A Synergistic Botanical Composition Increases Resting Energy Expenditure and Reduces Adiposity in High-Fat Diet-Fed Rats.

OBJECTIVE: An imbalance between dietary energy intake and energy expenditure may result in body fat gain or obesity. Increasing resting energy expenditure (REE) is an attractive strategy for managing body fat gain. The objective of the current study was to generate proof-of-concept data on a synergistic composition (LN19183) of Citrus aurantifolia fruit rind (CA) and Theobroma cacao seed (TC) extracts to increase REE and reduce body fat gain in a high-fat diet (HFD)-fed rats. METHOD: In in vitro cell-based experiments, CA, TC, or LN19183 were tested for fibroblast growth factor 21 (FGF-21) production from 3T3-L1 mouse adipocytes. Uncoupling protein 1 (UCP-1) and beta3-adrenergic receptor (ß3-AR) protein expressions in LN19183-treated 3T3-L1 lysates were also tested. The 56-day in vivo study in male Sprague Dawley (SD) rats (age: 12-14 weeks; body weight [b.w.]: 115-197 g) contained 2 phases of 28 days each of induction and supplementation. Seven rats received a regular rodent diet (RD) over 56 days. In the induction phase, 21 rats received HFD; in the supplementation phase, the obese rats (n = 7) received either HFD alone or in concurrence with a daily oral dose of either 100 or 250 mg/kg b.w. of LN19183 for 28 days. RESULTS: In 3T3-L1 adipocytes, LN19183 synergistically increased FGF-21 production and dose-dependently increased ß3-AR and UCP-1 protein expression. In HFD-fed rats, both doses of LN19183 supplementation significantly (p < 0.05) decreased the body weight gain, total fat mass, and liver weight and increased (p < 0.05) REE. High-dose LN19183 also significantly (p < 0.05) increased fat oxidation and UCP-1 protein expression in white fat tissue and reduced liver triglyceride (TG) level. LN19183-supplemented groups substantially reduced serum TG and glucose levels compared to the HFD rats. CONCLUSIONS: LN19183 reduces body fat mass and weight gain via increased REE and fat oxidation in HFD-fed obese rats.

A synergistic blend of Garcinia mangostana fruit rind and Cinnamomum tamala leaf extracts enhances myogenic differentiation and mitochondrial biogenesis in vitro and muscle growth and strength in mice.

Background: A proprietary combination of Garcinia mangostana fruit rind and Cinnamomum tamala leaf extracts (LI80020F4, CinDura®) improved the physical performance and muscle strength of resistance-trained adult males. Objective: This study assessed the underlying mechanisms of the ergogenic potential of LI80020F4 in in vitro and in vivo models. Methods: The individual extracts and their combination (LI80020F4) were assessed for nitrite production in EAhy926 human endothelial cells. Subsequent experiments evaluated the effect of LI80020F4 in myotube formation in C2C12 mouse myoblasts, expression of mammalian target of rapamycin (mTOR) signaling proteins, myogenic factors, and mitochondrial functions in L6 rat myoblasts.Moreover, adult male ICR mice were randomly assigned (n = 15) into vehicle control (G1), exercise alone (G2), oxymetholone-16 mg/kg body weight (bw) (G3), and 75 (G4)-, 150 (G5)-, or 300 (G6) mg/kg bw of LI80020F4, orally gavaged for 28 days. G1 and G2 mice received 0.5% carboxymethylcellulose sodium. Following completion, muscle strength and physical performance were assessed on forelimb grip strength and forced swimming test (FST), respectively. Gastrocnemius (GA), tibialis anterior (TA) muscle weights, muscle fiber cross-sectional area (CSA), levels of muscle, and serum protein markers were also determined. Results: LI80020F4 increased nitrite production in EAhy926 cells in a dose-dependent manner. LI80020F4 induced C2C12 myotube formation, increased mitochondrial biogenesis, upregulated the expressions of activated mTOR and other mitochondria and myogenic proteins, and mitigated H2O2-induced mitochondrial membrane depolarization in the myoblast cells. In the animal study, 75, 150, and 300 mg/kg bw LI80020F4 doses significantly (P < 0.05) increased the animals' forelimb grip strength. Mid- and high-dose groups showed increased swimming time, increased muscle weight, CSA, muscle growth-related, and mitochondrial protein expressions in the GA muscles. Conclusion: LI80020F4 increases nitric oxide production in the endothelial cells, mitochondrial biogenesis and function, upregulates skeletal muscle growth-related protein expressions and reduces oxidative stress; together, it explains the basis of the ergogenic potential of LI80020F4.

Toxicological Assessments of a Proprietary Blend of Punica granatum Fruit Rind and Theobroma cacao Seed Extracts: Acute, Subchronic, and Genetic Toxicity Studies.

LN18178 (Tesnor®) is a standardized, proprietary composition of aqueous ethanol extracts of Punica granatum fruit rind and Theobroma cacao seeds. The present study demonstrates a broad-spectrum toxicological evaluation of LN18178 utilizing in vitro and in vivo preclinical models following the Organization for Economic Cooperation and Development (OECD) guidelines for testing chemicals. Wistar rats did not show any clinical signs of toxicity and morbidity in acute oral and dermal toxicity tests with the median lethal dose (LD50) values of at least 5000 mg/kg and 2000 mg/kg body weight, respectively. LN18178 was nonirritating to the skin and eyes of the treated rabbits. In a ninety-day subchronic repeated oral dose toxicity study, the LN18178-treated Wistar rats did not show dose-related signs of toxicity on their body weight, food consumption, organ weights, hematology, and clinical chemistry parameters. The estimated no-observed-adverse-effect level (NOAEL) of LN18178 in male and female rats was 2500 mg/kg body weight. The observations from the bacterial reverse mutation test, in vitro chromosomal aberration assay, micronucleus assay in mouse bone marrow erythrocytes, and in vitro mouse lymphoma TK+/- gene mutation assay suggest that LN18178 is neither mutagenic nor clastogenic. In summary, the present study demonstrates that oral consumption of the herbal blend LN18178 does not show signs of toxicity; also it does not elicit genetic toxicity in the standard preclinical models.

Safety assessment of a novel water-soluble extract of Boswellia serrata gum resin: acute toxicity, 90-day sub-chronic toxicity, Ames' bacterial reverse mutation, and in vivo micronucleus assays.

Boswellia serrata gum resin extracts have demonstrated potential benefits in alleviating joint pain and discomfort of osteoarthritis. The major objective of the present study was to assess the safety of a water-soluble B. serrata gum resin extract (LI51202F1) in diverse models of acute oral, acute dermal, primary dermal irritation, eye irritation, and 90-day sub-chronic repeated dose toxicity studies, as well as Ames' bacterial reverse mutation assay and in vivo micronucleus assay. The acute oral and dermal toxicity studies in Sprague Dawley (SD) rats demonstrated that the median lethal dose (LD50) of LI51202F1 is >2000 mg/kg body weight (BW). The acute dermal and eye irritation tests in New Zealand white rabbits exhibited that LI51202F1 is non-irritating to the skin and mildly irritating to the eyes, respectively. The 90-days sub-chronic repeated oral dose study demonstrated that the LI51202F1-treated male and female SD rats did not show signs of toxicity on their BW, food intake, organ weights, thyroid hormones, and on the clinical pathology, gross pathology, and histopathological assessments. In male and female rats, the no-observed-adverse-effect level (NOAEL) of LI51202F1 was 500 mg/kg/day, the highest tested dose in the study. The results of the bacterial reverse mutation assay in Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2uvrA (pKM101) strains in the presence or absence of S9 metabolic activation system and a micro-nucleus assay in mouse bone marrow erythrocytes demonstrated that LI51202F1 is neither mutagenic nor clastogenic. In conclusion, under the conditions of these studies, LI51202F1 demonstrated broad-spectrum safety.

Combined extracts of Moringa oleifera, Murraya koeingii leaves, and Curcuma longa rhizome increases energy expenditure and controls obesity in high-fat diet-fed rats.

BACKGROUND: LI85008F is a proprietary combination of leaf extracts of Moringa oleifera, Murraya koeingii, and extract of Curcuma longa rhizome. This herbal extract combination is an effective weight loss supplement for overweight and obese subjects. The present study aimed to investigate the thermogenic potential of the LI85008F in high-fat diet (HFD)-induced obese Sprague Dawley rats. METHODS: Seven rats received a regular diet (RD), and twenty-one rats received a high-fat diet (HFD) for 56 days. On day 28, the HFD-fed rats were randomized into three groups (n = 7). Starting from day 29 through day 56, one HFD-fed group received daily oral gavage of 0.5% Carboxymethylcellulose Sodium (CMC) alone (HFD), and the remaining two groups received 100 and 250 mg/kg LI85008F (LI85008F-100 and LI85008F-250, respectively). Body weight, fat mass, fat cell size, liver weight, liver triglyceride were measured. The energy metabolism parameters were measured using indirect calorimetry. In serum, the metabolic and endocrine markers were analyzed. The adipogenic and thermoregulatory proteins expression in the white adipose tissue (WAT) were analyzed using an immunoblot assay. RESULTS: Supplementation with both doses of LI85008F significantly increased resting energy expenditure (REE) in the obese rats. The LI85008F-250 rats showed significant up-regulation of uncoupling protein-1 (UCP-1) expression, as compared with the HFD rats. LI85008F significantly reduced body weight gain, fat mass, fat cell size, liver weight, and hepatic triglycerides. Serum triglyceride, total cholesterol, glucose, leptin, and fat cell markers were significantly reduced in LI85008F-supplemented rats compared to the HFD rats. CONCLUSION: The present data suggest that LI85008F reduces body fat mass and controls body weight gain via increasing energy metabolism in combination with reduced lipogenesis in diet-fed obese rats.

Acute, Subacute, and Genotoxicity Assessments of a Proprietary Blend of Garcinia mangostana Fruit Rind and Cinnamomum tamala Leaf Extracts (CinDura®).

The present communication describes a battery of toxicity studies that include an acute oral toxicity, a subacute twenty-eight-day repeated oral dose toxicity, and genotoxicity studies on a herbal formulation CinDura® (GMCT). This proprietary herbal composition contains the extracts of the Garcinia mangostana fruit rind (GM) and the Cinnamomum tamala leaf (CT). The toxicological evaluations were performed following the Organization for Economic Cooperation and Development (OECD) guidelines. The acute oral toxicity study in Wistar rats suggests that the median lethal dose of CinDura® is at least 2000 mg/kg body weight. Acute dermal and eye irritation tests in New Zealand white rabbits indicate that the test item is nonirritant to the skin and eyes. A twenty-eight-day repeated dose oral toxicity study was conducted in male and female Wistar rats using daily doses of 250, 500, and 1000 mg/kg body weight, followed by a fourteen-day reversal period for two satellite groups. The CinDura®-supplemented animals did not show any sign of toxicity on their body weights, organ weights, and on the hematobiochemical parameters. The gross pathology and histopathological examinations indicated no treatment-related changes in the experimental animals. Overall, the no-observed-adverse-effect level (NOAEL) of the herbal blend is 1000 mg/kg body weight, the highest tested dose. Also, the results of the bacterial reverse mutation test and the erythrocyte micronucleus assay in mouse bone marrow suggest that CinDura® (GMCT) is neither mutagenic nor clastogenic.

An Anti-Inflammatory Composition of Boswellia serrata Resin Extracts Alleviates Pain and Protects Cartilage in Monoiodoacetate-Induced Osteoarthritis in Rats.

The boswellic acids, the active compounds in Boswellia serrata gum resin extract, are potent anti-inflammatory agents and are specific nonredox inhibitors of 5-Lipoxygenase (5-LOX). Here, we present the anti-osteoarthritis (OA) efficacy of LI13019F1 (also known as Serratrin®), a unique composition containing the acidic and nonacidic fractions of B. serrata gum resin. This composition strongly inhibited 5-LOX activity with the half-maximal inhibitory concentration (IC50) of 43.35 ± 4.90 µg/mL. Also, LI13019F1 strongly inhibited the leukotriene B4 (IC50, 7.80 ± 2.40 µg/mL) and prostaglandin E2 (IC50, 6.19 ± 0.52 µg/mL) productions in human blood-derived cells. Besides, LI13019F1 reduced TNF-α production with the IC50 of 12.38 ± 0.423 µg/mL. On average, 1, 2.5, and 5 µg/mL doses of LI13019F1 protected 34.62, 47.66, and 62.29% SW1353 human chondrosarcoma cells from IL-1ß induced SOX-9 depletion, respectively. Further, a 28-day preclinical proof-of-concept study evaluated the pain relief efficacy of LI13019F1 in monoiodoacetate- (MIA-) induced Sprague-Dawley rats. At the end of the study, 150 and 300 mg/kg doses of LI13019F1 supplemented rats showed significant improvements (55.17 ± 5.81 g (p < 0.05), and 66.22 ± 6.30 g (p < 0.05), respectively, vs. MIA: 31.22 ± 7.15 g) in body-weight-bearing capacities. Concurrently, LI13019F1-150 and LI13019F1-300 rats substantially (p < 0.05) increased the threshold of pain sensitivity to pressure (26.98 ± 2.36 and 28.06 ± 2.72-gram force, respectively; vs. 18.63 ± 5.82 in MIA) and increased (p < 0.05) the latent time to withdraw the paw after a thermal stimulus (23.61 ± 2.73 and 28.18 ± 1.90 sec, respectively; vs. 16.56 ± 1.22 sec. in MIA). Besides, the histological observations on Safranin-O green stained articular cartilage revealed that LI13019F1 also prevented the MIA-induced structural damage of the cartilage and reduced the loss of the extracellular matrix (ECM) components in the experimental rats. In conclusion, the present observations suggest that LI13019F1, a new composition of B. serrata gum resin extracts, reduces pain and protects articular cartilage from the damaging action of MIA in a rodent model.

Toxicological Assessment of a Standardized Boswellia serrata Gum Resin Extract.

The acidic and non-acidic fractions of Boswellia serrata gum resin extracts were combined to prepare a unique product, LI13019F1 (Serratrin). The present series of studies evaluated LI13019F1 for acute and subchronic (28-day) toxicity in Wistar rats and acute dermal and eye irritation in New Zealand white rabbits. The mutagenicity and clastogenicity of LI13019F1 were evaluated in bacteria and mouse bone marrow erythrocytes, respectively. All studies were performed following the Organization for Economic Co-operation and Development guidelines. Acute oral and acute dermal toxicity studies did not show mortality or signs of toxicity in Wistar rats at a limit dose of 2,000 mg/kg LI13019F1. LI13019F1 did not cause irritation to the skin or the eyes of New Zealand white rabbits. In a repeated dose 28-day oral toxicity study, LI13019F1-treated Wistar rats did not show dose-related signs of toxicity on their body weights, organ weights, and on the hematology and clinical chemistry parameters. The estimated no observed adverse effect level for LI13019F1 was 1,000 mg/kg/day in both male and female rats. The bacterial reverse mutation test and a micronucleus assay in mouse bone marrow erythrocytes revealed that LI13019F1 was neither mutagenic nor clastogenic. Together, the present observations demonstrate a broad-spectrum safety of LI13019F1.

Synthesis, in vitro and in silico evaluation of diaryl heptanones as potential 5LOX enzyme inhibitors.

A new series of diaryl heptanones (12a-q) were synthesized and their structures were confirmed by its 1H, 13C NMR and Mass spectral data. These analogs were evaluated for their anti-oxidant activity and potential to inhibit 5-lipoxygenase. Compounds 12k and 12o showed potent in vitro 5-lipoxygenase enzyme inhibitory activity with IC50 values of 22.2, 21.5 µM, which are comparable to curcumin (24.4 µM). Further they also have shown significant antioxidant activity. Molecular docking studies clearly showed correlation between binding energy and potency of these compounds.

Four new Sesquiterpenoids from Sphaeranthus indicus.

Isolation and characterisation of two new eudesmanolides, 5α-hydroperoxy-7α-hydroxy-isosphaerantholide (1) and (11α,13-dihydro-7α-hydroxyfrullanolide-13-yl)-adenine (2) from the flower heads of Sphaeranthus indicus are described. In addition, 5α-hydroxy-isosphaerantholide (3) and 11α,13-dihydro-eudesman-3,5,7-triene-6α-12-olide (4) are reported first time as a metabolite of S. indicus and as a natural product, respectively. The structures of these compounds were established from rigorous analysis of their high-resolution mass, IR, UV, 1H NMR, 13C NMR and 2D NMR spectral data.

Synthesis of new analogs of AKBA and evaluation of their anti-inflammatory activities.

A new series of 11-keto-ß-boswellic acid and 3-O-acetyl-11-keto-ß-boswellic acid analogs (5, 7, 8, 10, 13, 18a-d, 27a-c, 28a-d) were synthesized by modification of hydroxyl and acid functional moieties of boswellic acids. The structures of these analogs were confirmed by spectral data analysis (1H, 13C NMR and mass). Compounds 18b, 27a and 8 showed potent 5-lipoxygenase enzyme inhibitory activity (IC50: 19.53, 20.31 and 44.14µg/mL). The computational studies revealed that selectivity of AKBA is due to its fitment into the 5-LOX receptor, which is missing for the other enzymes like 12-LOX, COX-1 and COX-2. Our study found potentiating effects of 2-formyl and 3-keto substituents in reviving inactive AKBA analogues possessing essential COOH group at 4th position.

A New Flavanone from the Leaves of Chromolaena odorata.

Chromolaena odorata (Syn: Eupatorium odoratum) is a perennial plant belonging to the Asteraceae family. Extracts of C. odorata have shown strong anti-oxidant and moderate anti-adipogenenesis activities. Detailed phytochemical investigation of C. odorata led to the isolation of 21 secondary metabolites, which include a new flavanone (3), and a chalcone, 3,2'-dihydroxy-4,4',5',6'-tetramethoxychalcone (2), reported for the first time from a natural source, besides three known compounds 1, 4 and 5, all of which are reported for the first time from C. odorata. Some of these compounds exhibited potent anti-oxidant and anti-adipogenesis activities and these may be partly responsible for the activity of the extract.

Mitochondrial-targeted curcuminoids: a strategy to enhance bioavailability and anticancer efficacy of curcumin.

Although the anti-cancer effects of curcumin has been shown in various cancer cell types, in vitro, pre-clinical and clinical studies showed only a limited efficacy, even at high doses. This is presumably due to low bioavailability in both plasma and tissues, particularly due to poor intracellular accumulation. A variety of methods have been developed to achieve the selective targeting of drugs to cells and mitochondrion. We used a novel approach by conjugation of curcumin to lipophilic triphenylphosphonium (TPP) cation to facilitate delivery of curcumin to mitochondria. TPP is selectively taken up by mitochondria driven by the membrane potential by several hundred folds. In this study, three mitocurcuminoids (mitocurcuminoids-1, 2, and 3) were successfully synthesized by tagging TPP to curcumin at different positions. ESI-MS analysis showed significantly higher uptake of the mitocurcuminoids in mitochondria as compared to curcumin in MCF-7 breast cancer cells. All three mitocurcuminoids exhibited significant cytotoxicity to MCF-7, MDA-MB-231, SKNSH, DU-145, and HeLa cancer cells with minimal effect on normal mammary epithelial cells (MCF-10A). The IC50 was much lower for mitocurcuminoids when compared to curcumin. The mitocurcuminoids induced significant ROS generation, a drop in ΔØm, cell-cycle arrest and apoptosis. They inhibited Akt and STAT3 phosphorylation and increased ERK phosphorylation. Mitocurcuminoids also showed upregulation of pro-apoptotic BNIP3 expression. In conclusion, the results of this study indicated that mitocurcuminoids show substantial promise for further development as a potential agent for the treatment of various cancers.

Digalloylresveratrol, a novel resveratrol analog inhibits the growth of human pancreatic cancer cells.

Digalloylresveratrol (DIG) is a recently synthesized substance aimed to combine the effects of the natural polyphenolic compounds gallic acid and resveratrol, which both are excellent free radical scavengers with anticancer activity. In this study, we investigated the effects of DIG in the human AsPC-1 and BxPC-3 pancreatic adenocarcinoma cell lines. Treatment with DIG dose-dependently attenuated cells in the S phase of the cell cycle and led to a significant depletion of the dATP pool in AsPC-1 cells. The incorporation of (14)C-cytidine into nascent DNA of tumor cells was significantly inhibited at all DIG concentrations due to inhibition of ribonucleotide reductase, a key enzyme of DNA synthesis in tumor cells. Furthermore, Erk1/2 became inactivated and moderated p38 phosphorylation reflecting increased replication stress. DIG also activated ATM and Chk2, and induced the phosphorylation and proteasomal degradation of the proto-oncogene Cdc25A, which contributed to cell cycle attenuation. Taken together, DIG is an excellent free radical scavenger, strongly inhibits RR in situ activity, cell cycle progression, and colony formation in AsPC-1 and BxPC-3 cells thus warranting further investigations.

Synthesis and biological evaluation of nonaprenylsulfates.

A naturally occurring nonaprenylsulfate (1) and its synthetic analogue (2) were synthesized from substituted phenolic precursors in three steps with an overall yield of 40-45%. Both compounds exhibited potent anti-inflammatory activity against 5-lipoxygenase, and potent brine shrimp lethality. They also showed moderate anti-oxidant activity in the super oxide radical scavenging model. Nonaprenylsulfate (1) showed moderate inhibition of paw edema in Freund's Complete Adjuvant (FCA) induced model of arthritis, thus confirming its anti-inflammatory activity.

Combination effects of digalloylresveratrol with arabinofuranosylcytosine and difluorodeoxycytidine in human leukemia and pancreatic cancer cells.

Digalloylresveratrol (DIG) is a newly synthesized agent aimed to combine the biological effects of the natural compounds, gallic acid and resveratrol, which both are free radical scavengers exhibiting anticancer activity. In this study, we investigated the effects of DIG on the growth of human HL-60 leukemia cells and on the colony formation of human BxPC-3 and PANC-1 pancreatic cancer cells. DIG was applied alone and in combination with arabinofuranosylcytosine (Ara-C) or difluorodeoxycytidine (dFdC), depending on the cell line employed. All IC(50) values observed were in the low micromolar range rendering DIG a promising antitumor compound in vitro. Considering the combination experiments, DIG yielded additive effects with Ara-C in HL-60 cells and-to a lesser extent-with dFdC in BxPC-3 and PANC-1 cells. Owing to our results, DIG may be further investigated in vitro and in animals.

Cellular and molecular mechanisms of anti-inflammatory effect of Aflapin: a novel Boswellia serrata extract.

There is significant number of evidences suggesting the anti-inflammatory properties of gum resin extracts of Boswellia serrata containing 3-O-acetyl-11-keto-ß-boswellic acid (AKBA) and their promising potential as therapeutic interventions against inflammatory diseases such as osteoarthritis (OA). Unfortunately, the poor bioavailability of AKBA following oral administration might limit the anti-inflammatory efficacy of standardized Boswellia extract(s). To address this issue, we describe a novel composition called Aflapin, which contains B. serrata extract enriched in AKBA and non-volatile oil portion of B. serrata gum resin. Our observations show that the availability of AKBA in systemic circulation of experimental animals is increased by 51.78% in Aflapin-supplemented animals, in comparison with that of 30% AKBA standardized extract or BE-30 (5-Loxin(®)). Consistently, Aflapin confers better anti-inflammatory efficacy in Freund's Complete Adjuvant (FCA)-induced inflammation model of Sprague-Dawley rats. Interestingly, in comparison with BE-30, Aflapin(®) also provides significantly better protection from IL-1ß-induced death of human primary chondrocytes and improves glycosaminoglycans production in human chondrocytes. In Tumor necrosis factor alpha (TNFα)-induced human synovial cells, the inhibitory potential of Aflapin (IC(50) 44.736 ng/ml) on matrix metalloproteinase-3 (MMP-3) production is 14.83% better than that of BE-30 (IC(50) 52.528 ng/ml). In summary, our observations collectively suggest that both the Boswellia products, BE-30 (5-Loxin(®)) and Aflapin, exhibit powerful anti-inflammatory efficacy and anti-arthritic potential. In particular, in comparison with BE-30, Aflapin provides more potential benefits in recovering articular cartilage damage or protection from proteolytic degradation due to inflammatory insult in arthritis such as osteoarthritis or rheumatoid arthritis.

Neuroprotective and antiinflammatory properties of a novel demethylated curcuminoid.

A demethylated derivative of curcumin (DC; 67.8% bisdemethylcurcumin, 20.7% demethylmonodemethoxycurcumin, 5.86% bisdemethoxycurcumin, 2.58% demethylcurcumin) was prepared by using a 95% extract of curcumin (C(95); 72.2% curcumin, 18.8% monodemethoxycurcumin, 4.5% bisdemethoxycurcumin). DC increased glutathione and reduced reactive oxygen species (ROS) in HT4 neuronal cells. In a model of glutamate-induced death of HT4, DC was more effective than C(95) in neuroprotection. The protective effects of DC were retained even when DC was withdrawn from culture media after pretreatment. DC treatment, unlike an equal dose of C(95), completely spared glutamate-induced loss of cellular GSH. Both DC and C(95) prevented glutamate-induced elevation of cellular ROS but failed to attenuate glutamate-induced elevation of intracellular calcium. In human microvascular endothelial cells (HMECs) challenged with TNF-alpha, GeneChip analysis revealed that only a subcluster of 23 TNF-alpha-inducible genes were uniquely sensitive to C(95). In sharp contrast, 1,065 TNF-alpha-inducible genes were sensitive to DC but not to C(95), suggesting that DC was more effective in antagonizing the effects of TNF-alpha on HMECs. Functional analysis identified that the genes uniquely sensitive to DC belonged in four functional categories: cytokine-receptor interaction, focal adhesion, cell adhesion, and apoptosis. Real-time PCR as well as ELISA studies demonstrated that TNF-alpha-inducible CXCL10 and CXCL11 expression was sensitive to DC but not to C(95). Flow-cytometry studies recognized ICAM-1 and VCAM-1 as TNF-alpha-inducible adhesion molecules that were uniquely sensitive to DC. Taken together, DC exhibited promising neuroprotective and antiinflammatory properties that must be characterized in vivo.

A double blind, randomized, placebo controlled study of the efficacy and safety of 5-Loxin for treatment of osteoarthritis of the knee.

INTRODUCTION: 5-Loxin is a novel Boswellia serrata extract enriched with 30% 3-O-acetyl-11-keto-beta-boswellic acid (AKBA), which exhibits potential anti-inflammatory properties by inhibiting the 5-lipoxygenase enzyme. A 90-day, double-blind, randomized, placebo-controlled study was conducted to evaluate the efficacy and safety of 5-Loxin in the treatment of osteoarthritis (OA) of the knee. METHODS: Seventy-five OA patients were included in the study. The patients received either 100 mg (n = 25) or 250 mg (n = 25) of 5-Loxin daily or a placebo (n = 25) for 90 days. Each patient was evaluated for pain and physical functions by using the standard tools (visual analog scale, Lequesne's Functional Index, and Western Ontario and McMaster Universities Osteoarthritis Index) at the baseline (day 0), and at days 7, 30, 60 and 90. Additionally, the cartilage degrading enzyme matrix metalloproteinase-3 was also evaluated in synovial fluid from OA patients. Measurement of a battery of biochemical parameters in serum and haematological parameters, and urine analysis were performed to evaluate the safety of 5-Loxin in OA patients. RESULTS: Seventy patients completed the study. At the end of the study, both doses of 5-Loxin conferred clinically and statistically significant improvements in pain scores and physical function scores in OA patients. Interestingly, significant improvements in pain score and functional ability were recorded in the treatment group supplemented with 250 mg 5-Loxin as early as 7 days after the start of treatment. Corroborating the improvements in pain scores in treatment groups, we also noted significant reduction in synovial fluid matrix metalloproteinase-3. In comparison with placebo, the safety parameters were almost unchanged in the treatment groups. CONCLUSION: 5-Loxin reduces pain and improves physical functioning significantly in OA patients; and it is safe for human consumption. 5-Loxin may exert its beneficial effects by controlling inflammatory responses through reducing proinflammatory modulators, and it may improve joint health by reducing the enzymatic degradation of cartilage in OA patients. TRIAL REGISTRATION: ( CLINICAL TRIAL REGISTRATION NUMBER: ISRCTN05212803.).

Biosynthetic characterization and chemoenzymatic assembly of the cryptophycins. Potent anticancer agents from cyanobionts.

The lichen cyanobacterial symbiont Nostoc sp. ATCC 53789 and its close relative Nostoc sp. GSV 224 are prolific producers of natural products, generating >25 derivatives of the cryptophycin class of secondary metabolites. Cryptophycin 1, the prototypic member of the class, is a potent tubulin-depolymerizing agent, and several semisynthetic derivatives are being developed as anticancer therapeutics. Here we provide a detailed characterization of the cryptophycin metabolic pathway by stable-isotope labeling experiments and through cloning, sequencing, and annotating the cryptophycin biosynthetic gene cluster. A comparative secondary metabolomic analysis based on polyketide (PK)/non-ribosomal peptide gene clusters from the phylogenetically related, non-cryptophycin producing cycad symbiont, Nostoc punctiforme ATCC 29133, was used to identify the cryptophycin biosynthetic genes that encompass approximately 40 kb within the lichen symbiont Nostoc sp. ATCC 53789 genome. The pathway encodes a collinear set of enzymes, including three modular PK synthases, two non-ribosomal peptide synthetase modules, and an integrated adenylation/ketoreductase didomain for elaboration of the leucic acid subunit. In addition, genes encoding key tailoring steps, including a FAD-dependent halogenase and CYP450 epoxidase, were identified. The inherent flexibility of the cryptophycin biosynthetic enzymes was harnessed to generate a suite of new analogues by altering the pool of PK starter units and selected amino acid extender groups. Characterization of the cryptophycin CYP450 enabled development of the first stereospecific synthesis of cryptophycin 2, through a tandem chemoenzymatic synthesis from the natural seco-cryptophycin 4 chain elongation intermediate.