Crystallization of siamenoside i

Abstract

Crystal forms, compositions, and methods related to Siamenoside I are provided. An example crystal form of Siamenoside I has an orthorhombic crystal system with a space group of P212121. The crystal form of Siamenoside I is further characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (2θ) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°. An example method of making a solid of Siamenoside I includes providing / obtaining a crude solution of Siamenoside I, causing crystallization of Siamenoside I from the crude solution to obtain crystals of Siamenoside I as a precipitate, and isolating the crystals of Siamenoside I from the crude solution. An example method of making a composition containing Siamenoside I includes providing / obtaining a solid of Siamenoside I having the crystal form according to the present disclosure, and dispersing or dissolving the solid of Siamenoside I in a medium.

Description

CRYSTALLIZATION OF SIAMENOSIDE ICROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 657,102, filed June 6, 2024, which is hereby incorporated by reference in its entirety for all purposes.BACKGROUND OF THE DISCLOSURE

[0002] Siamenoside I is a compound of the Mogroside family, initially isolated from Siraitia siamensis (Kasai, R. et al., Agric. Biol. Chem. 53, 3347-3349 (1989)) and later, from Siraitia grosvenorii (Luo han guo) (Matsumoto. K. et al, Chem. Pharm. Bull. 38. 2030-2032 (1990)).

[0003] Four major compounds, Mogroside V, Mogroside IV, Siamenoside I, and 11- Oxomogroside V, have been identified from the fruits of Siraitia grosvenorii that are responsible for the sweetness of the fruits. Mogroside V is the most abundant of these four compounds at approximately 0.57 wt% of the dry fruit, followed by Mogroside IV and Siamenoside I, each of which contains four glucose moieties. (See, e.g., Takemoto, et al., Yakugaku Zasshi, 103, 1151-1154; 1155-1166; 1167-1173, (1983); Kasai, et al., Agric. Biol. Chem. 53, 3347-3349 (1989); Matsumoto. Chem. Pharm. Bull. 38, 2030-2032 (1990); and Prakash, et al., J. Carbohydrate Chem. 30. 16-26 (2011)).BRIEF SUMMARY OF THE DISCLOSURE

[0004] One embodiment of the present disclosure is related to a crystal form of Siamenoside I. The crystal form of Siamenoside I has an orthorhombic crystal system with a space group of P2i2i2i. The crystal form of Siamenoside I may be characterized by the following unit cell parameters measured by X-ray diffraction at about 100 K: a = 11.500(2) A, b = 13.160(3) A, c = 42.730(9) A, a = P = y = 90°, a unit cell volume is 6467(2) A3. The crystal form of Siamenoside I may be characterized by the following unit cell parameters measured by X-ray diffraction at about room temperature (e.g., about 290 K to about 300 K): a = 11.60 A, b = 13.37 A, c = 43.08 A, a = = y = 90°, a unit cell volume is 6681 A~.

[0005] The crystal form may be characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0.2° and 14.39°±0.2°. In some embodiments, the crystal form is characterized by a powder X-ray diffraction (PXRD) pattern131517313V.1including diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°. The PXRD pattern is acquired using Cu-Ka radiation with a wavelength of 1.54056 A.

[0006] Another embodiment of the present disclosure is related to a composition containing the crystal form of Siamenoside I described herein or Siamenoside I derived from the crystal form derived herein. In some embodiments, the composition is a sweetener composition or a beverage composition.

[0007] Another embodiment of the present disclosure is related to a method of making a composition containing the crystal form of Siamenoside I described herein or Siamenoside I derived from the crystal form described herein. In one example, a method includes providing / obtaining a solid of Siamenoside I having a crystal form described herein, and dispersing or dissolving the solid of Siamenoside I in a medium. In some embodiments, the medium includes a solvent, the crystal form of Siamenoside I is dissolved in the solvent to form a solution, and the method further includes performing spray drying on the solution to obtain an amorphous form of Siamenoside I.

[0008] Another embodiment of the present disclosure is related to a method of making a solid of Siamenoside I or improving purity of Siamenoside I. In one example, a method includes providing / obtaining a crude solution of Siamenoside I containing impurities, causing crystallization of Siamenoside I from the crude solution to obtain crystals of Siamenoside I as a precipitate, and isolating the crystals of Siamenoside I from the crude solution. The crystals of Siamenoside I have a higher purity compared with the crude solution.

[0009] In some embodiments, causing crystallization of Siamenoside I further includes at least one of: adding a seed crystal of Siamenoside I to the crude solution, adjusting a concentration of Siamenoside I in the crude solution to a saturated concentration before causing the crystallization, adjusting a pH of the crude solution, adding a solvent to the crude solution, performing one or more heating-cooling cycles, and adding a salt to the crude solution.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings are included to provide a further understanding of the disclosure. The drawings illustrate embodiments of the disclosure and together with the description serve to explain the principles of the embodiments of the disclosure.

[0011] FIG. 1 illustrates a molecular structure of an example crystal form of Siamenoside I, according to various embodiments of the present disclosure. The molecular structure is represented by a monochrome Oak Ridge Thermal Ellipsoid Plot Program (ORTEP) plot with 30% probability of ellipsoids. Only non-hydrogen atoms are labeled. For clarity, water molecules and disorder in one of the sugar rings have not been show n.

[0012] FIG. 2A illustrates a pow der X-ray diffraction (PXRD) pattern of an example crystal form of Siamenoside I measured at Cu-Ka wavelength (1.5406 A) from 5-80° (20). according to various embodiments of the present disclosure.

[0013] FIG. 2B illustrates a pow der X-ray diffraction (PXRD) pattern of an example crystal form of Siamenoside I measured at Cu-Ka wavelength (1.5406 A) from 8-32° (20), according to various embodiments of the present disclosure.

[0014] FIG. 3 illustrates a microscopic image of an example cry stal form of Siamenoside I, according to various embodiments of the present disclosure.

[0015] FIG. 4 is a flow diagram illustrating an example method for making a composition containing Siamenoside I, according to various embodiments of the present disclosure.

[0016] FIG. 5 is a flow diagram illustrating an example method for crystallization of Siamenoside I, according to various embodiments of the present disclosure.DETAILED DESCRIPTION OF THE DISCLOSURE

[0017] Siamenoside I is the one of the sweetest among all Mogroside compounds. (See, e.g., Ukiya, et al., J. Agric. Food Chem. 50, 6710-6715, (2022)). Because Siamenoside I has a highly preferred taste quality among the Mogroside compounds, it is desirable to isolate Siamenoside I with high purity.

[0018] Mogroside compounds with multiple sugar units (e.g., Mogroside V) can be soluble in water up to 50 wt%. The high solubility and rapid dissolution rates of key sweet Mogroside compounds, including Mogroside IIIE, Mogroside V, and Siamenoside I, in water and solvent mixtures provide both advantages and disadvantages for their commercialization in the food industry. On one hand, these properties are advantageous for food and beverage applications, as they facilitate faster processing times and potentially low er equipment costs. However, the high solubility poses challenges for purification and commercialization, as itoften necessitates costly chromatography methods or other intensive processes to achieve high purity.

[0019] One insight provided in the present disclosure is related to crystallization as a cost- effective alternative for purifying these highly soluble Mogroside compounds. Unlike other high-solubility Mogroside compounds such as Mogroside V and Mogroside IIIE, Siamenoside I was surprisingly found to exhibit excellent crystallization behavior at certain concentrations and under certain conditions. The present disclosure identifies the possibility of utilizing crystallization as a more economical option for purifying Siamenoside I to exclude impurities such as undesired Mogroside compounds. The compositions and methods provided in the present disclosure can significantly reduce the overall production costs associated with manufacturing and commercializing products containing Siamenoside I.Definition and interpretation of selected terms

[0020] As used herein, “weight percent,’’ “wt%,” “percent by weight,” “% by weight,” and variations thereof refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent," “%,” and the like are intended to be synonymous with “weight percent,” “wt%,” etc.

[0021] As used herein, “g” represents gram, “L” represents liter, “mg” represents milligram (10‘3gram), and “mL” or “cc” represents milliliter. The unit of temperature used herein is degree Celsius (°C) or Kelvin (K).

[0022] The term “about” is used in conjunction with numeric values to include normal variations in measurements as expected in the art, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial composition. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[0023] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes having two or more compounds that are either the same or different from each other. It should also be noted that the term “or” is generally employed in its sense including “and / or”unless the content clearly dictates otherwise. As used herein, ‘‘and / or"’ refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("‘or”).

[0024] In the interest of brevity and conciseness, any ranges of values set forth in this specification contemplate all values within the range and are to be construed as support for claims reciting any sub-ranges having endpoints which are real number values within the specified range in question. By way of example, a disclosure in this specification of a range of from 1 to 5 shall be considered to support claims to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4-5.

[0025] The term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. The term “substantially free” may refer to any component that the composition of the disclosure lacks or mostly lacks. When referring to “substantially free” it is intended that the component is not intentionally added to compositions of the disclosure. Use of the term “substantially free” of a component allows for trace amounts of that component to be included in compositions of the disclosure because they are present in another component. However, it is recognized that only trace or de minimus amounts of a component will be allowed when the composition is said to be “substantially free” of that component. In one embodiment, if a composition is said to be “substantially free” of a component, if the component is present in trace or de minimus amounts, it is understood that it will not affect the effectiveness of the composition. It is understood that if an ingredient is not expressly included herein or its possible inclusion is not stated herein, the disclosure composition may be substantially free of that ingredient. Likewise, the express inclusion of an ingredient allows for its express exclusion to allow a composition to be substantially free of that expressly stated ingredient.

[0026] The term “comprise,” “comprises,” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0027] As used herein, the transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. Thus, the term “consisting essentially of’ when used in a claim of this disclosure is not intended to be interpreted to be equivalent to “comprising.”

[0028] As used herein, the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced.” “enhancing.” and “enhancement” (and grammatical variations thereof) describe an elevation of at least about 1%, 5%, 10%, 15%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.

[0029] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and grammatical variations thereof), describe, for example, a decrease of at least about 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control. In particular embodiments, the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5% or even 1%) detectable activity' or amount.

[0030] As used herein, the term “peak error” in X-ray diffraction (XR.D) refers to the deviation of the observed diffraction peak position from the expected or theoretical position and is expressed in terms of angle deviation (e.g., in degrees such as “±0.2°”). This discrepancy can occur due to various factors such as instrumental errors, sample preparation issues, cry stal imperfections, or testing environmental conditions.

[0031] The term “beverage” as used herein means any drinkable liquid or semi-liquid, including for example water, flavored water, soft drinks, fruit drinks, tea-based drinks, juicebased drinks, gel drinks, carbonated or non-carbonated drinks, and alcoholic or non-alcoholic drinks. In some embodiments, a beverage powder may first be mixed with any drinkable liquid or semi-liquid to obtain a beverage.

[0032] The term “Mogroside”, as used herein, refers to a triterpene-glycoside and is recognized in the art and is intended to include the major and minor constituents of Mogroside extracts. Mogroside compounds are found in the fruit of luo han guo. Mogroside compounds can also be obtained in other organisms, including genetically engineered microorganisms or plants.

[0033] The term “Siamenoside I” as used herein refers to a compound having the chemical structure shown in Formula I. It has the chemical formula C54H92O24 and a molecular weight of 1125.29. Siamenoside I can be prepared by suitable methods as described in U.S. Patent Application No. 20220205010, the disclosure of which is incorporated by reference herein in its entirety.Crystal form of Siamenoside I

[0034] The present disclosure provides a cry stal structure (i. e. , crystal form or crystalline form) of Siamenoside I according to the following chemical structure of Formula I:(Formula I)

[0035] In some embodiments, the crystal form of Siamenoside I is a single crystal (i.e., monocrystal). In some embodiments, the crystal form of Siamenoside l is a polymorph containing multiple crystal forms. In some embodiments, the crystal form Siamenoside I is a crystallization product of a solution containing Siamenoside I. A molecular structure of an example crystal form is shown in FIG. 1.

[0036] In some embodiments, the crystal form of Siamenoside I according to the present disclosure is characterized by an orthorhombic crystal system with a space group of P2i2i2i. In some embodiments, the crystal form of Siamenoside I is characterized by the following unit cell parameters measured by X-ray diffraction at about 100 K: a = 11.500(2) A, b = 13.160(3) A, c = 42.730(9) A, a = [3 = y = 90°, a unit cell volume is 6467(2) A3.

[0037] In some embodiments, the crystal form of Siamenoside I is characterized by the following unit cell parameters measured by X-ray diffraction at about room temperature (e.g.,about 290 K to about 300 K): a = 11.60 A, b = 13.37 A, c = 43.08 A, a = (3 = y = 90°, a unit cell volume is 6681 A3.

[0038] In some embodiments, the crystal form is characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0.2° and 14.39°±0.2°. In some embodiments, the crystal form is characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, and 15.83±0.2°. In some embodiments, the crystal form is characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°. In some embodiments, the diffraction peak error is in a range of ±0.2°, ±0.1°, ±0.05°, ±0.01°, or ±0.001°. For example, the crystal form may be characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0.05° and 14.39°±0.05°. or characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0. 1°, 14.39°±0.1°, and 15.83±0.1°, or characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°. The X-ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of about 1.5406 A.

[0039] In some embodiments, the crystal form is further characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, 13.01±0.2°, 15.94±0.2°, 18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.11±0.2°, 10.87±0.2°, 10.52±0.2°, 12.77±0.2°. 11.81±0.2°, 13.95±0.2°, and 19.63±0.2°. In some embodiments, the diffraction peak error is in a range of ±0.2°, ±0.1°, ±0.05°, ±0.01°, or ±0.001°. The X-ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of about 1.5406 A.

[0040] In some embodiments, the crystal fonn is further characterized by an X-ray powder diffraction pattern including diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, 13.01±0.2°, 15.94±0.2°, 18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.1 1±0.2°, 10.87±0.2°, 10.52±0.2°, 12.77±0.2°, 11.81±0.2°, 13.95±0.2°, 19.63±0.2°, 15.57±0.2°, 15.28±0.2°, 11.17±0.2°, 25.00±0.2°, 22.23±0.2°, 21.73±0.2°, 22.75±0.2°, 18.58±0.2°, 15.42±0.2°, 17.14±0.2°, 24.77±0.2°. 17.58±0.2°, 13.22±0.2°, 28.07±0.2°, 27.32±0.2°, 12.32±0.2°, 13.85±0.2°, 23.49±0.2°. 41.44±0.2°, 14.59±0.2°. 36.75±0.2°, 21.14±0.2°, 34.88±0.2°, 32.79±0.2°, 17.34±0.2°, and 10.28±0.2°. In some embodiments, the diffractionpeak error is in a range of ±0.2°, ±0.1°, ±0.05°, ±0.01°, or ±0.001°. The X-ray powder diffraction patern is measured on a diffractometer using Cu-Ka radiation with a wavelength of about 1.5406 A.

[0041] In some embodiments, the crystal form of Siamenoside I may also be further characterized by the parameters as shown in Table 1 with a peak error in a range of ±0.2°, ±0.1°, ±0.05°, ±0.01°, or ±0.001°, the molecular structure of the crystal form shown in FIG.1, the X-ray diffraction patern shown in FIG. 2 A. and / or the X-ray diffraction patern shown in FIG. 2B.Table 1. A powder X-ray diffraction patern of an example crystal form of Siamenoside I according to the present disclosure.*”20” represents the diffraction angle, measured in degrees (°) , at which the diffraction peak occurs; “d” represents the d-spacing or the spacing between adjacent crystallographic planes; “Height” represents the height of the diffraction peak, measured in counts per second (cps) and indicating the intensity or amplitude of the diffraction signal; “FWHM” is the full width at half maximum (FWHM) representing the angular width of the diffraction peak at half of its maximum height; “Int. I.” is the integrated intensity, measured in counts per second perdegree (cps / °) and representing the total intensity of the diffraction peak over a specified angular range; “Int. W.” is the integrated width, measured in degrees (°) and representing the angular range over which the integrated intensity is calculated; '‘Norm. I.” is the normalized intensity, representing the intensity of the diffraction peak normalized to a background level.

[0042] In some embodiments, the crystal forms of Siamenoside I described herein can be further characterized by its appearance. As shown in FIG. 3, the crystals of Siamenoside I exhibit a rod-like morphology, characterized by elongated structures with small sizes in two dimensions. Despite their small sizes in two dimensions, the crystals are of high quality with sharp edges and excellent clarity. The quality and clarity indicate that the crystals of Siamenoside I may possess uniformity, well-defined crystal faces and edges, distinct crystallographic planes within the crystal lattice, and minimal defects or impurities.

[0043] In some embodiments, the crystal form of Siamenoside I is a solvate or a hydrate. A “solvate” refers to a crystalline structure that contains solvent molecules as an integral part of the crystalline structure. When the solvent is water, the solvate is specifically referred to as a “hydrate.” In some embodiments, the crystal form described herein includes both a nonsolvent (anhydrous) form and a solvate form. In some embodiments, the crystal form is partially solvated or partially hydrated.Crystallization products containing Siamenoside I

[0044] The present disclosure provides crystallization products of Siamenoside I. In some embodiments, a solid product is obtained from a crystallization process of a crude solution of Siamenoside I. The crystallization process may be used to purify a solution containing a crude source of Siamenoside I. The crude source of Siamenoside I may include Siamenoside I and one or more Mogroside compounds other than Siamenoside I, such as Mogroside IIIE, Mogroside IV, and / or Mogroside V, among others. The crude source of Siamenoside I may have a purity of Siamenoside I of 95% or less. 94% or less, 93% or less, 92% or less. 91% or less, 90% or less, 89% or less, 88% or less, 87% or less, 86% or less, 85% or less, 80% or less, 75% or less, 70% or less, or 65% or less. The purity may be measured by the weight percentage of Siamenoside I in the crude source, or from other measurement techniques such as the UV spectroscopy. The crude source of Siamenoside I may have a total content of Mogroside compounds of 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more. 13% or more, 14% or more. 15% or more, 20% or more, 25% or more, 30% or more, or 35% or more.

[0045] In some embodiments, the crude source of Siamenoside I may be a solid, which is dissolved in a medium to form the crude solution. The solid product obtained from crystallization of the crude solution includes crystals of Siamenoside I, and the crystals of Siamenoside I have the crystal form described herein. In some embodiments, the solid product consists of or consists essentially of Siamenoside I, without substantial presence of other Mogroside compounds (e.g.. Mogroside IIIE, Mogroside IV, Mogroside V. etc ). In some embodiments, the solid product has a purity of Siamenoside I of at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9%. In some embodiments, the solid product has a purity of Siamenoside I higher than the purity of Siamenoside I of the crude source. In some embodiments, a difference between the purity of Siamenoside I in the solid product and the purity of Siamenoside I in the crude source is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%. In some embodiments, a difference between the content of other Mogroside compounds in the crude source and the content of other Mogroside compounds in the solid product is at least 1%, at least 2%. at least 3%. at least 4%, at least 5%, at least 6%. at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, or at least 20%.

[0046] In some embodiments, the solid product of crystallization of the crude solution includes both Siamenoside I and one or more of other Mogroside compounds with a weight ratio of the Siamenoside I to the Mogroside compounds of at least 10: 1, at least 20: 1, at least 30: 1. at least 40: 1. or at least 50: 1. In some embodiments, the solid product includes a cocrystal of Siamenoside I and one or more Mogroside compounds other than Siamenoside I. The solid product may be used for making a composition such as a sweetener composition or a beverage composition as described below. In some embodiments, the solid product may undergo further purification (e.g., one or more subsequent recrystallization processes) to improve the purity of Siamenoside I as needed.

[0047] In some embodiments, the solid product includes crystals of Siamenoside I, the cry stals may include the crystal form of Siamenoside I described herein, the hydrate crystal form of Siamenoside I, the solvate crystal form of Siamenoside I, a co-crystal of Siamenoside I and another molecule such as a Mogroside compound other than Siamenoside I. The solid product may further include a crystal of a minor component such as a Mogroside compound other than Siamenoside I.Compositions including the crystal form of Siamenoside I

[0048] The present disclosure provides compositions including the crystal form of Siamenoside I described herein or other forms of Siamenoside I derived from the crystal form described herein. The compositions may be a solid composition, a semi-solid composition, a semi-liquid composition, a liquid composition, a solution, a syrup, or any other matter state. Solid compositions may include powders, tablets, capsules, or any other solid dosage forms containing the solid form Siamenoside I. Semi-solid compositions could include creams, ointments, gels, or pastes incorporating the solid form of Siamenoside I. Semi-liquid compositions might involve suspensions or thick liquids with a dispersed solid form of Siamenoside I. Liquid compositions could encompass solutions, suspensions, or emulsions containing a dissolved or dispersed solid form of Siamenoside I. Syrups could be formulated wi th the solid form Siamenoside I dissolved in a liquid medium.

[0049] In some embodiments, the composition is an amorphous form of Siamenoside I derived from the crystal form of Siamenoside I or the crystallization product of Siamenoside I according to the present disclosure. For example, the amorphous form of Siamenoside I can be obtained through a process involving the dissolution of the crystal form of Siamenoside I in a medium to create a high concentration solution or liquid. Subsequently, this solution or liquid is subjected to spray dry ing to produce an amorphous solid form. The spray drying can be performed by using a spray dryer, where the solution is atomized into fine droplets. The droplets are exposed to hot air or inert gas, causing rapid evaporation of the solvent or medium. As the solvent evaporates, the Siamenoside I molecules undergo a transition from a dissolved state to a solid state without sufficient time for crystallization, resulting in the formation of an amorphous solid. The amorphous form of Siamenoside I may be advantageous in manufacturing applications within the food industry.

[0050] In some embodiments, the crystal form of Siamenoside I described herein is used as an ingredient for making a sweetener composition or a beverage composition. For example, the sweetener composition may include the crystal form of Siamenoside I or an amorphous form of Siamenoside I derived from the crystal form, as well as additional ingredients such as additional sweeteners and additives. In some embodiments, the sweetener composition is a non-carbohydrate sweetener.

[0051] The beverage composition (i.e., beverage or beverage product) according to the present disclosure is a ready-to-drink beverage, a beverage concentrate, a beverage syrup, ora powdered beverage. Suitable ready -to-drink beverages include carbonated and noncarbonated beverages. Carbonated beverages include, but are not limited to, frozen carbonated beverages, enhanced sparkling beverages, cola, fruit-flavored sparkling beverages (e.g,. lemon-lime, orange, grape, strawberry and pineapple), ginger ale, soft drinks and root beer. Non-carbonated beverages include, but are not limited to, fruit juice, fruit-flavored juice, juice drinks, nectars, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, enhanced water drinks, enhanced water with vitamins, near water drinks (e.g., water with natural or synthetic flavorants), coconut water, tea type drinks (e.g., black tea, green tea, red tea, oolong tea), coffee, cocoa drink, beverages containing milk components (e.g., milk beverages, coffee containing milk components, cafe au lait, milk tea, fruit milk beverages), alcoholic beverages, carbonated or non-carbonated beverages, beverages containing cereal extracts and smoothies.

[0052] The beverage composition described herein can optionally include additives, functional ingredients and combinations thereof, as described herein. The beverage or beverage product may further comprise one or more other additives. In some embodiments, the beverage or beverage product contains additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, weighing agents, gums, antioxidants, colorants, flavonoids, alcohols, polymers and combinations thereof.

[0053] More examples of the sweetener composition and beverage composition can be found in U.S. Patent No. 11,653,680, the disclosure of which is incorporated by reference herein in its entirety.Method for preparing compositions containing Siamenoside I

[0054] The present disclosure provides methods for making compositions containing Siamenoside I. FIG. 4 is a flow diagram illustrating an example method 400 for making a composition containing Siamenoside I. The method 400 includes process blocks 402-412. Although the illustrated process blocks are in a sequential process, many of the process blocks can be performed in parallel or concurrently. In addition, the order of the process blocks may be rearranged in a suitable manner.

[0055] At 402, a source of Siamenoside I is obtained or provided. In some embodiments, the source of Siamenoside I is a crude solution of Siamenoside I. The crude solution includes dissolved Siamenoside I and one or more impurities. The impurity may be other Mogroside compounds that are undesired.

[0056] At 404, a crystallization process is performed to cause the dissolved Siamenoside I in the source to crystallize and generate a solid form of Siamenoside I. Examples of the crystallization process are described below with reference to FIG. 5. The solid form of Siamenoside I may precipitate from the crude solution.

[0057] At 406, the solid form of Siamenoside I is isolated from the source. The solid form of Siamenoside I may be further purified by washing with a medium followed by drying to yield a solid product of Siamenoside I. The solid form and solid product of Siamenoside I contain the crystal form of Siamenoside I described herein. The solid form of Siamenoside I is free or substantially free from the impurities of the source.

[0058] At 408, the solid form or solid product of Siamenoside I is redispersed or redissolved in a medium to form a solution. The medium may be a solid medium or a liquid medium or both. The solid medium may be a carrier, a stabilizer, or a texture modifier for incorporating active ingredients like the solid form of the Siamenoside I. The liquid medium may be water, an aqueous buffer, or a solvent system including two or more co-solvents. Examples of the solid medium include but are not limited to gelatin, pectin, Arabic gum, maltodextrin, starches or modified starches, cellulose or derivatives thereof, among others. Examples of the liquid medium include but are not limited to carbonate-based buffer, phosphate-buffered saline (PBS), citrate-phosphate buffer, tris-buffered saline (TBS), ethanol, propylene glycol, glycerol, among others.

[0059] At 410, one or more ingredients or additives are added to the solution. The additional ingredient or additive can be an additional ingredient or additive as described herein.

[0060] At 412. a composition of Siamenoside I is obtained. The composition may be further formulated for intended purposes, such as a sweetener composition, or a beverage composition, among others.

[0061] In some embodiments, the solid form of Siamenoside I from the crystallization process is further purified by one or more recrystallization cycles. An example of therecrystallization cycle is described below. The solid Siamenoside I obtained from the initial crystallization process is dissolved in a suitable solvent at an elevated temperature to form a solution. The solution may be filtered to remove insoluble impurities or any remaining solid particles that have not dissolved completely. The filtrate is allowed to cool to room temperature or below to cause the dissolved Siamenoside I to crystallize out of the solution. The resulting crystals are separated from the mother liquor through decanting, filtration, or centrifugation. The crystals may be washed with a small amount of cold solvent to remove any residual impurities adhering to the surface. The purified Siamenoside I crystals can be dried under vacuum or at ambient conditions to remove any traces of solvent.

[0062] In some embodiments, the solid form of Siamenoside I from the crystallization process is further treated to produce an amorphous form of Siamenoside I. An example process for producing the amorphous form of Siamenoside I is described below. Initially, the solid forms of Siamenoside I obtained from the initial crystallization process or the subsequent recry stallization processes are collected. The crystal forms are enriched with Siamenoside I and contain a higher purity of Siamenoside I compared to the original source (e.g., crude solution). The Siamenoside I crystals are re-dissolved in a suitable solvent or solvent mixture to form a high concentration solution or liquid. Water or a mixture of water and a co-solvent such as ethanol may be used as the solvent system. The high concentration solution or liquid containing Siamenoside I is subjected to spray dry ing. The solution / liquid is atomized into fine droplets using a spray nozzle, and then the droplets are exposed to hot air or inert gas in a drying chamber. As the droplets travel through the drying chamber, the solvent evaporates rapidly, where the solid particles of Siamenoside I remain an amorphous form. The amorphous particles of Siamenoside I are collected and may be further processed, if desired, to obtain the desired particle size or consistency.Method for crystallization of Siamenoside I

[0063] The present disclosure provides methods for crystallization of Siamenoside 1 from a crude source and improving purity of Siamenoside I. FIG. 5 is a flow diagram illustrating an example method 500 for cry stallization of Siamenoside I. The method 500 includes process blocks 502-518. Although the illustrated process blocks are in a sequential process, many of the process blocks can be performed in parallel or concurrently. In addition, the order of the process blocks may be rearranged in a suitable manner.

[0064] At 502, a crude solution of Siamenoside I is provided. The crude solution may be a solution of a crude source of Siamenoside I. In some embodiments, the crude solution includes Siamenoside I as well as one or more impurities. In some embodiments, the impurities may include an undesired Mogroside compound such as Mogroside IIIE, Mogroside IV, Mogroside V, or any combinations thereof.

[0065] At 504, the concentration of Siamenoside I is adjusted to a saturated level. For example, the crude solution can be concentrated to remove a portion of the solvent to achieve a concentration of Siamenoside I in a range from about 2 wt% to about 15 wt% at room temperature or an elevated temperature. As the solubility of Siamenoside I may increase at an elevated temperature, a saturated level of Siamenoside I may be achieved at the specific temperature.

[0066] At 506. the pH of the crude solution is adjusted. The pH may be in an acidic range (e.g., from 2 to below 6), a neutral range (e.g., from 6 to 8), or a basic range (e.g., from above 8 to 11). In some embodiments, the pH of the crude solution is adjusted to a range from 2 to 11, from 3 to 10, from 4 to 9, from 5 to 8, or from 6 to 7. In some embodiments, the pH of the crude solution is adjusted to a range from 7 to 11, from 8 to 10, or from 9 to 10. Various buffers may be used to control the pH. including but not limited to ammonia buffer, citric buffer, phosphate buffer, acetate buffer, tartrate buffer, lactate buffer, malate buffer, among others.

[0067] At 508, the temperature of the crude solution is adjusted. In some embodiments, one or more heating-cooling cycles are performed. Heating-cooling cycles involve alternating between heating the crude solution to elevate the temperature and cooling it to lower temperatures. The crude solution can be heated to a temperature from about 40 °C to about 60 °C for a time duration from about 5 minutes to about 1 hour. Heating the crude solution can increase the solubility of Siamenoside I to reach a saturated level before cry stallization. Cooling the solution after heating can induce the crystallization of Siamenoside I. The cooling rate can be controlled from about 0.01 °C / min to about 1 °C / min, or from about 0. 1 °C / min to about 1 °C / min. The crude solution can be cooled to room temperature (e.g., from about 18 °C to about 25 °C, or to a lower temperature of from about 4 °C to about 8 °C. The crude solution can be agitated after cooling for a duration of time (e.g., from 1 to 5 days).

[0068] In some embodiments, no heating-cooling cycle is performed. The crude solution is agitated at room temperature to allow the slow formation of crystals of Siamenoside I from the crude solution.

[0069] At 510, one or more solvents are added to the crude solution to facilitate crystallization. The solvent may be an organic solvent such as alcohol or other solvent commonly used in the food industry, such as ethanol, glycol, propylene glycol, and / or 1,3- propane diol, among others. In some embodiments, the solvent added to the crude solution is from about 5 wt% to about 50 wt%, from about 10 wt% to about 40 wt%, from about 15 wt% to about 30 wt%, or from about 20 wt% to about 25 wt%, relative to the total weight of the crude solution.

[0070] At 512, one or more salts can be added to the crude solution to facilitate crystallization. Examples of the salt include but are not limited to NaCl, KC1, NaHCOs, sodium citrate, K3PO4, CaCb, and / or MgCk, among others. The addition of salts can influence the solubility and crystallization behavior of Siamenoside I by modifying the ionic strength and / or pH of the crude solution.

[0071] At 514, a seed of Siamenoside I can be added to the crude solution to facilitate crystallization. The seed of Siamenoside I may be a trace amount of crystals of Siamenoside I used to induce crystallization of Siamenoside I from the crude solution.

[0072] At 516, the crystals of Siamenoside I are isolated from the crude solution as precipitates by using a suitable separation technique such as decanting, filtration, centrifugation, among others. In some embodiments, the crystals of Siamenoside I may be further purified by washing with a cold solvent followed by drying in vacuum or under ambient conditions at an elevated temperature or room temperature. As used herein, an elevated temperature refers to a temperature above room temperature (e.g., above about 25 °C).

[0073] At 518, one or more recry stallization processes may be performed to further improve the purity of the crystals of Siamenoside I obtained from the initial crystallization. Examples of the recrystallization processes are provided above with reference to FIG. 4.

[0074] The following non-limiting examples illustrate further embodiments of the present disclosure.EXAMPLESExample 1 - Crystal structure determination

[0075] A sample of Siamenoside I crystals was obtained from a 10 wt% solution of Siamenoside I in water-ethanol with a weight ratio of 80 / 20 at a pH of about 9.5 adjusted with dilute ammonia. A seed of Siamenoside I was added to the solution to facilitate crystallization. The crystals of Siamenoside I were isolated by decanting, washing with a small amount of water, and air-drying at room temperature. The morphology of the crystals is shown in FIG. 3.

[0076] The cry stal structure of the sample was measured on a synchrotron at a liquid nitrogen temperature (e.g., 100 K), and a single cry stal X-ray crystallography dataset of the sample was collected. The single crystal X-ray crystallography of the sample showed the unit cell parameters: a = 11.500(2) A, b = 13. 160(3) A, c = 42.730(9) A, a = 0 = y = 90°, a unit cell volume is 6467(2) A3.

[0077] The cry stal structure of the sample was further measured on an X-ray7diffractometer using Cu-Ka radiation with an X-ray wavelength of about 1.5406 A at ambient temperature (e.g., about 293 K). The acquired PXRD dataset showed the crystal structure of the sample with the following unit cell parameters: a = 11.60 A, b = 13.37 A, c = 43.08 A. a = 0 = y = 90°, a unit cell volume is 6681 A3. The PXRD measurement conditions include the following: the diffractor is HyPix-3000 (horizontal), the light source is CuK, the X-ray intensity is 40 KY / 30 mA, the scanning mode is ID (scan), the scanning angle range is 5°- 80°, the step width is 0.01°, and the scanning speed is 3.00°.

[0078] The unit cell parameters obtained from the single crystal X-ray crystallography and the PXRD dataset are shown in good consistency with each other, which supports the same crystal form. The slight difference may be attributed to the temperature difference - the single crystal X-ray crystallography dataset was collected at a lower temperature (100 K), which could lead to a contraction of the crystal lattice of Siamenoside I.

[0079] The PXRD diffraction pattern of the sample is shown in FIG. 2A and FIG. 2B, and the corresponding diffraction peak list is shown in Table 1. The sample is characterized by7one or more diffraction peaks with most intense signals at angles (20) of 16.48°±0.10°, 14.39°±0.10°, 15.83±0.10°, 13.01±0.10°, 15.94±0.10°, 18.43±0.10°, 21.40±0.10°, 12.22±0.10°. 18.11±0.10°. 10.87±0.10°. 10.52±0.10°. 12.77±0.10°. 11.81±0.10°.13.95±0.10°, 19.63±0.10°, 15.57±0.10°, 15.28±0.10°, 1 1.17±0.10°, 25.00±0.10°,22.23+0.10°, 21.73+0.10°, 22.75+0.10°, 18.58+0.10°, 15.42+0.10°, 17.14+0.10°. 24.77+0.10°. 17.58+0.10°. 13.22+0.10°. 28.07+0.10°. 27.32+0.10°. 12.32+0.10°. 13.85+0.10°, 23.49+0. 10°, 41.44+0. 10°, 14.59±0. 10°, 36.75+0. 10°, 21. 14+0. 10°, 34.88+0.10°, 32.79+0.10°, 17.34+0.10°, and 10.28+0.10°.Example 2 - Solubility Comparison of Siamenoside I, Mogroside V, and Mogroside HIE

[0080] Three solutions of Siamenoside I, Mogroside V, and Mogroside IIIE (Solution 1, Solution 2, and Solution 3. respectively) were prepared in three vials, and their solubilities were compared. The initial solutions respectively contained 10 wt% of Siamenoside I, 10 wt% Mogroside V, and 10 wt% of Mogroside IIIE. The vials were capped and allowed to undergo agitation at room temperature. Over time, an additional amount of substrate was added to each corresponding vial until a total concentration of the substrate reached 20 wt%. if no precipitate was observed.

[0081] Table 2 shows the observation of the three solutions. Solutions 2 and 3 respectively containing Mogroside IIIE or Mogroside V were found to have significantly better stability over time. In comparison, Solution 1 containing Siamenoside I had poor stability with heavyprecipitation after 3 days. These comparative results support that the water solubility of Siamenoside I is significantly less compared with Mogroside IIIE or Mogroside V. Accordingly, Siamenoside I is more inclined to undergo crystallization from solution. It is surprising that while Siamenoside I, Mogroside IIIE, and Mogroside V share structural similarities, their solubility and crystallization behavior are significantly different. Such differences in solubility and crystallization behavior may be attributed to the differences in their arrangement of functional groups or stereochemistry, crystalline nucleation and growth kinetics, interactions with the solvent molecules, among other properties.Table 2. Solutions of Siamenoside I, Mogroside V, and Mogroside IIIE and observations according to Example 2.

[0082] It should be noted that embodiments of the present disclosure identify a cost- effective method for purifying Siamenoside I by leveraging its low solubility and high ability to cry stallize. As mentioned above, crude products of Mogroside often include Siamenoside I as well as Mogroside compounds other than Siamenoside I. The crude products often have inferior taste quality than certain Mogroside compounds such as Siamenoside 1 in high purity. While most Mogroside compounds in high purity are obtained by chromatography due to their high solubility in water, chromatography-based purification methods can be costly, especially in the food industry where cost-effectiveness is a significant consideration. The embodiments of the present disclosure provide and show the low solubility of Siamenoside I, identity' its potential for crystallization to improve purity of Siamenoside I products, improve taste quality of the compositions containing Siamenoside I, as well as address the challenges associated with traditional purification methods.Example 3 - Purification of Siamenoside I by crystallization

[0083] Various crystallization conditions were tested on crude solutions of Siamenoside I. Three solutions of Siamenoside I, Solution 4, Solution 5, and Solution 6, were respectively prepared by dissolving 1.25 g of Siamenoside I with a purity of about 91% in 22 mL of water, with pH adjustment by addition of a base in a flask. Three different bases (NaOH, Na2COs, and NH4OH) were used respectively for the three solutions. Seed crystals of Siamenoside I (about 18 mg) were added to each solution to facilitate crystallization. Agitation was carried out at room temperature for about 24 hours with a rotary shaker (slow swirling). Spin-filtration was performed on the solution to obtain a mother liquid for analysis. Solids were collected using a filter paper (Whatman 541) to obtain a retentate (filter pad) and a filtrate (mother liquid). The flask was rinsed onto the filter pads with cold dilute ammonia (pH 9, 4.5 rnL), and the filter pad was rinsed again with cold dilute ammonia (pH 9, 4.5 rnL). Final drying of the solids was carried out under high vacuum. The purity of the isolated solids of Siamenoside I from each solution was measured by UV absorption, based on the area% of the UV absorption at 215 nm.

[0084] Table 3 shows the results of the crystallization of Siamenoside I from the three solutions. Crystallization of Siamenoside I was observed for all three solutions with a yield of over 50%. The purity of the isolated solids from all three solutions was found to be greater than 96% with a significant improvement compared with the starting purity. In addition, the purity of the filtrate (mother liquid) was also found to be significantly less than the purity of the starting Siamenoside I due to the exclusion of most minor impurities from the crystallization of Siamenoside I. All three bases were found to be effective in facilitating the crystallization without significant difference.Table 3. Results of the crystallization of Siamenoside I from the Solutions 4-6 according to Example 3.Example 4 - The purification of Siamenoside I in the presence of Mogroside V and Mogroside HIE by crystallization

[0085] Other components such as Mogroside compounds other than Siamenoside I (e.g., Mogroside V and Mogroside IIIE), are likely to be present as minor components and / or impurities in the preparation of Siamenoside I. Example solutions were prepared to mimic the crude solution of Siamenoside I with Mogroside V and Mogroside IIIE as minor components. Two solutions. Solution 7 and Solution 8 were prepared by mixing 1. 125 g of Siamenoside I (purity 97.8%), either 0.125 g of Mogroside V or 0. 130 g of Mogroside IIIE, and 22.5 mL of water, with pH adjustment by the addition of 1 wt% NH4OH. Cry stallization of Siamenoside I from the solutions was facilitated by adding about 18 mg of seed cry stals of Siamenoside I to the solution. Agitation was carried out at room temperature for about 24 hours with a rotary shaker (slow swirling). Spin-filtration was performed on the solution to obtain a mother liquid for analysis. Solids were collected using a filter paper (Whatman 541) to obtain a retentate (filter pad) and a filtrate (mother liquid). The flask was rinsed onto the filter pads with cold dilute ammonia (pH 9. 4.5 mL), and the filter pad w as rinsed again with cold dilute ammonia (pH 9. 4.5 mL). Final drying of the solids was carried out under high vacuum. The purity of the crude Siamenoside I and the isolated solids of Siamenoside I from each solution w as measured by UV absorption, based on the area% of the UV absorption at 215 nm.

[0086] Table 4 shows the results of cry stallization of Siamenoside I from Solution 7 and Solution 8 in the presence of other Mogroside compounds as minor components. Solution 4 initially contained about 10 wt% of Siamenoside I and about 10 wt% of Mogroside V relative to the total w eight of Siamenoside I and Mogroside V as minor component. After cry stallization, the isolated solids (Sample 4) from Solution 7 contained Siamenoside I with higher purity (e.g., 97.6 %) with only 1.5 % of Mogroside V. Most of the Mogroside V remained in the filtrate of Solution 4 (Sample 2), with a purity of Siamenoside I significantly less than the purity of the isolated solids (Sample 4). These results support the effectiveness of crystallization to obtain high purity of Siamenoside I solids and exclude Mogroside V as at least one minor component.Table 4. Results of crystallization of Siamenoside I from Solutions 7-8 according to Example 4.

[0087] Similarly, Solution 8 initially contained about 10 wt% of Siamenoside I and about 10 wt% of Mogroside IIIE relative to the total weight of Siamenoside I and Mogroside IIIE as a minor component. After crystallization, the isolated solids (Sample 5) from Solution 8 contained Siamenoside I with high purity (e.g., 92.6 %) and only 6.48 % of Mogroside IIIE. Most of the Mogroside IIIE impurity remained in the filtrate of Solution 8 (Sample 3), with a purity of Siamenoside I significantly less than the purity of the isolated solids (Sample 5). These results support the effectiveness of crystallization to obtain high purity of Siamenoside I solids and exclude Mogroside IIIE as at least one impurity'.

[0088] Crystallization of Siamenoside I from the crude solution can yield solid products with higher purity by effectively reducing the content of impurities or minor components initially present in the crude solution. During crystallization, minor components such as other Mogroside compounds (e.g.. Mogroside IIIE, Mogroside V) that are structurally similar to Siamenoside I can be effectively separated. In some embodiments, the impurity may include salts, buffers, non-Mogroside sweeteners, contaminants, and other undesired compounds and molecules initially present in the crude solution. These impurities can also be reduced or minimized during crystallization.Example 5 - Crystallization of Siamenoside I in NaCl solutions

[0089] Solutions 9-12 were prepared by mixing Siamenoside I. NaCl, and water, and the pH of each solution was adjusted by the addition of 1 wt% NH4OH, in a similar manner as Example 4. Solution 9 contained 22 wt% of NaCl and 3.7 wt% of Siamenoside I, based on the total weight of the solution. Solution 10 contained 13 wt% of NaCl and 4.2 wt% of Siamenoside I, based on the total weight of the solution. Solution 11 contained 23 wt% of NaCl and 1.5 wt% of Siamenoside I, based on the total weight of the solution. Solution 12 contained 13 wt% of NaCl and 1.7 wt% of Siamenoside I, based on the total weight of the solution.

[0090] Cry stallization of Siamenoside I from the solutions was facilitated by adding seed crystals of Siamenoside I to the solutions. Agitation was carried out at room temperature for about 24 hours with a rotary shaker (slow swirling). Spin-filtration was performed on the solution to obtain a mother liquid for analysis. Solids were collected using a filter paper (Whatman 541) to obtain a retentate (filter pad) and a filtrate (mother liquid). The flask was rinsed onto the filter pads with cold dilute ammonia (pH 9, 4.5 mL), and the filter-pad was rinsed again with cold dilute ammonia (pH 9, 4.5 mL). Final drying of the solids was carried out under high vacuum. The purity of the crude Siamenoside I and the isolated solids of Siamenoside I from each solution was measured by UV absorption, based on the area% of the UV absorption at 215 nm.

[0091] Table 5 shows the results of cry stallization of Siamenoside I from Solutions 9-12 in the presence of salts at various concentrations. Compared with Solutions 1-3 of Example 3, crystallization of Siamenoside I from Solutions 9-12 containing NaCl could significantly improve the yield (e.g., over 77%) as well as the purity of the isolated solids (e.g., over 98%), under diluted conditions (e.g., less than 5 wt% Siamenoside I in the crude solution). These results support the effectiveness of salt to facilitate the crystallization of Siamenoside I.Table 5. Results of crystallization of Siamenoside I from Solutions 9-12 according to Example 5.Example 6 - Solvent systems to improve the solubility of Siamenoside I

[0092] Various solvent systems were evaluated aiming to improve the solubility of Siamenoside I. Examples of solutions containing Siamenoside I and solvents were prepared according to Table 6, and the stability of solutions was observed over time.Table 6. Stability of solutions containing Siamenoside I and various solvents according to Example 6.*Highly viscous. The sample was heated at 40 °C for 10 minutes to form a clear solution.

[0093] As shown in Table 6. Samples 1-1 to 1-8 have a concentration of Siamenoside I of 5 wt%. Samples 2-1 to 2-18 have a concentration of Siamenoside I of 10 wt%, and Samples 3-1 to 3-3 have a concentration of Siamenoside I of 20 wt%.

[0094] Among the solvents tested, ethanol was found to demonstrate the best performance in solubilizing Siamenoside I and delaying cry stallization or recrystallization, with clear solutions maintained for extended periods even at higher concentrations (e.g., 20 wt%). The water-ethanol solvent system can stabilize Siamenoside I in solution at a broad ratio of water / ethanol. The delay in crystallization observed with ethanol-based solutions supports its effectiveness in stabilizing Siamenoside I and preventing the formation of precipitates.

[0095] Compared with ethanol, glycerol demonstrated the ability to delay crystallization of Siamenoside I, as indicated by the clear solution observed initially. However, the resulting solution became highly hazy or precipitated within one day, which suggests the limited effectiveness in maintaining stability over time. Propylene glycol could form clear solutions that were stable for extended periods, especially at higher content (e.g., 3: 1 ratio with buffer). However, at lower contents (e.g., 1:3 ratio with buffer), propylene glycol resulted in hazy or heavily precipitated solutions within one day. Similar to propylene glycol, 1,3-propanediol exhibited clear solutions that remained stable over time, particularly at higher contents (e.g.. 1 : 1 ratio with buffer). However, solutions with lower contents of 1,3-propanediol (e.g., 1 :3 ratio with buffer) resulted in hazy or heavily precipitated solutions within one day. In addition, solutions containing propylene glycol or 1,3-propanediol exhibited high viscosity at higher concentrations of Siamenoside I, which may limit the application of these solutions.

[0096] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of the foregoing illustrative embodiments, it will be apparent that variations, changes, modifications, and alterations may be applied to the composition, methods, and in the steps or in the sequence of steps of the methods described herein, without departing from the embodiments of the disclosure. In one embodiment, it will be apparent that certain agents, additives, and ingredients that are similar according to their physical, chemical, physiological, and / or gustative properties may be substituted for the agents, additives and ingredients described herein while the same or similar results would be achieved.

[0097] The following numbered clauses define further embodiments of the present disclosure:NUMBERED CLAUSES1. A cry stal form of Siamenoside I, the crystal form having an orthorhombic crystal system with a space group of P2I2I2L2. The crystal form of Siamenoside T according to clause 1 , wherein the crystal form has unit cell parameters: a = 11.50 A, b = 13. 16 A, c = 42.73 A, a = P = y = 90°, a unit cell volume is 6467 A3, wherein the unit cell parameters are obtained from single crystal diffraction at about 100 K.3. The cry stal form of Siamenoside I according to any one of the clauses 1-2, wherein the cry stal form has unit cell parameters: a = 11.60 A, b = 13.37 A, c = 43.08 A. a = = y = 90°, a unit cell volume is 6681 A3, wherein the unit cell parameters are obtained from powder X-ray diffraction (PXRD) at room temperature.4. The crystal form of Siamenoside I according to any one of the clauses 1-3, wherein the cry stal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°, wherein the X-ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of 1.5406 A.5. The crystal form of Siamenoside I according to clause 4, wherein the X-ray powder diffraction pattern further comprises diffraction peaks at angles (20) of 15.94±0.2°, 18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.11±0.2°, 10.87±0.2°, 10.52±0.2°, 12.77±0.2°, 11.81±0.2°, 13.95±0.2°, and 19.63±0.2°.6. The crystal form of Siamenoside I according to any one of the clauses 1-5, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in FIG. 2A.7. The crystal form of Siamenoside I according to any one of the clauses 1-5, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in FIG. 2B.8. The crystal form of Siamenoside I according to any one of the clauses 1-5, wherein the cry stal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in Table 1.9. The crystal form of Siamenoside I according to any one of the clauses 1-8, wherein the cry stal form is a partially solvated form.10. The cry stal form of Siamenoside I according to any one of the clauses 1-8, wherein the cry stal form is a partially hydrated form.11. The crystal form of Siamenoside I according to any one of the clauses 1-8, wherein the crystal form is a solvated form.12. The crystal form of Siamenoside I according to any one of the clauses 1-8, wherein the cry stal form is a hydrated form.13. A solid composition comprising the cry stal form of Siamenoside I according to any one of the clauses 1-12.14. The solid composition according to clause 13, wherein the solid composition is a sweetener composition or flavor composition.15. The solid composition according to any one of the clauses 1-14, wherein the solid composition comprises an amorphous form of Siamenoside I reconstructed from the crystal form of Siamenoside I.16. A composition comprising a medium and the cry stal form of Siamenoside I according to any one of the clauses 1-15, wherein the crystal form of the Siamenoside I is dispersed or dissolved in the medium.17. The composition according to clause 16. wherein the medium comprises an aqueous buffer.18. The composition according to clause 16, wherein the medium comprises an organic solvent.19. The composition according to clause 16, wherein the medium comprises an aqueous buffer and an organic solvent, and a weight ratio of the aqueous buffer to the organic solvent is from 10: 1 to 1 : 10.20. The composition according to any one of clauses 18-19, wherein the organic solvent is selected from the group consisting of glycerol, propylene glycol, 1,3-propane diol. 1,3-butanediol, ethanol, and any combination thereof.21. The composition according to any one of the clauses 17-20, wherein the composition has a concentration of Siamenoside I from 1 wt% to 25 wt%.22. The composition according to any one of the clauses 17-21, wherein the composition has a pH from 3 to 11.23. The composition according to any one of clauses 13-22, wherein the composition is substantially free from a Mogroside compound other than Siamenoside I.24. The composition according to clause 23, wherein the Mogroside compound is selected from the group consisting of Mogroside IIIE. Mogroside V, and a combination thereof.25. A method of making a composition comprising Siamenoside I, the method comprising: providing / obtaining a solid of Siamenoside I having a crystal form, wherein the crystal form has an orthorhombic crystal system with a space group of P2i2i2i; dispersing or dissolving the solid of Siamenoside I in a medium.26. The method according to clause 25, wherein the crystal form has unit cell parameters: a = 11.50 A, b = 13.16 A, c = 42.73 A, a = P = y = 90°, a unit cell volume is 6467 A3, wherein the unit cell parameters are obtained from single crystal diffraction at about 100 K.27. The method according to any one of clauses 25-26, wherein the crystal form has unit cell parameters: a = 11.60 A, b = 13.37 A, c = 43.08 A, a = = y = 90°, a unit cell volume is 6681 A3, wherein the unit cell parameters are obtained from powder X-ray diffraction (PXRD) at room temperature.28. The method according to any one of clauses 25-27, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°, wherein the X-ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of 1.5406 A.29. The method according to clause 28, wherein the X-ray powder diffraction pattern further comprises diffraction peaks at angles (20) of 15.94±0.2°, 18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.11±0.2°, 10.87±0.2°, 10.52±0.2°, 12.77±0.2°, 11.81±0.2°, 13.95±0.2°, and 19.63±0.2°, and 20.85°±0.2°.30. The method according to any one of clauses 25-29, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in FIG. 2A31. The method according to any one of clauses 25-29, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in FIG. 2B.32. The method according to any one of clauses 25-29, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in Table 1.33. The method according to any one of clauses 25-32, wherein the medium comprises a solvent, the crystal form of Siamenoside I is dissolved in the solvent to form a solution, and the method further comprises performing spray drying on the solution to obtain an amorphous form of Siamenoside I.34. The method according to any one of clauses 25-32, wherein the medium comprises an aqueous buffer.35. The method according to any one of clauses 25-32, wherein the medium further comprises an organic solvent.36. The method according to any one of clauses 25-32, wherein the medium comprises an aqueous buffer and an organic solvent, and a weight ratio of the aqueous buffer to the organic solvent is from 10: 1 to 1: 10.37. The method according to any one of clauses 35-36, wherein the organic solvent is ethanol.38. The method according to any one of clauses 25-37, wherein the composition has a concentration of Siamenoside I from 1 wt% to 25 wt%.39. The method according to any one of clauses 34-38, wherein the composition has a pH from 3 to 11.40. The method according to any one of clauses 25-39, wherein the composition is substantially free from a Mogroside compound other than Siamenoside I.41. The method according to clause 40, wherein the Mogroside compound is selected from the group consisting of Mogroside IIIE. Mogroside V, and a combination thereof.42. The method according to any one of clauses 25-41, wherein the composition is a solid composition.43. The method according to any one of clauses 25-42, wherein the composition is a sweetener composition or a flavor composition.44. The method according to any one of clauses 25-43, wherein the composition is a beverage.45. A method of making a solid of Siamenoside I, the method comprising: providing / obtaining a crude solution of Siamenoside I; causing crystallization of Siamenoside I from the crude solution to obtain crystals of Siamenoside I as a precipitate; and isolating the cry stals of Siamenoside I from the crude solution, wherein the crystals of Siamenoside I have a higher purity compared with the crude solution.46. The method according to clause 45, wherein causing crystallization of Siamenoside I further comprises: adding a seed crystal of Siamenoside I to the crude solution.47. The method according to any one of clauses 45-46, wherein causing crystallization of Siamenoside I further comprises: adjusting a concentration of Siamenoside I in the crude solution to a saturated concentration before causing the crystallization.48. The method according to any one of clauses 45-47, wherein causing crystallization of Siamenoside I further comprises: adjusting a pH of the crude solution to a range from 8 to 10.49. The method according to any one of clauses 45-48, wherein causing crystallization of Siamenoside I further comprises: adding a solvent to the crude solution, wherein the solvent is 30 wt% or less, based on a total weight of the crude solution.50. The method according to clause 49, wherein the solvent is an alcohol.51. The method according to clause 50, wherein the solvent is ethanol.52. The method according to any one of clauses 45-51, wherein causing crystallization of Siamenoside I further comprises: performing one or more heating-cooling cycles, each heating-cooling cycle further comprising: heating the crude solution to an elevated temperature above room temperature; and allowing the crude solution to cool to room temperature.53. The method according to any one of clauses 45-52, wherein causing crystallization of Siamenoside I further comprises: adding one or more salts to the crude solution.54. The method according to any one of clauses 45-53, wherein isolating the crystals of Siamenoside I further comprises: filtering the crude solution after crystallization to obtain a retentate and a filtrate, the retentate comprising the crystals of Siamenoside I; and washing and drying the retentate, wherein the retentate has a high purity of Siamenoside 1 compared with the filtrate.55. The method according to any one of clauses 45-54, further comprising: performing recrystallization on the isolated crystals of Siamenoside I.56. The method according to any one of clauses 45-55, wherein the crystals of Siamenoside I comprise a cry stal form characterized by an orthorhombic crystal system with a space group of P2i2i2i.57. The method according to clause 56, wherein the crystal form has unit cell parameters: a = 11.50 A, b = 13.16 A, c = 42.73 A, a = P = y = 90°, a unit cell volume is 6467 A3, wherein the unit cell parameters are obtained from single crystal diffraction at about 100 K.58. The method according to any one of clauses 56-57, wherein the crystal form has unit cell parameters: a = 11.60 A, b = 13.37 A, c = 43.08 A, a = = y = 90°, a unit cell volume is 6681 A3, wherein the unit cell parameters are obtained from powder X-ray diffraction (PXRD) at room temperature.59. The method according to any one of clauses 56-58, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°, wherein the X-ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of 1.5406 A.60. The method according to clause 59, wherein the X-ray powder diffraction pattern further comprises diffraction peaks at angles (20) of 15.94±0.2°, 18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.11±0.2°, 10.87±0.2°, 10.52±0.2°, 12.77±0.2°, 11.81±0.2°, 13.95±0.2°, and 19.63±0.2°.61. The method according to any one of clauses 56-60, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in FIG. 2A.62. The method according to any one of clauses 56-60, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in FIG. 2B.63. The method according to any one of clauses 56-60, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks as shown in Table. 1.64. The method according to any one of clauses 45-63, wherein the composition is substantially free from a Mogroside compound other than Siamenoside I.65. The method according to clause 64, wherein the Mogroside compound is selected from the group consisting of Mogroside IIIE, Mogroside V, and a combination thereof.66. A solid product obtained by a crystallization process of a solution of a crude source of Siamenoside I, wherein the crude source of Siamenoside I further comprises one or more Mogroside compounds, the solid product comprises a crystal form of Siamenoside I, and the crystal form is characterized by an orthorhombic crystal system with a space group of P2i2i2i.67. The solid product according to clause 66, wherein the crystal form has unit cell parameters: a = 11.50 A, b = 13.16 A. c = 42.73 A, a = P = y = 90°, a unit cell volume is 6467 A3, wherein the unit cell parameters are obtained from single crystal diffraction at about 100 K.68. The solid product according to any one of clauses 66-67, wherein the crystal form has unit cell parameters: a = 11 .60 A, b = 13.37 A, c = 43.08 A, a = P = y = 90°, a unit cell volume is 6681 A3, wherein the unit cell parameters are obtained from powder X-ray diffraction (PXRD) at room temperature.69. The solid product according to any one of clauses 66-68, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks at angles (29) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°, wherein the X-ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of 1.5406 A.70. The solid product according to clause 69, wherein the X-ray powder diffraction pattern further comprises diffraction peaks at angles (20) of 15.94±0.2°,18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.11±0.2°, 10.87±0.2°, 10.52±0.2°, 12.77±0.2°, 11.81±0.2°, 13.95±0.2°, and 19.63±0.2°.71. The solid product according to any one of clauses 66-70, wherein the solid product further comprises a co-crystal of Siamenoside I and the one or more Mogroside compounds.72. The solid product according to any one of clauses 66-71, wherein the solid product further comprises a crystal of the one or more Mogroside compounds.73. The solid product according to any one of clauses 66-72, wherein the solid product has a content of Siamenoside I of at least 90%, based on the total Mogroside compounds in the solid product.74. The solid product according to any one of clauses 66-73, wherein the solid product has a content of the one or more Mogroside compounds other than Siamenoside I of 10% or less, based on the total Mogroside compounds in the solid product.75. The solid product according to any one of clauses 66-74, wherein the solid product has a first purity of Siamenoside I, the crude source has a second purity of Siamenoside I, and the first purity' is higher than the second purity.76. The solid product according to any one of clauses 66-75, wherein the solid product has a first content of the one or more Mogroside compounds other than Siamenoside I, the crude source has a second content of the one or more Mogroside compounds other than Siamenoside I, and the second content is higher than the first content.77. The solid product according to any one of clauses 66-76, wherein the one or more Mogroside compounds comprises Mogroside IIIE, Mogroside V, or a combination thereof.78. A composition comprising the solid product according to any one of clauses 66-77.79. A composition comprising Siamenoside I reconstructed from the solid product according to any one of clauses 66-77.80. A method of preparing crystals of Siamenoside I, the method comprising:providing / obtaining a solution of Siamenoside I; causing crystallization of Siamenoside I from the solution to obtain crystals of Siamenoside I as a precipitate; and isolating the crystals of Siamenoside I from the solution, wherein the crystals of Siamenoside I comprise a cry stal form of Siamenoside I, and the crystal form is characterized by an orthorhombic cry stal system with a space group of P212121.81. The method according to clause 80, wherein causing cry stallization of Siamenoside I further comprises at least one of: adding a seed crystal of Siamenoside I to the solution; adjusting a concentration of Siamenoside I in the solution to a saturated concentration before causing the crystallization: adjusting a pH of the solution to a range from 8 to 10; adding a solvent to the solution, wherein the solvent is 30 wt% or less, based on a total weight of the solution; performing one or more heating-cooling cycles, each heating-cooling cycle further comprising: heating the solution to an elevated temperature above room temperature; and allowing the solution to cool to room temperature; and adding one or more salts to the solution.82. The method according to any one of clauses 80-81, wherein isolating the crystals of Siamenoside I further comprises: filtering the solution after cry stallization to obtain a retentate and a filtrate, the retentate comprising the crystals of Siamenoside I; and washing and drying the retentate, yvherein the retentate comprises the crystals of Siamenoside I.83. The method according to any one of clauses 80-82, yvherein the crystal form has unit cell parameters: a = 11.50 A, b = 13.16 A, c = 42.73 A, a = = y = 90°, a unit cell volume is 6467 A3, wherein the unit cell parameters are obtained from single crystal diffraction at about 100 K.84. The method according to any one of clauses 80-83, wherein the crystal form has unit cell parameters: a = 11.60 A, b = 13.37 A, c = 43.08 A, a = = y = 90°, a unit cell volume is 6681 A3, wherein the unit cell parameters are obtained from powder X-ray diffraction (PXRD) at room temperature.85. The method according to any one of clauses 80-84, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°, wherein the X-ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of 1.5406 A.86. The method according to clause 85, wherein the X-ray powder diffraction pattern further comprises diffraction peaks at angles (20) of 15.94±0.2°, 18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.11±0.2°. 10.87±0.2°, 10.52±0.2°, 12.77±0.2°, 11.81±0.2°, 13.95±0.2°, and 19.63±0.2°.

Claims

WHAT IS CLAIMED IS:

1. A crystal form of Siamenoside I, wherein the crystal form has an orthorhombic cry stal system with a space group of P2i2i2i.

2. The crystal form of Siamenoside I according to claim 1. wherein the crystal form has unit cell parameters: a = 11.50 A, b = 13.16 A, c = 42.73 A, a = P = y = 90°, a unit cell volume is 6467 A3, wherein the unit cell parameters are obtained from single crystal diffraction at about 100 K.

3. The crystal form of Siamenoside I according to claim 2, wherein the crystal form has unit cell parameters: a = 11.60 A. b = 13.37 A, c = 43.08 A, a = P = y = 90°, a unit cell volume is 6681 A3, wherein the unit cell parameters are obtained from powder X- ray diffraction (PXRD) at room temperature.

4. The cry stal form of Siamenoside I according to claim 1, wherein the cry stal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°. and 13.01±0.2°, wherein the X- ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of 1 .5406 A.

5. The crystal form of Siamenoside I according to claim 4. wherein the X-ray powder diffraction pattern further comprises diffraction peaks at angles (20) of 15.94±0.2°, 18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.1 1±0.2°, 10.87±0.2°, 10.52±0.2°, 12.77±0.2°, 11.81±0.2°, 13.95±0.2°, and 19.63±0.2°.

6. A composition comprising: a crystal form of Siamenoside I, wherein the crystal form having an orthorhombic cry stal system with a space group of P2i2r2r.

7. The composition according to claim 6. wherein the composition is a sweetener composition, flavor composition, food composition, or a beverage composition.

8. A method of making a composition comprising Siamenoside I, the method comprising: providing a solid of Siamenoside I having a crystal form, wherein the crystal form has an orthorhombic crystal system with a space group of P2r2r2r; anddispersing or dissolving the solid of Siamenoside I in a medium.

9. The method according to claim 8, wherein the crystal form has unit cell parameters: a = 11.50 A, b = 13.16 A, c = 42.73 A, a = P = y = 90°, a unit cell volume is 6467(2) A3, wherein the unit cell parameters are obtained from single crystal diffraction at about 100 K.

10. The method according to claim 8, wherein the crystal form has unit cell parameters: a = 11.60 A, b = 13.37 A, c = 43.08 A, a = p = y = 90°, a unit cell volume is 6681 A3, wherein the unit cell parameters are obtained from powder X-ray diffraction (PXRD) at room temperature.

11. The method according to claim 8, wherein the crystal form is characterized by an X-ray powder diffraction pattern comprising diffraction peaks at angles (20) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°. and 13.01±0.2°, wherein the X-ray powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of 1.5406 A.

12. The method according to claim 11, wherein the X-ray powder diffraction pattern further comprises diffraction peaks at angles (20) of 15.94±0.2°, 18.43±0.2°, 21.40±0.2°, 12.22±0.2°, 18.11±0.2°, 10.87±0.2°, 10.52±0.2°, 12.77±0.2°, 11.81±0.2°, 13.95±0.2°, and 19.63±0.2°.

13. The method according to claim 8, wherein the medium comprises a solvent, the cry stal form of Siamenoside I is dissolved in the solvent to form a solution, and the method further comprises performing spray drying on the solution to obtain an amorphous form of Siamenoside I.

14. The method according to claim 8, wherein the medium comprises a solvent selected from an aqueous buffer, a solvent, and a combination thereof.

15. The method according to claim 14, wherein the aqueous buffer has a pH from 3 to 11.

16. The method according to claim 14. wherein the solvent is selected from the group consisting of glycerol, propylene glycol, 1,3-propane diol, ethanol, and any combination thereof.

17. A method of making a solid of Siamenoside I, the method comprising: providing / obtaining a crude solution of Siamenoside I; causing crystallization of Siamenoside I from the crude solution to obtain crystals of Siamenoside I as a precipitate; and isolating the crystals of Siamenoside I from the crude solution, wherein the crystals of Siamenoside I have a higher purity compared with the crude solution.

18. The method according to claim 17, wherein causing crystallization of Siamenoside I further comprises at least one of: adding a seed crystal of Siamenoside I to the crude solution: adjusting a concentration of Siamenoside I in the crude solution to a saturated concentration before causing the crystallization; adjusting a pH of the crude solution to a range from 8 to 10; adding a solvent to the crude solution, wherein the solvent is 30 wt% or less, based on a total weight of the crude solution; performing one or more heating-cooling cycles, each heating-cooling cycle further comprising: heating the crude solution to an elevated temperature above room temperature; and allowing the crude solution to cool to room temperature; and adding one or more salts to the crude solution.

19. The method according to claim 17. wherein isolating the crystals of Siamenoside I further comprises: filtering the crude solution after crystallization to obtain a retentate and a filtrate, the retentate comprising the crystals of Siamenoside I; and washing and drying the retentate, wherein the retentate has a high purity of Siamenoside I compared with the filtrate.

20. The method according to claim 17. wherein a crystal form of the crystals of Siamenoside I has an orthorhombic crystal system with a space group of P212121 and is characterized by an X-ray powder diffraction pattern comprising diffraction peaks at angles (29) of 16.48°±0.2°, 14.39°±0.2°, 15.83±0.2°, and 13.01±0.2°, wherein the X-ray-powder diffraction pattern is measured on a diffractometer using Cu-Ka radiation with a wavelength of 1.5406 A.

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