenting a quantification thereof. The decreased anti-cancer effect with increased Vern plant extract concentration may result, without wishing to be bound by theory, from a protective activity of other compounds present in the extract, when present at higher concentrations at the low extract dilution.

[0116] The results also show that phytol at the concentration used (75 ^M) significantly inhibited tumor growth, yet to a lesser extent than by Vern plant extract at 1:500 dilution (Fig. 6B – Fig.6D).

[0117] Next, tumor-fixed paraffin-embedded sections were stained for Ki-67, a proliferation marker, showing that both Vern plant extract and phytol inhibited cell proliferation by about 80% (Fig. 6F). Representative confocal microscopy images are presented in Fig. 6E for the control (left panels), Vern plant extract (central panels) and for phytol-treated cells (right panels). Finally, apoptosis was analyzed by TUNEL staining as detailed above. As shown in the representative micro-images in Fig. 6G and in the quantification presented in Fig.6H, while no TUNEL-positive cells were apparent in control tumors, most of the cells were TUNEL-positive in the Vern plant extract-treated tumors and to a lesser extent in phytol-treated tumors, with staining co-localizing with PInuclear staining (Fig.6G, white arrows). Thus, Vern plant extract was more effective than phytol in cell death induction.

[0118] The results clearly indicate that the treatments induced apoptotic cell death and suggest that the marked decrease in tumor size in the Vern plant extract- and phytol- treated xenografts can be attributed to both inhibition of cell proliferation (decreased Ki- 67 staining) and cell death induction.

[0119] It has been previously shown that metabolic alterations occurring during malignant transformation involve a spectrum of functional aberrations and mutations that contribute to elevated glycolysis and increased expression levels of glucose transporters (Glut-1) and glycolytic enzymes, such as hexokinase (HK-I). The expression levels of these proteins and other proteins associated with metabolism, microenvironment, and cancer stem cells was evaluated by immunofluorescent (IF) staining. As shown in Figure 8A, IF of the control tumors derived from U-87MG cells showed high expression levels of Glut-1 and glyceraldehyde 3-phodphate dehydrogenase (GAPDH). Surprisingly, the expression levels of these proteins were decreased in animals bearing tumors treated with Vern plant extract or phytol, as shown in Fig.8A and in Fig.8B showing a quantification thereof. Similarly, the expression levels of VDAC1 and HK-I were decreased in the Vern plant extract- and phytol-treated animals bearing tumors, as shown by representative images in Figure 8C and by quantifications thereof provided in Figure 8D. The decreased expression levels of metabolism-related enzymes in the Vern plant extract- and phytol- treated tumors suggests decreased energy production in these treated tumors.

[0120] The effect of intra-tumorally administering Vern plant extract or phytol on the expression levels of proteins associated with angiogenesis was evaluated. As shown in Figure 9A, immunostaining of the endothelial cell marker CD-31 showed that in the Vern plant extract- or phytol-treated tumors, there was a significant decrease in the level of this cell marker, suggesting that the number of blood vessels significant decreased, with quantitation revealing about 70% and 60% decrease in Vern plant extract- and phytol-treated animals (tumors), respectively, relative to control tumors (Fig. 9B).

[0121] The effects of Vern plant extract and phytol on tumor microenvironment (TME) was further analyzed by IF-staining of the fibroblast marker alpha smooth muscle actin (α-SMA). As shown by the representative micrographs of Figure 9C and in the quantification thereof in Figure 9D, animals bearing tumors treated by either Vern plant extract or phytol showed decreased by 70 and 60%, respectively of α-SMA expression.

[0122] Accumulated recent evidence supports the cancer stem cell (CSC) hypothesis, which suggests that a sub-population of malignant cells exhibit the stem cell properties of self-renewal and differentiation. CSCs are resistant to conventional cytotoxic / anti-proliferative therapies. In glioblastoma multiforme (GBM), the condition wherefrom the cell line was derived, the proteins Sox2, CD133, SSEA1, CD49f, Musashi- 1, and Nestin are considered to be glioma stem cell CSC markers. The IF staining for Sox2 and Nestin of the tumor specimens demonstrated that in animals bearing tumors treated with Vern plant extract or phytol, the expression levels of these CSC markers was highly reduced, by about 70%, as shown by representative micrographs in Figure 10A and by a quantification thereof in Figure 9B. These results indicate that Vern plant extract and phytol treatment of U-87MG-derived tumors eliminated CSCs-associated with tumor recurrence. EXAMPLE 7 Identification of compounds in extracts of V. Nudiflora, B. trimera and P. major

[0123] The extracts have been analyzed using GC-MS / MS, as described above. The compounds identified are listed in the Table 5 (also referred herein as “Table A”), Table 6 (also referred herein as “Table AC”), Table 7 (also referred herein as “Table C”), Table 8 (also referred herein as “Table AB”), Table 9 (also referred herein as “Table B”), Table 10 (also referred herein as “Table ABC”) and Table 11 (also referred herein as “Table BC”) below. Additional information concerning the compounds is presented in Tables 5-11 below, listing the compound’s formula, calculated molecular weight (Calc. MW, Da), retention time (RT in minutes) and maximal chromatographic peak area in the relevant plant type (Area).Table 5 Compounds identified only in V. Nudiflora extract CompoundCompound NameFormula Calc. MW RT [min] Area ANo. A1 AsparagineC4 H8 N2 O3 132.05 0.652 90,024,032A2 11-methyl-2,7- C19 H20 O6 344.12 8.393 66,445,827 dimethylidene-6,12-dioxo- 5,14- dioxatricyclo[9.2.1.0â´,â¸]tetr adec-1(13)-en-9-yl 2- methylprop-2-enoate A3 Myristicin (4-methoxy-6- C11 H12 O3 192.07 8.752 125,507,328 (prop-2-en-1-yl)-2H-1,3- benzodioxole) A4 (3aS,4S,5S,11aR)-6-formyl-5- C19 H22 O7 362.13 9.978 604,917,241 hydroxy-10-(hydroxymethyl)- 3-methylidene-2-oxo- 2H,3H,3aH,4H,5H,8H,9H,11a H-cyclodeca[b]furan-4-yl 2- methylprop-2-enoate A5 Calealactone B C21 H26 O9 422.15 12.172 15,882,130,8 (1R,3R,4S,6S,8R,9R,10S,11S)- 10 9-(acetyloxy)-8-hydroxy-3,8- dimethyl-12-methylidene- 7,13-dioxo-5,14 dioxatricyclo[9.3.0.04,6]tetra decan-10-yl 2-methylprop-2- enoate A6 YangoninC15 H14 O4 258.08 12.172 1,244,465,484 A7 Flavone, 2-Phenyl-4H-1- C15 H10 O2 222.06 12.172 93,629,315 benzopyran-4one, 2- Phenylchromone A8 α-Lapachone, 3,4-Dihydro- C15 H14 O3 242.09 12.28 27,091,250 2,2-dimethyl-2H- naphtho[2,3-b]pyran-5,10- dione A9 Murrangatin 8-(1,2- C15 H16 O5 276.09 12.391 152,423,283 dihydroxy-3-methylbut-3-en- 1-yl)-7-methoxy-2H- chromen-2-one A10 (1R,4aS,5R,8aS)-5-(5- C20 H34 O3 322.25 14.517 4,378,871 hydroxy-3-methylpentyl)- 1,4a-dimethyl-6- methylidene- decahydronaphthalene-1- carboxylic acidTable 6 Compounds common to plant extracts of V. Nudiflora and P. major CompoundCompound nameFormula Calc. MW RT [min] Area Ext. A Area Ext. CNo. AC1 DL-CarnitineC7 H15NO3 161.10 0.708 386,595,363 188,858.350AC2 ValineC5 H11NO2 117.07 0.823 299,719,731 762,343,180AC3 L-NorleucineC6 H13NO2 131.09 1.986 105,168,196 776,052,439AC4 (2R,3S,4S,5R,6R)-2- C14 H20 O6 284.12 6.62 4,766,900,780 3,965,248,469 (hydroxymethyl)-6- (2- phenylethoxy)oxane -3,4,5-triol AC5 GenistinC21H20O10 432.10 7.887 46,850,468 19,272,245AC6 KuromaninC21H20O11 448.10 8.206 7,728,621 14,178,412AC7 NP-020760C21 H38 O8 418.25 8.509 169,216,326 286,281,319AC8 NP-008952C12 H20 O4 228.13 10.473 5,133,756 14,116,256AC9 Pelargonidin, C15 H10 O5 270.05 10.588 1,302,387,012 1,672,611,309 (3,4′,5,7- Tetrahydroxyflavyliu m) AC10 Aflatoxin B1C17 H12 O6 312.06 11.234 1,468,738 6,638,291AC11 2,20-dihydroxy-13- C25 H32 O8 460.20 12.586 3,232,602 14,795,380 methoxy- 4,7,17,22,22- pentamethy l5,10,21,23tetraoxa hexacyclo [18.2.1.0Aa,Aaa·.0a, Aaa.0a,Aaa.0a¸,Aa tricosa-6(14),7,12- trien-11-one AC12 (2R,3R,3aR,5R)-5,7- C22 H26 O7 402.16 12.843 125,072,984 114,632,747 dimethoxy-2- (7- methoxy-2H-1,3- benzodioxol-5-yl) - 3-methyl-3a-(prop- 2-en-1- yl)2,3,3a,4,5,6 - hexahydro-1- benzofuran-6-one AC13 OctadecanamineC18 H39 N 269.30 13.341 6,573,660 3,819,614AC14 OleamideC18 H35NO 281.27 15.712 2,177,872,750 1,919,921,186Table 7 Compounds identified only in plant extract of P. major CompoundCompound nameFormula Calc. MW RT [min] Area Ext. CNo. C1 1-O-(3,4,5- C16H22O10 374.12 3.875 148,046,091 Trimethoxybenzoyl)- beta-L-galactopyranose C2 3- C8 H8 O2 136.05 4.342 46,695,749 Methoxybenzaldehyde C3 (1S,4aS,7aR)-7-methyl- C21H30O13 490.16 5.707 29,114,110 1-{[(2S,3R,4S,5S,6R)- 3,4,5- trihydroxy-6- (hydroxymethyl)oxan- 2-yl]oxy}-4a- {[(2S,3R,4R,5R,6S)- 3,4,5-trihydroxy-6- methyloxan-2-yl]oxy}- 1H,4aH,5H,7aH- cyclopenta[c]pyran-5- one C4 3-(4-hydroxyphenyl)-7- C22H22O10 446.12 6.186 8,225,089 methoxy-5- {[(3R,4S,5S,6R)- 3,4,5- trihydroxy-6- (hydroxymethyl)oxan- 2-yl]oxy}- 4H-chromen- 4-one C5 1-(3-methoxy-4- C21H30O12 474.17 6.236 73,826,648 {[(2S,3R,4S,5S,6R)- 3,4,5-trihydroxy-6- ({[(2R,3R,4R,5R,6S)- 3,4,5-trihydroxy-6- methy-loxan -2- yl]oxy}methyl)oxan-2- yl]oxy} phenyl)ethan-1- one C6 (2R,3R,4S,5S,6R)-2- C16 H28 O7 332.18 6.99 399,856,237 {[(2E,6R)-6-hydroxy- 2,6-dimethylocta-2,7- dien-1-yl]oxy}-6- (hydroxymethyl)oxane- 3,4,5-triol C7 Solanid-5-en-3-yl 6- C45H73NO15 867.49 9.292 5,989,523 deoxy-alpha-L- mannopyranosyl-(1-2)- [beta-D- glucopyranosyl-(1-3)]- beta-D- mannopyranoside C8 NP-008274C15 H20 O4 264.13 10.291 6,514,510C9 FormononetinC16 H12 O4 268.07 11.904 11,361,966C10 SpiroxamineC18H35NO2 297.26 12.983 24,206,021Table 8 Compounds common to plant extracts of V. Nudiflora and B. trimera CompoundCompound nameFormula Calc. MW RT [min] Area Ext. A Area Ext. BNo. AB1 PhloroglucinolC6 H6 O3 126.03 0.707 41,675,260 276,902,261AB2 Caffeic acidC9 H8 O4 180.04 5.467 37,440,395 117,292,952AB3 (5S,6S)-5-hydroxy-4- C14H16O4 248.10 6.563 37,379,312 81,788,995 methoxy-6-(2-phe- nylethyl)-5,6-dihyd- ro-2H-pyran-2-one AB4 Quercetin-3β-D- C21H20O12 464.09 7.009 1,944,716,030 260,845,893 glucoside AB5 2-(3,4-dihydrox- C20H18O11 434.08 7.464 66,392,856 23,856,352 yphenyl)-5,7-dihyd- roxy-3-{[(2S,3R,4S ,5S)-3,4,5-trihydr- oxyoxan-2-yl]oxy}- 4H-chromen-4-one AB6 5,7-dihydroxy-2-(4- C22H22O12 478.11 8.694 1,997,194 26,272,176 hydroxy-3-{[(2S,3R, 4S,5S,6R)-3,4,5- trihydroxy-6-(hydro- xymethyl) oxan-2- yl]oxy}phenyl)-3- methoxy-4H- chromen-4-one AB7 4-HydroxycoumarinC9 H6 O3 162.03 9.875 2,384,008,330 5,413,658,494AB8 2-Hydroxycinnamic C9 H8 O3 164.04 11.409 78,128,321 271,611,189 acid AB9 19-norandrostenedioneC18 H24 O2 272.17 12.582 6,732,704 24,425,895Table 9 Compounds identified only in plant extract of B. trimera CompoundCompound nameFormula Calc. MW RT [min] Area Ext. BNo. B1 6-Pentyl-2H-pyran-2-oneC10 H14 O2 166.09 5.466 367,183,571B2 Myricetin 3-O-beta-D-galactopyranosideC21 H20 O13 480.08 6.353 54,473,499B3 5,7-Dihydroxy-2-(4-hydroxyphenyl)- C27 H30 O15 594.15 7.388 224,327,842 4-oxo-4H-chromen-3-yl 6-O-(6- deoxyhexopyranosyl)hexopyranoside B4 NP-003191 B5 (2R,3S,4S,5R,6R)-2-({[(2R,3R,4R, 5S)- C21 H36 O10 448.23 8.926 200,715,620 3,4-dihydroxy-5-(hydroxy methyl) oxolan- 2-yl]oxy}methyl)-6-{[(2E)- 3,7-dimethylocta-2,6-dien-1- yl]oxy}oxane-3,4,5-triol B6 2,6-Di-tert-butyl-1,4-benzoquinoneC14 H20 O2 220.14 9.197 116,434,233B7 4-MethoxycinnamaldehydeC10 H10 O2 162.06 10.772 47,596,601B8 SakuranetinC16 H14 O5 286.08 12.73 23,909,168B9 EstriolC18 H24 O3 288.17 13.532 22,924,076B10 TestosteroneC19 H28 O2 288.20 13.585 40,718,111B11 Nootkatone, (( 4,4a,5,6,7,8- C15 H22 O 218.16 14.248 706,868,081 hexahydronaphthalen-2(3H)-one) B12 2,4-DimethylbenzaldehydeC9 H10 O 134.07 14.304 80,005,955Table 10 Compounds common to plant extracts of V. Nudiflora, B. trimera and P. major Compound Compound name Formula Calc. RT Area Area Ext. B Area Ext. No. MW [min] Ext. A C ABC1 BetaineC5 H11NO2 117.07 0.694 3,530,740,11774,970,563 1,351,792,4 4 37 ABC2 ProlineC5 H9 N O2 115.06 0.759 2,596,224,604,829,925,97 476,675,06 0 3 0 ABC3 L-Pyroglutamic acidC5 H7 N O3 129.04 0.819 120,309,068 535,269,526 711,595,064 ABC4 L-PhenylalanineC9 H11NO2 165.07 3.256 46,652,027 119,879,831 229,319,024 ABC5 (1r,3R,4s,5S)-4-{[(2E)-3- C16 H18O9 354.09 5.054 717,332,175 1,566,476,22 22,570,076 (3,4-dihydroxyphenyl) 8 prop-2-enoyl]oxy}-1,3,5- trihydroxy cyclohexane-1- carboxylic acid; ABC6 (1S,3R,4R,5R)-1,3,4- C17 H20 O9 368.11 6.232 6,050,155 125,783,387 4,361,948 trihydroxy-5-{[(2E)-3-(4- hydroxy-3- methoxyphenyl)prop-2- enoyl]oxy}cyclohexane-1- carboxylic acid; ABC7 ScutellarinC21H18O12 462.07 7.162 9,544,332 3,987,959 399,151,403 ABC8 4,5-dicaffeoylquinic acid C25H24O12 516.12 7.949 717,332,175 1,566,476,22 22,570,076 (Isochlorogenic acid C) OK81R,3R,4S,5R)-3,4-bis({[(2E)- 3-(3,4- dihydroxyphenyl)prop-2- enoyl]oxy})-1,5-dihydroxy cyclohexane-1-carboxylic acid ABC9 5-hydroxy-2-(4- C22H22O11 462.11 8.116 5,934,396 6,322,103 48,852,430 hydroxyphenyl) -6- methoxy- 7{[(2S,3R,4S,5S,6R) -3,4,5- trihydroxy-6- (hydroxymethyl) oxan-2- yl]oxy} -4H-chromen-4-one ABC10 Eriodictyol [(S)-2-(3,4- C15 H12 O6 288.06 9.278 3,445,353 54,410,748 3,115,742 dihydroxyphenyl)- 5,7- dihydroxy-2,3- dihydrochromen-4-one] or 3',4',5,7-tetrahydroxy flavanone or 2,3- dihydroluteolin, ABC11 CyanidinC15 H10 O6 286.04 9.509 365,449,990 1,847,774,673 6,004,521,060 ABC12 DiosmetinC16 H12 O6 300.06 10.908 1,210,096,31778,882,279 3,374,118,7 3 43 ABC13 (±)13-HpODEC18 H32 O4 312.23 11.548 29,794,300 151,854,000 29,790,793ABC14 5,7-dihydroxy-2-(3- C18 H16 O8 360.08 11.704 1,918,047 15,810,934 5,900,261 hydroxy-4 methoxyphenyl) -3,6-dimethoxy-4H- chromen-4-oneCompound Compound name Formula Calc. RT Area Area Ext. B Area Ext. No. MW [min] Ext. A CABC15 GlyciteinC16 H12 O5 284.06 12.836 1,871,318,710 2,048,571,322 16,749,470ABC16 NobiletinC21 H22 O8 402.13 12.864 28,776,385 8,388,672 28,689,196ABC17 Bis(methylbenzylidene)sor C22 H26 O6 386.17 12.906 27,466,171,9 4,220,658,636 27,902,714, bitol 62 014ABC18 Aflatoxin B2C17 H14 O6 314.08 13.001 2,736,807,42874,589,672 9,950,100 7 ABC19 13,14-dihydro-15-keto- C16 H26 O5 298.17 13.965 50,183,155 6,591,428 16,587,611 tetranor Prostaglandin E2ABC20 12-Oxo phytodienoic acidC18 H28 O3 292.20 14.178 188,626,030 421,917,113 379,074,020 ABC21 (2S,4aS,6aS,6bR,10S,12aS, C30 H46 O4 470.34 14.492 10,734,543 41,053,407 164,719,75 14bS)-10-hydroxy- 4 2,4a,6a,6b,9,9,12a- heptamethyl-13-oxo- 1,2,3,4,4a ,5,6,6a,6b,7,8,8a ,9,10,11, 12,12a ,12b,13,14b- icosahydropicene-2- carboxylic acid ABC22 9-Oxo-10(E),12(E)- C18 H30O3 294.21 14.536 836,727,031 1,137,604,89 206,890,91 octadecadienoic acid91 0 8ABC23 α-Linolenoyl ethanolamideC20H35NO2 321.2614.584 96,331,653 144,983,358 22,559,247 64 3ABC24 Linoleoyl ethanolamideC20 H3 NO2 323.2815.011 382,279,845 622,398,515 292,070,98 19 2ABC25 Arachidonic acidC20 H32 O2 304.2315.208 421,592,785 746,792,985 756,456,21 98 1 ABC26 5-(1,2,4a,5-tetramethyl-7- C20 H32 O3 320.23 15.372 932,482,943 272,485,224 2,315,762,6 oxo-1,2,3,4,4a,7,8,8a-48 63octahydronaphthalen-1- yl)-3-methylpentanoic acid ABC27 8Z,11Z,14Z-Eicosatrienoic C20 H34 O2 306.25 15.482 71,268,301 45,589,288 65,609,194 acid 55ABC28 2,4-XylidineC8 H11 N 121.0816.933 536,746,096 140,381,883 414,932,25 93 3ABC29 QuercetinC15 H10 O7 302.04 8.303 906,000,000 370,000,000 9,436,007ABC30 SphingosineC18 H37N O2 299.28 13.293 115,704,693 33,337,083 3,739,707ABC31 Rhamnetin (2-(3,4- C16 H12 O7 316.05 12.088 3,999,606 487,756,323 2,123,057 dihydroxyphenyl)-3,5- dihydroxy-7-methoxy-4h- chromen-4-one ABC32 (1S,4S,5R,10S,13S,17S,19S, C30 H46 O3 454.34 14.413 6,081,044 38,975,281 345,271,32 20R)-10-hydroxy-54,5,9,9,13,19,20- heptamethyl-24- oxahexacyclo [15.5.2.0¹,¹⁸.0⁴,¹⁷.0⁵,¹⁴.0⁸,¹³] tetracos-15-en-23-oneTable 11 Compounds common to plant extracts of B. trimera and P. major CompoundCompound nameFormula Calc.RT [min] Area Ext. B Area Ext. C No. MW BC1 Trigonelline (N- C7 H7 NO2 137.04 0.742 262,043,732 106,297,400 Methylnicotinate) BC2 4-(4-hydroxy-2,6,6- C19 H32 O8 388.20 5.857 1,9167,772 488,977,805 trimethyl-3- {[(2R,3R,4S,5S,6R)-3,4,5- trihydroxy-6- (hydroxymethyl)oxan-2- yl]oxy}cyclohex-1-en-1- yl)butan-2-one BC3 5-(6-hydroxy-6- C13 H22 O3 226.15 5.868 23,817,617 27,604,558 methyloctyl)-2,5- dihydrofuran-2-one BC4 RutinC27H30O16 610.15 6.884 715,750,931 7,483,570BC5 [(2R,3S,4S,5R,6R)-6-[2-(3,4- C23 H26 478.14 7.165 9163,930 382,007,136 dihydroxyphenyl) ethoxy]- O11 3,4,5-trihydroxyoxan-2- yl]methyl (2E)-3-(3,4- dihydroxyphenyl)prop-2- enoate BC6 TrifolinC21H20O11 448.09 7.714 249,579,025 14,131,492BC7 (2S,3S,4S,5R,6S)-6-{[5,7- C21H18O12 462.07 8.204 1,664,311 81,408,493 dihydroxy-2- (4-hydroxyphenyl)-4-oxo- 4H-chromen-3-yl]oxy} -3,4,5-trihydroxyoxane-2- carboxylic acid BC8 ChrysinC15 H10 O4 254.05 9.082 2,016,251 28,745,233BC9 IsophoroneC9 H14 O 138.10 10.381 1,644,808,897 4,765,689BC10 (2R,5R,6R)-3-[(1E,3E)-hepta- C14 H22 O4 254.15 10.67 201,206,965 8,524,318 1,3-dien-1-yl] -5,6-dihydroxy-2- (hydroxymethyl)cyclohexan- 1-one BC11 CarvoneC10 H14 O 150.10 11.434 2,305,669,446 8,898,044BC12 3-Methoxy prostaglandin C21 H38 O6 386.26 12.769 214,114 20,526,597 F1α BC13 1,4-dihydroxy-1,4-dimethyl- C15 H24 O3 252.17 12.853 324,663,217 5,935,783 7-(propan-2-ylidene) -decahydroazulen-6-one BC14 5-hydroxy-6,7-dimethoxy-2- C17 H14 O5 298.08 14.032 272,839,318 2,225,655 phenyl-4H-chromen-4-one BC15 Bicyclo Prostaglandin E2C20 H30O4 334.21 14.454 17,867,376 27,812,813BC15 2,4,5-TrimethylanilineC9 H13N 135.10 16.8 83,080,637 56,870,246EXAMPLE 8 The effect of compounds identified in V. Nudiflora extract on cancer cells viability

[0124] In order to analyze the effect of some of the compounds identified in V. Nudiflora extract on cancer cells, cancer cells SHSY5Y were incubated (24 hours) with hydroethanolic Vern extract (1:1000) or with each one of the compounds listed in Table 12 below, at different concentrations, and then analyzed for induction of apoptosis by these compounds using propidium iodine staining (n=3). Figures 11, 12 and 13 show the results of cell death induction obtained by the various tested V. Nudiflora extract compounds.

[0125] In particular, the activity of V. Nudiflora extract compounds may be classified, without wishing to be bound by any theory, as being relatively effective in inducing cell death, as shown in Fig. 11A - Fig. 11I (e.g., as shown for Caffeic acid, Myricetin, Rutin, Quercetin, Keampferol, alpha-lapachone, 1,2:2,4-Di-p- methylbenyliedene and Myristicin in Fig. 11B - Fig. 11I, respectively), as being moderately effective in inducing cell death as shown in Fig. 12A – Fig. 12G (e.g., as shown for Quercetin-3-beta-glucoside, Diosmetin, Stachidrine chloride, Glyciteine and 8-hydroxyquinoline, 1,2:2,4-Di-p-methylbenyliedene sorbitol and Cyanidin chloride in Fig. 12A - Fig. 12G, respectively) or as being only marginally effective in inducing cell death, as shown in Fig.13A – Fig.13G (e.g., for Rosmarinic acid in Fig. 13E).

[0126] The compounds’ concentration yielding 50% cell death (IC50), the obtained maximal cell death and the concentration required for it are presented. In addition, the molecular mass (Mw) and empiric formula are indicated. The concentration in Vern extract was determined using a calibration curve obtained for each compound using a commercially available compound (ND - not determined).Compound Mw Empiric Maxim Conc. for Apop. Conc. in 1:1000 IC (Da) formula al cell maximal cell 50 diluted Vern (µM) death death (µM) extract (µM) (%) Vern Plant 99 1:1000 dilution (98% death) Rutin 610.52 C27H30O16 · 74 400 ~350 1.21 3H2O Keampferol 286.24 C15H10O672 300 100 0.93 Myricetin 318.24 C15H10O8 88 200 100 0.48 Caffeic acid 180.16 C9H8O4 90 700 500 122.3 Rosmarinic acid 360.31 C18H16O8 23 600< ~250 0.95 Yangonin 258.27 C15H14O4 23 100<< --- 4.34 Chlorogenic acid 354.31 C16H18O9 12 100<< --- 141.2 Betain 117.15 C5H11NO2 14 100<< --- 80.5 4- 162.14 C9H6O3 26 100<< --- 2.94 hydroxycoumarin Stachydrine 179.64 C7H14ClNO2 37 50 10 21.4 chloride Cyanidin chloride 322.7 C15H11ClO6 38 20 5 45.7 Glyciteine 284.26 C16H12O5 31 30 4 0 Diosmetin 300.26 C16H12O6 60 20 5 30.5 8- 145.16 C9H7NO 30 10 3 0.02 hydroxyquinoline Quercetin 302.24 C15H10O7 59 100 20 1.11 Quercetin-3-beta- 464.38 C21H20O12 34 50 20 90.9 glucoside Phytol 296.53 C20H40O 95 120 75 5.4 Ethyl linoleate 308.5 C20H36O2 34 ND 200<< 6.3 Myristicin 192.21 C11H12O3 93 2000 750 ND Nobiletin 402.399 C21H22O8 20 ND 150<< ND Murrangatin 276.28 C15H16O520 ND 150<< ND Alpha-lapachone 242.27 C15H14O390 300 150 ND 1,2:2,4-Di-p- 386.4 C22H26O6 100 200 40 ND methylbenyliedene Table 12 The effect on cell survival and apoptosis by isolated compoundsEXAMPLE 9 Fractionation of Vernonanthura nudiflora hydroethanolic extract by organic solvents and identification of specific compounds in the fractions obtained

[0127] Further to the above analyses, in order to identify the active compounds in Vernonanthura nudiflora hydroethanolic extract, samples of the Vernonanthura nudiflora hydroethanolic extract were fractionated using different organic solvents, as detailed below.

[0128] First, a Vernonanthura nudiflora hydroethanolic extract sample (100 ml) was air evaporated such that the concentration thereof was increased about 10-fold (i.e., to a final volume of 10 ml) and then subjected to fractionation using the solvents hexane, ethyl acetate, chloroform, and butanol according to the separating funnel method, as known in the art. Briefly, extraction was performed independently with each one of the solvents, by adding the solvent (10 ml) to the concentrated V. nudiflora hydroethanolic extract (7.5 ml), thoroughly shaking and separating into a solvent phase and a water phase. The above procedure was repeated (twice) to further extract any remaining agents from the water phase obtained. Next, each one of the extracts obtained was dried by evaporation (at 50°C), and the dry extract was then dissolved in 70% ethanol (0.2 ml of 70% ethanol). EXAMPLE 10 Analysis of cell death induction (apoptosis) by organic solvent extractions of V. nudiflora hydroethanolic extract

[0129] Next, the activity of the above-detailed solvent extracts in cell death induction was analyzed, in SHSY5Y and PC-3 cancer cells according to the procedures detailed above. The results shown in Table 13 and in Table 14 below for SHSY5Y and PC-3 cancer cells, respectively, indicate that all of the solvent extracts were active in inducing cell death.

[0130] Briefly, tables 13 and 14 below show the results of cell death induction by the various solvent extracts detailed above in, respectively, SHSY5Y cancer cells and inPC-3 cells (200,000 cells / well / 6 well plate) incubated (24 hour) with the indicated 1:400 dilutions of V. nudiflora hydroethanolic extract and the separated fractions thereof extracted by ethyl acetate, chloroform, butanol and hexane, and further subjected to analysis using propidium iodine (PI) staining as detailed above (n=3). Ethanol (70%) was used as control. Table 13 Cell death induction by solvent extracts of V. nudiflora hydroethanolic extract in SHSY5Y cancer cells Phase separation Volume (µl) Cell death, % Fraction (Final dilution) Control 2, 70% EtOH 7.74 5, 70% EtOH 6.59 Original plant extract A 2 (1:1000) 10.17 5 (1:400) 42.47 Hexane Fraction 2 (1:1000) 23.95 5 (1:400) 60.21 Ethyl acetate fraction 2 (1:1000) 12.4 5 (1:400) 10.57 Chloroform Fraction 2 (1:1000) 39.79 5 (1:400) 28.06 Butanol fraction Supernatant 2 (1:1000) 11.57 5 (1:400) 8.46 Butanol (Pellet 70% EtOH) 2 (1:1000) 10.03 5 (1:400) 12.81 H2O fraction supernatant 2 (1:1000) 12.02 5 (1:400) 8.68 H2O (Pellet 70% EtOH) 2 (1:1000) 11.65 5 (1:400) 12.25 Table 14 Cell death induction by solvent extracts of V. nudiflora hydroethanolic extract in PC-3 cancer cells. No. Sample Volume (µl) PI Assay Cell death (%) 1 Control 0 3.63 2 Original Extract 3 45.59 3 Original Extract 7 54.98 4 Hexane Fraction 3 19.49 5 7 26.95 6 Ethyl acetate Fraction 3 31.7 7 7 45.6 8 Chloroform upper fraction 3 45.34 9 7 46.32 10 Chloroform down fraction 3 36.61 7 40.94

[0131] The results of the cell death activity obtained with the solvents extraction of V. nudiflora hydroethanolic extract in cancer cells suggest that hexane, chloroform, and ethyl acetate, apparently showing a different collection of compounds, exhibit cell death activity, in particular hexane. EXAMPLE 11 – case reports Patient 1

[0132] This example describes a case report of treatment of a patient in need thereof by administering the composition according to Example 1. Briefly, the extracts of from Baccharis articulata, Plantago major, and Vernonanthura nudiflora, were mixed in proportion of 2:1:2, to obtain the mixed plant extract (also referred to herein as “MPE”).

[0133] A 69-year-old male presented for treatment prostate cancer with metastases Grade 4, Gleason factor 9, PSA 12.3 (ICD-11, 2C82). Immediate treatment with degarelix (240 mg) and bicalatumide (50mg) was initiated. Several weeks later the patient decided to try the MPE as well.

[0134] Following obtaining the informed consent, the patient was instructed and agreed to use the provided medication according to instruction or to return the unused medication. The patient started receiving the MPE. The dose was 15 mL, administered per os MPE diluted 1:8 in distilled water, four times a day.

[0135] After 25 days of therapy the patient demonstrated PSA 1.3. Degarelix was switched to darolutamide, and bicalatumide for leuprolide. After only 10 more days of treatment the patient demonstrated PSA score of 0.46.

[0136] According to treating professionals, such improvements are generally very rare, and are wholly unexpected because the improvement was so fast and the original patient’s condition has 5-year relative survival rate of 31%. Patient 2

[0137] A 34-year-old female has been suffering from Langerhans cell histiocytosis for about two years (ICD-11, 2B31.2) prior to being presented for treatment. Immediate treatment with vinblastine / prednisone, together with desmopressin andcabergoline to treat the symptoms, as well as senna glycosides as needed. Concomitantly, the patient decided to try the MPE as well.

[0138] Following obtaining the informed consent, the patient was instructed and agreed to use the provided medication according to instruction or to return the unused medication. The patient started receiving the MPE. The dose was 20 mL, administered per os MPE diluted 1:8 in distilled water, four times a day.

[0139] After six weeks of therapy the patient was in remission with no tumor observed. The treatment further constituted only MPE. Upon the recent follow-up after almost two years after diagnosis the patient remains in remission.

[0140] According to treating professionals, such improvements are generally very rare, and are wholly unexpected. Patient 3

[0141] A 34-year-old male has been diagnosed with testicular cancer (ICD-11, 2C80) mixed with germ cell malignant neoplasm, about a year before being presented for treatment of metastatic subpleural nodule. The initial treatment included resection and follow-up imaging. The metastasis has been observed first one year after the resection. No treatment for the metastatic pleural nodule has been given immediately after the diagnosis. Paraclinical examinations showed tumoral AFP marker on 22 ng / ml and nodule size 23x16 mm.

[0142] The patient decided to try the MPE prior to any conventional chemotherapy treatment. Following obtaining the informed consent, the patient wase instructed and agreed to use the provided medication according to instruction or to return the unused medication. The patient started receiving the MPE. The dose was 15 mL, administered per os MPE diluted 1:8 in distilled water, four times a day. Concomitantly,the patient received MPE intravenously, 6 mL of undiluted MPE administered in 500 mL of physiological solution, once a week.

[0143] One month later, the AFP marker was at 13 ng / mL, and the patient consented to conventional chemotherapy, without abandoning the MPE treatment. Conventional bleomycin, etoposide, and cisplatin protocol was given, 4 cycles every 21 days. The intravenous MPE was increased to twice a week.

[0144] After one month on the combined therapy, the AFP marker was at 2.4 ng / mL and the metastatic subpleural nodule was reduced by 30%. Upon completion of BEP, the patient continued with etoposide / cisplatin q21 days and MPE once a week, for another eight months. After this period, the markers returned to normal and no sign of metastatic nodules were present. The patient maintained peroral MPE, and upon the recent follow-up after almost two years after diagnosis the patient remains in remission.

[0145] According to treating professionals, such improvements were wholly unexpected and could be attributed to concomitant MPE treatment. Patient 4

[0146] A 47-year-old male has been diagnosed with testicular cancer (ICD-11, 2C80) with a large left para-aortic retroperitoneal metastatic mass (4.2x3.7x5.7 cm) consistent with adenopathy. A month later the patient underwent surgical dissection to remove the primary tumor. The patient decided to try the MPE four days before the surgery and prior to any conventional chemotherapy treatment. Following obtaining the informed consent, the patient was instructed and agreed to use the provided medication according to instruction or to return the unused medication. The patient started receiving the MPE. The dose was 2 mL, administered per os MPE diluted 1:8, four times a day.Concomitantly, the patient received MPE intravenously, 3 mL of undiluted MPE administered in 500 mL of physiological solution, twice a week.

[0147] Upon assessment made three weeks after the surgery, the metastatic mass was not identified. According to treating professionals, such improvements are extremely rare, and are wholly unexpected, given that no conventional treatment has been given. EXAMPLE 12 – Veterinary case reports Patient 1

[0148] A 12-year-old female Dachshund dog, 8 kg of weight, has been diagnosed with multiple inguinal breast tumors. The patient started receiving the MPE. The dose was 1 mL undiluted MPE, administered to the base of the tumors, once a week, which was elevated to 2 mL starting from week 2. Before the second injection, the clinical observation demonstrated slightly decreased size of the tumors and softer consistency. After fourth injection, the patient presented with significantly more vitality, with significantly decreased size of the tumors and mobility.

[0149] Fifth treatment, 2 mL, was performed after two weeks. The tumors were reduced even more in size. Skin in every one of them presented an ulcer wherefrom a viscous malodorous liquid was drained. After draining, the shape in the tumor area was planar, with no protrusions.

[0150] At the follow-up at three months thereafter, no tumor growth was observed and the subject maintained good vitality. Patient 2

[0151] A 13-year-old male Cimarron dog, 35 kg of weight, has been diagnosed with perianal tumor with possible hepatoid gland adenoma. The patient started receiving the MPE. The dose was 3 mL undiluted MPE, administered to the base of the tumors, once every two weeks. Two days after the second injection, treating veterinarian detectedthat tumor started to detach with profuse bleeding. Parts of the tumor detached from the original mass, which size also diminished significantly.

[0152] Upon follow-up one week after the second injection, the patient presented with more vitality, appetite, and weight and musculature gains. After the third injection the improvement was sustained and the patient presented with even more vitality

[0153] One week after the third injection, the remaining tumor mass was excised due to the significantly decreased size (about 20% of the original).

[0154] Four months post-surgery no relapse was observed. The patient maintained good appetite and vitality, and continued regaining musculature.

Claims

CLAIMS:

1. A composition comprising a hydroethanolic plant extract or any fraction thereof, wherein said plant is selected from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera, and / or Baccharis articulata, and any combination thereof.

2. The composition according to claim 1, wherein said extract is essentially identical to an extract obtained by a process comprising combining the biomass of at least one of said plant(s) with a hydroethanolic mixture comprising 20 to 50 volume percent of water, and extracting said biomass into said hydroethanolic mixture for a time interval of between about 3 and 35 days, preferably between about 18 and 23 days.

3. A composition comprising a hydroethanolic plant extract, said extract containing less than 5-20% by weight of the compounds listed in Table 1, or is essentially devoid of these compounds, wherein said plant is selected from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera, and / or Baccharis articulata, and any combination thereof.

4. The composition according to any one of the preceding claims, wherein said plant is Vernonanthura nudiflora or a combination of Vernonanthura nudiflora and Plantago major.

5. The composition according to any one of the preceding claims, wherein said extracting of said plant biomass into said hydroethanolic mixture is performed at a temperature range of between about 15°C and 40°C, preferably between 20°C and 25°C.

6. The composition according to any one of the preceding claims, wherein the concentration of said biomass being extracted is between about 15% and 25% weight by volume of total extraction solvent.

7. The composition according to any one of the preceding claims, wherein said process comprises drying of said plants prior to extracting.

8. The composition according to claim 7, wherein said drying is performed at a temperature of between about 15°C and 45°C, preferably between about 20°C and 25°C.

9. The composition according to claim 7 or claim 8, wherein said drying is carried out until a weight loss of about 40% to 60% by weight, of the initial weight of the plant is obtained.

10. The composition according to any one of the preceding claims, wherein said process comprises extracting the aerial parts of said plant(s), for example the aerial parts of Vernonanthura nudiflora.

11. The composition according to any one of the preceding claims, wherein said process comprises separating residues of said plant biomass from said hydroethanolic mixture to furnish said extract.

12. The composition according to claim 11, wherein said separating comprises filtering and / or centrifugation of the extraction mixture.

13. The composition according to any one of the preceding claims, wherein said extract is a dried hydroethanolic extract.

14. The composition according to any one of claims 1 to 12, wherein said extract is a liquid hydroethanolic extract.

15. The composition according to claim 14, wherein the concentration of plant- derived matter on dry basis in said liquid hydroethanolic extract is between 0.5% and 15.0%, preferably between 1.0% and 5.0% wt.

16. The composition according to claim 14 or claim 15, wherein the concentration plant-derived matter on dry basis in said liquid hydroethanolic extract is between 2.5% and 3.7% wt.

17. The composition according to any one of the preceding claims, wherein said hydroethanolic extract comprises between 40 and 80% of ethanol, the balance of the solvent consisting essentially of water.

18. The composition according to any one of the preceding claims, comprising a fraction of said hydroethanolic plant extract, wherein said fraction contains less than 5% by weight of the compounds listed in Table 1, or is essentially devoid of these compounds.

19. The composition according to claim 18, wherein said plant is Vernonanthura nudiflora or a combination of Vernonanthura nudiflora and Plantago major.

20. The composition according to claim 18, wherein said fraction is a sub- extraction of the hydroethanolic plant extract, preferably obtained by butanol, hexane, chloroform, or ethyl acetate extraction, or any combination thereof.

21. The composition according to any one of claims 18 to 20, comprising at least two compounds selected from the group consisting of rutin, keampferol, myricetin,stachydrine chloride, cyanidin chloride, glycitein diosmetin, 8-hydroxyquinoline, quercetin-3-beta-glucoside, phytol, myristicin, alpha-lapachone and 1,2:2,4-di-p- methylbenyliedene.

22. The composition according to any one of the preceding claims, for use in treating of a proliferative disorder in a subject in need thereof.

23. The composition for use according to claim 22, wherein said proliferative disease is cancer.

24. The composition for use according to claim 22 or claim 23, wherein said proliferative disorder or cancer is a primary or metastatic prostate cancer, Langerhans cell histiocytosis, testicular cancer, colon cancer, or breast cancer.

25. The composition for use according to any one of claims 22 to 24, wherein said composition is supplied to said subject in amounts of between 0.033 mg / kg and 8.6 mg / kg of extract by dry basis per kilogram weight of said subject.

26. The composition for use according to any one of claims 22 to 25, wherein said composition is supplied to said subject in amounts of between 20 and 500 mg of extract by dry basis per subject.

27. The composition for use according to any one of claims 22 to 26, wherein said composition is administered by a route selected from the group consisting of intravenous administration, oral administration, intramuscular administration, and intraperitoneal administration.

28. The composition for use according to any one of claims 22 to 27, wherein said subject concomitantly receives a treatment with an additional anti-cancer agent, preferably an apoptosis-inducing chemotherapy.

29. The composition according to any one of claims 1 to 21, for use in the manufacture of a medicament for treating a proliferative disorder in a subject in need thereof.

30. A method for the treatment of a proliferative disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a composition comprising a hydroethanolic plant extract or any fraction thereof, wherein said plant is selected from Vernonanthura nudiflora, Plantago major and a Baccharis family species selected from Baccharis crispa, Baccharis trimera, and / or Baccharis articulata, and any combination thereof, as defined in any one of the claims 1 to 21.

31. The method according to claim 30, wherein said proliferative disease is cancer.

32. The method according to claim 30 or claim 31, wherein said proliferative disorder or cancer is a primary or metastatic prostate cancer, Langerhans cell histiocytosis, testicular cancer, colon cancer, or breast cancer.

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