Compositions of lotus leaf extract, chrysanthemum morifolium extract, and PU'er tea extract, methods and uses of the same

Abstract

Described herein are compositions including at least one of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE) having an agonistic effect on glucagon-like peptide-1 receptor (GLP-1R). Also, described herein are compositions including a mixture of LLE, CME and PTE. Methods of using the described compositions, e.g., for supporting weight loss efforts in a subject in need of thereof are also described.

Description

COMPOSITIONS OF LOTUS LEAF EXTRACT, CHRYSANTHEMUM MORIFOLIUM EXTRACT, AND PU'ER TEA EXTRACT, METHODS AND USES OF THE SAMERELATED APPLICATIONS

[0001] The present patent document claims the benefit of the filing date of Chinese Patent Application Serial No. 202411287548.9, filed September 13, 2024, which is hereby incorporated by reference.BACKGROUND

[0002] Obesity has proven to be one of the largest public health concerns worldwide because it is a fundamental contributing factor to many chronic metabolic diseases, including type 2 diabetes mellitus (T2DM), hypertension, dyslipidemia, cardiovascular diseases, different types of cancer, and others.

[0003] Nelumbo nucifera Gaertn (lotus), Chrysanthemum morifolium (Chrysanthemum morifolium Ramat), and Camellia sinensis var. assamica (Pu'er tea) are traditional Chinese medicinal herbs. Extracts from these herbs have been shown to have significant hypoglycemic, lipid-lowering and weight loss bioactivities [He Y, Tao Y, Qiu L, et al. Lotus (Nelumbo nucifera Gaertn.) Leaf-Fermentation Supernatant Inhibits Adipogenesis in 3T3-L1 Preadipocytes and Suppresses Obesity in High-Fat Diet-Induced Obese Rats. Nutrients. 2022;14(20):4348; Lee MS, Kim Y. Chrysanthemum morifolium Flower Extract Inhibits Adipogenesis of 3T3-L1 Cells via AMPK / SIRT1 Pathway Activation. Nutrients. 2020;12(9):2726; Chu SL, Fu H, Yang JX, et al. A randomized double-blind placebo-controlled study of Pu'er tea extract on the regulation of metabolic syndrome. Chinese Journal of Integrative Medicine. 2011;17(7):492-8; Lin J, Wen J, Xiao N, et al. Anti-diabetic and gut microbiota modulation effects of sacha inchi (Plukenetia volubilis L.) leaf extract in streptozotocin-induced type 1 diabetic mice. J Sci Food Agric. 2022 Aug 15;102(10):4304-4312].

[0004] The extract derived from the lotus leaf (LLE) has previously shown lipid- lowering and weight-reduction properties [He Y, et al., Lotus (Nelumbo nucifera Gaertn.) Leaf-Fermentation Supernatant Inhibits Adipogenesis in 3T3-L1 Preadipocytes and Suppresses Obesity in High-Fat Diet-Induced Obese Rats. Nutrients. 2022;14(20):4348].

[0005] Chrysanthemum morifolium extract (CME) is widely used as herbal tea in many countries, it effectively prevents the build-up of lipids in adipocytes by downregulating the expression of genes associated with fat synthesis [Lee MS, Kim Y. Chrysanthemum morifolium Flower Extract Inhibits Adipogenesis of 3T3-L1 Cells via AMPK / SIRT1 Pathway Activation. Nutrients. 2020;12(9):2726].

[0006] Pu'er tea extract (PTE) has previously shown remarkable potential in combating central obesity, modulating blood lipid levels, reducing blood glucose, and offering antioxidant benefits [Chu SL, Fu H, Yang JX, et al. A randomized double-blind placebo-controlled study of Pu'er tea extract on the regulation of metabolic syndrome. Chinese Journal of Integrative Medicine. 2011 ; 17(7):492-8] . Also, numerous reports have highlighted its efficacy in promoting weight loss and reducing lipid levels.

[0007] The glucagon-like peptide- 1 receptor or “GLP-1R” belongs to class G protein- coupled receptors (GPCRs). It is a clinically proven target of multiple clinically approved peptide drugs for the treatment of type 2 diabetes, with select drugs also approved for obesity [Brown E, Cuthbertson DJ, Wilding JP. Newer GLP-1 receptor agonists and obesity-diabetes. Peptides. 2018; 100: 61-67; Nauck MA, Meier JJ. MANAGEMENT OF ENDOCRINE DISEASE: Are all GLP-1 agonists equal in the treatment of type 2 diabetes? European Journal of Endocrinology . 2019; 181(6): R211-R234].

[0008] Pancreatic lipase or “PL,” also known as triacylglycerol acyl hydrolase or triglyceride lipase, is an enzyme that catalyzes the hydrolysis of ester linkages of triglycerides. PL inhibitors can reduce the breakdown and absorption of dietary fat in the digestive organs, thereby improving metabolic diseases, such as obesity and hyperlipidemia. Therefore, screening PL inhibitors has become a hot topic in the field of weight loss product development [Birari RB, Bhutani KK. Pancreatic lipase inhibitors from natural sources: unexplored potential. Drug Discovery Today. 2007;12(19-20):879- 89; McClendon KS, Riche DM, et al. Orlistat: current status in clinical therapeutics.Expert Opinion on Drug Safety. 2009;8(6):727-44].

[0009] Nonetheless, safe and effective compositions and methods for weight loss management and obesity, and related conditions are still needed.SUMMARY

[0010] Described herein are compositions comprising at least one of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract that show significant agonism on GLP-lRs.

[0011] Also, descried herein are compositions comprising a combination of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract. Surprisingly, the combination of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract was found to have a greater effect on pancreatic lipase inhibition as compared to the individual extracts.

[0012] Certain embodiments relate to a composition comprising at least on of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the composition has an agonistic effect on glucagon-like peptide- 1 receptor (GLP-1R). In the composition, the LLE has nuciferine content not less than 0.2%, CME has chlorogenic acid content exceeding 1%, and PTE has tea polyphenol content surpassing 7% and caffeine levels exceeding 10%. In the composition, the concentration of LLE in the composition is in the range of 5-50 pg / mL, the concentration of CME in the composition is in the range of 5-50 pg / mL, and the concentration of PTE in the composition is in the range of 5-50 pg / mL. In the composition, the LLE exhibits an agonistic fold increase greater than 2 at the concentrations of 5 pg / mL or the LLE exhibits an agonistic fold increase greater than 2 at the concentrations of 10 pg / mL. In the composition, the CME exhibits an agonistic fold increase of 2.35 at the concentrations of 40 pg / mL or the CME exhibits an agonistic fold increase of 4.29 at the concentrations of 50 pg / mL. In the composition, the PTE exhibits an agonistic fold increase of 1.47 at the concentrations of 40 pg / mL or the PTE exhibits an agonistic fold increase of 2.54 at the concentrations of 50 pg / mL. The composition may be for supporting weight loss efforts in a subject in need of thereof, for reducing blood sugar levels in a subject in need of thereof, for managing blood lipids in a subject in need thereof, and / or for prevention and / or treatment of type 2 diabetes.

[0013] Certain other embodiments relate to a method for activating glucagon-like peptide- 1 receptor (GLP-1R) comprising providing a composition comprising at least oneof: lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE).

[0014] Certain further embodiments relate to a method for activating glucagon-like peptide- 1 receptor (GLP-1R) in a subject comprising administering to the subject a composition comprising at least one of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the LLE has nuciferine content is not less than 0.2%, CME has chlorogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%.

[0015] Certain further embodiments relate to a method for supporting weight loss efforts in a subject in need of thereof, comprising administering to the subject a composition comprising at least one of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the LLE has nuciferine content is not less than 0.2%, CME has chlorogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%.

[0016] Certain further embodiments relate to a method for reducing blood sugar levels in a subject in need of thereof, comprising administering to the subject a composition comprising at least one of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the LLE has nuciferine content is not less than 0.2%, CME has chlorogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%.

[0017] Certain further embodiments relate to a method for managing blood lipids in a subject in need thereof, comprising administering to the subject a composition comprising at least one of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the LLE has nuciferine content is not less than 0.2%, CME has chlorogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%.

[0018] Certain further embodiments relate to a method for prevention and / or treatment of type 2 diabetes, comprising administering to the subject a composition comprising at least one of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the LLE has nuciferine content is not less than 0.2%,CME has chi orogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%.

[0019] Yet, further embodiments relate to a composition comprising a mixture of: lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the mixture has a significantly greater effect on pancreatic lipase inhibition as compared to the individual extracts. In the composition, the LLE has nuciferine content not less than 0.2%, CME has chi orogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%. In the composition, the ratio of LLE to CME to PET in the composition is 3: 1 :7. In the composition, the total concentration of LLE, CME, and PTE is 10 mg / mL. The composition may further comprise a vehicle, a carrier, and / or an excipient. The composition may be formulated as a ready-to-drink beverage, a concentrate, a dry composition (e.g., powders, granules, or tablets that may be reconstituted with a liquid), a gel, a solid, a semi-solid (e.g., ice cream, pudding, or yogurt), a frozen liquid (e.g., ice pop), a lozenge or a hard candy, a dissolving strip, and chewing gum. In certain embodiments, the composition is in a form of a tablet. Certain further embodiments relate to the use of the composition for supporting weight loss efforts in a subject in need of thereof, for reducing blood sugar levels in a subject in need of thereof, and / or for managing blood lipids in a subject in need of thereof.

[0020] Yet, further embodiments relate to a method for inhibiting pancreatic lipase in a subject comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE). In the method, the ratio of LLE to CME to PET in the mixture is 3:1:7. In the method, the total concentration of LLE, CME, and PTE is 10 mg / mL.

[0021] Yet, further embodiments relate to a method for inhibiting pancreatic lipase in a subject comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the LLE has nuciferine content not less than 0.2%, CME has chlorogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%. In the method, the ratio of LLE to CME to PET in themixture is 3:1:7. In the method, the total concentration of LLE, CME, and PTE is 10 mg / mL.

[0022] Yet, certain further embodiments relate to method for supporting weight loss efforts in a subject in need of thereof, comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE). In the method, the ratio of LLE to CME to PET in the mixture is 3: 1 :7. In the method, the total concentration of LLE, CME, and PTE is 10 mg / mL.

[0023] Yet, certain further embodiments relate to method for reducing blood sugar levels in a subject in need of thereof, comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE). In the method, the ratio of LLE to CME to PET in the mixture is 3: 1 :7. In the method, the total concentration of LLE, CME, and PTE is 10 mg / mL.

[0024] Yet, certain further embodiments relate to method for managing blood lipids in a subject in need of thereof, comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE). In the method, the ratio of LLE to CME to PET in the mixture is 3: 1 :7. In the method, the total concentration of LLE, CME, and PTE is 10 mg / mL.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 depicts a bar graph showing cell viability of HEK293-GLP-lR-luc- cell following treatment with lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE) at the specified concentrations.

[0026] Figure 2 depicts a bar graph showing the agonistic fold change of GLP-1R by extracts following treatment with LLE, CME, and PTE at the specified concentrations.

[0027] Figure 3 depicts a graph showing a correlation between experimental and predicted PL inhibition rate.

[0028] Figure 4 depicts three-dimensional graphs produced by Box-Behnken design that clearly show the effect of extract and concentration on PL inhibition rate.DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERRED EMBODIMENTS

[0029] The present invention will now be described more fully herein after. For the purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Thus, before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified embodiments that may of course, vary.

[0030] Described herein are compositions comprising at least one of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract that show significant agonism on GLP-lRs and methods of us using the compositions.

[0031] Also, descried herein are compositions comprising a combination of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract. Surprisingly, a greater effect of lipase inhibition was observed with the combination of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract.

[0032] Unless otherwise defined, all terms technical and scientific used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention.

[0033] As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. The term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements.

[0034] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0035] As used herein, “around,” “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of agiven value or range. Numerical quantities given herein are approximate; meaning that the terms “around,” “about” or “approximately” can be inferred if not expressly stated. When the term “about” is used in describing a value or an endpoint of a range, the disclosure should be understood to include both the specific value and end-point referred to.

[0036] As used herein, the terms “comprising,” “including,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0037] The term “composition” is used herein to describe a formulation that includes at least one of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract, and in some embodiments, a combination of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract. The term refers to a comestible formulation that may be suitable for oral ingestion by the subject (e.g., the human female subject). Exemplary compositions that include at least one of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract, include but are not limited to: sprays (e.g., aerosols), powders, chewing gum, ingestible solids, gels, aqueous beverages, dry powder (e.g., a powder that can be directly consumed or that can be reconstituted with liquid to provide a beverage as defined herein), nutritional bars, lozenges, tablets, capsules, wafers, pastes, and the like. Other compositions are described herein. In certain preferred embodiments, the composition described herein is tablets.

[0038] An “extract” is the material which dissolves in a solvent after contacting a plant substance with a suitable solvent. Preferably, the plant substance is contacted for a sufficiently long period of time and at a suitable temperature so that substantially all of the soluble material is removed by the solvent. The solvent together with the dissolved soluble plant material is referred to as the “liquid extract.” The term “extract” refers to the soluble plant material remaining after removal of the solvent. The solvent can be removed by any suitable means, including evaporation, lyophilization, spray drying and the like.

[0039] The terms “lotus leaf extract” or “LLE” are used interchangeably herein and refer to one or more natural materials, in the form of a single material or ingredient (e.g., inorganic, organic, salt, etc.) or a mixture of natural materials obtained from lotus leaf (Nelumbo nucifera Gaertn).

[0040] The terms “Chrysanthemum morifolium extract” or “CME” are used interchangeably herein and refer to one or more natural materials, in the form of a single material or ingredient (e.g., inorganic, organic, salt, etc.) or a mixture of natural materials obtained from Chrysanthemum morifolium Ramat plant.

[0041] The terms “Pu'er tea extract” or “PTE” may be used interchangeably herein and refer to one or more natural materials, in the form of a single material or ingredient (e.g., inorganic, organic, salt, etc.) or a mixture of natural materials obtained from Camellia sinensis var. assamica plant.

[0042] As used herein, the expression “active combination” refers to the ability of the combination of the described extracts to exert a specified effect, as described herein. Neither of the components is regarded as an additive, such as carrier, diluent, or excipient.

[0043] As used herein, the terms “effective amount” or “pharmaceutically or therapeutically effective amount” refer to the amount of the active ingredient(s), the extract(s), to be administered orally to the subject to trigger the desired effect (e.g., reducing blood sugar levels, managing blood lipids, regulating appetite and food intake, slowing gastric emptying, and supporting weight loss efforts) without or causing minimal toxic adverse effect against the subject, and / or without undesirable side-effects. One skilled in the art should know that the effective amount can vary from one individual to another due to the external factors such as age, sex, diseased state, races, body weight, formulation of the composition, availability of other active ingredients in the formulation, and so on.

[0044] The term “an agonistic effect” with reference to the agonist(s) of the GLP-1R means that the composition, an extract or an ingredient thereof binds to and activates the GLP-1R on cells, causing a biological response, such as, for example reducing blood sugar levels, managing blood lipids, regulating appetite and food intake, slowing gastric emptying, and supporting weight loss efforts.

[0045] The agonistic fold increase may be at least 0.5-fold increase; at least 1.0-fold increase; at least 1.5-fold increase; at least 2.0-fold increase; at least 2.5-fold increase; at least 3-fold increase; at least 3.5-fold increase; at least 4.0-fold increase; at least 4.5-fold increase; at least 5.0-fold increase; at least 5.5-fold increase; at least 6-fold increase, of higher. Fold increases in-between the ones mentioned above are also contemplated.

[0046] The term “significant agonism” in the context of the effect on GLP-lRs means that compared with the “blank group,” the treatment group was at least 2-fold or more effective as GLP-1R agonist, and the statistical result P value was <0.001. For example, as shown herein, compared with “blank,” LLE at 10 ug / mL, CME and PTE at 50 ug / mL, LLE and PTE agonized GLP-1R 2-fold, and CME agonized GLP-1R 4-fold.

[0047] The terms “greater” or “better” in the context of “pancreatic lipase inhibition” means that the inhibitory effect of the mixture of the lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract on pancreatic lipase is greater in comparison to the effect of a single and / or any two of the three extracts combined. For example, the total concentration was 10 mg / mL, at the ratio of 3:1:7 of lotus leaf extract to Chrysanthemum morifolium extract to Pu'er tea extract, accordingly, the inhibitory effect of the mixture of the extracts on pancreatic lipase is greater (e.g., 89.35%) than a single extract or any two extracts combined.

[0048] The term “excipient” refers to a substance which helps to absorb any of the components of the product of the invention, stabilizes said components or helps in the preparation of the composition. Thus, excipients can have the function of keeping the components bound together, such as for example starches, sugars or celluloses, a sweetening function, a colorant function, the function of protecting the medicament from the external medium, such as for example isolating it from the air and / or moisture, a filler function for a tablet, capsule or any other form of formulation, such as for example di basic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term “excipient” is defined as the substance which, included in dosage forms, is added to the active substances or their associations in order to enable their preparation and stability, modify their organoleptic properties or determine the physical / chemical properties of the pharmaceutical composition and its bioavailability. The “pharmaceutically acceptable” excipient should not interact with the activity of the active compounds of the described composition. Examples of excipients are binding agents, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings and colorants. More specific, non-limiting examples of acceptableexcipients are starches, sugars, xylitol, sorbitol, calcium phosphate, steroid fats, talc, silica or glycerin, among others.

[0049] By “administering” and “administration” is meant a mode of delivery. A daily dosage can be divided into one, two, three or more doses in a suitable form to be administered one, two, three or more times throughout a time period, such as a day, week, month.

[0050] By ‘ ‘treating” or “ameliorating” or “alleviating” is meant administering a composition for therapeutic purposes or administering treatment to a subject already suffering from a disorder to improve the subject's condition. As compared with an equivalent untreated control, such amelioration or degree of treatment is at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as measured by any standard, suitable technique.

[0051] As described herein, the term “subject” is equivalent to the terms “individual” and “patient” whereby the terms can be used interchangeably. “Subject” means any animal belonging to any species. Examples of subjects include, but are not limited to, commercially bred animals such as birds (hens, ostriches, chickens, geese, partridges, etc.), rabbits, hares, domestic animals (dogs, cats, etc.), livestock such as sheep and goat livestock, pigs, wild boars, horses, ponies, etc., and cattle (bulls, oxen, etc.). In a particular embodiment, the subject is a mammal, preferably a primate, more preferably a human being of any race, sex or age.

[0052] Active Components

[0053] In certain embodiments, the described invention relates to a composition comprising at least one of: (i) lotus leaf extract (LLE), (ii) Chrysanthemum morifolium extract (CME), and (iii) Pu'er tea extract (PTE).

[0054] In certain other embodiments, the described invention relates to a composition comprising active combination of: (i) lotus leaf extract (LLE), (ii) Chrysanthemum morifolium extract (CME), and (iii) Pu'er tea extract (PTE).

[0055] (i) Nelumbo nucifera Gaertn. (A. nucifera) - Lotus

[0056] Nelumbo nucifera Gaertn. (N. nucifera), commonly known as lotus, sacred lotus, Indian lotus, water lily, and Chinese water lily, is famous for its extensive usage as a dietary and medicinal plant that is widely distributed throughout East Asia. As atraditional herbal medicine, the lotus, particularly the leaves, extract, fractions, and other constituents, has been proven to have diverse applications in both biology and pharmacology, i.e., antioxidant, antibacterial, antiviral, antifungal, immunomodulation, anti-inflammatory, anti-diarrheal, anti-thrombotic, anti-diabetic, cholesterol-lowering, anti-obesity, and anti-cancer properties, due to multiple bioactive compounds, including flavonoids, polyphenols, steroids, phenolic acids, polysaccharides, alkaloids, terpenoids, fatty acids, and glycosides. In Asia, lotus leaves have been used as a functional food and as a supplement with which to treat obesity. It is believed that the inhibitory effect of lotus leaves and their extract mainly impair the intestinal absorption of carbohydrates as well as lipids and increases energy expenditure, which may relieve chronic inflammation through ameliorating intestinal integrity and may modulate the gut microflora via specific metabolites such as short chain fatty acids. However, there are few reports on the antiobesity effect of fermented lotus leaves, with only some researchers having demonstrated that bioactive substances such as the total phenol and flavone content, as well as a variety of low-molecular- weight metabolites, were increased after fermentation [Hwang D., Charchoghlyan H., Lee J.S., Kim M. Bioactive compounds and antioxidant activities of the Korean lotus leaf (Nelumho nuciferd) condiment: Volatile and nonvolatile metabolite profiling during fermentation. Int. J. Food Sci. Technol. 2015;50:1988-1995; Kim J.-S., Wang S.-B., Kang S.-K., Cho Y.-S., Park S.-K. Quality properties of white lotus leaf fermented by mycelial Paecilomyces japonica. J. Korean Soc. Food Sci.Nutr. 2009;38:594-600],

[0057] The flavonol miquelianin, as well as the alkaloids (+)-(17?)-coclaurine and (-)- (IS)-norcoclaurine, can be found in the leaves of N. nucifera. The plant also contains nuciferine, neferine, and many other benzylisoquinoline alkaloids with medicinal properties.

[0058] In certain preferred embodiments, the nuciferine content in the LLE is not less than 0.2%.

[0059] In certain preferred embodiments, the nuciferine content in the LLE is not less than 0.2%; more preferably, not less than 0.3%; more preferably, not less than 0.4%; more preferably, not less than 0.5%; more preferably, not less than 0.6%; more preferably, not less than 0.7%; more preferably, not less than 0.8%; more preferably, notless than 0.9%; more preferably, not less than 1.0%; more preferably, not less than 1.2%; more preferably, not less than 1.3%; more preferably, not less than 1.4%; more preferably, not less than 1.5%; more preferably, not less than 1.6%; more preferably, not less than 1.7%; more preferably, not less than 1.8%; more preferably, not less than 1.9%; more preferably, not less than 2%; more preferably, not less than 3%; more preferably, not less than 4%; and more preferably, not less than 5%.

[0060] In certain other embodiments, the nuciferine content in the LLE is at least 0.2%.

[0061] (ii) Chrysanthemum morifolium

[0062] Chrysanthemum morifolium, a representative plant of the Asteraceae family, is a perennial plant that has long been used for edible or medicinal, as well as ornamental purposes. It contains components, such as, luteolin, apigenin, and chlorogenic acid, which have been previously shown to exhibit anti-obesity effects by suppressing weight gain. In particular, extracts of C. morifolium Ramat flower (CF) have demonstrated bioactive properties, including anti-oxidant, anti-diabetic, and hypolipidemic effects, and antiobesity effects.

[0063] In certain embodiments, chlorogenic acid in CME exceeds 1%.

[0064] In certain other embodiments, chlorogenic acid content in CME is at least exceeding 1%; more preferably, at least 2%; more preferably, at least 3%; more preferably, at least 4%; more preferably, at least 5%; more preferably, at least 6%; more preferably, at least 7%; more preferably, at least 8%; more preferably, at least 9%; more preferably, at least 10%; more preferably, at least 11%; more preferably, at least 12%; more preferably, at least 13%; more preferably, at least 14%; more preferably, at least 15%; more preferably, at least 16%; more preferably, at least 17%; more preferably, at least 18%; more preferably, at least 19%; and more preferably, at least 20%; or more.

[0065] (hi) Pu'er tea

[0066] Pu'er or pu-erh is a variety of fermented tea traditionally produced in Yunnan Province, China. In the context of traditional Chinese tea production terminology, fermentation refers to microbial fermentation (called “wet piling”), and is typically applied after the tea leaves have been sufficiently dried and rolled. As the tea undergoes controlled microbial fermentation, it also continues to oxidize, which is also controlled,until the desired flavors are reached. This process produces tea known as heicha, literally “black tea”, though the term is commonly translated to English as “dark tea” to distinguish it from the English-language black tea (hongcha, lit. “red tea” in Chinese).

[0067] Two main styles of pu'er production exist: a traditional, longer production process known as sheng (raw) pu'er; and a modern, accelerated production process known as shou (ripe) pu'er. Pu'er traditionally begins with a raw product called "rough" (mao) chd (lit. fuzzy / furry tea) and can be sold in this form or pressed into a number of shapes and sold as “ sheng cha ' (lit. “raw tea”). Both of these forms then undergo the complex process of gradual fermentation and maturation with time.

[0068] In certain embodiments, tea polyphenol content in PTE is surpassing 7% and caffeine levels exceed 10%.

[0069] In certain embodiments, tea polyphenol content in PTE is surpassing 5%; more preferably, surpassing 6%; more preferably, surpassing 7%; more preferably, surpassing 8%; more preferably, surpassing 9%; more preferably, surpassing 10%; more preferably, surpassing 11%; more preferably, surpassing 12%; more preferably, surpassing 13%; more preferably, surpassing 14%; more preferably, surpassing 15%; more preferably, surpassing 16%; more preferably, surpassing 17%; more preferably, surpassing 18%; more preferably, surpassing 19%; more preferably, surpassing 20%; or more.

[0070] (iv) Extract Preparation

[0071] The extract process and specification of LLE, CME and PTE, as follow:

[0072] The ground powder is submitted to reflux extraction with distilled water. Following extraction, the mixture undergoes centrifugation and the resulting supernatant is evaporated and subsequently lyophilized using a freeze-dryer to obtain the final extracts. The specifications for the extracts, nuciferine content not less than 0.2% in LLE, chlorogenic acid content exceeding 1 % in CME, along with tea polyphenol content surpassing 7% and caffeine levels exceeding 10% in PTE.

[0073] An application of a combined extract of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract was shown, surprisingly, to inhibit pancreatic lipase or “PL,” also known as triacylglycerol acyl hydrolase. Surprising and unexpectedly, the inhibition was shown to be greater as compared to the individual extracts.

[0074] Compositions

[0075] In certain embodiments, the described invention relates to a composition comprising at least one of: (i) lotus leaf extract (LLE), (ii) Chrysanthemum morifolium extract (CME), and (iii) Pu'er tea extract (PTE).

[0076] In certain other embodiments, the described invention relates to a composition comprising a mixture of: (i) lotus leaf extract (LLE), (ii) Chrysanthemum morifolium extract (CME), and (iii) Pu'er tea extract (PTE).

[0077] In certain embodiments, the concentration of LLE in the composition is in the range of 1-100 pg / mL; more preferably, 1-75 pg / mL; more preferably, 1-50 pg / mL; more preferably 5-50 pg / mL; more preferably.

[0078] In certain embodiments, the concentration of CME in the composition is in the range of 1-100 pg / mL; more preferably, 1-75 pg / mL; more preferably, 1-50 pg / mL; more preferably 5-50 pg / mL; more preferably.

[0079] In certain embodiments, the concentration of PTE in the composition is in the range of 1-100 pg / mL; more preferably, 1-75 pg / mL; more preferably, 1-50 pg / mL; more preferably 5-50 pg / mL; more preferably.

[0080] In certain preferred embodiments, the concentration of LLE in the composition is in the range of 5-50 pg / mL, the concentration of CME in the composition is in the range of 5-50 pg / mL, and the concentration of PTE in the composition is in the range of 5-50 pg / mL.

[0081] In certain embodiments, in the described composition, the total concentration at which the combination of LLE, CME, and PTE is most effective is 1 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 2 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 3 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 4 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 5 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 6 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE,CME, and PTE is most effective is 7 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 8 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 9 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 10 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 11 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 12 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 13 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 14 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 15 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 16 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 17 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 18 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 19 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 20 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 21 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 22 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 23 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 24 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 25 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 26 mg / mL of thecomposition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 27 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 28 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 29 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 30 mg / mL of the composition; more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 40 mg / mL of the composition; and more preferably, the total concentration at which the combination of LLE, CME, and PTE is most effective is 50 mg / mL of the composition.

[0082] In certain embodiments, in the described composition, the total concentration at which the combination of LLE, CME, and PTE is most effective is 1-50 mg / mL of the composition.

[0083] In certain preferred embodiments, the specific combination and concentration of LLE, CME, and PTE, effective for inhibiting phospholipase activity is 3 parts LLE to 1 part CME to 7 parts PTE, and the total concentration at which this combination is most effective is 10 mg / mL.

[0084] Non- Active Components of the Composition

[0085] In certain embodiments, the described composition may include at least one non- active ingredient or an extract.

[0086] In certain embodiments, the described composition may comprise a “vehicle” or “carrier,” which is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, to allow a better dosing and administration or to give consistency and shape to the composition. Therefore, the vehicle is a substance that is used to dilute any of the components of the described composition to a determined volume or weight, or even without diluting said components it is capable of allowing better dosing and administration or giving consistency and shape to the composition (e.g., nutraceutical composition). Due to all of this, a vehicle would be considered pharmaceutically or nutraceutically acceptable.

[0087] When the described composition is presented in liquid form, the vehicle is the diluent.

[0088] The compositions can also include one more excipients that are non-toxic and non-inflammatory in a subject.

[0089] In some embodiments, the excipient(s) can provide desirable or improved physical and / or chemical properties such as stability, flow, viscosity, rate of disintegration, taste, delivery, etc.

[0090] The composition described herein may additionally include, for example, electrolytes (e.g., potassium salt or other salts), sweeteners, flavoring and coloring agents, vitamins, minerals, preservatives, and antioxidants.

[0091] Viscosity modifiers may be added to the described compositions. Such viscosity modifiers include, for example, collagen, gellan gum, carbohydrate gel-forming polymers, carob bean gum, locust bean gum, carrageenan, alginates (e.g., alginic acid, sodium alginate, potassium alginate, ammonium alginate, and calcium alginate), agar, guar gum, xanthan aura, carboxymethyl cellulose, clear starch, pectin, gelatin, arrowroot, cornstarch, katakuri starch, potato starch, sago, tapioca, furcellaran, and sodium pyrophosphate. A viscosity modifier may be present in the composition in an amount of from about 0.01% to 10% by weight based on the total volume of the composition (e.g., 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%), though the viscosity modifier may be present in lower or higher concentrations.

[0092] In certain embodiment, electrolytes can be included in the described compositions. Exemplary electrolytes include potassium salts, chloride salts, bromide salts, sodium salts, magnesium salts, calcium salts, citrate salts, acetate salts, phosphate salts, salicylates, bicarbonate salts, lactate salts, sulphate salts, tartrate salts, benzoate salts, selenite salts, molybdate salts, iodide salts, oxides, and combinations thereof. An electrolyte may be present in a composition of the invention at a concentration range of about 0.01% to 10% by weight based on the total volume of the composition (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%), though an electrolyte may be present in lower or higher concentrations.

[0093] In certain embodiments, the described compositions can include potassium (e.g., potassium chloride). The concentration of potassium in the composition may be, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.5, 13, 4, 5, 6, or 7% or more by weight based on the total volume of the composition.

[0094] In certain embodiments, the described compositions may include magnesium (e.g., magnesium chloride). The concentration of magnesium in the composition may be, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, or 7% or more by weight based on the total volume of the composition.

[0095] Sweeteners may also be included in the described compositions. Exemplary sweeteners include high fructose corn syrup, mannose, maltose, glucose polymers, sucrose (e.g., cane sugar or beet sugar), glucose, dextrose, lactose, galactose, fructose, polysaccharides (e.g., malodextrins), rice syrup, honey, and natural fruit juices (e.g., orange juice, papaya juice, pineapple juice, apple juice, grape juice, apricot juice, pear juice, tomato juice, agave nectar, or cranberry juice). Additionally, non- or low-caloric sweeteners can be used in the compositions of the invention. Examples of such noncaloric or low-caloric sweeteners include, but are not limited to, saccharin, cyclamates, acetosulfam, sorbitol, sucralose, xylitol, erythritol, Stevia, Stevia extract, L-aspartyl-L- phenyl-alanine ester (e.g., aspartame), L-aspartyl-D-alanine alkyl amides, L-aspartyl-L-1- hydroxymethylalkaneamide, and L-aspartyl-1 -hydroxy ethylalkaneamide. Sweeteners may be present in the described composition at a concentration range of about 2% to 20% by weight based on the total volume of the composition (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%), though sweeteners may be present in lower or higher concentrations.

[0096] In certain other embodiment, flavoring and / or coloring agents may also be included in the described compositions. Exemplary flavoring agents include natural and synthetic flavoring agents, including almond oil, amaretto oil, anethole, anise oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seed, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, dextrose, eriodictyon, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, glycyrrhiza, grape, honey, lavender oil, lemon oil, lime, mannitol, methyl salicylate, myristica oil, orange oil, orange peel, orange syrup, peppermint, peppermint oil, peppermint water, phenylethyl alcohol, pineapple, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, sarsaparilla syrup, sorbitol, spearmint, spearmint oil, strawberry, sucrose, thyme oil, tolu balsam,tropical, vanilla, vanillin, and wild cherry syrup. Additional flavoring agents may be found in Food Chemicals Codex and Fenaroli's Handbook of Flavor Ingredients.

[0097] Small amounts of one or more coloring agents may be utilized in the described compositions. Coloring agents include, e.g., beta-carotene, riboflavin dyes, FD&C dyes (e.g., Yellow No. 5, Blue No. 1, Blue No. 2, and Red No. 40), FD&C lakes, chlorophylls and chlorophyllins, caramel coloring, annatto, cochineal, turmeric, paprika, and fruit, vegetable, and / or plant extracts (e.g., grape, black currant, aronia, carrot, beetroot, red cabbage, elderberry, and hibiscus extracts). The amount of coloring agent used will vary depending on the agents used in the composition and the color intensity desired in the finished product. The amount of coloring agent to be used can be readily determined by one skilled in the art.

[0098] In certain additional embodiments, vitamins and / or minerals may also be included in the described compositions. Non-limiting examples of vitamins and minerals that may be included in the described compositions include, e.g., choline bitartate, niacinamide, thiamin, folic acid, d-calcium pantothenate, biotin, vitamin A, vitamin C, vitamin Bi hydrochloride, vitamin B2, vitamin B3, vitamin Be hydrochloride, vitamin B12, vitamin D, vitamin E acetate, vitamin K, and salts of calcium, potassium, magnesium, zinc, iodine, iron, and copper. When included in the described composition, the composition contains at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% of the U.S. recommended daily intake (RDI) for such vitamins and minerals.

[0099] One or more preservatives may additionally be utilized in the described compositions. Exemplary preservatives include, for example, sorbate, benzoate, and polyphosphate preservatives (e.g., sorbic acid, benzoic acid, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and mixtures thereof), maltodextrin. When included in the described composition, the preservative may be included at levels from about 0.0005% to about 0.5% (e.g., 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, or 0.5%) by weight based on the total volume of the composition, though preservatives may be present in lower or higher concentrations.

[0100] One or more antioxidant agents may also be included in the described compositions. Exemplary antioxidants include vitamin C and vitamin E; beta-carotene, lutein, or other carotenoids; cyanidin, delphinidin, malvidin, or other anthocyanidins;apigenin, luteolin, or other flavones; hesperitin, naringenin, or other flavonones; isorhamnetin, quercetin, kaempferol or other flavonols; and epigallocatechin-3 -gallate, epicatechin, thearubigins, or other flavan-3-ols.

[0101] Additional components of the compositions described herein may include amino acids (e.g., leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), stimulants (e.g., caffeine), emulsifying agents, carbon dioxide (e.g., to carbonate a liquid composition), stabilizers, humectants, anticaking agents, or herbal extracts not previously mentioned.

[0102] Importantly, the additional components are pharmaceutically and or nutraceutically acceptable.

[0103] In certain embodiments, the composition may be lactose free.

[0104] In certain embodiments, the composition may be diary free.

[0105] In certain embodiments, the composition may be soy free.

[0106] In certain embodiments, the composition may be gluten free.

[0107] In certain embodiments, the composition may not contain artificial sweeteners.

[0108] In certain embodiments, the composition may not contain artificial preservatives.

[0109] In certain embodiments, the composition may not contain artificial flavors.

[0110] In certain embodiments, the described composition may not contain artificial colors.

[0111] Also, in certain embodiments, no refrigeration is necessary for the described composition (e.g., can be stored in a cool, dry place).

[0112] Furthermore, the described composition may not include irradiated ingredients.

[0113] In certain embodiments, the described composition may be Kosher and / or Hala certified.

[0114] In certain embodiments, the described composition may be vegetarian and / or vegan.

[0115] In certain embodiments, the described composition may be NSF certified.

[0116] In certain embodiments, the described composition may be free from peanuts, tree nuts, eggs, shellfish, and crustacean.

[0117] Formulations

[0118] The compositions described herein may be formulated as ready-to-drink beverages, concentrates (e.g., syrups), dry compositions (e.g., powders, granules, or tablets that may be reconstituted with a liquid (e.g., with water), gels, solids, semi-solids (e.g., ice cream, pudding, or yogurt), frozen liquids (e.g., ice pops), lozenges or hard candies, dissolving strips (e.g., an edible strip containing pullulan and compositions of the invention), and chewing gum.

[0119] In certain embodiments, compositions described herein may be formulated as a food supplement. The term “food supplement” is understood as the products marketed in the form of capsules, tablets, phials, herbal infusions, drinkable solutions, etc., intended to supplement the usual diet and which constitute a concentrated source of nutrients (such as vitamins, minerals, amino acids, essential fatty acids, fiber, etc.) or other substances that have a nutritional, or physiological effect.

[0120] In some embodiments, the compositions may be in the form of a dry powder, granule, or tablet that may be reconstituted in a specified amount of a liquid. The dried components may be mixed together and milled (e.g., to create a homogenous powder) or mixed in aqueous solution and dried by using methods known to one of skill in the art. Dried powders or granules may be “loose” or fashioned into tablets.

[0121] The compositions and solutions described herein can be ingested (e.g., through eating or drinking) at least daily for a minimum of one week, preferably, more than one week.

[0122] The compositions may be prepared using methods known to one of skill in the art. Such methods include dissolving, dispersing, or otherwise mixing all components singularly or in suitable combinations and agitating with, for example, a mechanical stirrer until all of the ingredients have been solubilized or adequately dispersed. Where a shelf-stable composition or solution is desired, the final mixture can be pasteurized, ultrapasteurized, sterilized, or filled aseptically at appropriate process conditions. Where required for mutual stability of two or more components (for example if a component is unstable at low pH), multiple components can be mixed shortly before ingestion.

[0123] Methods and Uses

[0124] Within the context of the present invention, methods of treatment and / or prevention equivalent to the uses described in the present description are also contemplated.

[0125] In certain embodiments, the described compositions may be used for activating the GLP-1R, which has been shown to play a significant role in reducing blood sugar levels, managing blood lipids, and supporting weight loss efforts.

[0126] In certain embodiments, the described composition may be used in the treatment and / or prevention of high blood sugar in a subject. As such, the invention relates to a method for treatment and / or prevention of high blood sugar in a subject, comprising administering to the subject the composition descried herein.

[0127] In certain embodiments, the described compositions may be used in the treatment and / or prevention of type 2 diabetes in a subject. As such, the invention relates to a method for treatment and / or prevention of type 2 diabetes in a subject, comprising administering to the subject the composition described herein.

[0128] In certain embodiments, the described compositions may be used in managing blood lipids in a subject. As such, the claimed invention relates to a method for managing blood lipids in a subject, comprising administering to the subject the described composition.

[0129] In certain embodiments, the described compositions may be used in supporting weight loss efforts in a subject. As such, the invention relates to a method for supporting weight loss efforts in a subject, comprising administering to the subject the described composition.

[0130] In certain embodiments, the described compositions may be used in the treatment and / or prevention of obesity in a subject in need of the treatment and / or prevention. As such, the invention relates to a method for treatment and / or prevention of obesity in a subject, comprising administering to the subject the described composition.

[0131] In further embodiments, the described composition may be used to inhibit pancreatic lipase (PL, also known as triacylglycerol acyl hydrolase). Specifically, the described composition may be used as PL inhibitor to reduce the breakdown and absorption of dietary fat in the digestive organs, thereby improving metabolic diseasessuch as obesity and hyperlipidemia. As such, in certain embodiment, the invention relates to a method for inhibiting pancreatic lipase in a subject, comprising administering to the subject a composition comprising LLE, CME, and PTE. Surprisingly, this inhibition is greater when LLE, CME, and PTE are administered in combination, as compared to the individual extracts.

[0132] In certain embodiments, the described compositions may be used to inhibit breakdown and absorption of dietary fat in the digestive organs of a subject. As such, the invention relates to a method inhibiting breakdown and absorption of dietary fat in the digestive organs of a subject, comprising administering to the subject the described composition. As such, in a preferred embodiment, the invention relates to a method for inhibiting breakdown and absorption of dietary fat in the digestive organs of a subject, comprising administering to the subject a composition comprising LLE, CME, and PTE. Surprisingly, this inhibition is greater when LLE, CME, and PTE are administered in combination, as compared to the individual extracts.

[0133] Combination Therapy

[0134] The described compositions may be used in combination with other actives used to achieve the same or similar effect(s) to the described compositions. For example, in certain embodiments, the described composition can be administered to a subject prior to, simultaneously, concurrently or subsequently to the administration of other active(s) used as lipid lowering active and / or active used for activating the GLP-1R.

[0135] Best way to practice the described compositions and methods are exemplified below.EXAMPLES

[0136] Example 1:

[0137] A method was designed to establish a HEK293-GLP-1R (luciferase) reporter cell, A luciferase assay was used to screen out the extract agonist(s) of the GLP- 1R.

[0138] A HEK293 stable cell line that overexpresses GLP-1R was developed, with tirzepatide serving as a positive control. Tirzepatide is a dual GIP and GLP-1R agonist, which had been previously shown to deliver superior glycemic control and weight loss ascompared to GLP-1R agonism in patients with type 2 diabetes [Samms RJ, Christe ME, Collins KA, et al. GIPR agonism mediates weight-independent insulin sensitization by tirzepatide in obese mice. Journal Of Clinical Investigation. 2021 ; 131 (12):el46353] .

[0139] Specifically, lentiviral plasmids containing GLP-1R were purchased from Hanbio Biotechnology Co. Ltd (Shanghai, China) and the volume of viral liquid was calculated using multiplicity of infection value 1. The GLP-1R lentivirus was added to the culture medium of HEK293 cells, allowing the viral particles to infect the cells. After 24 hours, the virus-containing medium was removed, and puromycin and blasticidin were added for selection. Only cells that had successfully integrated the GLP-1R gene survived and proliferated in the medium containing the selection drugs. The continuously proliferating cells were the HEK293 stable cell line that overexpressed GLP-1R.

[0140] Example 2

[0141] The process of preparing the extracts and specifications for lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE) are as follow:

[0142] The ground powder of each extract underwent reflux extraction with distilled water. Following the extraction, the mixture underwent centrifugation. The resulting supernatant was evaporated and subsequently lyophilized using a freeze-dryer to obtain the final extract.

[0143] The specifications for the extracts were as follows: nuciferine content not less than 0.2% in LLE, chi orogenic acid exceeding 1% in CME, along with tea polyphenol content surpassing 7% and caffeine levels exceeding 10% in PTE.

[0144] Example 3:

[0145] The agonistic activity on GLP-1R was evaluated using a luciferaseexpressing system. Specifically, the luciferase expressing system was used to screen out the agonistic effects on GLP-1R at certain concentrations of LLE, CME, and PTE.

[0146] HEK293 cells engineered to express GLP-1R (HEK293-GLP-lR-luc-cell) were plated in 96- well formats at a density of 8 x 10A3 cells per well and allowed to incubate for 12 hours. The subsequent day, treatments were applied across a range ofconcentrations (5-50 pg / mL) for LLE, CME and PTE, a control treatment without extracts, for a period of 48 hours.

[0147] Cell counting kit-8 (CCK-8) was used for cell toxicity assessment:

[0148] Post-treatment, each well received 10 pL of CCK-8 solution to facilitate the assessment of cell viability. The absorbance at 450 nm was measured using a microplate reader to determine the number of viable cells, calculated by subtracting the background optical density (O.D.).

[0149] The step for luciferase expressing system:

[0150] The medium was discarded, followed by the addition of 50 pL of cell lysate to each well. Subsequently, 20 pL of supernatant from each lysate was transferred to a new 96-well plate. The luciferase reaction was initiated by adding luciferase assay buffer to each well. Luminescence values associated with GLP-1R were then measured using a microplate reader.

[0151] The CCK-8 results showed no toxicity within 10 pg / mL for LLE, and within 50 pg / mL for CME and PEE. Figure 1 depicts the results of the cell viability assessment, which shows that LLE is non-toxic in the range of 5 pg / mL to 10 pg / mL, and CME and PEE are non-toxic in the range of 5 pg / mL to 50 pg / mL. Specifically, LLE in the range of 5-10 pg / mL, CME and PTE in the range of 5-50 pg / mL showed no cytotoxicity to HEK293-GLP-lR-luc-cell.

[0152] Further, surprisingly, luciferase assay findings revealed that LLE, CME, and PTE exhibit pronounced / significant agonistic actions on the GLP-1R receptor at specific concentrations. At concentrations of 5 and 10 pg / mL, LLE exhibited an agonistic fold increase greater than 2. For CME, at concentrations of 40 and 50 pg / mL, the agonistic fold increases were 2.35 and 4.29, respectively. For PTE, at concentrations of 40 and 50 pg / mL, the agonistic fold increases were 1.47 and 2.54, respectively, the specific data obtained are detailed in Figure 2. Compared with the “blank group,” there was a significant difference ( cO.OOl ).

[0153] These findings show that LLE, CME, and PTE extracts are each capable of activating the GLP-1R, which plays a significant role in reducing blood sugar levels, managing blood lipids, regulating appetite and food intake, slowing gastric emptying, and supporting weight loss efforts.

[0154] Example 4:

[0155] To study the effects of the composition comprising a mixture of lotus leaf extract, Chrysanthemum morifolium extract, and Pu'er tea extract, Box-Behnken design, a type of response surface methodology (RSM) was used, to model the multivariate quadratic equation with the pancreatic lipase inhibition rate as the response variable. This approach allowed to facilitate the determination of the optimal blend ratio for maximum efficacy in inhibiting pancreatic lipase.

[0156] Based on the Box Behnken method, the ratio of LLE, CME, and PTE were given as 3 parts LLE to 1 part CME to 7 parts PTE, and the total concentration at which this combination was most effective was 10 mg / mL.

[0157] RSM is a collection of mathematical and statistical techniques based on the fit of a polynomial equation to the experimental data, which describe the behavior of a data set with the objective of making statistical previsions [Bezerra MA, Santelli RE, Oliveira EP, et al. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Taianta. 2008;76(5):965-77].

[0158] The proportion and screening process were as follows:

[0159] RSM was employed to investigate the impact of process variables on PL inhibition rate. The experimental variables were explored across three levels: low (-1), medium (0), and high (+1). The scope and gradations of these independent variables are detailed in Table 1. The Box-Behnken design, along with its subsequent analysis, was conducted using Design-Expert version 13.0 software (Stat-Ease, Inc., Minneapolis, MN, USA).

[0160] Table 1. Actual and coded level of factors tested with Box-Behnken design.Code Factor Unit Coded levels

[0161] In the assay, a mixture of 15 pL of PL solution (1 mg / mL) and 50 pL of herbal extract compositions in phosphate-buffered saline (PBS) was prepared and incubated at 37°C for 10 minutes in a 96-well plate. Following this, 50 pL of the substrate 4-MU oleate (0.1 mmol / L) was added. The microplate was then incubated at 37°C for 30 minutes with moderate shaking. Each sample was tested in triplicate. The enzymatic hydrolysis of 4-MU oleate by lipase was quantified using a fluorescence microplate reader, measuring the fluorescence intensity at an excitation wavelength of 350 ± 10 nm and an emission wavelength of 450 nm.

[0162] The inhibition of PL activity was calculated as follows:Inhibition rate (%) = 100

[0163] To assess the impact of operational variables on the phospholipase (PL) inhibition rate resulting from the combination of LLE (A), CME (B), PTE(C) and total concentration (D), a series of 27 experiments were systematically conducted in accordance with RSM, as detailed in Table 2.

[0164] Table 2. Box-Behnken design matrix for optimization of variables and response values for yields of PL inhibition rate.Run A (LLE) B (CME) C (PTE) D (mass, mg) PL inhibition rate (%)1 5 1 5 10 90.842 9 9 5 6 75.083 1 5 5 2 53.814 9 5 5 10 83.145 9 5 1 6 72.596 5 1 1 6 72.737 5 5 5 6 77.438 5 5 9 10 90.519 9 1 5 6 76.2010 1 5 5 10 85.9511 1 5 5 10 77.8412 9 5 5 2 49.4413 5 5 1 10 82.2914 5 9 9 6 78.7115 1 5 1 6 75.3316 5 5 5 6 77.0917 5 1 9 6 79.6218 5 5 9 2 51.8519 5 1 5 2 51.7720 5 5 1 2 45.8421 1 1 5 6 80.5822 5 9 5 2 50.0423 5 5 5 6 76.6824 5 9 5 10 83.9225 1 9 5 6 78.6126 1 5 9 6 80.5527 5 9 1 6 73.45

[0165] The distribution of experimental and predicted values of response variable PL inhibition rate is shown in Figure 3, implying the fitness of proposed RSM. The summary statistics of the model emphasize the predicted R2and the adjusted R2values, which stand at 0.9667 and 0.9874, respectively.

[0166] Detailed statistics are provided in Table 3.

[0167] Table 3. Model summary statistics.Source statistics Source statisticsStd.Dev 1.48 R20.9942Mean 73.03 Adjusted R20.9874C.V.% 2.02 Predicted R20.9667Adeq Precision 37.9240

[0168] The analysis of variance (ANOVA) of RSM model is presented in Table 4.

[0169] Table 4. ANOVA for PL inhibition rate.„ Sum of ... Mean ,, , ,Source „ df F- value p- valueSquares SquareModel 4476.99 14 319.79 146.30 00001 SignificantA (LLE) 35.16 1 35.16 16.08 0.0017B (CME) 11.86 1 11.86 5.43 0.0381C (PTE) 113.16 1 113.16 51.77D (mass) 3812.77 1 2812.77 1744.29AB 0.1806 1 0.1806 0.0826 0.7787AC 0.0002 1 0.0002 0.0001 0.9921AD 0.6084 1 0.6084 0.2783 0.6074BC 0.6642 1 0.6642 0.3039 0.5916BD 6.73 1 6.73 3.08 0.1047CD 1.22 1 1.22 0.5586 0.4692A2 0.0324 1 0.0324 0.0148 0.9051B2 0.7769 1 0.7769 0.3554 0.5621C2 4.30 1 4.30 1.97 0.1859D2 397.86 1 397.86 182.02Residual 26.23 12 2.19Lack of Fit 25.95 10 2.59 18.40 0.0526 .N(°l+significantPure Error 0.2821 2 0.1410Cor Total 4503.22 26

[0170] ANOVA gives the significance of model and testing variables. A comparison of the F-values reveals the order of significance as D > C > A > B. The influence of PTE (C) and the total concentration (D) predominates over all other parameters in affecting PL inhibition rate. None of the interaction terms showed significance, with only the quadratic term for D (D2) being significant. Three-dimensional response surface plots were developed to identify the optimal levels of each factor that maximize PL inhibition rate. The graphical depiction of these response surfaces is illustrated in Figure 4. The quadratic equation representing the model's relationship with the operating variables is:Inhibition rate (%) = —1.7117 A - 0.9942B + 3.0708C + 17.8250D - 8.6372

[0171] According to the results of model fitting, under the ratio of LLE, CME, and PTE at 3: 1 :7, the inhibition rate of PL reached the optimal level when the total concentration was 10 mg / mL. Verification of these findings was conducted through empirical experiments, and the specific data obtained are detailed in Table 5.

[0172] Table 5. Actual experimental results.10 mg / mL PL inhibition SD rate / %3:1:7 89.35 0.240:1:7 87.19 0.413:0:7 87.88 0.23LLE 78.35 0.42CME 82.12 0.26PET 86.99 0,22

[0173] When evaluating the three extracts both individually and in combination, the optimal conditions identified through the Box-Behnken design (response surface method) were a ratio of 3:1:7 and a total concentration of 10 mg / mL. This specific combination of LLE, CME and PTE has a significant and unexpected lipid lowering effect.

[0174] Specifically, at the same concentration, the combination of lotus leaf extract, chrysanthemum extract, and Pu'er tea extract at a ratio of 3: 1 :7 had a greater inhibition on pancreatic lipase than individual extracts. Because in comparison, the mixture and individual extracts are at the same concentration of 10 mg / mL, the total amount of substance is the same.

[0175] Conclusion: Box-Behnken design predicted the optimal ratio of LLE, CME and PET combinations (3:1:7), which had a greater inhibition on PL when compared to the individual components of the composition.

Claims

CLAIMS1. A composition comprising a mixture of: lotus leaf extract (LLE),Chrysanthemum morifolium extract (CME), andPu'er tea extract (PTE), wherein the mixture has a greater effect on pancreatic lipase inhibition, as compared to the individual extracts.

2. The composition of claim 1, wherein the LLE has nuciferine content not less than 0.2%, CME has chlorogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%.

3. The composition of claim 1 or claim 2, wherein the ratio of LLE to CME to PET in the mixture is 3:1:7.

4. The composition of any of claims 1-3, wherein the total concentration of LLE, CME, and PTE in the composition is 10 mg / mL.

5. The composition of any of claims 1-4, further comprising a vehicle, a carrier, and / or an excipient.

6. The composition of any of claims 1-5, wherein the composition is formulated as a ready-to-drink beverage, a concentrate, a dry composition (e.g., powders, granules, or tablets that may be reconstituted with a liquid), a gel, a solid, a semi-solid (e.g., ice cream, pudding, or yogurt), a frozen liquid (e.g., ice pop), a lozenge or a hard candy, a dissolving strip, and chewing gum.

7. The composition of any of claims 1-5, wherein the composition is in a form of a tablet.

8. The composition of any of claims 1-7 for supporting weight loss efforts in a subject in need of thereof.

9. The composition of any of claims 1-7 for reducing blood sugar levels in a subject in need of thereof.

10. The composition of any of claims 1-7 for managing blood lipids in a subject in need of thereof.

11. A method for inhibiting pancreatic lipase in a subject comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the LLE has nuciferine content not less than 0.2%, CME has chlorogenic acid content exceeding 1%, and PTE has tea polyphenol content exceeding 7% and caffeine levels exceeding 10%.

12. The method of claim 11, wherein the ratio of LLE to CME to PET in the mixture is 3:1:7.

13. The method of claim 11 or claim 12, wherein the total concentration of LLE, CME, and PTE in the composition is 10 mg / mL.

14. A method for inhibiting pancreatic lipase in a subject comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), Chrysanthemum morifolium extract (CME), and Pu'er tea extract (PTE), wherein the ratio of LLE to CME to PET in the mixture is 3: 1 :7, and wherein the total concentration of LLE, CME, and PTE in the composition is 10 mg / mL.

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