Plant aqueous ethanol extract

Abstract

The present disclosure relates to an aqueous ethanol extract of the plant Vernonanthura nudiflora, Plantago major, and a species of the genus Baccharis, selected from the group consisting of Baccharis crispa, Baccharis trimera, and Baccharis articala, and a therapeutic use thereof, for example, in the treatment and prevention of cancer, and to the use of the aqueous ethanol extract of the plant Vernonanthura nudiflora, Plantago major, and of the species of the genus Baccharis selected from the group consisting of Baccharis crispa, Baccharis trimera, and Baccharis articala. In particular, the present disclosure relates to an aqueous ethanol extract of the plant Vernonanthura nudiflora.

Description

Technical Field

[0001] The present disclosure relates to hydroethanolic plant extracts and their therapeutic uses, such as 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, such as those found in V. nudiflora extracts. Background Art

[0002] Several natural products with anticancer activity are currently in clinical use, such as plant-derived paclitaxel and docetaxel. In addition, several plant species have demonstrated anticancer properties and are used as herbal remedies in developing countries.

[0003] Vernonanthura nudiflora is a perennial plant species in the Asteraceae family, which includes more than 23,500 species distributed in approximately 1600 genera. (Vernonanthura nudiflora (Less.) H. Rob. | Plants of the World Online | Kew Science". Plants of the World Online. Retrieved 2020-08-10) Distributed 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 genus Vernonanthura (Vernonia) contains more than 1000 species (Bremer, K. (1994) Asteraceae: Cladistics and Classification. Timber Press, Portland). Ramos, AVG et al. 2019 (Ramos, AVG et al., J. Braz. Chem. Soc. 2019, Vol. 30(8), 1728-1740) reported the antiproliferative and antioxidant activities of organic extracts of Vernonanthura nudiflora and some of its chemical constituents. In addition, some metabolites isolated from the flowers of Vernonanthura nudiflora showed antimicrobial activity (AVG Ramos et al., 2021, The chemistry of Vernonanthuranudiflora (Less.) H. Rob. flowers and its antimicrobial activities NaturalProductResearch, DOI: 10.1080 / 14786419.2021.1980790).

[0004] Plants from the Baccharis family have found important uses in traditional South American medicine. Among them, Baccharis trimera (B. trimera) is the most studied species and has been reported in traditional treatments of gastrointestinal disorders and liver diseases (Garcia et al. 2014 A comparative study of two clerodanediterpenes from Baccharis trimera (Less.) DC on the influx and mobilizationof 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. RevBras Farmacogn 19:931–936), and several biological activities have been reported for this plant, including antihepatotoxic, antidiabetic, schistosomal, antioxidant, antinociceptive, and anti-inflammatory effects, which have been attributed to flavonoids, diterpenes, triterpenes, saponins, essential oils, and caffeoylquinic acid (Abad MJ, Bermejo P (2007) Baccharis (Compositae): a review update. Arkivoc 7:76–96; Campos FR, BressanJ, Jasinski VC et al (2016) Baccharis (Asteraceae): chemical constituents and biological activities. Chem Biodivers 13: 1–17).A hydroethanolic extract of B. trimera has also been 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 aceticacid Naunyn Schmiedebergs Arch Pharmacol. 2016 Sep;389(9):985-98). Several species of Baccharis are used interchangeably, such as B. trimera, B. crispa, and B. articulata, with the first two being very difficult to distinguish.

[0005] In addition, Plantago major (P. major), commonly known as "great plantain," is a widely 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 L. 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 to treat various diseases such as constipation, cough, wounds, infections, fever, bleeding and inflammation (Aghili M. Makhzan-OL Advieh. Tehran: Tehran University of Medical Science Press; 2008. (3)). In addition, aqueous and ethanolic extracts of Plantago major L. showed anti-inflammatory activity against oral epithelial cells (Zubair et al. Journal of Traditional and Complementary Medicine 9 (2019) 169e171170).

[0006] Mitochondria are at the core of basic life functions that generate key components of cellular energy and biosynthetic pathways, and play a central role in apoptosis. During the transduction of apoptotic signals into cells, changes in mitochondrial membrane permeability occur, which allows the release of apoptotic proteins such as cytochrome c (Cyto c), 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 mitochondria, they all participate in a complex process that leads to the activation of proteases and nucleases, leading to DNA and protein degradation, and ultimately leading to apoptotic cell death. One of the mitochondrial proteins that controls both cell life and death as a mitochondrial gatekeeper is voltage-dependent anion channel 1 (VDAC1). As previously shown by some of the present inventors, VDAC1 occupies a key position in the cell, located in the outer mitochondrial membrane (OMM), thereby forming the main interface between mitochondria and cellular metabolism; VDAC1 is also a key regulator of adenine nucleotides and other metabolites as well as Ca. 2+VDAC1 is a key protein in the metabolic regulation of pathways for entering and exiting mitochondria, and is a key protein in the regulation of mitochondrial-mediated apoptotic cell death, and controls other biological and cellular functions, and is overexpressed in various cancer cell lines and different tumors, indicating its importance for their development and persistence, as well as being overexpressed 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 key role played by VDAC1 in regulating the metabolic and energy function of mitochondria in cancer cells was demonstrated by the following findings: downregulating VDAC1 expression by specific siRNA resulted in a decrease in energy production and cell growth and inhibited tumor growth (Arif, T. et al., Silencing VDAC1Expression by siRNA Inhibits Cancer Cell Proliferation and Tumor Growth InVivo. Mol Ther Nucleic Acids, 2014. 3: p. e159). Several mechanisms for the release of pro-apoptotic proteins have been proposed, including channels 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 deathinduction. Biochim Biophys Acta, 2014. 1843(10): p. 2270-81). Defects in apoptosis regulation are often associated with drug resistance and diseases such as cancer, in which apoptosis evasion is a hallmark of cancer (Hanahan, D. and RAWeinberg, Hallmarks of cancer: the next generation. Cell, 2011. 144(5): p.646-74).

[0007] It is known that all mitochondrial apoptotic proteins that are translocated to the cytoplasm after apoptotic stimulation are present in the mitochondrial intermembrane space (IMS). Therefore, only the permeability of the OMM needs to be changed for its release (Halestrap, AP et al., Thepermeability transition pore complex: another view. Biochimie, 2002. 84(2-3): p. 153-66). Therefore, VDAC1, as an OMM channel, can mediate the release of Cyto c. It has been previously shown that VDAC1 can exist in the form of oligomers, which mediate the release of Cyto c by forming large channels that can release pro-apoptotic proteins, leading to cell death (Keinan, N. et al., Oligomerization of the mitochondrial protein VDACis coupled to the induction of apoptosis, Molecular Cell Biol. 30(24), 5698-5709). Furthermore, it was shown that apoptosis induction by cisplatin, selenite, H2O2, UV light, etc. leads to apoptosis via induction of VDAC1 overexpression, shifting the equilibrium toward 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 utilize natural products, such as those produced by V. nudiflora and optionally by other plants, for effectively interacting with the VDAC1-mediated apoptotic pathway, particularly for use in treating cancer (alone or as an adjuvant to apoptosis-inducing chemotherapy), or for use in reducing the prevalence of cancer recurrence. Summary of the Invention

[0009] It has now been unexpectedly discovered that extracts of the plant V. nudiflora, alone or in combination with extracts of other plants already used in some complex herbal remedies (e.g., for general health improvement), can be used against cancer cells in culture, reducing their viability and inducing cell death. In mouse cancer models, V. nudiflora extracts were shown to inhibit tumor growth, with greater effectiveness at higher dilutions. However, unlike homeopathic pseudo-theories, and without being bound by a particular narrative, it is believed that the anti-apoptotic, pro-survival compounds reportedly present in the extract and contributing to its beneficial effects in other areas of human health (e.g., anti-oxidation and / or anti-aging) overcome the extract's pro-apoptotic effectors at the concentrations present in the extract, thereby counteracting their effects on malignant cells. Reducing the extract's concentration by higher dilutions reduces the effects of the extract's anti-apoptotic / pro-survival components, which may be less potent than the anti-cancer compounds present therein. It has further been unexpectedly discovered, as described in more detail below, that prolonged hydroethanolic extraction of plant material provides an extract characterized by a group of compounds, distinct from those obtained by exhaustive organic ethanolic extraction, that possess significant pro-apoptotic effects, likely mediated by the VDAC1 pathway. Additionally, as demonstrated in the Examples, administration of extracts containing Vernonantura to cancer patients, alone or as co-therapy with conventional chemotherapy, has resulted in prolonged remissions or complete responses.

[0010] Thus, in its first aspect, the present disclosure provides a composition comprising a hydroethanolic plant extract or any fraction thereof, wherein the plant is selected from the group consisting of Vernonanthura nudiflora, Plantago asiatica and a species of the family Baccharis selected from the group consisting of Baccharis crispa, Baccharis trimera and Baccharis articulata, and any combination thereof, e.g., a hydroethanolic plant extract of Vernonanthura nudiflora or a combination of Vernonanthura nudiflora and Plantago asiatica, or any fraction thereof. The extract is substantially the same as an extract obtained by a process comprising combining biomass of at least one of the plants with a hydroethanolic mixture comprising 20-50% by volume water, and extracting the biomass into the hydroethanolic mixture for a time interval of about 3-35 days, preferably about 18-23 days. In various embodiments, the extract is prepared by a process comprising combining biomass of the plant with a hydroethanolic mixture comprising 20 to 50% water by volume, and extracting the biomass into the hydroethanolic mixture for a time interval of about 3 to 35 days, such as about 3 to 28 days, or 10 to 30 days, preferably about 18 to 23 days.

[0011] In a second aspect of the present invention, a method for extracting bioactive compounds from the biomass of the above-mentioned plants, for example, from V. nudiflora biomass, is provided, by contacting the biomass with a water-ethanol mixture comprising 20 to 50% by volume of water in ethanol and extracting the biomass into the water-ethanol mixture for a time interval (period) of about 3 to 35 days, for example, about 3 to 28 days, or 10 to 30 days, preferably about 18 to 23 days. The method for extracting the plant biomass into the water-ethanol mixture can be carried out at a temperature range of about 15°C to 40°C, preferably 20°C to 25°C. The extraction method can further comprise harvesting the area (areal) parts of the plant (e.g., V. nudiflora), such as stems, branches, leaves, and optionally flowers, and optionally drying them to provide dried biomass, i.e., drying the plant before extraction. Drying can preferably be carried out at ambient temperature to slightly elevated temperature, for example, at a temperature of 15°C to 45°C, preferably about 20°C to 25°C. The drying step can preferably be carried out until a certain mass loss as water, for example, until a weight loss of about 40% to 60% of the initial weight of the plant (e.g., its aerial parts) is retained by weight. The method can further include grinding the dried biomass to provide a biomass suitable for extraction. The extraction can be carried out with a ratio of 1:2 to 1:8, for example 1:3 to 1:6, between the extracted biomass and the water-ethanol mixture, preferably about 1:4, for example about 13 to 33% by weight, or about 15 to 25% by weight, or about 18 to 22% by weight of the total volume of the biomass and water-ethanol solution. The method can further include separating the residue of the plant biomass from the water-ethanol mixture to provide the extract, for example, filtering the biomass to produce a crude liquid extract. The method can further include clarifying the crude liquid extract, for example, by centrifuging under suitable g-forces to achieve sedimentation of dispersed fine matter. In addition, what is obtained by the extraction method disclosed in the present invention is a dry hydroethanolic extract or a liquid hydroethanolic extract, in the latter case, the concentration of the plant-derived substance based on dry weight in the liquid hydroethanolic extract is 0.5%-15.0%, preferably between 1.0%-5.0%wt, or the concentration of the plant-derived substance based on dry weight in the liquid hydroethanolic extract is between 2.5%-3.7%wt. In a specific embodiment, the hydroethanolic extract comprises 40-80% ethanol, the remainder of the solvent being essentially composed of water. In certain embodiments, the method further comprises sub-extraction of the hydroethanolic plant extract, preferably by extraction with butanol, hexane, chloroform or ethyl acetate or any combination thereof, thereby obtaining sub-extracts (also referred to herein as fractions of the hydroethanolic extract).

[0012] By another aspect thereof, the present disclosure provides a composition comprising a hydroethanolic plant extract containing less than 5-20% by weight (e.g., less than 5%) of the compounds listed in Table 1 below, or being substantially free of these compounds, wherein the plant is from Vernonanthura nudiflora, Plantago asiatica, and a species of the Willow family selected from the group consisting of 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 asiatica. Additionally, in another aspect, provided herein is an artificial (i.e., non-natural) mixture, also referred to herein as a fraction of a component that may be present in at least one plant from Vernonanthura nudiflora, Plantago asiatica, and a species of the Willow family selected from the group consisting of Baccharis crispa, Baccharis trimera, and Baccharis articulata, and any combination thereof (e.g., V. nudiflora). For example, the fraction of the component 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 its another aspect, present disclosure provides compositions as defined herein, it is used for use in the proliferative disorder (for example, cancer) of the experimenter in need thereof for the treatment.Compositions as defined herein and the method for treating including described compositions are particularly suitable for treating proliferative disorder or cancer, and described proliferative disorder or cancer are primary or metastatic prostate cancer, Langerhans cell histiocytosis (Langerhans cell histiocytosis), testicular cancer, colon cancer or breast cancer. In some embodiments, compositions as defined herein are with the amount of 0.033 mg / kg to 8.6 mg / kg extract per kilogram weight of experimenter on dry basis, for example, with the amount of 20 to 500mg extract per experimenter on dry basis supplied to described experimenter. Compositions as defined herein can be administered by the approach selected from the group consisting of intravenous administration, oral administration, intramuscular administration and intraperitoneal administration. In addition, in certain embodiments, compositions as defined herein are applicable to the treatment of the experimenter receiving concomitantly other anticancer treatments (preferably apoptosis-inducing chemotherapy).

[0014] Furthermore, the compositions according to the present disclosure can be used in the preparation of a medicament for treating a proliferative disorder in a subject in need thereof.

[0015] The present disclosure further provides a method of treating a proliferative disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition as defined herein, i.e. a composition comprising a hydroethanolic plant extract or any fraction thereof, wherein the plant is from Vernonanthura nudiflora, Plantago asiatica and a species of the family Salix family selected from Bacchariscrispa, 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] Figure 1A- Figure 1F SHSY5Y cancer cells were incubated (24 h) with the indicated dilutions of hydroethanolic plant extracts of Vern (Vernonanthuranudiflora), Bac (Baccharis trimera), Pla (Pla plantain), or their mixture (40%:40%:20%, respectively). Figure 1A1 and Figure 1A2 are representative results of FACS analysis of propidium iodide (PI)-stained cells, showing the control ( Figure 1A1 ) and cells incubated with Vern plant extract for 24 h ( Figure 1A2, 1:500) of live and dead cells. Figure 1B Is a bar graph showing analysis of apoptosis (% cell death based on FACS analysis of propidium iodide (PI) stained cells) in cells incubated with extracts from Vern plants, Bac or Pla at the indicated dilutions. The results are means ± SEM of three independent experiments. Figure 1C is a graph showing the percentage of apoptosis in SHSY5Y cells incubated with the indicated dilutions of Vern plant extract (24 hours) and then analyzed using Annexin V / PI staining and FACS. Figure 1D-1F is a graph showing the viability of SHSY5Y cells revealed by XTT assay of cells incubated with different dilutions of the indicated plant extracts for 24, 48 or 72 hours.

[0018] Figure 2A-2F SHSY5Y and U-87MG cells were incubated with the indicated dilutions of Vern plant extracts for 24 h and then subjected to different analyses. Figure 2A This is an immunoblot performed using an anti-VDAC1 specific antibody, showing the expression levels of VDAC1 in cells treated with the indicated dilutions of Vern plant extract. Also shown is an immunoblot using actin as a loading control. VDAC1 levels are given in relative units (RU) below the immunoblot, indicating the increase in VDAC1 levels by the Vern plant extract. Figure 2B is a graph showing that samples of cells treated with plant Vern extract were analyzed for apoptosis, inducing more than 80% apoptosis. Figure 2C Immunoblot of SHSY5Y cells incubated with the indicated dilutions of plant extracts Vern, Bac, and Pla, or their mixtures (24 hours), followed by analysis of VDAC1 oligomerization by incubation with the cross-linking reagent EGS (100 μM), followed by immunoblotting with an anti-VDAC1 antibody. The locations of VDAC1 monomers, dimers, trimers, tetramers, and higher-order oligomers are shown. Figure 2D is a bar graph showing the levels of VDAC1 dimers as analyzed using Image J software and presented relative to their levels in control cells subjected to EGS. The results are the mean ± SEM of three independent experiments. Figure 2E : is an immunoblot showing SHSY5Y cells that were incubated with the indicated dilutions of plant extracts Vern, Bac and Pla or their mixtures without EGS treatment (24 hours) and immunoblotted. The positions of VDAC1 monomers, dimers, trimers and tetramers are shown. Figure 2F is a bar graph showing the levels of VDAC1 dimer as analyzed using Image J software and presented relative to its levels in control cells.

[0019] Figures 3A-3D . Figure 3A is a graph showing the percentage of MitoSox fluorescence in SHSY5Y cells that were incubated with the indicated dilutions of Vern plant extract for 24 hours and analyzed for reactive oxygen species (ROS) production using MitoSox Red reagent and FACS analysis. Figure 3B The intracellular [Ca 2+ ]i's picture. Figure 3C and Figure 3D Shown separately Figure 3B Representative Operetta imaging visualization of cells in the uterus and their quantification. Results are the mean ± SEM of three independent experiments.

[0020] Figures 4A-4C . Figure 4A is a photograph of TLC separation of hydroethanolic extracts of Vern plants, Bac and Pla and known amounts of phytol and ethyl linoleate using a solvent mixture of petroleum ether:diethyl ether:acetic acid (85:15:1, V:V:V) and developed by exposure to iodine vapor. Figure 4B is a bar graph showing the quantification of phytol and ethyl linoleate in plant extracts using compound calibration curves and Image J software (n=3). Figure 4C Figure 2 is a graph showing SHSY5Y cells incubated with and without the indicated concentrations of phytol or ethyl linoleate for 24 or 48 hours, followed by evaluation of cell death using PI staining and FACS analysis. Results are the mean ± SEM of three independent experiments.

[0021] Figure 5A- Figure 5D Figure 5A1 and Figure 5A2 is an immunoblot showing the expression of SH-SY5Y cells with and without the indicated concentrations of phytol (Fig. 5A1) or ethyl linoleate ( Figure 5A2 ) were incubated for 24 h, and then the VDAC1 expression level was analyzed by immunoblotting. Figure 5B is a graph showing the levels of VDAC1 in the cells detailed in FIG. 5A as relative units (RU), quantified using Image J software. Figure 5C is an immunoblot showing control and phytol- or ethyl linoleate-treated cells, which were also analyzed for VDAC1 oligomerization by incubation with the cross-linking reagent EGS (100 μM) followed by immunoblotting using an anti-VDAC1 antibody. The positions of VDAC1 monomers, dimers, trimers, and tetramers are shown. Figure 5D Yes Display Figure 5CGraph showing quantification of VDAC1 dimer levels in the experiments detailed in . Results are mean ± SEM (n = 3).

[0022] Figures 6A-6H . Figure 6A U-87MG cells (1.8 × 10 6 Schematic diagram of the experimental design in nude mice inoculated subcutaneously (sc) with 10 cells / mouse) of 50 μg / mL of TNF-α, 1 ... 3 At the time of inoculation, mice were divided into three groups with similar calculated mean volumes (5 or 6 mice per group). The three groups of mice were treated with the following: control (ethanol to a final concentration of 0.14%) or Vern plant extract to a final dilution of 1:250 or 1:500, and phytol to a final concentration of 75 μM. The injection volume was calculated based on the tumor volume and the stock solution used. Mice were monitored and sacrificed on day 34 (denoted by 34D) after cell inoculation. Figure 6B is a graph showing the calculated mean tumor volume in participating mice as a function of time, expressed as mean ± SEM (n = 5 or 6 mice). Figure 6C is a graph showing the tumor volume calculated before the mice were sacrificed (day 34) and expressed as % of control. Figure 6D is a graph showing the calculated mean tumor weight expressed as mean ± SEM. *p<0.05. Figure 6E Confocal images of representative immunofluorescence images of paraffin-embedded sections of U-87MG-derived tumors from mice treated with control, Vern plant extract (1:500), or phytol (75 μM) using an antibody against the proliferation marker Ki-67 are shown. Quantification of staining intensity is shown in Figure 6F The results are mean ± SEM (n = 3), ****p < 0.0001. Figure 6G Shown the microphotograph of TUNEL dyeing of the paraffin-embedded section that the tumor that handles from control tumor, Vern plant extract or phytol is cut.As described in the embodiment part, carry out the TUNEL dyeing of tumor section.Representative confocal image indication PI nuclear staining with red dyeing, and the cell indication TUNEL dyeing of green dyeing. Figure 6H is a graph showing the quantification of TUNEL-positive cells. The results are mean ± SEM (n = 3), *p < 0.0; ****p < 0.0001;

[0023] Figure 7A-7B . Figure 7A The results showed that U-87MG cells (1.8 × 10 6 The average tumor volume (mm) of nude mice inoculated sc with 10 cells / mouse3 ) as a function of the number of treatments. Tumor volume was monitored (using a digital caliper) and on day 14, when tumor volume was between 40 and 60 mm 3 Between 2 and 3 months, mice were divided into three groups with similar calculated mean tumor volumes (n = 5 or 6 mice per group). The three groups of mice were then treated with either control (ethanol to a final concentration of 0.14%) or Vern plant extract to a final dilution of 1:100 or 1:300, based on tumor volume. The calculated mean tumor volume as a function of time is expressed as mean ± SEM (n = 5 or 6 mice). Figure 7B is a graph showing calculated mean tumor weights, expressed as mean ± SEM. *P < 0.05.

[0024] Figures 8A-8D Confocal images of sections from U-87MG-derived tumors treated with control or Vern plant extract (1:500) or phytol (75 μM) and immunofluorescence using specific antibodies against glucose transporter 1 (Glut-1) and glyceraldehyde dehydrogenase (GAPDH) are shown in Figure 8A , or immunofluorescence of VDAC1 and hexokinase (HK-1) ​​was performed on Figure 8C The staining intensity was quantified using Image J software as a measure of relative protein levels, and for Figure 8A and Figure 8C The images detailed in Figure 8B and Figure 8D Results reflect mean ± SEM (n = 3), ****p ≤ 0.0001.

[0025] Figures 9A-9D Confocal images of sections from U-87MG-derived tumors immunofluorescently stained for CD-31 or α-SMA are shown in Figure 9A and Figure 9C The tumors were obtained from mice treated with control, Vern plant extract (1:500), or phytol (75 μM). Quantification of the above images is presented in Figure 9B (showing relative levels of CD-31) and Figure 9D (Relative levels of α-SMA are shown in the graph.) Results = Mean ± SEM (n = 3 mice) ****p ≤ 0.0001.

[0026] Figures 10A-10B . Figure 10AShown are representative IF staining of tumor sections from control or U-87MG-derived tumors treated with Vern plant extract (1:500) or phytol (75 μM) immunofluorescently stained with specific antibodies against the CSC markers Sox2 and Nestin. Figure 10B It shows Figure 10A Graphs showing quantitative analysis of images shown in. Results are mean ± SEM (n = 3 tumors), ****p ≤ 0.0001.

[0027] Figures 11A-11I Figure 2 shows the interaction of SHSY5Y cells with the indicated dilutions of Vern extract ( Figure 11A ) or with a specified concentration of the compound caffeic acid ( Figure 11B )、Myricetin( Figure 11C ), Rutin ( Figure 11D ), quercetin ( Figure 11E ), Kaempferol ( Figure 11F ), α-lapachone ( Figure 11G )、1,2:2,4-Di-p-Methylbenyliedene Sorbitol( Figure 11H ) and myristicin ( Figure 11I ) were incubated for 24 hours, and then cell death was assessed using PI staining and FACS analysis. The results are the mean ± SEM of three independent experiments.

[0028] Figures 12A-12G Figure SHSY5Y cells were treated with the indicated concentrations of quercetin-3-β-glucoside ( Figure 12A ), geranin ( Figure 12B )、Stachydrine Hydrochloride( Figure 12C )、Glycitein( Figure 12D ), 8-hydroxyquinoline ( Figure 12E ), di-p-methylbenzylidene sorbitol ( Figure 12F ) and cyanidin chloride ( Figure 12G Figure 3. Cell death was assessed by PI staining and FACS analysis after incubation with 5% paraformaldehyde (P < 0.05). Results are the mean ± SEM of three independent experiments.

[0029] Figures 13A-13G Figure SHSY5Y cells were treated with the indicated concentrations of Yangonin ( Figure 13A ), chlorogenic acid ( Figure 13B )、4-hydroxycoumarin ( Figure 13C )、Betaine Figure 13D), Rosmarinic acid ( Figure 13E )、Nobiletin( Figure 13F ) and Murrangatin ( Figure 13G Figure 3. Cell death was assessed by PI staining and FACS analysis after incubation with 5% paraformaldehyde (P < 0.05). Results are the mean ± SEM of three independent experiments. DETAILED DESCRIPTION

[0030] Extracts of Vernonanthura nudiflora have been prepared in the past by exhaustive organic ethanol extraction followed by fractionation into various organic solvents. Some compounds identified in these extracts, such as the pipocarphin sesquiterpene lactones, particularly 8α-tigloyloxy-10α-hydroxy-hirsutinolide, as reported in the aforementioned publication by Ramos A.VG et al., 2019, as well as some triterpenoids, glycosylated steroids, and some flavonoids, including velutin and chlorogenic acid derivatives, have also been tested for antiproliferative activity in cell lines with varying degrees of success. It has now been surprisingly discovered that, in contrast to organic ethanol extraction, subjecting V. nudiflora to hydroethanolic extraction for extended periods of time provides a distinct extract containing significantly greater amounts of these compounds. As demonstrated in the accompanying examples, in the hydroethanolic extracts according to some embodiments of the present invention, with the exception of chlorogenic acid, the vast majority of compounds reported in Ramos AVG et al., 2019 were not identified by gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS). The extracts prepared according to the present disclosure are not only capable of killing cancer cells by utilizing the VDAC1 pro-apoptotic pathway, but also show significant effects in xenograft mouse models and, notably, beneficial effects in the treatment of various cancers in human patients, particularly when administered as co-therapy.

[0031] Thus, by its first aspect, the present disclosure provides a composition comprising a hydroethanolic plant extract or any fraction thereof, wherein the plant is selected from the group consisting of Vernonanthura nudiflora, Plantago asiatica, and a species of the family Salix family selected from the group consisting of Baccharis crispa, Baccharis trimera, and Baccharis articulata, and any combination thereof (referred to herein as "plants"). For example, the plant species Vernonanthura nudiflora, Baccharistrimera, or Baccharis artuculata, and Plantago asiatica are individually extracted and a mixture thereof is prepared such that a mixture ratio of 40% Vernonanthura nudiflora, 40% Baccharis articulata / trimera, and 20% Plantago asiatica is obtained. As another example, the plant is Vernonanthura nudiflora or a combination of Vernonanthura nudiflora and Plantago asiatica.

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

[0033] As described above, the inventors have discovered that prolonged hydroethanolic extraction of plant material, as described below, provides an extract characterized by a group of compounds that are different from the group of compounds obtained by exhaustive organic ethanol extraction, and that have a significant pro-apoptotic effect. In specific embodiments, the hydroethanolic extract of at least one of the plants prepared by the extraction method described in detail below can be defined as comprising at least one of the compounds listed in Tables 5-11 below, for example, as comprising at least 60% (in the proportions recited therein) of the compounds, 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 one skilled in the art, for example by comparing the output of analytical methods such as mass spectrometry coupled to chromatography, e.g., LC-MS / MS, GC-MS / MS. The peak positions of the compounds, their molecular weights, their relative intensities (areas), and / or their fragmentation patterns can be used. In Tables 5-11, the compounds listed belong to their respective extracts as follows: A, B, C: compounds identified only in V. nudiflora, B. trimera and Plantago, respectively, and AB, AC, BC and ABC list compounds identified in two or more of the respective plants.

[0034] The compounds identified in Ramos AVG et al 2019 are presented in Table 1 below. By another aspect thereof, the present disclosure provides a composition comprising a hydroethanolic plant extract containing less than 5% to 20% by weight (e.g., less than 5%) of the compounds listed in Table 1 below, i.e., compounds 1-26 as disclosed in Ramos AVG et al., 2019 (incorporated herein by reference), or being substantially free of these compounds, wherein the plant is selected from Vernonanthura nudiflora, Plantago asiatica, and a species of the family Acanthaceae selected from Baccharis crispa, Baccharis trimera, and / or Baccharis articulata, and any combination thereof. Compounds 1-26 are identified in Ramos AVG et al., 2019, e.g., in FIG1 (compounds 1-17) and FIG3 (compounds 18-26). The names of compounds 1-26 are lupeol, α-amyrin, β-amyrin, taraxasterol, pseudotaraxasterol, piptocarphin A, piptocarphin B, piptocarphin D, 8α-tigloyloxy-10αhydroxy-hirsutinolide, velutin, apigenin, chrysoeriol, β-Sitosterol β-D-glucoside, and β-Sitosterol β-D-glucoside. β-D-glucoside)(sitosterol-3-β-OD-glucopyranoside), Stigmasterol-3-β-OD-glucopyranoside, 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-epoxycyclodecano[b]furan-4-yl(E)-2-methylbut-2-enoate(E)-3-(acetoxymethyl)-6-hydroxy-6,10-dimethyl-2-oxo-2,4,5,6,9,10-hexahydro-7,-10-epoxycyclodeca[b]furan-4-ylmethacrylate acrylate), 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 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-loyloxyhirsutinolide13-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-decahydrooxol[2',3':9,10]cyclodeca[1,2-b]furan-7-yl(E)-2-methylbut-2-enoate9-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 discovered that extended hydroethanolic extraction of plant matter, as described below, provides an extract that has a beneficial effect on treating patients, particularly those suffering from various cancers. The extracts or fractions thereof as defined herein can be obtained by the methods described in more detail herein. That is, the extracts or fractions thereof can be prepared by the method, or can be prepared by different methods, provided that the extracts or fractions obtained are substantially identical to the extracts or specific fractions thereof obtained by the methods described herein. Substantially identical extracts can have at least 60% similarity in composition to the extracts prepared by the methods described herein. Preferably, substantially identical extracts have at least 65% similarity, or at least 70% similarity, or 75, 80, 85, 90 or at least 95% similarity, preferably at least 80% similarity or more. Similarity can be determined as is conventionally known in the art, for example by comparing the outputs of analytical methods such as mass spectrometry chromatography, e.g., LC-MS / MS, GC-MS / MS. Similarity can be assessed using the peak positions, molecular weights, and / or fragmentation patterns of the compounds. In addition, suitable statistical and / or chemometric methods can be employed to determine the similarity and the extent thereof. As demonstrated in the accompanying Examples, fractions of the hydroethanolic extract prepared as described herein demonstrate anticancer properties comparable to or even superior to those of the original extract; thus, extract fractions may also be obtained by the methods described herein, including sub-extractions, or may be obtained in other ways, for example, directly by extracting plant material, in particular V. nudiflora, with solvents such as hexane, chloroform, ethyl acetate, or mixtures thereof, or equivalent solvents, provided that the extract obtained is substantially identical in the sense as described above to the sub-extracts prepared by the methods generally specified herein.

[0036] Furthermore, because the hydroethanolic extract contains a significantly different composition of compounds, a method for preparing such a hydroethanolic extract represents another aspect of the present invention. Thus, a method for preparing a plant extract of the plants described herein, also referred to herein as an extraction method, is provided, comprising combining biomass of V. nudiflora, Plantago major, and at least one species of the Willow family selected from Baccharis crispa, Baccharis trimera, and Baccharis articulata with a hydroethanolic mixture containing 20% ​​to 50% water by volume in ethanol. However, the extraction is performed over a very long time interval, between 3 and 35 days, but preferably between 18 and 23 days, such as three weeks. Without being bound by a particular theory, it is currently believed that this extraction process differs from a thorough organic ethanol extraction, for example, because it is performed in the presence of a large amount of water, which in turn allows for the extraction of more water-soluble compounds, and because some chemical modifications of compounds that may be sensitive to water and other environmental factors may occur during the extended extraction period, making the final extract stable over a long shelf life. Thus, preferably, the method comprises combining V. nudiflora biomass with a water-ethanol mixture and extracting said biomass for a time interval of 3 to 35 days, preferably 18 to 23 days.

[0037] Preferably, the extraction of the plant biomass into the water-ethanol mixture is carried out in a suitable container equipped with a stirring device and with intermittent stirring for a period of 24 to 48 hours at intervals of 1 to 2 hours. The extraction step can advantageously be carried out at ambient temperature, for example, between 15°C and 25°C. However, the extraction can be carried out at a slightly elevated temperature to facilitate and / or accelerate the extraction, for example, at a temperature between 25°C and 45°C. Thus, the temperature can be adjusted according to the needs of the method, between 15 and 45°C, but preferably, the temperature will be between 20 and 25°C. In a specific embodiment, the extraction of the plant biomass into the water-ethanol mixture is carried out at a temperature range of about 15°C to 40°C, preferably between 20°C and 25°C.

[0038] The water-ethanol mixture comprises ethanol in a concentration of 40-80% by volume. The remainder of the water-ethanol mixture is essentially composed of water. Preferably, the concentration of ethanol is 65-75% by volume. Further preferably, the concentration of ethanol is about 70%. Without being bound by a particular theory, it is currently believed that due to the antiseptic properties of the solvent, utilizing 70% ethanol as the extraction medium may be particularly advantageous, thereby contributing to the microbial stability of the extract in its liquid form.

[0039]

[0040]

[0041] Table 1 Compounds 1-26 identified in Ramos, AVG et al., 2019

[0042] As is known in the art, the plant Vernonanthura nudiflora (V. nudiflora) is a species in the genus Vernonia in the Asteraceae family, native to Uruguay, Brazil, and Argentina. It is a flowering subshrub 50-80 cm tall. Extracts of V. nudiflora are interchangeably referred to herein as "plant Vern" and "Vern plant" extracts.

[0043] V. nudiflora biomass and the biomass of plants Baccharis trimera / articulata and Plantago major (i.e., plant-based materials) can be obtained by harvesting the plants, checking the quality of the above-ground parts of the plants (i.e., the parts that reside above the soil, including stems, leaves, petioles, flowers, fruits, and seeds), cleaning and / or washing the plant material to remove loose dirt and contaminants, and drying the clean above-ground plant parts. In particular, the above-ground parts of the stems, leaves, petioles, and seeds of the plants can be used to prepare the extracts of the present disclosure. In some embodiments, the extraction method as defined herein includes drying the plant (e.g., V. nudiflora) before extraction. Drying is preferably carried out indoors at ambient temperature or a slightly elevated temperature (i.e., 15 to 45° C., preferably 20 to 25° C.). Drying is typically performed until at least 40% of the weight of the original plant material is lost. Drying can be continued until a weight loss of 40% to 60% by weight is achieved. This weight loss is typically achieved after 7-8 days at room temperature and after 3-4 days at a temperature of 45° C. The dried biomass can then be ground using a suitable size reduction device, such as a mill, for example, using an electric hammer mill to reduce the size of the dried biomass. The resulting dried ground biomass exhibits a particle size of 5.0-10.0 x 1.0-2.0 x 0.2-1.0 mm. The dried ground plant biomass (e.g., V. nudiflora) can then be used in the extraction step of the method.

[0044] Unlike the thorough extraction in which the extraction mixture is repeatedly exposed to a high volume of extraction solvent simultaneously or in smaller portions, the extraction process according to the present disclosure relies on the same amount of extraction solvent. Therefore, the concentration of the plant biomass can be controlled as needed. Typically, during the extraction step, the concentration of the plant biomass (e.g., V. nudiflora biomass) is 10-30% weight / volume of the extraction solvent, preferably 15-25% weight / volume of the extraction solvent. Preferably, the concentration of the biomass is 18-22% weight / volume, for example, about 20%. In other words, the extraction can be carried out in a ratio of 1:2 to 1:8, for example 1:3 to 1:6, preferably about 1:4, between the extracted biomass and the water-ethanol mixture.

[0045] After the extraction step is completed, that is, after the biomass is extracted into the water-ethanol mixture, the extract can be separated from the reduced biomass (i.e., the residue) to provide the extract. The separation of the extract can be carried out by decanting the extract from the biomass precipitate. Alternatively or additionally, the spent biomass can be separated by filtering through a suitable filter. The filtration can be carried out through cheesecloth, or can be carried out through a suitable filter with a defined pore size, such as a cellulose 15 μm filter. Alternatively, the filtration can be carried out by gravity, or using an electrical device with the above-mentioned pore size. Additionally or alternatively, the separation can also include centrifugation of the mixture to achieve complete sedimentation of the spent biomass and to enable easy decanting of the extract.

[0046] In some embodiments, the resulting extract as defined herein has a plant-derived solids content of 0.5% to 15.0%, preferably 1.0% to 5.0%, or preferably 1.8% to 4.5%, or 2.5 to 3.7% by weight per initial volume of the extract (the dry plant-derived content after evaporation of the solvent, interchangeably referred to herein as "dry basis," "dry extract," etc.). Exemplary dry basis values ​​specific to hydroethanolic extracts can be any of the following: 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 as a solid or dissolved solid with an additional solvent or other dilution medium (e.g., similar or identical to the extraction hydroethanolic mixture). In various embodiments, the original extract (i.e., containing dissolved solids) can be further diluted, for example, by diluting 1 part extract with 9 parts distilled water or 2 parts extract with 8 parts distilled water. Another suitable dilution is 1 part extract to 8 parts distilled water.

[0047] Obviously, this method provides liquid hydroethanolic plant extracts (e.g., V. nudiflora). However, the extract can be dried using a suitable drying method, such as under reduced pressure (e.g., as illustrated below), to provide a dry hydroethanolic extract. The dry ethanolic extract can further be adsorbed onto a suitable inert carrier for use as a powder. Alternatively, or additionally, the liquid hydroethanolic extract can be dried in the presence of at least a portion of an inert carrier to provide a dry hydroethanolic extract of adsorption.

[0048] In another aspect, provided herein is a hydroethanolic extract prepared as described herein and in particular as described above.

[0049] As shown in the Examples section of this article, the hydroethanolic extract exhibits unexpected anti-cancer activity properties in cultured cells and intratumoral injection of mouse xenograft models. Surprisingly, the efficacy of cancer cell eradication increases with increasing dilution of the crude extract. As mentioned above, plant extracts are complex mixtures comprising substances with a variety of possible pharmacological activities. Therefore, on the other hand, the present disclosure provides an artificial mixture (i.e., a mixture that is not necessarily present in nature, also referred to herein as a "fraction" of the compounds present in the extract), which is essentially composed of the compounds present in the hydroethanolic extract defined herein, and contains less than 5%-20% (e.g., less than 5%) by weight of the compounds listed in Table 1 above (i.e., compounds 1-26 disclosed in Ramos, AVG et al., 2019), or is substantially free of these compounds. In various embodiments, the compound fraction can be based solely on the compounds present in Vernonanthura nudiflora or a combination of Vernonanthura nudiflora and Plantago major. In a further embodiment, the fractions may be sub-extractions of the hydroethanolic plant extract, preferably with butanol, hexane, chloroform or ethyl acetate or any combination thereof.

[0050] However, the artificial mixture may further comprise plant-derived compounds, which are not necessarily present only in the V. nudiflora extract, provided that they exhibit at least some antiproliferative activity, for example, as demonstrated in the accompanying examples. Thus, in other embodiments, the artificial mixture according to the present disclosure comprises at least two compounds selected from the group consisting of rutin, kaempferol, myricetin, stachydrine hydrochloride, cyanidin chloride, glycitein, diosmetin, 8-hydroxyquinoline, quercetin-3-β-glucoside, phytol, myristyl ether, α-lapachone and 1,2:2,4-di-p-methylbenzylidene.

[0051] As shown in the examples below, the hydroethanolic extract has demonstrated significant pro-apoptotic activity, inducing apoptosis and mitochondrial dysfunction mediated by VDAC1. Furthermore, the accompanying examples provide evidence of the beneficial therapeutic effects of compositions comprising the extract as defined herein when administered in combination with standard of care therapy or even independently. Thus, in another aspect thereof, the present invention provides a method for treating a proliferative disorder (e.g., cancer), or for reducing the prevalence of its recurrence or occurrence, or for alleviating the symptoms associated therewith, in a patient in need thereof, by administering to said patient a therapeutically effective amount of a composition comprising said hydroethanolic extract as generally described herein or an artificial mixture as described herein.

[0052] As will be understood, the terms "treatment," "treating," or "treat" are intended to include improving one or more clinical manifestations of disease activity in a subject suffering from a proliferative disorder (e.g., cancer), or reducing the prevalence of the disease or condition, its recurrence or occurrence, or at least alleviating the symptoms associated therewith. As is known in the art, a "proliferative disorder" is characterized by excessive proliferation of cells and turnover of the cellular matrix, such as cancer. As used herein to describe the present invention, "cancer" and "tumor" refer equivalently to a malignant proliferative disorder of a tissue or organ. As generally used herein, cancer is preferably a malignant proliferative disorder classified in the 11th revision of the World Health Organization's International Classification of Diseases. th Diseases or conditions in subclass 02 [Neoplasms] of the International Classification of Disease (ICD-11) of the revised World Health Organization International Classification of Disease (hereinafter: "ICD-11").

[0053] In general, the methods and compositions of the present invention can be used to treat solid tumors and / or non-solid tumors and solid tumors. The efficacy of treating a disease or condition can be assessed, for example, using clinical trials as known in the art. Preferably, the clinical trial is a controlled clinical trial. Depending on the purpose of the clinical study and its objectives, the control group for measuring the effects of a clinical trial involving cancer patients can be a group receiving supportive care or a known standard of care, depending on the severity of the condition. Clinical studies are typically randomized clinical studies in which the control group receives one of the control treatments and the treatment group receives varying doses of a composition comprising a hydroethanolic extract as generally described herein. The range of test doses can be determined based on preclinical data in previous pilot clinical studies and obtained in other ways as known in the art. A clinical study can have one or more primary endpoints, i.e., measurable results, and can further have one or more secondary endpoints, i.e., further measurable results. A clinical study can be considered successful if at least one primary and / or at least one secondary outcome is achieved, i.e., the measurable result of the treatment group is statistically significantly different from the measurable result of the control group and is within a predetermined range of difference. A sufficient number of patients can be enrolled in the clinical study, the number of which is controlled by the desired outcome and the expected difference to meet the required statistical power. Enrolled patients must meet the inclusion criteria for the disease or disorder, condition and / or symptom and must not have any of the exclusion criteria defined as not allowing certain patients who may not benefit from treatment or who may be at risk of developing side effects or otherwise have their medical safety compromised by treatment.

[0054] The present disclosure also provides a composition comprising the hydroethanolic extract as defined herein (e.g., in a 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.

[0055] By way of example, a proliferative disorder or cancer as defined herein is primary or metastatic prostate cancer, Langerhans cell histiocytosis, testicular cancer, colon cancer or breast cancer.

[0056] The present invention relates to the treatment of subjects in need thereof (also referred to herein as patients). "Patient" or "subject in need thereof" refers to any organism, such as a mammal, such as a human, that may be affected by the conditions described above and is in need of the treatment methods described herein. It is within the skill of the clinician to diagnose a disease or condition in a subject for whom the compositions and methods described herein are useful.

[0057] Administration according to the present disclosure is by a route selected from the group consisting of oral administration, intravenous administration, intramuscular administration, and intraperitoneal administration.

[0058] The term "therapeutically effective amount" is intended to mean the amount of a composition as defined herein, or any portion thereof, that elicits a desired biological or medical response, which can be determined by such means known in the art, for example, by preclinical and clinical trials, for example, such as those described herein. Treatment of a patient in need thereof as disclosed herein requires administration of an effective amount of the hydroethanolic extract to the patient. The dosage (i.e., therapeutically effective amount) can vary between 0.008 mL / kg (kg - patient body weight) and 2 mL / kg of a diluted 1:8 hydroethanolic extract, i.e., between 0.001 mL / kg and 0.25 mL / kg of concentrated mL, which means between 0.033 mg / kg (dry weight / kg patient) and 8.6 mg / kg (dry weight / kg patient). Preferably, the dosage (i.e., therapeutically effective amount) can vary between 0.048 mL / kg (kg patient body weight) and 1 mL / kg of a diluted 1:8 hydroethanolic extract, i.e., between 0.006 mL / kg and 0.125 mL / kg of concentrated mL, which translates to between 0.2 mg / kg (dry weight / kg patient) and 4.3 mg / kg (dry weight / kg patient). As shown in the accompanying examples, the amount administered orally can be 8 to 80 mL of a 1:8 diluted extract per day, and the amount administered intravenously can be 6 mL of undiluted extract once per week. The dosage is preferably expressed on a dry basis of the extract. Thus, in some embodiments, the dosage can vary between 25 and 500 mg per day.

[0059] The specific dosage for a particular disease or condition may be adjusted according to the severity of the condition, patient factors, comorbidities, and the desired therapeutic outcome.

[0060] The oral daily dose as described herein can be administered in a variety of regimens, depending on the patient's mental state, general health, and desired outcome. These regimens include once-daily administration, i.e., the entire daily dose is administered once a day; twice-daily administration, i.e., the total daily dose is divided into two preferably equal doses and administered at reasonable intervals, preferably every 12 hours; three times a day, i.e., the total daily dose is divided into three preferably equal doses and administered at reasonable intervals, preferably every 8 hours; or four times a day, i.e., the total daily dose is divided into four preferably equal doses and administered at reasonable intervals, preferably every 6 hours. When administered orally, the hydroethanolic extract can be administered with or without food, preferably without food.

[0061] In some embodiments, the treatment method or use of the hydroethanolic extract is in addition to standard of care therapy, such as conventional chemotherapy, particularly in combination with cell proliferation inhibition chemotherapy or apoptosis inducing chemotherapy. Since the effects of the hydroethanolic extract as described herein are directly mediated by VDAC1, without being bound by theory, it is hypothesized that the disclosed hydroethanolic extract may also be effective in cancers that overexpress anti-apoptotic cellular factors.

[0062] In some other embodiments, the hydroethanolic extract can be provided with a composition comprising it. For administration by parenteral route, the extract can be provided as a liquid for injection. Preferably, for example, for intravenous administration, the liquid comprising the extract is diluted in a large volume of physiological solution such as used in medicine. For this purpose, the hydroethanolic extract (or its dry form) can be combined with an inert ingredient (such as a pharmaceutical excipient). For the principles of formulation, the technical staff refers to Remington's Pharmaceutical Sciences, 18th edition, 1990, ISBN 9780912734040 or a suitable textbook.

[0063] The hydroethanolic extract can be formulated into a liquid dosage form. The liquid dosage form can be the original hydroethanolic extract, in which case it will be in the form of a tincture. The dry basis concentration of the tincture can be adjusted by dilution to enable more precise dosing. The extract can also be in the form of a diluted extract, with a dilution greater than 1:5. The diluted extract can contain the same solvent as the original hydroethanolic extract, or it can have a higher amount of water. If the concentration of ethanol in the diluted extract is less than 20% by volume, the diluted extract may further contain a preservative.

[0064] The hydroethanolic extract can be further formulated into a medicated syrup, for example by mixing the original tincture with a predetermined amount of syrup. To maintain a hydroethanolic composition, the tincture can be further formulated into an elixir, for example by mixing the original tincture with a predetermined amount of syrup diluted with ethanol.

[0065] Hydroethanolic extracts, particularly dried or lyophilized hydroethanolic extracts, can be formulated into capsules, such as gelatin capsules or hypromellose capsules. The liquid hydroethanolic extract can be adsorbed onto a carrier and then carefully evaporated to dryness to provide a powder suitable for filling capsules. As known in the art, the dried extract or adsorbed extract can be further compressed into tablets. The tablets or capsules can be further coated, for example with a protective coating, and packaged in suitable containers, such as bottles or blister packs.

[0066] Inactive ingredients suitable for use in the dosage forms described herein can be found in Handbook of Pharmaceutical Excipients, 9th edition, 2020, ISBN 9780857113757, or equivalent texts known to those skilled in the art.

[0067] ***

[0068] Unless otherwise specified, all scientific and technical terms used herein have the meanings commonly used in the art. The definitions provided herein are for ease of understanding certain terms frequently used herein and are not meant to limit the scope of the present disclosure.

[0069] As used herein, the term "about" indicates that a value may deviate from the recited value by up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20%, with the deviation range including integer values ​​and, if applicable, non-integer values, constituting a continuous range. As used herein, the term "about" refers to ±10%.

[0070] The terms "comprises," "comprising," "includes," "including," "having," and their cognates mean "including but not limited to." This term encompasses the terms "consisting of" and "consisting essentially of.

[0071] Various embodiments and aspects of the present invention as described above and as claimed in the claims section below find experimental support in the following examples. The following examples are representative of the techniques employed by the inventors in implementing aspects of the present invention. It should be understood that while these techniques are illustrative of preferred embodiments for practicing the present invention, those skilled in the art will recognize, in light of this disclosure, that many modifications may be made without departing from the spirit and intended scope of the present invention.

[0072] It will be appreciated that certain features of the invention that are described, for the sake of brevity, in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, combinations of various features of the invention that are described, for the sake of clarity and illustration, in the context of a single embodiment may also be provided separately or in any suitable subcombination, or as appropriate in any other described embodiment of the invention.

[0073] 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.

[0074] Various other features according to the present invention as described herein for aspects of methods of treating conditions apply mutatis mutandis to compositions and / or dosage forms for use in these methods according to the teachings herein, and vice versa. As used herein, the terms "composition" and the like are used interchangeably with hydroethanolic plant extracts as described herein, and with compositions comprising at least a portion of a hydroethanolic extract, e.g., a composition ready for administration to a patient in need thereof, or with dosage forms as described herein. Example

[0075] 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.

[0076] Cell lines and culture

[0077] U-87MG (human glioblastoma) and SH-SY5Y (human neuroblastoma) cells were maintained at 37° C. and 5% CO 2 in recommended culture medium supplemented with 10% FBS, 1 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0078] Table 2. Antibodies used in the experiments

[0079]

[0080] Material

[0081] 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 reagent JetPRIME was from PolyPlus (Illkirch, France). Annexin-V (FITC) was obtained from Alexis Biochemicals (Lausen, Switzerland). Dulbecco's modified Eagle's medium (DMEM), 7.5% BSA, 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 obtained from Invitrogen (Waltham, MA). TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) kit was obtained from Promega (Madison, WI). XTT cell viability assay kit was obtained from Biological Industries (Beit Haemek, Israel). Table 2 above lists the primary and secondary antibodies used against the indicated proteins, their catalog numbers, sources, and dilutions used in immunoblotting (WB) and immunofluorescence (IF) experiments. TLC silica gel 60 F254 plates were obtained from Merck (Darmstadt, Germany).

[0082] Freeze-drying of hydro-ethanolic plant extracts

[0083] The dried product of the water-ethanol plant extract produced as detailed above was obtained by freeze drying as follows. First, the plant extract sample (1 liter) in the water-ethanol phase was removed from the mother liquor by the ethanol phase. This step was carried out by applying a vacuum of 750mbar at 45°C over a period of 80 hours. After completing the drying step, the volume of the sample was reduced to 450mL, so that all the ethanol originally present was obviously completely eliminated. A starch-based carrier (N-Zorbit 2144, Ingredion) was then added and homogenized to the final product by stirring for three (3) minutes at a ratio of 1 gr of modified starch per 5mL of the original extract (i.e., 200 g per liter of the original extract before vacuum drying). The sample was then frozen at -80°C for 72 hours (by introducing the sample into a super freezer that reached the temperature mentioned). Once the sample was frozen, it was freeze-dried at -50°C and 1 MPa vacuum for 96 hours. A uniform and dry solid (234 gr) was obtained. Once collected, the sample was stored at room temperature and kept in the dark.

[0084] Cell treatment and cell death analysis using plant extracts

[0085] Cells (6 × 10 5 100 μL of culture medium (500 μL / ml, 70-80% confluent) were incubated with ethanol extracts of plants A (V. nudiflora), B (B. trimera), or C (Plantago major), or mixtures thereof, prepared as described above, at the indicated dilutions in culture plates containing 500 μL of culture medium (each). 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 adding PI (6.25 μg / ml) to the cells, followed by immediate analysis using an EC800 Eclipse flow cytometer or a fluorescence-activated cell sorter (FACS) caliber (Beck-ton-Dickinson, San Jose, CA) and BD CellQuest Pro software.

[0086] For apoptosis analysis by PI and Annexin V-FITC staining, cells (2 × 10 5 ), washed, and resuspended 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 resuspended in binding buffer (200 μL), to which PI was immediately added. Flow cytometry analysis was then performed using an EC800 Eclipse Flow Cytometer. At least 10,000 events were collected and recorded on a dot plot.

[0087] Cell viability assay

[0088] The effects of various plant extracts on SH-SY5Y cell survival were determined using an XTT-based kit (Biological Industries, Beit Haemek, Israel) according to the manufacturer's protocol. Briefly, cells were seeded in 96-well plates and incubated at 37°C with 5% CO2, and 24 hours later were treated with different concentrations of extracts for the times specified in the figure legends. XTT reagent (2,3-bis-(2-methoxy-4-nitro-5-sulfinyl)-(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.

[0089] Reactive oxygen species and intracellular Ca 2+ Determination of levels

[0090] To measure the accumulation of reactive oxygen species (ROS) in mitochondria, SH-SY5Y cells were seeded in 6-well plates (1 × 10 5 The cells were treated with the specified plant extracts for 24 hours and then incubated with MitoSOX-Red (a mitochondrial superoxide indicator for live cell imaging) at 37°C for 10 minutes. Fluorescence was measured using flow cytometry (iCyt, Sony Biotechnology, San Jose, CA). At least 10,000 events were recorded on the FL2 detector, expressed as a histogram, and analyzed with ec800 software (Sony Bio-technology, San Jose, CA). Positive cells showed a shift toward an enhanced level of green fluorescence (FL2).

[0091] Cytosolic Ca was analyzed using Fluo-4-AM (Invitrogene, Grand Island, NY) as follows 2+ Level [Ca 2 + ]i. After appropriate treatment, cells were harvested (1 × 10 6Cells were collected by centrifugation (1,500 x g, 10 min), 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 μM Fluo-4 in 200 μL HBSS(+) buffer for 30 min at 37°C in the dark. After washing away the remaining dye, the cells were incubated with 200 μL HBSS(+) buffer and [Ca] was immediately measured by FACS. 2+ The cells were analyzed using an EC800 Eclipse flow cytometer (Sony Biotechnology). Positive cells showed a shift toward increased levels of green fluorescence (FL1).

[0092] Cellular Ca2+ levels in living cells were monitored using the high-content Operetta screening system (Perkin-Elmer, Hamburg, Germany). 2+ In each well, ten fields were imaged using a 20× wide-field objective with an excitation filter of 520–550 nm and an emission filter of 560–630 nm.

[0093] Cross-linking experiments

[0094] To assess the amount of VDAC1 protein dimers formed, cells were treated with plant extracts for the indicated times and concentrations, harvested, washed with PBS, pH 8.3, and incubated with the cross-linking reagent EGS (ethylene glycol bis(succinimidyl succinate)) (Pierce chemicals TX, USA) at a ratio of 3 mg protein / ml / 300 μM EGS for 15 minutes. 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).

[0095] Gel electrophoresis and immunoblotting

[0096] 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 lysate was then centrifuged at 12,000 x g (10 minutes at 4°C) and the protein concentration was determined. Aliquots (10-20 μg of protein) were subjected to SDS-PAGE and immunoblotting using various primary antibodies (sources and dilutions are provided in Table 2 above), followed by incubation 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 intensity of the appropriate β-actin signal, which served as a loading control.

[0097] Gas chromatography-mass spectrometry (GC-MS) analysis

[0098] CG-MS analysis of plant extracts A, B, and C was performed using a 7890B mass detector; 5977A, Agilent Technologies; Column 5MS UI. Compounds were identified using library name W 10N 14L (NIST MS Search 2.2). The individual compound names, identification masses (maximum value 100%), and peak areas (Ab*s) for each compound are shown in Table 4 below.

[0099] Liquid chromatography-mass spectrometry (LC-MS / MS) analysis

[0100] The plant extract compounds were identified by 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 modes.

[0101] The Q Exactive is an instrument featuring an API source with S-lens ion optics, a quadrupole mass filter for precursor ion selection, and a collision cell for performing MS / MS experiments. It is equipped with an Orbitrap analyzer.

[0102] Chromatographic separation of the compounds was performed using an ACQUITY UPLC® HSS T3 Waters column (2.1 × 100 mm, particle size 1.8 μm) (Waters). The column temperature was set at 40°C, and the sample injection volume was 5 μL. 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.

[0103] The ESI source was operated in positive and negative ion modes with the following parameters: capillary voltage of 3.5 kV, sheath drying and auxiliary gas (N2) flow rates of 60 L / min and 20 L / min, respectively, capillary temperature of 275 °C, and drying gas temperature of 300 °C.

[0104] The collision-induced dissociation was carried out with ultrahigh purity (99.999%) nitrogen at a flow rate of 0.5 ml / min.

[0105] The major compounds of the plant extracts were identified by comparing their retention times (RT) and mass spectrometric (Q Exactive; Thermo Scientific) fragmentation patterns with 25 nce (collision energy normalized to m / z 500). The mass spectrometer was operated in positive and negative modes, and the fragmentation patterns of the compounds were obtained. The precursor ion for the positive mode was [M+H] + , for negative mode [MH] - .

[0106] Data were processed using the associated X-Calibur and Compound Discoverer programs to identify matching compounds. MS data, MS / MS fragmentation spectra, and molecular formulas were generated using predicted compositions*, M / Z Cloud, and ChemSpider, and compared with literature data and several databases to annotate the compounds in the extracts. Molecular formulas generated from the predicted compositions in the MS experiments were compared with literature data, with a maximum error of 2 ppm.

[0107] Thin layer chromatography (TLC) separation

[0108] TLC silica gel 60 F254 plates (Merck, 20 × 20 cm) were used to separate the hydroethanolic plant extracts of A, B, and C, as well as the 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.

[0109] Xenograft mouse model

[0110] On day 0 (0D), U-87MG cells (3 × 10 6 10 cells / mouse) were inoculated subcutaneously (sc) into the hind flank of athymic eight-week-old male nude mice (Envigo). Tumor size was measured daily using a digital caliper and the volume was calculated. 3 At 14 days (on day 14, "14D"), mice were randomly divided into several groups (5 mice / group). One group was injected intratumorally with HBSS (5.33 mM KCl, 0.44 mM KH2PO4, 138 mM NaCl, 4 mM NaHCO3, 0.3 mMNa2HPO4 and 5.6 mM glucose, pH 7.3) containing 0.14% ethanol (control, untreated), and the 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 μM. Xenografts were injected three times a week. Mice were sacrificed 34 days after cell inoculation and tumors were excised. The experimental design is schematically presented in Figure 6A Tumors were fixed in 4% buffered formaldehyde, embedded in paraffin, and processed for immunofluorescence (IF). These experimental protocols were approved by the Institutional Animal Care and Use Committee of Ben-Gurion University.

[0111] Immunofluorescence (IF) of tumor tissue sections

[0112] Formalin-fixed, paraffin-embedded sections (5 μm thick) of U-87MG cell-derived tumors from control and V. nudiflora extract- or phytol-treated tumors were deparaffinized by placing the slides at 60°C for 1 hour and deparaffinized with xylene, followed by rehydration with a graded ethanol series (100%–50%). Antigen retrieval was performed in 0.01 M citrate buffer (pH 6.0) at 95°C–98°C for 20 minutes. After washing in PBS, pH 7.4, sections were incubated in 10% normal goat serum for 2 hours and then incubated with primary antibodies overnight at 4°C (as detailed in Table 2). Sections were washed thoroughly with PBST, incubated with fluorescently labeled secondary antibodies (as detailed in Table 2) for 2 hours, washed five times with PBST, and coverslipped with fluoroshield mounting medium (Immunobioscience, Mukilteo, WA). Fluorescence images were observed with an Olympus IX81 confocal microscope. Quantification of protein levels, as reflected by staining intensity, was analyzed across the entire area of ​​the sections using Image J™ software.

[0113] TUNEL assay

[0114] Paraffin-embedded fixed tumor sections (5 μm thick) were processed for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay using a dead-end fluorescent TUNEL system according to the manufacturer's instructions. The sections were deparaffinized, balanced in PBS, permeabilized with proteinase K (20 μg / ml in PBS), post-fixed in 4% paraformaldehyde, and incubated in TdT reaction mixture at 37°C in the dark for 1 hour. The slides were then washed in saline-sodium citrate buffer, counterstained with PI (1 μg / ml), and coverslipped with fluoroshield mounting medium (Immunobioscience, Mukilteo, WA). Fluorescent images of apoptotic cells (green) and nuclei (red) were captured using a confocal microscope (Olympus IX81). Quantitative analysis of stained slides was performed using the Image J program.

[0115] Statistics and data analysis

[0116] Results obtained from three independent experiments are presented as mean ± SE. Statistical significance is reported as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****).

[0117] Example 1

[0118] Preparation of extracts of Vernonanthura nudiflora, Baccharis trimera and Plantago major

[0119] The following extraction procedures were performed on each of the specified plants. Wild-growing plants of each of the plant species Vernonanthuranudiflora, Baccharis trimera and Plantago asiatica were harvested and selected based on the overall condition of the plant material (also referred to herein as "biomass"), for example, the absence of parasites, the presence of agricultural chemicals nearby, etc. Next, the plant material of each of the plants was carefully cleaned before extraction, leaving only the above-ground parts (i.e., the plant parts residing above the soil, including stems, leaves and petioles). The plant material was washed with plenty of water and then air-dried indoors for about 7-8 days at room temperature or in a heating chamber (45°C) for about 3-4 days until its total mass was reduced by 50%. The dried plant material was then ground to a final size of about 5.0-10.0 mm × 1.0-2.0 mm × 0.2-1.0 mm using a mechanical grinder. The ground plant biomass was then extracted over a 21-day period in a reactor containing a hydroethanolic solution (70% ethanol in water, interchangeably referred to herein as a hydroethanolic solution) wherein the concentration of the plant material was 0.2 g / ml. The solution was periodically agitated for approximately 1-2 hours every 24-48 hours. The extract obtained was then filtered by gravity through a filter having a pore size of 15 μm and packaged in an opaque and airtightly sealed container, which was stored at room temperature in a cool and ventilated place to prevent exposure to light. In order to prepare a mixture of the three extracts, plant parts of the plant species Vernonanthura nudiflora, Baccharis trimera, and Plantago asiatica were individually extracted and a mixture thereof was prepared to obtain a mixing ratio of 40% Vernonanthura nudiflora, 40% Baccharis trimera, and 20% Plantago asiatica. The extract obtained was stored at 4°C and was active within one year.

[0120] Example 2

[0121] Study on apoptosis induced by plant ethanol extracts

[0122] To investigate the effects of hydroethanolic extracts from plants of Vernonanthura nudiflora, Baccharis trimera and Plantago asiatica and mixtures thereof on cancer cell survival and apoptotic cell death, cells of the cancer cell line SHSY5Y were incubated in the presence of various dilutions of extracts from Vernonanthura nudiflora, Baccharis trimera and Plantago asiatica or a 2:2:1 (V:V:V) mixture thereof as described above.

[0123] First, the activity of three plant hydroethanolic extracts and their mixtures in inducing cell death as a function of their amount was analyzed by propidium iodide (PI) staining and flow cytometric analysis. PI is a fluorescent intercalating agent that stains nucleic acids by binding to DNA and intercalating between bases, has little or no sequence preference, and is not membrane permeable, making it useful for distinguishing apoptotic cells from intact healthy cells based on membrane integrity. Figure 1A1 and Figure 1A2 Shown are controls ( Figure 1A1 ) and plant Vern extract ( Figure 1A2 )-induced cell death.

[0124] Figure 1B The results presented in clearly showed that the extract of V. nudiflora (Vern plant) was the most active in triggering massive cell death, followed by the extract of Plantago asiatica (Pla plant), with the extract of B. trimera (Bac plant) being the least active. Figure 1B As shown, extract Vern at a dilution of 1:500 induced higher cell death than mixed extract at a dilution of 1:100 or plant Bac extract at a dilution of 1:166, indicating 5-fold and 3-fold higher activity, respectively.

[0125] To determine whether the induced cell death was related 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 the extracellular side of the plasma membrane in apoptotic cells. Figure 1C The results presented clearly show that Vern extract induces apoptotic cell death.

[0126] Next, the effects of plant extracts on cell viability were analyzed using the XTT assay as described above. Figure 1D -F, for experiments with Bac and Pla extracts of Vern plants, respectively. Vern extracts highly reduced cell viability in a dilution-dependent manner after 24, 48, or 72 hours of incubation ( Figure 1D), while Bac extracts diluted 1:500 showed some decreased cell viability after 48 and 72 h of incubation ( Figure 1E ), and Pla plant extracts showed no decrease in cell survival at all tested dilutions and incubation times ( Figure 1F Considering the fact that the XTT assay relies on the reduction of NADH produced in mitochondria, these results suggest that the Vern extract, but not the Bac or Pla plant extracts, induces mitochondrial dysfunction. Furthermore, without wishing to be bound by theory, the results suggest that the induction of cell death by the three plant extracts involves different active compounds and modes of action.

[0127] Furthermore, as shown in Table 3 below, the cell death-inducing activity of the Vernonanthura nudiflora plant extract remained stable even when the extract was heated. This was demonstrated by incubating the Vernonanthura nudiflora plant extract at dilutions of 1:500, 1:1000, and 1:2000 at 4°C, 45°C, 60°C, and 80°C for 10 minutes and then determining (as described above) the ability of the extract to induce cell death in SH-SY5Y cells when the cells were incubated (24 hours) in the presence of the heated extract dilutions.

[0128] Table 3 The cell death activity of Vern plant extracts is stabilized after heating the extracts

[0129]

[0130] Example 3

[0131] Hydroethanolic plant extracts induce VDAC1 overexpression and oligomerization

[0132] It has been previously shown that apoptotic triggers, such as chemotherapeutic drugs, stress conditions, and radiation, induce VDAC1 overexpression, thereby shifting the equilibrium from monomers to VDAC1 oligomers, followed by the release of pro-apoptotic proteins from mitochondria and apoptosis. Therefore, the effects of plant extracts on VDAC1 expression levels and its oligomerization were next tested in SHSY5Y and U-87MG cells. These cells were incubated with different dilutions of plant extracts for 24 hours, and then VDAC1 expression levels were analyzed using immunoblotting, as detailed above.

[0133] Figure 2A Vern plant extract was demonstrated to induce VDAC1 overexpression in two different cell lines tested, the neuroblastoma-derived cell line SHSY5Y and the glioblastoma-derived U-87MG cell line. In both cell lines, Vern extract highly increased the expression level of VDAC1 by up to 3-fold ( Figure 2A ), which is accompanied by pro-apoptotic activity, e.g. Figure 2B Shown are the levels of cell death as a function of Vern extract dilution (IC 50 = 1:800). In addition, all three extracts increased VDAC1 oligomeric forms in a concentration-dependent manner, which were stabilized by chemical cross-linking using EGS and monitored by immunoblotting, as detailed above ( Figure 2C ). It is clear that the highest VDAC1 oligomerization level was induced by Vern plant extract ( Figure 2C 、 Figure 2D ), consistent with its highest cell death-inducing activity.

[0134] Interestingly, in the absence of chemical cross-linking, VDAC1 oligomers were observed even after exposing the cells to high detergent concentrations (i.e., 1% SDS) and heating at 70°C for 5 min (results shown in Figure 2E 、 Figure 2F In this case, too, when cells were treated with the Vern plant extract, the levels of oligomeric VDAC1 were highest, as seen with VDAC1 overexpression, oligomerization, and apoptosis induction. This suggests that the VDAC1 oligomers induced by the plant extract are very stable.

[0135] Without being bound by theory, the above results suggest that the active compounds in the Vern plant extract act by enhancing VDAC1 expression levels, leading to VDAC1 oligomerization and apoptosis.

[0136] Example 4

[0137] Vernonanthura nudiflora extract increases intracellular Ca 2+ and reactive oxygen species (ROS) production

[0138] Reactive oxygen species (ROS) are known to be involved in the induction of apoptosis. Therefore, we next measured mitochondrial ROS after treatment with Vern plant extracts, as detailed above.

[0139] like Figure 3A As shown, treatment of cells with Vern plant extracts induced the production of ROS. It has been previously shown that calcium ions (Ca 2+ ) levels are involved in apoptosis induction, and Ca 2+ is required for VDAC1 overexpression and VDAC1 oligomerization induced by apoptotic stimuli. Therefore, the effects of Vern plant extracts on cellular Ca2+ expression were analyzed using Flu-4 and FACS or by Operetta as described above. 2+ Levels ([Ca 2+ ])'s effect( Figure 3B-3D ). Both assays demonstrated that the extract significantly increased cellular [Ca2+ ]level.

[0140] Example 5

[0141] Analysis of phytol and ethyl linoleate identified in plant extracts and their effects on cell death and on VDAC1 expression and oligomerization

[0142] In order to identify at least some of the chemical compounds present in the hydroethanol extract, they are carried out gas chromatography-mass spectrometry (GC-MS) analysis, as mentioned above.In these extracts, multiple compounds have been identified, as listed in Table 4. Two kinds of abundant compounds identified in the extract have been tested, i.e. the cell death inducing activity of phytol and ethyl linoleate (ethyl linoleate) (ethyl linoleate (linoleate acid ethyl ester)).First, the relative amount ( Figure 4A In the extracts of Vern (Vernonanthura nudiflora), Bac (Baccharis trimera), and Pla (Plantago major), the amounts of phytol in the three plant extracts were approximately 1800, 800, and 1200 nmol / ml (or μM), respectively ( Figure 4B , the concentration of ethyl linoleate in these extracts is also shown).

[0143]

[0144]

[0145]

[0146] Table 4. Compounds identified by GC-MS in plant extracts—Vern, Bac, and Pla

[0147] CG-MS analysis was performed using a 7890B mass detector, an Agilent Technologies 5977A, and a Column 5MS UI. Compounds were identified using the library name W 10N 14L (NIST MS Search 2.2). The names, identification masses (maximum value 100%), and peak areas (Ab*s) for each compound are presented.

[0148] Next, the activity of phytol and ethyl linoleate in inducing cell death was analyzed by incubating SHSY5Y cells with different concentrations (50-200 μM) of each compound for 24 or 48 h. Figure 4CAs shown, phytol induced cell death, with maximal cell death being 100% and half-maximal cell death (IC 50 Ethyl linoleate showed weak cell death activity, which increased from about 15% in untreated cells to about 40% at 200 μM ethyl linoleate ( Figure 4C ).

[0149] Next, VDAC1 expression levels and its oligomerization were tested in cells incubated with phytol or ethyl linoleate. As shown in Figure 5A1, phytol, but not ethyl linoleate ( Figure 5A2 ), especially at the high concentrations used, induces VDAC1 overexpression ( Figure 5B ) and VDAC1 oligomerization ( Figure 5C , Figure 5D ).like Figure 5B and Figure 5D As shown, phytol induces VDAC1 overexpression and oligomerization in a concentration-dependent manner and is associated with a consequently higher activity in cell death induction.

[0150] Notably, the Vern plant extract induced cell death at dilutions of 500-1,000, with phytol concentrations in these dilutions ranging from 2.5 to 5 μM. When used on its own, phytol exhibited cell death at concentrations exceeding 50 μM, suggesting, without wishing to be bound by theory, that extract components other than phytol were involved in the cell death induction. Without being bound by a particular theory, it is believed that neither phytol nor ethyl linoleate were responsible for the observed activity of V. nudiflora extract A.

[0151] Example 6

[0152] Antitumor activity of Vern plant extracts and phytol in a mouse xenograft model

[0153] Next, as detailed above and as Figure 6A The effects of two dilutions of Vern plant extracts and the effect of phytol on tumor growth were tested in a xenograft GBM mouse model schematically shown in FIG. Briefly, U-87MG malignant glioma cells were subcutaneously (sc) inoculated into the hind flank of 7-week-old male athymic nude mice. When the tumor volume was approximately 50 mm 3At 4 hr, mice were divided into four groups with similar average volumes and treated with Vern plant extract (at two different final concentrations of 1:500 and 1:250) or phytol (at a concentration of 75 μM). Control tumors were injected with PBS containing 5% ethanol (the final concentration in the tumor was 0.14%). Treatment was given three times a week, and tumor growth was monitored three times a week. All mice were sacrificed 34 days after cell inoculation, tumors were excised, weighed and fixed, and sections were immunofluorescently stained for selected proteins.

[0154] Figure 6B The results shown indicate that the tumors in the controls grew exponentially over time and grew in a similar manner when the tumors were injected with the Vern plant extract to a final dilution of 1:250. Figure 6B-6D As shown in Figure 2, tumors treated with a higher dilution of Vern plant extract (i.e., 1:500) showed approximately a 70% reduction in tumor volume and weight. The results indicate that the higher concentration (1:250) of Vern plant extract was not as effective as the 1:500 dilution. Similar results with essentially no effect on tumor growth were obtained using 1:100 and 1:300 dilutions of Vern plant extract, as presented in the quantitative Figure 7A and Figure 7B Without wishing to be bound by theory, the decreasing anticancer effect with increasing concentration of the Vern plant extract may be due to the protective activity of other compounds present in the extract when present in higher concentrations at low extract dilutions.

[0155] The results also showed that phytol at the concentration used (75 μM) significantly inhibited tumor growth, but to a lesser extent than the 1:500 dilution of the Vern plant extract ( Figure 6B-6D ).

[0156] Next, fixed paraffin-embedded sections of the tumors were stained for Ki-67, a proliferation marker, indicating that both Vern plant extract and phytol inhibited cell proliferation by approximately 80% ( Figure 6F Representative confocal microscopy images of control (left panel), Vern plant extract (middle panel), and phytol-treated cells (right panel) are presented in Figure 6E Finally, apoptosis was analyzed by TUNEL staining as described above. Figure 6G Representative micro-images and Figure 6H As shown in the quantification presented in Figure 3, while there were no obvious TUNEL-positive cells in control tumors, the majority of cells were TUNEL-positive in Vern plant extract-treated tumors and, to a lesser extent, in phytol-treated tumors, with staining colocalizing with PI nuclear staining ( Figure 6G, white arrows). Therefore, Vern plant extracts were more effective than phytol in inducing cell death.

[0157] The results clearly demonstrated that these treatments induced apoptotic cell death and indicated that the significant reduction in tumor size in Vern plant extract and phytol treated xenografts could be attributed to the inhibition of cell proliferation (reduced Ki-67 staining) and the induction of cell death.

[0158] It has been previously shown that metabolic changes that occur during malignant transformation involve a series of functional abnormalities and mutations that contribute to elevated glycolysis and increased expression levels of glucose transporter (Glut-1) and glycolytic enzymes such as hexokinase (HK-1). The expression levels of these proteins and other proteins related to metabolism, microenvironment, and cancer stem cells were assessed by immunofluorescence (IF) staining. Figure 8A As shown, IFs of control tumors derived from U-87MG cells showed high expression levels of Glut-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, the expression levels of these proteins were reduced in tumor-bearing animals treated with Vern plant extracts or phytol, as shown in Figure 2. Figure 8A and Figure 8B Similarly, the expression levels of VDAC1 and HK-1 were decreased in tumor-bearing animals treated with Vern plant extracts and phytol, as shown in Figure 8C Representative images and Figure 8D The quantification thereof is shown in Figure 2. The decreased expression levels of metabolism-related enzymes in Vern plant extract- and phytol-treated tumors indicated a decrease in energy production in these treated tumors.

[0159] The effects of intratumoral administration of Vern plant extracts or phytol on the expression levels of proteins associated with angiogenesis were evaluated. Figure 9A As shown, immunostaining for the endothelial cell marker CD-31 showed that the level of this cell marker was significantly reduced in tumors treated with Vern plant extract or phytol, indicating a significant decrease in the number of blood vessels, wherein quantification showed a decrease of approximately 70% and 60% in Vern plant extract and phytol treated animals (tumors) respectively relative to control tumors ( Figure 9B ).

[0160] The effects of Vern plant extracts and phytol on the tumor microenvironment (TME) were further analyzed by IF staining of the fibroblast marker α-smooth muscle actin (α-SMA). Figure 9C Representative micrographs and Figure 9D As shown in the quantification, tumor-bearing animals treated with Vern plant extract or phytol showed a 70% and 60% reduction in α-SMA expression, respectively.

[0161] Accumulating recent evidence supports the cancer stem cell (CSC) hypothesis, which suggests that a subpopulation of malignant cells exhibits stem cell properties of self-renewal and differentiation. CSCs are resistant to conventional cytotoxic / antiproliferative therapies. In glioblastoma multiforme (GBM), the condition from which this cell line was derived, the proteins Sox2, CD133, SSEA1, CD49f, Musashi-1, and nestin are considered to be glioma stem cell CSC markers. IF staining of tumor specimens for Sox2 and nestin showed that in tumor-bearing animals treated with Vern plant extract or phytol, the expression levels of these CSC markers were highly reduced by approximately 70%, as shown in Figure 2. Figure 10A Representative micrographs and Figure 9B These results indicate that Vern plant extract and phytol treatment of U-87MG-derived tumors eliminated CSCs associated with tumor recurrence.

[0162] Example 7

[0163] Identification of Chemicals in Extracts of V. nudiflora, B. trimera, and Plantago asiatica

[0164] As described above, the extracts were analyzed using GC-MS / MS. The identified compounds are listed in Table 5 (also referred to herein as "Table A"), Table 6 (also referred to herein as "Table AC"), Table 7 (also referred to herein as "Table C"), Table 8 (also referred to herein as "Table AB"), Table 9 (also referred to herein as "Table B"), Table 10 (also referred to herein as "Table ABC"), and Table 11 (also referred to herein as "Table BC") below. Additional information about the compounds is presented in Tables 5-11 below, which list the compound's molecular formula, calculated molecular weight (Calc. MW, Da), retention time (RT, in minutes), and maximum chromatographic peak area (Area) in the relevant plant type.

[0165] Table 5 Compounds identified only in V. nudiflora extracts

[0166]

[0167] Table 6 Compounds shared by plant extracts of V. nudiflora and Plantago major

[0168]

[0169] Table 7 Compounds identified only in the plant extracts of Plantago asiatica

[0170]

[0171] Table 8 Compounds common to plant extracts of V. nudiflora and B. trimera

[0172]

[0173] Table 9 Compounds identified only in B. trimera plant extracts

[0174]

[0175] Table 10 Compounds common to plant extracts of V. nudiflora, B. trimera and Plantago asiatica

[0176]

[0177]

[0178]

[0179] Table 11. Compounds common to plant extracts of B. trimera and Plantago asiatica

[0180]

[0181]

[0182] Example 8

[0183] Effects of compounds identified in V. nudiflora extracts on cancer cell viability

[0184] To analyze the effects of some compounds identified in V. nudiflora extracts on cancer cells, SHSY5Y cancer cells were incubated (24 hours) with hydroethanolic Vern extract (1:1000) or with each of the compounds listed in Table 12 below at different concentrations, and then analyzed for apoptosis induction by these compounds using propidium iodide staining (n=3). Figures 11, 12, and 13 show the results of cell death induction obtained by various tested V. nudiflora extract compounds.

[0185] In particular, without wishing to be bound by any theory, the activities of the V. nudiflora extract compounds can be classified as being relatively effective in inducing cell death, such as Figures 11A-11I As shown (for example, Figures 11B-11I caffeic acid, myricetin, rutin, quercetin, kaempferol, α-lapachone, 1,2:2,4-di-p-methylbenzylidene and myristyl ether) were moderately effective in inducing cell death, as shown in Figures 12A-12G As shown (for example, Figures 12A-12G quercetin-3-β-glucoside, diosmetin, stachydrine hydrochloride, glycitein and 8-hydroxyquinoline, 1,2:2,4-di-p-methylbenzylidene sorbitol and cyanidin chloride) were only slightly effective in inducing cell death, or as shown in Figures 13A-13G As shown (for example, Figure 13E rosmarinic acid).

[0186] The concentration of compound that produced 50% cell death (IC 50 ), the maximum cell death achieved and the concentration required. In addition, the molecular weight (Mw) and empirical formula are indicated. The concentration in the Vern extract was determined using a calibration curve obtained for each compound using commercially available compounds (ND - not determined).

[0187]

[0188] Table 12 Effects of isolated compounds on cell survival and apoptosis

[0189] Example 9

[0190] Fractionation of hydroethanolic extracts of Vernonanthura nudiflora by organic solvents and identification of specific compounds in their fractions

[0191] In addition to the above analyses, in order to identify the active compounds in the hydroethanolic extract of Vernonanthura nudiflora, samples of the hydroethanolic extract of Vernonanthura nudiflora were fractionated using different organic solvents as described below.

[0192] First, a sample of the hydroethanolic extract of Vernonanthura nudiflora (100 ml) was air evaporated to increase its concentration approximately 10-fold (i.e., to a final volume of 10 ml), and then fractionated using the solvents hexane, ethyl acetate, chloroform, and butanol according to the separatory funnel method known in the art. Briefly, the solvent (10 ml) was added to the concentrated hydroethanolic extract of V. nudiflora (7.5 ml), shaken thoroughly, and separated into a solvent phase and an aqueous phase, and each of the solvents was extracted independently. The above procedure was repeated (twice) to further extract any remaining agent from the obtained aqueous phase. Next, each of the obtained extracts was dried by evaporation (at 50°C), and the dried extract was then dissolved in 70% ethanol (0.2 ml of 70% ethanol).

[0193] Example 10

[0194] Analysis of cell death induction (apoptosis) by organic solvent extracts of hydroethanolic extracts of V. nudiflora

[0195] Next, according to the protocol detailed above, the activity of the solvent extracts detailed above in cell death induction was analyzed in SHSY5Y and PC-3 cancer cells. The results for SHSY5Y and PC-3 cancer cells shown in Tables 13 and 14 below, respectively, showed that all solvent extracts had cell death induction activity.

[0196] Briefly, Tables 13 and 14 below show the results of the various solvent extracts described in detail above inducing cell death in SHSY5Y cancer cells and PC-3 cells (200,000 cells / well / 6-well plate), respectively, which were incubated (24 hours) with the specified 1:400 dilution of the V. nudiflora hydroethanolic extract and its fractions isolated by ethyl acetate, chloroform, butanol, and hexane extraction, and further subjected to analysis using propidium iodide (PI) staining as described in detail above (n=3). Ethanol (70%) was used as a control.

[0197] Table 13 Cell death induction by solvent extracts of V. nudiflora hydroethanolic extracts in SHSY5Y cancer cells

[0198]

[0199] Table 14 Cell death induction in PC-3 cancer cells by solvent extracts of V. nudiflora hydroethanolic extracts.

[0200]

[0201] The results obtained for the cell death activity in cancer cells using solvent extracts of the hydroethanolic extract of V. nudiflora showed that hexane, chloroform and ethyl acetate (which clearly showed different sets of compounds) exhibited cell death activity, especially hexane.

[0202] Example 11 - Case Report

[0203] Patient 1

[0204] This example describes a case report of a patient in need thereof treated by administering a composition according to Example 1. Briefly, extracts of Baccharis articulata, Plantago asiatica, and Vernonanthura nudiflora were mixed in a ratio of 2:1:2 to obtain a mixed plant extract (also referred to herein as "MPE").

[0205] A 69-year-old man presented for treatment with metastatic prostate cancer, grade 4, Gleason factor 9, and PSA 12.3 (ICD-11, 2C82). Immediate treatment with degarelix (240 mg) and bicalutamide (50 mg) was initiated. A few weeks later, the patient decided to also try MPE.

[0206] After obtaining informed consent, the patient was instructed and agreed to use the provided medication according to the instructions or return the unused medication. The patient began treatment with MPE. The dose was 15 mL per os, diluted 1:8 in distilled water, administered orally four times daily.

[0207] After 25 days of treatment, the patient showed a PSA of 1.3. Degarelix was switched to darolutamide. Bicalutamide He was switched to leuprolide and after 10 days of retreatment, the patient showed a PSA score of 0.46.

[0208] According to treating professionals, such improvements are typically very rare and were completely unexpected because the improvement was so rapid and the original patient condition had a 31% 5-year relative survival rate.

[0209] Patient 2

[0210] A 34-year-old woman had been diagnosed with Langerhans cell histiocytosis (ICD-11, 2B31.2) for approximately two years prior to presentation. She was treated with immediate vinblastine / prednisone with desmopressin and cabergoline to manage symptoms, and sennosides as needed. Concurrently, she decided to also try MPE.

[0211] After obtaining informed consent, the patient was instructed and agreed to use the provided medication according to the instructions or return any unused medication. The patient began treatment with MPE. The dose was 20 mL, orally administered four times daily in a 1:8 dilution of MPE in distilled water.

[0212] After 6 weeks of treatment, the patient was in remission with no tumor observed. The treatment further included only MPE. At the most recent follow-up, almost two years after diagnosis, the patient remained in remission.

[0213] According to treating professionals, such improvements are often very rare and completely unexpected.

[0214] Patient 3

[0215] A 34-year-old man was diagnosed with testicular cancer (ICD-11, 2C80) with mixed germ cell malignancy approximately one year prior to presentation for treatment of a metastatic subpleural nodule. Initial treatment consisted of resection and follow-up imaging. Metastases were observed within the first year after resection. No treatment was initiated immediately after diagnosis for the metastatic pleural nodule. Clinical examination revealed a tumor AFP marker of 22 ng / mL and a nodule measuring 23 x 16 mm.

[0216] The patient decided to try MPE before any conventional chemotherapy. After obtaining informed consent, the patient was instructed and agreed to use the provided medication according to the instructions or return any unused medication. The patient began receiving MPE. The dose was 15 mL, administered orally four times daily in a 1:8 dilution of MPE in distilled water. Concomitantly, the patient received MPE intravenously, with 6 mL of undiluted MPE in 500 mL of physiological solution administered once a week.

[0217] One month later, the AFP marker was 13 ng / mL, and the patient agreed to conventional chemotherapy without abandoning MPE treatment. A conventional regimen of bleomycin, etoposide, and cisplatin was administered every 21 days for four cycles. Intravenous MPE was increased to twice weekly.

[0218] After one month of combined therapy, the AFP marker was 2.4 ng / mL and the number of metastatic subpleural nodules had decreased by 30%. After completing BEP, the patient continued etoposide / cisplatin every 21 days and MPE once a week for an additional eight months. After this period, the markers returned to normal and there was no evidence of metastatic nodules. The patient continued oral MPE and remained in remission at the most recent follow-up, almost two years after diagnosis.

[0219] According to the treating professionals, such improvements were completely unexpected and can be attributed to concomitant MPE treatment.

[0220] Patient 4

[0221] A 47-year-old man was diagnosed with testicular cancer (ICD-11, 2C80) with a large left paraaortic retroperitoneal metastatic mass (4.2 × 3.7 × 5.7 cm) that met criteria for adenopathy. One month later, the patient underwent surgical resection to remove the primary tumor. The patient decided to try MPE four days before surgery and before any conventional chemotherapy treatment. After obtaining informed consent, the patient was instructed and agreed to use the provided medication according to the instructions or return the unused medication. The patient was started on MPE. The dose was 2 mL, administered orally in a 1:8 dilution, four times a day. Concomitantly, the patient received MPE intravenously, administering 3 mL of undiluted MPE in 500 mL of physiological solution twice a week.

[0222] When evaluated three weeks after surgery, no metastatic masses were identified. According to the treating professionals, these improvements are extremely rare and completely unexpected, as no conventional treatment was given.

[0223] Example 12 - Veterinary Case Report

[0224] Patient 1

[0225] A 12-year-old female Dachshund dog weighing 8 kg was diagnosed with multiple inguinal mammary tumors. The patient began receiving MPE. The dose was 1 mL of undiluted MPE applied to the tumor base once a week, which was increased to 2 mL starting in week 2. Before the second injection, clinical observations showed that the tumors were slightly smaller and softer in consistency. After the fourth injection, the patient showed significantly greater vitality, with a significant decrease in tumor size and activity.

[0226] Two weeks later, the fifth treatment, a 2 mL dose, was performed. The tumors had decreased even further. Ulcers appeared on the skin at each site, emitting a thick, foul-smelling fluid. After drainage, the tumor areas were flat, with no protrusions.

[0227] During the three-month follow-up, no tumor growth was observed and the subject remained in good health.

[0228] Patient 2

[0229] A 13-year-old male Cimarron dog, weighing 35 kg, was diagnosed with a perianal tumor with possible hepatoid adenoma. The patient was started on MPE. A dose of 3 mL of undiluted MPE was applied to the base of the tumor every two weeks. Two days after the second injection, the treating veterinarian detected tumor shedding accompanied by heavy bleeding. Part of the tumor had detached from the original mass, and its size had also decreased significantly.

[0230] At follow-up one week after the second injection, the patient showed more energy, appetite, and increased weight and muscle tissue. After the third injection, the improvements continued and the patient showed even more energy.

[0231] One week after the third injection, the remaining tumor mass was resected due to a significant reduction in size (approximately 20% of the original).

[0232] No recurrence was observed 4 months after surgery. The patient maintained a good appetite and energy and continued to regain muscle tissue.

Claims

1. A composition comprising a hydroethanolic plant extract or any fraction thereof, wherein the plant is selected from the group consisting of Vernonanthura nudiflora, Plantago asiatica and a species of the family Salix family selected from the group consisting of Baccharis crispa, Baccharis trimera and / or Baccharis articulata, and any combination thereof.

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

3. A composition comprising a hydroethanolic plant extract containing less than 5-20% by weight of the compounds listed in Table 1, or being substantially free of these compounds, wherein the plant is selected from the group consisting of Vernonanthuranudiflora, Plantago, and a species of the family Willow family selected from the group consisting of Baccharis crispa, Baccharis trimera, and / or Baccharisarticulata, and any combination thereof.

4. A composition according to any one of the preceding claims, wherein the 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 the extraction of the plant biomass into the water-ethanol mixture is carried out at a temperature ranging from about 15°C to 40°C, preferably from 20°C to 25°C.

6. The composition of any one of the preceding claims, wherein the biomass is extracted at a concentration of about 15% to 25% weight per total extraction solvent volume.

7. A composition according to any one of the preceding claims, wherein the method comprises drying the plant prior to extraction.

8. The composition according to claim 7, wherein the drying is carried out at a temperature of about 15°C to 45°C, preferably about 20°C to 25°C.

9. A composition according to claim 7 or claim 8, wherein the 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. A composition according to any preceding claim, wherein the method comprises extracting aerial parts of the plant, such as aerial parts of Vernonanthura nudiflora.

11. A composition according to any one of the preceding claims, wherein the process comprises separating the residue of the plant biomass from the water-ethanol mixture to provide the extract.

12. The composition of claim 11, wherein the separating comprises filtering and / or centrifuging the extraction mixture.

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

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

15. The composition according to claim 14, wherein the concentration of the plant-derived substance in the liquid hydroethanolic extract is from 0.5 wt% to 15.0 wt%, preferably from 1.0 wt% to 5.0 wt% on a dry weight basis.

16. A composition according to claim 14 or claim 15, wherein the concentration of the plant-derived material in the liquid hydroethanolic extract is from 2.5 wt% to 3.7 wt% on a dry weight basis.

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

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

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

20. The composition according to claim 18, wherein the fraction is a sub-extract 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, kaempferol, myricetin, stachydrine hydrochloride, cyanidin chloride, glycitein, diosmetin, 8-hydroxyquinoline, quercetin-3-β-glucoside, phytol, myristyl ether, α-lapachone and 1,2:2,4-di-p-methylbenzylidene.

22. A composition according to any preceding claim for use in treating a proliferative disorder in a subject in need thereof.

23. The composition for use of claim 22, wherein the proliferative disorder is cancer.

24. A composition for use according to claim 22 or claim 23, wherein the proliferative disorder or cancer is 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 the composition is supplied to the subject in an amount of 0.033 mg / kg to 8.6 mg / kg of extract per kilogram weight of the subject on a dry basis.

26. The composition for use according to any one of claims 22 to 25, wherein the composition is supplied to the subject in an amount of 20 to 500 mg of extract per subject on a dry basis.

27. The composition for use according to any one of claims 22 to 26, wherein the 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 the subject is concomitantly receiving treatment with an additional anticancer agent, preferably apoptosis-inducing chemotherapy.

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

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

31. The method of claim 30, wherein the proliferative disorder is cancer.

32. The method of claim 30 or 31 , wherein the proliferative disorder or cancer is primary or metastatic prostate cancer, Langerhans cell histiocytosis, testicular cancer, colon cancer, or breast cancer.

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