Hepatoprotective effect of silyamarin in individuals chronically exposed to hydrogen sulfide; modulating influence of TNF-α cytokine genetic polymorphism

Background and the purpose of the study

Silymarin, a standardized extract of the milk thistle (Silybum marianum), is believed to exert some of its hepatoprotective effects though inhibition of free radicals and inflammation. In this study the effect of some pro- and anti-inflammatory cytokines and also antioxidant genes polymorphisms on the hepatoprotective effects of silymarin in the occupationally exposed individuals to hydrogen sulfide (H₂S) in the sour natural gas refinery was investigated.

Methods

We genotyped seven polymorphisms in six genes reported by others as modifiers of oxidative stress (NQO1, mEPXH1, GSTT1 and GSTM1) and inflammation (TNF-α and TGF-β1) for an association in effect of decreasing in liver function tests (LFTs). The LFTs of 77 sour gas refinery workers were measured before and after administration of silymarin (140 mg, three times per day for 1 month).

Results

A significant reduction of blood AST, ALT and ALP was observed after 30 days of consumption (p < 0.001). The decreasing effect of silymarin on ALT in the subjects with high producer genotype (A allele carriers) was less than low producers. There were no significant associations between TGF-β1 and the studied genes of oxidative stress pathway and the effectiveness of silymarin.

Conclusion

This is the first report about the effectiveness of silymarin in the subjects exposed chronically to H₂S. Meanwhile, the modulatory effect of TNF-α on the effectiveness of silymarin might be used for individualize therapy.     

Silymarin alleviates bleomycin-induced pulmonary toxicity and lipid peroxidation in mice

Context: The application of bleomycin is limited due to its side effects including lung toxicity. Silymarin is a flavonoid complex isolated from milk thistle [Silybum marianum L. (Asteraceae)] which has been identified as an antioxidant and anti-inflammatory compound.

Objective: This study evaluates the effect of silymarin on oxidative and inflammatory parameters in the lungs of mice exposed to bleomycin.

Materials and methods: BALB/c mice were divided into four groups of control, bleomycin (1.5?U/kg), bleomycin plus silymarin (50 and 100?mg/kg). After bleomycin administration, mice received 10?d intraperitoneal silymarin treatment. On 10th day, blood and lung samples were collected for measurement of oxidative and inflammatory factors.

Results: Silymarin led to a decrease in lung lipid peroxidation (0.19 and 0.17?nmol/mg protein) in bleomycin-injected animals. Glutathione-S-transferase (GST) which was inhibited by bleomycin (32.4?nmol/min/mg protein) induced by higher dose of silymarin (41?nmol/min/mg protein). Silymarin caused an elevation in glutathione (GSH): 2.6 and 3.1?µmol/g lung compare with bleomycin-injected animals 1.8?µmol/g lung. Catalase (CAT) was increased due to high dose of silymarin (65.7?µmol/min/ml protein) compare with bleomycin treated-mice. Myeloperoxidase (MPO) which was induced due to bleomycin (p?p?Conclusions: Silymarin attenuated bleomycin induced-pulmonary toxicity. This protective effect may be due to the ability of silymarin in keeping oxidant–antioxidant balance and regulating of inflammatory mediator release.     

The effect of silymarin (Silybum marianum) on human skin fibroblasts in an in vitro wound healing model

Context: Silymarin, a flavonolignan from Silybum marianum (L.) Gaertn. (Asteraceae), has been reported to have antioxidant and anti-inflammatory properties. Therefore, it may be worthwhile to study the effect of silymarin on wound healing.

Objective: To evaluate the effect of silymarin on human fibroblast cells in an in vitro model of wound healing.

Materials and methods: Human fibroblast cells were treated with different concentrations (4.5, 9, 18, 36 µg/mL) of silymarin. The effects of silymarin on cell viability, proliferation, collagen synthesis, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2′-deoxy-uridine, hydroxyproline analysis and real-time PCR, respectively. The effect of silymarin on cellular antioxidant status was determined by protection against hydrogen peroxide (H₂O₂)-induced cell injury and free radical scavenging activity (ABTS assay) of the cells.

Results: Results of the present study indicate that pretreatment of fibroblast cells with silymarin significantly protected cells against H₂O₂-induced injury (p < 0.05). After an 18?h treatment of cells with 36 µg/mL silymarin, total antioxidant capacity of cells significantly increased (p < 0.05). Furthermore, pretreatment of human fibroblast cells with silymarin significantly inhibited lipopolysaccharide (LPS)-induced COX-2 mRNA expression (p < 0.001). There was no significant difference in fibroblast proliferation and collagen synthesis between treatment and control groups (p > 0.05).

Discussion and conclusion: Silymarin may be useful as a therapeutic agent for the treatment of cutaneous wounds through its antioxidation and anti-inflammation effects.     

Inhibition of cyclooxygenase-2 and inducible nitric oxide synthase by silymarin in proliferating mesenchymal stem cells: comparison with glutathione modifiers

Silymarin, a mixture of flavonolignans, is extracted from milk thistle (Silybum marianum) and has a strong antioxidant activity and exhibits anticarcinogenic, anti-inflammatory, and cytoprotective effects. In this study we attempted to determine whether silymarin and the glutathione modifiers, buthionine sulfoxamine (BSO) and N-acetylcysteine (NAC), are involved in regulation of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in proliferating mesenchymal stem cells (MSCs). Cellular glutathione was manipulated during a 14-day culture using BSO, NAC and silymarin. At intervals of 2, 7 and 14 days, cells were collected and COX-2 and iNOS levels were measured. In parallel, generation of cellular H₂O₂ and glutathione were measured. Supplementation of the culture media with BSO caused a dose-dependent decrease in MSC proliferation, whereas NAC or silymarin elevated the proliferation (p < 0.05). Treatment of MSC with NAC or silymarin caused a significant decrease in COX-2 levels. However, COX-2 levels in cells treated with high levels of NAC (1.0 mM) were significantly lower than those in MSCs treated with high levels of silymarin (100 μM). BSO (1.0 and 5.0 μM) caused a significant increase in COX-2 on days 2, 7 and 14. BSO caused a significant increase in iNOS, whereas NAC or silymarin decreased cellular iNOS. Overall result show that glutathione, iNOS and COX-2 in proliferating MSCs are affected by silymarin treatment. It appears that glutathione is the main target of silymarin, and in consequence iNOS and COX-2 are affected in response to silymarin treatment.     

Pharmacological Antagonism of Fumonisin B1 Cytotoxicity in Porcine Renal Epithelial Cells (LLC‐PK1): A Model for Reducing Fumonisin‐Induced Nephrotoxicity in vivo

Abstract: Fumonisin B₁ is a mycotoxin commonly found on corn. It is hepatotoxic and nephrotoxic in domestic and experimental animals, and causes equine leukoencephalomalacia and porcine pulmonary oedema. It is a potent inhibitor of ceramide synthase. Inhibition leads to accumulation of free sphingoid bases in cells and tissues. In pig kidney epithelial cells (LLC‐PK₁), fumonisin B₁ induces increased tumour necrosis factor α (TNFα) expression independent of the accumulation of sphingoid bases. The objective of this study was to investigate pharmacological approaches for intervening in fumonisin B₁ toxicity using the LLC‐PK₁ cell model. The toxicity of fumonisin B₁ was assayed using cell viability and lactate dehydrogenase (lactate dehydrogenase) release. Pretreatment of cells with myriocin, preventing sphinganine accumulates, prevented the fumonisin B₁‐induced decrease in cell viability and increased lactate dehydrogenase release. Modulation of adenosine receptor activity did not reduce the fumonisin B₁ cytotoxicity. As with myriocin, silymarin pretreatment prevented the fumonisin B₁‐induced effects on cell viability and lactate dehydrogenase release. When added 6 or 24 hr after treatment of cells with fumonisin B₁, both myriocin and silymarin reversed the decreased cell viability and suppressed the increased lactate dehydrogenase release. Myriocin, but not silymarin, blocked the accumulation of sphinganine in fumonisin B₁‐treated cells. Silymarin, unlike myriocin, induced expression of TNFα to an extent similar to fumonisin B₁, but pretreatment with silymarin decreased the fumonisin B₁‐induced TNFα expression in LLC‐PK₁ cells. Results suggest that the mechanisms by which myriocin and silymarin protect renal cells are different, and silymarin potentially prevents fumonisin B₁‐induced toxicity by modulating TNFα expression or signals downstream of the inhibition of ceramide synthase.     

Novel phenanthridine (PHE-4i) derivative inhibits carrageenan-induced rat hind paw oedema through suppression of hydrogen sulfide

This study was conducted to assess the anti-inflammatory effect of a novel synthesized phenanthridine alkaloid (PHE-4i) and to examine the possible involvement of hydrogen sulfide (H₂S) in anti-inflammatory mechanism. The synthesized phenanthridine derivative PHE-4i (2, 5, and 10 mg/kg) was administered intraperitoneally to rats. One hour following treatment, inflammation was induced by intraplantar injection of carrageenan (1 %), in the hind paw. Paw volume as the index of inflammation was measured before and after carrageenan injection. Neutrophil sequestration into the hind paw was quantified by measuring tissue myeloperoxidase (MPO) activity and was compared for the inhibition of H₂S production. Pretreatment with PHE-4i significantly reduced carrageenan-induced hind paw weight, MPO activity, leukocyte infiltration, and H₂S production in a dose-dependent manner (p < 0.001). These results indicate that the anti-inflammatory effect of PHE-4i on carrageenan-induced rat paw oedema could be via the inhibition of the gaseous mediator H₂S.     

Effect of Bakuchiol on Leukocyte Functions and Some Inflammatory Responses in Mice

The effects of bakuchiol, a meroterpenoid isolated from the leaves of Psoralea glandulosa L., on phospholipase A₂ (PLA₂) activity from different sources, human neutrophil responses, zymosan air pouch and topical inflammation in mice, were investigated. This natural product was a weak inhibitor of secretory and intracellular PLA₂ but dose-dependently reduced the formation of LTB₄ and TXB₂ by human neutrophils and platelet microsomes, respectively. In addition, bakuchiol inhibited degranulation in human neutrophils, whereas superoxide generation was not affected. In mice, bakuchiol decreased cell migration, myeloperoxidase activity and eicosanoid levels in the air pouch inflammation induced by zymosan. After topical administration, this compound was effective as an inhibitor of oedema and myeloperoxidase activity in the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear oedema and significantly reduced the PGE₂ content and ear oedema in the arachidonic acid-induced response. Bakuchiol is a natural anti-inflammatory agent able to control leukocytic functions such as eicosanoid production, migration and degranulation in the inflammatory site.     

Flavonolignan Production in Cell Suspension Culture of Silybum marianum

The fruits of Silybum marianum (L.) Gaertn (Compositae) contain silymarin, an isomeric mixture of flavonolignans (silybin, silychristin, and silydianin). Silymarin acts as a strong anti-hepatotoxic. Silybum marianum cell cultures represent an alternate source of flavonolignans, but the yields of these compounds in such cultures are very low. In this study, in vitro cell culture systems of Silybum marianum were used to monitor the effect of elicitation (picloram, jasmonic acid, and light) on cell growth and production of silymarin. The presence of silymarin was established by high performance liquid chromatography and the greatest growth rates dry weight (32 mg) and silymarin content (0.413 mg g^{? 1} DW) were obtained with 3 mg l^{? 1} picloram and 2 mg l^{? 1} jasmonic acid in darkness after 28 days. The light grown cell suspension cultures showed reduced growth in term of dry weight (24 mg) and silymarin content. The data presented in this study demonstrate that media supplemented with 3 mg l^{? 1} picloram with JA in dark conditions can be useful for production of silymarin and growth in cell suspension cultures of Silybum marianum.     

Hepatoprotective effects of the nitric oxide donor isosorbide-5-mononitrate alone and in combination with the natural hepatoprotectant, silymarin, on carbon tetrachloride-induced hepatic injury in rats

The aim of this study was to investigate the effect of the nitric oxide donor isosorbide-5-mononitrate (5-ISMN) alone or in combination with the natural hepatoprotectant with anti-oxidant activity silymarin on the carbon tetrachloride (CCl₄)-induced hepatic injury in rats. 5-ISMN (1.8, 3.6 or 7.2 mg/kg), silymarin (25 mg/kg) or 5-ISMN (1.8, 3.6 or 7.2 mg/kg) combined with silymarin was given once daily orally simultaneously with CCl₄ and for 15 days thereafter. Liver damage was assessed by determining serum enzyme activities and hepatic histopathology. 5-ISMN given at the above doses conferred significant protection against the hepatotoxic actions of CCl₄ in rats, reducing serum alanine aminotransferase (ALT) levels by 31.2, 39.3 and 61.6%, respectively, when compared with controls. Serum aspartate aminotransferase (AST) levels decreased by 19.8, 22.7 and 59.4%, respectively, while alkaline phosphatase (ALP) decreased by 26.1 and 32.6% by the drug at 3.6 and 7.2 mg/kg, respectively. When silymarin was added to 5-ISMN (1.8, 3.6 or 7.2 mg/kg), ALT decreased by 32.8, 59.6, 70.2% and AST by 28.7, 50.3, 60%, when compared with CCl₄ control group levels. Silymarin in combination with 3.6 or 7.2 mg/kg 5-ISMN resulted in 37.5 and 39.2% reductions in ALP when compared with CCl₄ control group. Meanwhile, silymarin alone reduced ALT, AST and ALP levels by 65.9, 52 and 62.3%, respectively. Blood levels of reduced glutathione were markedly decreased in CCl₄-treated rats. Reduced glutathione levels were increased by the administration of 5-ISMN and restored to near normal values by silymarin treatment. Histopathological alterations by CCl₄ were markedly reduced after treatment with 5-ISMN alone or in combination with silymarin. Histopathologic examination of the livers of CCl₄-treated rats administered 5-ISMN at 7.2 mg/kg showed marked restoration of the normal architecture of the liver tissue and minimal fibrosis. Silymarin co-administered with 5-ISMN resulted in further improvement of the histologic picture. These results indicates that treatment with 5-ISMN protects against hepatocellular necrosis induced by CCl₄. The study suggests a potential therapeutic use for 5-ISMN in combination with silymarin in liver injury.     

The Effects of a Triterpene Fraction Isolated from Crataegus monogyna Jacq. on Different Acute Inflammation Models in Rats and Mice. Leucocyte Migration and Phospholipase A2 Inhibition

The plant Crataegus monogyna has action against cardiac insufficiency, angina and arrhythmia. The antiinflammatory properties of the cycloartenol fraction from this plant have been investigated. Chromatographic fractionation of the hexane extract of Crataegus monogyna Jacq. (Rosaceae) furnished a triterpene fraction containing cycloartenol as the main component (80.87%). The anti-inflammatory activity of the fraction was tested against hind-paw oedema induced by carrageenan in rats. At the highest oral dose (40 mg kg^?1) inhibition was 61.5 and 52.5% at 3 and 5 h respectively. In the mouse carrageenan peritonitis test, the triterpene fraction given orally inhibited peritoneal leucocyte infiltration (41.9, 64.7 and 89.4% at 10, 20 and 40 mg kg^?1, respectively). The fraction also showed weak inhibition of phospholipase A₂ (PLA₂) in-vitro. These results suggest that the fraction containing cycloartenol as the main component exerts an important anti-inflammatory action in-vivo by reducing the oedema.     

Evaluation of Some Pharmacological Activities of ethanol extracts of seeds, pericarp and leaves of Eugenia Jamolana in Rats

The present study investigated the effect of ethanol extracts of seeds, pericarp and leaves of Eugenia Jamolana (E. Jamolana) on inflammation, gastric ulcer, anti-oxidants and hepatoprotective in rats. The acute inflammation was induced by intra-plantar injection of carrageenan (100 μl of 1 %) in the rat hind paw. Gastric ulcer was evoked by indomethacin (25 mg/kg) oral administration. Liver damage was induced by given CCL₄ (2.5 ml/kg) orally. The median lethal (LD₅₀) of the ethanol extract of both seeds and pericarp were determined and revealed that the investigated extracts of seeds and pericarp were non toxic up to 5g/kg. The anti-inflammatory results showed that the oral administration of ethanol extract of E. Jamolana seeds (250,500 mg/kg) showed significant inhibition of oedema formation in dose-dependent manner by −27.86, −41.23, −44.73, −51.78 % and by −63.16, −37.77, −47.04, −55.36 % at 1, 2, 3 and 4 h at 1, 2, 3 and 4 h, respectively. While the pericarp given at dose (500 mg/kg) exhibited significant inhibition of the oedema formation by −34.64, −21,8, 19.23 and −33.47 % at 1, 2, 3 and 4 h, respectively post carrageenan injection as compared with saline control group. E. Jamolana leaves fraction 1 given orally at dose of 25 mg/kg, induced non significant change on oedema, while the oedema response was significantly inhibited by −25.14, −33.4, −20.57 and −26.46 % at 1, 2, 3 and 4 h, respectively in group of rats that received leaves fraction 2 at the same dose. Rats were given leaves fraction 3 extract showed inhibition of oedema formation by −4.48 % at 1^st h post- carrageenan injection, while at 2^nd, 3^rd and 4^th h showed non significant change on oedema formation. The acute gastric mucosal lesions was significantly reduced by given ethanol extract of E. Jamolana seeds, pericarp (250, 500 mg/kg) and leaves fractions 1, 2 and 3 (25 mg/kg) respectively in dose dependent manner, as compared with indomethacin treated group (control group). All tested extracts showed significant reduction in elevated serum ALT, AST and ALP levels as compared with CCl ₄ treated group. The ethanol extract of E. Jamolana seeds, pericarp and leaves fractions 1, 2, 3 showed significant elevation of blood GSH level and significant reduction in elevated plasma lipid peroxides (MDA) as compared with CCl₄ treated group. In conclusion we can see that the ethanol extracts of E. Jamolana of seeds, pericarp and leaves fractions showed anti-inflammatory, anti-ulcer, hepatoprotective and anti-oxidants activity. Received 15 June 2008; accepted 11 November 2008     

Anti-inflammatory activity of aqueous leaf extract of Chromolaena odorata

The anti-inflammatory activity of the aqueous extract of Chromolaena odorata was investigated in rats using the carrageenan-induced oedema, cotton pellet granuloma and formalin-induced oedema methods. The extract was administered orally at doses of 25, 50, 100 and 200 mg/kg. In the carrageenan method the paw oedema was significantly reduced by all the doses of the extract administered, with the 200 mg/kg dose producing the highest oedema inhibition (80.5%). In the cotton pellet method, granuloma weight was significantly reduced from 14 ± 0.1 to 9.0 ± 0.1 mg, while in the formaldehyde induced arthritis the extract inhibited the oedema during the 10-day period. In conclusion, this study has established the anti-inflammatory activity of C. odorata and, thus, justifies the traditional uses of the plant in the treatment of wounds and inflammation.     

Influence of silymarin components on keratinocytes and 3D reconstructed epidermis

Silymarin is a flavonoid complex isolated from the plant Silybum marianum which is well known for its antioxidant, hepatoprotective and immunomodulatory effects. Since little is known about its anti-inflammatory properties and healing effects, our study focused on whether or not silymarin components reduce inflammation and support epidermis regeneration. Lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS) were used to induce inflammation in normal human epidermal keratinocytes (NHEKs) and reconstructed epidermis (RHE), respectively. The expression of pro-inflammatory cytokines (IL-1, IL-6 and IL-8) in NHEKs and RHE was measured by enzyme – linked immunosorbent assay (ELISA). The expression of cytokeratin 14 and loricrin in RHE was detected by immunofluorescent analysis. Hematoxylin and eosin staining was used for the morphological evaluation of RHE. It was determined that 2, 3 – dehydrosilybin (DHSB) downregulated the production of selected pro-inflammatory cytokines produced by NHEKs. Although all layers of RHE displayed full thickness, when SDS was applied, cell detachment was seen in the stratum corneum and loricrin expression was diminished.     

Anti-inflammatory,analgesic and antioxidant activities of 3,4-oxo-isopropylidene-shikimic acid

Context 3,4-Oxo-isopropylidene-shikimic acid (ISA) is an analog of shikimic acid (SA). SA is extracted from the dry fruit of Illicium verum Hook. f. (Magnoliaceae), which has been used for treating stomachaches, skin inflammation and rheumatic pain.

Objective To investigate the anti-inflammatory, analgesic and antioxidant activities of ISA.

Materials and methods Analgesic and anti-inflammatory activities of ISA were evaluated using writhing, hot plate, xylene-induced ear oedema, carrageenan-induced paw oedema and cotton pellets-induced granuloma test, meanwhile the prostaglandin E₂ (PGE₂) and malondialdehyde (MDA) levels were assessed in the oedema paw tissue. ISA (60, 120 and 240?mg/kg in mice model and 50, 120 and 200?mg/kg in rat model) was administered orally, 30?min before induction of inflammation/pain. Additionally, ISA was administered for 12 d in rats from the day of cotton pellet implantation. The active oxygen species scavenging potencies of ISA (10^?3–10^?5 M) were evaluated by the electron spin resonance spin-trapping technique.

Results ISA caused a reduction of inflammation induced by xylene (18.1–31.4%), carrageenan (7.8–51.0%) and cotton pellets (11.4–24.0%). Furthermore, ISA decreased the production of PGE₂ and MDA in the rat paw tissue by 1.0–15.6% and 6.3–27.6%, respectively. ISA also reduced pain induced by acetic acid (15.6–48.9%) and hot plate (10.5–28.5%). Finally, ISA exhibited moderate antioxidant activity by scavenging the superoxide radical and hydroxyl radical with IC₅₀ values of 0.214 and 0.450?μg/mL, respectively.

Discussion and conclusion Our findings confirmed the anti-inflammatory, analgesic and antioxidant activities of ISA.     

The roles of Akt and MAPK family members in silymarin's protection against UV-induced A375-S2 cell apoptosis

Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) and has anti-inflammatory, cytoprotective as well as anticarcinogenic effects [Manna, S.K., Mukhopadlhyay, A., Van, N.T., Aggarwal, B., Silymarin suppresses TNF-induced activation of NF-kappaB, c-Jun N-terminal kinase, and apoptosis. J. Immunol. 1999; 163, 6800-6809.]. In this study, we assessed the effect of silymarin on ultraviolet light (UV)-induced cell apoptosis in human malignant melanoma, A375-S2 cells. Silymarin pre-treatment reversed the effect of UV irradiation on the expression of phosphorylated Akt and phosphorylated p53 (regulated by Akt activation), followed by down-regulation of Bax and up-regulated expressions of Bcl-2 and Bcl-xL proteins in UV-irradiated A375-S2 cells. Akt inhibitor decreased the viability of UV-irradiated cells which was treated with silymarin. In addition, the effect of UV irradiation on the phosphorylation of mitogen-activated protein kinase (MAPK) family members [extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK)] was also reversed by silymarin. Moreover, ERK inhibitor (PD98059) and p38 inhibitor (SB203580) augmented UV-induced apoptosis in silymarin treated A375-S2 cells. Consequently, silymarin partially reduced UV-induced apoptosis by activating the Akt pathway, and silymarin's protective effect was also exerted by MAPK family members.     

Effects of silymarin nanoemulsion against carbon tetrachloride-induced hepatic damage

Silymarin is a complex mixture of four flavonolignan isomers (silybin, isosilybin, silydianin and silychristin) obtained from ‘milk thistle’ (Silybum marianum). This plant compound is used almost exclusively for hepatoprotection. Because of its low and poor oral bioavailability, silymarin was formulated as a nanoemulsion to increase its solubility (and so its oral bioavailability) as well as therapeutic activity. The present study assessed the hepatoprotective activity on Wistar rats by determining biochemical parameters and histopathological properties of the nanoemulsion formulation of silymarin against carbon tetrachloride (CCl₄)-induced hepatotoxicity. Hepatoprotective activity was evaluated by the activity of serum alkaline phosphatase, alanine transaminase and aspartate transaminase; antioxidative defence markers (concentration of reduced glutathione); oxidative stress parameter (thiobarbituric acid reactive substances) and liver histopathology. The nanoemulsion-treated group showed significant decreases in glutamate oxaloacetate transaminase, pyruvate transaminase, alkaline phosphotase, total bilirubin and tissue lipid peroxides and increased total protein, albumin, globulin and tissue glutathione as compared to toxicant. The results indicate an excellent potential of the nanoemulsion formulation for the reversal of CCl₄-induced liver toxicity in rats as compared to standard silymarin.     

Anti-inflammatory activity of standardized dichloromethane extract of Salvia connivens on macrophages stimulated by LPS

Context: A previous study demonstrated that the chloroform extract of Salvia connivens Epling (Lamiaceae) has anti-inflammatory activity.

Objective: Identification of the active components in the dicholorometane extract (DESC), and, standardization of the extract based in ursolic acid.

Material and methods: DESC was prepared by percolation with dichlromethane and after washed with hot hexane, its composition was determined by CG-MS and NMR, and standardized by HPLC. The anti-inflammatory activity was tested on acute TPA-induced mouse ear oedema at doses of 2.0?mg/ear. The cell viability of macrophages was evaluated by MTT method, and pro- and anti-inflammatory interleukin levels were measured using an ELISA kit.

Results: Ursolic acid, oleanolic acid, dihydroursolic acid and eupatorin were identified in DESC, which was standardized based on the ursolic acid concentration (126?mg/g). The anti-inflammatory activities of DESC, the acid mixture, and eupatorin (2?mg/ear) were 60.55, 57.20 and 56.40% inhibition, respectively, on TPA-induced ear oedema. The IC₅₀ of DESC on macrophages was 149.4?μg/mL. DESC (25?μg/mL) significantly reduced TNF-α (2.0-fold), IL-1β (2.2-fold) and IL-6 (2.0-fold) in macrophages stimulated with LPS and increased the production of IL-10 (1.9-fold).

Discussion: Inflammation is a basic response to injuries, and macrophages are involved in triggering inflammation. Macrophage cells exhibit a response to LPS, inducing inflammatory mediators, and DESC inhibits the biosynthesis of the pro-inflammatory and promote anti-inflammatory cytokines.

Conclusion: DESC has an anti-inflammatory effect; reduced the levels of IL-1β, Il-6 and TNF-α; and increases IL-10 in macrophages stimulated with LPS. Ursolic acid is a good phytochemical marker.     

Anti-inflammatory properties of BHUx,a polyherbal formulation to prevent atherosclerosis

BHUx is a polyherbal formulation consisting of water-soluble fractions of five medicinal plants (Commiphora mukul, Terminalia arjuna, Boswellia serrata, Semecarpus anacardium and Strychnos nux vomica). The present study was undertaken to evaluate its antioxidant and anti-inflammatory effects. BHUx, standardized by HPLC fingerprinting and filtered through 0.2 μm filter paper, was employed for different studies under in vivo and in vitro conditions. Under in vivo conditions, BHUx significantly reduced inflammation in the carrageenan-induced rat paw oedema model of inflammation, suggesting its anti-inflammatory properties. In order to test the mechanism of action of BHUx, further in vitro studies were undertaken on cumene-hydroperoxide-induced lipid peroxidation (CHP) in liver homogenate, LPS-induced NO production in peritoneal macrophages and on key enzymes of arachidonic acid cascade, involved in the mediation of inflammation. Under the conditions, BHUx showed concentration-dependent inhibition of CHP-induced lipid peroxidation in liver homogenate, suggesting its antioxidant properties. Similarly the potent anti-inflammatory effects of BHUx are evident by (a) preferential inhibition of COX-2 (IC₅₀ for COX-2 = 80 μg/ml and IC₅₀ for COX-1 = 169 μg/ml), (b) low ratios in the IC₅₀ values of COX-2/COX-1 (0.47), (c) decreased production of NO in LPS-induced peritoneal macrophages and (d) inhibition of 5-LOX (IC₅₀ = 795 μg/ml). BHUx also showed a preference for inhibiting 15-lipoxygenase (IC₅₀ = 44 μg/ml), a key enzyme implicated in LDL oxidation. These studies suggest that BHUx is acting mainly at three levels, i.e., as a potent natural antioxidant, by reduction of key inflammatory mediators of arachidonic acid cascade and by preventing 15-LOX-mediated LDL oxidations, to prevent atherosclerosis.     

Preparation of tritium‐labeled Silybin—a protectant for common liver diseases

Silymarin, the seed extract of milk thistle plant, Silybum marianum, has been used traditionally for the treatment of liver diseases and bile duct infection. Silybin 1 is the main bioactive components of silymarin, consisting a pair of diastereomers: Silybin A and Silybin B. In this article, we report the preparation of tritium‐labeled Silybin, which was accomplished by protection of Silybin as tritylated compound 2 and followed by oxidation of the primary alcohol to its corresponding aldehyde 3 . Subsequent reduction with NaB[³H]₄ and deprotection of the trityl group provided the tritium‐labeled Silybin 4 . Copyright © 2006 John Wiley & Sons, Ltd.