Efficient separation and preparation method of compound theanine diglucoside TFG

Abstract

The invention relates to the field of natural medicinal chemistry, and discloses an efficient separation and preparation method of a compound theanine diglucoside TFG. Although TFG structures are reported, separation from tea leaves is not disclosed. According to the invention, an efficient separation and purification method is established for the first time, and TFG is accurately extracted and identified from tea leaves by combining an HPLC-ELSD (High Performance Liquid Chromatography-Evaporative Light Scattering Detector) guided multi-stage column chromatography technology with nuclear magnetic resonance and mass spectrometry. Experiments prove that the TFG activates an antioxidant pathway, improves H2O2-induced HUVEC cell and HT22 cell damage, protects the vascular endothelial barrier function and neuronal activity, and is suitable for developing antioxidant drugs for treating Alzheimer's disease. Compared with the prior art, the invention fills the blank of separation of the tea-derived TFG, explores the new application of the tea-derived TFG in the field of cell protection, and provides a new thought for natural product development and functional food research.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicinal chemistry, and specifically relates to a method for efficiently separating and preparing the compound theanine disodium glycoside TFG, and its application in preparing vascular endothelial and neuroprotective drugs or functional health products based on the antioxidant activity of tea-derived TFG. Background Art

[0002] L-Theanine, a characteristic functional component of tea, accounts for over 50% of the total free amino acids in tea and is a key determinant of tea quality and bioactivity. During tea heat processing, the Maillard reaction in which theanine participates is a key pathway for the formation of characteristic flavor compounds. However, research on its use as a precursor to generate derivatives through glycosylation reactions presents significant technical gaps. Existing technologies primarily focus on the antioxidant and neuroprotective biological activities of theanine itself. However, systematic research on the isolation, identification, and functional exploration of glycoside derivatives, particularly disaccharide glycosides, generated through the Maillard reaction with sugars has yet to be conducted.

[0003] Existing literature indicates that theanine can react with monosaccharides (such as glucose) under heat treatment to form theanine monoglycoside derivatives, and these compounds have been isolated and reported in tea. Previous studies have revealed the chemical structures of two theanine disaccharide isomers (Theanine-fructosyl-glucose, TFG) (see: AmorePacific Co., Ltd. Theanine Derivatives, Preparation Methods, and Applications in Acne Relief: CN201180038302.2 [P]. 2014-12-31). Their structures are shown in Formulas (I) and (II), respectively. However, their natural occurrence in tea leaves, their specific structural characteristics (such as glycosidic linkage), and their potential for biological activity remain unclear. Although the theanine core structure possesses significant antioxidant and cytoprotective properties, experimental evidence remains lacking as to whether disaccharide modification can enhance or expand functional activities through structural changes. Currently, there are no public reports on the separation and identification of theanine disaccharide with a specific glycosidic bond connection pattern from the complex component system of tea. This technical gap has seriously restricted the application of this type of compound in the field of functional food and drug development.

[0004] In view of the above technical status, the present invention has established a multi-stage column chromatography separation method based on HPLC-ELSD detection for the first time, combined with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis technology, and successfully separated and identified TFG formula (I) from tea leaves, hereinafter referred to as TFG, and clarified that its molecular formula is C 19 H 34 N2O 13and a unique disaccharide glycoside bond connection structure. Experiments further confirmed that the tea-derived TFG significantly improved hydrogen peroxide (H2O2)-induced damage to human umbilical vein endothelial cells (HUVEC) by activating specific intracellular antioxidant signaling pathways, effectively protecting the vascular endothelial barrier function; at the same time, based on its improvement effect on damage to HUVEC and hippocampal neuronal cells (HT22), the compound is suitable for the development of antioxidant drugs for the treatment of Alzheimer's disease, in which TFG achieves synergistic protection of the neuron-vascular endothelial system by inhibiting oxidative stress response. The present invention fills the dual gaps in the isolation and identification of natural theanine disaccharide glycosides in tea and their functional applications, providing an innovative technical path for the in-depth development of theanine derivatives and the high-value utilization of tea active ingredients. Summary of the Invention

[0005] The present invention is the first to separate and identify theanine disaccharide (TFG) from the complex components of tea leaves. By using HPLC-ELSD detection-guided multi-stage column chromatography separation technology combined with nuclear magnetic resonance and mass spectrometry analysis, its molecular formula was confirmed to be C 19 H 34 N2O 13 The structural characteristics are consistent with those reported in the literature, confirming for the first time the natural existence of this compound in tea.

[0006] The tea-derived TFG in the present invention can significantly improve hydrogen peroxide (H2O2)-induced oxidative damage in human umbilical vein endothelial cells (HUVEC) by activating specific intracellular antioxidant signaling pathways, effectively protecting the barrier function of vascular endothelial cells, and revealing for the first time its biological activity in the fields of antioxidant and vascular endothelial protection.

[0007] To address the technical difficulties in separating TFG from tea leaves, the present invention establishes an efficient separation method comprising impurity removal using a macroporous adsorption resin, enrichment using a cation exchange resin, and fine purification using a polyacrylamide column. Combined with detection technology, the target component is precisely located, achieving highly selective acquisition of high-purity TFG from tea leaves.

[0008] The present invention not only fills the gap in the separation and identification of theanine disaccharide compounds in tea, but also provides a new material basis and technical path for the development of TFG-based antioxidant drugs, functional foods and vascular protection preparations through its improvement effect on oxidative stress damage. The compound theanine disaccharide has the molecular formula: C 19 H 34 N2O 13 , the structural formula is as follows (I): (I)

[0009] The structural formula of the isomers is as follows (II): (II)

[0010] The method for separating the compound of the present invention comprises the following steps:

[0011] Tea powder was extracted twice with distilled water, combined, and concentrated to 10 g / mL. Tea polyphenols and other macromolecular pigments were then eluted through a macroporous resin, and the filtrate was collected and lyophilized. The lyophilized powder was redissolved in distilled water, passed through a 732 cation exchange resin, and the ninhydrin-positive fraction was collected and lyophilized. Finally, the lyophilized powder was dissolved in 0.2% aqueous acetic acid, passed through a polyacrylamide column (Bio-gel P-2 column), and the fraction with an Rf = 0.480 was collected and lyophilized to obtain compound TFG. Compound TFG is a white flocculent solid. HPLC-ELSD analysis shows an absorption peak retention time of 23.83 min. The present invention further provides the use of the compound in the preparation of antioxidant drugs, specifically, in the preparation of drugs for preventing, treating, or ameliorating H2O2-induced oxidation in human umbilical vein endothelial cells.

[0012] The present invention further provides a pharmaceutical composition, which optionally includes one or more excipients, wherein the excipients are selected from one or more of microcrystalline cellulose, lactose, starch, pregelatinized starch, magnesium stearate, talc, cross-linked polyvinylpyrrolidone, and cross-linked sodium carboxymethyl cellulose.

[0013] This study isolated theanine-fructosyl-glucose (TFG) from tea leaves for the first time. Using HPLC-ELSD-guided multistage column chromatography combined with nuclear magnetic resonance and mass spectrometry analysis, the unique disaccharide linkage structure was confirmed. Experiments confirmed that this tea-derived TFG significantly ameliorated hydrogen peroxide (H2O2)-induced oxidative damage in human umbilical vein endothelial cells (HUVECs) by activating specific intracellular antioxidant signaling pathways, effectively protecting the vascular endothelial cell barrier function. This study not only fills the gap in the isolation and identification of natural TFG from tea leaves but also, based on its well-established antioxidant cytoprotective activity, provides a novel material foundation and technological path for the development of antioxidant drugs, functional foods, and vascular protection-related preparations.

[0014] Alternatively, the application is preferably an application in the preparation of a drug for preventing, treating or improving hydrogen peroxide (H2O2)-induced oxidative stress in mouse hippocampal neuronal cells.

[0015] Based on the above, TFG is suitable for the development of antioxidant drugs for the treatment of Alzheimer's disease, and at the same time provides a new material basis for antioxidant functional foods and vascular protection preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The properties of TFG of the present invention are

[0017] Figure 2 This is the TLC analysis chart of TFG of the present invention

[0018] Figure 3 Schematic diagram of HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector) determination of TFG of the present invention

[0019] Figure 4 The TFG of the present invention 13 C-NMR spectrum

[0020] Figure 5 The TFG of the present invention 1 H-NMR spectrum

[0021] Figure 6 The HMBC spectrum of TFG of the present invention is

[0022] Figure 7 HSQC spectrum of TFG of the present invention

[0023] Figure 8 HR-ESI-MS diagram of TFG of the present invention DETAILED DESCRIPTION

[0024] The separation and purification method of the present invention will be further described below with reference to specific examples, but the scope of protection of the present invention is not limited thereto.

[0025] Example 1 Isolation and Preparation of the Compound TFG of the Present Invention

[0026] The structural formula of the compound TFG of this embodiment is as follows: (I)

[0027] 1. The method for separating and preparing the compound TFG comprises the following steps:

[0028] A. Extraction: Weigh 100 g of tea powder (passed through a 100-mesh sieve) and add 50-fold the volume of distilled water. Extract at room temperature for 8 h (solid-to-liquid ratio: g / mL, rotation speed: 500 rpm). Repeat the distilled water extraction twice. Combine the extracts and concentrate under reduced pressure at 30°C to 1 / 15 of the original volume to obtain the tea water extract concentrate.

[0029] B. Removal of tea polyphenols and other macromolecular pigments: Utilize a macroporous adsorption resin column, elute with distilled water, and collect the eluted portion for concentration and freeze-drying.

[0030] C. Dissolve the above lyophilized powder in double-distilled water (concentration: 1 g / mL). Pass it through a 732 cation exchange resin and elute with 1.5 mol / L ammonia. Collect the eluates, combine the eluates rich in TFG components, collect the ninhydrin-positive fractions, and concentrate and lyophilize them.

[0031] D. Dissolve the above lyophilized powder in 0.2% acetic acid (volume ratio) to a 1 g / mL solution. Pass the solution through a polyacrylamide (Bio-gel P-2) column and elute with 0.2% acetic acid (volume ratio) at a flow rate of 0.3 mL / min. Collect the fraction with Rf = 0.480 and lyophilize to obtain the compound TFG.

[0032] Compound TFG is a white flocculent solid. Figure 1 , as analyzed by thin layer chromatography Figure 2 , the Rf of the compound is 0.480. Figure 3 The retention time of the absorption peak of this compound is 23.83 min.

[0033] 2. The structure of the monomer compound was identified using modern nuclear magnetic resonance technology and high-resolution mass spectrometry HR-ESI-MS.

[0034] According to one-dimensional nuclear magnetic resonance (1D NMR) spectrum analysis, the hydrogen spectrum (1HNMR) of theanine disaccharide (TFG) of the present invention shows 25 hydrogen proton signals, which are mainly concentrated in the chemical shift range of 3.0~4.0ppm ( Figure 5 ); Combined with the carbon spectrum (13CNMR) test results, it was confirmed that the compound contains 19 carbon atoms ( Figure 4 ). The specific signal assignments are detailed in Table 1. The sugar moiety exhibits 11 carbon signals, and the characteristic peaks at chemical shifts of 100.57 and 95.41 ppm clearly indicate the presence of two sugar units. Through heteronuclear multiple bond correlation spectroscopy (HMBC) analysis, a long-range correlation signal between H-3′ and C-1′′ was observed, based on which the connection mode between sugar moieties was determined ( Figure 6 and Figure 7 ). Comprehensive NMR and mass spectrometry data ( Figure 8 ), confirming that the molecular formula of the compound is C 19 H 34 N2O 13 The structural characteristics are consistent with theanine disaccharide reported in the literature, but it is the first time that it is isolated from tea.

[0035] Table 1 Compound TFG 1 H-NMR and 13 C-NMR chemical shift and HMBC data (D2O)

[0036] As shown in Table 2, although the synthetic method is simple in steps, it uses theanine and maltose as raw materials and reacts with methanol reflux, resulting in an unseparated mixture. However, Example 1 uses tea leaves as a natural source and, through multi-stage purification (resin adsorption → ion exchange → gel chromatography) combined with modern analytical techniques (HPLC, NMR, HR-MS), not only obtains high-purity monomeric TFG but also confirms for the first time the natural presence of this compound in tea leaves.

[0037] Table 2 Comparison of the TFG separation method of this patent and the method of patent CN201180038

[0038] Example 2

[0039] The compound TFG of the present invention has a protective effect on H2O2-induced human umbilical vein endothelial cells HUVEC cells.

[0040] 1. Drug preparation

[0041] TFG (structural formula shown in Formula I, previously referred to as TFG, hereinafter referred to as TFG) and TFG (structural formula shown in Formula II, reported in CN201180038302.2) were dissolved in DMEM high-glucose medium to prepare a 1 mM stock solution. The stock solution was then diluted to concentrations of 1.25, 2.5, 5, and 10 μM, respectively. H₂O₂ was diluted in DMEM high-glucose medium to a 100 mM solution. TFG is inherently unstable and should be prepared freshly before use. The entire process must be performed under sterile conditions. After dissolution, the solution must be filtered through a 0.22 μM filter membrane before use.

[0042] 2. Cell culture

[0043] HUVEC (human umbilical vein endothelial cell) cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in a thermostat at 37°C and 5% CO2. The culture medium used was Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. Formal experiments were performed when HUVEC cells exhibited a normal karyotype, adhered to the cell wall and were in the logarithmic growth phase, reaching a cell density of 80–90%.

[0044] 3. MTT assay to test the effect of TFG on cell viability

[0045] HUVEC cells were seeded in 96-well culture plates and treated with H₂O₂ and varying concentrations of TFG. Then, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation at 37°C for 3.5 hours, dark blue formazan crystals formed. The supernatant was discarded, and 150 μL of DMSO solution was added to each well to dissolve the formazan crystals. The plate was shaken and mixed for 5 minutes. The absorbance was then measured at 490 nm using a DR-200B microplate reader. The viability of the group treated with DMEM alone was normalized to 100%.

[0046] 4. Antioxidant activity (MDA, SOD, CAT, GSH-Px) detection

[0047] HUVEC cells were seeded in 6-well culture plates and treated with H2O2 and different concentrations of TFG. The cells were then mixed with RIPA lysis buffer (with protease inhibitors and phosphatase inhibitors added in proportion) for protein extraction. The MDA, SOD, CAT, and GSH-Px detection kits were followed according to their instructions. The absorbance was measured using a DR-200B microplate reader, and the antioxidant activity was calculated based on the absorbance.

[0048] 5. Data processing

[0049] All data were expressed as mean ± standard deviation (mean ± SD) established in different experiments and analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Statistical graphs were generated by using GraphPad Prism 8.0.2 software (GraphPad Software, Inc, San Diego, USA). p <0.05, p <0.01 or p The difference was statistically significant when the value was <0.001.

[0050] 6. Results

[0051] The protective effect of TFG on H2O2-induced oxidative stress in human umbilical vein endothelial cells (HUVEC) is shown in Tables 2 and 3. According to the MTT results, the MTT assay was used to determine the effect of TFG at different concentrations (1.25, 2.5, 5, and 10 μM) on HUVEC cell viability with or without H2O2 addition. After H2O2 treatment, cell viability was significantly decreased ( *** p < 0.001), TFG showed better protective activity ( ### p < 0.001, # p< 0.05), while TFG (Ⅱ) ( # p < 0.05) was weaker than TFG. MDA, SOD, CAT and GSH-Px assays were used to test the effects of different concentrations (1.25, 2.5, 5 and 10 μM) of TFG and TFG (II) on HUVEC cells with or without H2O2 treatment. The results showed that H2O2 treatment significantly increased the MDA level in HUVEC cells ( *** p < 0.001), SOD, CAT and GSH-Px levels were significantly decreased ( *** p <0.001); after TFG intervention, the abnormal changes of the above indicators were significantly improved. Among them, TFG (10 μM) showed the best protective effect and could significantly alleviate the oxidative stress damage of HUVEC cells induced by H2O2 (compared with the H2O2 treatment group, ### p <0.001; ## p < 0.01; # p < 0.05). The experimental data showed that TFG can effectively reduce H2O2-induced oxidative stress damage in HUVEC cells. It can be concluded that TFG has a better advantage in protecting H2O2-induced oxidative stress in HUVEC cells. However, the biological activity of TFG (Ⅱ) (compared with the H2O2 treatment group, ## p < 0.01; # p < 0.05) was significantly lower than that of TFG.

[0052] Table 2 Effect of TFG on H2O2-induced HUVEC cell viability

[0053] Table 3 Effects of TFG on basic oxidative indices of HUVEC cells induced by H2O2 (MDA / SOD / CAT / GSH-Px)

[0054] Example 3

[0055] The compound TFG of the present invention has a protective effect on H2O2-induced mouse hippocampal neuronal cells HT22 cells.

[0056] 1. Drug preparation

[0057] TFG (structural formula shown in Formula I, previously referred to as TFG, hereinafter referred to as TFG) and TFG (structural formula shown in Formula II, reported in CN201180038302.2) were dissolved in DMEM high-glucose medium to prepare a 1 mM stock solution. The stock solution was then diluted to concentrations of 1.25, 2.5, 5, and 10 μM, respectively. H₂O₂ was diluted in DMEM high-glucose medium to a 100 mM solution. TFG is inherently unstable and should be prepared freshly before use. The entire process must be performed under sterile conditions. After dissolution, the solution must be filtered through a 0.22 μM filter membrane before use.

[0058] 2. Cell culture

[0059] HT22 cell lines (mouse hippocampal neurons) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in a thermostat at 37°C and 5% CO2. The culture medium used was DMEM supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. For formal experiments, HT22 cells exhibited a normal karyotype under a microscope, were adherent and in the logarithmic growth phase, and reached a confluence of 80–90%.

[0060] 3. MTT assay to test the effect of TFG on cell viability

[0061] HT22 cells were seeded in a 96-well culture plate and treated with H₂O₂ and various concentrations of TFG. Then, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation at 37°C for 3.5 hours, dark blue formazan crystals formed. The supernatant was discarded, and 150 μL of DMSO solution was added to each well to dissolve the formazan crystals. The plate was shaken and mixed for 5 minutes. The absorbance was then measured at 490 nm using a DR-200B microplate reader. The viability of the group treated with DMEM alone was normalized to 100%.

[0062] 4. Antioxidant activity (MDA, SOD, CAT, GSH) detection

[0063] HT22 cells were seeded in 6-well culture plates and treated with H2O2 and different concentrations of TFG. The cells were then mixed with RIPA lysis buffer (with protease inhibitors and phosphatase inhibitors added in proportion) for protein extraction. The MDA, SOD, CAT, and GSH detection kits were followed according to their instructions. The absorbance was measured using a DR-200B microplate reader, and the antioxidant activity was calculated based on the absorbance.

[0064] 5. Data processing

[0065] All data were expressed as mean ± standard deviation (mean ± SD) established in different experiments and analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Statistical graphs were generated by using GraphPad Prism 8.0.2 software (GraphPad Software, Inc, San Diego, USA). p <0.05, p <0.01 or p The difference was statistically significant when the value was <0.001.

[0066] 6. Results

[0067] The protective effects of TFG and TFG (II) on oxidative stress in mouse hippocampal neuronal cells HT22 induced by H2O2 are shown in Tables 4 and 5. According to the MTT results, the MTT assay was used to determine the effect of TFG at different concentrations (1.25, 2.5, 5, and 10 μM) on the viability of HT22 cells with or without the addition of H2O2. The cell viability was significantly decreased after H2O2 treatment ( *** p <0.001), TFG showed better protective activity ( ### p < 0.001, # p < 0.05). TFG (Ⅱ) administered at the same dose showed lower protective activity ( # p < 0.05). Subsequently, the effects of different concentrations (1.25, 2.5, 5, and 10 μM) of TFG and TFG (II) on HT22 cells with or without H2O2 treatment were detected using MDA, SOD, CAT, and GSH assays. The results showed that H2O2 treatment significantly increased the MDA level in HT22 cells ( *** p < 0.001), SOD, CAT and GSH levels were significantly decreased ( *** p < 0.001); after TFG intervention, the abnormal changes of the above indicators were significantly improved. Among them, TFG (10 μM) showed the best protective effect and could significantly alleviate the oxidative stress damage of HT22 cells induced by H2O2 (compared with the H2O2 treatment group, ### p < 0.001; ## p < 0.01; # p< 0.05). The experimental data showed that TFG can effectively reduce the oxidative stress damage of HT22 cells induced by H2O2. It can be concluded that TFG has a better advantage in protecting HT22 cells from oxidative stress induced by H2O2. However, the biological activity of TFG (Ⅱ) (compared with the H2O2 treatment group, ## p < 0.01; # p < 0.05) was lower than that of TFG.

[0068] It can be concluded that the present reaction TFG has a better advantage in protecting HT22 cells from oxidative stress induced by H2O2.

[0069] Table 4 Effect of TFG on H2O2-induced HT22 cell viability

[0070] Table 5 Effects of TFG on basic oxidative indices (MDA / SOD / CAT / GSH) of HT22 cells induced by H2O2

[0071] The present invention has been described in detail above with reference to preferred embodiments and exemplary examples. However, it should be noted that these specific embodiments are merely illustrative explanations of the present invention and do not constitute any limitation on the scope of protection of the present invention. Various improvements, equivalent substitutions, or modifications may be made to the technical content of the present invention and its embodiments without departing from the spirit and scope of protection of the present invention, and all of these fall within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the appended claims.

Claims

1. An efficient separation and preparation method for the compound Theanine disaccharide TFG, the compound is named Theanine-fructosyl-glucose, and the molecular formula is: C 19 H 34 N2O 13 , the structural formula is as follows: ; It is characterized in that, The following steps are involved: A. Extraction: Pass tea powder through a 100-mesh sieve. Add distilled water at a material-liquid ratio of 1g:50mL. Extract at room temperature for 8 hours at 500 rpm. Repeat the distilled water extraction twice. Combine the extracts and concentrate under reduced pressure at 30°C to 1 / 15 of the original volume to obtain the tea water extract concentrate. B. Impurity Removal: Pass the concentrated solution obtained in step A through a macroporous adsorption resin column, elute with distilled water, collect the eluate, concentrate it, and then freeze-dry it; C. Dissolve the above lyophilized powder in double-distilled water to a concentration of 1 g / mL. Pass it through a 732 cation exchange resin and elute with 1.5 mol / L ammonia. Collect the eluates, combine the eluates rich in TFG components, collect the ninhydrin-positive fraction, and concentrate and lyophilize. D. Dissolve the above lyophilized powder in 0.2% acetic acid (volume ratio) to a 1 g / mL solution. Pass the solution through a polyacrylamide Bio-gel P-2 column and elute with 0.2% acetic acid (volume ratio) at a flow rate of 0.3 mL / min. Collect the fraction with Rf = 0.480 and lyophilize to obtain the compound TFG.

2. A compound TFG prepared by the method of claim 1, characterized in that: It is a white flocculent solid that easily absorbs moisture and decomposes at room temperature. It needs to be sealed and stored at -20°C. The retention time detected by HPLC-ELSD is 23.83 minutes. Nuclear magnetic resonance and mass spectrometry analysis confirms its unique structure derived from tea.

3. Use of the compound TFG according to claim 2 in the preparation of a medicine or health product for preventing or treating oxidative stress-related diseases.

4. The use according to claim 3, characterized in that The oxidative stress-related disease is selected from neurodegenerative diseases or vascular diseases; preferably, the disease is Alzheimer's disease.

5. The use according to claim 3 or 4, characterized in that The TFG protects human vascular endothelial cells HUVEC and mouse hippocampal neuronal cells HT22 damaged by hydrogen peroxide H2O2 by activating intracellular antioxidant signaling pathways and inhibiting oxidative stress reactions, thereby maintaining vascular endothelial barrier function and neuronal activity.

6. A pharmaceutical composition, characterized in that The invention comprises the compound TFG according to claim 2 and a pharmaceutically acceptable excipient; the excipient is selected from one or more of microcrystalline cellulose, lactose, starch, pregelatinized starch, magnesium stearate, talc, cross-linked polyvinylpyrrolidone, and cross-linked sodium carboxymethyl cellulose.

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