Glycyrrhizic acid analog

Based on the SAR analysis of glycyrrhizin, 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) with strong inhibition against LPS-induced NO and IL-6 production in RAW264.7 cells was discovered. Western blotting and immunofluorescence results showed that 18α-GAMG reduced the expression of iNOS, COX-2, and MAPKs, as well as activation of NF-κB in the LPS-stimulated RAW264.7 cells. [1]

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To evaluate the anti-inflammatory effect of the glycyrrhizin analogs, Griess reagent was used to detect the level of the LPS-induced NO release in the RAW264.7 cells. Excessive release of NO is regarded as an important factor in inflammatory responses. As shown in Fig. 2, after treatment with glycyrrhizin analogs, the increase in the LPS-induced NO release was significantly alleviated in the RAW264.7 cells. SAR analysis showed that (i) the anti-inflammatory activity of 18α-epimer of the oleanane-type aglycone was superior to that of 18β-epimer (18α-GA > 18β-GA, 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) > 18β-GAMG, 18α-GCCS > 18β-GCCS) and (ii) the number of glucuronic acids at the C-3 position had an effect on the anti-inflammatory activity (mono-glucuronide > aglycone > bis-glucuronide, such as 18β-GAMG > 18β-GCCS > 18β-GA, 18α-GAMG > 18α-GCCS > 18α-GA). Glycyrrhizin analogs displayed preferable anti-inflammatory activity; among them, 18α-GAMG exhibited the strongest activity, and the NO inhibition rate exceeded 70% at a concentration of 40 μM. [1]

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To further evaluate the effects of glycyrrhizin analogs on the LPS-induced IL-6 production, RAW264.7 cells were cultured with LPS (1 μg mL–1) in the presence of glycyrrhizin analogs for 20 h, and the levels of IL-6 in the supernatant were determined via ELISA. As shown in Fig. 3, the LPS-induced IL-6 production decreased after treatment with the glycyrrhizin analogs, and inhibitory effects were observed with the following conclusions: (i) for the inhibition of the IL-6 production, 18α-epimer was better than 18β-epimer (18α-GA > 18β-GA, 18α-GAMG > 18β-GAMG, 18α-GCCS ≈ 18β-GCCS); (ii) the number of glucuronic acids had an effect on the anti-inflammatory activity (mono-glucuronide > aglycone > bis-glucuronide, such as 18β-GAMG > 18β-GCCS > 18β-GA, 18α-GAMG > 18α-GCCS > 18α-GA). Thus, combining the anti-inflammatory activity and cytotoxicity of the glycyrrhizin analogs, 18α-GAMG was selected to further explore the mechanisms of the anti-inflammatory effect. [1]

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18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) was most effective in inhibiting the NO and IL-6 production. Thus, it was used to investigate the expression of the inflammation-related proteins.32,33 The expression of nitric oxide synthase (iNOS) and COX-2 was examined in the LPS-stimulated RAW264.7 cells. Western blotting results showed that 18α-GAMG strongly attenuated the expression of iNOS and COX-2 in the LPS-stimulated RAW264.7 cells in a dose-dependent manner (Fig. 4). These preliminary results demonstrated that 18α-GAMG may participate in signaling pathways activated by LPS in macrophages. [1]

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NF-κB is a well-known transcription factor that positively regulates inflammatory genes, such as iNOS, COX-2, and IL-6, in response to inflammatory stimuli.34 NF-κB activation is controlled by phosphorylation and degradation of IκB-α, a cognate regulatory subunit of NF-κB.35 Therefore, western blotting was used to examine the effects of 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG)on the NF-κB pathways in the LPS-stimulated RAW264.7 cells. As shown in Fig. 5A, LPS significantly increased the levels of phosphorylated NF-κB p65 and IκBα, and 18α-GAMG treatment could attenuate the activation of these proteins to varying degrees. Furthermore, the nuclear translocation of NF-κB was examined in the LPS-stimulated RAW264.7 cells. Immunofluorescence analysis showed that 18α-GAMG clearly inhibited NF-κB p65 nuclear translocation from the cytosol to the nucleus (Fig. 5B). These results further confirmed that 18α-GAMG might regulate the expression of pro-inflammatory proteins through the inhibition of the NF-κB signaling pathways. [1]

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The mitogen-activated protein kinase (MAPK) transduction pathway is activated by NF-κB in mammalian cells.36,37 Inhibition of the activation of MAPK down-regulates the expression of the inflammatory mediators and thus improves the outcome of the experimental inflammatory diseases.38,39 To determine the role of 18α-GAMG in modulating MAPK activation in LPS-stimulated RAW264.7 cells, the expression of ERK, JNK, and p38 was examined. As expected (Fig. 6), the levels of phosphorylation of p38, JNK, and ERK increased after LPS-stimulated treatment for 30 min. 18α-GAMG dose-dependently (10, 20, and 30 μM) inhibited LPS-induced phosphorylation of ERK, but had little effect on the phosphorylation of JNK or p38 in RAW264.7 cells. These results suggested that the anti-inflammatory activity of 18α-GAMG might be associated with its negative effects on ERK activation. [1]

To further verify the anti-inflammatory activity of 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) in vivo, a mice model of CCl4-induced hepatic fibrosis was established in this study to investigate its effect on hepatic fibrosis.40 Healthy C57BL6 mice (SPF, male, 20 ± 2 g) were purchased from the Experimental Animal Center of Anhui Medical University. Animals were housed in a temperature (22 ± 2 °C) and relatively humidity (50%)-controlled room under a 12 h light/dark cycle, given free access to food and water, and acclimatized for at least one week prior to use. All the animal experiments were performed in accordance with the Regulations of the Experimental Animal Administration issued by the State Committee of Science and Technology of China. Efforts were made to minimize the number of animals used and their suffering. Animals were maintained in accordance with the Guides of Center for Developmental Biology, Anhui Medical University for the Care and Use of Laboratory Animals, and in all the experiments, protocols approved by the institutions' subcommittees on animal care were used. [1]
As shown in Fig. 7, in the control group, the structure of the liver was clear, and the size of hepatocytes was constant. The hepatic lobule was intact, without denaturation or necrosis (Fig. 7A and F). In the model group, the amount of blue collagen fibers was obviously increased. Fatty degeneration was apparent and ballooning degeneration of the hepatocyte can be seen in the model group (Fig. 7B and G). The extent of inflammatory cell infiltration, blue collagen fibers and fibrosis of liver in the colchicine group (0.1 mg kg–1), the high-dose 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) group, and the low-dose 18α-GAMG were significant decreased, and the high-dose group was better than the low-dose group (Fig. 7C, D, E, H, I and J). These results showed that 18α-GAMG could significantly improve the pathological changes of the CCl4-induced hepatic fibrosis. [1]

Assay for NO Production [1]
RAW264.7 cells were inoculated at 1×105 cells per well in 24-well plate and cultured for 20 h. The cells were then pre-treated with 50 µM compounds which were prepared in serum-free medium for 2 h before stimulation with LPS (1 µg/mL). After stimulated for 24 h by LPS. The NO production was determined by detecting the nitrite level using Griess reagent according to the manufacturer’s instructions. then measured absorbance of the samples at 540 nm (OD540) in a microplate reader. NO inhibition rate = [control (OD540) - compound (OD540)] / [control (OD540) - blank (OD540)] × 100%. Control: treated with LPS only. Compound: treated with LPS and compounds. Blank: cultured with fresh medium only. [1]

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Cell Cytotoxicity [1]
Cell cytotoxicity was evaluated by methyl thiazolyl tetrazolium (MTT) assay. RAW264.7 cells were inoculated at 6 × 103 cells per well in 96-well plate. After cultured for 24 h, the cells were treated with different compounds which were diluted in DMEM for 24 h. Then 20 µL of 0.5 mg/mL MTT reagent was added into the cells and incubated for 4 h. After 4 h, cell culture was removed and then 150 µL DMSO was added to dissolve the formazan. The optical density was measured at 570 nm (OD570). Cell viability was calculated from three independent experiments. The density of formazan formed in blank group was set as 100% of viability. Cell viability (%) = compound (OD570 / blank (OD570) × 100% Blank: cultured with fresh medium only. Compound: treated with compounds or LPS. [1]

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Measurement of IL-6 [1]
RAW264.7 cells (7 × 104 cells/well) were cultured in 24-well plate. After cultured for 24 h, and pretreated with 10 and 40 µM of compounds for 2 h, and then LPS was added. The production of IL-6 was stimulated by the addition of 1 µg/mL LPS and incubated for 20 h. The levels of IL-6 in the supernatant were determined using the mouse ELISA kit which is operated according to the manufacturer’s instructions. [1]

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Western Blot Analysis [1]
RAW264.7 cells were seeded into a 6-well culture plate at a density of 2 × 106 cells per well, and then cultured for 24 h. Then, the culture medium was replaced by fresh medium containing 10、20 and 30 µM compounds, and 1 µg/mL LPS was added. After cultured for another 30 min or 20 h, the cells were harvested and lysed with IP buffer supplemented with 1 mM phenylmethanesulfonyl fluoride and incubated on ice for 30 min. The cell lysates were centrifuged at 14,000 × g for 10 min at 4 ºC to remove insoluble materials and the supernatant was collected. Total protein concentration was determined using a BCA protein assay kit. Each protein sample was mixed with a quarter volume of 5X SDS-PAGE sample loading buffer (100 mmol/L Tris-HCl pH 6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol, and 0.1% bromophenol blue) and 7 S boiled for 10 min. Equal amounts of total cellular protein were loaded per well in 12.5% precast SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Bio-Rad) for over 60 min at 300 mA. The membranes were blocked with 5% non-fat dry milk in TBS plus 0.1% Tween 20 (TBST) for 2 h at room temperature, washed 3 times in TBST for 5 min each, incubated with the primary antibody (anti-phosphorylation of SAPK/JNK, anti-SAPK/JNK, anti-phosphorylation of ERK1/2, anti-ERK1/2, anti-phosphorylation of p38, anti-p38, anti-phosphorylation of IkBa , anti-IkBa, antiphosphorylation of NF-kB p65, and anti-NF-κB p65) at 4 ºC overnight (all the primary antibodies were diluted at the ratio of 1:1000),washed 3 times in TBST for 5 min each, incubated with anti-rabbit or anti-mouse secondary antibody (1:5000 in TBST) for 90 min, washed in TBST and exposed to ECL reagents [1]

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Immunofluorescence Assay [1]
RAW264.7 cells (7 × 104 cells/well) were cultured in 24-well plate. After cultured for 24 h, and pretreated with 20 and 30 µM of 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) for 2 h, and then treated with LPS (1 µg/mL) for 3 h. The cells were washed twice with cold PBS, fixed with 4% formaldehyde for 15 min, and then permeabilized with 0.3% Triton X-100 in PBS for 10 min. After that, the cells were blocked for 0.5 h with 5% BSA. Cells were later incubated with primary antibody anti-NF-κB p65 antibody for overnight, followed by Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody. After a wash step, stained with DAPI for 5 min and the images were acquired.

18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) Alleviated CCl4-induced Hepatic Fibrosis [1]
Healthy male C57BL6 mice were randomly divided into 4 groups: control group, model group, high-dose 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) group and low-dose 18α-GAMG group, and each with 10. All mice except normal group were given 20% CCl4 olive oil (2 mL/kg) by hypodermic injection，two times a week for 4 weeks. Control group was given 0.9% sodium chloride by hypodermic injection, two times a week for 4 weeks. 18α-GAMG (200 mg/kg and 100 mg/kg) was given by intra-gastric administration to high-dose 18α-GAMG group and low-dose 18α-GAMG, two times a week for 4 weeks at beginning of third week. All mice were killed at 4th week. Pathological changes in hepatic tissue were observed by HE and Masson staining. [1]

158471 mouse LD50 intravenous 912 mg/kg LUNGS, THORAX, OR RESPIRATION: RESPIRATORY STIMULATION; LUNGS, THORAX, OR RESPIRATION: OTHER CHANGES Zhongguo Yaoxue Zazhi. Chinese Pharmacuetical Journal., 28(215), 1993
158471 mouse LD50 oral 6460 mg/kg LUNGS, THORAX, OR RESPIRATION: RESPIRATORY STIMULATION; LUNGS, THORAX, OR RESPIRATION: OTHER CHANGES Zhongguo Yaoxue Zazhi. Chinese Pharmacuetical Journal., 28(215), 1993

[1].18α-Glycyrrhetinic acid monoglucuronide as an anti-inflammatory agent through suppression of the NF-κB and MAPK signaling pathway. Medchemcomm. 2017 Jun 2;8(7):1498–1504.

[2].Mechanism of antihepatotoxic activity of glycyrrhizin. I: Effect on free radical generation and lipid peroxidation. Planta Med. 1984 Aug;50(4):298-302.

alpha-Glycyrrhizin has been reported in Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata with data available.

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In conclusion, the clear structure–activity relationships of glycyrrhizin with anti-inflammatory activity was explained; among them, the glucuronide unit and the 18-α/β-stereoisomer were important factors. Among these compounds, 18α-glycyrrhetinic acid monoglucuronide (18α-GAMG) was found to exhibit strongest inhibition. Western blotting and immunofluorescence results showed that 18α-GAMG decreased the expression of iNOS, COX-2, and MAPKs, as well as the activation of NF-κB in the LPS-stimulated RAW264.7 cells. Overall, 18α-GAMG exerted its anti-inflammatory activity through the inhibition of NO generation as a result of inhibition of the NF-κB and MAPKs-related inflammatory signaling pathways. In addition, the in vivo results showed that 18α-GAMG could significantly improve the pathological changes of CCl4-induced hepatic fibrosis. Therefore, 18α-GAMG may be clinically useful for the reduction of inflammation in the future. [1]

C42H62O16

822.93

822.404

83896-44-0

158471

White to off-white solid powder

2.245

8

16

7

58

1730

19

CC1(C2CCC3(C(C2(CCC1OC4C(C(C(C(O4)C(=O)O)O)O)OC5C(C(C(C(O5)C(=O)O)O)O)O)C)C(=O)C=C6C3(CCC7(C6CC(CC7)(C)C(=O)O)C)C)C)C

LPLVUJXQOOQHMX-IOHDZAKGSA-N

InChI=1S/C42H62O16/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)/t19-,21+,22+,23+,24+,25+,26+,27-,28+,29+,30-,31-,34+,35+,38-,39+,40+,41-,42-/m1/s1

(2S,3S,4S,5R,6R)-6-[(2S,3R,4S,5S,6S)-2-[[(3S,4aR,6aR,6bS,8aS,11S,12aS,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

alpha-Glycyrrhizin; 83896-44-0; 18alpha-Glycylrrhizin; Isoglycyrrhizinic acid; 18; A-Glycyrrhizic acid; (2S,3S,4S,5R,6R)-6-[(2S,3R,4S,5S,6S)-2-[[(3S,4aR,6aR,6bS,8aS,11S,12aS,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; 80ARS2076G;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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