Plant fractions with anti-pathogenic properties

Abstract

The present invention includes methods of using improved compositions and improved compositions comprising thermally stable plant juice soluble vegetable proteins for resisting pathogenic effects; the improvements include that the composition comprising heat stable plant juice soluble vegetable proteins is essentially a water insoluble concentrated substance free of components that are more soluble in plant juice, and is enriched in heat stable proteins of plant juice; dosage forms prepared from the composition are useful for adiponectin agonist action for reducing the risk of one or more conditions selected from the group consisting of type II diabetes, hyperlipidemia, atherosclerosis, hypertension, heart disease and obesity, and are useful for antimicrobial action for reducing the risk of one or more conditions selected from the group consisting of type II diabetes, hyperlipidemia, atherosclerosis, hypertension, heart disease and obesity. The present invention relates to antimicrobial agents for reducing the risk of or treating microbial infections in plants and animals with or without synergy with other antimicrobial agents. The present invention includes a composition substantially characterized by the infrared absorption spectrum provided in the present invention.

Description

[0001] This case is a divisional application of the invention patent application filed on April 1, 2017, with application number 201780022147.2 and invention title "Plant fraction with anti-pathogenic properties". Technical Field

[0002] This invention relates to osmotin-like proteins, compositions containing them, and methods for preparing the compositions from plants. The invention also relates to the use of osmotin-like proteins, comprising impure or semi-purified compositions containing osmotin or osmotin-like proteins, together with other components derived from green leaves in fractions prepared from plants, for the treatment of human diseases and conditions caused by adiponectin deficiency. Such diseases and conditions include type II diabetes, insulin resistance, hyperlipidemia, arteriosclerosis, and heart disease. Background Technology

[0003] Singh et al. (1987) (NARENDRA K. SINGH, CHARLES A. BRACKER, PAUL M. HASEGAWA, AVTAR K. HANDA, SCORR BUCKEL, MARK A. HERMODSON, ED PFANKOCH, FRED E. REGNIER, and RAYA. BRESSAN* Plant Physiol. (1987) 85, 529-536) reported specific alterations in gene expression in salt-adapted tobacco cells, as several novel proteins were produced by the adapting cells, including a major 26kD protein. This protein was named “osmolyte” by Singh et al. because it is synthesized and accumulated by cells that undergo stepwise osmotic regulation of salt or drying stress, rather than by osmotic shock. Singh et al. further added that the osmolyte is a cationic protein with a pI² value > 8.2. Singh et al. also observed that an immunologically relevant 26kD protein with a lower pI was synthesized in unstressed, unadapted cells, but it did not accumulate under normal growth conditions. It has been reported that the accumulation of osmolysins depends on the regulation of cells by NaCl or water stress. The isolation method described by Singh et al. involves homogenizing tobacco cells after cold acetone washing to adapt them to 25 g / L NaCl, (a) extracting them in a detergent-free buffer and collecting the supernatant as fraction I after centrifugation, and (b) extracting cell particles with an extraction buffer containing detergent and collecting the supernatant as fraction II. Singh et al. (1987) also isolated osmolysins by ammonium sulfate precipitation. After discarding 40% of the precipitate, fraction I was collected as an 80% ammonium sulfate precipitate by centrifugation at 10,000 g at 4 °C. It was resuspended in extraction buffer I and dialyzed for 20 hours at 44 °C, changing the water five times with 4 L of distilled water. The supernatant of fraction II was precipitated with 80% ammonium sulfate, and a 10,000 g float was collected (due to the presence of detergent). This float was resuspended in extraction buffer I and dialyzed separately as done for fraction I. After dialysis, fraction I was centrifuged separately at 10,000g for 10 minutes, and the clear supernatant was collected. Fraction II containing turbid precipitate was dissolved by adding an equal volume of resuspension buffer (40 mM potassium phosphate buffer [pH 6.0], 8 M urea, and 0.2% NP-40) to fraction II.

[0004] In CM- Fractions I and II were further purified by A-25 and SCX-polyamide HPLC to obtain osmomodulation protein I and osmomodulation protein II, respectively. They were then dialyzed with water and suspended in buffer to obtain single 26kD proteins as shown by SDS-PAGE.

[0005] Represents porous dextran gels used as molecular sieves, wherein when loaded into After being loaded onto the gel, molecules with the largest molecular size are eluted first, and molecules with the smallest molecular size are eluted last. The remaining molecules are eluted in order of their molecular size, with larger molecules eluted earlier than smaller molecules.

[0006] Oromin II has been found to be resistant to most proteolytic enzymes. Oromin I is more susceptible; however, its N-terminal portion has been found to be resistant to proteolytic enzymes. Oromin II is inherently hydrophobic and granular.

[0007] Singh et al. (1987) pointed out that there was no functional test for osmolytes, and therefore identification was based solely on a uniform band of osmolytes at approximately 26 kD in SDS-PAGE electrophoresis.

[0008] US 5,389,609 points out that tobacco osmolysins "show large amino acid sequence identity, as well as similarity to a 24kD osmolysin-like protein from tomato (Lycopersicon esculentum), and similarity to other proteins including kiwifruit sweet protein from winged arrowroot (Thaumatococcus daniellii), pathogenesis-associated protein S (PR-5) from tobacco, and bifunctional maize trypsin / α-amylase inhibitors. Among these, similarities are found in millet, soybean, carrot (Daucus carota), cotton, potato (Solanum tuberosum) (Singh et al., (1987), PNASUSA 84, 739-743), and alfalfa (Medicago)." Saturnia sativa and soybean (Phaseolus) (King et al., (1986), above) described osmolysin-like proteins that are serologically correlated and have molecular weights corresponding to those of osmolysins from tobacco and tomato. For a long time, the only presumed relevance of osmolysins was their function of providing osmotic tolerance to plants upon exposure to low water potential. However, US 5,389,609 discloses that these PR-proteins have antifungal activity, inhibiting fungal growth. US 5,389,609 also discloses a general method for obtaining antifungal proteins with activity against oomycetes, described as “osmolysins.” US 5,389,609 observes that "antifungal activity appears to be thermally unstable. The active component from tobacco was identified as a hydrophobic protein with a molecular weight of approximately 24 kD and a basic isoelectric point (pI), and is referred to as AP20 (also known as AP24). After purification and determination of the N-terminal amino acid sequence of AP20, this moiety appears to be identical to the corresponding moiety of an osmolyte (a protein known to occur in tobacco (Singh et al. 1987))," and "belongs to a group of proteins named osmolytes or alternatively osmolyte-like proteins." US 5,389,609 states that the accumulation of osmolyte-like proteins appears to depend on the continuous presence of an osmolyte. No accumulation occurs in the presence of (some) non-osmolytes such as cadmium chloride (King et al., (1986), PlantMol. Biol. 7, 441-449). US 5,389,609 also points out PR-proteins (pathogenesis-related proteins), a class of osmotic protein-like proteins with the same molecular weight range that can be induced to form plant responses to threats from pathogens or stress-related conditions.US 5,389,609 discloses a method for recovering antifungal proteins from an extract fraction, comprising: inoculating 7- to 8-week-old tobacco (Nicotiana tabacum cv. Samsun NN) leaves with tobacco mosaic virus (TMV), harvesting the leaves after 7 days, homogenizing them at 40°C in a buffer containing 2-mercaptoethanol and activated charcoal using a Waring stirrer, filtering the homogenate on cheesecloth, centrifuging the filtrate at 5,000 g, collecting the supernatant and centrifuging at 22,000 g for 50 min, desalting on a Sephadex G25 column (Pharmacia), and equilibrating it in 20 mM NaOAc at pH 5.2. The protein-containing fraction is then centrifuged at 22,000 g for 45 min. The supernatant is passed through an S-agarose gel "fast flow" (Pharmacia) column, equilibrated with 20 mM NaOAc at pH 5.2, at a flow rate of approximately 15 ml per min. Unbound proteins were collected. Bound proteins were eluted using a linear NaCl gradient (0 to 500 mM) increased in the buffer solution described above and a flow rate of 3 ml per minute; 4.2 ml fractions were collected. Each fraction was analyzed by electrophoresis using a 12.5% ​​polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS), with molecular weight markers (20–66 kD) as a reference. The antifungal activity of individual fractions of the same fraction was tested. 400 μg of antifungal protein (95% purity) or 200 μg of antifungal protein (99% purity) could be isolated from 400 g of leaf material.

[0009] US 5,521,153 discloses isolated and substantially purified Zeamatin having a molecular weight of about 22 kilodaltons under reducing conditions and an N-terminal amino acid sequence as disclosed in SEQ.ID No.:1, wherein said Zeamatin reduces the minimum inhibitory concentration (MIC) of nicotinic acid against strains of Candida albicans by about 10 to about 100 times in in vitro tests. Zeamatin can be separated from corn flour protein extract by a purification procedure comprising the following steps: (1) separating corn protein fractions precipitated between 30% and 55% saturated ammonium sulfate; (2) applying the fraction precipitated by the 30%-55% saturated ammonium sulfate to a cross-linked carboxymethyl dextran column with dry beads of 40-125 micrometers, washing the column to remove unbound protein, eluting the protein fraction from the column using a linear salt gradient and separating a first eluted protein fraction exhibiting SAFP (synergistic antifungal protein) activity; and (3) applying the first eluted protein fraction to a hydrophobic column, such as a porous, interconnected phenyl agarose column with wet beads of 45-165 micrometers in diameter, eluting the protein fraction from the column using a low-salt aqueous solution and separating a second eluted protein fraction exhibiting SAFP activity; the second eluted protein fraction comprising the zeamatin.

[0010] Therefore, osmolysin and proteins homologous to them have long been classified as having antifungal and stress-resistant activities.

[0011] Narasimhan et al. (2005) (Narasimhan ML, Coca MA, Jin J, Yamagichi T, Ito Y, Kadowaki T., Kim KK, Pardo JM, Damsz B., Hasegawa PM, Yun DJ, Bressan RA, Osmotin is a homolog of mammalian adiponectin and controls apoptosis in yeast through ahomolog of mammalian adiponectin receptor. IN Mol Cell, 2005 Jan 21; 17(2):171-80.) first disclosed that osmotin and adiponectin do not share sequence similarity; however, the β-barrel domains of the two proteins can overlap, and osmotin, like adiponectin, activates AMP kinase in C2C12 myocytes through the adiponectin receptor. This discovery gives osmotin a highly important role compared to the function attributed to osmotin since Narasimhan et al. Odoromones, or odoromones-like proteins, are now potential alternatives to adiponectin (a protein hormone). Human adiponectin deficiency is associated with a range of lifestyle conditions, including insulin resistance, and further with conditions such as type 2 diabetes, hyperlipidemia, atherosclerosis, hypertension, heart disease, and obesity. Odoromones have been shown to act as adiponectin agonists in mammalian systems. Therefore, odoromones and their homolog PR-protein could be used to develop treatments for mammalian conditions, in which adiponectin receptor-mediated therapies are possible.

[0012] In fact, the discovery of adiponectin itself and the lack of recognition of its association with type 2 diabetes, hyperlipidemia, atherosclerosis, hypertension, heart disease, and obesity have increased its potential for use in treating these diseases / conditions. However, obtaining sufficient quantities of adiponectin in natural compositions is inconceivable, and its large-scale synthesis is far too complex.

[0013] With the discovery by Narsimhan et al., osmolysin was first identified as an adiponectin agonist and attracted the attention of researchers.

[0014] US 7,803,854 discloses a method for treating mammals suffering from a condition selected from the group consisting of type 2 diabetes, insulin resistance, hyperlipidemia, arteriosclerosis, and heart disease, the method comprising: administering an isolated PR-5 protein to the mammal, wherein the type 2 diabetes, insulin resistance, hyperlipidemia, arteriosclerosis, or heart disease is treated. The PR-5 protein is specifically described as an osmotic protein. The scope of the claims in US 7,803,854 specifically excludes unisolated PR-5 proteins associated with vegetables.

[0015] Citing Narasimhan et al. (2005) and Lele (2010) “RD Lele; Pro-insulin, C Peptide, Glucagon, Adiponectin, TNFα, AMPK: Neglected Players in Type 2 Diabetes Mellitus”, JAPI January 2010, Vol. 58, pp 30-36, it is noted that the beneficial effects of a diet containing 400g of fruits and vegetables can be partly attributed to the osmoregulatory protein content in addition to antioxidants and high fiber and low fat content. Vandit et al. (2012) [VR Trivedi, M.Chorawala, GB Shah, ANTIATHEROSCLEROTIC ACTIVITY OF OSMOTIN, AADIPONECTIN AGONIST IN ATHEROGENIC DIET INDUCED HYPERTRIGLYCERIDEMIA AND HYPERCHOLESTEROLEMIA IN WISTAR RATS; ARPB, 2012; Vol 2(II)] have shown that osmodin-I and osmodin-II produced from salt-adapted tobacco cell suspension (Nicotiana tabacum L) by Singh et al. (1987) resulted in reductions in body weight, serum triglycerides, total serum cholesterol, and atherosclerotic lesion area. These effects are comparable to those produced by atorvastatin.

[0016] However, consuming 400 grams of leafy green vegetables or 400 grams of fruit daily does not appear to be a feasible way to obtain significantly sufficient osmotonic proteins in the diet. Given how vegetables are cooked and consumed, there is reason to believe that the release of osmotonic proteins from the leaves is expected to be no more than 3-10%, as osmotonic proteins are large protein molecules. If small molecules such as β-carotene are used as indicator molecules, the same molecules are released, as has been shown in experiments by Lopez et al. (2010) [(Armando Carrillo-Lopez,1Elhadi M.Yahia and1,2Gabriela K.Ramirez-Padilla.Bioconversion of Carotenoids in Five Fruits and Vegetables to Vitamin A Measured by Retinol Accumulation in RatLivers.American Journal of Agricultural and Biological Sciences 5(2):215-221,2010)]. In fact, even without rat feeding, the low release of phytonutrients from leafy green vegetables is corroborated by the fact that the usual way of cooking and eating fenugreek leafy greens produces a slight bitterness, which is practically acceptable to most people who enjoy this vegetable. However, if the same vegetables are pulped in a kitchen blender before or after cooking, the same vegetables produce a very strong, unpleasant bitterness because a much larger proportion of the bitter substances in fenugreek are released from the cells due to mechanical pulping. Furthermore, considering the volume, eating 400 grams of leafy green vegetables daily is a difficult task. Therefore, eating vegetables in the diet is not a feasible way to obtain all the potential benefits. As Lopez et al. have shown, fruits do offer better bioavailability than the phytochemicals they contain. However, many of them are associated with their sugar / energy content, which is unacceptable to a segment of the population, including those with diabetes. Therefore, making plant fractions free of undesirable factors but containing a large amount of bioavailable osmolytes (which is suitable for standardizing their osmolyte content) is crucial for considering the practicality of vegetables and / or fruits as feasible sources of osmolytes.

[0017] However, it is difficult to obtain sufficient amounts of atomodulatory proteins or atomodulatory protein-like proteins from their currently known sources using methods known in the prior art, as it requires subjecting the cultured cells or plants to pathogenic or abiotic stress and involves several steps: precipitation with ammonium sulfate at 10,000 g or centrifugation or both, followed by carboxymethyl dextran (CM) Gradient elution on SCX-polyamide HPLC. US 5,389,609 reports the yield of osmolyte-like protein by following the above steps, as 400 μg of antifungal protein with a purity of 95% or 200 μg of antifungal protein with a purity of 99% from 400 g of leaf material. Furthermore, these methods are not readily applicable to large-scale manufacturing. A much higher yield of isolated osmolyte from stressed plant material will be needed for methods using osmolyte as a functional homolog of adiponectin or as an adiponectin agonist for the treatment of diseases and symptoms caused by adiponectin deficiency.

[0018] To address the feasibility of considering osmolytes or osmolyte-like proteins as adiponectin agonist component in pharmaceutical formulations for the treatment of diseases and conditions caused by adiponectin deficiency, a simpler method suitable for scalability is needed. Existing technologies have disclosed that such diseases and conditions include at least type II diabetes, insulin resistance, hyperlipidemia, arteriosclerosis, and heart disease. Given the current knowledge, this is a very challenging task because osmolytes or osmolyte-like proteins constitute a very small fraction of the total protein content in plants, and the separation of osmolytes or osmolyte-like proteins from a large number of protein impurities has so far been only achievable by methods based on Narasimhan et al. (1987) or modifications thereof, which involve several steps including precipitation with ammonium sulfate at 10,000 g or centrifugation or both, followed by gradient elution on carboxymethyl dextran (CM Sephadex) or SCX-polyamide HPLC; and also yielding meager yields of 400 μg of 95% pure antifungal protein or 200 μg of 99% pure antifungal protein from 400 g of leaf material. However, for it to be used in therapeutic applications, the required yield of osmolysin far exceeds the current level of 200-400 μg.

[0019] Savangikar & Savangikar (2014) (WO2014 / 195975) disclose a method for using heat-stable plant sap-soluble plant proteins for antipathogenic effects; the entire contents of that disclosure are incorporated herein by reference. The composition is standardized with parameters relating to its antipathogenic properties or the content of protein molecules with a molecular weight of 26 kilodaltons (kD) or smaller. Also disclosed is an antipathogenic composition comprising heat-stable plant sap soluble plant protein, wherein the heat-stable plant sap soluble plant protein is obtained as a sap soluble by heat-treating the plant or plant sap to precipitate the protein, a large amount of the protein can be thermally coagulated together with a group of phytochemicals that co-coagulate with heat-precipitable proteins in the plant, leaving other plant components in the sap carrying a small amount of heat-stable sap soluble protein to produce a sap soluble, and wherein the composition is standardized with respect to parameters relating to the properties of the composition, which are related to the antipathogenic properties or the content of protein molecules with a molecular weight of 26 kilodaltons (kD) or smaller. A method / process for preparing a composition comprising heat-stable plant juice soluble plant protein is also disclosed, the process comprising the following steps: (a) heating and precipitating the plant juice and separating the macromolecules, including proteins coagulated upon heating, from the soluble components to obtain a juice soluble containing heat-stable plant juice soluble plant protein; (b) quantifying the heat-stable plant juice soluble plant protein by a method relating to the content of protein molecules with antipathogenic properties or a molecular weight of 26 kDaltons or less; (c) concentrating or diluting to achieve a figure of a property parameter determined / selected as a standard; and, optionally, further adding an additive ingredient selected from the group consisting of: pharmaceutically active substances, pharmaceutical excipients, cosmeceutical actives, cosmeceutical excipients, nutraceutical actives, nutraceutical excipients, plant protective active substances, food additives, or other food ingredients.A plant material composition free of heat-stable proteins is also disclosed; wherein the composition is prepared by: pulping the plant material, removing juice from the solid residue, heating the juice to remove coagulating proteins and non-protein substances as coagulations, removing the coagulations, then removing heat-stable proteins from the resulting composition, optionally treating the resulting composition to obtain a further composition comprising non-protein hydrophilic plant juice solubles or non-protein lipophilic plant juice solubles or both in the same composition; and optionally an additive component selected from the group consisting of: pharmaceutically active substances, pharmaceutical excipients, cosmeceutical active substances, cosmeceutical excipients, nutritional pharmaceutically active substances, nutritional pharmaceutical excipients, plant protective active substances, food additives or other food ingredients.

[0020] Savangikar & Savangikar (2014) demonstrated that concentrated plant juice solubles could effectively replace the daily dose of three 500mg metformin tablets for type 2 diabetes patients to lower postprandial blood glucose levels. However, serum postprandial blood glucose remained above 200 mg / dL on most days. Furthermore, there were practical difficulties in adapting the composition to regular use, consuming two to three tablespoons of the concentrated juice solubles two to three times daily, as the concentrated juice soluble syrup was extremely unpalatable, highly bitter and astringent, and difficult to carry, dispense, and accurately measure. Therefore, improvements are needed. Furthermore, the composition was observed to be not storage stable, as evidenced by the gradual decrease in potency during storage. Summary of the Invention

[0021] The present invention includes a method of using an improved composition comprising heat-stable plant sap soluble plant protein for antipathogenic effects; the improvement comprising: the composition comprising heat-stable plant sap soluble plant protein is essentially a water-insoluble concentrate free of components that are more readily soluble in plant sap, and is rich in heat-stable proteins from plant sap.

[0022] The present invention also includes improved compositions of heat-stable plant juice soluble plant proteins; wherein the improvements include: (a) the concentrate is inherently water-insoluble, (b) the concentrate is inherently free of components that are more readily soluble in plant juice, and (c) being rich in heat-stable proteins compared to compositions of plant juice solubles; including compositions of heat-stable plant juice soluble plant proteins that are inherently water-insoluble concentrates free of components that are more readily soluble in plant juice and rich in heat-stable proteins of plant juice.

[0023] The compositions of the present invention also include dosage forms prepared from the compositions of claim 2.

[0024] In one aspect of the invention, parameters relating to the properties of the composition are standardized, which are related to antipathogenic properties or the content of protein molecules with a molecular weight of 26 kilodaltons (kD) or smaller.

[0025] On the other hand, the antipathogenic effects of the above-mentioned compositions include antimicrobial effects on organisms including microorganisms or effects as adiponectin agonists in mammals.

[0026] Methods of using the compositions of the present invention include adiponectin agonist effects in mammals, further comprising reducing the risk of one or more conditions selected from type II diabetes, hyperlipidemia, atherosclerosis, hypertension, heart disease, and obesity.

[0027] Methods using the compositions of the present invention described above, wherein the antimicrobial effect includes reducing the risk of microbial infection in plants and animals or treating microbial infection, with or without synergy with other antimicrobial agents.

[0028] The present invention also includes: an improved antipathogenic composition comprising heat-stable plant sap soluble plant proteins, wherein the heat-stable plant sap soluble plant proteins are obtained as sap solubles by heat-treating the plant or plant sap to precipitate proteins, a large amount of the protein being thermally coagulated together with a group of phytochemicals that co-coagulate with heat-precipitable proteins in the plant, leaving other plant components in the sap carrying small amounts of heat-stable sap soluble proteins to produce sap solubles, and wherein parameters relating to the properties of the composition are standardized to antipathogenic properties or to the content of protein molecules with a molecular weight of 26 kilodaltons (kD) or less; the improvement includes: the composition comprising heat-stable plant sap soluble plant proteins is essentially a water-insoluble concentrate free of components more readily soluble in plant sap and rich in heat-stable plant sap proteins.

[0029] The compositions of the present invention can be standardized with respect to parameters of the composition properties, which are determined based on one or more analytical parameters selected from the group consisting of: (a) the dry matter percentage of the composition, (b) the nitrogen percentage of the dry matter of the composition, (c) the content of casein or gallic acid, (d) by molecular weight determination, or (e) by Fourier transform infrared spectroscopy.

[0030] This invention comprises compositions of heat-stable plant juice soluble plant proteins, which may or may not be used with: pharmaceutically active substances, pharmaceutical excipients, cosmeceutical active substances, cosmeceutical excipients, nutritional pharmaceutically active substances, nutritional pharmaceutical excipients, plant protective active substances, food additives or other food ingredients.

[0031] On the other hand, the present invention includes a method / process for preparing a composition comprising heat-stable plant juice-soluble plant proteins, the process comprising the following steps:

[0032] a. Heating precipitates the plant juice and separating the macromolecules, including proteins that coagulate during heating, from the soluble components to obtain a juice soluble concentrate containing heat-stable plant juice soluble proteins.

[0033] b. Quantification of heat-stable plant sap soluble proteins by means of methods relating to their antipathogenic properties or the content of protein molecules with a molecular weight of 26 kDa or smaller.

[0034] c. Concentrate or dilute to achieve the number of parameters for the properties determined / selected as standards, and

[0035] d. Further optionally add an additive ingredient selected from the group consisting of: pharmaceutically active substances, pharmaceutical excipients, cosmeceutical active substances, cosmeceutical excipients, nutritional pharmaceutically active substances, nutritional pharmaceutical excipients, phytoprotective active substances, food additives or other food ingredients;

[0036] in,

[0037] Compositions including heat-stable plant juice soluble plant proteins are essentially water-insoluble concentrates that do not contain components that are more easily soluble in plant juices and are rich in heat-stable proteins from plant juices.

[0038] The compositions of the present invention are also embodied in concentrated syrups comprising improved compositions, chewable or orally dissolving tablets, granules, powders, immediate delivery tablets, intermediate delivery tablets, controlled delivery tablets, capsules, and dosage forms with or without microemulsions.

[0039] The present invention includes a composition characterized by an infrared absorption spectrum comprising peaks with a transmittance of 35-60 near a wavelength of 3365, a transmittance of 60-80 near a wavelength of 2975, a transmittance of 60-80 near a wavelength of 1612, a transmittance of 65-70 near a wavelength of 1306, a transmittance of 70-90 near a wavelength of 1280, a transmittance of 20-40 near a wavelength of 1080, a transmittance of 70-98 near a wavelength of 887, and a transmittance of 85-98 near a wavelength of 825; wherein the composition comprises heat-stable plant sap soluble plant protein, which is essentially a water-insoluble concentrate free of components readily soluble in plant sap and rich in heat-stable proteins from plant sap. Attached image description:

[0040] Figure 1 Fourier transform infrared (FT-IR) spectrum of improved composition 6F.

[0041] Figure 2 Fourier transform infrared (FT-IR) spectrum of improved composition 7F.

[0042] Figure 3 Fourier transform infrared (FT-IR) spectra of prior art compositions by Savangikar and Savangikar (2014) DPJ.

[0043] Figure 4 Fourier transform infrared (FT-IR) spectrum of the improved composition UF. Detailed Implementation

[0044] Savangikar & Savangikar (2014) Figure 3 The composition (which exhibits antipathogenic properties by lowering blood glucose levels to or even below those achieved with metformin) is a concentrated leaf juice soluble extract containing "heat-stable plant sap soluble protein" plus all soluble components of the leaf juice. Throughout the day, when metformin is consumed along with the concentrated whole-juice soluble extract, urinary glucose levels remain at zero. Figure 3Given the fact that metformin alone showed almost four times higher blood glucose levels in the absence of juice-soluble components, it demonstrated an additional blood glucose-lowering effect (Savangikar & Savangikar, 2014, p. 3, Table 1). Therefore, it is concluded that although the leaf juice was heat-treated to coagulate and remove high molecular weight proteins from the juice, leaving only a juice-soluble component containing heat-stable proteins and other juice-soluble components, the fully concentrated leaf juice-soluble component did indeed retain their activity. Therefore, in the context of the prior art, it was observed that it was no longer necessary to isolate PR-5 protein into a 95-95% purity isolate to increase reasonable adiponectin agonist activity, and a composition containing an effective level of adiponectin agonist protein was prepared for the first time, and its use as an antipathogenic composition was confirmed. However, Savangikar & Savangikar (2014) observed that two-year-old juice syrups did not exhibit any adiponectin agonist activity, while activity was observed once freshly prepared juice-soluble syrups were administered, indicating that the activity of heat-stable juice-soluble proteins in concentrated juice solubles is unstable. This was also observed with freshly prepared juice-soluble syrups, as continued observation after the time period reported by Savangikar & Savangikar (2014) did show a slow decline in the activity of the juice-soluble syrups, and metformin was restarted after the syrup was depleted, this time at 500 mg each before breakfast, lunch, and bedtime. In this dosing regimen, based on regular urine tests using the Benedict reagent on combined urine samples taken 2 hours after a meal and at each time interval between two food intake events (morning fruit and occasional dessert, breakfast, lunch, dinner; and nighttime snacks to the next morning), it was observed that blood glucose levels were consistently below the renal threshold of 180 mg / dl in the combined urine samples taken after dinner and between the nighttime snacks and the next morning.

[0045] Savangikar and Savangikar (2014) chose to use concentrated juice solubles for antipathogenic effects, where the activity requirements would be minimal, i.e., the heat-stable juice-soluble proteins would retain their integrity even during boiling and concentration. As shown, this is achieved through the hypoglycemic effect of freshly prepared concentrated juice solubles. However, given the problems with concentrated syrups of leaf juice solubles, including unpalatability, a slow decline in storage stability, and difficulty in converting them into convenient dosage forms, further improvements are necessary.

[0046] For the desired improvement, it seems necessary to improve the relative content of heat-stable juice-soluble proteins by removing non-protein-soluble components from the concentrated juice of Savangikar and Savangikar (2014) to improve palatability, and to further concentrate it to provide a concentrate of heat-stable juice-soluble proteins. The processing conditions used should not adversely affect the antipathogenic activity of the heat-stable juice-soluble proteins, and the production cost should be within practical limits. Such a composition of heat-stable juice-soluble protein concentrates should be suitable for preparing convenient dosage compositions.

[0047] In the prior art (Singh et al. (1987), US 5,389,609, US 5,521,153) used to prepare active formulations of PR-5 protein with antipathogenic activity, the methods employed involve extremely mild processing conditions involving gel filtration on a column followed by affinity chromatography of the eluted components on an adsorbent in the column, resulting in very low yields. In fact, this is typical of any composition of any type of bioactive protein molecule. Against this backdrop, a surprising embodiment of the present invention is that the compositions of the heat-stable plant sap-soluble plant proteins of the present invention, possessing antipathogenic activity, are essentially water-insoluble compositions. It has been found that water-insoluble compositions containing heat-stable plant sap-soluble plant proteins are also effective for antipathogenic use, and simpler methods can be used to separate and concentrate compositions rich in these proteins, essentially free of soluble components that are not protein sap-soluble.

[0048] Therefore, in one aspect of the invention, the invention includes a method of using an improved composition comprising heat-stable plant sap soluble plant protein for antipathogenic effects; the improvement comprising: the composition comprising heat-stable plant sap soluble plant protein is essentially a water-insoluble concentrate free of components that are more readily soluble in plant sap, and rich in heat-stable proteins from plant sap.

[0049] On the other hand, the present invention includes an improved composition of heat-stable plant juice soluble plant protein; wherein the improvements include: (a) the concentrate is inherently water-insoluble, (b) the concentrate is inherently free of components that are more readily soluble in plant juice, and (c) it is rich in heat-stable protein compared to compositions of plant juice solubles.

[0050] On the other hand, the present invention includes dosage forms prepared from the above-described improved compositions. In a further aspect, dosage forms include concentrated syrups, chewable or orally dissolving tablets, granules, powders, direct-delivery tablets, intermediate-delivery tablets, controlled-delivery tablets, capsules, and orally soluble films with or without microemulsions of the above-described improved compositions of the present invention.

[0051] The present invention also includes a method / process for preparing the composition of the present invention by means of the following steps: adding an amount of ascorbic acid to prevent oxidation reaction to a sap soluble material prepared by coagulating the sap of green leafy vegetation, followed by one or more of the following steps: (a) allowing the sap soluble material to stand for a period of time to allow insoluble solids to appear, and (i) allowing the solids to precipitate at the bottom and decanting the supernatant or (ii) centrifuging the insoluble solids from the sap soluble material before or after precipitation, or (b) concentrating the sap soluble material by ultrafiltration or boiling, and further decanting or filtering or centrifuging away the insoluble matter from the retentate.

[0052] It has been found that the Fourier transform infrared (FT-IR) spectra of the improved compositions of the present invention (UF, 6F, and 7F described in the examples below) prepared by variations of the above methods are essentially identical, thus confirming that the compositions / products formed by variations of the above methods produce the same products, and that the compositions are reproducible in their properties and structure. It has been observed that when consumed as dietary supplements, all of them result in a reduction of postprandial blood glucose and can completely replace the three-times-daily 1500mg tablet dose of metformin taken as a snack with breakfast, lunch, and bedtime. Improvement of very low high-density lipoprotein to normal levels has also been observed, and reductions in very low-density lipoprotein (VLDL), low-density lipoprotein, and triglycerides to normal levels have also been observed. High blood pressure has also returned to normal levels. This indicates that the compositions of the present invention do indeed possess adiponectin agonist properties, which can be reasonably contributed by PR-5 protein (i.e., osmolyte or osmolyte-like protein).

[0053] Analysis of the compositions of the present invention (6F and 7F) showed that they contained 9% protein. When suspended in water, much of them precipitated at the bottom, but some golden-yellow substances gave the supernatant a clear golden-yellow color. Therefore, it appears that the process of preventing a portion of the juice-soluble matter from dissolving includes both proteins and non-protein substances, wherein the protein portion dissolves again when sufficient water is provided, and it is this portion of the composition that provides the antipathogenic activity. Therefore, it is possible that this soluble component can also be suitable for concentration without impairment, and this can facilitate obtaining a further concentrated composition, and the composition and dosage forms derived from it are also included within the scope of the present invention.

[0054] The antipathogenic activity of the PR-5 protein family is well-known and established in plant science. Therefore, the compositions of the present invention can also be used alone or in combination with other antimicrobial agents having additional or synergistic effects for antimicrobial purposes; and such applications of the compositions of the present invention are also included within the scope of the present invention.

[0055] The syrup of the improved composition of the present invention was used in a dropper bottle, wherein the concentration of the dry matter was calibrated to dispense a known amount of the improved composition per drop or spoon. Immediate-release tablets of 40 mg and 80 mg of the improved composition were prepared and taken four times a day, and two 320 mg sustained-release tablets were taken 12 hours apart each day. Whether as a syrup or tablet, the pharmaceutical preparation of the composition of the present invention was taken four times a day: once immediately upon waking with morning fruit, and once each at breakfast, lunch, and as a nighttime snack before bedtime. It was observed that when provided as adjunctive therapy for patients taking metformin, patients experienced a sharp drop in postprandial blood glucose levels on almost the same day as administration of the pharmaceutical preparation of the present invention. Metformin in the preparation could be gradually removed in a stepwise manner over a 30-day span; and even after one year of continued use of the composition of the present invention alone without metformin, blood glucose levels slowly increased again, but never exceeded the blood glucose levels recorded before the start of use of the pharmaceutical preparation of the composition of the present invention as adjunctive therapy. Therefore, it can be concluded that the composition of the present invention can at least replace metformin; and compared to metformin, it provides additional effects, namely, that the composition of the present invention also normalizes blood lipids and triglycerides and reduces the dosage of antihypertensive drugs.

[0056] In this invention, it was also observed that, in syrup form, no difference in efficacy was noted when the dosage was gradually increased from 80 mg / tablet to the aforementioned 320 mg / tablet. No improvement was also noted when using sustained-release tablets at 320 mg / tablet. However, a significant improvement was observed when using immediate-release tablets at 40 mg / tablet. This observation is also surprising, contrary to the expectation that drying the active protein for tablet preparation would reduce the activity and efficacy of the compositions of this invention.

[0057] The invention of a water-insoluble form of a heat-stable plant sap-soluble plant protein for providing an active composition with antipathogenic effects represents a technological advancement. This product can be readily prepared and recovered from sap-soluble materials using inexpensive equipment and a developable, economically feasible method that does not require expensive chemicals / adsorbents, and the active composition can be readily formulated into tablets with optimal efficacy. Given its excellent stability without the need for buffers, its application in plant protection compositions is also envisioned. As a sufficient precaution, the slurry of the insoluble composition recovered from the sap-soluble material is adjusted to pH 6.5 by adding dipotassium hydrogen phosphate and potassium dihydrogen phosphate, making its potassium buffer relative to pH 6.5 20 mM, and potassium sorbate and sodium benzoate in a 50:50 ratio are added to make its volume relative to the slurry 300 ppm for their preservative effect. However, the necessary levels of buffer, pH adjuster, and preservative in the slurry have not yet been determined.

[0058] "Sap-soluble matter" is understood to be plant-derived sap that contains heat-stable proteins and does not contain heat-coagulating proteins.

[0059] "Sap-soluble matter" is defined as an aqueous liquid composition prepared from plants from which heat-coagulating components have been removed, and the composition includes plant-derived components that do not coagulate upon heating. In the examples provided in this specification, "sap-soluble matter" is derived from the juice obtained from pulped green leafy plants / vegetables by pressing or centrifugation, and the resulting green juice is heated to 80°C or higher to coagulate heat-coagulating proteins and remove coagulated proteins and co-coagulated nutrients from the heated brown juice. This composition can also be obtained from the sap of green leafy plants by heating the sap of green leafy plants to 80°C or higher to coagulate heat-coagulating proteins and remove insoluble juice components from the substance to obtain a brown liquid juice containing soluble matter.

[0060] The importance of this invention stems from the crucial role adiponectin, as a protein hormone, plays in maintaining human health. Adiponectin deficiency in humans is associated with a range of lifestyle disorders, including insulin resistance, and further, type II diabetes, hyperlipidemia, atherosclerosis, hypertension, heart disease, and obesity. Because adiponectin is a protein molecule that cannot be synthesized to date, the conditions caused by its deficiency require treatment with as many medications as the diseases it causes as individual illnesses, each with its own side effects and drug interactions, adversely impacting quality of life. The multiple medications required daily are themselves an additional risk factor for dependence in patients of all ages. Lifestyle modifications to reduce the risk of lifestyle diseases are considered to have limited utility. The availability of effective and affordable adiponectin agonists will revolutionize the nature of treatments available for managing lifestyle disorders. It will provide a single composition that will significantly reduce the risk of all the aforementioned lifestyle diseases and conditions caused by adiponectin deficiency, eliminate or significantly reduce the need for multiple chemical doses required to treat and manage lifestyle diseases and conditions, and will be the most logical first-line response to lifestyle conditions, where multiple chemical doses are only required to the extent that the disease or condition persists even after administration of an adiponectin agonist. The adiponectin agonist concentrate of the present invention is exemplified herein as an extract prepared from the leafy green vegetable fenugreek (Methi) (fenugreek, Trigonella foenum-graecum L.). However, osmolytes and osmolyte-like proteins are known to be ubiquitous in all plant species (US 7,803,854; column 9, lines 38-40), therefore, the compositions of the present invention can be prepared from leafy vegetation of any plant species other than fenugreek.

[0061] The following describes examples illustrating compositions comprising osmolysins or osmolysin-like proteins (also known as PR-5 proteins, which satisfy the aforementioned long-unmet needs) and methods for preparing such compositions. Those skilled in the art will readily understand from the pervasive nature of this protein family that, although illustrated using fenugreek leafy green vegetables, the scope of the invention covers all plant species from which fractions of heat-soluble proteins can be obtained and isolated as concentrates using the methods described herein.

[0062] Savangikar and Savangikar (2014) describe a "composition comprising heat-stable plant sap-soluble plant proteins" with antipathogenic activity, a concentrated sap-soluble syrup, which is a composition of a substance different from the "improved composition of heat-stable plant sap-soluble plant proteins" of the present invention; wherein the improvements include (a) the inherently water-insoluble nature of the concentrated substance, (b) the inherent absence of components more readily soluble in plant sap, and (c) a higher content of heat-stable proteins compared to the composition of plant sap-soluble substances. This is largely reflected in the dried sap-soluble substance (Savangikar & Savangikar (2014) "composition comprising heat-stable plant sap-soluble plant proteins") Figure 3 ) and the improved composition of the present invention (i.e., an improved composition of heat-stable plant juice soluble plant protein; wherein the improvements include (a) the inherently water-insoluble nature of the concentrate, (b) the inherent absence of components more readily soluble in plant juice, and (c) a higher content of heat-stable protein compared to compositions of plant juice solubles) Figure 1 , Figure 2 and Figure 4 The FT-IR (Fourier Transform Infrared) spectra are significantly different.

[0063] The following are non-limiting embodiments of the work of the present invention. Obvious and equivalent variations of these embodiments will be apparent to those skilled in the art, and such variations are also included within the scope of the present invention.

[0064] Example

[0065] Example 1:

[0066] A syrup for preparing a water-insoluble concentrated composition comprising heat-stable plant juice-soluble plant proteins, free of components more readily soluble in plant juices, and rich in heat-stable proteins from plant juices:

[0067] In one embodiment of a further improved method for use in treating pathogenicity, the juice soluble protein is prepared by pulping shoots of leafy fenugreek (Trigonella foenum-graecum L.) by adding an ascorbic acid solution containing approximately 380 mg of ascorbic acid per kg of vegetables, and extracting juice from the resulting pulp. The juice is coagulated by heating at 80-100 degrees Celsius and filtered through a cloth to obtain a green coagulated material on the cloth and a filtrate collected below, which is the juice soluble protein (referred to as "juice soluble protein" for the purposes of this specification). Additional ascorbic acid is added to the filtrate / "juice soluble protein" until it turns from brown to golden yellow. Typically, this corresponds to approximately 200 grams of additional ascorbic acid per 150 liters of juice soluble protein. This amount of ascorbic acid can vary depending on the batch of vegetables, as it will depend on the phenolic content of the vegetables. The aim is to avoid the oxidation of phenols, as well as protein-phenol interactions and any other oxidation reactions. During pulping, the volume of the ascorbic acid solution is adjusted so that the weight of the soluble juice at the stage of adding additional ascorbic acid is approximately the same as the weight of the pulped plant.

[0068] The juice-soluble substances are further processed in the following different ways:

[0069] a) In one embodiment, the juice-soluble material is passed through a 1kD cutoff ultrafiltration membrane to separate species with molecular weights smaller than 1kD from the remaining juice-soluble material in the filtrate, to obtain a protein fraction of 26kD or smaller in the juice-soluble material in a retention fraction concentrated approximately six times. In illustrative experiments, a 1kD cutoff membrane is used because it is readily available. Alternatively, two cutoff membranes may be used, one with a 20kD cutoff and the other with a 26kD cutoff, to obtain a fraction of the retained product; the fraction retained on the ultrafiltration membrane and unable to pass through the membrane is in the permeate / filtered-off fraction. The retained product is a fraction concentrated six times (modified composition, labeled: IC1), containing high molecular weight proteins and phenols contained in the juice-soluble material, and showing a dramatic reduction in postprandial glucose when administered as an adjunct to metformin treatment with three 500mg tablets daily, one tablet with each main meal (breakfast, lunch, and dinner). A concentrated fraction containing heat-stable proteins, prepared from the juice-soluble material, is obtained from the retentate as a sediment by centrifuging the juice-soluble material at a speed sufficient to separate the precipitate from the supernatant for a period of time. In the illustrative experiment, the precipitate as a sediment was separated using 5000 rpm for 5 minutes. This sediment will contain proteins of interest with a molecular weight of 26 kDa and below, and these proteins are resuspended in water to a total volume reduction of 16.6 times relative to the volume of the juice from which the sediment was obtained; that is, a 16.6-fold concentration of high molecular weight species in the juice-soluble material containing the proteins of interest. When filled into a nozzle-containing bottle that provides droplets of contents upon inversion and squeezing, 25 drops constitute a 1 ml composition. Therefore, it is also convenient to dispense measured amounts of active ingredients from this device. It is also highly likely that resuspension in a smaller amount of water will be possible, thus requiring fewer droplets to more conveniently dispense the same amount of the proteins of interest. As a precaution, 0.5 g of ascorbic acid was added per liter of water to the water used for resuspending the sediment after centrifugation. K₂HPO₄ and KH₂PO₄ were added to the final volume of resuspended sediment so that the total volume was 20 mM relative to the resulting pH 6.5 phosphate buffer. The resulting composition is a pale yellow suspension and should be shaken before use as a dropper bottle (modified composition: IC₂).

[0070] (b) In another embodiment, the juice-soluble material is concentrated to approximately four times or more by heating to boiling and allowed to stand to allow the precipitate to settle. The precipitate collected at the bottom of the container is collected by decanting most of the supernatant, with the last portion of the supernatant removed by centrifugation, or by washing three times with resuspended water, adding 500 mg ascorbic acid per liter of water, allowing it to stand, precipitating and discarding the supernatant, and collecting the slurry of the precipitate (two separately prepared batches of these improved compositions are labeled 6F and 7F). By reasoning, the precipitate will contain small amounts of protein that is soluble in the juice-soluble material but precipitates upon concentration, and the molecular weight will be 26 kD or less. To obtain a syrup for consumption, a portion of the slurry is suspended in water containing K₂HPO₄ and KH₂PO₄ to a volume of 20 mM relative to a phosphate buffer at pH 6.5, to contain the required dry matter content per spoonful. To obtain a solid for tablet preparation or for chemical composition analysis, the slurry was coated onto polyethylene sheets and dried at room temperature (approximately 25°C) under a fan (from batches labeled 6F and 7F).

[0071] c) In the third embodiment, the juice-soluble material is centrifuged at 16000g using a decanter centrifuge to obtain a sediment containing a high molecular weight component (greater than 10 kD) soluble in the juice-soluble material and a juice-soluble material substantially free of the high molecular weight component. The sediment (modified composition, labeled UF) is approximately 45% in dry matter, and based on dry matter, the yield of this component is approximately 0.15-0.2% of the weight of fresh green fenugreek vegetation. The sediment is resuspended in water, and KH₂PO₄ and K₂HPO₄ are added to make the total volume 20 mM relative to a phosphate buffer at pH 6.5, and to make each spoonful (4.5 ml) have the desired dry matter concentration of precipitated solids.

[0072] d) Tablets are prepared from a dried powder of the improved composition labeled 7F. They have the following composition:

[0073] a. Immediate-release tablets with 40 mg and 80 mg of active ingredient per tablet: Each of these tablets contains: a 7F slurry providing a dose equivalent to 40 mg or 80 mg per tablet, as appropriate; 93 mg of microcrystalline cellulose; and Povidone. TM (Polyvinylpyrrolidone, viscosity 44000-54000cp) 30mg, xanthan gum 0.5mg, and whey protein concentrate, bringing the total to 500mg. Talc is used as a lubricant.

[0074] b. Extended-release tablets: Each of these tablets contains: a slurry providing the equivalent of 320 mg of 7F, Carbopol TM100 mg of a high molecular weight polymer of acrylic acid crosslinked with pentaerythritol allyl ether, 93 mg of microcrystalline cellulose, 30 mg of povidone, and whey protein concentrate, bringing the total to 500 mg. Talc is used as a lubricant.

[0075] The slurry from 7F is mixed with whey protein concentrate as a diluent / filler, and water is added as needed to prepare a paste. For sustained-release tablets, Carbopol is also used. TM Add to the paste. Mix the paste in a household Waring blender / mixer to prepare a well-mixed, smooth paste. Dry it as a film on a polyethylene sheet under a fan at room temperature (approximately 25°C) and then pulverize it in a household blender.

[0076] The dried powder mixture of the active ingredient and whey protein concentrate was sieved through a 40-mesh sieve and further thoroughly mixed with microcrystalline cellulose, then sieved through another 40-mesh sieve. The mixture was granulated using a Povidone solution. For 320 mg sustained-release tablets, the granulated mixture could be easily compressed into 1-gram tablets. For 40 mg and 80 mg immediate-release tablets, the tablets did not achieve sufficient hardness. Therefore, a xanthan gum solution prepared in water was further added to the granulated mixture, and the mixture was thoroughly mixed in a mixer / grinder by brief agitation to ensure that the granules did not break and that only the xanthan gum solution was uniformly coated on the granules. The granules were dried at room temperature under a fan until the granule surface was dry and they did not stick together, and tablets were prepared.

[0077] Example 2:

[0078] An improved composition 6F has been provided. Figure 1 ), 7F ( Figure 2 ), UF ( Figure 4 Fourier transform infrared (FT-IR) spectra of ) were compared with those of concentrated juice solubles (DPJ) by Savangikar and Savangikar (2014). Figure 3 The FT-IR spectra of the two instruments were compared.

[0079] Example 3:

[0080] Effects of modified compositions of heat-stable plant juice soluble plant proteins on patients with type II diabetes:

[0081] This case study includes self-monitoring and self-reported observations of urinary glucose in a 65-year-old, 5-foot-6-inch tall, 65-kilogram male human subject with type 2 diabetes (Subject 1). He was also an expert in the laboratory techniques required for urinary glucose testing using the Benedict reagent and had an office connected to his residence, including a fixed desk workspace. This person is the same person to whom Savangikar and Savangikar (2014) described the data on the efficacy of the antipathogenic composition. Urinary glucose was monitored by evaluating urine samples almost daily between waking and breakfast, between breakfast and lunch, between lunch and dinner, between dinner and evening nap (approximately 21.15 hours), between evening nap and late at night, and at wakefulness. Between February 2016 and March 2017, urinary glucose was qualitatively tested in the above samples using the Benedict reagent, except when leaving the workstation.

[0082] After the experimental period reported by Savangikar and Savangikar (2014), Subject 1 continued to consume the freshly prepared juice concentrate syrup until it was depleted, and then resumed a dose of one 500 mg metformin tablet as a snack at breakfast, lunch, and evening nap. The diet and exercise program was as follows: wake up at approximately 5:00 AM, eat a medium-sized banana (with peel) weighing approximately 90-100 grams (or, if bananas were unavailable, sweet laddu, i.e., chickpea flour balls fried in ghee, with 20 grams of sugar added to each laddu), take amlodipine tablets, work at a desk in the home office, drink unsweetened tea at approximately 7:00 AM, and have breakfast at 8:45 AM. Breakfast consisted of two pancakes made with 30 grams of whole wheat flour and 100 ml of milk respectively, and a boiled egg. This is followed by six repetitions of an exercise called "Suryanamaskar," each consisting of a series of 12 postures, including forward bends, backward bends, leg extensions, and prone balancing on the arms. Each posture is held for four breaths. Then, small lateral flexions are performed on both sides of the spine, up to 15 breaths on each side, followed by 30 repetitions of rolling the arms laterally over the head, and 30 forceful leg lifts. This is followed by a 30-minute morning walk on a 2km jogging track, starting 30 minutes after breakfast. This is then followed by desk work until 1:15 PM, at which point lunch is eaten, consisting of two flatbreads, 18g of lentil soup (dahl), a boiled egg, and a serving of vegetables. Desk work continues until 5:00 PM, during which time unsweetened tea or coffee is consumed, followed by desk work again until 6:00 PM, at which point breakfast and dinner are eaten, consisting of two flatbreads, 18g of lentil soup, and a serving of vegetables. Next came six “Suryanamaskars” and a 30-minute evening walk of 2 kilometers 30 minutes after dinner. Then I worked at my desk again until 9:15 p.m., at which point I ate one and a half pancakes, 18 grams of lentil soup, and one egg without the yolk, and went to bed around 10:00 p.m.

[0083] In this procedure, it was observed that on most days, regardless of whether metformin or the syrup of the improved composition of the present invention was used, the urine glucose level was always zero at the time of morning (fasting) sample, at the intervals from waking up to breakfast, dinner to evening bedtime snack, and evening bedtime snack to late night urine sample. Urine samples from breakfast to lunch and from lunch to dinner contained glucose.

[0084] Prior to administration of the improved composition of the present invention, Subject 1 had resumed a dosage regimen of three 500 mg metformin tablets, one at breakfast, lunch, and bedtime snack. Blood counts were 138 mg / dL fasting and 224 mg / dL under the long-term adherence to this dosage regimen. Therefore, the blood count was stable for the aforementioned dosage regimen.

[0085] Surprisingly, when 50 ml of the modified composition labeled IC1 (equivalent to approximately 80 mg of dry matter from 5 ml of 6-fold concentrated ultrafiltered fenugreek (Trigonella foenum-graecum L.) – a juice soluble in leafy green vegetables) was administered as adjunctive therapy after meals, urine glucose tests were negative from the same afternoon, indicating that postprandial blood glucose levels had decreased below the renal threshold of 180 mg / L. IC1 is highly palatable as it has no bitter or astringent taste and no unpleasant flavor. This treatment was also repeated at lunch and dinner, and urine glucose tests in combined urine samples after lunch and up to the next morning were also negative. Even with metformin, there had been no negative urine glucose tests after breakfast and lunch at any time in the years prior to this observation. The rapidity of action is also surprising. It is clear that there is no need for a high concentration of the active ingredient to accumulate in the blood. Urine samples were combined after breakfast, lunch, and dinner, late at night, and after a small snack at bedtime around 9:15 PM. Urine samples were tested negative after three consecutive days of single administration of 5 ml of a 6-fold concentrated extract. On the fourth day, blood glucose levels were examined in the pathology laboratory; the fasting level was 130.4 mg / dL, and the postprandial level was 177.1 mg / dL.

[0086] Following the unexpectedly favorable results of the IC-1 composition, to explore the convenience of administering smaller volumes, a six-fold ultrafiltration concentrate fraction of the juice solubles prepared through a 1kD filter was centrifuged at 7000 rpm for approximately 2.5 minutes. The collected precipitate was resuspended in an aqueous solution containing 0.5% ascorbic acid, achieving a concentration equivalent to 16.6 times the volume of the concentrated juice solubles. Therefore, the concentration achieved by this fraction, starting from the juice solubles from each kg of vegetables, was 6 × 16.6 = 100 times. K₂HPO₄ and KH₂PO₂ were added to this concentrated solution to bring the liquid phase relative to phosphate buffer to 20 mM, and it was determined that 0.3 ml of this composition was administered instead of the initial 5 ml dose. This dose also maintained a negative urine glucose test, thus confirming that the blood glucose level was at least below the renal threshold within 24 hours. Therefore, this is by far the simplest known method for obtaining compositions of leafy green vegetables containing less than 26 kDa of heat-stable plant juice soluble plant proteins (osmolytes or osmolyte-like proteins) with antipathogenic effects / efficacy described in the context of lowering postprandial blood glucose levels. It is also surprising to note that this fraction, which appears to be a precipitate and is generally considered an inactive protein composition, retains its activity / efficacy intact even after precipitation without any loss, and exhibits activity as an adiponectin agonist for blood glucose level control when resuspended in water. Indeed, the components of ascorbic acid and phosphate buffer are added to such an aqueous suspension prior to serving as a sufficient precaution against inactivation due to interactions with other plant juice components (including, but not limited to, protein-phenol interactions); however, it has not been confirmed whether these steps are essential. Dry, inert solids may also be reasonably added, resulting in the preparation of a dried composition; such formulations are also included within the scope of this invention. It was also noted that the concentrated juice-soluble matter also exhibited white sediment after a period of time. This sediment is simply the 26kD or smaller molecular weight fraction of the juice-soluble matter, which can be separated from the soluble liquid above it by decantation, and the sediment can be recycled. Alternatively, the precipitate of proteins with a molecular weight below 26kD can be directly recovered by sending the juice-soluble matter, after treatment to precipitate / insoluble proteins with a molecular weight below 26kD, directly to a decanter centrifuge and collecting the sediment.

[0087] Replace metformin with IC1 compositionThe experience is that from the first day of consuming this IC1 composition, or by preparing the liquid composition by suspending sediment collected from this 6x concentrated liquid concentrate (which is a 100x concentrated composition relative to the original juice from which the sediment originated (100LC)), hunger returns earlier than usual at all three times of the day, stimulating the consumption of more food to achieve satiety. However, despite the feeling of satiety after eating more food, the experience is that fatigue is felt in the afternoon and evening when walking. Metformin is known to work through its effects on cells, which slows the rate of glucose release between meals when energy is needed for maintenance and activity. It seems that if the composition, including juice-soluble proteins below 26kD, clears excess glucose to storage sites quickly enough, then the recovery of glucose utilization in the metabolic cycle may be slowed by metformin, and in this case, a dose of metformin that would not be excessive without consuming IC1 now becomes excessive and needs to be reduced. Therefore, it was decided to discontinue the evening 500mg metformin tablet in the three daily metformin tablets administered. For two days, all urine samples remained negative for 24-hour urine tests. However, thereafter, as indicated by the Benedict's reagent test, glucose began to occasionally appear in the urine after lunch, reaching at least approximately 0.5% in the urine, indicating that postprandial blood glucose remained slightly above the renal threshold, sometimes below the threshold, and occasionally slightly above the threshold and appeared in the urine. Instead of resuming metformin, it was explored whether increasing the dose of the 100LC composition and the frequency of consumption of the composition to four times daily would compensate for the full effect of the omitted metformin. The timing of metformin administration was also changed from the earlier plan of one tablet at breakfast and a second tablet after lunch in the afternoon to one tablet approximately two hours before breakfast with an early morning fruit snack, and another tablet with dinner in the evening, creating an interval of approximately 12 hours between two metformin administrations.

[0088] During the exploration, the dosage of the improved compositions IC1, IC2, and 100LC was gradually increased from 80 mg per dose to 160 and 320 mg per dose, respectively. However, some glycosuria continued to occur; however, all metformin tablets were gradually withdrawn, initially the nighttime and morning doses, and finally the lunchtime dose, and postprandial blood glucose levels, although exceeding 180 mg / dL, were consistently kept below 224 mg / dL (the blood glucose level recorded when using only full-dose metformin), with an additional postprandial blood glucose record of 238 mg / dL prior to the initiation of the latest series of administration of the improved compositions of the present invention. Two months after complete discontinuation of metformin, blood glucose levels were 108.8 mg / dL fasting and 193.5 mg / dL postprandial, and approximately 11 months later, these levels were 123 mg / dL fasting and 209 mg / dL postprandial. It is noteworthy that even 11 months after complete discontinuation of metformin, blood glucose levels did not exceed the fasting level of 138 mg / dl and the postprandial level of 224 mg / dl; thus providing evidence that the improved composition is more effective than metformin and can replace it.

[0089] When it appeared that this provided the highest level of efficacy of the improved composition, immediate-release tablets of the improved composition 7F containing 40 mg and 80 mg, and a 320 mg extended-release tablet prepared from 7F, were prepared and evaluated. The 320 mg extended-release tablet was initially evaluated by Subject 1 by taking it twice at 12-hour intervals over 24 hours. However, no perceptible improvement was observed. Subsequently, the 40 mg tablet was evaluated four times daily (once upon waking, once at breakfast, once at lunch, and once at bedtime snack); and surprisingly, after a long period, urine glucose was again negative in all four time intervals, indicating better optimal release of the active ingredient compared to the syrup (IC1, IC2, and 100 LC) and the 320 mg extended-release tablet. Based on this finding, after four consecutive days of taking the 40 mg tablet, the dose was reduced to half the dose of 40 mg each time; and this also helped to maintain urine glucose levels at zero throughout. The 80 mg tablet was finally evaluated, four times daily at the times shown for the 40 mg tablet, and the results were the same as for the 40 mg tablet. All of this suggests that the threshold level for efficacy may be 40 mg or less, and there is still room to determine the correct optimal dose, as well as to explore ways to release the active agent more effectively so that the dose can be further reduced.

[0090] In 2013, when metformin was administered alone to a patient, Subject 1's HBA1C count deteriorated to 9.56. In 2016, when the improved composition of the present invention was started, the HBA1C count was approximately 8.6, and after approximately 11 months of complete discontinuation of metformin and use of the improved composition of the present invention alone, it was 8.16. Based on the fact that negative urine glucose tests at any time indicated that the tablets were more effective in optimally releasing the active substance (even by quantitative Benedict's reagent testing), this treatment can contribute to a slight improvement in HBA1C count, bringing it to an acceptable level, in adults aged 65 years who have had diabetes for at least the past 11 years.

[0091] HDL cholesterol levels increased from 30 mg / dl to 50 mg / dl.

[0092] It was also observed that when the improved composition was started in March 2016, high blood pressure also slowly rose to 160 / 90 each evening, causing headaches. This decreased to about 140 / 80 and quickly reached 130 / 72, forcing a reduction in the amlodipine dose from 5 mg daily to 2.5 mg daily.

[0093] Within a month of treatment with the improved composition, which began in March 2016, the fatigue that had gradually increased to chronic disease levels in mid-2016 disappeared, and the quality of life improved.

[0094] Therefore, Subject 1's health condition was comprehensively improved.

[0095] In Subject 2 (a 58-year-old woman, also mentioned by Savangikar and Savangikar (2014)), blood glucose levels, as indicated by urine glucose levels, remained within control (fasting 144 mg / dl and postprandial 207 mg / dl) after a full year of administration of the composition of the present invention as a complete replacement for previously used metformin.

[0096] In the third subject diagnosed with glucose intolerance, all of the above compositions were administered twice daily at a dose of 80 mg; once with breakfast and once with dinner. His blood glucose levels returned to normal (105 mg / dL fasting and 129 mg / dL postprandial).

Claims

1. Use of a composition for manufacturing an antipathogenic dosage form, said composition comprising a water-insoluble concentrate that is inherently free of components more readily soluble in plant sap than said water-insoluble concentrate, and said water-insoluble concentrate being prepared from plant sap solubles, said water-insoluble concentrate being enriched with heat-stable proteins of said plant sap solubles.

2. Use of the composition of claim 1, wherein the parameters relating to the properties of the composition are standardized and are related to antipathogenic properties or the content of protein molecules with a molecular weight of 26 kilodaltons (kD) or less.

3. Use of the composition of claim 1, wherein the antipathogenic effect comprises antimicrobial activity against organisms including microorganisms or as an adiponectin agonist in mammals.

4. Use of the composition of claim 3, wherein the adiponectin agonist effect in mammals includes reducing the risk of one or more conditions selected from type II diabetes, hyperlipidemia, atherosclerosis, hypertension, heart disease, and obesity.

5. Use of the composition of claim 3, wherein the antimicrobial effect comprises reducing the risk of microbial infection in plants and animals or treating said microbial infection, with or without synergy with other antimicrobial agents.

6. A composition comprising a water-insoluble concentrate, the water-insoluble concentrate being essentially free of components more readily soluble in plant juices than the water-insoluble concentrate, and the water-insoluble concentrate being prepared from plant juice solubles, the water-insoluble concentrate being enriched with heat-stable proteins of the plant juice solubles.

7. The composition of claim 6, further comprising a dosage form prepared from the composition of claim 6.

8. The composition of claim 7, wherein the dosage form comprises a concentrated syrup of the composition, a chewable or orally soluble tablet, granules, powder, a direct-delivery tablet, an intermediate-delivery tablet, a controlled-delivery tablet, a capsule, or an orally soluble film with or without a microemulsion.

9. A method for preparing a water-insoluble concentrate, wherein the water-insoluble concentrate inherently does not contain components more readily soluble in plant juices than the water-insoluble concentrate itself, and the water-insoluble concentrate is prepared from plant juice-soluble substances, wherein the water-insoluble concentrate is enriched with heat-stable proteins of the plant juice-soluble substances. This method involves heat-treating plants or plant sap to precipitate proteins, thereby obtaining heat-stable plant sap-soluble proteins as sap-soluble substances. A significant amount of these proteins can be thermally coagulated along with a group of phytochemicals that co-coagulate with heat-precipitable proteins in the plant. Other plant components, carrying small amounts of heat-stable, juice-soluble proteins, are left in the juice to produce juice-soluble substances, and in which... Add an amount of ascorbic acid to the juice-soluble components to prevent oxidation. This is followed by one or more of the following steps: (a) Allow the soluble parts of the juice to stand for a period of time to allow insoluble solids to appear, and (i) Allow the solid to settle at the bottom, then decant the supernatant, or (ii) Centrifuging to separate the insoluble solids from the juice solubles before or after precipitation, or (b) Concentrate the soluble components of the juice by ultrafiltration or boiling, and further decant, filter, or centrifuge away the insoluble components from the retentate. Furthermore, parameters relating to the properties of the composition are standardized, which are related to antipathogenic properties or the content of protein molecules with a molecular weight of 26 kilodaltons (kD) or smaller.

10. The method of claim 9, wherein the standard for the parameters relating to the properties of the composition is determined according to one or more analytical parameters selected from the group consisting of: (a) the dry matter percentage of the composition, (b) the nitrogen percentage of the dry matter of the composition, (c) the content of casein or gallic acid, (d) by molecular weight determination, or (e) by Fourier transform infrared spectroscopy.

11. The method of claim 9, wherein the heat-stable plant sap soluble plant protein is used with or not used with: pharmaceutically active substances, pharmaceutical excipients, cosmeceutical active substances, cosmeceutical excipients, nutritional pharmaceutically active substances, nutritional pharmaceutical excipients, plant protective active substances, food additives or other food ingredients.

12. A composition comprising a water-insoluble concentrate, said water-insoluble concentrate being inherently free of components more readily soluble in plant juices than said water-insoluble concentrate, and said water-insoluble concentrate being prepared from plant juice solubles, said water-insoluble concentrate being enriched with heat-stable proteins of said plant juice solubles. Optionally, one or more additive ingredients selected from the group consisting of: pharmaceutically active substances, pharmaceutical excipients, cosmeceutical active substances, cosmeceutical excipients, nutritional pharmaceutically active substances, nutritional pharmaceutical excipients, plant protective active substances, food additives or other food ingredients may be added.

13. A composition characterized by an infrared absorption spectrum comprising a peak with a transmittance of 35-60 near a wavelength of 3365, a peak with a transmittance of 60-80 near a wavelength of 2975, a peak with a transmittance of 60-80 near a wavelength of 1612, a peak with a transmittance of 65-70 near a wavelength of 1306, a peak with a transmittance of 70-90 near a wavelength of 1280, a peak with a transmittance of 20-40 near a wavelength of 1080, a peak with a transmittance of 70-98 near a wavelength of 887, and a peak with a transmittance of 85-98 near a wavelength of 825. The composition comprises a heat-stable plant juice soluble protein, which is essentially a water-insoluble concentrate, the water-insoluble concentrate containing no components more soluble in the plant juice than the water-insoluble concentrate, and the water-insoluble concentrate is prepared from plant juice solubles, the water-insoluble concentrate being enriched with the heat-stable protein of the plant juice solubles.

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