Natural triterpenoid saponinl; anti-inflammatory; antiallergic; antigastriculcer; and antihepatitis

ammonium glycyrrhizate (AG) Counteracted High Glucose-Induced Cell Death [3]
One of the causes of DPN is death of Schwann cells due to prolonged exposure to high glucose and consequent oxidative stress. On this basis, we investigated the effect of GLU by using SH-SY5Y neuroblastoma as a model cell line. First of all, to determine the effect of high glucose on viability of SH-SY5Y cells, we treated cells with increasing concentrations of GLU or MAN (0–300 mM) for different time points (24, 48 and 72 h). We observed that, starting from 48 h, GLU was able to induce an appreciable cytotoxic effect. The results obtained at this timepoint using Calcein-AM showed that the percentage of dead cells increased along with increasing concentrations of GLU, being about 30% at a concentration of 300 mM (Figure 1A). Importantly, percentage of cell death induced by MAN was significantly lower than that induced by the same concentration of GLU and not significantly different from control untreated cells (Figure 1A). The antiapoptotic and anti-inflammatory effect of AG has been reported in various contexts. Thus, we tested two different concentrations (500 μg/mL and 1000 μg/mL) of AG to verify its ability to inhibit the cytotoxic effects induced by 300 mM GLU (Figure 1B). We observed a protective effect exerted by AG already at 500 μg/mL, which became significantly more marked by increasing the concentration of AG to 1000 μg/mL. On the basis of these preliminary experiments, we selected 300 mM as the concentration of GLU inducing an appreciable cytotoxic effect (also indicated as high glucose through the text, HG) and 1000 μg/mL as the concentration of AG capable of significantly inhibiting it (Figure 1B). Note that both fall within the range of concentrations used, as reported in the literature.

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ammonium glycyrrhizate (AG) Counteracted HG-Induced Apoptosis and Mitochondrial Alterations [3]
Through flow cytometry analysis after double cell staining with Annexin V (AV)/propidium iodide (PI), we analyzed apoptosis in SH-SY5Y neuroblastoma cells growing in HG conditions for 48 h. We found that HG induced cell death by apoptosis in about 30% of SH-SY5Y cells and that 1000 μg/mL AG was able to almost completely inhibit HG-induced apoptosis (Figure 2A). Neither MAN (considered as an internal control) nor AG-treated cells showed any significant difference when compared to untreated control cells. It is known that hyperglycemia induces mitochondrial dysfunction. We therefore analyzed the MMP by flow cytometry, after cell staining with JC-1 probe. HG induced a significant increase in the percentage of cells with high MMP, i.e., hyperpolarized mitochondria (boxed area, in Figure 2B). According to apoptosis data, AG was able to significantly (p < 0.01) reduce the percentage of cells with high MMP induced by HG, and MAN did not induce any alteration of MMP (boxed areas in Figure 2B). Overlapping results were obtained using TMRM as a probe for the study of the MMP (Figure S1).
The accumulation of evidence indicates that the mitochondrial fragmentation and fission represent important contributing factors to alterations of mitochondrial membrane and ATP production. Thus, we also evaluated the mitochondrial network organization by immunofluorescence analysis after cell staining with antimitochondrial import receptor subunit TOM20 (red) and counterstaining with Hoechst (blue) in cells grown under HG conditions. We found that GLU induced mitochondrial fragmentation (Figure 3A), which is normally associated with dysfunctional mitochondria. The administration of AG to cells growing under HG restored normal mitochondrial morphology, as also revealed by morphometric analysis performed by using ImageJ to measure average mitochondrial area in cells stained with an anti-TOM20 antibody (Figure 3B).

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ammonium glycyrrhizate (AG) Counteracted Inflammation Induced by HG [3]
Diabetic patients have been found to increased HMGB1 and RAGE levels. HMGB1 is normally expressed in the nucleus. However, following signals of stress, injury, or tissue damage, the protein was released into the extracellular space. HMGB1 can bind the Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE), leading to increased inflammation commonly through nuclear factor kappa beta (NFκB). On these bases, we investigated the possible anti-inflammatory activity of AG in our cell model analyzing by Western blot HMGB1, which is a ubiquitous nuclear protein that promotes inflammation when extruded from the cell after stress, damage, or death, and p65-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), which is a critical regulator of immune and inflammatory responses. According to the literature’s data, HG condition induced a significant increase in the expression levels of both HMGB1 and NFκB. Administration of AG in cells subjected to HG effectively decreased the level of both proinflammatory proteins (Figure 5).

When Monoammonium glycyrrhizinate (MAG) was administered at high and medium doses (10 and 30 mg/kg), the rise in lung W/D weight ratio was much lessened. The generation of TNF-α and IL-1β was effectively decreased by pretreatment with MAG (10 and 30 mg/kg). MAG (10, 30 mg/kg) dramatically decreased the expression of NF-κB p65 protein when compared to LPS. In contrast, MAG (10 and 30 mg/kg) dramatically boosted I���B-��� expression compared with the LPS group, while LPS considerably decreased IκB-α protein expression [1]. At the 14- and 21-day time intervals, AST, ALT, TBIL, and TBA levels were significantly lower in the low-dose and high-dose MAG treatment groups compared to the RIF and INH groups. This suggests that MAG has a protective effect against liver injury generated by RIF and INH. The MAG-treated group of RIF and INH-treated rats showed a protective effect against RIF, as evidenced by the significant decrease in MDA levels at the 14- and 21-day time points and the increase in hepatic GSH levels at the 7, 14, and 21-day time points. INH-related liver damage [2].

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The present study aimed to investigate the therapeutic effect of Monoammonium glycyrrhizinate (MAG) on lipopolysaccharide- (LPS-) induced acute lung injury (ALI) in mice and possible mechanism. Acute lung injury was induced in BALB/c mice by intratracheal instillation of LPS, and MAG was injected intraperitoneally 1 h prior to LPS administration. After ALI, the histopathology of lungs, lung wet/dry weight ratio, protein concentration, and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were determined. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the BALF were measured by ELISA. The activation of NF-κB p65 and IκB-α of lung homogenate was detected by Western blot. Pretreatment with MAG attenuated lung histopathological damage induced by LPS and decreased lung wet/dry weight ratio and the concentrations of protein in BALF. At the same time, MAG reduced the number of inflammatory cells in lung and inhibited the production of TNF-α and IL-1β in BALF. Furthermore, we demonstrated that MAG suppressed activation of NF-κB signaling pathway induced by LPS in lung. The results suggested that the therapeutic mechanism of MAG on ALI may be attributed to the inhibition of NF-κB signaling pathway. Monoammonium glycyrrhizinate may be a potential therapeutic reagent for ALI. [1]

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Results: Liver function, histopathological analysis, and oxidative stress factors were significantly altered in each group. The expression of Mrp2 was significantly increased 230, 760, and 990% at 7, 14, and 21 time points, respectively, in RIF- and INH-treated rats. Compared with the RIF and INH groups, Mrp2 was reduced and Ntcp was significantly elevated by 180, 140, and 160% in the Monoammonium glycyrrhizinate (MAG) high-dose group at the three time points, respectively. The immunoreaction intensity of Oatp1a4 was increased 170, 190, and 370% in the MAG low-dose group and 160, 290, and 420% in the MAG high-dose group at the three time points, respectively, compared with the RIF and INH groups. Discussion and conclusion: These results indicated that MAG has a protective effects against RIF- and INH-induced hepatotoxicity. The underlying mechanism may have correlation with its effect on regulating the expression of hepatobiliary membrane transporters. [2]

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ammonium glycyrrhizate (AG) Induced Anti-Hyperalgesic Effect in Diabetic Mice [3]
To test the efficacy of AG in preventing or mitigating diabetic neuropathy induced by hyperglycemia in vivo, we used STZ as inducers of diabetes in mice. The results of experiments performed in diabetic mice are reported in Figure 6. STZ resulted in increased thermal hyperalgesia—a reduction in the withdrawal threshold to a noxious stimulus—when compared to baseline recorded before diabetes as observed 13 days after STZ treatment. When AG was first administered 15 days after STZ, a nonsignificant increase in paw withdrawal latencies was observed (Figure 6). AG was administered again at days 17 and 19 after STZ injection, and at this time, a strong increase in paw latency was recorded. Thus, our data demonstrated that a short-repeated treatment with AG is able to induce an anti-hyperalgesic effect in diabetic mice.

The human neuroblastoma cell line SH-SY5Y was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4500 mg/L glucose, sodium pyruvate, and sodium bicarbonate; 10% fetal bovine serum, and penicillin and streptomycin at 100 U/mL penicillin and 100 mg/mL streptomycin, and kept at 37 °C in a humidified 5% CO2 incubator. SH-SY5Y cells were obtained from ATCC, and all experiments were carried out up to 12 passages. In total, 80,000 cells/well were seeded on 12-well tissue culture plates. After 24 h, cells were treated with different concentrations of D-Glucose (GLU) (75, 100, 150, 200, 250, and 300 mM) for 24, 48, and 72 h to find out the maximum concentration that induce cytotoxicity. At the same time, different concentrations of ammonium glycyrrhizate (AG) (200 μg/mL) were used to select the concentration able to counteract the effects of high glucose. As control, we used cells treated with mannitol, an osmotic sugar alcohol that is metabolically inert in humans, at the same concentration as GLU. [3]

LPS-Induced ALI in Mice [1]
Mice were randomly divided into five groups: control group, LPS group, and LPS + Monoammonium glycyrrhizinate (MAG) (3, 10, and 30 mg/kg) groups. Each group contained eight mice. Mice were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg). Before inducing acute lung injury, the mice were given intraperitoneal injection with Monoammonium glycyrrhizinate (MAG) (3, 10, and 30 mg/kg). One hour later, LPS (5 mg/kg) was instilled intratracheally to induce acute lung injury. Normal mice were given PBS. Twenty-four hours after LPS administration, lung tissues and BALF were collected.

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Experimental design [2]
Rats were randomly divided into four groups, i.e., control group, RIF and INH group, Monoammonium glycyrrhizinate (MAG) low-dose group, and Monoammonium glycyrrhizinate (MAG) high-dose group, each group had 15 rats. Rats in the RIF and INH group received RIF (60 mg/kg) and INH (60 mg/kg) by gavage administration once daily; rats in Monoammonium glycyrrhizinate (MAG) groups were pretreated with Monoammonium glycyrrhizinate (MAG) at the doses of 45 or 90 mg/kg, RIF (60 mg/kg) and INH (60 mg/kg) were given 3 h after Monoammonium glycyrrhizinate (MAG) administration; rats in the control group were treated with saline. To evaluate the dynamic effect of drugs, rats in each group were sacrificed on 7, 14, and 21 d after drug administration. At each time point, five rats were randomly selected and anesthetized with ether, blood was collected by abdominal aortic puncture, and serum was obtained for biochemical analysis. The livers were harvested immediately, a portion of liver was fixed in 10% formaldehyde for histological analysis, the remaiders were frozen with liquid nitrogen and stored at −80 °C for GSH and MDA measurements as well as western blot analysis.

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Biochemistry parameters [2]
Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and total bile acids (TBA) were determined using kits according to the protocols of the manufacturer. The determination was performed using the standard clinical method by Automatic Biochemistry Analyzer.

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It was reported that a high dose of streptozotocin/STZ is directly toxic to pancreatic β-cells, rapidly causing diabetes, with blood glucose levels of >500 mg/dl within 48 h in mice. Thus, STZ dissolved in saline was administered at 200 mg/kg (1.0 mL/100 g) by a single intraperitoneal (i.p.) injection to induce diabetes. Control mice were injected with vehicle alone. Measures of blood glucose were performed using a One Touch Basic blood glucose monitoring system to ensure hyperglycemia. Body weight was also monitored. Only mice with blood glucose concentration exceeding 500 mg/dL were considered diabetic and used for the study. Notably, 15, 17, and 19 days after the STZ administration, diabetic mice received i.p. injection of saline (10 mL/kg, control mice) or ammonium glycyrrhizate (AG) in saline (50 mg/kg, 10 mL/kg).[3]
Paw thermal withdrawal latency (PWL) was used to measure thermal hyperalgesia and performed by using an infrared generator. Mice were gently restrained using a glove, and after placing the mouse footpad in contact with the radiant heat source paw, withdrawal latency was measured. A timer initiated automatically when the heat source was activated, and a photocell stopped the timer when the mouse withdrew its hind paw. An intensity of 30 and a cut-off time of 15 s were used of the heat source on the plantar apparatus to avoid tissue damage. The PWL, in terms of seconds, of each animal in response to the plantar test was determined. Baseline paw thermal withdrawal latencies were determined before saline or ammonium glycyrrhizate (AG) administration.[3]

[1]. Anti-Inflammatory Effects of Monoammonium Glycyrrhizinate on Lipopolysaccharide-Induced Acute Lung Injury in Mice through Regulating Nuclear Factor-Kappa B Signaling Pathway. Evid Based Complement Alternat Med. 2015;2015:272474.

[2]. Monoammonium glycyrrhizinate protects rifampicin- and isoniazid-induced hepatotoxicity via regulating the expression of transporter Mrp2, Ntcp, and Oatp1a4 in liver. Pharm Biol. 2016;54(6):931-7.

[3]. Ammonium Glycyrrhizinate Prevents Apoptosis and Mitochondrial Dysfunction Induced by High Glucose in SH-SY5Y Cell Line and Counteracts Neuropathic Pain in Streptozotocin-Induced Diabetic Mice. Biomedicines. 2021 May 26;9(6):608.

Monoammonium glycyrrhizinate is an organic molecular entity.

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Our findings showed that Monoammonium glycyrrhizinate (MAG) could attenuate lung histopathological changes, reduce wet/dry weight ratio of the lung, and inhibit protein extravasation into alveolar space. The protective effects of MAG on ALI were correlated with the ability of reducing neutrophil infiltration and the production of TNF-α and IL-1β by suppressing the activation of NF-κB signaling pathway. This indicates that MAG may be an agent for preventing and treating ALI. [1]

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This study comprehensively characterized the notable alterations in hepatobiliary transporters Mrp2, Ntcp, and Oatp1a4 expressions, in the protective effect of MAG on RIF- and INH-induced hepatotoxicity for the first time. This study indicated that the protective effects of MAG on RIF- and INH-induced liver injuries have close correlation with its effect on regulating the expression of hepatobiliary membrane transporters. The coordinated reduction of efflux transporter expression (e.g., Mrp2) together with a corresponding increase of uptake carriers expression (e.g., Oatp1a4) suggest a protective mechanism of MAG for maintaining bilirubin and BAs homeostasis in RIF- and INH-induced hepatotoxicity. A better understanding on the altered expression of Ntcp (decreased at 7 d time point and increased at 14 and 21 d time points, respectively) in the RIF and INH group is necessary to address the functional contribution of transport mechanisms to the changed hepatic of xenobiotics during injury as well as conferring resistance to subsequent toxicant exposure.[2]

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Glycyrrhiza glabra, commonly known as liquorice, contains several bioactive compounds such as flavonoids, sterols, triterpene, and saponins; among which, glycyrrhizic acid, an oleanane-type saponin, is the most abundant component in liquorice root. Diabetic peripheral neuropathy is one of the major complications of diabetes mellitus, leading to painful condition as neuropathic pain. The pathogenetic mechanism of diabetic peripheral neuropathy is very complex, and its understanding could lead to a more suitable therapeutic strategy. In this work, we analyzed the effects of ammonium glycyrrhizinate, a derivate salt of glycyrrhizic acid, on an in vitro system, neuroblastoma cells line SH-SY5Y, and we observed that ammonium glycyrrhizinate was able to prevent cytotoxic effect and mitochondrial fragmentation after high-glucose administration. In an in vivo experiment, we found that a short-repeated treatment with ammonium glycyrrhizinate was able to attenuate neuropathic hyperalgesia in streptozotocin-induced diabetic mice. In conclusion, our results showed that ammonium glycyrrhizinate could ameliorate diabetic peripheral neuropathy, counteracting both in vitro and in vivo effects induced by high glucose, and might represent a complementary medicine for the clinical management of diabetic peripheral neuropathy.[3] Considering the absence of cytotoxicity, even at high concentrations, and the good pharmacological tolerability in rodents and humans after acute or subchronic treatment, ammonium glycyrrhizate (AG) may represent a complementary medicine in the clinical management of DPN with the added advantage of providing a multitarget effect on the various etiological factors underlying the pathophysiology of DPN, such as inflammation and mitochondrial damage.[3]

C42H65NO16

839.97

839.43

C, 60.06; H, 7.80; N, 1.67; O, 30.48

53956-04-0

Glycyrrhizic acid;1405-86-3;Dipotassium glycyrrhizinate;68797-35-3

62074

White to off-white solid powder

1.43g/cm3

971.4ºC at 760mmHg

209ºC

288.1ºC

49 ° (C=1.5, EtOH)

0.328

9

17

7

59

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

ILRKKHJEINIICQ-OOFFSTKBSA-N

InChI=1S/C42H62O16.H3N/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);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

Ammonium glycyrrhizate; Ammonium glycyrrhizate; AMMONIUM GLYCYRRHIZINATE; 53956-04-0; Glycamil; ammonium glycyrrhizate (AG); Monoammonium glycyrrhizinate; Glycyrram; Monoammonium glycyrrhizate (MAG); Ammoniated glycyrrhizin; 18β-Glycyrrhizic acid monoammonium salt; (+)-Glycyram

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, avoid exposure to moisture.

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 (~119.05 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.98 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 (2.98 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 (2.98 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.)