Boswellic acids: a group of medicinally important compounds.

This review, containing over 276 references, covers the progress made in the chemistry and bioactivity of this important group of triterpenoids. Though initially known for their anti-inflammatory and anti-arthritic activities through a unique 5-LO inhibition mechanism, boswellic acids have recently attained significance due to their anti-cancer properties. The phytochemistry and chemical modifications, including mechanism of action, are discussed.

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.

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.

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.

A triterpenediol from Boswellia serrata induces apoptosis through both the intrinsic and extrinsic apoptotic pathways in human leukemia HL-60 cells.

A triterpenediol (TPD) comprising of isomeric mixture of 3alpha, 24-dihydroxyurs-12-ene and 3alpha, 24-dihydroxyolean-12-ene from Boswellia serrata induces apoptosis in cancer cells. An attempt was made in this study to investigate the mechanism of cell death by TPD in human leukemia HL-60 cells. It inhibited cell proliferation with IC50 approximately 12 microg/ml and produced apoptosis as measured by various biological end points e.g. increased sub-G0 DNA fraction, DNA ladder formation, enhanced AnnexinV-FITC binding of the cells. Further, initial events involved massive reactive oxygen species (ROS) and nitric oxide (NO) formation, which were significantly inhibited by their respective inhibitors. Persistent high levels of NO and ROS caused Bcl-2 cleavage and translocation of Bax to mitochondria, which lead to loss of mitochondrial membrane potential (Deltapsim) and release of cytochrome c, AIF, Smac/DIABLO to the cytosol. These events were associated with decreased expression of survivin and ICAD with attendant activation of caspases leading to PARP cleavage. Furthermore, TPD up regulated the expression of cell death receptors DR4 and TNF-R1 level, leading to caspase-8 activation. These studies thus demonstrate that TPD produces oxidative stress in cancer cells that triggers self-demise by ROS and NO regulated activation of both the intrinsic and extrinsic signaling cascades.

Boswellic acids and glucosamine show synergistic effect in preclinical anti-inflammatory study in rats.

The present study revealed the synergistic effect of boswellic acid mixture (BA) and glucosamine for anti-inflammatory and anti-arthritic activities in rats. Two studies were conducted, that is, acute anti-inflammatory by carrageenan edema and chronic anti-arthritic by Mycobacterium-induced developing arthritis. Five groups of animals were included in each of the study: the vehicle control, positive control (ibuprofen 100mg/kg), boswellic acids (250 mg/kg), glucosamine (250 mg/kg) and a combination of boswellic acids (125 mg/kg) and glucosamine (125 mg/kg). BA when administered at 250 mg/kg in rats, carrageenan-induced paw edema and Mycobacterium-induced developing arthritis were significantly inhibited. In comparison to boswellic acids, glucosamine when administered at 250 mg/kg showed a mild effect in carrageenan-induced edema and moderate inhibition of paw swelling against developing arthritis. Although the combination of boswellic acids and glucosamine did not affect the acute inflammation to a greater extent yet a significant anti-arthritic activity was observed in rats. In conclusion, a synergistic effect was observed in chronic inflammatory conditions when two chemical entities were administered in combination in preclinical study.

Immunomodulation exhibited by piperinic acid through suppression of proinflammatory cytokines.

Piper longum (PL) has been reported for its varied pharmacological activities including bio-enhancer and anti-inflammatory activities in traditional medicine. Here the premise of the study was to investigate the immunoregulatory potential of PL and piperinic acid, one of its active constituent, in Balb/C mice (in vivo) and human PBMCs (in vitro) models. Piperinic acid moderated the proinflammatory mediators and cytokines in our experiments. At doses of 10, 20, 40 and 80 mg/kg p.o. PL showed a dose dependent decrease of lymphocytes (CD4+ and CD8+ T cells) and cytokine levels in sensitized Balb/C mice with a marked inhibition at 40 mg/kg. At an in vitro dose of 20 mug/ml of PL and 5 mug/ml of piperinic acid, there was a significant inhibition of mitogen induced human PBMC proliferation, mRNA transcripts of IL-2 (ConA) and TNFalpha, IL-1beta and iNOS (LPS) respectively under stimulated conditions in time dependent (6 h, 12 h and 24 h respectively) expression studies. In parallel, induced nitric oxide production was also reduced by stimulated macrophages. Our observations rationalize the traditional use of PL and also validate the immunoregulatory potential of piperinic acid.

Anti-arthritic activity of a biopolymeric fraction from Euphorbia tirucalli.

The present study was undertaken to investigate the anti-arthritic activity of a biopolymeric fraction (BET) from plant Euphorbia tirucalli Boiss (Euphorbiaceae). The fraction showed dose dependent anti-arthritic activity and also showed in vivo immunomodulatory capacity being a major component in inhibiting arthritis. It caused suppression of CD4(+) and CD8(+) T cells, inhibition of intracellular Interleukin-2 (IL-2) and Interferon-gamma (IFN-gamma) by flowcytometry. It inhibited vascular permeability and the migration of leucocytes at the site of the insult. The oral LD(0) in both rats and mice was more than 2000 mg/kg.

Selective Th1 up-regulating activity of Withania somnifera aqueous extract in an experimental system using flow cytometry.

Withania somnifera (Ashwagandha) is reported to be immunoprotective and immunoadjuvant. We studied its roots aqueous extract on T helper (Th) immunity using flow cytometry. This extract was standardized with six withanolides as marker compounds using HPLC. Once daily dose ranging from 25 to 400 mg/kg/p.o. was used to study effect on Th1: IFN-gamma, IL-2 and Th2: IL-4 cytokine modulation. We also studied effect on CD4 and CD8 in normal and immunesuppressed mice. The results indicate that extract at 100 mg/kg resulted significant selective up-regulation of Th1 response. Treatment with extract showed significant increase in CD4 and CD8 counts as compared to control and cyclopsorin A, with a faster recovery of CD4+ T cells in immunesuppressed animals. Under immunesuppressed conditions, potentiation of cellular and humoral immune responses of extract was comparable to levamisole. This study indicates the selective Th1 up-regulating effect of extract and suggests its use for selective Th1/Th2 modulation.

Protection of the hemopoietic system by Podophyllum hexandrum against gamma radiation-induced damage.

A semi-purified extract of low-altitude Podophyllum hexandrum (REC-2001) containing a relatively low content of podophyllotoxin (3.25 %) exhibited potent antioxidant ability in lipid media (at 1000 microg/mLagainst 0.25 kGy) and significant (p < 0.05) hydroxyl ion scavenging potential (78.83 % at 500 microg/mL). In vitro investigations revealed the ability of REC-2001 to significantly (p < 0.05) reduce radiation-induced hemolysis (2 microg/mL; 46.184 %) and nitric oxide scavenging levels (IC (50): 792 +/- 1.25 microg/mL). Protection of the hemopoietic system of Strain 'A' mice administered 20 mg/kg BW REC-2001 30 min prior to lethal irradiation (10 Gy) was recorded and was mediated by free radical scavenging and lowering of lipid oxidation. Further studies investigating the effects of REC-2001 on stem cell modulation are warranted.

Phytochemical analysis and genetic characterization of six Hypericum species from Serbia.

The secondary metabolite contents and genetic profiles of six Hypericum species (H. barbatum Jacq., H. hirsutum L., H. linarioides Bosse, H. maculatum Crantz, H. rumeliacum Boiss. and H. tetrapterum Fries), collected from different locations in Serbia, have been analyzed. Methanol extracts of the aerial parts of the plants were obtained by accelerated solvent extraction (ASE) at 40 degrees C and 100 bar, and analyzed for five pharmacologically important standard constituents (hyperoside, quercitrin, pseudohypericin, hyperforin and hypericin) by LC-MS/MS. The highest content of hypericin and pseudohypericin was observed in the H. barbatum extract, while the highest content of hyperforin and quercitrin was found in the H. tetrapterum extract and the highest content of hyperoside in the H. maculatum extract. A literature survey shows that the above six Hypericum species, with the exception of H. maculatum, have not been previously genetically profiled. In order to correlate the chemical constituents of the species under investigation with their genetic factors, genetic profiling of these species was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) profiles of the above selected plants. Among the 52 random primers used for the initial screening, only 10 yielded polymorphic RAPD profiles. A total of 111 polymorphic markers were generated using these primers. The SSR analysis shows that 8 out of the 10 primers used were polymorphic. The correlation among the species under investigation using the two genetic markers was performed using Jaccuard's coefficients of similarity and a high correlation (r=0.99) was obtained. The main conclusion from the above data is that there exists a stronger correlation for secondary metabolite contents with RAPD data than with SSR data among the six Hypericum species from Serbia.

3-O-beta-D-Galactopyranoside of quercetin as an active principle from high altitude Podophyllum hexandrum and evaluation of its radioprotective properties.

The aqueous-ethanolic extract (AEE) of high altitude Podophyllum hexandrum has earlier been reported to render a radioprotective effect against lethal gamma radiation in in vitro model. AEE has also been reported to possess metal chelating and DNA protecting properties. The present study was undertaken to isolate and characterize the bioactive principle present in AEE and investigate its role in radiation protection. A novel molecule was found to be present in AEE and was assigned as 3-O-beta-D-galactoside of quercetin by acid hydrolysis, LC-MS, LC-APCI-MS/MS and 13C NMR spectra. Various biological activities were investigated at in vitro level. The antioxidant potential of AEE in lipid and aqueous phase was determined against numerous stresses. AEE was found to be significantly (p < 0.05) protective, i.e., against Fe2+ and Cu2+-induced linoleic acid degradation, respectively. Radiation-induced lipid oxidation studies revealed that AEE maximally works at a [lignan]/0.25 kGy ratio 400 (ratio of concentration of AEE divided by the radiation dose, i.e., 0.25 kGy) and no drug-induced lipid oxidation at all concentrations tested was found. In a time-dependent study, total antioxidant activity was maximally exhibited at 1 mg/ml. The site-specific and non-site-specific deoxyribose degradation assay exhibited a dose-dependant hydroxyl scavenging potential of AEE (0.05-500 microg/ml). The anti-lipid peroxidation ability of AEE against radiation (0.25 kGy)-induced lipid peroxidation was higher in case of neural tissue homogenate as compared to kidney homogenate [activity ratio: 0.039 (brain) < 0.24 (kidney)]. The protein protection study using bovine serum albumin was also done for two time intervals (2 h and 4 h) and significant (p < 0.05) protection was observed at 500 microg/ml (> 97%). This study implies that 3-O-beta-D-galactoside present in AEE renders radioprotection by protecting lipids, proteins in renal and neural model system against supra-lethal (0.25 kGy) gamma radiation.

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: role in radiation protection.

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2'-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389 +/- 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 microg/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 microg/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.

Radioprotective and antioxidant properties of low-altitude Podophyllum hexandrum (LAPH).

The development of nontoxic yet effective radioprotectors is needed because of the increasing risk of human exposure to ionizing radiation. We have reported that high-altitude Podophyllum hexandrum (HAPH) confers a radioprotective effect in in vitro and in vivo models. The present study reports on the antioxidant and radioprotective properties of low-altitude Podophyllum hexandrum (LAPH), from which the toxic compound podophyllotoxin has been partially removed during the extraction process. Using HPLC,we estimated the relative content of two marker compounds, podophyllotoxin and podophyllotoxin glycoside, in low-altitude Podophyllum extract (LAPE) and found them to be 23.3% and 9.50%, respectively. The ferrous ion chelation potential of LAPE was estimated using the 2,2 bipyridyl assay, and the activity was found to be increased concomitantly with the increase in its concentration, with a maximal inhibition at 25 microg/mL (42.20%) as compared to quercetin (34.9%). The electron donation potential of LAPE was also evaluated, because the antioxidant activities of natural products are known to bear a direct correlation with their ability to donate electrons. The concentration required to attain unit absorbance values at 700 nm were 0.230541+/-0.09 and 0.041+/-0.06 for butylated hydroxyl toluene and LAPE, respectively, indicating a higher antioxidant activity of LAPE. The free radical scavenging ability of LAPE was also assessed and exhibited a dose-dependant increase (1-100 microg/mL), comparable to that of quercetin at 25 microg/mL. The role of LAPE in protecting DNA was evaluated, and it was found that LAPE (30 microg/mL) rendered its maximum radioprotection against the 250 Gy-induced damage in the plasmid (pBR322) relaxation assay. LAPE significantly inhibited radiation-induced, iron/ascorbate- and combined stress (iron/ascorbate and radiation)-induced formation of TBARS (p<0.05). We conclude that LAPH, with its easy accessibility, ease of cultivation, multifarious radioprotective properties, and role as a renewable source of bioactive constituents, along with its low associated toxicity (due to partial removal of podophyllotoxin), enhances its possible use for human clinical applications.