Patent Description:
The written disclosure herein describes illustrative embodiments. Reference is made to certain of such illustrative embodiments that are depicted in the figures, in which:.

Trees in the genus Cecropia (in the Urticaceae family) are generally dioecious, with few branches (usually with a candelabrum-like branching system), and a hollow trunk. These trees can have stilt roots, fully amplexicaul stipules, peltate blades with one to two trichilia at the base of the petioles, and inflorescences arranged in digitate clusters (or a single inflorescence), usually enveloped by a spathe until anthesis. In such trees, the interfloral bracts are generally absent, the flowers have two stamens, and the trees have small, dry fruits enveloped by a tubular greenish perianth (<NPL>); <NPL>)). Trees within this genus are widespread and abundant. For instance, these generally fast-growing trees are distributed across the tropical and subtropical rainforests of Mexico, Central America, and South America at elevations below <NUM> (<NPL>). The genus includes <NUM> species (<NPL>), including species popularly known, among other folk names, as "yarumo," "guarumo," "guarumbo," "embaúba," "ambay," "torém," and "trumpet tree. " (<NPL>); <NPL>); <NPL>); <NPL>).

Trees within the genus Cecropia are of ecological significance. For instance, due to their rapid rate of growth, such trees are often the primary colonizers of deforested tropical areas (Monro et al. , available at http://www. org/science/tropamerica/ neotropikey/families/Urticaceae. htm (<NUM>)) and act as invasive species in non-native regions. (<NPL>); GISD, available at http://www. org/gisd/species. php?sc=<NUM>. ) In addition, most species within the genus Cecropia are ant-plants or myrmecophytes. In other words, such trees may live in a mutualistic relationship with a colony of symbiotic ants, especially ants of the genus Azteca. They possess specialized structures for offering shelter and food to ants in exchange for protection against natural enemies. (<NPL>); <NPL>).

Medicinal claims related to the genus Cecropia have been advanced in several Latin American countries. Material from such plants has been used as a diuretic, an antioxidant, an antitussive, an expectorant, and for the treatment of several ailments or diseases such as cough, asthma, diabetes, inflammation, anxiety, and depression. (<NPL>); <NPL>); <NPL>). Reports have also been made purporting the efficacy of plant-derived material in wound healing, pain relief, and antimicrobial activity. The therapeutic properties of these plants have generally been attributed to compounds in the plants, such as flavonoids, proanthocyanidins (<NPL>)), terpenoids, steroids (<NPL>)), chlorogenic- and caffeic acid (<NPL>)), and other phenolic compounds (<NPL>)).

Flavonoids are a large group of secondary metabolites. The carbon structure of such compounds can be abbreviated C6-C3-C6, with two aromatic rings and a cycled oxygen. They can be found as aglycones, glycosides, and methyl derivatives at different positions of their core structure. The most abundant flavonoid glycosides are the flavones O/C glycosides and the flavonol O-glycosides. The glycosidic bond is normally found at position <NUM> or position <NUM>, and the carbohydrate unit can be a glucoside, galactoside, ramnoside, or arabinoside. The biological activities of these compounds can depend not only on the structural differences of the core but also on their glycosylation patterns. Document <NPL>.

discloses the activity of aqueous leaf extracts of Cecropia on arterial pressure in rats.

This disclosure is related to bioactive compositions, formulations, that involve compounds that can be extracted from plants of the genus Cecropia, such as flavonoid compositions.

It will be readily understood that the embodiments, as generally described herein, are exemplary. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order of specific steps or actions may be modified.

Amounts, concentrations, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also all the individual numerical values or sub-ranges encompassed within that range, as if each numerical value and sub-range were explicitly recited. For example, an amount of from <NUM> to <NUM> should be interpreted to include not only the explicitly recited limits of <NUM> and <NUM>, but also individual amounts such as <NUM>, <NUM>, <NUM>, and sub-ranges such as <NUM> to <NUM>, <NUM> to <NUM>, etc. Unless otherwise stated, all ranges include both endpoints.

According to the present invention, there is provided a formulation for use in the treatment of hypertension, as defined in claim <NUM>. Some embodiments of the present disclosure relate to formulations for use in methods of perturbing (e.g., acting as an agonist or antagonist for) one or more G-protein coupled receptors in a mammalian cell according to the claims. In these embodiments, such methods involve obtaining and/or producing a formulation that comprises one or more flavonoids selected from the group consisting of isovitexin-<NUM>"-O-rhamnoside, isovitexin-<NUM>"-O-glucoside, isovitexin-<NUM>"-O-xyloside, isovitexin-O-xyloside, and isoorientin-<NUM>"-O-xyloside. Such flavonoid(s) may be derived from plant material of the genus Cecropia.

For example, in some embodiments, the formulation is obtained by extracting the one or more flavonoids from plant material of the genus Cecropia. In some embodiments, the flavonoids from the plant material are isolated from exclusively (or in greater abundance) from pistillate flowers (female) plants of the genus Cecropia. The extraction may be carried out via any suitable solvent. For example, in some embodiments, the solvent used to carry out the extraction may be selected from the group consisting of water, alcohols, ketones, esters, ethers, polyhydric alcohols, chlorine-containing solvents, and mixtures of at least two of the aforementioned solvents. Stated differently, in some embodiments plant material from the Cecropia genus may be mixed with one or more solvents to extract a portion of the plant material into the one or more solvents. In some embodiments, the solvent or solvent system is an organic solvent. In some embodiments, the solvent comprises or consists of an alcohol, such as methanol, ethanol, or butanol (e.g., alcohols with four or fewer carbon atoms). The extracted portion of the plant material can be isolated from the remainder of the plant material. Subsequently, at least a portion of the solvent(s) may be removed to obtain a first composition. In some embodiments, the extracted components are further purified (e.g., via chromatography or a fractionated liquid/liquid or liquid/solid extraction technique).

In some embodiments, the plant material from the genus Cecropia may be macerated prior to extracting the one or more flavonoids from the plant material. In some embodiments, the plant material is exclusively from aerial parts of the plant (e.g., the leaves and stems) and does not include roots. In some embodiments, the plant material is from one or more plants of the species Cecropia obtusifolia.

In some embodiments, the first composition obtained by extraction may be combined with a second composition to form a formulation for administration to mammalian cells and/or a mammalian subject. In some embodiments, the first composition is between <NUM>% and <NUM>% of the formulation by weight, between <NUM>% and <NUM>% of the formulation by weight, and/or between <NUM>% and <NUM>% of the formulation by weight.

In some embodiments, the one or more flavonoids are between <NUM>% and <NUM>%, such as between <NUM>% and <NUM>% of the formulation by weight. In some embodiments, the formulation includes two or more, three or more, four or more, or all five of the flavonoids selected from the group consisting of isovitexin-<NUM>"-O-rhamnoside, isovitexin-<NUM>"-O-glucoside, isovitexin-<NUM>"-O-xyloside, isovitexin-O-xyloside, and isoorientin-<NUM>"-O-xyloside. In some embodiments, the flavonoids of the formulation include or consist of isovitexin-<NUM>"-O-xyloside and isovitexin-<NUM>"-O-rhamnoside. In some embodiments, the formulation consists essentially of the one or more flavonoids. In other words, the formulation may include one or more flavonoids that are configured to perturb one or more G-protein coupled receptors, but does not include other compounds or substances that materially affect the ability of the formulation to perturb the G-protein coupled receptors. In some embodiments, the formulation consists essentially of isovitexin-<NUM>"-O-xyloside and isovitexin-<NUM>"-O-rhamnoside. In some embodiments, the formulation includes one or more of a dispersant, a humectant, a carrier, an antistatic agent, a filler, or a diluent. In some embodiments, the formulation includes a total Cecropia extract, which includes all active extractable components of the plant. In other embodiments, the extract is further purified to obtain a fraction that includes crude, semi-purified, or purified flavonoids. In some embodiments, only the active components of the extract are used in the formulation.

The formulation may be delivered to one or more mammalian cells to perturb one or more G-protein coupled receptors. In other words, the mammalian cells may be contacted with an effective amount of the formulation. For example, in some embodiments, a cell may be identified in which perturbation of one or more G-protein coupled receptors is desired. For example, in some embodiments, the formulation is delivered to cells in vitro. In other embodiments, the formulation is delivered in an effective amount to a mammalian (e.g., human) patient, such as a patient in which perturbation of G-protein coupled receptor is desired. In some embodiments, such patients may have been selected for treatment based on a diagnosis of high blood pressure or an elevated risk of high blood pressure. The one or more G-protein coupled receptors that are perturbed by the formulation may be selected from the group consisting of (<NUM>) angiotensin II receptor, type <NUM>, (<NUM>) angiotensin II receptor, type <NUM>, and (<NUM>) endothelin receptor type B. In some embodiments, the one or more flavonoids of the formulation are, individually or collectively, configured to perturb all three of (<NUM>) the angiotensin II receptor, type <NUM>, (<NUM>) the angiotensin II receptor, type <NUM>, and (<NUM>) the endothelin receptor type B. In some embodiments, the formulation acts as an agonist on the AT<NUM> and ETB receptors, and antagonizes the AT<NUM> receptor. In some embodiments, perturbation of the one or more G-protein coupled receptors causes vasodilation in a mammalian subject. Stated differently, administration of the formulation to a patient may cause a decrease in blood pressure. In other or further embodiments, not claimed, administration to a patient may provide neuroprotective, cardioprotective, and/or vasculoprotective effects. In some embodiments that include two or more flavonoids, the flavonoids may provide a synergistic effect on the one or more G-protein coupled receptors relative to the same quantity of each of the two or more flavonoids alone.

Leaves and stems of dried Cecropia obtusifolia were ground to obtain <NUM> of pulverized plant material. The pulverized plant material was then exhaustively extracted with three liters of methanol. The resulting alcohol extract was washed with <NUM> of hexane and two liters of dichloromethane under agitation. The solvent was then removed under reduced pressure at temperatures of less than <NUM> to afford a dry solid.

The dry alcohol fraction from C. Obtusifolia (<NUM>) was submitted to a column chromatography over Sephadex LH-<NUM> (<NUM> × <NUM> i. d, <NUM>), using a mobile phase of water:ethanol (<NUM>:<NUM>). Ten fractions were collected. Fraction <NUM> and fraction <NUM> were pooled ("Fraction <NUM>-<NUM>") based on their HPLC profile, yielding <NUM> of solid material.

The chemical compounds of the solid material from Fraction <NUM>-<NUM> were then isolated by semi-preparative reverse phase liquid chromatography (HPLC) to yield five pure compounds. More particularly, to isolate the chemical compounds of the solid material from Fraction <NUM>-<NUM>, the sample was applied using a manual injector and separated on a C18 column (<NUM> × <NUM> i. ; <NUM>) at <NUM>. The mobile phase was a gradient generated by combining solvent A (<NUM>% acetic acid, adjusted to pH <NUM>) and solvent B (acetonitrile) as follows: <NUM>-<NUM>, linear gradient from A:B (<NUM>:<NUM> v/v) to A:B (<NUM>:<NUM> v/v); <NUM>-<NUM> isocratic A:B (<NUM>:<NUM> v/v). The flow rate was <NUM>/min. For detection, a chromatogram was recorded at <NUM> using a diode array detector (DAD) while the UV spectrum was monitored over a range of <NUM>-<NUM>.

The five compounds that were separated by HPLC were identified based on their mass spectrometry data, nuclear magnetic resonance (NMR) spectrums (<NUM>H NMR, <NUM>C NMR), and by comparison with a reference standard and/or the available literature data. The data used for identification of these compounds is set forth below.

As shown in both <FIG> and Table <NUM>, when subjected to ESI/MS (positive ion mode), compound P9 displayed a molecular ion (MI) at m/z <NUM> [M+H]+ and a base peak (BP) at m/z <NUM> [M+Na]+. Its molecular formula was deduced as C<NUM>H<NUM>O<NUM>. Fragmentations of the molecular ion (<FIG>) and the base peak (i.e., <FIG>) produced abundant ions Yo+; <NUM> [(M+H)-<NUM>]+ and <NUM> [(M+Na)-<NUM>]+. Such masses are attributed to loss of a neutral sugar moiety (deoxyhexose) from glycosylated hydroxyl groups. <NPL>); <NPL>). Ions typical of C-hexosyl flavones (see <FIG> and Table <NUM>) were also observed: <NUM>,<NUM>X<NUM>+: <NUM> [(M+Na)-<NUM>-<NUM>]+, and <NUM>,<NUM>X<NUM>+: <NUM> [(M+Na)-<NUM>-<NUM>]+, and <NUM> [(M+H)-<NUM>-<NUM>]+ (<NPL>); <NPL>)).

Compound P9 was also characterized by both <NUM>H NMR and <NUM>C NMR. The experimental results are shown in Table <NUM> alongside published data for isovitexin-<NUM>"-O-rhamnoside (<NPL>)).

Since the NMR chemical shifts were in agreement with reported values, Compound P9 was identified as isovitexin-<NUM>"-O-rhamnoside.

As shown in <FIG>, when subject to ESI/MS (positive ion mode), compound P7 displayed a molecular ion (MI) at m/z <NUM> [M+H]+ and a base peak (BP) at m/z <NUM> [M+Na]+. Its molecular formula was deduced as C<NUM>H<NUM>O<NUM>. Fragmentations of molecular ion (<FIG>) and base peak (<FIG>) produced abundant ions Yo+; <NUM> [(M+H)-<NUM>]+ and <NUM> [(M+Na)-<NUM>]+. Such masses are attributed to loss of a neutral sugar moiety (hexose) from glycosylated hydroxyl groups (<NPL>); <NPL>)). Ions typical of C-hexosyl flavones (see <FIG> and Table <NUM>) were also observed: <NUM>,<NUM>X<NUM>+: <NUM> [(M+Na)-<NUM>-<NUM>]+ and <NUM> [(M+H)-<NUM>-<NUM>]+; and <NUM>,<NUM>X<NUM>+: <NUM> [(M+Na)-<NUM>-<NUM>]+ and <NUM> [(M+H)-<NUM>-<NUM>]+ (<NPL>)).

Compound P7 was also characterized by <NUM>H NMR and <NUM>C NMR. The <NUM>H NMR spectrum shows that a hexose is linked to position <NUM> (flavone) since H-<NUM> is displayed as a singlet at δ <NUM> ppm. See Table <NUM> (providing NMR data for compound P7).

The HMBC spectra (not shown) of compound P7 indicate that compound P7 has a hexose link through an O-glycosidic bond at position <NUM>". The compound P7 is identified as isovitexin-<NUM>"-O-glucoside.

As shown in <FIG>, when subject to ESI/MS (positive ion mode), compound P8 displayed a molecular ion (MI) at m/z <NUM> [M+H]+ and a base peak (BP) at m/z <NUM> [M+Na]+. Its molecular formula was deduced as C<NUM>H<NUM>O<NUM>. Fragmentations of molecular ion (<FIG>) produced an abundant ion Yo+; <NUM> = [(M+H)-<NUM>]+, which can be attributed to a loss of a neutral sugar moiety (pentose) from glycosylation on hydroxyl groups (<NPL>); <NPL>)). The fragmentation pattern of the base peak is shown in <FIG>. Ions typical of C-hexosyl flavones (see <FIG> and Table <NUM>) were also observed: <NUM>,<NUM>X<NUM>+: <NUM> [(M+Na)-<NUM>-<NUM>]+, <NUM>,<NUM>X<NUM>+: <NUM> [(M+Na)-<NUM>-<NUM>]+ and <NUM> [(M+H)-<NUM>-<NUM>]+ (<NPL>).

Additionally, as shown in <FIG>, compound P8 displayed fragment ions associated to a pentose moiety neutral loss and a hexose cleavage: <NUM> [(M+H)-<NUM>-H<NUM><NUM>]+, <NUM> [(M+H)-<NUM>-<NUM><NUM><NUM>]+, <NUM> [(M+H)-<NUM>-<NUM>]+→<NUM>,<NUM>X+-<NUM><NUM>O, <NUM> [(M+H)-<NUM>-<NUM>]+→<NUM>,<NUM>X+-<NUM><NUM>O y <NUM> [(M+H)-<NUM>-<NUM>]+→<NUM>,<NUM>X+.

Compound P8 was also characterized by <NUM>H NMR and <NUM>C NMR. The experimental results are shown in Table <NUM> alongside published date for isovitexin-<NUM>"-xyloside (Zielińska-Pisklak et al. , <NUM> J Pharm Biomed Anal <NUM>-<NUM> (<NUM>)).

As the NMR chemical shifts were in agreement with reported values (see Table <NUM>), compound P8 was identified as isovitexin-<NUM>"-O-xyloside (Zielińska-Pisklak et al. , <NUM> J Pharm Biomed Anal <NUM>-<NUM> (<NUM>).

As shown in <FIG> and Table <NUM>, when subjected to ESI/MS (positive ion mode), compound P6 displayed a molecular ion (MI) at m/z <NUM> [M+H]+ and a base peak (BP) at m/z <NUM> [M+Na]+ that closely correspond with values obtained for compound P8. The molecular formula of compound P6 was deduced to be C<NUM>H<NUM>O<NUM>. Fragmentations of molecular ion (<FIG>) produced an abundant ion Yo+; <NUM> = [(M+H)-<NUM>]+, attributed to loss of a neutral sugar moiety (pentose) from glycosylated on hydroxyl groups (<NPL>); <NPL>)). Ions typical of C-hexosyl flavones (see <FIG> and <FIG>) were also observed: <NUM>,<NUM>X<NUM>+: <NUM> [(M+Na)-<NUM>-<NUM>]+, <NUM>,<NUM>X<NUM>+: <NUM> [(M+Na)-<NUM>-<NUM>]+ and <NUM> [(M+H)-<NUM>-<NUM>]+ (<NPL>)).

Additionally, as shown in <FIG>, compound P6 displayed fragment ions associated to a pentose moiety neutral loss and a hexose cleavage: <NUM> [(M+H)-<NUM>-H<NUM><NUM>]+, <NUM> [(M+H)-<NUM>-<NUM><NUM><NUM>]+, <NUM> [(M+H)-<NUM>-<NUM>]+→<NUM>,<NUM>X+-<NUM><NUM>O, <NUM> [(M+H)-<NUM>-<NUM>]+→<NUM>,<NUM>X+-<NUM><NUM>O and <NUM> [(M+H)-<NUM>-<NUM>]+→<NUM>,<NUM>X+.

Compound P6 was also characterized by both <NUM>H NMR. The experimental results are shown in <FIG> and Table <NUM>.

The <NUM>H NMR spectrum displayed similar chemical shifts to compound P8. These results suggest that compound P6 is an isomer of compound P8. Accordingly, compound P6 is identified as isovitexin-O-xyloside.

As shown in both <FIG> and Table <NUM>, when subjected to ESI/MS (positive ion mode) compound P4 displayed a molecular ion (MI) at m/z <NUM> [M+H]+ and a base peak (BP) at m/z <NUM> [M+Na]+. Its molecular formula was deduced as C<NUM>H<NUM>O<NUM>. Fragmentation of molecular ion (<FIG>) produced an abundant ion Yo+; <NUM> = [(M+H)-<NUM>]+, which was attributed to loss of a neutral sugar moiety (pentose) from glycosylated hydroxyl groups (<NPL>); <NPL>)). As shown in Table <NUM> and <FIG> and <FIG>, ions typical of C-hexosyl flavones were also observed: E<NUM>: <NUM> [(M+H)-Xyl-H<NUM>O]+, E<NUM>: <NUM> [(M+H)-Xyl-<NUM><NUM>O]+, E<NUM>: <NUM> [(M+H)-Xyl-<NUM><NUM>O]+, <NUM>,<NUM>X+-<NUM><NUM>O: <NUM>[(M+H)-Xyl-<NUM>]+, <NUM>,<NUM>X-<NUM><NUM>O: <NUM> [(M+H)-Xyl-<NUM>]+ and <NUM>,<NUM>X+: <NUM> [(M+H)-Xyl-<NUM>]+ (<NPL>).

Compound P4 was also characterized by <NUM>H NMR. The experimental results are shown in Table <NUM> alongside published data for isoorientin-<NUM>"-O-xyloside (<NPL>)).

The <NUM>H NMR spectrum shows that a hexose is attached to position <NUM> of the flavone since H-<NUM> appeared as a singlet at δ <NUM> ppm (<NPL>)). In addition, <NUM>-OH is displayed as a broad singlet at δ <NUM> ppm (<NPL>); <NPL>)). The compound P4 is identified as isoorientin-<NUM>"-O-xyloside.

As shown in <FIG>, when subjected to ESI/MS (positive ion mode), compound P2 displayed a base peak (BP) at m/z <NUM> [M+Na]+. Its molecular formula was deducted as C<NUM>H<NUM>O<NUM>. The compound P2 is identified as chlorogenic acid by direct comparison (retention time) with an analytical standard (<FIG>).

Angiotensin II, receptor type <NUM> ("AT<NUM>"), angiotensin II, receptor type <NUM> ("AT<NUM>"), and endothelin receptor type B ("ETB") are G-protein coupled receptors that play a role in blood pressure regulation and vascular remodeling. Activation of the AT<NUM> receptor can lead to vasoconstriction, aldosterone synthesis and secretion, increased vasopressin secretion, cardiac hypertrophy, augmentation of peripheral noradrenergic activity, vascular smooth muscle cells proliferation, decreased renal blood flow, renal renin inhibition, renal tubular sodium reuptake, modulation of central sympathetic nervous system activity, cardiac contractility, central osmocontrol, and extracellular matrix formation (<NPL>). Activation of the AT<NUM> receptor can induce vasodilation in multiple vascular beds, enhance natriuresis, prevent vascular remodeling by decreasing collagen deposition, and attenuate arterial stiffening. Activation of the ETB receptor can lead to vasodilation and clearance of endothelin <NUM> from the systemic blood.

To investigate the vasculoprotective, neuroprotective, and/or antihypertensive properties of Cecropia extracts, Chinese hamster ovary cells that had been stably transfected with aequorin (CHO-AEQ cells) were transiently transfected with various G-protein coupled receptors.

More particularly, CHO-AEQ cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing <NUM> % fetal bovine serum as previously described (<NPL>)). At <NUM> % confluency, these cells were transiently transfected with pcDNA3. <NUM> + plasmid DNA into which had been inserted the coding region for human AT<NUM>, AT<NUM>, or ETB receptor. The transient transfection was performed with Fugene HD® following the manufacturers' instructions (<NUM>µg DNA to <NUM>µl Fugene HD® in Opti-MEMO medium. After transfection, the cells were cultured for an additional day, after which they were harvested and loaded with coelenterazine-h.

Intracellular calcium can be detected based on the interaction of calcium ions with the calcium binding bioluminescent complex aequorin. Upon calcium binding, aequorin oxidizes coelenterazine-h into coelenteramide with production of CO<NUM> and emission of light (<NUM>) (<NPL>). The CHO-AEQ cells that had been transiently transfected with human AT<NUM>, human AT<NUM> or human ETB receptors were used in a bioluminescence assay to evaluate whether-and to what extent-material isolated from Cecropia obtusifolia could perturb G-protein coupled receptor(s).

Confluent CHO-AEQ cells that were grown and transfected in <NUM><NUM> culture flasks were harvested by a brief treatment with a trypsin/EDTA solution, centrifuged, and resuspended in DMEM-F12 cell culture medium containing <NUM> % BSA and denoted as assay buffer, at a cell density of <NUM> × <NUM><NUM> cells/ml. Subsequently, coelenterazine-h was added to the cell suspension to a final concentration of <NUM>. Cells were then incubated between <NUM> and <NUM> at room temperature in the dark under gentle shaking. After this loading step, <NUM> of assay buffer was added to the cells and then centrifuged for <NUM> at <NUM> rpm in a swinging bucket centrifuge. The resulting cell pellets were gently resuspended in assay buffer containing <NUM> coelenterazine-h at a cell density of <NUM> × <NUM><NUM> cells/ml. After a further incubation of <NUM> at room temperature, the cell suspension was distributed at <NUM>µl/well in white Cellstar <NUM>-well plates.

To assess the activity of the compounds derived from Cecropia obtusifolia extracts, lyophilized crude extract (and compounds that had been identified in the extract) were dissolved in DMSO at <NUM>/ml, and then further diluted in assay buffer to <NUM>µg/ml (final concentration) (<NPL>)). A <NUM> aliquot was then added to each well containing the transiently transfected cells. For comparison, control peptides were diluted in assay buffer to a final concentration of <NUM> angiotensin II or <NUM> endothelin-<NUM>. These control peptides are known to cause a transient rise in cytosolic calcium. The agonist solutions were loaded in the cell injector of a Victor spectrophotometer, and a <NUM>-well plate was inserted.

Measurements were initiated by injecting <NUM>µl solution/well, and the bioluminescence was recorded every <NUM> for <NUM> cycles. To account for slight variations in cell number, a final concentration of <NUM> ATP was added by a second injector to the cells (<NUM>µl/well) and the resulting response was measured.

The effects were quantified by calculation of the area under the curves by integration of the obtained transient responses using GraphPad Prism <NUM>™. The activation of the AT<NUM>, AT<NUM>, or ETB receptors was expressed as a fractional response. This is obtained by dividing the agonist response by the sum of the agonist and ATP response for each well (i.e., agonist response/(ATP+agonist response)). The fractional response is used to normalize the agonist response in each well for the amount of living cells in the well (that is proportional to the ATP response) (<NPL>)).

The effect of the evaluated extracts was calculated as percent inhibition of the control responses and is given as the average ± standard error of three determinations. In order to validate the three receptor assays, the agonist responses were measured after pre-incubation with <NUM> BQ788 or <NUM> losartan, which are selective antagonists for the AT<NUM>, ETB, and AT<NUM> receptors, respectively.

The results are shown in <FIG>. In each figure, the Y-axis represents the amount of cytoplasmic calcium expressed as a fraction from the total intracellular content.

More particularly, <FIG> shows cytoplasmic calcium release after binding of the compounds or extracts to AT<NUM> receptors expressed on CHO-AEQ cells. Extract of the Cecropia genus containing compounds P2, P4, P6, P7, P8 and P9 showed a higher degree of activation of the AT<NUM> receptor than the AT<NUM> endogenous peptide angiotensin II. Compounds P2, P4, P6, P7, P8 and P9, individually tested, showed approximately half of the activity of the extractsuggesting synergism of the individual components of Cecropia genus active extract on the activation of the AT<NUM> receptor.

<FIG> shows cytoplasmic calcium release after binding of the compounds or extracts to the ETB receptors expressed on CHO-AEQ cells. Fraction <NUM>-<NUM>-which is composed mainly of compounds P9 and P8-induced a higher cytoplasmic calcium release than <NUM> of the endogenous agonist endothelin <NUM> (ET-<NUM>). In this experiment, compound P6, which is not the primary component of the extract, is the most active sample tested, followed by P4, P7, P8, P9 and P2.

<FIG> shows that Fraction <NUM>-<NUM> inhibits the agonistic effect of <NUM> angiotensin II on the AT<NUM> receptor with the same intensity as the selective AT<NUM> receptor antagonist losartan. Flavonoids P2, P4, P7, P8 and P9 from this fraction inhibited angiotensin II effect on the AT<NUM> receptor with different intensities. Their effect is less than half of the effect of Fraction <NUM>-<NUM> when tested individually, suggesting a synergistic effect of these components.

Any methods disclosed herein include one or more steps or actions for performing the described method. The method steps and/or actions may be interchanged with one another. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order and/or use of specific steps and/or actions may be modified.

Similarly, it should be appreciated by one of skill in the art with the benefit of this disclosure that in the above description of embodiments, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure. This method of disclosure, however, is not to be interpreted as reflecting an intention that any claim requires more features than those expressly recited in that claim. Rather, as the following claims reflect, inventive aspects lie in a combination of fewer than all features of any single foregoing disclosed embodiment.

Claim 1:
Formulation for use in the treatment of hypertension, where the treatment involves:
obtaining a composition comprising one or more flavonoids selected from the group consisting of isovitexin-<NUM>"-O-rhamnoside, isovitexin-<NUM>"-O-glucoside, isovitexin-<NUM>"-O-xyloside, isovitexin-O-xyloside, and isoorientin-<NUM>"-O-xyloside; and
administering an effective amount of the composition to a mammalian patient;
wherein administration of an effective amount of the composition causes dilation of a blood vessel.