Methods for concentrating polyphenolic compounds

Abstract

Described herein is a resin-free method for extracting a polyphenol from a polyphenol- containing biomass and polyphenol-containing extracts made by the method. Also described is a method for extracting a polyphenol from a polyphenol-containing biomass, the method comprising: prefiltering an extracted biomass to remove large impurities and collecting a prefiltered permeate; ultrafiltering the prefiltered permeate and collecting an ultrafiltered permeate; concentrating the ultrafiltered permeate and collecting the concentrated retentate. Various methods and uses of such compositions as natural health products and pharmaceutical products are provided herein.

Description

[0001] METHODS FOR CONCENTRATING POLYPHENOLIC COMPOUNDS

[0002] FIELD

[0003] The present invention relates to the preparation of polyphenol-containing extracts. More specifically, the present invention relates to resin-free extraction methods and related compositions.

[0004] BACKGROUND

[0005] Polyphenols, a group of compounds found in a variety of plants, have garnered significant attention in recent years due to their potential health benefits. These naturally occurring chemicals are abundant in an array of foods like fruits, vegetables, legumes, whole grains, tea, red wine, and. Polyphenols are characterized by the presence of multiple phenol (aromatic) rings and are represented by different classes, for instance, flavonoids (which include subgroups such as flavonols, flavones, flavanones, isoflavones, and anthocyanins); stilbenoids, phenolic acids, and lignans'1’.

[0006] These bioactive compounds have been explored for decades and are known to exhibit antioxidant, and anti-inflammatory activities, reducing the risk of developing chronic diseases, including cardiovascular diseases, cancer, diabetes, infections, asthma, aging, and neurodegenerative disorders'2’3). The pleiotropic beneficial effects of dietary plant polyphenols, particularly of flavonoids and stilbenoids, in promoting human health and preventing various chronic diseases have prompted extensive research and commercial interest in their extraction from medicinal plants.

[0007] Cannabis and hemp also contain a variety of bioactive phytochemicals, including noncannabinoid polyphenols'4 6), each with unique properties that hold promise for improving human health. While further research is necessary to unlock the full potential of these compounds, they offer exciting avenues for developing new therapeutic interventions and enhancing our understanding of how these plants can positively impact human well-being. Bioactive flavonoids and stilbenoids found in cannabis have gained significant attention in recent years due to their diverse and potentially significant effects on human health'78).

[0008] Several extraction methods have been utilized to isolate polyphenols from botanical sources'10). Hot water and organic solvent extraction are commonly used to release polyphenols from the plant material, usually combined with maceration or ultrasound-assisted extraction. Other conventional methods of purification include supercritical fluid extraction utilizing supercritical carbon dioxide; and solid-phase extraction involving passing a liquid polyphenol-containing extract through a solid-phase column, selectively capturing polyphenols by precipitation-adsorption or other chromatographic techniques. The latter approaches, while effective to some extent, often involve multiple steps and suffer from drawbacks such as consuming a significant amount of time and accommodating only small sample volumes. Moreover, certain methods entail a substantial initial investment and result in low polyphenol yields.

[0009] Several patents and patent applications have described various extraction techniques, aiming to enhance the efficiency, yield, and purity of polyphenols from specific plant materials. U.S. Patent No. 10155176 describes a process for the production of a concentrated cannabinoid product optionally also containing an array of terpenes, flavonoids, and other phytoconstituents from a cannabinoid-containing extract feed. The cannabinoid-containing extract feed is contacted with an adsorbent to produce a pre-treated extract which is then subjected to one or more filtration steps, and recovering the concentrated cannabinoid product utilizing an extractant or an evaporator.

[0010] U.S. Patent No. 11344596 describes a method for making a caffeoylquinic composition from a botanical source. The method may include chromatographing an extract of biomass on an ion exchange stationary phase and obtaining an eluent comprising a caffeoylquinic composition. The biomass may be Stevia or yerba mate, for example, whereas the caffeoylquinic composition includes one or more of monocaffeoylquinic acid, dicaffeoylquinic acid, and salts of the foregoing.

[0011] U.S. Patent No. 7682637 describes a product made by a process which results in compositions enriched in total phenols, including purification steps not involving the addition of bisulfite ions. The enriched compositions are characterized as containing monomeric, oligomeric and polymeric phenols, total phenol concentration >12% and exhibiting in vitro COX-2 inhibitory activity enhanced by a factor of at least 2.5 over a standard aspirin dose of 660 pg / ml_.

[0012] U.S. Patent No. 6171638 describes the production of isoflavone-enriched fractions from soy protein extracts. The temperature-sensitive differential of the solubilities of various isoflavone fractions is used to initially separate the fractions by heating an aqueous soy molasses or soy whey feed stream. The temperature of the feed stream is selected according to the temperature at which a desired isoflavone fraction or fractions become soluble. Then, the heated feed stream is passed through an ultrafiltration membrane or reverse osmosis in order to concentrate the solids. The resulting permeate is put through a resin adsorption process carried out in at least one liquid chromatography column to further separate the desired isoflavone fractions. Various processes are described for drying and crystallizing the isoflavone fractions to a powder. A solvent is then added to the isoflavone fraction to dissolve impurities and rehydrate the dry powder which is used as an ingredient in a food ingredient or food product. At various points in the process, a selected amount of isoflavones may or may not be blended with the powder in order to bring the isoflavone to a desired characteristic specification or to produce a food ingredient or food product.

[0013] U.S. Patent Application Publication No. 2021 / 0330732 describes methods of making an extract of fruit of the Solanaceae family wherein fruit is processed to optimise the platelet aggregation inhibiting activity of the extract. The methods involve preparing a start mix of homogenised fruit; separating a water-soluble fraction from fruit solids; filtration of the water-soluble fraction; and concentration of active agents in the filtration permeate. Also described are fruit extracts manufactured by such methods, and also fruit extracts containing glycosylated phenolic acid or a phenolic ester, or derivatives thereof; a glycosylated flavonoid; and a nucleoside. The extracts are described as being useful as medicaments for the treatment or prevention of a medical condition characterised by inappropriate platelet aggregation. In particular, the medicaments may be of use in maintaining heart health by reducing platelet aggregation; benefiting the circulation; and / or normalizing or otherwise benefiting blood flow. International Patent Application Publication No. 2005115160 describes a process for producing cocoa polyphenol concentrate comprising the steps of: (a) subjecting unfermented cocoa beans to a blanching step in water at a temperature in the range from 85-100°C for a time period in the range from 3 to 15 minutes to give unfermented cocoa beans with reduced polyphenol oxidase activity; (b) drying the unfermented cocoa beans with reduced polyphenol oxidase activity at a temperature of less than 85°C to obtain dried unfermented cocoa beans with a moisture content of no more than 15%; (c) subjecting the dried unfermented cocoa beans to a particle size reduction step to obtain dry unfermented cocoa bean intermediate whereby at least 99 wt% of the product has a particle size less than or equal to 300pm; (e) extracting polyphenols from the dry unfermented cocoa bean intermediate to give a cocoa polyphenol extract and extracted solids; (d) concentrating the cocoa polyphenol extract to yield a cocoa polyphenol concentrate; and wherein the process further includes a defatting step which is carried out prior to step (d).

[0014] U.S. Patent Application Publication No. 20160031924 describes methods for purifying steviol glycosides, including Rebaudioside X. Sweetener and sweetened containing Rebaudioside X are also provided. Methods of improving the flavor and / or temporal profile of sweetenable compositions, such as beverages, are also provided.

[0015] U.S. Patent Application Publication No. 20200308211 describes a method for purifying sialylated oligosaccharides from a fermentation broth, cell-lysate or biocatalytic reaction mixture for obtaining high amounts of desired sialylated oligosaccharides in high purity. The method is particularly suitable for the large-scale economic purification of sialylated human milk oligosaccharides (such as 3'-sialyllactose, 6'-sialyllactose or sialylated lacto-N-tetraose derivatives) from microbial fermentation, using recombinant bacterial cells or yeast cells. The obtained material is of high purity and can be used for food or medical applications such as medical nutrition products, infant formula, dietary supplements, and general nutrition products.

[0016] U.S. Patent Application Publication No. 20210212335 relates to a method for the purification of a sialyllactose from other carbohydrates, characterized in that the method comprises the steps of subjecting an aqueous solution containing the sialyllactose to two membrane filtration steps using different membranes, a membrane having a molecular weight cut-off of between about 300 Dalton to about 500 Dalton and a membrane having a molecular weight cut-off of between about 600 Dalton to about 800 Dalton.

[0017] International Patent Application Publication No. WO 2021 / 119826describes a cannabis extract enriched in polyphenolic compounds. Also described is a method for enriching a composition with polyphenolic compounds from cannabis as well as compositions made by the method. Various cosmetic and pharmaceutical products, methods, and uses are provided as well as natural health products.

[0018] There remains a need for improved techniques that offer, for example, higher purity, increased efficiency, and / or reduced environmental impact in the extraction of bioactive polyphenols, especially flavonoids and stilbenoids from plant material, and / or to provide a useful alternative. DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further understood from the following description concerning the Figures, in which:

[0020] Figure 1. Process flow diagram of the steps for preparing a polyphenol-enriched extract or composition according to the general method disclosed herein. The numbers indicate the main steps described in the methods.

[0021] Figure 2. Schematic representation of the equipment setup for the extraction of polyphenols adapting filtration steps. The numbers indicate the main steps listed in Figure 1.

[0022] Figure 3. Recovery of (A) cannflavin A and (B) cannflavin B after ultrafiltration tests with different membrane sizes (0.22 micron, 300 KDa, and 50 KDa MWCO), and with dilution of the ethanol with water in the extract feed. The extraction process did not include the use of capturing resin. The concentration of the cannflavins was determined via HPLC-DAD.

[0023] Figure 4. Recovery of cannflavin A from a crude extraction with or without the use of resin to capture polyphenols and adapting a filtration step to recover the flavonoid for the removal of the resin. (A) The bars represent the percentage of recovery (+ / - SD) of the flavonoid in the permeate after the filtration step (with 0.22-micron membrane), or in the final extract represented in relation to the initial feed (60% crude extract : 40% water) (a) or in relation to the permeate after the first filtration (b) when the extract is passed through a nano filter (NF270) without the use of resin. (B) Filtration test with NF270 membrane showing the effect of omitting the use of resin on the concentration of cannflavin A recovered in the final output after the filtration.

[0024] Figure 5. Effect of the temperature on the extraction efficiency of flavonoids from a hemp extract. The crude extract was adjusted to 60% ethanol and at the described temperature (4°C or 20°C) before loading into an ultrafiltration (0.22 micron) 8-channel multi-filter. The concentration of cannflavin A (A) and cannflavin B (B) was measured in the input (feed), permeate of the ultrafiltration stage, and final extract. UQL, under quantification limit.

[0025] Figure 6. Comparison of two membrane types for the nanofiltration stage. The extraction efficiency of flavonoids from a hemp extract was determined when the crude extract was adjusted to 60% ethanol and at 20°C before loading into an ultrafiltration (0.22 micron) 8-channel multi-filter. The nanofiltration stage consisted of a 15 KDa ceramic filter (with a 30:70 ethanokwater extract feed) or the NF270 organic membrane (with a 60:40 ethanokwater extract feed). Measurements included total cannflavin mass in the retentate estimated for 8 L of crude extract (A), cannflavin recovery rate in relation to the permeate (B), and cannflavin concentration in the retentate in relation to total solids (C). The concentration of cannflavins in the permeate was below the quantification limit.

[0026] Figure 7. Comparison of the membrane used in the nanofiltration stage, and their efficiency in the extraction of flavonoids from a hemp extract. The crude extract was adjusted to a 60:40 ethanokwater ratio before loading into the nanofiltration organic membrane (NF270) or XN-45. Measurements of (A) the recovery efficiency, and (B) the concentration (purity) of cannflavin A were performed in the retentate.

[0027] Figure 8. Impact of the ethanol ratio before nanofiltration. The extraction efficiency of flavonoids from a hemp extract was determined when the crude extract was adjusted to the specified ethanokwater ratio before loading into the nanofiltration organic membrane (NF270) at the temperatures tested (4°C and 20°C). Measurements of (A) the recovery efficiency, and (B) and concentration of cannflavin A were performed in the retentate.

[0028] Figure 9. Effect of ethanokwater ratio and nanofiltration membrane on the efficiency of flavonoid purification. (A) Concentration of cannflavin A; (B) concentration of cannflavin B; (C) cannflavin A purity (expressed as a concentration in the total solid in the retentate); (D) concentration factor of cannflavin A extracted (in retentate vs. crude extract).

[0029] Figure 10. Effect of ethanokwater ratio and type of nanofiltration membrane on the filtration efficiency. (A) Final volume after nanofiltration; (B) filtration time.

[0030] Figure 11. Comparison of cannflavins yield and purity obtained with two extraction methods at a pilot scale. The extraction efficiency of flavonoids from a hemp extract was determined by measuring (A) total cannflavin yield and (B) total final cannflavin concentration after performing the extraction process at a pilot scale using the previously developed resin-based protocol and the method described herein based on tandem filtrations stages.

[0031] Figure 12. Effect of the acidity of the solvent on the filtration efficiency. (A) Filtration time of the ultrafiltration stage with a 0.22-micron multi-channel ceramic filter. (B) Filtration time of the nanofiltration stage using an XN-45 organic membrane. (C) Final volume obtained after the nanofiltration step. Tests were performed with different ethanokwater dilution of the solvent and adjusting the pH to 3.0 (+HCI).

[0032] Figure 13. Effect of the acidity of the solvent and the ethanol dilution on the efficiency of flavonoid purification. (A) Concentration of cannflavin A obtained with ethanol dilution previous nanofiltration (using the XN-45 membrane). (B) Effect of the ethanol dilution (50% ethanol with HCL, pH 3) on cannflavin A purity (expressed as concentration in the total solid). (C) Estimation of the concentration factor of cannflavin A extracted (in retentate vs. crude extract). (D) Effect of the ethanol dilution on the concentration factor of cannflavin A extracted.

[0033] SUMMARY

[0034] In accordance with an aspect, there is provided a resin-free method for extracting a polyphenol from a polyphenol-containing biomass.

[0035] In an aspect, the method comprises a filtration step.

[0036] In an aspect, the filtration step comprises tandem filtration.

[0037] In an aspect, the tandem filtration is carried out in batch mode or continuous mode.

[0038] In an aspect, the filtration step comprises microfiltration, ultrafiltration, nanofiltration, or any combination thereof.

[0039] In an aspect, the filtration step comprises, in order, microfiltration, ultrafiltration, and nanofiltration.

[0040] In an aspect, the method further comprises a pre-filtration step.

[0041] In an aspect, the pre-filtration step comprises microfiltration to remove particulate material and produce a microfiltered permeate comprising the polyphenol. In an aspect, the microfiltration uses a microfilter comprising a bag filter, a filter cloth, or a combination thereof.

[0042] In an aspect, the microfiltration uses a microfiltration solvent with a neutral pH.

[0043] In an aspect, the microfiltration uses a microfiltration solvent with an acidic pH.

[0044] In an aspect, the microfiltration solvent has a pH of about 3.0, about 4.0, about 5.0, or about 6.0.

[0045] In an aspect, the microfiltered permeate is diluted with water, such as to about 50% to about 65% ethanol, prior to further filtration steps.

[0046] In an aspect, the filtration step comprises an ultrafiltration step.

[0047] In an aspect, the ultrafiltration step uses an ultrafilter that removes high molecular weight impurities from the biomass and / or from the microfiltered permeate to produce an ultrafiltered permeate comprising the polyphenol.

[0048] In an aspect, the ultrafilter has a tangential flow filtration configuration of a single or multichannel filter.

[0049] In an aspect, the ultrafilter comprises a ceramic membrane.

[0050] In an aspect, the membrane has a 0.1 to 0.5 pm pore size.

[0051] In an aspect, the membrane has a molecular weight cut-off (MWCO) of between 50 KDa and 400 KDa.

[0052] In an aspect, the ultrafiltration is operated at a pressure from about 1 psi to about 20 psi.

[0053] In an aspect, the ultrafiltration is operated at a temperature of about 20° C to about 25° C.

[0054] In an aspect, the ultrafiltration retentate is collected as a waste product or diluted with water to be recycled through the ultrafiltration step again.

[0055] In an aspect, the ultrafiltered permeate is diluted with ethanol, such as with about an equal amount of ethanol, such as with 100% ethanol, prior to further filtration steps.

[0056] In an aspect, the filtration step comprises a nanofiltration step.

[0057] In an aspect, the nanofiltration step uses a nanofilter to concentrate the polyphenols in the biomass and / or microfiltered permeate and / or ultrafiltered permeate to produce a nanofiltered retentate comprising the polyphenol.

[0058] In an aspect, the nanofiltration step comprises tangential flow filtration (TFF).

[0059] In an aspect, the TFF uses a single membrane with a tubular, flat sheet, or spiral-wound configuration.

[0060] In an aspect, the nanofilter comprises an organic thin composite membrane.

[0061] In an aspect, the membrane comprises a polyamide layer deposited on top of a polyethersulfone porous layer on top of a non-woven fabric support sheet.

[0062] In an aspect, the membrane has a MWCO between 150 daltons and 500 daltons.

[0063] In an aspect, the nanofiltration step is operated at a pressure from about 100 psi to about 500 psi.

[0064] In an aspect, the nanofiltration step is operated at a temperature of about 20° C to about 25° In an aspect, the nanofiltered permeate is collected as a waste product or recycled through the nanofiltration step again.

[0065] In an aspect, the extracted polyphenol is further subjected to further separation to provide desired polyphenols of interest.

[0066] In an aspect, the further separation comprises flash chromatography, preparative HPLC, semi-preparative HPLC, or low-pressure vacuum liquid chromatography.

[0067] In an aspect, the extracted polyphenol is concentrated, for example into a concentrated liquid or powder.

[0068] In an aspect, the extracted polyphenol is diafiltrated with water and lyophilized, freeze-dried, spray-dried, or dehydrated.

[0069] In an aspect, the extracted polyphenol is diafiltrated with ethanol and evaporated, for example in a rotary evaporator or ethanol evaporator, to remove the ethanol.

[0070] In an aspect, the final product is a semi-solid deposit.

[0071] In an aspect, the final product is a resin or an oily substance.

[0072] In an aspect, the resin or oily substance is formulated with carrier oils to maintain the oily consistency or with solid excipients to generate a product with a powdery texture.

[0073] In an aspect, the method results in a concentrated polyphenol product comprising from about 10% to about 95% w / w of total polyphenols.

[0074] In an aspect, the polyphenol-containing biomass comprises cannabis.

[0075] In an aspect, the cannabis comprises Cannabis sativa L. marijuana or hemp.

[0076] In an aspect, the polyphenol-containing biomass comprises a leaf, a root, a stem, a branch, a flower, an inflorescence, a fruit, a seed, a cell, a tissue culture, or any combination thereof.

[0077] In an aspect, the polyphenolic compounds comprise flavonoids and / or stilbenoids.

[0078] In an aspect, the flavonoids comprise cannflavin A, cannflavin B, cannflavin C, apigenin, luteolin, orientin, vitexin, isovitexin, chrysoeriol, quercetin, kaempferol, rutin, catechin, or epicatechin, in their glycosylated or aglicone forms, or in a prenylated form, or any combination thereof.

[0079] In an aspect, the stilbenoids comprise canniprene, resveratrol, dihydroresveratrol, cannabistilbene I, or any combination thereof.

[0080] In accordance with an aspect, there is provided a method for extracting a polyphenol from a polyphenol-containing biomass, the method comprising:

[0081] - optionally extracting a milled biomass;

[0082] - prefiltering the extracted biomass to remove large impurities and collecting a prefiltered permeate;

[0083] - ultrafiltering the prefiltered permeate, optionally by tangential flow filtration and collecting an ultrafiltered permeate;

[0084] - concentrating the ultrafiltered permeate, optionally by nanofiltration and collecting the concentrated retentate.

[0085] In an aspect, the method further comprises drying the concentrated retentate.

[0086] In an aspect, the method does not include the use of a capturing resin. In an aspect, the method results in increased extraction efficiency as compared to a method using capture resin.

[0087] In an aspect, the extraction efficiency is greater than about 80%.

[0088] In an aspect, the extraction efficiency is greater than about 99%.

[0089] In an aspect, the polyphenol yield is at least 2 times greater than the yield of a method using capture resin.

[0090] In an aspect, the yield is at least about 5 ug / g.

[0091] In accordance with an aspect, there is provided a polyphenol composition produced by the method described herein.

[0092] In an aspect, the composition comprises a higher concentration of polyphenols as compared to a method comprising the use of adsorption resins.

[0093] In an aspect, the composition is formulated as capsules, pills, liquids, or tinctures alone or combined with excipients.

[0094] In an aspect, the composition is for use as a natural health product, a botanical drug, or a pharmaceutical.

[0095] In accordance with an aspect, there is provided a use of the polyphenol composition described herein for the treatment and / or prevention of inflammatory responses such as rheumatoid arthritis, osteoarthritis, cardiovascular disease, neuroinflammation, and respiratory illnesses.

[0096] In accordance with an aspect, there is provided a use of the polyphenol composition described herein for neuroprotection and / or preventing progression of neurodegenerative and motor neuron disorders such as amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s diseases.

[0097] In accordance with an aspect, there is provided a use of the polyphenol composition described herein for treating and / or preventing cancer.

[0098] In accordance with an aspect, there is provided a method for the treatment and / or prevention of inflammatory responses such as rheumatoid arthritis, osteoarthritis, cardiovascular disease, neuroinflammation, and respiratory illnesses, the method comprising administering the polyphenol composition described herein to a subject in need thereof.

[0099] In accordance with an aspect, there is provided a method for neuroprotection and / or preventing progression of neurodegenerative and motor neuron disorders such as amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s diseases, the method comprising administering the polyphenol composition described herein to a subject in need thereof.

[0100] In accordance with an aspect, there is provided a method for treating and / or preventing cancer, the method comprising administering the polyphenol composition described herein to a subject in need thereof.

[0101] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description. DETAILED DESCRIPTION

[0102] Described herein is a method that represents a substantial advancement in the field of polyphenol extraction by offering, in aspects, a more sustainable, efficient, and / or economically viable approach, setting it apart from existing methods. In aspects, this approach aims to streamline a scaled-up extraction process, enhancing the extraction efficiency and purity of polyphenols from medicinal plants such as cannabis. The various embodiments described herein offer solutions to the challenges within this field and hold distinct advantages. Additionally, this innovation described herein brings forth further benefits that become evident upon reviewing the present disclosure.

[0103] In aspects, described herein are methods for producing a polyphenol-concentrated extract derived from cannabis biomass, where the concentrated extract has an increased concentration of at least one flavonoid and / or at least one other polyphenol molecule relative to the original biomass. Methods described herein generate enriched products that have a variety of uses, most notably as a natural health product or as starting ingredients that can be used to produce botanical drugs, with consistencies from batch to batch.

[0104] Definitions

[0105] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the present invention, the following terms are defined below.

[0106] "Isolated" refers to a molecule that has been purified from its source or has been prepared by recombinant or synthetic methods and purified. Purified flavonoids are substantially free of contaminating components, such as waxes, lipids, pigments, fiber, and / or other phytochemicals, for example.

[0107] "Substantially free" herein means less than about 5%, typically less than about 2%, more typically less than about 1 %, even more typically less than about 0.5%, most typically less than about 0.1% contamination, such as with THC, cannabinoids, and / or terpenes.

[0108] The term “resin-free” in reference to the methods described herein means that the method is, in embodiments, carried out without the use of a capturing resin, such as an adsorption resin or a macroporous adsorption resin.

[0109] As used herein, the term "treatment" refers to an approach aimed at achieving positive clinical outcomes. These outcomes encompass, but are not restricted to, symptom relief, reduction of disease severity, maintenance of a stable disease state (i.e. , preventing deterioration), slowing down the progression of the disease, improvement or palliation of the disease condition, and achieving either partial or complete remission, whether detectable or not. Consequently, "treatment" denotes an intentional intervention designed to modify the pathology of a disorder. More specifically, the intervention may directly impede, decelerate, or otherwise mitigate the pathological processes associated with a disease or disorder, such as inflammation. Alternatively, it might enhance the responsiveness of inflammation to treatment or therapy with other therapeutic agents.

[0110] The terms "effective amount," or "effective dose" signify a quantity that, when administered to a subject, including a mammal like a human, can bring about a desired outcome, such as an amount effective in addressing inflammation. The effective amounts of the polyphenolic compounds detailed here may vary based on factors like the subject's disease state, age, sex, and weight. Dosages or treatment regimens can be adapted to achieve the optimal therapeutic response, as comprehended by a skilled individual. Similarly, an "effective amount" of the polyphenolic compounds mentioned herein denotes an amount adequate to fulfill the intended purpose, such as the treatment of inflammation.

[0111] The term "in combination with" as applied to administration encompasses both simultaneous (concurrent) and consecutive delivery in any sequence, involving one or more additional therapeutic agents.

[0112] The phrase "pharmaceutically acceptable" denotes that the compound or combination of compounds is compatible with the other ingredients in a pharmaceutical formulation and is generally safe for human administration in accordance with established governmental standards, including those set forth by the United States Food and Drug Administration.

[0113] "Carriers" as used herein refer to cosmetically or pharmaceutically acceptable substances, such as carriers, excipients, or stabilizers, that are non-toxic when exposed to cells or subjects at the specified dosages and concentrations. Frequently, the pharmaceutically acceptable carrier takes the form of an aqueous pH-buffered solution. Examples of acceptable carriers include buffers like phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins like serum albumin, gelatin, or immunoglobulins; hydrophilic polymers like polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, and dextrins; chelating agents like EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™; lipid excipients such as triglycerides, mixed glycerides and polar oils, mixed with co-solvents such as ethanol, propylene glycol, glycerol, and polyethylene glycols, or with water-soluble and insoluble surfactants such as Kholliphor®, Maisine®, Peceol®, or Transcutol®.

[0114] The terms "a," "an," "the," and "said" in this context signify one or more elements within the scope of the current application. Similarly, the use of "comprising" and its derivatives indicates inclusivity, allowing for the presence of specified features, elements, components, groups, integers, and / or steps, while not excluding the possibility of other undisclosed elements. This inclusivity extends to synonymous terms such as "including" and "having" and their derivatives.

[0115] It's important to note that embodiments described as "comprising" certain components may also fall under the categories of "consisting of' or "consisting essentially of." The former implies a closed-ended or restrictive composition, while the latter means including the specified components, excluding others except for materials present as impurities, those unavoidably generated during the manufacturing process, and additional components serving a purpose other than the technical effect of the invention. For instance, when a composition is defined as "consisting essentially of," it encompasses any known pharmaceutically acceptable additive, excipient, diluent, carrier, etc. Typically, such a composition will contain less than 5% by weight, often less than 3%, and more typically less than 1% of non-specified components.

[0116] It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation. For example, in embodiments, THC, cannabinoids, and / or terpenes are explicitly excluded from the compositions and methods described herein. In embodiments, the method for extracting a polyphenol as described herein does not use an adsorption resin.

[0117] The provided ranges mentioned herein encompass both their endpoints and any intermediary points within the range, whether expressly mentioned or not.

[0118] Finally, terms denoting degree, such as "substantially," "about," and "approximately," as employed herein, mean an acceptable level of variance in the modified term, ensuring that the outcome remains largely unaffected. In the context of these degree terms, a deviation of at least ±5% from the modified term should be considered, provided such a deviation does not undermine the intended meaning of the term.

[0119] Compositions

[0120] Described herein are cannabis extracts enriched in polyphenolic compounds, such as flavonoids, and related molecules. Also described herein are polyphenol compositions produced by the methods described herein. Typically, the polyphenol compositions comprise a higher concentration of polyphenols as compared to a method comprising the use of adsorption resins. The compositions may be formulated as capsules, pills, liquids, or tinctures alone or combined with excipients and may be used as a natural health product, a botanical drug, or a pharmaceutical.

[0121] Usually, the extracts contain a minimum of 50% polyphenolic compounds, with variations such as at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% polyphenolic compounds. These extracts described herein are typically devoid of THC and contain low concentrations of other cannabinoids (such as cannabidiol), and terpenes. Such extract compositions may have concentrations below 20%, 15%, 10%, 5%, 2%, 1%, 0.5%, 0.1%, or 0.01% of cannabinoids by weight.

[0122] The extracts discussed herein are sourced from various cannabis species, encompassing marijuana and hemp. Cannabis sativa, Cannabis indica, Cannabis ruderalis, and their combinations are potential origins of the extract. Cannabis sativa L. is a common source, and the use of individual strains or combinations within any species is also considered. Moreover, it is essential to note that the extracts may originate from any part of the plant, including leaves, roots, stems, branches, flowers, inflorescences, fruits, seeds, cells, tissue cultures, or a combination thereof.

[0123] The distribution of flavonoids and other polyphenolic compounds in cannabis varies among strains and across different plant tissues®. Consequently, various parts of the cannabis plant can be explored for the extraction and isolation of polyphenols including flavonoids. Flavonoids identified in cannabis include apigenin, luteolin, quercetin, kaempferol, cannflavin A, cannflavin B, vitexin, isovitexin, and orientin'4’5). These compounds appear as methylated and prenylated aglycones or as conjugated O-glycosides or C-glycosides. Additionally, cannabis has other groups of phenolic molecules which include stilbene-related structures such as bibenzyls, dihydrostilbenes, phenanthrenes, and 9,10-dihydriphenanthrenes, and that can also be present as prenylated, geranylated, and glycosylated derivatives. Examples include cannabistilbene I, cannabistilbene Ila, cannabistilbene lib, canniprene, and 3,4’-dihydroxy-5,3’-dimethoxy-5’ isoprenyl bibenzy7).

[0124] Therefore, typically, the extracts described herein comprise flavonoids and stilbenoids, and may include the examples of the compounds described above. In specific aspects, the extracts are enriched with a concentration of cannflavins.

[0125] The compositions described herein are, in aspects, made by the methods described below.

[0126] The compositions are designed for administration to a subject, which may include mammals, including humans. The compositions encompass polyphenolic compounds described herein, with the active ingredient ranging from approximately 0.001% to about 99% by weight, and can span various increments within this range, from about 0.001%, and progressing through increments such as about 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 99.99%. For example, typical dosages may range from about 0.1 pg to 100 pg of the specified molecules per 300 mg dose. This encompasses quantities like 0.5 pg, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 25 pg, 50 pg, or 75 pg per 300 mg dose. Alternatively, dosages may fall within specific ranges, such as 0.1 pg to 10 pg, 1 pg to 5 pg, or 1 pg to 2 pg per 300 mg dose, along with all related increments and weight percentages.

[0127] The described compositions can be utilized in various amounts, typically falling within the range of about 1 to about 10000 ng / kg. Examples include doses ranging from about 1 to about 1000 ng / kg, about 1 to about 500 ng / kg, about 10 to about 250 ng / kg, or about 50 to about 100 ng / kg. Specific values within these ranges could be, for instance, about 1 , about 10, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, or about 500 ng / kg. Alternatively, the compositions may be administered in doses ranging from about 1 to about 10000 mg per dose. Examples of such doses include about 1 to about 1000 mg, about 1 to about 500 mg, about 10 to about 250 mg, or about 50 to about 100 mg. Specific values within these ranges could be, for instance, about 1, about 10, about 25, about 50, about 75, about 100, about 150, about 200, about 250, about 300, about 400, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg. For example, in certain cases, cannflavin A at about 300 mg per dose, and cannflavin B at about 100 mg per dose.

[0128] In various respects, the formulations outlined in this context are administered to achieve a target concentration in the blood of a human, ranging from about 0.01 pM to about 100 pM. This includes concentrations like about 0.01 pM, 0.05 pM, 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, or 90 pM, up to about 100 pM.

[0129] The duration of administering these compositions can vary greatly, ranging from hours to months, depending on several factors including the severity and type of inflammation or other conditions, the possibility of recurrence, and the goal of preventing inflammation or other illnesses. The method of administration may be consistent, such as through a continuous infusion spread over hours, days, weeks, or months. Alternatively, an intermittent approach is also possible, where molecules are administered periodically, such as once daily over several days or once hourly over several hours, based on a suitable schedule.

[0130] The compositions outlined herein can be formulated using established methods for creating compositions suitable for pharmaceutical or cosmetic use, ensuring they can be safely administered to subjects. These compositions involve combining an effective quantity of the active substance with a pharmaceutically acceptable vehicle. The selection of these vehicles can draw from well-known options, as detailed in sources like the "Handbook of Pharmaceutical Additives" (compiled by Michael and Irene Ash, Gower Publishing Limited, Aidershot, England, 1995). The compositions, though not limited to this, may include solutions of substances in conjunction with one or more pharmaceutically acceptable vehicles, excipients, or diluents. These solutions may exist in buffered forms with an appropriate pH level and / or maintain iso-osmotic properties with physiological fluids.

[0131] Those proficient in the field are familiar with pharmaceutically acceptable carriers, which encompass well-known substances such as sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, cannabis oil, and water. Additionally, the composition may include stabilizers like carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, dextrin, and glucose), proteins (e.g., albumin or casein), and buffers such as alkaline phosphates. The compositions presented here may be administered through various routes, including but not limited to parenteral, intravenous, subcutaneous, intradermal, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, intrarectal, intravaginal, aerosol, oral, topical, or transdermal methods. Generally, oral or topical administration is preferred, directly targeting sites of inflammation or incorporating the composition into cosmetic oils, lotions, creams, or gels for specific applications on the skin.

[0132] Professionals in the field recognize that the compositions described herein can be employed in conjunction with established therapies for preventing and / or treating inflammation in subjects. Additionally, they may be combined with formulations designed to combat aging signs or other cosmetic products. Furthermore, these compositions may be blended with other cannabis-derived products, including cannabinoids. In various embodiments, the compositions may be administered in combination, concurrently, or sequentially with conventional treatments for inflammation or other conditions, such as non-steroidal anti-inflammatory drugs. Formulating the compositions together with these conventional treatments is a viable option when deemed appropriate.

[0133] Because of their high content of polyphenolic compounds, the compositions discussed here are expected to exhibit various effects typically linked with these compounds. Consequently, these compositions might display a range of properties, such as anti-inflammatory, antioxidant, neuroprotective, anticancer, antimicrobial, and antiviral activities.

[0134] Methods of Production

[0135] Described herein are resin-free methods for extracting a polyphenol from a polyphenolcontaining biomass. Generally, the methods involve a series of filtration steps, which can be carried out in series or in tandem. When tandem filtration is used, it can be carried out in batch mode or continuous mode. It will be understood that any type of filtration can be used, alone or in combination and that each type of filtration can be used singly or repeatedly. For example, typically, the filtration steps include microfiltration, ultrafiltration, nanofiltration, or any combination thereof. Typically, the filtration steps include, in order, microfiltration, ultrafiltration, and nanofiltration.

[0136] Typically, the methods include a pre-filtration step. The pre-filtration step is generally carried out with a microfilter to remove particulate material. Any microfilter can be used, examples include a bag filter, a filter cloth, or a combination thereof. The microfiltration solvent typically has a neutral or acidic pH, such as from about 3.0 to about 7.0, such as a pH of about 3.0, about 4.0, about 5.0, about 6.0, or about 7.0.

[0137] The retentate is discarded or recycled through the microfilter again, usually after being diluted. The microfiltered permeate is retained and contains the polyphenol. Typically, the microfiltered permeate is diluted prior to further filtration steps. For example, it can be diluted with water, such as to about 50% to about 65% ethanol, prior to further filtration steps.

[0138] Typically, the methods include an ultrafiltration step, which uses an ultrafilter that removes high molecular weight impurities from the biomass and / or from the microfiltered permeate. The ultrafilter typically has a tangential flow filtration configuration of a single or multichannel filter. In aspects, the ultrafilter comprises a ceramic membrane. The pore size of the ultrafilter can be selected to filter out desired impurities and is, typically, in the range of from about 0.1 to about 0.5 pm. Similarly, the ultrafilter can be selected to have a desired molecular weight cut-off (MWCO), which is typically between about 50 KDa and about 400 KDa.

[0139] Any of the filtration steps can be operated at a desired pressure and temperature. Typically, the ultrafiltration step is operated at a pressure from about 1 psi to about 20 psi and at a temperature of from about 20° C to about 25° C.

[0140] Similar to the microfiltration step, the retentate is either discarded or recycled through the ultrafiltration step again after being diluted, typically with water. The ultrafiltered permeate is retained and contains the polyphenol.

[0141] Prior to further filtration steps, typically the ultrafiltered permeate is diluted, for example with ethanol, such as with about an equal amount of ethanol, such as with 100% ethanol.

[0142] Typically, the methods include a nanofiltration step, which uses a nanofilter to concentrate the polyphenols. In this case, the nanofiltered retentate contains the polyphenol and the nanofiltered permeate is collected as a waste product or recycled through the nanofiltration step again.

[0143] While any nanofiltration can be used, as will be understood, generally, the nanofiltration step comprises tangential flow filtration (TFF), which typically uses a single membrane with a tubular, flat sheet, or spiral-wound configuration. Typically, the nanofilter comprises an organic thin composite membrane. Any such membrane can be used and typically comprises a polyamide layer deposited on top of a polyethersulfone porous layer optionally on top of a non-woven fabric support sheet.

[0144] As noted above, the membrane can be chosen to have any desired MWCO. Typically, a MWCO of between about 150 daltons and about 500 daltons is chosen. Likewise, any desired pressure and temperature can be chosen for the nanofiltration step. Generally, the nanofiltration step is operated at a pressure from about 100 psi to about 500 psi and at a temperature of about 20° C to about 25° C.

[0145] Thus, in some specific aspects, provided herein is a method for extracting a polyphenol from a polyphenol-containing biomass. The method comprises: prefiltering an extracted biomass to remove large impurities and collecting a prefiltered permeate; ultrafiltering the prefiltered permeate, optionally by tangential flow filtration and collecting an ultrafiltered permeate; concentrating the ultrafiltered permeate, optionally by nanofiltration and collecting the concentrated retentate. Typically, the method includes an initial step of extracting a milled biomass. Typically, the concentrated retentate is dried although it can be used in liquid or semi-solid form. In specific aspects, the method does not include the use of a capturing resin.

[0146] In aspects, the methods include steps of further separating the extracted polyphenol composition in order to further isolate specific desired polyphenols of interest. This further separation can be by any known method. Typically, the further separation comprises flash chromatography, preparative HPLC, semi-preparative HPLC, or low-pressure vacuum liquid chromatography.

[0147] The extracted polyphenol is concentrated, for example into a concentrated liquid or powder. For example, in aspects, the extracted polyphenol is diafiltrated with water and lyophilized, freeze- dried, spray-dried, or dehydrated. More specifically, in aspects, the extracted polyphenol is diafiltrated with ethanol and evaporated, for example in a rotary evaporator or ethanol evaporator, to remove the ethanol.

[0148] The final product may take any form. Typically, however, it is a semi-solid deposit, a resin, or an oily substance. Typically, the resin or oily substance is formulated with carrier oils to maintain the oily consistency or with solid excipients to generate a product with a powdery texture.

[0149] In aspects, the methods described herein result in a concentrated polyphenol product comprising from about 10% to about 95% w / w of total polyphenols.

[0150] The polyphenol-containing biomass can be from any source. Typically, it is a plant source and, more typically it comprises cannabis, such as Cannabis sativa L. marijuana or hemp. For example, the polyphenol-containing biomass comprises a leaf, a root, a stem, a branch, a flower, an inflorescence, a fruit, a seed, a cell, a tissue culture, or any combination thereof.

[0151] Typically, the sought-after polyphenolic compounds comprise flavonoids and / or stilbenoids. For example, the flavonoids may comprise cannflavin A, cannflavin B, cannflavin C, apigenin, luteolin, orientin, vitexin, isovitexin, chrysoeriol, quercetin, kaempferol, rutin, catechin, or epicatechin, in their glycosylated or aglicone forms, or in a prenylated form, or any combination thereof. The stilbenoids may comprise canniprene, resveratrol, dihydroresveratrol, cannabistilbene I, or any combination thereof.

[0152] In typical aspects, the method advantageously results in increased extraction efficiency as compared to a method using capture resin. For example, the extraction efficiency is greater than about 80% or greater than about 99%. In additional or alternative aspects, the polyphenol yield is at least 2 times greater than the yield of a method using capture resin. For example, in aspects, the yield is at least about 5 ug / g.

[0153] The methods described herein are explained in more detailed below. Extraction. Plant material, such as hemp or cannabis plant material in raw form (fresh or dried) or previously extracted plant material (including flowers, leaves, stems, stalks, etc.) used as starting material for preparing the polyphenol-containing extract may be subjected to conventional extraction processes known in the art (STEP 1 , Figure 1 ). These include extraction with solvents such as ethanol, isopropyl alcohol, methanol, carbon dioxide, butane, hexane, ethyl acetate, and ionic liquids; however, less toxic solvents are generally preferred. For example, in one embodiment the solvent used to prepare the polyphenol-rich extract comprises ethanol. To create a polyphenol extract, first, the cannabis plant material is milled or ground and then introduced into an extraction vessel. Subsequently, a solvent such as ethanol, is typically introduced into the extraction vessel, and the contents are then stirred, often mechanically with an impeller to substantially reduce the plant material to a mixture containing a suspension of finely particulate plant material. This agitation process typically spans from approximately 1 to 4 hours, though variations in duration are possible. The resulting mixture from the extraction vessel serves as the polyphenol extract feed for the process described herein. When utilizing ethanol for extraction, specifically 200-proof USP grade anhydrous ethanol, the preparation of the polyphenol extract involves incorporating around 10 L of ethanol per kilogram of raw plant material into the extraction vessel. The ethanol introduced into the extraction vessel can be cooled to temperatures ranging from approximately -20° C to about +20° C. Subsequently, the temperature of the polyphenol-containing extract may be elevated to create optimal conditions for downstream processes.

[0154] Pre-filtration. Depending on the amount of fiber present in the extraction mix, typically a first pre-filtration step is helpful to remove particulate material. In this first filtration step of the process (STEP 2, Figure 1 ), various filtration techniques can be applied. One method involves utilizing a bag filter for batch extraction, composed of polyethylene or polypropylene, which can be placed in line with a collecting vessel or tank as described in Figure 2. Some variations of this pre-filtration entail utilizing filter paper, filter cloth, or a combination thereof. The crude extract might undergo pressurization (e.g., maintaining a pressure ranging from about 1 psi to about 10 psi) to facilitate the filtration process. Generally, these filters have an average particle size of less than about 500 pm, less than about 250 pm, less than about 100 pm, less than about 50 pm or less than about 10 pm. For example, from about 10 pm to about 100 pm, from about 75 pm to about 250 pm, or from about 100 pm to about 400 pm. Alternative filtration approaches include passing the crude extract through a sand bed or a 30- micron polypropylene filter, diatomaceous earth (silica), or cellulose, and then passing through a filter aid bed. These filter types aim to eliminate precipitated solids and are not designed as size-exclusion filters.

[0155] We have previously described a method to capture polyphenols based on macroporous resins in International Patent Application Publication No. WO 2021 / 119826, which is incorporated herein by reference in its entirety. Described herein is a substantial and surprising improvement over the previously described method, which involves the implementation of a separation technique using filtration processes to produce a concentrated polyphenol extract. The present method includes, for example, microfiltration, ultrafiltration, and nanofiltration. The filtration processes outlined herein can be carried out either in batch mode or continuous mode. Continuous filtration, in particular, benefits from filters such as tangential flow or cross-flow filters, and is configured as single-pass or multi-pass modules, with a variety of forms including tubular, capillary, or spiral-wound. Irrespective of the type, the filters employed in the methods described herein typically have a molecular weight cut-off, which determines the particle size retained within the filter. Consequently, those skilled in the field can select the suitable filter for the desired end use based on the procedures outlined herein. In several embodiments, the ultrafiltration and nanofiltration separation filters or membranes typically consist of ceramic materials, organic polymers, or organic nanostructures. In specific embodiments, the filters are made of a ceramic material, for example, filters comprising multi-channel ceramic elements.

[0156] TFF Ultrafiltration. In one aspect of the method shown in Figure 1 , the filtered extract from STEP 2 is diluted with water to about 50%, about 55%, about 60%, and about 65% ethanol before loading to the ultrafiltration column (STEP 3 in Figure 1). The objective of this filtration step is to remove undesired non-phenolic materials from the crude extract such as high molecular weight waxes, high molecular weight biological fragments, and combinations thereof, as well as various impurities, such as cellulose fragments, pesticides, chlorophyll, and other pigments, which is evidenced by the lighter tone of the resulting permeate. Additionally, this step diminishes the necessity for winterization, a process usually employed to eliminate waxes, fats, and other undesired substances from plant extraction techniques.

[0157] Typically, the solvent used to prepare the polyphenol extract is neutral, with the pH of the solvent being from about 6.5 to about 7.5. In one variant, the diluted extract feed comprises an acid, such as hydrochloric acid, sulfuric acid, or acetic acid that is added to the diluting water to adjust the pH of the extract feed to about 3.0, to about 4.0, to about 5.0, or to about 6.0. Typically, the extract feed that will be subject to ultrafiltration is diluted with deionized water without adjusting pH or with acidified water to a final pH of 3.0.

[0158] Referring to STEP 3 described in Figure 1 , the ultrafiltration process can be performed as tangential flow filtration (TFF), which has the advantages of minimizing fouling, offering high product recovery rates, allowing process scalability and continuous operation. In some embodiments, the column consists of one housing unit that has one, about three, about five, about seven, about ten, or about 20 ceramic channels. Typically, a single 0.2 pm cut-off filter can run at a rate of 30 to 60 L per hour per m2, and a column with seven filters at approximately 400 L per hour. This permeate comprises polyphenols and other valuable phytochemicals, including flavonoids and cannabinoids. Typically, the permeate recovers about 80% of the flavonoids and cannflavins present in the extract feed.

[0159] Separation filters for use in the ultrafiltration stage may be constructed from materials such as organic polymers and inorganic ceramic materials, including, for example, and without limitation, filters comprising oxide-based ceramic, titanium dioxide (TO2), polyethersulfone, cellulose triacetate, polyacrylonitrile, or polypiperazine-amide thin-film composite. Generally, these membranes are rated by their molecular weight cut-off (MWCO) of less than about 400 KDa, less than about 300 KDa, less than about 150 KDa, less than about 50 KDa, less than about 10 KDa, or less than about 5 KDa. Examples of suitable separation membranes for use in this stage include, for example and without limitation, Filtanium 0.2 Inside Ceram (Tami), KleanSep™ (Orelis), UF-PES, UF-CTA, and Hydrostart (Sartorius), UH030, UP005, and FB-02-FC (Mycrodin Nadir), HFM-116 / 100, HFM-180 / 513, and HFK- 618 (Koch Inc.), XT, MT, and VT (Synder Filtration, Inc.), UF-GK, UF-GH, UF-PT, and UF-GE (GE Osmonics), UF UA60 (TriSep Corp), UF10706, and UF03705 (SolSep BV), and PVDF20, (Novamen Ltd.).

[0160] This ultrafiltration stage typically operates at a reduced pressure, which can be adjusted contingent on the filter's type, configuration, and composition. Operating pressures usually do not exceed about 30 psi, about 20 psi, or about 10 psi. Typically, this ultrafiltration stage is conducted at an operating pressure from about 1 psi to about 20 psi, or from about 5 psi to about 10 psi. This filtration process is typically carried out at room temperature, from about 20° C to about 25° C. The filtration can also be conducted at a temperature from about 4° C to about 50° C. In one embodiment, the retentate is collected as a waste product or diluted with water to be recycled to the same ultrafiltration stage (STEP 3) to further separate larger molecules from the desired lower molecular weight components such as polyphenols, cannabinoids, and other desirable phytochemicals that pass through that filter stage. In another embodiment, the permeate is collected and subjected to the next filtration stage to obtain a concentrated polyphenol product as detailed below.

[0161] TFF nanofiltration. In one aspect of the method shown in Figure 1 , the permeate from the ultrafiltration stage (STEP 3) is brought into contact with a nanofiltration column (STEP 4 in Figure 1 ) to obtain a new retentate and another permeate. This retentate comprises the desired polyphenols and other phytochemicals molecules of similar mass, while the permeate contains other impurities of small molecular weight present in the permeate from STEP 3. Typically, the permeate from STEP 3 is directly passed through this new filter at room temperature and without further adjustment of pH. In one variant, the permeate from STEP 3 is diluted with an equal volume of 100% ethanol before applying it to the filter in STEP 4. Regarding this nanofiltration stage, this process can be performed as tangential flow filtration (TFF) which allows scalability of the process and continuous operation. In some embodiments, the column consists of organic polymer filters that have a molecular weight cutoff (MWCO) of about 150 daltons, about 200 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or about 700 daltons, allowing the pass of ethanol with small molecular weight impurities which are substantially removed from the polyphenol-containing retentate, resulting in a higher concentration of the polyphenols in the retentate than in the permeate. Separation filters used in the nanofiltration stage may be constructed from non-ceramic organic materials, including, for example, thin-film composite, thin-film polypiperazine, polyamide thin-film composite, polyethersulfone thin-film composite, and can be of different configurations such as hollow fiber, tubular, flat sheet, and spiralwound. Examples of appropriate separation membranes for utilization in the nanofiltration stage encompass, among other possibilities, FILMTEC™ NF270, and NF90 (Dupont), XN-45 and TS80 (TriSep Corp.), NFS, NFX, NFW, NFG, and NDX (Synder Filtration Inc.), NF090801, NF03705, NF080105, NF08105, and NF10706 (SolSep), Puramem (Evonik), TS40 and NP030 (Microdyn- Nadir), Desai DK (GE Water & Process Technologies), PVDF20 and PEEK 1000 (Novamen Ltd), TFC-RO, TFC-S, and TFC-SRD3 (Koch Inc), and NF4, and NF8 (Nanostone Water Inc.).

[0162] This filtration stage typically operates at a moderate pressure, which can be adjusted depending on the membrane characteristics and composition. Operating pressures usually do not exceed about 500 psi, about 300 psi, or about 100 psi. Typically, this nanofiltration stage is conducted at an operating pressure from about 200 psi to about 500 psi, and the flow rate ranges between 5 and 20 L per hour per m2Typically, an input extract of 5 L can produce a polyphenol-concentrated retentate of approximately 250 ml_ with a 99% recovery of flavonoids and cannflavins present in the extract feed (permeate from STEP 3). This filtration process is typically carried out at room temperature, from about 20° C to about 25° C. The filtration can also be conducted at a temperature from about 4° C to about 50° C. In one embodiment, the permeate is collected as a waste product or recycled to the same nanofiltration stage (STEP 4) to further recover polyphenols and other desirable phytochemicals.

[0163] Separation. One skilled in the art will recognize that the composition of the concentrated polyphenol product recovered from the retentate will depend on a variety of factors including the chemical attributes of the biomass, composition of the extract feed, and conditions of the whole extraction process. Therefore, in one embodiment of the method shown in Figure 1 , the retentate from STEP 4 could be further partitioned to separate desired flavonoids or other polyphenols of interest from the rest of the components of the retentate (STEP 5). Suitable separation methods include using flash chromatography, preparative high-performance liquid chromatography (HPLC), semi-preparative HPLC, or low-pressure vacuum liquid chromatography (STEP 5, Figures 1, 2). Typical chromatography methods involve the use of mobile phases comprising organic solvents such as methanol, or acetonitrile, and acidic additives to adjust an acidic pH, such as formic acid, acetic acid, and trifluoroacetic acid. The organic solvents can be used in various concentrations and combinations and concentration gradients, and run at varying flow rates. Acetonitrile concentration in the mobile phase can be below 90%, below 80%, below 70%, below 60%, below 50%, below 40%, below 30%, or below 20%, methanol concentration in the mobile phase can be below 90%, below 80%, below 70%, below 60%, below 50%, below 40%, or below 30%. Commonly, trifluoroacetic acid, formic acid, and acetic acid are used in aqueous solutions at concentrations below 0.1% v / v, below 0.05% v / v, or below 0.01% v / v. A typical chromatographic separation of flavonoids (e.g. cannflavins) from unwanted phytochemicals (e.g. cannabinoids) comprises loading the extract and resuspended in methanol onto a C18 cartridge and using a mobile phase of methanol with 0.1% formic acid and water + 0.1% formic acid in a 10 min 78% to 90% gradient mode and run at a 45 mL / min flow rate. Such separation process can yield exclusion of above 80%, or above 85%, or above 90% or above 95%, or above 99% of cannabinoids or other unwanted phytochemicals. Those skilled in the field can understand that such chromatographic separations can also provide capabilities to characterize other constituents of interest in the polyphenol-rich extract known to be present in cannabis such as stilbenes and other flavonoids.

[0164] Drying. In certain embodiments of the method shown in Figure 1 , a concentrated polyphenol product may be recovered from the retentate from STEP 4 without the need for separation, to be further concentrated to a final product (powder or liquid) depending on the final purpose, intended formulation and use. After the nanofiltration, the retentate in 60:40 ethanokwater solvent ratio can be diafiltrated with water or ethanol, depending on the subsequent drying method to be used. For example, if ethanol is chosen, then the retentate may be transferred to a rotary evaporator or ethanol evaporator, or similar device wherein the solvent is removed and the semi-solid deposit generated is collected as the concentrated polyphenol product (STEP 6, Figures 1 , 2). Alternatively, if water is chosen, then the retentate may be transferred to a spray drying equipment, lyophilizer, freeze dryer or dehydrator. Because of the lipophilic properties of flavonoids and some polyphenols, the retentate is more soluble in higher ethanol concentrations; therefore, the preferred drying method is a rotary evaporator. Typically, temperatures for the evaporative recovery of a concentrated polyphenol product are above about 40°C, above about 50°C, to about 60°C, under reduced pressure. The final product will likely be in the form of a resin or semi-solid oily sediment to an oily substance. A skilled person in the field will understand that such a product can be formulated with carrier oils to maintain the oily consistency or with solid excipients to generate a product with a powdery texture, depending on the intended use. The process described herein typically results in a concentrated polyphenol product comprising at least about 10% w / w, at least about 20% w / w, at least about 50% w / w, at least about 70% w / w, at least about 90% w / w, or at least about 95% w / w total polyphenols.

[0165] The above-described process is suitable for many purposes. For example, preparing compositions sufficiently enriched in total phenols for use as natural health products or botanical drugs. The polyphenol-enriched composition described herein can be crafted into capsules, pills, liquids, or tinctures. When formulating these compositions, a diverse array of excipients can be employed, selected based on the intended method of application of the composition. Excipients may include preservatives, carriers, thickening agents, suspending agents, stabilizers, emulsifiers, and flavoring agents. Specifically, examples encompass ethyl alcohol, water, glycerol, sorbitol, propylene glycol, vegetable triglycerides, fatty acid esters, starch, cellulose derivatives such as methylcellulose, hydroxypropyl methylcellulose, and carboxymethyl cellulose; lauryl sulfate, dicalcium phosphate, lecithin, pectin, gums, polyvinylpyrrolidone, among others.

[0166] The polyphenol-enriched compositions described herein possess a range of biological activities. For example, the compositions described herein can be found to have antioxidant, antiinflammatory, anti-cancer, and neuroprotective properties'7’8>. For example, cannflavins have been investigated as anti-inflammatory substances due to their inhibition of mPGESI activity'9’. In aspects, the compositions described herein containing hemp-derived polyphenols or flavonoid-enriched compounds are used for the treatment of inflammatory ailments in humans and mammals by administering a therapeutically effective dose of these compositions. The compositions can be applied alone or in conjunction with other anti-inflammatory medications to diminish or prevent inflammatory reactions. These reactions may arise from various conditions or diseases, including but not limited to rheumatoid arthritis, osteoarthritis, cardiovascular disease, respiratory illnesses, neuritis, neurodegenerative and motor neuron disorders such as amyotrophic lateral sclerosis, Alzheimer's, and Parkinson's diseases, as well as cancer progression. Moreover, they can provide support for conditions featuring inflammatory responses like infectious diseases, and support the immune system and gastrointestinal health.

[0167] Thus also provided herein are uses of the extracted polyphenols for the treatment and / or prevention of inflammatory responses such as rheumatoid arthritis, osteoarthritis, cardiovascular disease, neuroinflammation, and respiratory illnesses. Further provided are uses of the extracted polyphenols for neuroprotection and / or preventing progression of neurodegenerative and motor neuron disorders such as amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s diseases. Also provided are uses of the extracted polyphenols for treating and / or preventing cancer.

[0168] Also provided herein are methods for the treatment and / or prevention of inflammatory responses such as rheumatoid arthritis, osteoarthritis, cardiovascular disease, neuroinflammation, and respiratory illnesses, the methods comprising administering the extracted polyphenols described herein to a subject in need thereof. Also provided herein are methods for neuroprotection and / or preventing progression of neurodegenerative and motor neuron disorders such as amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s diseases, the methods comprising administering the extracted polyphenols described herein to a subject in need thereof. Further provided herein are methods for treating and / or preventing cancer, the methods comprising administering the extracted polyphenols described herein to a subject in need thereof.

[0169] The preceding disclosure outlines the current invention. Further insight can be gleaned from the specific Examples provided below. These Examples serve purely as illustrations and do not seek to restrict the scope of the invention. Modifications in form and the substitution of equivalents are anticipated as deemed appropriate or expedient. While certain terminology is used herein, it is intended descriptively rather than as restrictive terminology.

[0170] EXAMPLES

[0171] Example 1

[0172] This example describes a process that incorporates a filtration step in a method described in a previous patent application!11) based on the use of an absorption resin that captures polyphenols, and where such filter does not interfere with the collection of such phytochemicals but separates unwanted impurities from the extract. To test this, a membrane filtration assay was set up with three different ceramic filters with different MWCO (0.22 microns, 300 KDa, and 50 KDa) and feeding 4 L of the extract (at a rate of 30 L / H) in 100% ethanol or 50% ethanol.

[0173] Quantification of cannflavins A and B by HPLC-DAD analysis shows that in all three cases, most of the flavonoids remained in the permeate, denoting minimal loss after filtration (Figures 3A, 3B).

[0174] Example 2

[0175] This example describes the incorporation of a filtration step in a method described in a previous patent application!11) that uses an absorption resin to capture polyphenols and its effect on the recovery efficiency of the hemp flavonoid cannflavin A. The conditions of the experiment were: extraction of 5 kg of biomass with denatured ethanol (10 L EtOH / kg of biomass), run at 4°C for 1 hour; dilution of the extract with water to 60% ethanol and passed through a tangential filter (TFF) system that uses a 0.22-micron filter to remove impurities, at a flow of 30 L / Hin at 5 psi of pressure. Six liters of the permeate was mixed with or without a PVPP resin to capture the polyphenols in the extract. A TFF step with the same specifications was used to recover the resin and after elution of the bound polyphenols with 100% ethanol, a third TFF step was used to remove the resin from the isolated compounds. The observed recovery of cannflavin A was not superior to that obtained when the extract was subjected to a nanofiltration stage consisting of passing the permeate of the first TFF step through an NF270 membrane and without the use of the absorption resin (Figure 4). In this nanofiltration step, 2 L of the permeate was allowed to pass at 20 L / H at 200 psi and room temperature, while 6 L of 100% ethanol was used as diafiltration to remove excess water from the extract and recover the flavonoids in ethanol in a final volume of 0.3 L (retentate).

[0176] Up to 47% recovery was obtained with the resin with the filtration method when the amount of flavonoids recovered was compared with the amount present in the initial feed to the filtration stage vs. 73% obtained with the filtration method without the resin. Moreover, the nanofiltration membrane was able to recover over 99% of the flavonoids when compared with the filtration permeate (Figure 4A).

[0177] The average yield of cannflavin A in this example was estimated by calculating the concentration in the samples that were subjected to HPLC analysis. Calculation of the concentration of cannflavins in the final extract when comparing both methods, shows similar results (Figure 4B), suggesting that eliminating the use of the resin would not affect the development of an efficient resin- free extraction method.

[0178] Example 3

[0179] This example describes the effect of the temperature on the efficiency of an ultrafiltration step used to extract flavonoids from a hemp extraction. The crude extract (8 liters) was adjusted to 60% ethanol and at 4°C or 20°C before loading it onto an 8-channel TFF filter system with a 0.22-micron ceramic filter. The TFF conditions were 30 L / H and run under 7 psi or pressure. Concentrations of the flavonoids were analyzed in the input (feed), in the ultrafiltration permeate, and final extract.

[0180] An increase in the purity of cannflavins A and B was observed at 4°C. While extraction of cannflavin A was more efficient at 4°C, extraction of cannflavin B seems to be more effective at 20°C (Figure 5).

[0181] Example 4

[0182] This example describes the efficacy of two types of filters in concentrating flavonoids in the nanofiltration step described in this disclosure. Five kg of hemp biomass was used to perform an extraction with denatured ethanol (10 L EtOH / kg of biomass) for 1 hour at 4°C. Eight liters of the extract was then diluted with water to 60% ethanol and passed through an 8-channel TFF filter system that uses a 0.22-micron filter to remove impurities, at a flow of 30 L / H at 7 psi of pressure. Three liters of the collected permeate was then subject to a nanofiltration stage consisting of a TFF step using a 15 KDa ceramic filter with a 30:70 EtOH:water extract feed, or using an NF270 organic membrane with a 60:40 EtOH:water extract feed. The permeate was allowed to pass at 20 L / H at 250 psi and room temperature, while 6 L of 100% ethanol was used as diafiltration to remove excess water from the extract and recover the flavonoids in ethanol in a final volume of 0.8 L (retentate).

[0183] Quantification of cannflavins A and B in the retentate shows that the NF270 organic membrane was more efficient than the 15 KDa filter in recovering both flavonoids when measured by mass (Figure 6A), recovery rate (Figure 6B) or final concentration in relation to total solids (Figure 6C).

[0184] Example 5

[0185] This example describes the efficacy of two organic membranes in concentrating flavonoids in the nanofiltration step described in this disclosure. This test followed the same protocol as described in Example 4. The nanofiltration stage consists of a TFF step using an NF270 or an XN-45 organic membrane with a 60:40 EtOH:water extract feed. The permeate was allowed to pass at 20 L / H at 400 psi and room temperature, while 6 L of 100% ethanol was used as diafiltration to remove excess water from the extract and recover the flavonoids in ethanol in a final volume of 0.8 L (retentate). Quantification of cannflavin A in the retentate shows similar recovery efficiency with both membranes (Figure 7A); however, the NF270 membrane yielded about 20% more flavonoid concentration in the retentate when compared to the XN-45 membrane (Figure 7B).

[0186] Example 6

[0187] This example describes the efficiency of the nanofiltration step to recover cannflavins when the ethanol ratio is changed before the filtration stage. This test followed the same protocol as described in Example 4. The nanofiltration stage consists of a TFF step using an NF270 organic membrane with a 60:40 EtOH:water or an 80:20 EtOH:water extract feed. The permeate was allowed to pass at 20 L / H at 250 psi and two different temperatures, 4°C and 20°C, while 6 L of 100% ethanol was used as diafiltration to remove excess water from the extract and recover the flavonoids in ethanol in a final volume of 0.8 L (retentate). Quantification of cannflavin A in the retentate showed better recovery efficiency with the 60:40 EtOH:water feed (Figure 8) when the filtration was run at 4°C, while a 60:40 EtOH:water feed resulted in better flavonoids recovery at room temperature or 20°C (Figures 8A and 8B).

[0188] Example 7

[0189] This example describes the efficiency of the nanofiltration step to recover cannflavins with different concentrations of ethanol and with two different organic membranes. This test also followed the same protocol as described in Example 4. The nanofiltration stage consisting of a TFF step using the NF270 or the XN-45 organic membrane with an extract feed with EtOH:water at 60:40, 55:45, and 50:50 ratios. 2 L of permeate was allowed to pass at 30 L / H at 250 psi and room temperature, while 2 L of 100% ethanol was used as diafiltration to remove excess water from the extract and recover the flavonoids in ethanol in a final volume of approximately 0.3 L (retentate). The 60:40 ratio was less specific to cannflavin (reduced purity and more contaminants). This may be due to the smaller pore size of the NF270 membrane which causes some clogging. The concentration of the flavonoids was more difficult than with the XN-45 membrane (Figures 9A, 9B). The 50:50 EtOH:water ratio appeared to be a better solvent ratio to increase cannflavin purity with the NF270 membrane, although the final concentration was similar to that with the XN-45 membrane (Figure 9C). With this solvent ratio, fewer contaminants and no significant clogging were observed, however recovery of the flavonoids was lower most likely due to more precipitation of the prenylated flavonoids in water (Figures 9A, 9B, 9D). Finally, the 55:45 ratio appeared as a good compromise between yield and selectivity for the flavonoids (Figures 9A, 9B, 9D).

[0190] Example 8

[0191] This example describes the effect on the nanofiltration time and volume produced in the same test described in Example 7 which used the NF270 or the XN-45 organic membrane with an extract feed with EtOH:water at 60:40, 55:45, and 50:50 ratios. The 55:45 and 50:50 EtOH:water ratios resulted in less volume generated in the retentate with either organic membrane, while the 60:40 ratio only worked optimally with the XN-45 membrane (Figure 10A). The filtration time was more favorable with the 60:40 solvent ratio (Figure 10B).

[0192] Example 9

[0193] This example illustrates the advantages of the filtration-based polyphenol extraction described in the present disclosure over the previously disclosed resin-based protoco|(11>. The extraction test using an absorption resin is described in Example 2, while the extraction method used based on ultrafiltration and nanofiltration steps is described in Example 7, with a 60:40 ethanol:water ratio as extract feed for the nanofiltration step. Both tests were performed at a pilot scale Comparison of the yield of both cannflavins shows that the filtration method generates almost 3 times more flavonoids in the retentate compared to the resin-based method (Figure 11 A). These results suggest that despite both methods rendering similar purity (Figure 11 B), the filtration-based method is more efficient in extracting the desired polyphenols from the hemp extract. In addition, the method disclosed herein is simpler to operate, quicker, and more cost-effective.

[0194] Example 10

[0195] This example describes the effect of changing the pH of the solvent on the filtration times and volumes of the filtration-based extraction process. Five kg of hemp biomass was used to perform an extraction with denatured ethanol (10 L EtOH / kg of biomass) for 1 hour at 4°C. Six liters of the extract was then diluted with water with or without HCL (adjusted to pH 3) to 60% ethanol or 50% ethanol and passed through an 8-channel TFF filter system that uses a 0.22-micron filter to remove impurities, at a flow of 30 L / H. Two liters of the collected permeate is then subject to a nanofiltration stage consisting of a TFF step using an XN-45 organic membrane. The permeate was allowed to pass at 8 mL / H at 200 psi and room temperature, while 2 L of 100% ethanol was used as diafiltration to remove excess water from the extract and recover the flavonoids in ethanol in a final volume of approximately 0.3 L (retentate). The addition of acidic water to the extract feed reduced significantly the filtration time regardless of the ethanokwater ratio used during the ultrafiltration stage (Figure 12A), while it did not have a major effect on the filtration time of the nanofiltration step (Figure 12B). However, using an acidic solvent increased the volume of the retentate obtained after the nanofiltration with both solvent ratios (Figure 12C).

[0196] Example 11

[0197] This example describes the effect of changing the pH of the solvent and the use of ethanol dilution step on the efficiency of flavonoid purification with the filtration-based extraction process. In the same test described in Example 10, quantification of cannflavin A in the retentate shows that the addition of acidic water to the extract feed resulted in small changes in the purity of the flavonoid (Figure 13A), however, increased the concentration factor of the compound with the use of either ethanol:water ratio (Figure 13C). Dilution with 50% ethanol at pH 3 during the nanofiltration stage did not affect significantly the purity of the cannflavin (Figure 13B), but increased the concentration factor (retentate vs. crude extract) of the flavonoid by 25% (Figure 13D). While dilution of the ethanol may not have a great effect on the efficiency of the filtration, these results suggest that using an acidified solvent may favor the recovery of polyphenols.

[0198] The publications, patents, and patent applications mentioned above are fully incorporated by reference herein, to the same extent as if each one was specifically and individually indicated for complete incorporation by reference.

[0199] REFERENCES

[0200] 1. Rasouli H et al. (2017) Polyphenols and their benefits: A review. Int J Food Properties 20: S1700- S1741.

[0201] 2. Kumar S, Pandey AK (2013) Chemistry and biological activities of flavonoids: an overview Sci. World J 2013: 162750.

[0202] 3. Kikuchi H et al. (2019) Chemopreventive and anticancer activity of flavonoids and its possibility for clinical use by combining with conventional chemotherapeutic agents. Am J Cancer Res 9: 1517- 1535.

[0203] 4. Andre CM et al. (2016) Cannabis sativa: The plant of the thousand and one molecules. Front Plant Sci 7: 19.

[0204] 5. Flores-Sanchez I J, Verpoorte R (2008) Secondary metabolism in cannabis. Phytochem Rev 7: 615-639.

[0205] 6. McPartland JM, Russo EB (2014) Non-phytocannabinoid constituents of cannabis and herbal synergy. In: Pertwee, R.G. ed. Handbook of cannabis. Oxford, U.K.: Oxford University Press, 280- 295.

[0206] 7. Pollastro F et al. (2018) Cannabis Phenolics and their Bioactivities. Curr Med Chem 25: 1160- 1185.

[0207] 8. Baron EP (2018) Medicinal properties of cannabinoids, terpenes, and flavonoids in cannabis, and benefits in migraine, headache, and pain: An update on current evidence and cannabis science, headache. J Head Face Pain 58: 1139-1186.

[0208] 9. Barrett ML et al. (1985) Isolation from Cannabis sativa L. of cannflavin - a novel inhibitor of prostaglandin production. Biochem Pharmacol 34: 2019-2024.

[0209] 10. Watson RS (2014) Polyphenols in plants: Isolation, purification and extract preparation. Academic Press, Oxford, UK.

[0210] 11. International Patent Application Publication No. WO 2021 / 119826 (2021-06-24) Extracts enriched with polyphenolic compounds and related methods.

Claims

CLAIMS1. A resin-free method for extracting a polyphenol from a polyphenol-containing biomass.

2. The method of claim 1 , wherein the method comprises a filtration step.

3. The method of claim 2, wherein the filtration step comprises tandem filtration.

4. The method of claim 2, wherein the tandem filtration is carried out in batch mode or continuous mode.

5. The method of any one of claims 2 to 4, wherein the filtration step comprises microfiltration, ultrafiltration, nanofiltration, or any combination thereof.

6. The method of claim 5, wherein the filtration step comprises, in order, microfiltration, ultrafiltration, and nanofiltration.

7. The method of any one of claims 2 to 6, further comprising a pre-filtration step.

8. The method of claim 7, wherein the pre-filtration step comprises microfiltration to remove particulate material and produce a microfiltered permeate comprising the polyphenol.

9. The method of claim 8, wherein the microfiltration uses a microfilter comprising a bag filter, a filter cloth, or a combination thereof.

10. The method of claim 8 or 9, wherein the microfiltration uses a microfiltration solvent with a neutral pH.11 . The method of claim 8 or 9, wherein the microfiltration uses a microfiltration solvent with an acidic pH.

12. The method of claim 11 , wherein the microfiltration solvent has a pH of about 3.0, about 4.0, about 5.0, or about 6.0.

13. The method of any one of claims 8 to 12, wherein the microfiltered permeate is diluted with water, such as to about 50% to about 65% ethanol, prior to further filtration steps.

14. The method of any one of claims 2 to 13, wherein the filtration step comprises an ultrafiltration step.

15. The method of claim 14, wherein the ultrafiltration step uses an ultrafilter that removes high molecular weight impurities from the biomass and / or from the microfiltered permeate to produce an ultrafiltered permeate comprising the polyphenol.

16. The method of claim 15, wherein the ultrafilter has a tangential flow filtration configuration of a single or multichannel filter.

17. The method of claim 15 or 16, wherein the ultrafilter comprises a ceramic membrane.

18. The method of claim 17, wherein the membrane has a 0.1 to 0.5 pm pore size.

19. The method of claim 17 or 18, wherein the membrane has a molecular weight cut-off (MWCO) of between 50 KDa and 400 KDa.

20. The method of any one of claims 17 to 19, wherein the ultrafiltration is operated at a pressure from about 1 psi to about 20 psi.

21. The method of any one of claims 17 to 20, wherein the ultrafiltration is operated at a temperature of about 20° C to about 25° C.

22. The method of any one of claims 17 to 21, wherein the ultrafiltration retentate is collected as a waste product or diluted with water to be recycled through the ultrafiltration step again.

23. The method of any one of claims 17 to 22, wherein the ultrafiltered permeate is diluted with ethanol, such as with about an equal amount of ethanol, such as with 100% ethanol, prior to further filtration steps.

24. The method of any one of claims 2 to 23, wherein the filtration step comprises a nanofiltration step.

25. The method of claim 24, wherein the nanofiltration step uses a nanofilter to concentrate the polyphenols in the biomass and / or microfiltered permeate and / or ultrafiltered permeate to produce a nanofiltered retentate comprising the polyphenol.

26. The method of claim 24 or 25, wherein the nanofiltration step comprises tangential flow filtration (TFF).

27. The method of claim 26, wherein the TFF uses a single membrane with a tubular, flat sheet, or spiral-wound configuration.

28. The method of any one of claims 24 to 27, wherein the nanofilter comprises an organic thin composite membrane.

29. The method of claim 28, wherein the membrane comprises a polyamide layer deposited on top of a polyethersulfone porous layer on top of a non-woven fabric support sheet.

30. The method of claim 28 or 29, wherein the membrane has a MWCO between 150 daltons and 500 daltons.31 . The method of any one of claims 24 to 30, wherein the nanofiltration step is operated at a pressure from about 100 psi to about 500 psi.

32. The method of any one of claims 24 to 31 , wherein the nanofiltration step is operated at a temperature of about 20° C to about 25° C.

33. The method of any one of claims 24 to 32, wherein the nanofiltered permeate is collected as a waste product or recycled through the nanofiltration step again.

34. The method of any one of claims 1 to 33, wherein the extracted polyphenol is further subjected to further separation to provide desired polyphenols of interest.

35. The method of claim 34, wherein the further separation comprises flash chromatography, preparative HPLC, semi-preparative HPLC, or low-pressure vacuum liquid chromatography.

36. The method of any one of claims 1 to 35, wherein the extracted polyphenol is concentrated, for example into a concentrated liquid or powder.

37. The method of claim 36, wherein the extracted polyphenol is diafiltrated with water and lyophilized, freeze-dried, spray-dried, or dehydrated.

38. The method of claim 36, wherein the extracted polyphenol is diafiltrated with ethanol and evaporated, for example in a rotary evaporator or ethanol evaporator, to remove the ethanol.

39. The method of any one of claims 36 to 39, wherein the final product is a semi-solid deposit.

40. The method of any one of claims 36 to 39, wherein the final product is a resin or an oily substance.

41. The method of claim 40, wherein the resin or oily substance is formulated with carrier oils to maintain the oily consistency or with solid excipients to generate a product with a powdery texture.

42. The method of any one of claims 1 to 41 , resulting in a concentrated polyphenol product comprising from about 10% to about 95% w / w of total polyphenols.

43. The method of any one of claims 1 to 42, wherein the polyphenol-containing biomass comprises cannabis.

44. The method of claim 43, wherein the cannabis comprises Cannabis sativa L. marijuana or hemp.

45. The method of claim 43 or 44, wherein the polyphenol-containing biomass comprises a leaf, a root, a stem, a branch, a flower, an inflorescence, a fruit, a seed, a cell, a tissue culture, or any combination thereof.

46. The method of any one of claims 1 to 45, wherein the polyphenolic compounds comprise flavonoids and / or stilbenoids.

47. The method of claim 46, wherein the flavonoids comprise cannflavin A, cannflavin B, cannflavin C, apigenin, luteolin, orientin, vitexin, isovitexin, chrysoeriol, quercetin, kaempferol, rutin, catechin, or epicatechin, in their glycosylated or aglicone forms, or in a prenylated form, or any combination thereof.

48. The method of claim 46, wherein the stilbenoids comprise canniprene, resveratrol, dihydroresveratrol, cannabistilbene I, or any combination thereof.

49. A method for extracting a polyphenol from a polyphenol-containing biomass, the method comprising:- optionally extracting a milled biomass;- prefiltering the extracted biomass to remove large impurities and collecting a prefiltered permeate;- ultrafiltering the prefiltered permeate, optionally by tangential flow filtration and collecting an ultrafiltered permeate;- concentrating the ultrafiltered permeate, optionally by nanofiltration and collecting the concentrated retentate.

50. The method of claim 49, further comprising drying the concentrated retentate.

51. The method of claim 49 or 50, wherein the method does not include the use of a capturing resin.

52. The method of any one of claims 1 to 51 , wherein the method results in increased extraction efficiency as compared to a method using capture resin.

53. The method of claim 52, wherein the extraction efficiency is greater than about 80%.

54. The method of claim 53, wherein the extraction efficiency is greater than about 99%.

55. The method of any one of claims 1 to 54, wherein the polyphenol yield is at least 2 times greater than the yield of a method using capture resin.

56. The method of claim 55, wherein the yield is at least about 5 ug / g.

57. A polyphenol composition produced by the method of any one of claims 1 to 56.

58. The polyphenol composition of claim 57, comprising a higher concentration of polyphenols as compared to a method comprising the use of adsorption resins.

59. The polyphenol composition of claim 57 or 58, formulated as capsules, pills, liquids, or tinctures alone or combined with excipients.

60. The polyphenol composition of any one of claims 57 to 59 for use as a natural health product, a botanical drug, or a pharmaceutical.61 . Use of the polyphenol composition of any one of claims 57 to 60 for the treatment and / or prevention of inflammatory responses such as rheumatoid arthritis, osteoarthritis, cardiovascular disease, neuroinflammation, and respiratory illnesses.

62. Use of the polyphenol composition of any one of claims 57 to 60 for neuroprotection and / or preventing progression of neurodegenerative and motor neuron disorders such as amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s diseases.

63. Use of the polyphenol composition of any one of claims 57 to 60 for treating and / or preventing cancer.

64. A method for the treatment and / or prevention of inflammatory responses such as rheumatoid arthritis, osteoarthritis, cardiovascular disease, neuroinflammation, and respiratory illnesses,the method comprising administering the polyphenol composition of any one of claims 57 to 60 to a subject in need thereof.

65. A method for neuroprotection and / or preventing progression of neurodegenerative and motor neuron disorders such as amyotrophic lateral sclerosis, Alzheimer’s, and Parkinson’s diseases, the method comprising administering the polyphenol composition of any one of claims 57 to 60 to a subject in need thereof.

66. A method for treating and / or preventing cancer, the method comprising administering the polyphenol composition of any one of claims 57 to 60 to a subject in need thereof.

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