JNK; Akt; FXR; Caspase;
Z/E-Guggulsterone targets multiple receptors and enzymes. It is an antagonist of the farnesoid X receptor (FXR) . It acts as a broad-spectrum steroid receptor ligand: mineralocorticoid receptor antagonist (Ki = 37 nM for Z-isomer), progesterone receptor antagonist (Ki = 224 nM), glucocorticoid receptor antagonist (Ki = 252 nM), and weak androgen receptor agonist (Ki = 315 nM) . It also inhibits the VEGF-VEGF-R2-Akt signaling axis . Regarding cytochrome P450 enzymes, Z/E-Guggulsterone is a substrate for CYP3A4, CYP2C19, and CYP2D6 isoforms, and inhibits CYP2C19 with an IC50 of 2.1 μM .

GugguLsterone (0.5 -20 μM; 24 hours) suppresses the production of TREM-1, TLR4 and TNF-α and the phosphorylation of IκBα and NF-κB p65 by LPS [2].

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Guggulsterone suppressed LX-2 cell growth in a dose- and activation-dependent manner. This growth suppression was due to the induction of HSC apoptosis, which was mediated by the activation of c-Jun N-terminal kinase and mitochondrial apoptotic signaling. Additionally, guggulsterone regulated phosphorylation of Akt and adenosine monophosphate-activated protein kinase, which were subsequently proven responsible for the guggulsterone-induced HSC growth suppression. Guggulsterone inhibited NF-κB activation in LX-2 cells, which is one of the major mediators in HSC activation. Indeed, guggulsterone decreased collagen α1 synthesis and α-smooth muscle actin expression in these cells. [3]

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Z/E-Guggulsterone inhibits the growth of a wide variety of tumor cells and induces apoptosis through downregulation of antiapoptotic gene products (IAP1, xIAP, Bfl-1/A1, Bcl-2, cFLIP and survivin), modulation of cell cycle proteins (cyclin D1 and c-Myc), activation of caspases and JNK, and inhibition of Akt . It inhibits angiogenesis by suppressing VEGF-VEGF-R2-Akt signaling: treatment with 10-20 μM (Z)-guggulsterone for 24-48 hours causes reduction in VEGF-R2 protein levels in HUVEC cells and inhibits capillary-like tube formation and cell migration . Z/E-Guggulsterone also reduces ACE2 expression and SARS-CoV-2 infection in multiple cell types through FXR-mediated ACE2 regulation . In vitro metabolism studies using human liver microsomes (HLM) have identified nineteen phase I and II metabolites, with hydroxylation as the major metabolic pathway; both isomers and their hydroxylated metabolites form adducts with glutathione (GSH) and N-acetylcysteine (NAC), indicating electrophilic reactive metabolite formation .

GugguLsterone (mouse; 100 mg/kg once daily for 8 days) significantly increased the incidence of TNBS-induced cystitis in wild-type mice [2].

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Compared with the control mice or mice treated with a low dose of guggulsterone , high dose of guggulsterone significantly decreased the extent of collagen deposition and the percentage of activated HSCs undergoing apoptosis. Conclusions: These results demonstrate that guggulsterone suppressed HSC activation and survival by inhibiting NF-κB activation and inducing apoptosis. Therefore, guggulsterone may be useful as an antifibrotic agent in chronic liver diseases.[3]

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In a DU145 xenograft mouse model, oral administration of (Z)-Guggulsterone (1 mg, five times per week) significantly reduced tumor volume and wet tumor weight . The compound inhibited in vivo angiogenesis as evidenced by a statistically significant decrease in tumor burden, microvessel area (staining for factor VIII and CD31), and VEGF-R2 protein expression . Guggulsterone has been shown efficacious for lipid disorders, inflammation, osteoarthritis, cancer, cardiovascular diseases, and obesity in various preclinical studies .

Triggering receptor expressed on myeloid cells 1 (TREM-1)-expressing intestinal macrophages are significantly increased in the colons of patients with inflammatory bowel disease (IBD). We focused here on the effects of guggulsterone on macrophage modulation in colitis as a potential therapeutic molecule in human IBD and explore the underlying mechanisms. Gene expression in macrophages was examined and wound-healing assay using HT-29 cells was performed. Colitis in wild-type and IL-10-, Toll-like receptor 4 (TLR4)-, and myeloid differentiation primary response 88 (MyD88)-deficient mice was induced via the administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the colon. In both in vitro and in vivo experiments, guggulsterone suppressed intestinal inflammation amplified by TREM-1 stimulation, in which the suppression of NF-κB, activating protein-1, and proteasome pathways was involved. In the TNBS-induced colitis model, guggulsterone reduced disease activity index scores and TREM-1 expression, stimulated IL-10 production, and improved survival in wild-type mice. These effects were not observed in IL-10-, TLR4-, and MyD88-deficient mice. Guggulsterone also suppressed M1 polarization, yet induced the M2 phenotype in macrophages from IBD patients as well as from mice. These findings indicate that guggulsterone blocks the hyperactivation of macrophages via TREM-1 suppression and induces M2 polarization via IL-10 mediated by the TLR4 signaling pathway. Furthermore, this study provides a new rationale for the therapeutic potential of guggulsterone in the treatment of IBD. NEW & NOTEWORTHY We found that guggulsterone attenuates triggering receptor expressed on myeloid cells 1 (TREM-1)-mediated hyperactivation of macrophages and polarizes macrophages toward the M2 phenotype. This was mediated by IL-10 and partly Toll-like receptor 4 signaling pathways. Overall, these data support that guggulsterone as a natural plant sterol modulates macrophage phenotypes in colitis, which may be of novel therapeutic importance in inflammatory bowel disease treatment[2].

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The following experimental protocols are described in the literature: CYP inhibition assay – Z/E-Guggulsterone inhibits CYP2C19 with an IC50 value of 2.1 μM . Plasma protein binding – Determined by equilibrium dialysis method; both isomers are highly bound to plasma proteins in human, monkey, rabbit, and rat plasma (>96% bound), with binding to human serum albumin at ∼72% and to α-acid glycoprotein at ∼25% . In vitro metabolic stability assay – Using liver microsomes (rat or human), intrinsic clearance (CLint) is determined: for E-isomer in rat liver microsomes, CLint = 33.34 ± 0.51 μL/min/mg protein; for Z-isomer, CLint = 39.23 ± 8.12 μL/min/mg protein . In human liver microsomes, unbound intrinsic clearance is 0.029 ± 0.0009 mL/min/mg protein for E-isomer and 0.027 ± 0.008 mL/min/mg protein for Z-isomer . Reactive metabolite trapping assay – Incubations with human liver microsomes are conducted in the presence of glutathione (GSH) and N-acetylcysteine (NAC) as trapping agents; samples are analyzed using UHPLC-Orbitrap mass spectrometry to detect adduct formation .

Western Blot Analysis[1]
Cell Types: RAW 264.7 murine macrophage
Tested Concentrations: 0.5, 5, 10, 20 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: LPS inhibits TREM-1, TLR4 and TNF-α expression as well as IκBα and NF-κB Phosphorylation of p65.

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The following cell-based assay protocols are described: HUVEC angiogenesis assay – Human umbilical vein endothelial cells (HUVEC) are treated with (Z)-guggulsterone (10-20 μM for 24-48 hours) to assess VEGF-R2 protein levels by Western blot, capillary-like tube formation, and cell migration . Prostate cancer cell migration assay – DU145 cells are treated with guggulsterone to assess inhibition of migration; knockdown of VEGF-R2 protein level increases the inhibitory effect, while ectopic expression of constitutively active Akt confers protection . Western blot analysis – Cells are lysed, proteins resolved by electrophoresis, transferred to membranes, and probed with antibodies against targets such as VEGF-R2, Akt, and other signaling proteins . ACE2 expression assay – Primary airway and intestinal organoids are treated with (Z)-guggulsterone (10 μM for 24 hours) to assess ACE2 levels and SARS-CoV-2 infection .

Animal/Disease Models: TNBS-induced colitis in wild-type mice [2]
Doses: 100 mg/kg
Route of Administration: po (po (oral gavage)) one time/day; for 8 days
Experimental Results: Improved survival of wild-type mice with TNBS-induced colitis Rate.

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Thirty-five white male mice of six weeks’ old were used. Their weights ranged between 20-30 gm. The animals were housed in well-ventilated plastic cages, and were maintained under conditions of relatively controlled temperature, humidity, and 12 hrs. light / dark cycle. With a free access to a standard commercial diet that was purchased from the local market and tap water ad libitum. The animals randomly were divided into five groups of 7 animals in each group, as the following: Group I: mice utilized in this group were fed standard commercial diet and considered to be the control group. Group II: mice utilized in this group were fed a specially formulated high-fat diet (HFD) for 12 weeks to induce nonalcoholic liver disease. Group III: mice utilized in this group were fed a high-fat diet that contains Guggulsterone at a concentration of 500 ppm for 12 weeks. Group IV: mice utilized in this group were fed a high-fat diet that contains guggulsterone at a concentration of 1000 ppm for 12 weeks. Group V: mice utilized in this group were fed a high-fat diet that contains Guggulsterone at a concentration of 2000 ppm for 12 weeks. The animal’s weight of all groups was measured routinely once weekly and at zero time. The animals of each group at the end of the experiment after overnight fasting were anesthetized using diethyl ether, and blood was collected from the heart by cardiac puncture, then the animals were sacrificed and the liver were obtained and weighed for calculating liver index (by dividing liver weight in mg by the last body weight. [4]

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Non-alcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases worldwide, which is characterized by steatosis, inflammation, and fibrosis. The aim of this designed study is to evaluate the ability of guggulsterone to prevent high fat diet induced steatohepatitis in mice. Five groups of male mice were selected and treated as the following: group I, mice had free access to standard commercial diet and considered as control group, group II, mice were fed a specially formulated high-fat diet for 12 weeks to induce non-alcoholic liver disease, while groups III, IV and V the mice were administered high fat diet containing guggulsterone at 500, 1000 and 2000 ppm concentration respectively for 12 weeks. Maintaining mice on fat rich diet only resulted in inducing the metabolic and histological changes related to NAFLD. While the treatment with guggulsterone significantly improves the evaluated markers. These results demonstrate guggulsterone may be useful in preventing the development of steatohepatitis.[4]

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The following animal experimental protocols are described: DU145 xenograft model – Male nude mice (5-6 weeks old) are subcutaneously implanted with Matrigel plugs containing DU145 human prostate cancer cells. (Z)-Guggulsterone is administered orally at 1 mg per mouse, five times per week. Tumor volume and wet tumor weight are measured to assess efficacy . In vivo angiogenesis assessment – Matrigel plug assay is performed; plugs are harvested, sectioned, and stained for angiogenic markers (factor VIII and CD31) to assess microvessel area, and VEGF-R2 protein expression is evaluated . Pharmacokinetic study in rats – Male Sprague Dawley rats are administered guggulsterone orally; plasma samples are collected at various time points for LC-MS/MS analysis .

Z/E-Guggulsterone is orally active . Both isomers are highly bound to rat plasma proteins (>95% bound) . In human, monkey, rabbit, and rat plasma, protein binding exceeds 96% . Binding to human serum albumin is ∼70-72%, and to α-acid glycoprotein is ∼25% . Following oral administration in rats, plasma concentration decreases rapidly; terminal half-life is 0.63 ± 0.25 h for E-isomer and 0.74 ± 0.35 h for Z-isomer . Clearance (CL) is 2.79 ± 0.73 L/h/kg for E-isomer and 3.01 ± 0.61 L/h/kg for Z-isomer . In vitro intrinsic clearance in rat liver microsomes: 33.34 ± 0.51 μL/min/mg protein (E-isomer) and 39.23 ± 8.12 μL/min/mg protein (Z-isomer) . In human liver microsomes, unbound intrinsic clearance is low at 0.029 ± 0.0009 (E) and 0.027 ± 0.008 (Z) mL/min/mg protein . First-pass metabolism appears responsible for low bioavailability . Z/E-Guggulsterone is a substrate for CYP3A4, CYP2C19, and CYP2D6 . ADME prediction shows no violation of Lipinski and Veber rules . The compound may inhibit CYP2C19 and CYP2C9 but cannot inhibit CYP1A2, CYP2D6, and CYP3A4 .

Reported side effects of guggulipid (containing Z/E-Guggulsterone) include: widespread erythematous papules in a morbilliform pattern and macules localized to the arms; swelling and erythema of the face with burning sensation; pruritis; and bullous lesions on the lower legs with associated headaches, myalgia, and itching . In male mice, guggulipid administration has been reported to cause histological abnormalities in liver, increased alanine aminotransferase levels, lower hepatic scavenger receptor class B type I content, hypercholesterolemia, endothelial dysfunction, enhanced atherosclerosis, and accelerated death in animals with severe ischemic heart disease . The toxicity is hypothesized to arise from formation of electrophilic reactive metabolites of guggulsterone isomers, which form adducts with cellular proteins . In silico prediction indicates that guggulsterone has no hepatotoxicity, cytotoxicity, or mutagenicity effects .

[1]. Shishodia S, et al. Guggulsterone for Chemoprevention of Cancer. Curr Pharm Des. 2016;22(3):294-306.
[2]. Che X, et al. Protective effects of guggulsterone against colitis are associated with the suppression of TREM-1 and modulation of macrophages. Am J Physiol Gastrointest Liver Physiol. 2018 Jul 1;315(1):G128-G139.
[3]. Guggulsterone attenuates activation and survival of hepatic stellate cell by inhibiting nuclear factor kappa B activation and inducing apoptosis. J Gastroenterol Hepatol. 2013 Dec;28(12):1859-68.
[4]. Possible Amelioration of the Severity of Nutritional Steatohepatitis by Guggulsterone in Mice. Iraqi J Pharm Sci, Vol.28(1) 2019. DOI: https://doi.org/10.31351/vol28iss1pp17-23; https://bijps.uobaghdad.edu.iq/index.php/bijps/article/view/801; https://pdfs.semanticscholar.org/fed5/b2b899b009d9a009406450b1d111bb67f233.pdf

Gucoustin is a 3-hydroxysteroid with androgenic activity. It has been reported that gucoustin is found in myrrh (Commiphora mukul) and white myrrh (Commiphora wightii), and relevant data are available for reference.

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Z/E-Guggulsterone (CAS: 95975-55-6) has molecular formula C21H28O2 and molecular weight 312.45 . It is a racemic mixture of (E)- and (Z)-guggulsterone, which are stereoisomers arising from asymmetric carbons at the seventeen position of the guggulsterone ring . Guggulipid (containing guggulsterone) was approved as a hypolipidemic drug in India in 1986, but a clinical study in the United States in 2003 found it ineffective in Western populations . Z/E-Guggulsterone is classified as "for research use only, not for human or veterinary use" and is not considered a hazardous substance according to safety data sheets . The compound is stable for up to 3 years when stored as a powder at -20°C . Guggulsterone has been investigated for chemoprevention of cancer and has demonstrated activity in various cancer cell lines, including head and neck squamous cell carcinoma, prostate cancer, and colon cancer .

C21H28O2

312.4458

312.208

C, 80.73; H, 9.03; O, 10.24

95975-55-6

39025-23-5 (Z-Guggulsterone); 39025-24-6 (E-Guggulsterone)

6450278

Typically exists as White to off-white solids at room temperature

1.1±0.1 g/cm3

463.3±45.0 °C at 760 mmHg

172.3±25.7 °C

0.0±1.1 mmHg at 25°C

1.557

Commiphora wightii

3.65

0

2

0

23

640

5

C[C@@]12C(=CC)C(=O)C[C@H]1[C@@H]1CCC3=CC(CC[C@]3(C)[C@H]1CC2)=O

WDXRGPWQVHZTQJ-BVEOMGIMSA-N

InChI=1S/C21H28O2/c1-4-16-19(23)12-18-15-6-5-13-11-14(22)7-9-20(13,2)17(15)8-10-21(16,18)3/h4,11,15,17-18H,5-10,12H2,1-3H3/t15?,17?,18?,20-,21+/m0/s1

(10R,13S)-17-ethylidene-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthrene-3,16-dione

Z/E-Guggulsterone; Guggulsterone; 95975-55-6; (10R,13S)-17-ethylidene-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthrene-3,16-dione

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)

DMSO : ~20.83 mg/mL (~66.67 mM)

Solubility in Formulation 1: 1.25 mg/mL (4.00 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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 2: ≥ 1.25 mg/mL (4.00 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10 mg/mL (32.01 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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