Device for determining the coexistence of lupeol and antidiabetic compound in ethyl acetate and n-hexane extracts

Abstract

A device (100) for determining the coexistence of lupeol and an antidiabetic compound in ethyl acetate and n-hexane extracts, wherein the device (100) comprises: a container (102) for collecting parts of the plant A. leiocarpus; a pulverizer (104) which is operationally connected to the container to pulverize the collected plant parts; a cold maceration vessel (106) which is operationally connected to the pulverizer to obtain ethanolic extracts from the pulverized sample using 95% v / v ethanol; a rotary evaporator (108) which is operationally connected to the cold maceration vessel to separate and concentrate the obtained ethanolic extracts into ethyl acetate and aqueous fractions; a column chromatography unit (110) consisting of a glass column filled with silica gel and mixed with 95% n-hexane and 5% ethyl acetate for eluting and concentrating fractions; a thin-layer chromatography (TLC) unit (112) which is operationally connected to the column chromatography unit consists of a TLC plate which is coated with silica gel, dried and immersed in a solvent for fluorescence observation; and a nuclear magnetic resonance (NMR) spectrometer (114) containing the solvents DMSO-d6, CDCl3 and acetone-d6 for determining the structure of purified compounds.

Description

Field of the invention:

[0001] The present invention relates to the field of identifying properties of herbal extracts. In particular, the present invention describes a device for determining the coexistence of lupeol and an antidiabetic compound in ethyl acetate and n-hexane extracts. Background of the invention:

[0002] Inflammation is a vascular and cellular response to injury in living tissue characterized by five main symptoms: (i) hyperemia (redness) and (ii) warmth (heat), resulting from increased blood flow to the affected area; (iii) swelling caused by hypoxia due to blood stasis and cell aggregation; (iv) pain; and (v) loss of function of the affected body part. Regardless of the cause of injury—whether cuts, burns, infections (bacterial, fungal, protozoan, metazoan, viral, and toxins), chemical poisons, mechanical or thermal trauma, or immune reactions—there is increased vascular permeability and inflammatory edema. This is accompanied by the production of inflammatory biomarkers, such as elevated cytokines and high-sensitivity C-reactive protein (hs-CRP), which is particularly evident in individuals with type 2 diabetes mellitus (T2DM).Similar elevations have been observed in dogs with alloxan-induced type 1 diabetes (T1DM). These biomarkers are associated with injury caused by chronic hyperglycemia, which contributes to microvascular and macrovascular damage and results in severe complications such as cardiovascular disease, nephropathy, neuropathy, and retinopathy, often leading to blindness.

[0003] Increased blood flow to the inflamed site further increases levels of inflammatory cytokines, as red blood cells act as dynamic cytokine reservoirs. Diabetes mellitus (DM) remains a significant global health problem, affecting over 425 million people and imposing a significant economic burden. It involves metabolic disorders related to carbohydrate, protein, and lipid metabolism, leading to chronic hyperglycemia. Both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) contribute to this condition, with T2DM projected to be a leading cause of death by 2030, according to the WHO.

[0004] Complications of diabetes, including nephropathy, retinopathy, and delayed wound healing, pose significant health risks. Current treatments such as sulfonylureas, meglitinides, biguanides, and thiazolidinediones have shown limitations and side effects, particularly in developing countries where resources are scarce.

[0005] To overcome the above-mentioned limitations, there is therefore a need to investigate the medicinal properties of traditional medicinal plants for the treatment of diabetes. This study investigates the properties of Anogeissus leiocarpus in the treatment of diabetes and the restoration of electrolyte and acid-base balance in alloxan-diabetic dogs.

[0006] The technical advances disclosed by the present invention overcome the limitations and disadvantages of existing and conventional systems and methods. Summary of the invention:

[0007] The present invention relates to a device for determining the simultaneous presence of lupeol and an antidiabetic compound in ethyl acetate and n-hexane extracts.

[0008] An object of the present invention is to evaluate the properties of herbal plants.

[0009] Another aim of the present invention is to achieve a modulating effect on the erythrocyte surface and free sialic acids in serum.

[0010] Another aim of the present invention is to effectively detect the elevated serum sialic acids as an effective biomarker and

[0011] Another object of the present invention is to study the structure of the purified product, in particular to elucidate its mechanism of action, including its requirements for environmental soil fertility.

[0012] A device for determining the co-presence of lupeol and an antidiabetic compound in ethyl acetate and n-hexane extracts, the device comprising: a container for collecting parts of the plant A. leiocarpus; a pulverizer operatively connected to the container for pulverizing the collected plant parts; a cold maceration vessel operatively connected to the pulverizer for obtaining ethanolic extracts from the pulverized sample using 95% v / v ethanol; a rotary evaporator operatively connected to the cold maceration vessel for separating and concentrating the obtained ethanolic extracts into ethyl acetate and aqueous fractions; a column chromatography unit consisting of a glass column packed with silica gel and mixed with 95% n-hexane and 5% ethyl acetate for eluting and concentrating fractions;A thin-layer chromatography (TLC) unit, operatively connected to the column chromatography unit, consists of a TLC plate coated with silica gel, dried, and immersed in a solvent for fluorescence observation; and a nuclear magnetic resonance (NMR) spectrometer with the solvents DMSO-d6, CDCl3, and acetone-d6 for determining the structure of purified compounds.

[0013] To further clarify the advantages and features of the present invention, a more detailed description of the invention will be given with reference to specific embodiments thereof, which are illustrated in the accompanying drawings. It should be noted that these drawings represent only typical embodiments of the invention and are therefore not to be considered limiting its scope. The invention will be described and explained in additional detail and in greater detail with reference to the accompanying drawings. Short description of the figures:

[0014] These and other features, aspects, and advantages of the present invention will become more readily understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Fig. Figure 1 shows a device for determining the coexistence of lupeol and an antidiabetic compound in ethyl acetate and n-hexane extracts. Fig. Figure 2 illustrates a structure of lupeol .

[0015] Furthermore, those skilled in the art will appreciate that elements in the drawings are shown for convenience and may not necessarily be drawn to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Moreover, with respect to device construction, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawings with details that would be readily apparent to those skilled in the art who would benefit from the description herein. Detailed description:

[0016] To facilitate an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and described in specific language. It is to be understood, however, that this is not intended to limit the scope of the invention, as such changes and further modifications to the illustrated system and such further applications of the principles of the invention as illustrated therein are contemplated as would normally occur to one skilled in the art to which the invention pertains.

[0017] It will be understood by one skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0018] References in this specification to "one aspect," "another aspect," or similar expressions mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, the occurrences of the terms "in one embodiment," "in another embodiment," and similar expressions throughout this specification may or may not all refer to the same embodiment.

[0019] The terms "comprises," "comprising," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps not only includes those steps, but may also include other steps not expressly listed or inherent in such process or method. Likewise, one or more devices or subsystems or elements or structures or components preceded by "comprises...a" does not preclude, without further limitation, the existence of other devices or other subsystems or other elements or other structures or other components or additional devices or additional subsystems or additional elements or additional structures or additional components.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0021] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0022] Fig.Figure 1 illustrates an apparatus (100) for determining the co-presence of lupeol and an antidiabetic compound in ethyl acetate and n-hexane extracts, the apparatus (100) comprising: a container (102) for collecting parts of the plant A. leiocarpus; a pulverizer (104) operatively connected to the container for pulverizing the collected parts of the plant; a cold maceration vessel (106) operatively connected to the pulverizer for obtaining ethanolic extracts from the pulverized sample using 95% v / v ethanol; a rotary evaporator (108) operatively connected to the cold maceration vessel for separating and concentrating the obtained ethanolic extracts into ethyl acetate and aqueous fractions;a column chromatography unit (110) consisting of a glass column packed with silica gel and mixed with 95% n-hexane and 5% ethyl acetate to elute and concentrate fractions; a thin-layer chromatography (TLC) unit (112) operatively connected to the column chromatography unit consisting of a TLC plate coated with silica gel, dried, and immersed in a solvent for fluorescence observation; and a nuclear magnetic resonance (NMR) spectrometer (114) comprising the solvents DMSO-d6, CDCl3, and acetone-d6 for determining the structure of purified compounds.

[0023] In one embodiment, the column chromatography unit (110) comprises: a glass column (110a) packed with 500 grams of silica gel (70-230 mesh) mixed with 95% n-hexane and 5% ethyl acetate; a rotary evaporator (110b) for concentrating the ethyl acetate fraction; a water bath (110c) for concentrating the aqueous fraction; a collection unit (110d) for collecting and concentrating fractions to dryness at 40°C; a capillary tube (110e) for applying spots of concentrated fractions to a TLC plate; a solvent unit (110f) for developing the TLC plate; and a UV light source (110g) for detecting fluorescence and a sulfuric acid spray system for visualizing specific compounds.

[0024] In one embodiment, the plant part is selected as stem bark.

[0025] In one embodiment, several phytochemical components are detected in the ethanolic extract.

[0026] In one embodiment, the ethanolic extracts are partitioned with ethyl acetate so that the ethyl acetate fraction is concentrated using a rotary evaporator and the aqueous fraction is concentrated in a water bath prior to storage at 20 °C.

[0027] In one embodiment, the column chromatography unit removes bubbles and pours them into a glass column, with fractions being collected in vials and concentrated to dryness by evaporation at 40°C.

[0028] In one embodiment, the positions of compounds are observed by fluorescence under UV light and sprayed with sulfuric acid for the specific compounds and fractions with similar TLC profiles.

[0029] In one embodiment, the dryness of the sample and the soil condition are measured. The drying process of the sample and the soil fertility are normal. The phytochemical components are identical. The ethanolic extraction from the stem bark yielded 451.58 g.

[0030] In one embodiment, ethyl acetate fractions and yields (weights / percentages) from column chromatography and detailed TLC are evaluated. An evaluation of the ethyl acetate fractions and total yields (weights and percentages) against the purified and ethanolic extracts used to prepare for NMR analyses is shown in Table 1: Table: Ethyl acetate fraction and yield (weight / percent) from column chromatography S / No. or letter identity fractions Weight (g) Percentage of purified extracts Percentage of ethanolic extract 1 interface 5.10 71.83 1.13 A 28-37 0.70 9.86 0.16 B 38-58 0.60 8.45 0.13 C 59-80 0.70 9.86 0.16 In total 7.10 100.0 1.58

[0031] Fig. Figure 2 illustrates a structure of lupeol.

[0032] Soil fertility assessments and phytochemical analyses of the ethanolic extracts from the stem bark of *A. leiocarpus* complied with the recommended standards for non-deficient soils. Fraction A, weighing 0.70 grams from 451.58 grams of the ethanolic extract, was isolated and purified by column chromatography and TLC using ethyl acetate and n-hexane. This fraction represents only 9.86% of the total purified fractions and a mere 0.16% of the ethanolic extract (Table 1).

[0033] NMR analysis confirmed that fraction A is lupeol, a pentacyclic triterpenoid known for its pharmacological activity. It is hypothesized that only a small amount of lupeol may be required to treat the inflammatory aspects of diabetes mellitus (DM). This hypothesis is supported by the efficacy of crude ethanol extracts from the stem bark of A. leiocarpus in treating alloxan-induced diabetes in dogs and rats.

[0034] Crystallization with ethyl acetate and n-hexane during the final purification steps, in conjunction with TLC, indicates the purity of each of the four different compounds. The global prevalence of type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) underscores the need for unconventional treatments given the far-reaching social and health impacts associated with these diseases.

[0035] The identification of lupeol in the purified extract supports claims of its efficacy in treating DM complications, such as improved wound healing and reduced liver and kidney damage in diabetic animals. It is suggested that A. leiocarpus, due to its lupeol content, could effectively treat type 2 diabetes and its associated damage, highlighting the benefits of crude ethanol extracts. The anti-inflammatory effects of lupeol, coupled with its role in alleviating diabetic inflammation—as evidenced by elevated cytokines and high-sensitivity C-reactive protein (hs-CRP) in type 2 diabetes patients—support this notion.

[0036] The presence of lupeol in A. leiocarpus offers additional benefits in the treatment of diabetes-related inflammatory pain, such as diabetic neuropathic pain. Elevated serum sialic acid, a biomarker of diabetes-related inflammation, further underscores the importance of lupeol in this context. Furthermore, some triterpenes, including those identified in the ethanolic extracts of the stem bark of A. leiocarpus, have been associated with antidiabetic effects.

[0037] However, the lupeol yield (9.86% of the ethanolic extracts) is insufficient for comprehensive bioassay studies on its antidiabetic activity or toxic effects. Nevertheless, toxicity studies have shown that lupeol is well tolerated, with no adverse effects observed in rats and mice at doses up to 2 grams / kg body weight. Likewise, the interfacial fraction of the purified extracts, which constitutes 71.83% of the ethanolic extract, is also safe when administered to Wistar rats at a dose of 2000 mg / kg body weight.

[0038] It is plausible that lupeol, contained in the crude ethanol extracts of the stem bark of A. leiocarpus, contributes significantly to the observed hepatorenal and dyslipidemic healing effects, as well as its antioxidant activity and accelerated wound healing in diabetic dogs. This synergy is supported by reports that lupeol scavenges peroxyl radicals, enhances antioxidant defenses, reduces lipid peroxidation, and improves antioxidant enzyme activity.

[0039] The drawings and the foregoing description provide examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of the processes described herein may be changed and are not limited to the manner described herein. Furthermore, the actions of any flowchart need not be implemented in the order shown; nor do all actions necessarily need to be performed. Also, those actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and material use, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0040] Advantages, other benefits, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, benefits, solutions to problems, and any components that may cause an advantage or solution to occur or become more apparent are not to be construed as a critical, required, or essential feature or component of any or all of the claims. REFERENCES 100 A device for determining the coexistence of lupeol and antidiabetic compound in ethyl acetate and n-hexane extracts. 102 containers 104 pulverizers 106 cold maceration vessel 108 rotary evaporators 110 Column Chromatography Unit 110a glass column 110b rotary evaporator 110c water bath 110d collection unit 110e capillary tubes 110f solvent unit 110g UV light source 112 Thin-layer chromatography (TLC) unit 114 nuclear magnetic resonance spectrometers (NMR)

Claims

[1] A device (100) for determining the coexistence of lupeol and an antidiabetic compound in ethyl acetate and n-hexane extracts, the device (100) comprising: a container (102) for collecting parts of the plant A. leiocarpus ; a pulverizer (104) operatively connected to the container for pulverizing the collected plant parts; a cold maceration vessel (106) operatively connected to the pulverizer for obtaining ethanolic extracts from the pulverized sample using 95% v / v ethanol; a rotary evaporator (108) operatively connected to the cold maceration vessel for separating and concentrating the resulting ethanolic extracts into ethyl acetate and aqueous fractions; a column chromatography unit (110) consisting of a glass column filled with silica gel and mixed with 95% n-hexane and 5% ethyl acetate for eluting and concentrating fractions; a thin-layer chromatography (TLC) unit (112) operatively connected to the column chromatography unit, consisting of a TLC plate coated with silica gel, dried, and immersed in a solvent for fluorescence observation; and a nuclear magnetic resonance (NMR) spectrometer (114) containing the solvents DMSO-d6, CDCl 3 and acetone-d6 for determining the structure of purified compounds. [2] The apparatus of claim 1, wherein the column chromatography unit (110) comprises: a glass column (110a) packed with 500 g of silica gel (70-230 mesh) mixed with 95% n-hexane and 5% ethyl acetate; a rotary evaporator (110b) for concentrating the ethyl acetate fraction; a water bath (110c) for concentrating the aqueous fraction; a collection unit (110d) for collecting and concentrating fractions to dryness at 40°C; a capillary tube (110e) for applying spots of concentrated fractions to a TLC plate; a solvent unit (110f) for developing the TLC plate; and a UV light source (110 g) for fluorescence detection and a sulfuric acid spray system for visualizing specific compounds. [3] Device according to claim 1, characterized by that the plant part is selected as stem bark. [4] Device according to one of claims 1 to 3, characterized by that a variety of phytochemical components are detected in the ethanolic extract. [5] The device according to claim 1, wherein the ethanolic extracts are partitioned with ethyl acetate such that the ethyl acetate fraction is concentrated by rotary evaporation and the aqueous fraction is concentrated in a water bath prior to storage at 20°C. [6] The apparatus of claim 1, wherein the column chromatography unit removes bubbles and pours them into a glass column, fractions are collected in vials and concentrated to dryness by evaporation at 40°C. [7] The device of claim 1, wherein the positions of the compounds are observed by fluorescence under UV light and sprayed with sulfuric acid for the specific compounds and fractions with similar TLC profiles.

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