Device for the identification of betulinic acid and trimethoxyellaginic acid in purified extract from the stem bark of Anogeissus leiocarpus

Abstract

A device (100) for identifying betulinic acid and trimethoxyellagic acid as antidiabetic compounds in purified extract from the stem bark of Anogeissus leiocarpus, wherein the device (100) comprises: a collection unit (102) for collecting stem bark samples of Anogeissus leiocarpus; a processing unit (104) connected to the collection unit (102) for pulverizing the collected samples, wherein the pulverized samples are subjected to cold maceration with 90-100% v / v ethanol to extract ethanolic extracts; a segmentation unit (106) connected to the processing unit (104) for splitting the ethanolic extract using ethyl acetate and n-hexane solvents; a column chromatography unit (108) connected to the segmentation unit (106) for triggering precipitation by adding cold acetone to the ethanolic extract; A solution and adsorption unit (110) comprises a container (110a) connected to the column chromatography unit (108) for dissolving the precipitate with a mixture of dichloromethane (DCM) and methanol, and a glass column (110b) for adsorbing the dissolved precipitate; an evaporation unit (112) connected to the solution and adsorption unit (110) to evaporate the solvent mixture and obtain a free-flowing powder from the dissolved precipitate; and a plurality of evaluation units (114) connected to the evaporation unit (112) for identifying properties of the stem bark of Anogeissus leiocarpus.

Description

[0001] Field of the invention:

[0002] The present invention relates to the field of thermal extract production. In particular, the present invention discloses the identification of properties of herbal extracts. More specifically, the present invention discloses a device for identifying betulinic acid and trimethoxyellagic acid as antidiabetic compounds in purified extract from the stem bark of Anogeissus leiocarpus. Background of the invention:

[0003] The global prevalence of diabetes mellitus (DM), including type 1 (T1DM) and type 2 (T2DM), has increased significantly and poses a serious threat to public health. Complications such as cardiovascular disease, nephropathy, retinopathy, and blindness are contributing factors to DM being projected to be a leading cause of death by 2030. According to the World Health Organization (WHO), an even more alarming scenario is expected by 2035. Diabetes-related risk factors, particularly their impact on COVID-19 prognosis and mortality, also have significant implications for national budgets and the global economy.

[0004] Obesity, a major predisposing factor for diabetes, exacerbates the disease by contributing to delayed or non-healing wounds known as diabetic ulcers, which result from chronic hyperglycemia. This creates an additional social burden that is difficult to quantify. In Africa, the age distribution and prevalence of undiagnosed diabetes pose particular challenges.

[0005] In response to the growing threat of diabetes to humans and animals, demand for research into non-conventional treatments for diabetes and its complications has increased. Cattle, which normally have low blood sugar levels due to their dependence on volatile fatty acids for energy metabolism, are also showing signs of diabetes mellitus. The focus is now on alternative treatments using medicinal plants and their phytoconstituents.

[0006] To meet the above-mentioned requirements, there is therefore a need to investigate the properties of traditional medicinal plants for the treatment of diabetes. The properties of Anogeissus leiocarpus in treating diabetes and preventing the worsening of existing anemia are being investigated only within the first 24 hours.

[0007] The technical advances disclosed by the present invention overcome the limitations and disadvantages of existing and conventional systems and methods. Summary of the invention:

[0008] The present invention relates to a device for identifying betulinic acid and trimethoxyellagic acid as antidiabetic compounds in the purified extract of the stem bark of Anogeissus leiocarpus .

[0009] An object of the present invention is to evaluate the properties of herbal extracts.

[0010] Another aim of the present invention is to establish elevated serum sialic acids as an effective biomarker for diabetes mellitus.

[0011] Another aim of the present invention is to accelerate wound healing in diabetes mellitus types 1 and 2.

[0012] Another object of the present invention is to prevent worsening of existing anemia within the first 24 hours.

[0013] An apparatus for identifying betulinic acid and trimethoxyellagic acid as antidiabetic compounds in purified Anogeissus leiocarpus stem bark extract, the apparatus comprising: a collection unit for collecting Anogeissus leiocarpus stem bark samples; a processing unit connected to the collection unit for pulverizing the collected samples, wherein the pulverized samples are subjected to cold maceration with 95% v / v ethanol to extract ethanolic extracts; a segmentation unit connected to the processing unit for partitioning the ethanolic extract using ethyl acetate and n-hexane solvents; a column chromatography unit connected to the segmentation unit for inducing precipitation by adding cold acetone to the ethanolic extract;A dissolution and adsorption unit connected to the column chromatography unit, comprising a container for dissolving the precipitate with a mixture of dichloromethane (DCM) and methanol and a glass column for adsorbing the dissolved precipitate; an evaporation unit connected to the dissolution and adsorption unit for evaporating the solvent mixture and obtaining a free-flowing powder from the dissolved precipitate; a silica gel fractionation column connected to the evaporation unit for fractionating the free-flowing powder into several fractions; a thin-layer chromatography (TLC) unit operatively connected to the fractionation column for ignoring gummy fractions and visualizing light brown powder; and a nuclear magnetic resonance (NMR) spectrometer containing the solvents DMSO-d6, CDCl3, and acetone-d6 for determining the structure of purified compounds.

[0014] 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:

[0015] 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 identifying betulinic acid and trimethoxyellagic acid as antidiabetic compounds in the purified extract from the stem bark of Anogeissus leiocarpus; Fig. 2 shows the structure of betulinic acid; and Fig.3 illustrates a structure of trimethoxyellagic acid. 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. Furthermore, 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 identifying betulinic acid and trimethoxyellagic acid as antidiabetic compounds in purified Anogeissus leiocarpus stem bark extract, the apparatus (100) comprising: a collection unit (102) for collecting Anogeissus leiocarpus stem bark samples; a processing unit (104) connected to the collection unit (102) for pulverizing the collected samples, wherein the pulverized samples are subjected to cold maceration with 90-100% v / v ethanol to extract ethanolic extracts; a segmentation unit (106) connected to the processing unit (104) for dividing the ethanolic extract using ethyl acetate and n-hexane solvents; a column chromatography unit (108) connected to the segmentation unit (106) for inducing precipitation by adding cold acetone to the ethanolic extract;a dissolution and adsorption unit (110) connected to the column chromatography unit (108) comprising a container (110a) for dissolving the precipitate with a mixture of dichloromethane (DCM) and methanol and a glass column (110b) for adsorbing the dissolved precipitate; an evaporation unit (112) connected to the dissolution and adsorption unit (110) for evaporating the solvent mixture and obtaining a free-flowing powder from the dissolved precipitate; a fractionation column (114) containing silica gel connected to the evaporation unit (112) for fractionating the free-flowing powder into a plurality of fractions; and a plurality of evaluation units (114) connected to the evaporation unit (112) for identifying properties of the stem bark of Anogeissus leiocarpus.

[0023] In one embodiment, several phytochemical components are detected in the ethanolic extract.

[0024] In one embodiment, the mixture of DCM and methanol for dissolving the precipitate is selected in a ratio of 9:1.

[0025] In one embodiment, the free-flowing powder is fractionated over 50 grams of silica gel to produce 40 fractions.

[0026] In one embodiment, the purified compound is measured. The purified compound is approximately 100 mg.

[0027] In one embodiment, the plurality of evaluation units (114) comprise: a fractionation column (114a) containing silica gel to fractionate the free-flowing powder into multiple fractions; an extract thin-layer chromatography (TLC) unit (114b) operatively connected to the fractionation column (114) to ignore gummy fractions and visualize light brown powder; and a nuclear magnetic resonance (NMR) spectrometer (114c) comprising the solvents dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and acetone-d6 to determine the structure of purified compounds.

[0028] Fig.Figure 2 shows the structure of betulinic acid. The strong chemical reaction between betulinic acid and glucose produces the end products betulinic acid glycoside, an ester, and water. The reactions occur through glycosidic bonds with the reactive hydroxyl group (OH) at position C of two glucose molecules, which are linked to the carboxyl groups (COOH) and the hydroxyl groups (OH) of betulinic acid.

[0029] Fig. Figure 3 shows the structure of trimethoxyellagic acid. The strong chemical reaction between trimethoxyellagic acid and glucose produces the end products trimethoxyellagic acid glycoside, an ether, and water.

[0030] Similarly, the reaction occurs via the glycosidic bond with the reactive hydroxyl group (OH) at position C1 of glucose and the hydroxyl group (OH) of trimethoxyellagic acid.

[0031] The Fig. 2 and Fig.3 illustrate the removal of glucose molecules from the blood of diabetics and animals.

[0032] In one embodiment, the proton and carbon NMR chemical shift data of betulinic acid and trimethoxyellagic acid are analyzed and presented in Table 1 and Table 2: Table 1: NMR data table of betulinic acid Betulinic acid Betulinic acid continued position 13 C (δ ppm) 1 H (δ ppm) position 13 C (δ ppm) 1 H (δ ppm) 1 38.8 (t) 1.64, 0.89 16 32.2 (t) 2.30, 1.21 2 27.4 (t) 1.59 17 57.2 (S) - 3 79.1 letter d 3.20 18 46.9 (d) 1.57 4 38.9 (S) - 19 49.3 (d) 3.00 5 55.4 (d) 0.68 20 150.4 (seconds) - 6 18.3 (t) 1.53, 1.38 21 29.7 (t) 2.00, 1.44 7 34.4 (t) 1.41 22 37.1 (t) 2.34, 1.14 8 40.8 (seconds) - 23 28.0 (q) 0.99 9 50.6 (d) 1.29 24 15.4 (q) 0.76 10 37.3 (numbers) - 25 16.1 Letter q 0.85 11 20.9 (t) 1.48, 1.18 26 16.2 (q) 1.26 12 25.6 (t) 1.63, 1.06 27 14.7 (q) 0.97 13 38.5 (d) 1.64 28 180.4 (seconds) - 14 42.5 (seconds) - 29 109.7 (t) 4.74, 4.61 15 30.6 (t) 1.66, 1.06 30 19.4 (q) 1.69 Table 2: NMR data table of trimethoxyellagic acid Experimental values Literary values position 1 H ppm (mult.) 13 °C ( ~ ppm) 1 H ppm (mult.) 13 °C ( ~ ppm) 1 - 114.6 (C) - 2 - 139.7 (C) - 3 - 141.3 (C) - 4 - 140.9 (C) - 5 7.52 (1 hour, s) 112.0 (CH) 7.53 (1 hour, s) 6 - 111.8 (C) - 7 - 158.2 (C) - 1' - 114.6 (C) - 2' - 139.7 (C) - 3' - 140.4 (C) - 4' - 140.9 (C) - 5' 7.44 (1 hour, s) 112.6 (CH) 7.25 (1 hour, s) 6' - 111.8 (C) - 7' - 158.2 (C) - 3-OCH3 3.90 (3 hours, seconds) 61.7 (CH3 ) 3.96 (3 hours, seconds) 61.6 (CH3 ) 4-OCH3 4.04 (3 hours, seconds) 61.0 (CH3 ) 4.02 (3 hours, sec.) 57.0 (CH3 ) 4' - OCH3 4.04 (3 hours, seconds) 61.3 (CH3 ) 4.02 (3 hours, sec.) 56.5 (CH3 )

[0033] In one embodiment, betulinic acid is isolated as a white powder. The combination of 1H and 13C NMR data indicates the presence of a carboxylic acid carbonyl group and a C=C double bond, which together account for two of the seven degrees of unsaturation. Thus, the remaining five degrees of unsaturation are attributable to four hexacyclic rings and one pentacyclic core within the compound. The 1H NMR spectrum shows six tertiary methyl groups, two exomethylene protons (each 1H), one carbonyl proton, and several methine signals. Accordingly, the 13C NMR spectrum shows signals for thirty carbons, categorized as six methyl groups, eleven methylene groups, six methine groups, and seven quaternary carbons. Trimethoxyellagic acid is isolated in this study as a pale yellow amorphous powder; the structure of the compound is determined by comprehensive one- and two-dimensional NMR analysis.The 1H NMR spectrum shows five signals: two aromatic protons at δ 7.52 (s, 1H) and δ 7.44 (s, 1H) and three methyl groups at δ 3.90 (s, 3H), 4.04 (s, 3H), and 4.04 (s, 3H). The 13C NMR spectrum shows seventeen carbon signals, six of which are assigned to a sugar moiety and eleven to the aglycone residue, which are assigned to two methyl signals, two methine signals, two carbonyl carbons, and five quaternary carbons.

[0034] The identification of betulinic acid, a pentacyclic triterpenoid, and trimethoxyellagic acid in the stem bark extracts of A. leiocarpus purified with ethyl acetate and n-hexane in the present study is of extraordinary importance for elucidating the mechanisms of action of the antihyperglycemic and wound healing effects of A. leiocarpus in diabetes mellitus type 1 and 2 and the associated life-threatening complications.

[0035] The presence of betulinic acid and trimethoxyellagic acid in A. leiocarpus, with their tremendous medicinal properties, offers additional benefits in the treatment of type 1 and type 2 diabetes mellitus. Betulinic acid and trimethoxyellagic acid are therapeutically useful, synergistically, directly or as industrially synthesized products for remarkable enhancement of their activities. This hypothesis is enormously supported by the synthesis of betulinic acid derivatives through stepwise structure optimization, which exhibit enhanced anti-alpha-glycosidase activity as a treatment tool for type 2 diabetes mellitus. This latter glucose management process involves the inhibition of alpha-glycosidase, thus preventing carbohydrate hydrolysis and regulating blood glucose in postprandial hyperglycemia and the risk of diabetic complications.The discovery of trimethoxyellagic acid in the stem bark extracts of A. leiocarpus purified with ethyl acetate and n-hexane is quite remarkable overall, especially because, in the present study, it is also present in the interfacial precipitates from the purification processes. This interfacial precipitation is reproducible, as demonstrated by its production in enormous quantities in a previous study, which led to its use for bioassay studies and a strong identification of the erythropoietic, thrombopoietic, and hypoglycemic, and thus antidiabetic, activities in the interface. This discovery of trimethoxyellagic acid in the interface of the purified stem bark of A. leiocarpus is comprehensive and remarkable for its potential as a pharmacological antidiabetic compound.

[0036] 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 use of materials, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0037] 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 Device for the identification of betulinic acid and trimethoxyellagic acid as antidiabetic compounds in purified extract from the stem bark of Anogeissus leiocarpus. 102 collection unit 104 processing unit 106 Segmentation unit 108 Column Chromatography Unit 110 Dissolution and adsorption unit 110a container 110b glass column 112 Evaporation unit 114 Plurality of evaluation units 114a Fractionation column 114b Thin-layer chromatography (TLC) unit 114c Nuclear Magnetic Resonance Spectrometer (NMR)

Claims

[1] A device (100) for identifying betulinic acid and trimethoxyellagic acid as antidiabetic compounds in purified extract from the stem bark of Anogeissus leiocarpus, the device (100) comprising: a collection unit (102) for collecting stem bark samples of Anogeissus leiocarpus; a processing unit (104) connected to the collection unit (102) for pulverizing the collected samples, wherein the pulverized samples are subjected to cold maceration with 90-100% v / v ethanol to extract ethanolic extracts; a segmentation unit (106) connected to the processing unit (104) for dividing the ethanolic extract using ethyl acetate and n-hexane solvents; a column chromatography unit (108) connected to the segmentation unit (106) for inducing precipitation by adding cold acetone to the ethanolic extract; a dissolving and adsorption unit (110) comprising a container (110a) connected to the column chromatography unit (108) for dissolving the precipitate with a mixture of dichloromethane (DCM) and methanol, and a glass column (110b) for adsorbing the dissolved precipitate; an evaporation unit (112) connected to the dissolving and adsorption unit (110) for evaporating the solvent mixture and obtaining a free-flowing powder from the dissolved precipitate; and a plurality of evaluation units (114) connected to the evaporation unit (112) for identifying properties of the stem bark of Anogeissus leiocarpus. [2] Device according to claim 1, characterized by that a variety of phytochemical components are detected in the ethanolic extract. [3] Device according to claim 1 or 2, characterized bythat the mixture of DCM and methanol in a ratio of 9:1 is chosen to dissolve the precipitate. [4] Device according to claim 1, wherein the free-flowing powder is fractionated into 40 fractions over 50 grams of silica gel. [5] Device according to claim 1, wherein the plurality of evaluation units (114) comprises: a fractionation column (114a) containing silica gel for fractionating the free-flowing powder into several fractions; Extract - thin layer chromatography (TLC) (114b) operatively connected to the fractionation column (114a) to ignore gummy fractions and visualize light brown powder; and a nuclear magnetic resonance (NMR) spectrometer (114c) consisting of the solvents dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3) and acetone-d6 for determining the structure of purified compounds.

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