Composition of lotus leaf extract, chrysanthemum extract and Pu'er tea extract, method and application thereof

Abstract

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

Description

Background Technology

[0001] Obesity has been proven to be one of the world’s biggest public health problems because it is a fundamental contributing factor to many chronic metabolic diseases, including type 2 diabetes (T2DM), hypertension, dyslipidemia, cardiovascular disease, and various types of cancer.

[0002] Lotus (Nelumbo nucifera Gaertn, lotus), chrysanthemum (Chrysanthemum morifolium, Chrysanthemum morifolium Ramat), and large-leaf tea (Camellia sinensis var. assamica, Pu'er tea) are traditional Chinese medicinal materials. Extracts of these herbs have been shown to have significant bioactivities in lowering blood sugar, lowering blood lipids, and reducing weight [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 modulationeffects of sacha inchi(Plukenetia volubilis L.)leaf extract instreptozotocin-induced type 1diabetic mice.J Sci Food Agric.2022Aug15;102(10):4304-4312].

[0003] Lotus leaf extract (LLE) has previously shown lipid-lowering and weight-loss 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].

[0004] Chrysanthemum morifolium extract (CME) is widely used as an herbal tea in many countries. It effectively prevents the accumulation of lipids in adipocytes by downregulating the expression of genes associated with adipose 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].

[0005] Pu'er tea extract (PTE) has previously shown significant potential in combating central obesity, regulating blood lipid levels, lowering blood sugar, and providing 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]. Furthermore, numerous reports have highlighted its efficacy in promoting weight loss and lowering blood lipid levels.

[0006] The glucagon-like peptide-1 receptor (GLP-1R) belongs to the G protein-coupled receptor (GPCR) class. It is a clinically proven target for many clinically approved peptide drugs for the treatment of type 2 diabetes, and some drugs are also approved for the treatment of 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].

[0007] Pancreatic lipase, or "PL," also known as triacylglycerol acyl hydrolase or triglyceride lipase, is an enzyme that catalyzes the hydrolysis of ester bonds in 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 for 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 therapyeutics. Expert Opinion on Drug Safety. 2009; 8(6): 727-44].

[0008] Nevertheless, there is still a need for safe and effective compositions and methods for weight loss management, obesity, and related conditions. Summary of the Invention

[0009] This article describes a composition comprising at least one of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE), which exhibits a significant agonistic effect on GLP-1R.

[0010] Furthermore, this article describes a composition comprising a combination of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract. Surprisingly, the combination of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract was found to have a greater inhibitory effect on pancreatic lipase than the individual extracts.

[0011] Some embodiments involve compositions comprising at least one of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE), wherein the composition has an agonistic effect on glucagon-like peptide-1 receptor (GLP-1R). In the composition, LLE has a content of not less than 0.2% lotus leaf alkaloid, CME has a content of more than 1% chlorogenic acid, and PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%. In the composition, the concentrations of LLE, CME, and PTE are in the range of 5-50 μg / mL. In the composition, LLE exhibits an agonistic increase of greater than 2 at a concentration of 5 μg / mL, or an agonistic increase of greater than 2 at a concentration of 10 μg / mL. In the composition, CME showed a 2.35-fold increase in activation at a concentration of 40 μg / mL, or a 4.29-fold increase in activation at a concentration of 50 μg / mL. In the composition, PTE showed a 1.47-fold increase in activation at a concentration of 40 μg / mL, or a 2.54-fold increase in activation at a concentration of 50 μg / mL. The composition can be used to support weight loss efforts in subjects with this need, to lower blood glucose levels in subjects with this need, to manage blood lipids in subjects with this need, and / or for the prevention and / or treatment of type 2 diabetes.

[0012] Some other embodiments relate to a method for activating glucagon-like peptide-1 receptor (GLP-1R), which includes providing a composition comprising at least one of the following: lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE).

[0013] Some 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 extract (CME), and Pu-erh tea extract (PTE), wherein the LLE has a content of not less than 0.2% lotus leaf alkaloid, the CME has a content of more than 1% chlorogenic acid, and the PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%.

[0014] Some further embodiments relate to a method for supporting a subject’s weight loss efforts in need, comprising administering to the subject a composition comprising at least one of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE), wherein the LLE has a content of not less than 0.2% lotus leaf alkaloid, the CME has a content of more than 1% chlorogenic acid, and the PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%.

[0015] Some further embodiments relate to a method for lowering blood glucose levels in a subject who requires this, comprising administering to the subject a composition comprising at least one of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE), wherein the LLE has a content of not less than 0.2% lotus leaf alkaloid, the CME has a content of more than 1% chlorogenic acid, and the PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%.

[0016] Some further embodiments relate to a method for managing blood lipids in subjects who require such management, comprising administering to the subject a composition comprising at least one of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE), wherein the LLE has a content of not less than 0.2% lotus leaf alkaloid, the CME has a content of more than 1% chlorogenic acid, and the PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%.

[0017] Some further embodiments relate to a method for the prevention and / or treatment of type 2 diabetes, comprising administering to a subject a composition comprising at least one of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE), wherein the LLE has a content of not less than 0.2% lotus leaf alkaloid, the CME has a content of more than 1% chlorogenic acid, and the PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%.

[0018] However, further embodiments involve compositions comprising a mixture of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE), wherein the mixture has a significantly greater effect on pancreatic lipase inhibition compared to the individual extracts. In the composition, LLE has a content of not less than 0.2% lotus leaf alkaloid, CME has a content of more than 1% chlorogenic acid, and PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%. In the composition, the ratio of LLE to CME to PTE 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 medium, a carrier, and / or excipients. The composition may be formulated as a ready-to-drink beverage, a concentrate, a dry composition (e.g., a powder, granules, or tablet that can be reconstituted with a liquid), a gel, a solid, a semi-solid (e.g., ice cream, pudding, or yogurt), a frozen liquid (e.g., popsicles), a tablet or hard candy, a dissolving strip, and chewing gum. In some embodiments, the composition is in tablet form. Some further embodiments relate to the use of the composition for supporting weight loss efforts in subjects with such needs, for lowering blood glucose levels in subjects with such needs, and / or for managing blood lipids in subjects with such needs.

[0019] However, a further embodiment relates 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 extract (CME), and Pu-erh tea extract (PTE). In this method, the ratio of LLE to CME to PET in the mixture is 3:1:7. In this method, the total concentration of LLE, CME, and PTE is 10 mg / mL.

[0020] However, certain further embodiments involve 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 extract (CME), and Pu-erh tea extract (PTE), wherein the LLE has a content of not less than 0.2% lotus leaf alkaloid, the CME has a content of more than 1% chlorogenic acid, and the PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%. In the method, the ratio of LLE to CME to PTE in the mixture is 3:1:7. In the method, the total concentration of LLE, CME, and PTE is 10 mg / mL.

[0021] However, some further embodiments involve methods for supporting weight loss efforts in subjects with this need, comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE). In this method, the ratio of LLE to CME to PET in the mixture is 3:1:7. In this method, the total concentration of LLE, CME, and PTE is 10 mg / mL.

[0022] However, some further embodiments involve a method for lowering blood glucose levels in subjects with this need, comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE). In this method, the ratio of LLE to CME to PTE in the mixture is 3:1:7. In this method, the total concentration of LLE, CME, and PTE is 10 mg / mL.

[0023] However, a further embodiment relates to a method for managing blood lipids in a subject with this need, comprising administering to the subject a composition comprising a mixture of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE). In this method, the ratio of LLE to CME to PET in the mixture is 3:1:7. In this method, the total concentration of LLE, CME, and PTE is 10 mg / mL. Attached Figure Description

[0024] Figure 1 A bar graph was plotted to show the cell viability of HEK293-GLP-1R-luc- cells after treatment with specified concentrations of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE).

[0025] Figure 2 A bar graph was plotted showing the change in the activating factor of GLP-1R through the extract after treatment with specified concentrations of LLE, CME, and PTE.

[0026] Figure 3 A graph depicting the correlation between experimental and predicted PL inhibition rates is presented.

[0027] Figure 4 A three-dimensional plot generated by the Box-Behnken design was depicted, which clearly shows the effect of extract and concentration on PL inhibition rate.

[0028] Detailed description of the accompanying drawings and the current preferred embodiments

[0029] The invention will now be described more fully below. For the purposes of the following detailed description, it should be understood that the invention may take various alternative variations and sequences of steps unless explicitly indicated to the contrary. Therefore, before describing the invention in detail, it should be understood that the invention is not limited to the embodiments of the specific examples, which are of course subject to variation.

[0030] This article describes a composition comprising at least one of lotus leaf extract, chrysanthemum extract and Pu-erh tea extract, and a method of using the composition, which exhibits a significant stimulating effect on GLP-1R.

[0031] Furthermore, this article describes a composition comprising a combination of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract. Surprisingly, a greater lipase inhibitory effect was observed with the combination of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract.

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

[0033] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates 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 "ideally" refer to embodiments of the invention that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are useless, nor is it intended to exclude other embodiments from the scope of the invention.

[0035] As used herein, “about,” “approximately,” or “approximately” generally means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. The numerical values ​​given herein are approximate; it is meant that unless explicitly stated otherwise, the terms “about,” “approximately,” or “approximately” can be inferred. When the term “about” is used to describe an endpoint of a value or range, this disclosure should be understood to include both the specific value mentioned and the endpoint.

[0036] As used herein, the terms “comprising,” “including,” “having,” “containing,” “involving,” etc., should be understood as open-ended, meaning including but not limited to.

[0037] The term "composition" is used herein to describe formulations comprising at least one of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract, and in some embodiments, to describe formulations comprising a combination of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract. The term refers to an edible formulation suitable for oral ingestion by a subject (e.g., a human female subject). Exemplary compositions comprising at least one of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract include, but are not limited to: sprays (e.g., aerosols), powders, chewing gum, ingestible solids, gels, aqueous beverages, dry powders (e.g., powders that can be directly consumed or can be reconstituted with liquid to provide a beverage as defined herein), nutrition bars, lozenges, tablets, capsules, wafers, pastes, etc. Other compositions are described herein. In some preferred embodiments, the compositions described herein are tablets.

[0038] An "extract" is a plant material that dissolves in a suitable solvent after contact with the plant material. Preferably, the plant material is contacted with the solvent at a suitable temperature for a sufficient time to allow substantially all soluble material to be dissolved. The solvent, along with the dissolved soluble plant material, is referred to as a "liquid extract." The term "extract" refers to the soluble plant material remaining after the solvent has been removed. The solvent can be removed by any suitable means, including evaporation, freeze-drying, spray drying, etc.

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

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

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

[0042] As used herein, the term "active combination" refers to the ability of the combination of described extracts to exert a specific effect, as described herein. No single component is considered an additive, such as a carrier, diluent, or excipient.

[0043] As used herein, the term "effective amount" or "pharmaceutical or therapeutically effective amount" refers to the amount of an active ingredient or extract that, when orally administered to a subject to produce the desired effect (e.g., lowering blood glucose levels, managing blood lipids, regulating appetite and food intake, slowing gastric emptying, and supporting weight loss efforts) without causing or minimal adverse toxicity to the subject, and / or without undesirable side effects. Those skilled in the art will recognize that effective amounts can vary from person to person due to external factors such as age, sex, disease state, race, weight, formulation of the composition, availability of other active ingredients in the formulation, etc.

[0044] The term “agonist effect” in relation to GLP-1R agonists refers to a composition, its extract or component binding to and activating GLP-1R on cells, evoking a biological response such as, for example, lowering blood glucose levels, managing blood lipids, regulating appetite and food intake, slowing gastric emptying, and supporting weight loss efforts.

[0045] The increase in stimulant multiplier can be at least 0.5 times; at least 1.0 times; at least 1.5 times; at least 2.0 times; at least 2.5 times; at least 3 times; at least 3.5 times; at least 4.0 times; at least 4.5 times; at least 5.0 times; at least 5.5 times; at least 6 times, or higher. Multiple increases between the above multipliers are also considered.

[0046] In the context of the effects on GLP-1R, the term "significant agonist effect" means that the treatment group is at least 2-fold or more effective as a GLP-1R agonist compared to the "blank group," and the statistical result has a p-value < 0.001. For example, as shown in this paper, compared to the "blank," LLE and PTE agonized GLP-1R 2-fold at 10 μg / mL, and CME agonized GLP-1R 4-fold at 50 μg / mL.

[0047] In the context of “pancreatic lipase inhibition,” the terms “greater” or “better” mean that the mixture of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract has a greater inhibitory effect on pancreatic lipase than any two of the three extracts alone and / or in combination. For example, at a total concentration of 10 mg / mL, with a ratio of lotus leaf extract to chrysanthemum extract to Pu-erh tea extract of 3:1:7, the mixture of extracts showed a greater inhibitory effect on pancreatic lipase than the single extract or any two of the extracts in combination (e.g., 89.35%).

[0048] The term "excipient" refers to any substance that facilitates the absorption of a component of the product of the present invention, stabilizes said component, or contributes to the preparation of the composition. Thus, excipients may have functions such as keeping components bound together (e.g., starch, sugar, or cellulose), sweetening functions, coloring functions, protecting the drug from external media (e.g., isolating it from air and / or moisture), filling function for tablets, capsules, or any other form of formulation (e.g., calcium dibasic phosphate), or disintegration functions that promote the dissolution of components and their absorption in the intestine, but other types of excipients not mentioned in this paragraph are not excluded. Therefore, the term "excipient" is defined as a substance included in dosage forms, added to active substances or their associations to enable their preparation and stabilization, altering their sensory properties, or determining the physical / chemical properties and bioavailability of a pharmaceutical composition. "Pharmaceutically acceptable" excipients should not interact with the activity of the active compound in the composition. Examples of excipients are binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, and coloring agents. More specific, non-limiting examples of acceptable excipients are starch, sugar, xylitol, sorbitol, calcium phosphate, steroidal fats, talc, silicon dioxide, or glycerol.

[0049] The term "administering" (or "administration") refers to the mode of delivery. Daily doses can be divided into one, two, three, or more doses in a suitable manner, to be administered once, twice, three times, or more times over a period of time (such as daily, weekly, or monthly).

[0050] The term "treatment," "improvement," or "relief" refers to the application of the composition or treatment to a subject already suffering from a condition for therapeutic purposes to improve the subject's condition. Such improvement or treatment is at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to an equivalent untreated control, as measured by any standard, suitable technique.

[0051] As described herein, the term "subject" is equivalent to the terms "individual" and "patient," and thus these terms are used interchangeably. "Subject" refers to any animal belonging to any species. Examples of subjects include, but are not limited to, commercially raised animals such as birds (hens, ostriches, chickens, geese, partridges, etc.), rabbits, hares, livestock (dogs, cats, etc.), livestock (e.g., sheep and goats, pigs, wild boars, horses, ponies, etc.), and cattle (bulls, castrated bulls, etc.). In certain embodiments, the subject is a mammal, preferably a primate, and more preferably a human of any race, sex, or age.

[0052] Active components

[0053] In some embodiments, the described invention relates to compositions comprising at least one of the following: (i) lotus leaf extract (LLE), (ii) chrysanthemum extract (CME), and (iii) Pu-erh tea extract (PTE).

[0054] In some other embodiments, the described invention relates to compositions comprising the following active ingredients: (i) lotus leaf extract (LLE), (ii) chrysanthemum extract (CME), and (iii) Pu-erh tea extract (PTE).

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

[0056] Lotus (N. nucifera), commonly known as lotus, holy lotus, Indian lotus, water lily, and Chinese water lily, is widely distributed throughout East Asia and is renowned for its extensive use as a culinary and medicinal plant. In traditional herbal medicine, lotus, particularly its leaves, extracts, fractions, and other components, has been shown to have diverse biological and pharmacological applications due to its numerous bioactive compounds, including flavonoids, polyphenols, steroids, phenolic acids, polysaccharides, alkaloids, terpenes, fatty acids, and glycosides, exhibiting antioxidant, antibacterial, antiviral, antifungal, immunomodulatory, anti-inflammatory, antidiarrheal, antithrombotic, antidiabetic, cholesterol-lowering, anti-obesity, and anticancer properties. In Asia, lotus leaves have been used as functional foods and supplements for treating obesity. The inhibitory effects of lotus leaves and their extracts are believed to primarily impair the absorption of carbohydrates and lipids in the gut and increase energy expenditure, which can alleviate chronic inflammation by improving intestinal integrity and can regulate the gut microbiota through specific metabolites such as short-chain fatty acids. However, there are few reports on the anti-obesity effect of fermented lotus leaves. Only some studies have shown that the bioactive substances in lotus leaves, such as total phenols, flavonoids, and a variety of low molecular weight metabolites, increase after fermentation [Hwang D.,Charchoghlyan H.,Lee JS,Kim M.Bioactive compounds and antioxidant activities of the Korean lotus leaf (Nelumbo nucifera) condiment:Volatile and nonvolatile metabolite profiling during fermentation.Int.J.FoodSci.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] Flavonol miquelianin, as well as the alkaloids (+)-(1R)-codone and (-)-(1S)-norcodone, were found in lotus leaves. The plant also contains nuciferine, neferine, and many other benzylisoquinoline alkaloids, which have medicinal value.

[0058] In some preferred embodiments, the lotus leaf alkaloid content in the LLE is not less than 0.2%.

[0059] In some preferred embodiments, the lotus leaf alkaloid 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, not less 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 some other embodiments, the lotus leaf alkaloid content in the LLE is at least 0.2%.

[0061] (ii) Chrysanthemum (Chrysanthemum morifolium)

[0062] Chrysanthemum, a representative plant of the Asteraceae family, is a perennial plant that has long been used for food, medicine, and ornamental purposes. It contains components such as luteolin, apigenin, and chlorogenic acid, which have previously been shown to exhibit anti-obesity effects by inhibiting weight gain. In particular, extracts of chrysanthemum (C. morifolium ramat flower) (CF) have shown bioactive properties, including antioxidant, anti-diabetic, lipid-lowering, and anti-obesity effects.

[0063] In some implementations, the chlorogenic acid content in CME exceeds 1%.

[0064] In some other embodiments, the chlorogenic acid content in the CME is at least 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%; more preferably, at least 20%; or more.

[0065] (iii) Pu-erh tea

[0066] Pu'er (or pu-erh) is a 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 has been fully dried and kneaded. When the tea undergoes controlled microbial fermentation, it also continues to oxidize, and this process is also controlled until the desired flavor is achieved. The tea produced by this process is called black tea (hēichá), literally meaning "black tea", although this term is usually translated into English as "dark tea" to distinguish it from English black tea (hóngchá), which literally means "red tea" in Chinese.

[0067] There are two main styles of Pu'er production: the traditional, longer production process, called raw Pu'er (shēng (raw) pu'er); and the modern, accelerated production process, called ripe Pu'er (shóu (ripe) pu'er). Traditionally, Pu'er starts from a primary product called "coarse" (mao) tea (literally meaning hairy / fluffy tea) and can be sold in this form or pressed into various shapes and sold as "raw tea" (literally meaning "raw tea"). Both forms will undergo a complex process of gradual fermentation and maturation over time.

[0068] In certain embodiments, the content of tea polyphenols in PTE exceeds 7% and the caffeine level exceeds 10%.

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

[0070] (iv) Extract preparation

[0071] The extraction processes and descriptions of LLE, CME, and PTE are as follows:

[0072] The ground powder was extracted by reflux with distilled water. After extraction, the mixture was centrifuged, the supernatant was evaporated, and then freeze-dried to obtain the final extract. The extract specifications were as follows: LLE contained at least 0.2% lotus leaf alkaloids, CME contained more than 1% chlorogenic acid, and PTE contained more than 7% tea polyphenols and more than 10% caffeine.

[0073] The combined application of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract surprisingly showed inhibition of pancreatic lipase, or "PL," also known as triacylglycerol acylhydrolase. Surprisingly and unexpectedly, this inhibition was shown to be stronger than that of the individual extracts.

[0074] Composition

[0075] In some embodiments, the described invention relates to compositions comprising at least one of the following: (i) lotus leaf extract (LLE), (ii) chrysanthemum extract (CME), and (iii) Pu-erh tea extract (PTE).

[0076] In some other embodiments, the described invention relates to a composition comprising a mixture of (i) lotus leaf extract (LLE), (ii) chrysanthemum extract (CME), and (iii) Pu-erh tea extract (PTE).

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

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

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

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

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

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

[0083] In some preferred embodiments, the specific combination and concentration of LLE, CME and PTE that effectively inhibit phospholipase activity is 3 parts LLE to 1 part CME to 7 parts PTE, and the most effective total concentration of this combination is 10 mg / mL.

[0084] Inactive components of the composition

[0085] In some embodiments, the composition may contain at least one inactive ingredient or extract.

[0086] In some embodiments, the composition may contain a "carrier" or "propellant," which is preferably an inert substance. The function of the carrier is to facilitate the incorporation of other compounds to allow for better administration or to impart consistency and form to the composition. Thus, a carrier is a substance used to dilute any component of the composition to a defined volume or weight, or which enables better administration or to impart consistency and form to the composition even without dilution of the component (e.g., a nutritional supplement composition). For all these reasons, the carrier will be considered pharmaceutically or nutritionally acceptable.

[0087] When the composition is presented in liquid form, the medium is a diluent.

[0088] The composition may also contain one or more excipients that are non-toxic and non-inflammatory to the subject.

[0089] In some implementations, excipients can provide desired or improved physical and / or chemical properties, such as stability, flowability, viscosity, disintegration rate, taste, delivery, etc.

[0090] The compositions described herein may additionally include, for example, electrolytes (e.g., potassium salts or other salts), sweeteners, flavorings and colorings, vitamins, minerals, preservatives, and antioxidants.

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

[0092] In some embodiments, the electrolyte may be included in the composition. Exemplary electrolytes include potassium salts, chloride salts, bromide salts, sodium salts, magnesium salts, calcium salts, citrates, acetates, phosphates, salicylates, bicarbonates, lactates, sulfates, tartrates, benzoates, selenites, molybdates, iodides, oxides, and combinations thereof. Based on the total volume of the composition, the electrolyte may be present in the compositions of the invention at a concentration ranging from about 0.01% to 10% by weight (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%), although the electrolyte may be present at lower or higher concentrations.

[0093] In some embodiments, the composition may contain potassium (e.g., potassium chloride). Based on the total volume of the composition, the concentration of potassium in the composition may be, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.5%, 13%, 4%, 5%, 6%, or 7% or higher by weight.

[0094] In some embodiments, the composition may contain magnesium (e.g., magnesium chloride). Based on the total volume of the composition, the concentration of potassium in the composition may be, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or higher by weight.

[0095] Sweeteners may also be included in the 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., maltodextrin), 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, calorie-free or low-calorie sweeteners may be used in the compositions of the present invention. Examples of such calorie-free or low-calorie sweeteners include, but are not limited to, saccharin, cyclamate, acesulfame potassium, sorbitol, sucralose, xylitol, erythritol, stevia, stevia extract, L-aspartic-L-phenylalanine esters (e.g., aspartame), L-aspartic-D-alanine alkylamide, L-aspartic-L-1-hydroxymethylalkanamide, and L-aspartic-1-hydroxyethylalkanamide. Based on the total volume of the composition, the sweetener may be present in the compositions of the present invention at a concentration ranging from about 2% to 20% by weight (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%), although the sweetener may be present at lower or higher concentrations.

[0096] In some other embodiments, flavoring agents and / or coloring agents may also be included in the composition. Exemplary flavoring agents include natural and synthetic flavoring agents, including almond oil, bitter almond oil (amaretto) Oil), anethole, fennel oil, benzaldehyde, blackberry, black walnut oil, blueberry, caraway, caraway oil, cardamom oil, cardamom seeds, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, coriander oil, glucose, sage, ethyl acetate, ethyl vanillin, fennel oil, ginger, glucose, glycerin, licorice, grape, honey, lavender oil, lemon oil, lime, mannitol, methyl salicylate, nutmeg 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, toluene, tropical, vanilla, vanillin, and wild cherry syrup. Other flavorings can be found in Food Chemicals Codex and Fenaroli's Handbook of Flavor Ingredients.

[0097] One or more colorants may be used in small amounts in the composition. Colorants include, for example, β-carotene, riboflavin dyes, FD&C dyes (e.g., Yellow 5, Blue 1, Blue 2, and Red 40), FD&C lakes, chlorophyll and chlorophyllin, caramel coloring, annatto, cochineal, turmeric, paprika, and fruit, vegetable, and / or plant extracts (e.g., grape, blackcurrant, chokeberry, carrot, beetroot, red cabbage, elderberry, and hibiscus extracts). The amount of colorant used will vary depending on the reagents used in the composition and the desired color intensity in the finished product. Those skilled in the art can readily determine the amount of colorant used.

[0098] In some additional embodiments, vitamins and / or minerals may also be included in the composition. Non-limiting examples of vitamins and minerals that may be included in the composition include, for example, choline bitartate, nicotinamide, thiamine, folic acid, calcium d-pantothenate, biotin, vitamin A, vitamin C, vitamin B1 hydrochloride, vitamin B2, vitamin B3, vitamin B6 hydrochloride, and vitamin B6. 12 Vitamins D, vitamin E acetate, vitamin K, and calcium, potassium, magnesium, zinc, iodine, iron, and copper salts. When included in the 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) of such vitamins and minerals.

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

[0100] One or more antioxidants may also be included in the composition. Exemplary antioxidants include vitamin C and vitamin E; beta-carotene, lutein or other carotenoids; anthocyanins, malvidin, or other anthocyanins; apigenin, luteolin or other flavonoids; hesperidin, naringenin or other dihydroflavonoids; isorhamnetin, quercetin, kaempferol or other flavonols; and epigallocatechin-3-gallate, epicatechin, thearubigin or other flavan-3-ols.

[0101] Other 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), emulsifiers, carbon dioxide (e.g., for carbonizing the liquid composition), stabilizers, humectants, anticaking agents, or herbal extracts not previously mentioned.

[0102] Importantly, the other components are pharmaceutically and / or nutritionally acceptable.

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

[0104] In some embodiments, the composition may be dairy-free.

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

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

[0107] In some embodiments, the composition may be free of artificial sweeteners.

[0108] In some embodiments, the composition may be free of artificial preservatives.

[0109] In some embodiments, the composition may be free of artificial flavorings.

[0110] In some embodiments, the composition may be free of artificial colorants.

[0111] Moreover, in some embodiments, the composition does not require refrigeration (e.g., it can be stored in a cool, dry place).

[0112] Furthermore, the composition may not contain any irradiated components.

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

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

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

[0116] In some embodiments, the composition may be free of peanuts, nuts, eggs, shellfish, and crustaceans.

[0117] Preparations

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

[0119] In some embodiments, the compositions described herein may be formulated as food supplements. The term "food supplement" should be understood as a product sold in the form of capsules, tablets, vials, herbal infusions, drinking solutions, etc., intended to supplement the daily diet and constitute a concentrated source of nutrients (e.g., vitamins, minerals, amino acids, essential fatty acids, fiber, etc.) or other substances with nutritional or physiological effects.

[0120] In some embodiments, the composition may be in the form of a dry powder, granules, or tablets, which may be reconstituted in a specified amount of liquid. The dried components may be mixed together and ground (e.g., to produce a uniform powder) or mixed in an aqueous solution and dried using methods known to those skilled in the art. The dried powder or granules may be "loose" or formulated into tablets.

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

[0122] Compositions can be prepared using methods known to those skilled in the art. Such methods include dissolving, dispersing, or otherwise mixing all components individually or in a suitable combination and stirring, for example, with a mechanical stirrer until all components are dissolved or sufficiently dispersed. When a shelf-stable composition or solution is desired, the final mixture can be pasteurized, ultrapasteurized, sterilized, or aseptically filled under appropriate processing conditions. When the mutual stability of two or more components is required (e.g., if one component is unstable at low pH), multiple components can be mixed shortly before ingestion.

[0123] Methods and uses

[0124] In the context of this invention, treatment and / or prevention methods equivalent to those described in this specification are also contemplated.

[0125] In some embodiments, the composition can be used to activate GLP-1R, which has been shown to play an important role in lowering blood glucose levels, managing blood lipids, and supporting weight loss efforts.

[0126] In some embodiments, the composition can be used to treat and / or prevent hyperglycemia in a subject. Therefore, the present invention relates to a method for treating and / or preventing hyperglycemia in a subject, comprising administering the composition described herein to the subject.

[0127] In some embodiments, the composition can be used to treat and / or prevent type 2 diabetes in a subject. Therefore, the present invention relates to a method of treating and / or preventing type 2 diabetes in a subject, comprising administering the composition described herein to the subject.

[0128] In some embodiments, the composition can be used to manage blood lipids in a subject. Therefore, the claimed invention relates to a method for managing blood lipids in a subject, comprising administering the composition to the subject.

[0129] In some embodiments, the composition can be used to support a subject's weight loss efforts. Therefore, the present invention relates to a method for supporting a subject's weight loss efforts, comprising administering the composition to the subject.

[0130] In some embodiments, the composition can be used to treat and / or prevent obesity in a subject requiring treatment and / or prevention. Therefore, the present invention relates to a method of treating and / or preventing obesity in a subject, comprising administering the composition to the subject.

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

[0132] In some embodiments, the composition can be used to inhibit the breakdown and absorption of dietary fat in the digestive organs of a subject. Therefore, the present invention relates to a method for inhibiting the breakdown and absorption of dietary fat in the digestive organs of a subject, comprising administering the composition to the subject. Therefore, in a preferred embodiment, the present invention relates to a method for inhibiting the 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 compared to the individual extracts.

[0133] combination therapy

[0134] The composition may be used in combination with other active agents that achieve the same or similar effects as the composition. For example, in some embodiments, the composition may be administered to the subject before, simultaneously, in parallel, or subsequently before the administration of other active agents used as lipid-lowering agents and / or active agents used to activate GLP-1R.

[0135] The following examples illustrate the best way to implement the described composition and method. Example

[0136] Example 1:

[0137] A method was designed to establish HEK293-GLP-1R (luciferase) reporter cells, and luciferase assays were used to screen for extract agonists of GLP-1R.

[0138] A stable HEK293 cell line overexpressing GLP-1R was developed, with tirzepatide serving as a positive control. Tirzepatide is a dual GIP and GLP-1R agonist that has previously been shown to provide better glycemic control and weight loss in patients with type 2 diabetes compared to GLP-1R agonists [Samms RJ, Christe ME, Collins KA, et al. GIP agonism mediates weight-independent insulin sensitization by tirzepatide inobese mice. Journal of Clinical Investigation. 2021; 131(12):e146353].

[0139] Specifically, lentiviral plasmids containing GLP-1R were purchased from HanbioBiotechnology Co., Ltd. (Shanghai, China), and the viral volume was calculated using a multiplicity of infection (MOI) of 1. GLP-1R lentivirus was added to the culture medium of HEK293 cells to infect the cells. After 24 hours, the virus-containing medium was removed, and puromycin and blastomycin were added for selection. Only cells that successfully integrated the GLP-1R gene survived and proliferated in the medium containing the selection drugs. The continuously proliferating cells were the stable HEK293 cell lines overexpressing GLP-1R.

[0140] Example 2

[0141] The preparation processes and specifications of lotus leaf extract (LLE), chrysanthemum extract (CME), and Pu-erh tea extract (PTE) are as follows:

[0142] The ground powders of each extract were extracted by reflux with distilled water. After extraction, the mixture was centrifuged. The resulting supernatant was evaporated and then freeze-dried to obtain the final extract.

[0143] The specifications for the extracts are as follows: LLE contains no less than 0.2% lotus leaf alkaloids, CME contains more than 1% chlorogenic acid, and PTE contains more than 7% tea polyphenols and more than 10% caffeine.

[0144] Example 3:

[0145] The agonistic activity of GLP-1R was evaluated using a luciferase expression system. Specifically, the agonistic effects of certain concentrations of LLE, CME, and PTE on GLP-1R were screened using the luciferase expression system.

[0146] Engineered HEK293 cells expressing GLP-1R (HEK293-GLP-1R-luc-cells) were seeded at a density of 8 x 10^3 cells per well in 96-well plates and allowed to incubate for 12 hours. The following day, a series of treatments (5–50 μg / mL) were applied to LLE, CME, and PTE, with an extract-free control, for a period of 48 hours.

[0147] Cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8).

[0148] After treatment, 10 μL of CCK-8 solution was added to each well to facilitate cell viability assessment. The absorbance at 450 nm was measured using a microplate reader, and the number of viable cells was determined by subtracting the background optical density (OD).

[0149] Steps of the luciferase expression system:

[0150] Discard the culture medium and add 50 μL of cell lysis buffer to each well. Then, transfer 20 μL of supernatant from each lysate to a new 96-well plate. Initiate the luciferase reaction by adding luciferase assay buffer to each well. Then, measure the luminescence values ​​associated with GLP-1R using a microplate reader.

[0151] The CCK-8 results showed that LLE was non-toxic up to 10 μg / mL, and CME and PEE were non-toxic up to 50 μg / mL. Figure 1The results of cell viability assessment were presented, showing that LLE was non-toxic in the range of 5 μg / mL to 10 μg / mL, and that CME and PEE were non-toxic in the range of 5 μg / mL to 50 μg / mL. Specifically, LLE showed no cytotoxicity to HEK293-GLP-1R-luc- cells in the range of 5–10 μg / mL, and CME and PTE in the range of 5–50 μg / mL.

[0152] Furthermore, surprisingly, luciferase assays revealed that LLE, CME, and PTE exhibited significant agonistic effects on the GLP-1R receptor at specific concentrations. At concentrations of 5 and 10 μg / mL, LLE showed an activation fold increase greater than 2. For CME, the fold increases were 2.35 and 4.29 at concentrations of 40 and 50 μg / mL, respectively. For PTE, the fold increases were 1.47 and 2.54 at concentrations of 40 and 50 μg / mL, respectively. Detailed data can be found in [link to relevant documentation]. Figure 2 There was a significant difference compared to the "blank group" (P<0.001).

[0153] These findings indicate that LLE, CME, and PTE extracts can all activate GLP-1R, which plays an important role in lowering blood glucose levels, managing blood lipids, regulating appetite and food intake, slowing gastric emptying, and supporting weight loss efforts.

[0154] Example 4:

[0155] To investigate the effects of compositions containing a mixture of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract, a Box-Behnken design (a type of response surface methodology (RSM)) was used to model a multivariate quadratic equation with pancreatic lipase inhibition rate as the response variable. This method helps determine the optimal mixing ratio to maximize the inhibition of pancreatic lipase activity.

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

[0157] Response surface methodology (RSM) is a collection of mathematical and statistical techniques based on fitting experimental data with polynomial equations. These techniques describe the performance of the dataset and aim to make statistical predictions [Bezerra MA, Santelli RE, Oliveira EP, et al. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta. 2008; 76(5):965-77].

[0158] The proportions and screening process are as follows:

[0159] RSM was used to study the effect of process variables on PL inhibition rate. Experimental variables were divided into three levels: low (-1), medium (0), and high (+1). The ranges and levels of these independent variables are detailed in Table 1. The Box-Behnken design and subsequent analysis were performed using Design-Expert version 13.0 software (Stat-Ease, Inc., Minneapolis, MN, USA).

[0160] Table 1. Actual and coded levels of factors used in testing with Box-Behnken design.

[0161]

[0162] In the assay, a mixture of 15 μL PL solution (1 mg / mL) and 50 μL of the herbal extract composition in phosphate-buffered saline (PBS) was prepared and incubated in a 96-well plate at 37 °C for 10 min. Subsequently, 50 μL of the substrate 4-MU oleate (0.1 mmol / L) was added. The microplate was then incubated with gentle shaking at 37 °C for 30 min. Each sample was tested in triplicate. The enzymatic hydrolysis of 4-MU oleate by lipase was quantified using a fluorescence microplate reader, and the fluorescence intensity at excitation wavelength of 350 ± 10 nm and emission wavelength of 450 nm was measured.

[0163] The inhibition of PL activity was calculated as follows:

[0164]

[0165] To assess the effect of the manipulated variables on the inhibition rate of phospholipase (PL) caused by the combination of LLE (A), CME (B), PTE (C), and total concentration (D), a series of 27 experiments conforming to the RSM were systematically performed, as detailed in Table 2.

[0166] Table 2. Box-Behnken design matrix for optimizing the response values ​​generated by the variable and the PL suppression rate.

[0167]

[0168] Figure 3 The distribution of experimental and predicted values ​​for the suppression rate of the response variable PL is shown, indicating a good fit to the proposed RSM. The summary statistics of the model highlight the predicted and adjusted R² values, which are 0.9667 and 0.9874, respectively.

[0169] Detailed statistics are provided in Table 3.

[0170] Table 3. Summary statistics of the model.

[0171]

[0172] The analysis of variance (ANOVA) for the RSM model is presented in Table 4.

[0173] Table 4. ANOVA of PL inhibition rate.

[0174]

[0175] Analysis of variance (ANOVA) revealed the significance of the model and test variables. Comparison of F-values ​​showed a significance order of D > C > A > B. The effects of PTE (C) and total concentration (D) were superior to all other parameters in influencing the PL inhibition rate. All interaction terms were insignificant, with only the quadratic term of D (D²) being significant. Three-dimensional response surface plots were developed to identify the optimal levels of each factor maximizing the PL inhibition rate. Graphical representations of these response surfaces are illustrated in [the figure]. Figure 4 The quadratic equation representing the relationship between the model and the operated variables is:

[0176] Inhibition rate (%) = -1.7117A - 0.9942B + 3.0708C + 17.8250D - 8.6371D 2

[0177] Based on the model fitting results, the inhibition rate of PL reached its optimal level at a total concentration of 10 mg / mL, with an LLE, CME, and PTE ratio of 3:1:7. These findings were validated through empirical experiments, and the detailed data are shown in Table 5.

[0178] Table 5. Actual experimental results.

[0179]

[0180] When evaluating the three extracts individually and in combination, the optimal conditions identified by Box-Behnken design (response surface methodology) were a 3:1:7 ratio and a total concentration of 10 mg / mL. This specific combination of LLE, CME, and PTE exhibited a significant and unexpected lipid-lowering effect.

[0181] Specifically, at the same concentration, the combination of lotus leaf extract, chrysanthemum extract, and Pu-erh tea extract in a 3:1:7 ratio showed greater inhibition of pancreatic lipase than the individual extracts. This was because the concentrations of the mixture and the individual extracts were the same (10 mg / mL), resulting in the same total amount of substance.

[0182] Conclusion: The Box-Behnken design predicted the optimal ratio (3:1:7) of LLE, CME and PET combination, which showed greater inhibition of PL compared to the individual components of the composition.

Claims

1. A composition comprising a mixture of the following: Lotus leaf extract (LLE) Chrysanthemum extract (CME) and Pu-erh tea extract (PTE), The mixture described therein has a greater effect on inhibiting pancreatic lipase compared to the extracts alone.

2. The composition according to claim 1, wherein the LLE has a content of not less than 0.2% lotus leaf alkaloid, the CME has a content of more than 1% chlorogenic acid, and the PTE has a content of more than 7% tea polyphenols and a caffeine level of more than 10%.

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

7.

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

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

6. The composition according to any one of claims 1-5, wherein the composition is formulated as a ready-to-drink beverage, a concentrate, a dry composition (e.g., a powder, granules or tablet that can be reconstituted by a liquid), a gel, a solid, a semi-solid (e.g., ice cream, pudding or yogurt), a frozen liquid (e.g., popsicles), a tablet or hard candy, a dissolving strip and chewing gum.

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

8. The composition according to any one of claims 1-7, for supporting the weight loss efforts of a subject in need.

9. The composition according to any one of claims 1-7, used to lower the blood glucose level of a subject in need of doing so.

10. The composition according to any one of claims 1-7, for managing blood lipids in a subject with such need.

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 extract (CME), and Pu-erh tea extract (PTE). The LLE contains at least 0.2% lotus leaf alkaloid, the CME contains more than 1% chlorogenic acid, and the PTE contains more than 7% tea polyphenols and more than 10% caffeine.

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 according to 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 extract (CME), and Pu-erh tea extract (PTE). The ratio of LLE to CME to PET in the mixture is 3:1:7, and The total concentration of LLE, CME and PTE in the composition is 10 mg / mL.

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