Natural anti-inflammatory agent

Compared to the acetic acid control, both Diammonium Glycyrrhizinate (DG) and dexamethasone showed a significant anti-inflammatory effect (P < 0.01). The expression of NF-kappaB, TNF-alpha and ICAM-1 in colonic mucosa was significantly lower in the Diammonium Glycyrrhizinate group and dexamethasone group than in the acetic acid group. Conclusion: Diammonium Glycyrrhizinate could reduce inflammatory injury in a rat model of ulcerative colitis. This may occur via suppression of NF-kappaB, TNF-alpha and ICAM-1 in colonic mucosa. [1]

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DAI, morphologic injury, histological changes, and MPO activity [1]
1-2 d after colonic infusion of acetic acid, rats displayed diarrhea, pyemic stool, and reduced body weight. Morphologically, a dilated lumen, thickened wall, and brown or black color was observed continuously in the injured bowel. Edema, erosions, necrosis, superficial ulcerations, crypt abscesses, and inflammatory infiltration into the lamina propria were observed in the injured segment by light microscopy. In Table 3, according to DAI, scores of morphological and histological changes, and MPO activity, the colon showed significant pathogenic changes in the Diammonium Glycyrrhizinate (DG), dexamethasone, and acetic acid control groups compared to the normal control group that received saline alone (P < 0.01), which demonstrated that acetic acid infusion results in injuries that are comparable to those seen in humans with ulcerative colitis. These inflammatory indices were significantly improved by DG and dexamethasone (P < 0.01). The anti-inflammatory effect of DG was significantly lower than that of dexamethasone (P < 0.01).

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Expression of NF-κB p65, TNF-α and ICAM-1 in injured colon [1]
In rats that received acetic acid, NF-κB p65 was positive mainly in nuclei of most endothelial cells, epithelial cells and mononuclear cells, especially in the mucosa and submucosa. TNF-α and ICAM-1 were positive mainly in the cytoplasm, membrane and rarely in nuclei. ICAM-1 was positive in most endothelial cells and macrophages. TNF-α positive cells, including mononuclear cells, macrophages and neutrophils, were located densely in lamina propria and in proximity to the muscularis. The percentage of cells positive for these three molecules was significantly correlated with the degree of inflammatory injury (Table 4), and these markers were rarely expressed in samples taken from the normal control group. The positive percentage and density of NF-κB p65, TNF-α and ICAM-1 in injured colon was significantly higher than that in normal control. After Diammonium Glycyrrhizinate (DG) or dexamethasone treatment, the positive percentage and density of these molecules were reduced significantly, which indicates that both DG and dexamethasone may inhibit the expression of these molecules. Also, the expression of these molecules was significantly lower in DG treated samples than in dexamethasone treated samples (P < 0.01).

Spragur-Dawley female rats were divided into four groups: Diammonium Glycyrrhizinate (DG) group, dexamethasone group, acetic acid control and normal control group. Colonic inflammation was evaluated by disease activity index, gross morphologic damage, histological injury and colonic myeloperoxidase activity. Immunohistochemistry was used to detect the expression of NF-kappaB, TNF-alpha and ICAM-1 in colonic mucosa. [1]

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Preparation of animal model: Forty SD rats were divided into four groups: Diammonium Glycyrrhizinate (DG) group, dexamethasone group, acetic acid control and normal control group. All rats were fasted for twenty-four hour. Before the colonic infusion of acetic acid, 0.3 mL (30 mg/kg) Natrium pentobarbital was injected peritoneally. A polyethylene catheter was put into the colon extending a distance of eight centimeters beyond the anus. For DG, dexamethasone, and the acetic acid control groups, 1 mL of 10% (v/v) acetic acid was infused into the colon through this catheter, held in place for 30 s, and then flushed with 5 mL normal saline. Only normal saline was infused into the colon in the normal control group. In the DG group, 40 mg/kg DG was injected intraabdominally every day for one week; in dexamethasone group, 0.2 mg/kg dexamethasone was injected intraabdominally cavity daily for one week; in the acetic acid control and normal control groups, equal volumes of normal saline were injected into the abdominal cavity daily for one week. [1]

Absorption, Distribution and Excretion
Glycyrrhizic acid is absorbed in the small intestine of rats; glycyrrhetinic acid is not detected in the blood after rapid intravenous injection of glycyrrhizic acid via the portal vein; glycyrrhetinic acid is detectable in the blood after oral administration. SAKYA et al.; Bulletin of Chemical Pharmacy 27(5) 1125 (1979)
High performance liquid chromatography (HPLC) can rapidly and accurately determine glycyrrhizic acid (GZA) and glycyrrhetinic acid (GRA) in the body fluids and tissues of laboratory animals and humans. Glycyrrhizic acid and glycyrrhetinic acid can be extracted from plasma and tissues, and the extracts can be directly used for HPLC analysis. Extraction and determination of glycyrrhizic acid and glycyrrhetinic acid from bile or urine are difficult due to interference from endogenous compounds and the binding of glycyrrhetinic acid with glucuronide or sulfate. Methods for extracting glycyrrhizic acid and glycyrrhetinic acid from urine or bile include extraction with organic solvents or solid-phase extraction after ion-pair binding. Glycyrrhetinic acid conjugates can be determined by chromatographic separation or pretreatment with β-glucuronidase. The pharmacokinetics of glycyrrhetinic acid and glycyrrhizic acid can be described using a biphasic elimination model, which shows elimination from the central compartment followed by a dose-dependent second elimination phase. Depending on the dose, the half-life of glycyrrhizic acid in the second elimination phase in humans is 3.5 hours, while the half-life of glycyrrhetinic acid is 10–30 hours. Both glycyrrhizic acid and glycyrrhetinic acid are largely excreted via bile. Glycyrrhizic acid can be excreted in its unmetabolized form and undergoes enterohepatic circulation; while glycyrrhetinic acid is conjugated with glycyrrhetinic acid glucuronide or sulfate before bile excretion. Orally administered glycyrrhizic acid is almost completely hydrolyzed by intestinal bacteria and enters systemic circulation as glycyrrhetinic acid. PMID:8191540
Glycyrrhizic acid currently has clinical application value in the treatment of chronic hepatitis. It is also used as a sweetener in food and chewing tobacco. Serious side effects such as hypertension and electrolyte disturbances have been reported in some high-exposure populations. To analyze the health risks of exposure to this compound, this study quantitatively assessed the kinetics of glycyrrhizic acid and its active metabolites. Glycyrrhizic acid and its metabolites are subject to complex kinetic processes, including enterohepatic circulation and first-pass metabolism. Detailed information on these processes is often difficult to obtain in humans. Therefore, this study developed a model to describe the systemic and gastrointestinal kinetics of glycyrrhizic acid and its active metabolite, glycyrrhetinic acid, in rats. Because this model is based on physiological structures, it directly integrates previous in vitro and in vivo data on absorption, enterohepatic circulation, and first-pass metabolism. The model shows that glycyrrhizic acid and its metabolites are efficiently transported from plasma to bile, possibly involving the hepatic transporter 3α-hydroxysteroid dehydrogenase. Following glycyrrhizic acid reabsorption, the bile-excreted metabolites are hydrolyzed by bacteria, resulting in the observed delayed terminal plasma clearance of glycyrrhizic acid. These mechanistic findings, derived from the analysis of experimental data using a physiologically based pharmacokinetic model, can ultimately be used to quantitatively assess the health risks of human exposure to products containing glycyrrhizic acid. Copyright 2000 Academic Press. PMID: 10652246
To assess the diversity of bile excretion of xenobiotic conjugates, this study investigated the bile excretion of glycyrrhizic acid (glycyrrhizin) in rats after intravenous injection of 10 mg/kg glycyrrhizic acid, followed by intravenous infusion of the inhibitors dibromosulfonphthalein and indocyanine green, respectively. Indocyanine green did not affect the bile excretion of glycyrrhizic acid, while dibromosulfonphthalein significantly reduced its excretion. Dibromosulfonphthalein sodium increased plasma glycyrrhizic acid concentration, while indocyanine green had no such effect. In Eisai hyperbilirubinemia rats, bile excretion of glycyrrhizic acid was severely impaired, leading to elevated plasma concentrations. The results indicate that bile excretion of glycyrrhizic acid is mediated by a system shared by glycyrrhizin glucuronide and dibromosulfonphthalein sodium, rather than indocyanine green, and that this system is genetically defective in Eisai hyperbilirubinemia rats. PMID:8987080

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Metabolism/Metabolites
Following rapid intravenous injection of glycyrrhizic acid into the portal vein of rats, the level of a substance in the blood increased, which appears to be a glucuronide conjugate formed from the metabolism of glycyrrhizic acid. SAKYA et al.; Chemical Pharmacy Bulletin 27(5) 1125 (1979)

Interactions
Adding 10⁻⁶ M glycyrrhizic acid in the presence of 10⁻⁸ M aldosterone significantly stimulated short circuits compared to control skin treated with aldosterone alone. PMID: 6973465
Administration of glycyrrhizic acid inhibited the effect of injected cortisone on glycogen storage and enhanced the immunosuppressive effect of cortisone. KUMAGAI A; TAISHA 10 632 (1973)
To confirm whether oral glycyrrhizic acid (GL) affects the metabolism of prednisolone in humans, we investigated the pharmacokinetics of total prednisolone and free prednisolone (PSL) in six healthy men with or without oral glycyrrhizic acid (GL) pretreatment. Each subject received an intravenous injection of 0.096 mg/kg prednisolone succinate (PSL-HS), and some subjects received a pre-oral injection of 50 mg glycyrrhizic acid four times. Blood samples were collected from peripheral veins at 5, 10, 15, 30, and 45 minutes and 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 hours after the start of prednisolone-HS infusion. The concentration of total prednisolone in plasma was analyzed by high-performance liquid chromatography (HPLC), and the concentration of free prednisolone was determined by isocolloid osmotic equilibrium dialysis. Pharmacokinetic parameters of prednisolone were determined using a non-compartmental model analysis. The study found that oral administration of glycyrrhizic acid significantly increased the concentration of total prednisolone at 6 and 8 hours after prednisolone hemisuccinate infusion, as well as the concentration of free prednisolone at 4, 6, and 8 hours. Furthermore, oral administration of glycyrrhizic acid altered the pharmacokinetics of both total and free prednisolone. After oral administration of glycyrrhizic acid, the area under the curve (AUC) significantly increased, the total plasma clearance (CL) significantly decreased, and the mean residence time (MRT) significantly prolonged. However, no significant change was observed in the volume of distribution (Vdss). This indicates that oral administration of glycyrrhizic acid increases the plasma concentration of prednisolone and affects its pharmacokinetics by inhibiting its metabolism rather than affecting its distribution. PMID:1752235
To clarify whether glycyrrhizic acid (aqueous extract of licorice root, a drug used to treat chronic active hepatitis) can prevent liver injury induced by carbon tetrachloride, allyl formate, and endotoxins, this study was conducted in rats. Administration of glycyrrhizic acid 20 hours before carbon tetrachloride administration protected against periportal hepatocyte necrosis. Administration of glycyrrhizic acid 2 hours before allyl formate administration also inhibited periportal hepatocyte necrosis. However, glycyrrhizic acid did not protect against endotoxin-induced focal and random hepatocyte necrosis. These results suggest that glycyrrhizic acid has no protective effect against liver injury caused by endotoxin-induced sinusoidal circulation disturbance, but it can protect hepatocytes from hepatotoxic damage caused by direct effects of hepatotoxins such as carbon tetrachloride and allyl formate. PMID:2767217
This study aims to investigate the effect of Potenlini on the binding activity of nuclear factor-κB (NF-κB) in the liver of an animal model of liver cirrhosis, and to elucidate the molecular mechanism of Potenlini's biological activity. Methods: Male SD rats were randomly divided into a normal control group, a model control group, and a Potenlini group. The latter two groups of rats were treated with carbon tetrachloride and ethanol solutions, respectively, to induce chronic liver injury. The rats in the Potenlini group were treated with Potenlini at the same time. All rats were sacrificed at week 9 after administration of carbon tetrachloride (CCl4). Serum and liver specimens were collected to assess serum alanine aminotransferase (ALT) activity and histological changes. Nuclear extracts of liver tissue were prepared and gel retardation assays were performed to assess nuclear factor-κB (NF-κB) activity. Results: (1) Compared with the model control group rats with significantly elevated ALT levels, the serum ALT level of the Potenlini treatment group rats was significantly reduced. (2) Histological examination showed that the liver of rats in the model group was severely steatotic and fibrotic, while the degree of steatotic and fibrotic liver of rats in the Potenlini treatment group was significantly reduced. (3) Compared with normal liver, the NF-κB binding activity in the liver specimens of rats in the model control group was significantly increased, while the NF-κB binding level in the liver of rats in the Potenlini treatment group was close to normal. Conclusion: Potenlini can inhibit the binding activity of NF-κB in chronic liver injury induced by carbon tetrachloride and ethanol, which may be part of the mechanism by which Potenlini protects the liver from hepatotoxic-induced liver injury and cirrhosis. PMID: 10366987

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Human toxicity excerpt
14 volunteers took 100-200 g of licorice daily, equivalent to 0.7-1.4 g of glycyrrhizic acid, for 1-4 weeks. The plasma potassium concentration of all subjects decreased; the plasma renin and urinary aldosterone levels were inhibited.
... It is speculated that the oral toxicity of this compound and its derivatives is low. Licorice and its derivatives
Some people may experience potentially serious metabolic disorders after consuming small amounts of licorice daily for less than a week.
Glycyrrhizic acid completely inhibited the growth and cytopathic effects of vaccinia virus, herpes simplex virus type 1, Newcastle disease virus, and vesicular stomatitis virus in human aneuploid HEP2 cell cultures. POMPEI R et al.; Nature (London) 281(5733):689(1979)

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Non-human toxicity excerpt
In a 10-week dietary exposure, weight gain in rats showed a dose-response effect. The number of dead implants during the first week of reproduction was significantly increased after adding 40,000 ppm to the rat diet.
Glycyrrhizic acid promotes sodium retention by inhibiting corticosteroids in the liver and inhibits the metabolism of progesterone and aldosterone.
Glycyrrhizic acid and its derivatives inhibited the 5β-reduction of cortisol, aldosterone, and testosterone in rat liver preparations in vitro, with a greater degree of inhibition than 5α-reduction.
In rat and mouse studies, results from single parenteral and oral administrations of ammonium glycyrrhizinate indicated that the compound was associated with near-non-toxicity. Repeated (30 times) intragastric administration at the maximum daily therapeutic dose (7 mg/kg) and four times the dose (28 mg/kg) did not induce toxic symptoms, significant changes in hematological and comprehensive indicators, alterations in serum enzyme activity, or morphological changes in visceral organ cell structure. A second administration at a dose of 28 mg/kg resulted in alterations in the activity of certain enzymes in the brain and hepatic dystrophy, subsequently progressing to eosinophilic necrosis with signs of regeneration. Under subacute experimental conditions, the maximum daily therapeutic dose of ammonium glycyrrhizinate can be considered practically non-toxic.

[1]. Anti-inflammatory effect of Diammonium Glycyrrhizinate in a rat model of ulcerative colitis. World J Gastroenterol. 2006 Jul 28;12(28):4578-81.

Diammonium glycyrrhizate is the diammonium salt of glycyrrhizic acid, the active ingredient of the traditional Chinese medicine licorice (Glycyrrhiza uralensis, also known as Chinese licorice or licorice root), which has anti-inflammatory, antioxidant, and hepatoprotective effects. Diammonium glycyrrhizate (DG) is slowly metabolized intracellularly to glycyrrhizic acid, which inhibits enzymes controlling cortisol metabolism, thereby exerting its anti-inflammatory effect. Although its exact mechanism of action is not fully elucidated, DG may prevent or reduce hepatotoxicity by scavenging free radicals. Furthermore, this drug can upregulate the expression of the transcriptional coactivator PGC-1α and regulate the activity of liver enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase, and glutathione peroxidase. Diammonium glycyrrhizate is a widely used anti-inflammatory drug isolated from licorice root. It is metabolized to glycyrrhizic acid, which inhibits 11β-hydroxysterol dehydrogenase and other enzymes involved in corticosteroid metabolism. Therefore, glycyrrhizic acid (the main sweet component of licorice) has been studied for its ability to induce hyperchlorocinemia (with sodium retention and potassium loss), edema, increased blood pressure, and inhibition of the renin-angiotensin-aldosterone system.
See also: Glycyrrhizic acid (note moved to).

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Therapeutic Uses
Lubricant, mild laxative; expectorant; used to mask the taste of medicines
In Japan, the active ingredient of SNMC (potent neominophen C), glycyrrhizic acid (a saponin extracted from licorice), has been used to improve liver function. To evaluate the efficacy of interferon (IFN) combined with a more potent Neominophagen C in patients unresponsive to interferon monotherapy, we studied 28 patients histologically diagnosed with CAH 2B after 12 weeks of interferon treatment. Fifteen patients continued interferon monotherapy (Group A), and 13 patients received interferon combined with a more potent Neominophagen C after 12 weeks of interferon treatment (Group B). In groups A and B, 33.3% and 64.3% of patients, respectively, had their serum ALT levels return to normal. In groups A and B, 13.3% and 38.5% of patients, respectively, had absent serum HVC RNA, but these differences were not statistically significant. According to the Knodel HAI score, there was no significant difference in histological improvement between groups A and B, but reversal of histological grade (European grade) was more common in group B. The article also reports a case of hepatitis C following a blood transfusion, which was exacerbated by interferon treatment. After interferon administration, HLA class I antigen expression in liver tissue was significantly enhanced. In this case, enhanced cellular immunity was considered the cause of the exacerbation, and interferon combined with the more potent Neominophagen C therapy helped improve liver function. PMID:7521424
Licorice (Glycyrrhiza glabra) is a Mediterranean plant that has been used in China for generations as a traditional medicine, including antidotes, skin moisturizers, and elixirs. The main water-soluble component of licorice is glycyrrhizic acid (GL), which has been shown to possess a variety of pharmacological activities. This study demonstrates that oral administration of glycyrrhizic acid significantly protects Sencar mice from skin tumors induced by 7,12-dimethylbenzo[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Compared to animals not fed glycyrrhizic acid, animals fed glycyrrhizic acid showed a significantly prolonged latency period before tumor development, resulting in a significant reduction in the number of tumors per mouse during and at the end of the experiment. Addition of glycyrrhizic acid to drinking water also inhibited the binding of topically applied [3H]benzo[a]pyrene and [3H]DMBA to epidermal DNA. A possible mechanism of its antitumor initiation activity is that glycyrrhizic acid acts as a carcinogen metabolism inhibitor, thereby inhibiting the formation of DNA adducts. Our results suggest that glycyrrhizic acid possesses significant antitumor activity and may contribute to the prevention of certain types of human cancers. PMID:1907733
Hepatocellular carcinoma (HCC) occurs in patients with chronic liver disease who are positive for hepatitis C virus RNA. Drug therapy is crucial for the prevention of HCC. Methods: This retrospective study evaluated the long-term preventive effect of potent neominofen C (SNMC) on the development of HCC. Potent neominofen C is a Japanese drug commonly used to treat patients with chronic hepatitis C to lower serum alanine aminotransferase (ALT) levels. This retrospective analysis included 453 patients with chronic hepatitis C admitted to our hospital between January 1979 and April 1984, of whom 84 (Group A) received potent neominofen C. The dosing regimen of potent neominofen C was 100 mL daily for 8 weeks, followed by 2–7 times weekly for 2–16 years (median 10.1 years). Another group of 109 patients (Group B) were unable to receive high-potency minofen C or interferon therapy for a long period (median 9.2 years) and therefore received other traditional Chinese medicine treatments (such as vitamin K). These patients were retrospectively followed up, and the cumulative incidence and risk factors of hepatocellular carcinoma were analyzed. Results: The cumulative incidence of hepatocellular carcinoma at year 10 in Group A and Group B was 7% and 12%, respectively, and at year 15, it was 12% and 25%, respectively. Cox regression analysis showed that the relative risk of developing hepatocellular carcinoma in patients who did not receive high-potency minofen C therapy (Group B) was 2.49 times that in patients who received high-potency minofen C therapy (Group A). Conclusion: This study demonstrates that long-term use of high-potency minofen C to treat chronic hepatitis C can effectively prevent the occurrence of liver cancer. As mentioned earlier, NF-κB is a key molecule in the initiation and progression of inflammatory responses. Activated NF-κB translocates to the cell nucleus, inducing the expression of pro-inflammatory cytokines, adhesion molecules, and chemokines. In this rat model, both DG and dexamethasone inhibited NF-κB expression, suggesting that the anti-inflammatory mechanism of DG may be similar to that of dexamethasone. Although the efficacy of DG is not as good as that of dexamethasone, its side effects are expected to be milder. In summary, DG is effective in treating experimental ulcerative colitis in rats with mild side effects. These results suggest that DG may be a promising candidate drug for the treatment of ulcerative colitis. [1]

C42H68N2O16

856.9931

856.456

79165-06-3

656656

White to off-white solid powder

1021.4ºC at 760 mmHg

2.893

10

18

7

60

1730

19

C[C@]12CC[C@](C[C@H]1C3=CC(=O)[C@@H]4[C@]5(CC[C@@H](C([C@@H]5CC[C@]4([C@@]3(CC2)C)C)(C)C)O[C@@H]6[C@@H]([C@H]([C@@H]([C@H](O6)C(=O)O)O)O)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)C(=O)O)O)O)O)C)(C)C(=O)O.N.N

SPPIIOPGDLITJE-VLQRKCJKSA-N

InChI=1S/C42H62O16.2H3N/c1-37(2)21-8-11-42(7)31(20(43)16-18-19-17-39(4,36(53)54)13-12-38(19,3)14-15-41(18,42)6)40(21,5)10-9-22(37)55-35-30(26(47)25(46)29(57-35)33(51)52)58-34-27(48)23(44)24(45)28(56-34)32(49)50;;/h16,19,21-31,34-35,44-48H,8-15,17H2,1-7H3,(H,49,50)(H,51,52)(H,53,54);2*1H3/t19-,21-,22-,23-,24-,25-,26-,27+,28-,29-,30+,31+,34-,35-,38+,39-,40-,41+,42+;;/m0../s1

(2S,3S,4S,5R,6R)-6-[(2S,3R,4S,5S,6S)-2-[[(3S,4aR,6aR,6bS,8aS,11S,12aR,14aR,14bS)-11-carboxy-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-2,3,4a,5,6,7,8,9,10,12,12a,14a-dodecahydro-1H-picen-3-yl]oxy]-6-carboxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid;azane

Diammonium glycyrrhizinate; 79165-06-3; UNII-A9ZZD585U6; A9ZZD585U6; Glycyrrhizic acid, diammonium salt; 18beta-Glycyrrhizic acid diammonium salt; glycyrrhizin; (2S,3S,4S,5R,6R)-6-[(2S,3R,4S,5S,6S)-2-[[(3S,4aR,6aR,6bS,8aS,11S,12aR,14aR,14bS)-11-carboxy-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-2,3,4a,5,6,7,8,9,10,12,12a,14a-dodecahydro-1H-picen-3-yl]oxy]-6-carboxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid;azane;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~116.69 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.92 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.92 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.92 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)