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

甘草酸铵(AG)对抗高糖诱导的细胞死亡[3]
DPN的原因之一是雪旺细胞由于长期暴露于高葡萄糖和随之而来的氧化应激而死亡。在此基础上，我们以SH-SY5Y神经母细胞瘤为模型细胞系，研究GLU的作用。首先，为了确定高糖对SH-SY5Y细胞活力的影响，我们在不同的时间点（24、48和72 h）分别用增加浓度的GLU或MAN （0-300 mM）处理细胞。我们观察到，从48 h开始，GLU能够诱导明显的细胞毒性作用。Calcein-AM在该时间点获得的结果显示，死细胞百分比随着GLU浓度的增加而增加，在浓度为300 mM时约为30%（图1A）。重要的是，MAN诱导的细胞死亡百分比明显低于相同浓度GLU诱导的细胞死亡百分比，与对照未处理细胞无显著差异（图1A）。AG的抗凋亡和抗炎作用已在各种情况下被报道。因此，我们测试了两种不同浓度（500 μg/mL和1000 μg/mL）的AG，以验证其抑制300 mM GLU诱导的细胞毒作用的能力（图1B）。我们观察到AG浓度在500 μg/mL时已具有保护作用，当AG浓度增加到1000 μg/mL时，这种保护作用更加明显。在这些初步实验的基础上，我们选择300 mM作为GLU诱导明显细胞毒作用的浓度（也通过文本表示为高葡萄糖，HG）， 1000 μg/mL作为AG能够显著抑制它的浓度（图1B）。请注意，正如文献中报道的那样，两者都在使用浓度范围内。

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甘草酸铵(AG)抵消hg诱导的细胞凋亡和线粒体改变[3]
通过膜联蛋白V (AV)/碘化丙啶（PI）双细胞染色后的流式细胞术分析，我们分析了在HG条件下生长48 h的SH-SY5Y神经母细胞瘤细胞的凋亡情况。我们发现，HG诱导约30%的SH-SY5Y细胞凋亡，1000 μg/mL AG几乎可以完全抑制HG诱导的细胞凋亡（图2A）。与未处理的对照细胞相比，MAN（被认为是内部对照）和ag处理的细胞都没有显示出任何显著差异。众所周知，高血糖会引起线粒体功能障碍。因此，在JC-1探针对细胞进行染色后，我们用流式细胞术分析了MMP。HG诱导具有高MMP的细胞百分比显著增加，即线粒体超极化（图2B中框区）。根据细胞凋亡数据，AG能够显著（p < 0.01）降低HG诱导的高MMP细胞百分比，而MAN没有引起MMP的任何改变（图2B中框区）。使用TMRM作为探针来研究MMP得到重叠结果（图S1） 越来越多的证据表明，线粒体断裂和裂变是线粒体膜改变和ATP产生的重要因素。因此，在HG条件下生长的细胞中，用抗线粒体进口受体亚基TOM20（红色）和Hoechst（蓝色）对细胞进行反染色后，我们也通过免疫荧光分析来评估线粒体网络组织。我们发现GLU诱导线粒体断裂（图3A），这通常与线粒体功能障碍有关。AG处理在HG下生长的细胞恢复了正常的线粒体形态，使用ImageJ测量抗tom20抗体染色细胞的平均线粒体面积进行形态计量学分析也显示了这一点（图3B）。

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甘草酸铵(AG)可拮抗HG引起的炎症[3]
发现糖尿病患者HMGB1和RAGE水平升高。HMGB1通常在细胞核中表达。然而，随着应激、损伤或组织损伤的信号，这种蛋白质被释放到细胞外空间。HMGB1可以结合toll样受体4 （TLR4）和晚期糖基化终产物受体（RAGE），通常通过核因子κ b （NFκB）导致炎症增加。在此基础上，我们通过Western blot分析了AG在细胞模型中可能的抗炎活性，HMGB1是一种普遍存在的核蛋白，在应激、损伤或死亡后从细胞中挤出时促进炎症，p65-活化B细胞的核因子kappa-轻链增强子（NFκB）是免疫和炎症反应的关键调节因子。根据文献数据，HG状态诱导HMGB1和NFκB表达水平显著升高。在受HG影响的细胞中给予AG有效地降低了这两种促炎蛋白的水平（图5）。

当Monoammonium glycyrrhizinate (MAG)以高剂量和中剂量（10 和 30 mg/kg）给药时，肺 W/D 重量比的上升幅度大大减轻。 MAG（10 和 30 mg/kg）预处理可有效减少 TNF-α 和 IL-1β 的产生。与 LPS 相比，MAG (10, 30 mg/kg) 显着降低 NF-κB p65 蛋白的表达。相比之下，MAG（10 和 30 mg/kg）显着提高了 I���B-���与LPS组相比，LPS显着降低IκB-α蛋白表达[1]。在 14 天和 21 天的时间间隔内，低剂量和高剂量 MAG 治疗组的 AST、ALT、TBIL 和 TBA 水平显着低于 RIF 和 INH 组。这表明MAG对RIF和INH引起的肝损伤具有保护作用。 MAG 治疗组的 RIF 和 INH 治疗组的大鼠表现出对 RIF 的保护作用，这通过第 14 天和 21 天时间点的 MDA 水平显着降低以及第 7 天和 14 天时间点的肝脏 GSH 水平增加来证明。 ，和 21 天的时间点。 INH 相关的肝损伤[2]。

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本研究旨在探讨甘草酸单铵(MAG)对脂多糖（LPS）诱导的小鼠急性肺损伤（ALI）的治疗作用及其可能机制。通过气管内灌注LPS诱导BALB/c小鼠急性肺损伤，并在LPS给药前1 h腹腔注射Monoammonium glycyrrhizinate (MAG)。ALI后，检测肺组织病理学、肺干湿比、蛋白浓度、支气管肺泡灌洗液（BALF）炎症细胞。采用ELISA法检测BALF中肿瘤坏死因子-α (TNF-α)和白细胞介素-1β (IL-1β)水平。Western blot检测肺匀浆中NF-κB p65和i -κB -α的活化情况。MAG预处理可减轻LPS引起的肺组织病理损伤，降低肺干湿比和BALF蛋白浓度。同时，MAG可减少肺内炎性细胞数量，抑制BALF中TNF-α和IL-1β的产生。此外，我们还证明了MAG抑制LPS诱导的肺NF-κB信号通路的激活。提示MAG对ALI的治疗机制可能与抑制NF-κB信号通路有关。甘草酸一铵可能是一种潜在的治疗ALI的试剂。[1]

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结果：各组小鼠肝功能、组织病理学、氧化应激因子均有明显改变。在RIF和inh处理的大鼠中，Mrp2的表达分别在7、14和21个时间点显著增加230、760和990%。与RIF和INH组比较，Monoammonium glycyrrhizinate (MAG)高剂量组在三个时间点Mrp2降低，Ntcp显著升高，分别为180%、140%和160%。在三个时间点，与RIF和INH组相比，MAG低剂量组Oatp1a4的免疫反应强度分别提高了170、190、370%，MAG高剂量组提高了160、290、420%。 讨论与结论：这些结果表明MAG对RIF和inh诱导的肝毒性具有保护作用。其作用机制可能与其调节肝胆膜转运蛋白的表达有关。[2]

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甘草酸铵(AG)诱导的糖尿病小鼠抗痛觉作用[3]
为了验证AG在体内预防或减轻高血糖引起的糖尿病性神经病变的作用，我们将STZ作为小鼠糖尿病的诱导剂。在糖尿病小鼠中进行的实验结果见图6。与糖尿病前相比，STZ治疗后13天观察到，STZ导致热痛觉过敏增加——对有害刺激的戒断阈值降低。当STZ后15天首次给药AG时，观察到足部戒断潜伏期无显著增加（图6）。在STZ注射后第17天和第19天再次给药AG，此时记录到足部戒断潜伏期明显增加。因此，我们的数据表明，AG短期重复治疗能够诱导糖尿病小鼠的抗痛觉过敏作用。

人神经母细胞瘤细胞系SH-SY5Y在Dulbecco改良Eagle培养基（DMEM）中培养，DMEM中含有4500 mg/L葡萄糖、丙酮酸钠和碳酸氢钠；10%胎牛血清，青霉素和链霉素浓度分别为100u /mL青霉素和100mg /mL链霉素，在5% CO2加湿培养箱中37℃保存。SH-SY5Y细胞从ATCC获得，所有实验均进行至12代。在12孔组织培养板上共接种8万个细胞/孔。24 h后，用不同浓度的d -葡萄糖（GLU）（75、100、150、200、250和300 mM）处理细胞24、48和72 h，找出诱导细胞毒性的最大浓度。同时，采用不同浓度ammonium glycyrrhizate (AG)(200 μg/mL)来选择能够抵消高糖作用的浓度。作为对照，我们使用甘露醇处理的细胞，甘露醇是一种渗透性糖醇，在人体中代谢惰性，浓度与GLU相同。[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

272.7

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (2.98 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (2.98 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (2.98 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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