Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R.

AIMS: This study aimed to investigate the potential therapeutic applications of stigmasterol for treating neuropathic pain. METHODS: Related mechanisms were investigated by DRG single-cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting. RESULTS: Cell communication analysis by single-cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL-34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. In vitro studies demonstrated that stigmasterol can reduce the secretion of IL-34 in LPS-induced RSC96 Schwann cells; stigmasterol treatment of LPS-induced Schwann cell-conditioned medium (L-S-CM) does not induce the proliferation and migration of RAW264.7 macrophages; L-S-CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. In vivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model; stigmasterol can reduce IL-1ß, IL-6, TNF-α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that stigmasterol can reduce the levels of IL-34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats. CONCLUSION: Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R axis.

The elucidation of structure-activity and structure-permeation relationships for the cutaneous delivery of phytosterols to attenuate psoriasiform inflammation.

Phytosterols have been reported to exert anti-inflammatory activity. This study aimed to investigate the capacity of campesterol, ß-sitosterol, and stigmasterol on the mitigation of psoriasiform inflammation. We also tried to establish structure-activity and structure-permeation relationships for these plant sterols. To support this study, we first approached the in silico data of the physicochemical properties and the molecular docking of phytosterols with stratum corneum (SC) lipids. The anti-inflammatory activity of the phytosterols was explored in the activated keratinocytes and macrophages. Using the activated keratinocyte model, a significant inhibition of IL-6 and CXCL8 overexpression by phytosterols was detected. A comparable inhibition level was found for the three phytosterols tested. The macrophage-based study showed that the anti-IL-6 and anti-CXCL8 activities of campesterol were greater than those of the other compounds, which indicated that a phytosterol structure without a double bond on C22 and with methyl moiety on C24 was more effective. The conditioned medium of phytosterol-treated macrophages decreased STAT3 phosphorylation in the keratinocytes, suggesting the inhibition of keratinocyte hyperproliferation. ß-sitosterol was the penetrant with the highest pig skin absorption (0.33 nmol/mg), followed by campesterol (0.21 nmol/mg) and stigmasterol (0.16 nmol/mg). The therapeutic index (TI) is a parameter measured by multiplying the cytokine/chemokine suppression percentage with skin absorption for anticipating the anti-inflammatory activity after topical delivery. ß-sitosterol is a potential candidate for treating psoriatic inflammation due to having the greatest TI value. In this study, ß-sitosterol attenuated epidermal hyperplasia and immune cell infiltration in the psoriasis-like mouse model. The psoriasiform epidermis thickness could be reduced from 92.4 to 63.8 µm by the topical use of ß-sitosterol, with a downregulation of IL-6, TNF-α, and CXCL1. The skin tolerance study manifested that the reference drug betamethasone but not ß-sitosterol could generate barrier dysfunction. ß-sitosterol possessed anti-inflammatory activity and facile skin transport, showing the potential for development as an anti-psoriatic agent.

Stigmasterol alleviates allergic airway inflammation and airway hyperresponsiveness in asthma mice through inhibiting substance-P receptor.

CONTEXT: Stigmasterol has significant anti-arthritis and anti-inflammatory effects, but its role in immune and inflammatory diseases is still unclear. OBJECTIVE: The potential advantages of stigmasterol in asthma were explored in IL-13-induced BEAS-2B cells and asthmatic mice. MATERIALS AND METHODS: The optimal target of stigmasterol was confirmed in asthma. After detecting the cytotoxicity of stigmasterol in BEAS-2B cells, 10 µg/mL and 20 µg/mL stigmasterol were incubated with the BEAS-2B cell model for 48 h, and anti-inflammation and antioxidative stress were verified. Asthmatic mice were induced by OVA and received 100 mg/kg stigmasterol for 7 consecutive days. After 28 days, lung tissues and BAL fluid were collected for the following study. To further verify the role of NK1-R, 0.1 µM WIN62577 (NK1-R specific antagonist), and 1 µM recombinant human NK1-R protein were applied. RESULTS: NK1-R was the potential target of stigmasterol. When the concentration of stigmasterol is 20 µg/mL, the survival rate of BEAS-2B cells is about 98.4%, which is non-toxic. Stigmasterol exerted anti-inflammation and antioxidant stress in a dose-dependent manner and decreased NK1-R expression in IL-13-induced BEAS-2B. Meanwhile, in vivo assay also indicated the anti-inflammation and antioxidant stress of stigmasterol after OVA challenge. Stigmasterol inhibited inflammation infiltration and mucus hypersecretion, and NK1-R expression. DISCUSSION AND CONCLUSIONS: The protective effect of stigmaterol on asthma and its underlying mechanism have been discussed in depth, providing a theoretical basis and more possibilities for its treatment of asthma.

Stigmasterol attenuates inflammatory response of microglia via NF-κB and NLRP3 signaling by AMPK activation.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Although its pathogenesis remains unclear, studies have indicated microglia-mediated neuroinflammation playing an important role. Phytosterols are a class of natural compounds presented in food, and have anti-inflammatory abilities. Recent studies suggested that phytosterols can traverse the blood-brain barrier and enter the brain, however, it remains largely unknown that whether phytosterols affect neuroinflammation in the AD pathogenesis. Here, we used APPswe/PS1dE9 mice as the animal model of AD, and found that stigmasterol treatment attenuated cognitive deficits, and decreased Aß42 concentration in cortex and hippocampus. Stigmasterol treatment also suppressed neuroinflammation, by reducing pro-inflammatory cytokine levels and microglia activation. Next, we simulated BV2 cells with Aß42 oligomers, which induced inflammatory responses of microglia. Stigmasterol protected BV2 cells against Aß42 oligomers induced inflammation, and mediated secretion of pro-inflammatory cytokines via NF-κB and NLRP3 signaling pathways by AMPK activation. Stigmasterol also alleviated the M1 polarization of BV2 cells. In general, our study demonstrates that stigmasterol ameliorated neuroinflammation in APP/PS1 mice, and suppressed inflammatory response of microglia to Aß42 oligomers via AMPK/NF-κB and AMPK/NLRP3 signaling, which provides a mechanistic insight for stigmasterol in anti-inflammation and AD therapy.

The inhibitory role of stigmasterol on tumor growth by inducing apoptosis in Balb/c mouse with spontaneous breast tumor (SMMT).

BACKGROUND: Breast cancer is one of the most common types of cancer in women worldwide. Anti-apoptotic activity of cancer cells is considered the main reason for drug resistance in BC which reduces the 5-year survival rate of patients and is still considered the main obstacle for cancer therapy. Stigmasterol (SS) is natural phytosterols compound in the plant which has been proved to play an important role to lower cholesterol and inducing anti-inflammatory, and anticancer properties. METHODS: In this, study, we aimed to evaluate the effect of SS on the expression of anti-apoptotic genes (Bcl-2 and BCL-XL), and also evaluate its effects on cell apoptosis and cell viability using MCF-7 cell line as well as evaluating its effect on tumor growth of spontaneous breast tumor (SMMT) in vivo. RESULT: SS significantly decreased the expression of Bcl-2 and BCL-XL genes (*P < 0.05), induced apoptosis, and reduced cell proliferation in MCF-7 cell lines. Our in vivo study also indicated that SS could inhibit tumor size after treatment with (0, 10, 20 µM) compared to the normal control. CONCLUSION: SS can be suggested as a potential agent in BC cancer treatment or as an adjuvant based on its ability to decrease the expression of Bcl-2 and BCL-XL genes and induce apoptosis.

Evaluation of the Antidepressant-like Effect of Total Sterols Fraction and Stigmasterol Isolated from Leaves of Aegle marmelos and Possible Mechanism( s) of Action Involved.

BACKGROUND: Aegle marmelos Corr. (Rutaceae) commonly known as 'Indian Bael' has been used as a brain tonic traditionally. However, despite this traditional use, not enough scientific report is present that can confirm the use of this plant in neurological disorders. Thus, the total sterols fraction and stigmasterol from the leaves of Aegle marmelos were investigated for antidepressant-like effect along with their possible mechanism(s) of action by primarily performing acute toxicity study of total sterols. METHODS: An acute toxicological study was carried out at a single oral dose of 2000 mg/kg. Sign of toxicity was observed by estimating biochemical and performing histopathological analysis. For the antidepressant-like effect, different doses of total sterols (50-200 mg/kg, p.o. for seven days) and stigmasterol (5- 20 mg/kg, i.p. acute) were administered in mice using TST and FST models. To evaluate the mechanism of action, mice were pretreated with GABA, 5-HT, DA, adrenergic antagonists, and glutamate agonists. Furthermore, a neurochemical study was performed following TST and molecular docking study was also performed to determine the binding affinity of stigmasterol. RESULTS: Total sterols fraction presents no sign of toxicity up to the oral dose of 2000 mg/kg. Oral treatment of total sterols and acute intraperitoneal treatment of stigmasterol (except 5 mg/kg) reduced the immobility time significantly. Pretreatment with pCPA (5-HT synthesis inhibitor) and NMDA (an agonist of the glutamate site) effectively reversed the immobility time of total sterols and stigmasterol (except pCPA) in TST. However, bicuculline (competitive GABA antagonist), haloperidol (D2 dopaminergic antagonist) and prazosin (α1 adrenergic antagonist) could not reverse the immobility time. Meanwhile, total sterols also effectively altered the hippocampus 5-HT and Glu levels. Also, the result of the molecular docking study depicted that stigmasterol has an affinity to the NMDA receptor. CONCLUSIONS: The present study suggests that the total sterols fraction did not produce any acute toxicity in rats. Also, we reported that total sterols, stigmasterol and sub-effective stigmasterol coadministration with fluoxetine significantly reduced the time of immobility in TST and FST confirmed the antidepressant-like effect of total sterols fraction and stigmasterol. Moreover, further findings suggest that the antidepressant-like effect of total sterols might be mediated by the serotonergic and glutamatergic systems. Whereas only the glutamatergic system was involved in the antidepressant activity of stigmasterol.

Phytosterols: Potential Metabolic Modulators in Neurodegenerative Diseases.

Phytosterols constitute a class of natural products that are an important component of diet and have vast applications in foods, cosmetics, and herbal medicines. With many and diverse isolated structures in nature, they exhibit a broad range of biological and pharmacological activities. Among over 200 types of phytosterols, stigmasterol and ß-sitosterol were ubiquitous in many plant species, exhibiting important aspects of activities related to neurodegenerative diseases. Hence, this mini-review presented an overview of the reported studies on selected phytosterols related to neurodegenerative diseases. It covered the major phytosterols based on biosynthetic considerations, including other phytosterols with significant in vitro and in vivo biological activities.

Saringosterol from Sargassum fusiforme Modulates Cholesterol Metabolism and Alleviates Atherosclerosis in ApoE-Deficient Mice.

Dysregulation of cholesterol homeostasis is a major risk factor of atherosclerosis, which can lead to serious health problems, including heart attack and stroke. Liver X receptor (LXR) α and ß are transcription factors belonging to the nuclear receptor superfamily, which play important roles in cholesterol homeostasis. Selectively activating LXRß provides a promising strategy for the treatment of atherosclerosis. Here, we employed atherosclerotic apoE-knockout mice to evaluate the effects of saringosterol, a phytosterol with potent and selective action for LXRß, which we identified previously in edible marine seaweed Sargassum fusiforme. We found that saringosterol treatment reduced the atherosclerotic plaque burden without having undesirable adverse hepatic effects in apoE-deficient mice fed an atherogenic diet. Meanwhile, reduced serum levels of cholesterol, accompanied by altered expression of LXR-regulated genes involved in cholesterol absorption, transport, efflux, excretion, and elimination, were observed in apoE-knockout mice after saringosterol treatment. Together, our study not only establishes saringosterol as an effective cholesterol-lowering and anti-atherogenic phytosterol but also provides insights into the underlying mechanism.

Carrier-Free Triterpene Prodrugs with Glutathione Response and Biosafety for Synergistically Enhanced Photochemotherapy.

Carrier-free pure drug self-assembled nanosystems have been proposed as a promising strategy for synergetic anticancer therapy. Herein, we purposefully designed and synthesized disulfide-modified glutathione (GSH)-responsive natural pentacyclic triterpene betulinic acid (BA) with better biodegradability and biocompatibility to construct carrier-free photosensitive prodrugs BA-S-S/Ce6 NPs for synergistically enhanced and biosafe photochemotherapy. The molecular dynamics simulation elucidates the possible coassembly mechanism that the coplanar arrangement of BA-S-S dimeric may be primarily responsible for the formation of a long lamella-like or spherical morphology. The density functional theory calculations demonstrate that the reduced energy gap (ΔEST) of Ce6 facilitates the improved singlet oxygen generation of BA-S-S/Ce6 nanoparticles (NPs). The assembled prodrugs exhibited remarkable GSH-responsive property and multiple favorable therapeutic features, leading to enhanced synergistic antitumor efficacy without noticeable toxicity. Additionally, evaluation of the antitumor efficacy of another tetracyclic triterpene stigmasterol (ST)-mediated ST-S-S/Ce6 NPs further confirmed the effectiveness of this rational design. This work provides a promising insight for exploring the pure drug self-assembly behavior and construction of GSH-responsive carrier-free triterpenoid prodrugs toward improved multiple combination antitumor therapies.

Saringosterol acetate isolated from Hizikia fusiforme, an edible brown alga, suppressed hepatocellular carcinoma growth and metastasis in a zebrafish xenograft model.

Saringosterol acetate (SSA) was isolated from an edible brown alga Hizikia fusiforme. In this study, we developed an adult zebrafish human hepatocellular carcinoma (HCC) xenograft model to confirm that SSA inhibits tumor growth and metastasis. Established Hep3B cells labeled with the fluorescent tracker CM-Dil were xenografted into the abdominal cavity of zebrafish once every three days for one month. Compared with the control group, the fish injected with Hep3B showed a significant increase in α-fetoprotein (AFP) and factors related to tumor growth and metastasis (IL-6, TNF-α, TGFß, MMP2, and MMP9). Using the model, it was proven that SSA affected survival rate, AFP production, and the levels of factors related to tumor growth and metastasis via the PI3K/AKT/mTOR and TGFß/Smad pathways. In conclusion, this HCC model can be used for in vivo experiments to investigate the inhibition of cancer, and SSA may be useful for the treatment of cancer.

Fucosterol Suppresses the Progression of Human Ovarian Cancer by Inducing Mitochondrial Dysfunction and Endoplasmic Reticulum Stress.

Ovarian cancer is difficult to diagnose early and has high rates of relapse and mortality. Therefore, the treatment of ovarian cancer needs to be improved. Recently, several studies have been conducted in an attempt to develop anticancer drugs from naturally derived ingredients. Compared to traditional chemotherapy, natural compounds can overcome drug resistance with lower side effects. Fucosterol, a phytosterol present in brown algae, reportedly possesses many bioactive effects, including anticancer properties. However, the anticancer effects of fucosterol in ovarian cancer remain unexplored. Therefore, we investigated the effects of fucosterol on progression in human ovarian cancer cells. Fucosterol inhibited cell proliferation and cell-cycle progression in ovarian cancer cells. Additionally, fucosterol regulated the proliferation-related signaling pathways, the production of reactive oxygen species, mitochondrial function, endoplasmic reticulum stress, angiogenesis, and calcium homeostasis. Moreover, it decreased tumor formation in a zebrafish xenograft model. These results indicate that fucosterol could be used as a potential therapeutic agent in ovarian cancer.

Stigmasterol protects rats from collagen induced arthritis by inhibiting proinflammatory cytokines.

Rheumatoid arthritis (RA) is an autoimmune disorder, in which imbalance in synthesis and production of inflammatory cytokines promotes cartilage and bone destruction. Out of the numerous factors contributing to RA prognosis, the transcription factor NF-kBp65 and p38 mitogen-activated protein kinase (p38MAPK) signaling module has been well implicated as a key regulator of inflammation and downstream signaling events in RA. Stigmasterol (STG) is a natural plant based product exhibiting anti-inflammatory activity, however, the mechanism through which it exhibits anti-inflammatory activity has not been completely understood. The current study aimed to understand the mechanisms underlying the anti-inflammatory effect of STG in the treatment of RA in collagen-induced arthritic (CIA) model of arthritis. Our results showed that STG improved the clinical severity in CIA rats compared to control. The therapeutic effects were related with reduced joint destruction and improved histological alterations. Furthermore, treatment of STG also significantly suppresses the expression of proinflammatory mediators (TNF-α, IL-6, IL-1ß, iNOS and COX-2) and increases the expression of anti-inflammatory cytokine (IL-10) through down-regulating the expression of NF-kBp65 (inhibiting p-IKB-α activation) and p38MAPK in joints. In agreement with our results, we can suggest that high therapeutic efficacy of STG against CIA induced inflammation in rats are attributed through the suppressing proinflammatory cytokines.

Stigmasterol alleviates cerebral ischemia/reperfusion injury by attenuating inflammation and improving antioxidant defenses in rats.

BACKGROUND/AIMS: The paper aimed to investigate the effects of Stigmasterol on inflammatory factors, antioxidant capacity, and apoptotic signaling pathways in brain tissue of rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The neurological deficits of the rats were analyzed and HE staining was performed. The cerebral infarct volume was calculated by means of TTC staining, and neuronal apoptosis was detected by TUNEL staining. At the same time, the contents of glutathione peroxidase, glutathione, superoxide dismutase (SOD), nitric oxide, and malondialdehyde in brain tissue were measured. The expression of the relevant protein was detected by means of Western blotting. RESULTS: The results showed that the neurological deficit score and infarct area of the I/R rats in the soy sterol treatment group were significantly lower than those in the I/R group. Moreover, the levels of carbon monoxide and malondialdehyde in the soysterol group were significantly lower than those in the I/R group, and the expressions of cyclooxygenase-2 (Cox-2) and NF-κB (p65) in the soysterol group were also significantly lower than those in the I/R group. The expression of Nrf2 (nucleus) and heme oxygenase-1 (HO-1) increased significantly, and the activities of antioxidant enzymes and SOD were increased. In addition, the stigmasterol treatment can inhibit apoptosis, down-regulate Bax and cleaved caspase-3 expression, and up-regulate Bcl-Xl expression. CONCLUSION: Stigmasterol protects the brain from brain I/R damage by reducing oxidative stress and inflammation.

Stigmasterol as a potential biomarker for amphotericin B resistance in Leishmania donovani.

BACKGROUND: Leishmania donovani, a protozoan parasite, is the primary causative agent for visceral leishmaniasis. Toxicity and increased resistance to existing drugs have led to an urgent need for identifying new drugs and drug targets. Understanding the risks and mechanisms of resistance is of great importance. Amphotericin B (AmB) is a polyene antimicrobial, the mainstay therapy for visceral leishmaniasis in several parts of India. OBJECTIVES: In the present study, we established a line of AmB-resistant L. donovani promastigotes in vitro and demonstrated the molecular basis of resistance to AmB. METHODS: AmB-resistant promastigotes were generated and characterized to evaluate the mechanism of resistance to AmB. We examined the sterol composition of the promastigotes and the axenic amastigotes derived from the WT and AmB-resistant promastigotes. The role of the plant-like C-22 desaturase responsible for stigmasterol production was also evaluated in the AmB-resistant strain. RESULTS: The IC50 for resistant cells was four times higher than for the WT. AmB-resistant promastigotes showed an increase in the conversion of ß-sitosterol into stigmasterol. The presence of higher amounts of stigmasterol in resistant promastigotes, as well as in axenic amastigotes, signifies its role in AmB resistance in Leishmania. The resistant strain showed reduced infectivity in vitro. CONCLUSIONS: We have elucidated the mode of action and resistance mechanisms to the drug. However, further work is required to validate the potential role of stigmasterol in resistance and to help develop a diagnostic kit that can assist in diagnosing potentially resistant lines in the field.

Seaweed Secondary Metabolites with Beneficial Health Effects: An Overview of Successes in In Vivo Studies and Clinical Trials.

Macroalgae are increasingly viewed as a source of secondary metabolites with great potential for the development of new drugs. In this development, in vitro studies are only the first step in a long process, while in vivo studies and clinical trials are the most revealing stages of the true potential and limitations that a given metabolite may have as a new drug. This literature review aims to give a critical overview of the secondary metabolites that reveal the most interesting results in these two steps. Phlorotannins show great pharmaceutical potential in in vivo models and, among the several examples, the anti-dyslipidemia activity of dieckol must be highlighted because it was more effective than lovastatin in an in vivo model. The IRLIIVLMPILMA tridecapeptide that exhibits an in vivo level of activity similar to the hypotensive clinical drug captopril should still be stressed, as well as griffithsin which showed such stunning results over a variety of animal models and which will probably move onto clinical trials soon. Regarding clinical trials, studies with pure algal metabolites are scarce, limited to those carried out with kahalalide F and fucoxanthin. The majority of clinical trials currently aim to ascertain the effect of algae consumption, as extracts or fractions, on obesity and diabetes.

Elucidation of the Chemopreventive Role of Stigmasterol Against Jab1 in Gall Bladder Carcinoma.

BACKGROUND: Plant sterols have proven a potent anti-proliferative and apoptosis inducing agent against several carcinomas including breast and prostate cancers. Jab1 has been reported to be involved in the progression of numerous carcinomas. However, antiproliferative effects of sterols against Jab1 in gall bladder cancer have not been explored yet. OBJECTIVE: In the current study, we elucidated the mechanism of action of stigmasterol regarding apoptosis induction mediated via downregulation of Jab1 protein in human gall bladder cancer cells. METHODS: In our study, we performed MTT and Trypan blue assay to assess the effect of stigmasterol on cell proliferation. In addition, RT-PCR and western blotting were performed to identify the effect of stigmasterol on Jab1 and p27 expression in human gall bladder cancer cells. We further performed cell cycle, Caspase-3, Hoechst and FITC-Annexin V analysis, to confirm the apoptosis induction in stigmasterol treated human gall bladder cancer cells. RESULTS: Our results clearly indicated that stigmasterol has up-regulated the p27 expression and down-regulated Jab1 gene. These modulations of genes might occur via mitochondrial apoptosis signaling pathway. Caspase-3 gets activated with the apoptotic induction. Increase in apoptotic cells and DNA were confirmed through annexin V staining, Hoechst staining, and cell cycle analysis. CONCLUSION: Thus, these results strongly suggest that stigmasterol has the potential to be considered as an anticancerous therapeutic agent against Jab1 in gall bladder cancer.

Anti-nociceptive effect of stigmasterol in mouse models of acute and chronic pain.

Stigmasterol is a common sterol found in plants, but the anti-nociceptive effect of this compound and its mechanism of action are not fully explored. Thus, in the present study, the anti-nociceptive effect of stigmasterol was investigated in acute and chronic models of pain and its mechanism of action. We used adult male albino Swiss mice (25-35 g) to observe the anti-nociceptive effect of stigmasterol in acetic-acid writhing test or in complete Freund's adjuvant injection, surgical incision in hind paw, or partial sciatic nerve ligation. Moreover, we investigate the involvement of opioid receptors (naloxone, 2 mg/kg, intraperitoneally) in stigmasterol anti-nociceptive effect and stigmasterol action on acetylcholinesterase activity. Some possible adverse effects caused by stigmasterol were also investigated. Stigmasterol (0.3-3 mg/kg, orally) exhibited an anti-nociceptive effect on acetic-acid-induced writhing test. Furthermore, it markedly attenuated the mechanical allodynia caused by surgical incision (after acute treatment with stigmasterol, preventive and curative effects were observed) and partial sciatic nerve ligation (after acute treatment with stigmasterol) and complete Freund's adjuvant (after acute or repeated treatment with stigmasterol). The anti-nociceptive effect of stigmasterol was not reversed by naloxone. Moreover, stigmasterol did not alter in vitro acetylcholinesterase activity in spinal cord or brain samples. Also, stigmasterol did not cause gastric ulcers or alter the gastrointestinal transit of mice. Taken together, these results support the potential anti-nociceptive effect of stigmasterol in different models of pain.

Buddleja thyrsoides Lam. crude extract presents antinociceptive effect on an arthritic pain model in mice.

Arthritis is a chronic inflammatory disease which reduces the life quality of affected individuals. Therapeutic tools used for treating inflammatory pain are associated with several undesirable effects. Buddleja thyrsoides Lam., known as 'Barbasco' or 'Cambara', is mostly used in several disorders and possesses antirheumatic, anti-inflammatory, and analgesic properties. Here, we investigated the antinociceptive and anti-inflammatory effects of the B. thyrsoides crude extract applied orally and topically in acute pain models and an arthritic pain model induced by complete Freund's adjuvant (CFA) paw injection in male mice (25-30 g). The high-performance liquid chromatography (HPLC) of the B. thyrsoides extract crude revealed the presence of the lupeol, stigmasterol, and ß-sitosterol. The stability study of the B. thyrsoides gel did not show relevant changes at low temperatures. The oral treatment with the B. thrysoides extract prevented the capsaicin-induced spontaneous nociception and the acetic acid-induced abdominal writhing, but did not alter the thermal threshold in the tail immersion test. The B. thyrsoides antinociceptive effect was not reversed by naloxone in the capsaicin test. The B. thyrsoides oral or topical treatment reversed the CFA-induced mechanical allodynia and thermal hyperalgesia with maximum inhibition (Imax) of 69 ± 6 and 68 ± 5% as well as 78 ± 15 and 87 ± 12%, respectively. Moreover, the topical but not oral treatment inhibited the CFA-induced cell infiltration, but did not reduce the paw edema significantly. The oral treatment with B. thyrsoides did not cause adverse effects. These findings suggest that the oral or topical treatment with B. thyrsoides presents antinociceptive actions in an arthritic pain model without causing adverse effects.

Biodistribution and In Vivo Antileishmanial Activity of 1,2-Distigmasterylhemisuccinoyl-sn-Glycero-3-Phosphocholine Liposome-Intercalated Amphotericin B.

1,2-Distigmasterylhemisuccinoyl-sn-glycero-3-phosphocholine (DSHemsPC) is a new lipid in which two molecules of stigmasterol (an inexpensive plant sterol) are covalently linked via a succinic acid to glycerophosphocholine. Our previous study revealed that liposome (Lip)-intercalated amphotericin B (AMB) prepared from DSHemsPC (DSHemsPC-AMB-Lip) possesses excellent colloidal properties and in vitro antifungal and antileishmanial activities similar to those of the liposomal AMB preparation AmBisome. The aim of this study was to determine the biodistribution and evaluate the antileishmanial effects of DSHemsPC-AMB-Lip in Leishmania major-infected BALB/c mice. The serum profile and tissue concentrations of AMB were similar in DSHemsPC-AMB-Lip- and AmBisome-treated mice after intravenous (i.v.) injection. Multiple i.v. doses of the micellar formulation of AMB (Fungizone; 1 mg/kg of body weight), DSHemsPC-AMB-Lip (5 mg/kg), and AmBisome (5 mg/kg) were used in L. major-infected BALB/c mouse models of early and established lesions. In a model of the early lesions of cutaneous leishmaniasis (CL), the results indicated that the level of footpad inflammation was significantly (P < 0.001) lower in mice treated with DSHemsPC-AMB-Lip and AmBisome than mice treated with empty liposomes or 5% dextrose. The splenic and footpad parasite load was also significantly (P < 0.001) lower in these groups of mice than in control mice that received 5% DW or free liposome. The in vivo activity of DSHemsPC-AMB-Lip was comparable to that of AmBisome, and both provided improved results compared to those achieved with Fungizone at the designated doses. The results suggest that systemic DSHemsPC-AMB-Lip administration may be useful for the treatment of leishmaniasis, and because it costs less to produce DSHemsPC-AMB-Lip than AmBisome, DSHemsPC-AMB-Lip merits further investigation.

Stigmasterol retards the proliferation and pathological features of Trypanosoma congolense infection in rats and inhibits trypanosomal sialidase in vitro and in silico.

Stigmasterol has been reported to possess antitrypanosomal activity using in vitro model but information on the in vivo antitrypanosomal effects which is necessary in drug development process has not been evaluated. Hence, the present study investigates the in vivo effects of stigmasterol against T. congolense in addition to its inhibitory effects of trypanosomal sialidase. Stigmasterol, at 100mg/kg BW, did not significantly (p>0.05) reduce the progression of T. congolense infection in animals but a 200mg/kg BW stigmasterol treatment significantly (p<0.05) reduced the parasitemia, although, it did not completely eliminate the parasite from the bloodstream of infected animals. However, the stigmasterol treatments significantly (p<0.05) ameliorated the T. congolense induced anemia as well as hepatic and renal damages. Furthermore, the T. congolense-associated increase in free serum sialic acid with a corresponding decrease in membrane bound sialic acid were prevented, though insignificantly (p>0.05), by the 200mg/kg BW treatment. Subsequently, in vitro enzyme kinetic studies revealed that stigmasterol is an uncompetitive inhibitor of a partially purified bloodstream T. congolense sialidase with an inhibition binding constant of 356.59µM. Using molecular docking studies, stigmasterol formed a single hydrogen bonding interaction with a major residue (D63) at the catalytic domain of T. rangeli sialidase with a predicted binding free energy of -24.012kcal/mol. We concluded that stigmasterol could retard the proliferation and the major pathological features of T. congolense infection whilst the anemia amelioration was mediated via inhibition of sialidase.