Subjective biosafety assessment of bisdemethoxycurcumin from the rhizomes of Curcuma longa.

Introduction/Background: Curcuma longa, a plant native to the Indian subcontinent has a variety of biological activities. Curcumin is the most abundant and biologically active compound with many therapeutic properties. Demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) - the two other bioactive components present in Curcuma longa, besides curcumin, are collectively termed curcuminoids. Apart from the well-known curcumin, BDMC also has been reported to possess promising biological and pharmacological effects, but very little scientific evidence on its safety assessment has been published.Objective: The present study was undertaken to determine the safety of pure BDMC from Curcuma longa extract in rodents which comprises of general toxicity (both four weeks and three months duration), reproductive/developmental toxicity and genotoxicity studies.Methods: The Good Laboratory Practice studies were carried out in accordance with the test guidelines established by the Organization for Economic Cooperation and Development.Results: No treatment-related adverse findings were seen in general toxicity testing and a no observed adverse effect level (NOAEL) of 1000 mg/kg/day was established after four weeks (sub-acute) and three-months (sub-chronic) dosing. Evaluation of fertility, embryo-fetal, and post-natal reproductive and developmental parameters also showed no adverse findings with a NOAEL of 1000 mg/kg/day established. The results of genotoxicity as evaluated by in vitro reverse mutation assay, and in vivo micronucleus test in mice indicate that BDMC did not induce any genotoxic effects.Conclusion: Oral administration of BDMC is safe in rodents and non-mutagenic, with no adverse effects under experimental conditions.

Multifunctional neuroprotective effect of Withanone, a compound from Withania somnifera roots in alleviating cognitive dysfunction.

Alzheimer's disease (AD) is a chronic disorder that slowly worsens and impairs the person's memory, learning, reasoning, judgment, communication and familiar tasks with loss of orientation. AD is characterized clinically by cognitive deficit and pathologically by the deposition of ß amyloid plaques, neurofibrillary tangles, associated with degeneration of the cholinergic forebrain. Withanone (WS-2), a compound isolated from root extract of Withania somnifera at doses administered orally/day to wistar rats for duration of 21 days showed significant improvement in the cognitive skill by inhibiting amyloid ß-42 and attenuated the elevated levels of pro-inflammatory cytokines like TNF alpha, IL-1 beta, IL-6, MCP-1, Nitric oxide, lipid peroxidation and both ß- and γ- secretase enzymatic activity. Administration of WS-2 also significantly reversed the decline in acetyl choline and Glutathione (GSH) activity. None of the treatments that are available today alter the underlying causes of this terminal disease. Few preliminary clinical treatments have demonstrated that some plant medicines do ameliorate and improve memory and learning in patients with mild-to-moderate AD. WS-2 showed promise in AD treatment because of cognitive benefits and more importantly, mechanisms of action with respect to the fundamental pathophysiology of the disease, not limited to the inhibition of AChE, but also include the modification of Aß processing, protection against oxidative stress and anti-inflammatory effects.

Anti-arthritogenic effect of Saponin-1 by alteration of Th1/Th2 cytokine paradigm in arthritic mice.

OBJECTIVE & DESIGN: Investigation was carried out on Saponin 1 (SAP-1), a novel molecule isolated from Parthenium hysterophorus, on proinflammatory (Th1) & anti-inflammatory (Th2) cytokines in blood of arthritic balb/c mice. METHODS: Adjuvant induced developing inflammatory arthritis was induced in mice which were treated with SAP-1 in graded oral doses. The molecular markers were determined using Flow Cytometry which uses sensitivity of amplified fluorescence detection to measure soluble analytes in particle based immune assay. The T-helper (Th1) deviated cells produce detectable level of Tumor necrosis factor (TNF-alpha), interleukin-2 (IL-2) & interferon-gamma (IFN-gamma), while the Th2 deviated cells produce significant amount of interleukin-4 (IL-4) and interleukin-5 (IL-5). RESULTS: SAP-1 at graded oral doses inhibited expression of IFN-gamma & TNF-alpha in serum & correspondingly increased expression of IL-4 significantly. SAP-1 also inhibited IL-17 and CD4(+)CD25(+) cell population showing to have suppressive effect on Th-17 pathway as well as T-regulatory cells. It also suppressed the increased levels of pro-inflammatory mediators like IL-1ß and NO. Inhibitors of Cox-2 and MCP-1 provide effective improvements in signs and symptoms of Rheumatoid Arthritis. SAP-1 decreased the elevated concentration of both COX-2 and MCP-1 in arthritic animals. CONCLUSIONS: SAP-1 diminishes Th1 immunity activation, a primary cause of arthritis, in favour of Th2 dominance, which reduces arthritic condition in mice displaying immune-modulatory potential.

Immunosuppressive effects of Euphorbia hirta in experimental animals.

Euphorbia hirta L. (Euphorbiaceae) (E. hirta) is a tree locally used as a traditional medicine in Africa and Australia to treat numerous diseases such as hypertension, respiratory ailments, tumors, wounds, antipyretic, anti-inflammatory activities, etc. Therefore, we undertook to investigate their immunomodulatory effect on T lymphocytes (CD3+, CD4+ and CD8+ receptors) and Th1 cytokines (IL-2, TNF-α, IFN-γ) in a dose-dependent manner. E. hirta ethanol extract at 25, 50, 100 and 200 mg/kg doses was given orally for 7 days from the day of immunization. E. hirta maximum inhibition at 100 and 200 mg/kg p.o. was found to significantly block the production of the cell-mediated immune response, (CD3+, CD4+ and CD8+ receptors) and (IL-2, TNF-α, IFN-γ) and also prolongs graft rejection. E. hirta also showed a decrease of delayed hypersensitivity (DTH) response and dose-related decrease in the primary antibody response, respectively. Based on the data, it can be suggested that E. hirta is a potent and non-toxic immunosuppressor, which can be further explored for the development of potent immunosuppressor.

TNF-α inhibitory effect of Euphorbia hirta in rats.

CONTEXT: Euphorbia hirta L. (Euphorbiaceae) (E. hirta) is a tree locally used as a traditional medicine in Africa and Australia to treat numerous diseases such as hypertension, respiratory ailments, tumors, and wounds, and it has reported antiallergic, antipyretic, anti-inflammatory activities, etc. OBJECTIVE: This study evaluated the ability of fresh leaves of E. hirta ethanol extract to inhibit the intracellular tumor necrosis factor α (TNF-α) level in the synovial fluid and neutrophils in lipopolysaccharide (LPS)-induced inflamed rat knees. MATERIALS AND METHODS: Female Wister albino rats 140-160 g were used. E. hirta ethanol extract was given orally at 25, 50, 100, and 200 mg/kg, 2 h before an intra-articular (i.a.) injection of LPS. Two and three hours later, synovial fluid and neutrophils levels of intracellular TNF-α production were measured. RESULTS: In the time course of the experiment, E. hirta maximum inhibition at 100 and 200 mg/kg (p.o.) dose showed 16.5 ± 1.34 and 14.4 ± 1.30% of synovial fluid, 4.26 ± 0.36 and 3.78 ± 0.29% of neutrophils levels of intracellular TNF-α productions at 2 h after LPS injection. LPS control displayed 22.97 ± 1.61 and 6.78 ± 0.34% of synovial fluid and neutrophils levels of intracellular TNF-α at 2 h after LPS injection. Intracellular TNF-α was also estimated at 3 h after LPS injection. DISCUSSION AND CONCLUSION: The LPS-injected rat knee model gives a comparative study of acute anti-inflammatory responses. E. hirta inhibition of proinflammatory intracellular cytokine TNF-α production with LPS-induced inflamed rat knee is of great importance in defining the anti-arthritic potential of E. hirta.

Assessment of Safety Profile of Activated Curcumin C3 Complex (AC3®), Enriched Extract of Bisdemethoxycurcumin from the Rhizomes of Curcuma longa.

The present work was carried out to investigate the toxic effects of Activated Curcumin C3 Complex (AC3®) through the methods of acute, subacute, subchronic, reproductive/developmental toxicity, and genotoxicity when administered orally in experimental rodents. The studies were carried out in line with OECD principles of good laboratory practice. A single-dose acute oral toxicity study was conducted on female Wistar rats that produced no toxic effects after 14 days (the observation period) of treatment. Subacute, subchronic, and reproductive/developmental studies were conducted in Wistar rats, divided equally into vehicle control, 125, 250, and 500 mg/kg dose groups along with recovery groups for vehicle control and high dose. In all the studies, there were no abnormal clinical signs/behavioral changes, reproductive and developmental parameters, or gross and histopathological changes. Likewise, no alteration was found in the body weight, hematology, and other biochemical parameters. Also, it did not show mutagenicity in the in vitro AMES test or clastogenicity and aneugenicity in the in vivo micronucleus test, indicating that AC3® did not induce any genotoxic effects. This revealed that oral administration of AC3® is safe in rodents, nonmutagenic, and had no observed adverse effects under experimental conditions.

Anti-arthritic activity of agnuside mediated through the down-regulation of inflammatory mediators and cytokines.

OBJECTIVE AND DESIGN: The purpose of this study was to elucidate the probable mechanism for the anti-arthritic activity of agnuside (AGN), a compound isolated from the leaf extract of Vitex negundo. METHODOLOGY: The anti-inflammatory activity of AGN within a dose range of 1.56-12.50 mg/kg in normal and adrenalectomized rats was evaluated against different inflammagens. An array of pro-inflammatory mediators (PGE(2) and LTB(4)) and T-cell-mediated cytokines (IL-2, TNF-α, IFN-γ, IL-4, IL-10, IL-17) was assayed using flow cytometry, in arthritic paw tissue homogenate and splenocytes of treated animals. RESULTS: Significant anti-arthritic activity was observed in the polyarthritis test in rats and this was associated with significant suppression of inflammatory mediators and T-cell-mediated cytokines (Th1/Th2). The anti-inflammatory activity in adrenalectomized rats confirmed that the effect of AGN is not mediated by the pituitary-adrenal axis. AGN also showed inhibition of vascular permeability and leukocyte migration in vivo. CONCLUSION: The study suggests the possible development of AGN as a therapeutic agent in the treatment of arthritis by the modulation of the host immune response.

Differential effects of chlorogenic acid on various immunological parameters relevant to rheumatoid arthritis.

Despite chlorogenic acid (CGA) being widely present in nature, particularly in the human diet, there is very little information regarding its pharmacological activities. The present investigation was carried out to investigate the antiarthritic activities of this compound in adjuvant induced-arthritis in male Wistar rats, and to explore the underlying mechanisms of actions in view of immunological responses. We observed that CGA effectively controlled the total (CD3) and differentiated (CD4 and CD8) T cells count at the dose of 40 mg/kg. We also assessed the effect on co-stimulatory molecules (CD28, CD80/86) and found that CGA efficiently suppressed CD80/86 but failed to bring any changes in the CD28 count, whereas ibuprofen (standard drug) resulted in highly significant inhibition of both. We next examined the effect on CD4⁺ T cells specific Th1/Th2 cytokines by flow cytometry and observed that CGA suppressed the Th1 cytokines in a highly significant manner but elevated Th2 cytokines with dose dependence. Results of the present investigation suggest that CGA is a potent antiarthritic agent.

The Anti-Obesity Potential of Cyperus rotundus Extract Containing Piceatannol, Scirpusin A and Scirpusin B from Rhizomes: Preclinical and Clinical Evaluations.

PURPOSE: Obesity is a complex medical problem that increases the risk of other diseases like diabetes, cardiovascular diseases, and fatty liver disease. The present study evaluated the efficacy and safety of Cyperus rotundus rhizome extract (CRE), standardized to contain Piceatannol, Scirpusin A, and Scirpusin B (5% total Stilbenoids) in overweight individuals. The mechanism of activity was evaluated in a diet-induced mice model of obesity and adipocytes in vitro. MATERIALS AND METHODS: The efficacy, safety, and tolerability of CRE were evaluated in 30 obese individuals with a BMI of 30 to 40 kg/m2 for 90 days in a randomized, double-blind, parallel-group, placebo-controlled study. In vitro studies were carried out in differentiated 3T3 L1 adipocytes, and the therapeutic efficacy was evaluated in high-fat diet-induced obese mice. RESULTS: The pilot clinical study showed a reduction in body weight with a significant decrease in waist circumference and BMI. The serum lipid profile showed a significant improvement in CRE-treated individuals. The extract was well tolerated, and no adverse effects were reported at the end of the study. CRE showed a dose-dependent adipogenesis reduction in vitro with an IC50 value of 9.39 µg/mL, while oral administration of CRE reduced weight gain in diet-induced obese mice. The efficacy in mice was associated with reduced levels of leptin, corticosteroids, and serum lipid levels, with no adverse effects. CONCLUSION: CRE has anti-adipogenic properties, is safe for human consumption, and effectively manages weight and hypercholesterolemia in overweight individuals.

Psilostachyin, acetylated pseudoguaianolides and their analogues: preparation and evaluation of their anti-inflammatory potential.

Naturally occurring acetylated pseudoguaianolides and psilostachyin including their analogues were synthesized. The structure of semi-synthetic psilostachyin was also confirmed by X-ray crystallography. The anti-inflammatory potential of all the derivatives has been evaluated through in vitro expression of TNF-α, IL-1ß and IL-6 in murine neutrophils.

Selective Th2 Upregulation by Crocus sativus: A Neutraceutical Spice.

The immunomodulatory activity of an Indian neutraceutical spice, saffron (Crocus sativus) was studied on Th(1) and Th(2) limbs of the immune system. Oral administration of alcoholic extract of Crocus sativus (ACS) at graded dose levels from 1.56-50 mg/kg p.o. potentiated the Th(2) response of humoral immunity causing the significant increases in agglutinating antibody titre in mice at a dose of 6.25 mg/kg and an elevation of CD19(+) B cells and IL-4 cytokine, a signature cytokine of Th(2) pathway. Appreciable elevation in levels of IgG-1 and IgM antibodies of the primary and secondary immune response was observed. However, ACS showed no appreciable expression of the Th(1) cytokines IL-2 (growth factor for CD4(+) T cells) and IFN-γ (signature cytokine of Th(1) response). A significant modulation of immune reactivity was observed in all the animal models used. This paper represents the selective upregulation of the Th(2) response of the test material and suggests its use for subsequent selective Th(2) immunomodulation.

Modulation of Th1/Th2 cytokines and inflammatory mediators by hydroxychavicol in adjuvant induced arthritic tissues.

The present study was undertaken to investigate the anti-arthritic activity of hydroxychavicol (HC) a major phenolic compound isolated from the aqueous extract leaves of plant Piper betle (Piperaceae). The compound showed significant lowering of pro-inflammatory (Th1) cytokine levels in arthritic paw tissue homogenate supernatant viz. IL-2, IFN-gamma, and TNF-alpha with maximum inhibition at higher dose levels of 2 and 4 mg/kg p.o. and enhanced the production of anti-inflammatory (Th2) cytokines IL-4 and IL-5 estimated by cytometric bead array immunoassay. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate five cytokines in a 50-microl sample volume. The T-helper (Th1) deviated cells produce detectable level of tumor necrosis factor (TNF-alpha), interleukin-2 (IL-2), and interferon-gamma (IFN-gamma), while the Th2 deviated cells produce significant amount of interleukin-4 (IL-4) and interleukin-5 (IL-5). HC at graded doses also significantly decreased the expression of IL-1beta, PGE(2), LTB(4), and nitric oxide levels showing significant inhibition of these parameters. Elevated levels of CD4(+) T cell specific interferon-gamma (IFN-gamma) in splenocytes of arthritic animals was also inhibited in treated animals. The oral LD(0) in both mice and rats was more than 1000 mg/kg.

Protective effect of Labisia pumila on stress-induced behavioral, biochemical, and immunological alterations.

The aim of the present study was to investigate the antistress potential of LABISIA PUMILA aqueous extract (LPPM/A003) using a battery of tests widely employed in different stressful situations. Pretreatment of experimental animals with LPPM/A003 caused an increase in the swimming endurance and hypoxia time and also showed the recovery of physical stress-induced depletion of neuromuscular coordination and scopolamine induced memory deficit. LPPM/A003 at graded doses reversed the chronic restraint stress (RST), induced depletion of CD4 (+) and CD8 (+) T lymphocytes, NK cell population, and corresponding cytokines expression besides downregulating the stress-induced increase in plasma corticosterone, a major stress hormone. In addition, LPPM/A003 reversed the chronic stress-induced increase in adrenal gland weight, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and hepatic lipid peroxidation (LP) levels and augmented the RST induced decrease in hepatic glutathione (GSH), thymus and spleen weight. Thus, we conclude that LPPM/A003 has the ability to reverse the alterations produced by various stressful stimuli and therefore restores homeostasis.

Downregulation of pro-inflammatory cytokines by lupeol measured using cytometric bead array immunoassay.

The objective of the study was to investigate the activity of Lupeol (LUP) on proinflammatory and anti-inflammatory cytokines in the pleural exudate from male swiss albino mice. We applied Cytometric bead array technology for simultaneously measurement of these cytokines in pleurisy induced mice treated with lupeol in graded oral doses. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate 5 cytokines in a 50 microlitre sample volume. Oral administration of LUP at doses of 25, 50, 100 and 200 mg/kg p.o. produced dose related inhibition of IL-2, IFN-gamma and TNF-alpha in the pleural exudate with the most significant effect at 100 mg/kg oral dose. LUP had a non significant inhibitory effect on the levels of IL-4 and IL-5.

Evaluation of the antimicrobial, antioxidant, and anti-inflammatory activities of hydroxychavicol for its potential use as an oral care agent.

Hydroxychavicol isolated from the chloroform extraction of aqueous extract of Piper betle leaves showed inhibitory activity against oral cavity pathogens. It exhibited an inhibitory effect on all of the oral cavity pathogens tested (MICs of 62.5 to 500 microg/ml) with a minimal bactericidal concentration that was twofold greater than the inhibitory concentration. Hydroxychavicol exhibited concentration-dependent killing of Streptococcus mutans ATCC 25175 up to 4x MIC and also prevented the formation of water-insoluble glucan. Interestingly, hydroxychavicol exhibited an extended postantibiotic effect of 6 to 7 h and prevented the emergence of mutants of S. mutans ATCC 25175 and Actinomyces viscosus ATCC 15987 at 2x MIC. Furthermore, it also inhibited the growth of biofilms generated by S. mutans and A. viscosus and reduced the preformed biofilms by these bacteria. Increased uptake of propidium iodide by hydroxychavicol-treated cells of S. mutans and A. viscosus indicated that hydroxychavicol probably works through the disruption of the permeability barrier of microbial membrane structures. Hydroxychavicol also exhibited potent antioxidant and anti-inflammatory activities. This was evident from its concentration-dependent inhibition of lipid peroxidation and significant suppression of tumor necrosis factor alpha expression in human neutrophils. Its efficacy against adherent cells of S. mutans in water-insoluble glucan in the presence of sucrose suggests that hydroxychavicol would be a useful compound for the development of antibacterial agents against oral pathogens and that it has great potential for use in mouthwash for preventing and treating oral infections.

Semi-synthetic analogs of pinitol as potential inhibitors of TNF-alpha cytokine expression in human neutrophils.

Semi-synthetic analogs of pinitol were subjected to screening by determining TNF-alpha expression in human neutrophils using flowcytometry. Among the tested compounds, three derivatives displayed more than 50% inhibition of TNF-alpha cytokine secretion in LPS induced stimulated neutrophils and can be considered as potent anti-inflammatory moieties.

Saponins as novel TNF-alpha inhibitors: isolation of saponins and a nor-pseudoguaianolide from Parthenium hysterophorus.

Two novel saponins and a 13-nor-pseudoguaianolide designated as hysterolactone were isolated from Parthenium hysterophorus. The two saponins were found to be potent inhibitors of TNF-alpha. Their mode of inhibition was studied through molecular modeling. The wet lab results were in concordance with the data obtained from docking experiments.

Immunomodulatory activity of Asparagus racemosus on systemic Th1/Th2 immunity: implications for immunoadjuvant potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Roots of Asparagus racemosus Willd (Shatavari in vernacular) are widely used in Ayurveda as Rasayana for immunostimulation, galactogogue as also in treatment of conditions like ulcers and cancer. Various studies have indicated immunomodulatory properties of Shatavari root extracts and formulations. AIM OF THE STUDY: To study the effect of standardized Asparagus racemosus root aqueous extract (ARE) on systemic Th1/Th2 immunity of SRBC sensitized animals. MATERIALS AND METHODS: We used HPTLC to quantify steroidal saponins (Shatavarin IV, Immunoside) and flow cytometry to study effects of ARE on Th1/Th2 immunity. SRBC specific antibody titres and DTH responses were also monitored as markers of Th2 and Th1 responses, respectively. We also studied lymphocyte proliferation. Cyclosporin, cyclophosphamide and levamisole were used as controls. RESULTS: Treatment with ARE (100mg/(kg b.w.p.o.)) resulted in significant increase of CD3(+) and CD4/CD8(+) percentages suggesting its effect on T cell activation. ARE treated animals showed significant up-regulation of Th1 (IL-2, IFN-g) and Th2 (IL-4) cytokines suggesting its mixed Th1/Th2 adjuvant activity. Consistent to this, ARE also showed higher antibody titres and DTH responses. ARE, in combination with LPS, Con A or SRBC, produced a significant proliferation suggesting effect on activated lymphocytes. CONCLUSION: The study suggests mixed Th1/Th2 activity of ARE supports its immunoadjuvant potential.

Amelioration of adjuvant induced arthritis by apocynin.

The premise of the study was to investigate the antiarthritic potential of apocynin (APO) in Balb/c mice (in vivo). The experiment showed a dose-dependent decrease in oedema and showed a suppression of proinflammatory cytokines such as TNF-alpha and IL-1beta and mediators such as prostaglandin E(2) (PGE(2)) and LTB(4). At oral doses of 0.5, 1.0, 2.0 and 4.0 mg/kg once daily during the course of the experiment, APO induced an inhibition of T cell mediated immune response causing suppression of CD4+ and CD8+ T cells and of intracellular interferon-gamma (IFN-gamma) by flow cytometry in arthritic mice. In parallel there was a dose-dependent inhibition in vascular permeability causing an inhibition in the migration of leucocytes and exudate volume at the site of the inflammatory reaction. These observations validate the immunoregulatory potential of apocynin.

Immunomodulatory activity of isoflavones isolated from Iris germanica (Iridaceae) on T-lymphocytes and cytokines.

The immunomodulatory activities of two isoflavones, 5,7-dihydroxy-6,4'-dimethoxyisoflavone (irisolidone) (1) and 5,4'-dihydroxy-6,7-methylenedioxyisoflavone (irilone) (2) isolated from Iris germanica (Iridaceae) is reported. Their influence on production of T-lymphocytes (CD4+ and CD8+ cells) and T-cell cytokines, namely Th1: IL-2, IFN-gamma and Th2: IL-4 and IL-5 in a dose-dependent manner was studied by flow cytometric method in Balb/c mice. Oral administration of drugs at doses of 0.1-0.8 mg/kg per oral dose showed 1 to possess stimulatory activity on T-cells and Th1 cytokine production, while as 2 acted as an immunosuppressant for both cells and cytokines. The methylated products of 1 and 2 showed a similar trend to that of their parent compounds but their activity was drastically decreased revealing the importance of free phenolic groups for their immunomodulating activities.