Comparison of Inhibitory Effects of Safflower Decoction and Safflower Injection on Protein and mRNA Expressions of iNOS and IL-1ß in LPS-Activated RAW264.7 Cells.

OBJECTIVE: Safflower has antioxidant and anti-inflammatory activities. The two forms of preparations for safflower which are widely used in China are injection and decoction. The first step of the process for preparing an injection involves extracting safflower with water, which actually yields a decoction. This study is intended to investigate how the preparation process influences the anti-inflammatory activity of safflower in vitro. METHODS: Five samples, including a decoction (sample 1) and an injection (sample 5) of safflower, were prepared according to the national standard WS3-B-3825-98-2012 and were analyzed by the oxygen radical absorbance capacity (ORAC) method and the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) method for comparison. Sample 1 and sample 5 were further tested by the Griess assay and ELISA for their effects on nitric oxide (NO) production and interleukin- (IL-) 1ß content in lipopolysaccharide- (LPS-) activated RAW264.7 cells. The protein and mRNA levels of inducible nitric oxide synthase (iNOS) and IL-1ß were measured by Western blotting and real-time quantitative PCR. RESULTS: Sample 5 showed a significantly higher ORAC value and a lower half inhibitory concentration (IC50) for DPPH scavenging activity as compared to the other four samples (p < 0.05). LPS significantly upregulated the mRNA and protein expressions of iNOS and IL-1ß as compared to the solvent control (p < 0.01). As compared to sample 1, sample 5 significantly decreased NO production, iNOS protein expression, and the contents of IL-1ß mRNA and IL-1ß protein at both 100 µg/ml and 200 µg/ml (all: p < 0.05) and significantly downregulated iNOS mRNA expression at 100 µg/ml (p < 0.05). CONCLUSIONS: Results of this study demonstrate that the safflower injection prepared according to the national standard has a significant effect of suppressing protein and mRNA expressions of iNOS and IL-1ß as compared to its traditional decoction.

Are the Therapeutic Effects of Huangqi (Astragalus membranaceus) on Diabetic Nephropathy Correlated with Its Regulation of Macrophage iNOS Activity?

OBJECTIVE: To investigate the correlation between the clinical effects of Huangqi (Astragalus membranaceus) on different stages of diabetic nephropathy (DN) and the pharmacological effect of Huangqi on the activity of inducible nitric oxide synthase (iNOS) in macrophages in different states. METHODS: The PubMed, China National Knowledge Infrastructure, and Wanfang databases were searched. Clinical data was sourced from papers on treatment of different stages of DN with Huangqi, and pharmacological data was from papers on the effects of Huangqi on the iNOS activity of macrophages in a resting or an activated state. RESULTS: Meta-analysis of Huangqi injections on stages III and III-IV DN and randomized controlled trials on other stages showed that Huangqi had therapeutic effects on different stages of DN and on macrophages in different states: inducing normal macrophages in a resting state to generate nitric oxide (NO), tumor necrosis factor-α, and so forth upon iNOS activation; inhibiting NO generation by normal lipopolysaccharide- (LPS-) activated macrophages; and enhancing NO generation by LPS-induced macrophages from patients with renal failure. CONCLUSIONS: Huangqi can regulate iNOS activity of macrophages in different states in vitro. These biphasic or antagonistic effects may explain why Huangqi can be used to treat different stages of DN.

Different Proportions of Huangqi (Radix Astragali Mongolici) and Honghua (Flos Carthami) Injection on α-Glucosidase and α-Amylase Activities.

Objective. To study the effect of different proportions of Huangqi (Radix Astragali Mongolici) and Honghua (Flos Carthami) injection on α-glucosidase and α-amylase activity simultaneously. Methods. The injections were prepared according to the standards of the China Food and Drug Administration. The assay for potential α-glucosidase inhibitors was based on the hydrolysis of 4-methylumbelliferyl-α-D-glucopyranoside (4-MUG). The α-amylase EnzChek assay kit was used to determine potential α-amylase inhibitors. Acarbose was the positive control. Results. The half maximal (50%) inhibitory concentration (IC50) of acarbose against α-glucosidase and α-amylase was (1.8 ± 0.4) µg/mL and (227 ± 32) µg/mL, respectively. Honghua showed significant inhibition of α-glucosidase activity compared with Huangqi (P < 0.01). Honghua inhibited α-amylase activity, but Huangqi did not. IC50s for α-glucosidase inhibition by mixtures at 10 : 1, 5 : 1, and 2 : 1 were significantly lower than those at the 20 : 1 mixture (P < 0.01). α-Amylase inhibition by the 2 : 1 mixture was significantly higher than that by the 20 : 1, 10 : 1, and 5 : 1 mixtures at 500 µg/mL and 1000 µg/mL (P < 0.01), with 5 : 1 significantly higher than 20 : 1 and 10 : 1 at 1000 µg/mL (P < 0.01). Conclusion. Honghua significantly inhibited α-glucosidase activity compared with Huangqi (P < 0.01). For simultaneous inhibition of α-glucosidase and α-amylase activities, the mixtures at 2 : 1 and 5 : 1 exhibited significant effects compared with those at 20 : 1 (P < 0.01).

Compounds from Geijera parviflora with prostaglandin E2 inhibitory activity may explain its traditional use for pain relief.

ETHNOPHARMACOLOGICAL RELEVANCE: Australian Aboriginal people used crushed leaves of Geijera parviflora Lindl. both internally and externally for pain relief, including for toothache (Cribb and Cribb, 1981). This study tested the hypothesis that this traditional use might be at least in part explained by the presence of compounds with anti-inflammatory activity. MATERIALS AND METHODS: A crude extract (95% EtOH) was prepared from powdered dried leaves. From the CH3Cl fraction of this extract compounds were isolated by bioassay-guided fractionation and tested for: (1) cytotoxicity in RAW 264.7 murine leukemic monocyte-macrophages, (2) prostaglandin E2 (PGE2) inhibitory activity in 3T3 Swiss albino mouse embryonic fibroblast cells, as well as (3) nitric oxide (NO) and (4) tumour necrosis factor alpha (TNFα) inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Isolated compounds were also tested for (5) antibacterial activity against a panel of Gram-positive (Staphylococcus aureus ATCC 29213 and ATCC 25923, Staphylococcus epidermidis ATCC 35984, biofilm-forming) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) strains by broth microdilution. RESULTS: Eleven compounds were isolated, including one new flavone and one new natural product, with a further four compounds reported from this species for the first time. Some of the compounds showed good anti-inflammatory activity in vitro. In particular, flindersine (1) and N-(acetoxymethyl) flindersine (3) inhibited PGE2 release with IC50 values of 5.0µM and 4.9µM, respectively, without any significant cytotoxicity. Several other compounds showed moderate inhibition of NO (5, 6, 7) and TNF-α (6), with IC50 in the low micromolar range; however much of this apparent activity could be accounted for by the cytotoxicity of these compounds. None of the compounds showed anti-bacterial activity. CONCLUSIONS: The inhibition of PGE2, an important mediator of inflammation and pain, by flindersine and a derivative thereof, along with the moderate anti-inflammatory activity shown by several other compounds isolated from Geijera parviflora leaf extract, support the traditional use of this plant for pain relief by Australian Aboriginal people.

Effect of honghua (Flos Carthami) on nitric oxide production in RAW 264.7 cells and alpha-glucosidase activity.

OBJECTIVE: To study the effects of extracts from Honghua (Flos Carthami) on lipopolysaccharide induced nitric oxide (NO) production in RAW 264.7 cells and the influence of the extracts on yeast a-glucosidase activity. The total flavonoid content of the extracts was also determined. METHODS: Cytotoxicity of the extracts to RAW 264.7 cells was evaluated by the ATPlite method. Inhibitory effects of the extracts on NO production were evaluated by Griess assay. Curcumin was used as a positive control. Screening of extracts for potential a-glucosidase inhibitors was done by a fluorometric assay. The assay was based on the hydrolysis of 4-methylumbelliferyl-a-D-glucopyranoside to form the fluorescent product, 4-methylumbelliferone. Acarbose was used as a positive control. The total flavonoid content was tested using kaempferol as the standard. RESULTS: There were significant inhibitory effects on NO production when the extracts were 25-100 microg/ mL (P < 0.05) and curcumin was 2-4 microg/mL (P < 0.001). The extracts showed an inhibitory effect on alpha-glucosidase activity at the concentrations of 15.6-125 microg/mL with a half maximal (50%) inhibitory concentration (IC50) of (32.8 +/- 5.7) microg/mL, compared with the IC50 of acarbose at (1.8 +/- 0.4) microg/mL. There was a significant difference between the two IC50 values (P < 0.001). The total content of flavonoids per gram of dried herb was 1.14 mg. CONCLUSION: Honghua (Flos Carthami) showed inhibitory effects on NO production in activated RAW 264.7 macrophage cells and an inhibitory effect on yeast alpha-glucosidase. There might be a relationship between these pharmacological effects and its flavonoid content.

Antibacterial anthranilic acid derivatives from Geijera parviflora.

Five anthranilic acid derivatives, a mixture I of three new compounds 11'-hexadecenoylanthranilic acid (1), 9'-hexadecenoylanthranilic acid (2), and 7'-hexadecenoylanthranilic acid (3), as well as a new compound 9,12,15-octadecatrienoylanthranilic acid (4) together with a new natural product, hexadecanoylanthranilic acid (5), were isolated from Geijera parviflora Lindl. (Rutaceae). Their structures were elucidated by extensive spectroscopic measurements, and the positions of the double bonds in compounds 1-3 of the mixture I were determined by tandem mass spectrometry employing ozone-induced dissociation. The mixture I and compound 5 showed good antibacterial activity against several Gram-positive strains.

Parvifloranines A and B, two 11-carbon alkaloids from Geijera parviflora.

Two novel alkaloids (parvifloranines A and B), possessing an unusual 11-carbon skeleton linked with amino acids, were isolated from Geijera parviflora, an endemic Australian Rutaceae. Their structures were elucidated by extensive spectroscopic measurements including 2D NMR analyses. Parvifloranine A was found to be a mixture of two enantiomers, (S)-1 and (R)-1, in a ratio of 1:4, based on their separation using a chiral column. Parvifloranine B is also believed to be a mixture of enantiomers. Proposed biosynthetic pathways are discussed. Parvifloranine A inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages with an IC50 value of 23.4 µM.

Biologically active dibenzofurans from Pilidiostigma glabrum, an endemic Australian Myrtaceae.

In an effort to identify new anti-inflammatory and antibacterial agents with potential application in wound healing, five new dibenzofurans, 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di(2-methylbutanoyl)dibenzofuran (1), 1,3,7,9-tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2-methylpropionyl)dibenzofuran (2), 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di(2-methylpropionyl)dibenzofuran (3), 1,3,7,9-tetrahydroxy-4,6-dimethyl-2-(2-methylbutanoyl)-8-(2-methylpropionyl)dibenzofuran (4), and 1,3,7,9-tetrahydroxy-4,6-dimethyl-2,8-di(2-methylpropionyl)dibenzofuran (5), were isolated from the leaves of Pilidiostigma glabrum together with one previously described dibenzofuran. Structure elucidation was achieved by way of spectroscopic measurements including 2D-NMR spectroscopy. Compounds with 2,8-acyl substitutions had potent antibacterial activity against several Gram-positive strains (MIC in the low micromolar range), while compounds with 4,6-acyl substitutions were less active. All compounds except 3 inhibited the synthesis of nitric oxide in RAW264 macrophages with IC(50) values in the low micromolar range. Compounds with 2,8-acyl substitutions also inhibited the synthesis of PGE(2) in 3T3 cells, whereas 4,6-acyl-substituted compounds were inactive. None of the compounds inhibited the synthesis of TNF-α in RAW264 cells. The compounds showed variable but modest antioxidant activity in the oxygen radical absorbance capacity assay. These findings highlight that much of the Australian flora remains unexplored and may yet yield many new compounds of interest. Initial clues are provided on structure/activity relationships for this class of bioactives, which may enable the design and synthesis of compounds with higher activity and/or selectivity.

Antioxidant activity of 45 Chinese herbs and the relationship with their TCM characteristics.

Here, 45 Chinese herbs that regulate blood circulation were analyzed for antioxidant activity using the oxygen radical absorbance capacity (ORAC) assay. A recent publication by Ou et al. identified a close relationship between in vitro antioxidant activity and classification of Chinese herbs as yin or yang. The 45 Chinese herbs in this study could be assigned the traditional characteristics of natures (cold, cool, hot and warm), flavors (pungent, sweet, sour, bitter and salty) and functions (arresting bleeding, promoting blood flow to relieve stasis, nourishing blood and clearing away heat from blood). These characteristics are generalized according to the theory of yin and yang. We identified a broad range, 40-1990 micromol Trolox Equivalent/g herbs, of antioxidant activity in water extracts. There was no significant correlation between ORAC values and natures or functions of the herbs. There was a significant relationship between flavors and ORAC values. Bitter and/or sour herbs had the highest ORAC values, pungent and/or sweet herbs the lowest. Other flavors had intermediate values. Flavors also correspond with the yin/yang relationship and our results are supportive of the earlier publication. We reported for the first time antioxidant properties of many Chinese herbs. High antioxidant herbs were identified as Spatholobus suberectus vine (1990 micromol TE/g), Sanguisorba officinalis root (1940 micromol TE/g), Agrimonia pilosa herb (1440 micromol TE/g), Artemisia anomala herb (1400 micromol TE/g), Salvia miltiorrhiza root (1320 micromol TE/g) and Nelembo nucifera leaf (1300 micromol TE/g). Antioxidant capacity appears to correlate with the flavors of herbs identified within the formal TCM classification system and may be a useful guide in describing their utility and biochemical mechanism of action.

Effects and potential mechanisms of Danzhi Xiaoyao Pill on proliferation of MCF-7 human breast cancer cells in vitro.

OBJECTIVE: To investigate the effects of 50% ethyl alcohol (EtOH) extracts from Danzhi Xiaoyao Pill (, DXP) on the proliferation of MCF-7 human breast cancer cells and potential mechanisms. METHODS: ATP-Lite assay was performed to test the proliferation of the MCF-7 breast cancer cell line; and antioxidant activity was measured by the oxygen radical absorbance capacity (ORAC). The effects of DXP on nitric oxide (NO) production were tested by lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages using the Griess reaction. RESULTS: The 50% EtOH DXP extracts displayed a cytotoxic response on MCF-7 cells at 0.10, 0.25 and 0.50 mg/mL dose-dependently with the proliferation inhibited by more than 85%. The ORAC value of the DXP was 820 micro moL Trolox equivalent/g, about 40% of the vitamin C value. DXP extracts had significant inhibitory effect on NO production at the concentration from 0.0625 mg/mL to 0.5 mg/mL (P<0.05, P<0.01). CONCLUSION: The extracts of DXP could significantly inhibit the proliferation of MCF-7 cells, with the effect possibly related to its antioxidant activity and the inhibition of NO production.

Echinacea alkylamides modulate induced immune responses in T-cells.

The effects of Echinacea and several of its phytochemical components on NFkappaB expression by Jurkat cells (a human T-cell line) were investigated in vitro. In the absence of stimulation, Echinacea and its components exerted no significant effect on basal NFkappaB expression levels. In the presence of endotoxin (LPS), NFkappaB expression was decreased. However, this decrease was significantly reversed by treatment with cichoric acid, an Echinacea root extract (prepared from both Echinacea angustifolia and Echinacea purpurea) and the alkylamide fraction derived from this combination. For the phorbol myristate acetate stimulation of Jurkat cells, effects on NFkappaB expression were mixed. Depending on the concentration, cichoric acid and a 2,4-diene alkylamide significantly induced NFkappaB levels, whereas a 2-ene alkylamide caused a significant inhibition. In contrast, both the Echinacea and the mixed alkylamide fraction exerted no effect. The alkylamide results indicate that the two basic forms of these compounds present in Echinacea may have opposing effects. These opposing effects demonstrate the importance of a knowledge, not only of the phytochemical make-up of a herbal preparation, but also of the actions of each component and the consequences of differing relative amounts in the preparation being investigated.

Elucidation of danzhixiaoyao wan and its constituent herbs on antioxidant activity and inhibition of nitric oxide production.

Danzhixiaoyao Wan (DW) is a common 10 herbs formulation in China for regulating several clinical conditions affecting women. This research tried to explain one of DW's functions, purging heat, using in vitro pharmacological analyses. The whole formulation and each single herb of DW were compared based on antioxidant activity with the oxygen radical absorbance capacity (ORAC) assay, and for their inhibitory effect (IE) on nitric oxide (NO) production by lipopolysaccharide (LPS)-activated RAW 264.7 macrophages with the Griess assay. The results showed that DW as a whole formulation had both antioxidant activity and an IE on NO production, while the individual herb component of DW varied in their ORAC values and inhibition of NO production. The ORAC value of the whole DW was 450 mumol TE g(-1). The order of antioxidant (ORAC) activity of the single herbs was: Mentha haplocalyx (1352 mumol TE g(-1)) > Glycyrrhiza uralensis (1184 mumol TE g(-1)) > Gardenia jasminoides (1129 mumol TE g(-1)) > Paeonia suffruticosa (465 mumol TE g(-1)), with the contributions being additive rather than synergistic. The production of nitrite by stimulated RAW 264.7 murine macrophages (unstimulated: 0.5 +/- 0.1 muM versus LPS: 38.9 +/- 2.3 muM) was significantly inhibited (P < 0.05) by M. haplocalyx, G. jasminoides, Bupleurum chinense and Paeonia lactiflora. DW as a whole had an IE on NO production, but this was not significant. The single herb M. haplocalyx had the highest ORAC value and the highest IE on NO production, followed by G. jasminoides. Both of these herbs have the 'purging heat' property in the theory of traditional Chinese medicine and this property of the samples may be correlated with the antioxidant activity and IE on NO production.

Cytotoxic clerodane diterpenes from Glossocarya calcicola.

Three clerodane diterpenes were isolated and identified from leaf extract of Glossocarya calcicola. Compound has been characterised as (rel)-10betaH-trans-12xi-(2-methylbut-2(E)-enoyl)-1beta-(isobutanoyl)-6alpha,13xi-dihydroxyclerodan-4(20),8(18)-dien-7,15-dione-15,16-oxide, to which we have assigned the trivial name calcicolin-A. The other two compounds had the same skeletal structure and C-12 substituent but in compound, the C-1 esterifying group becomes 2-methylbut-2(E)-enoic acid and in it becomes 2-methylbutanoic acid. Although anti-insect activity was not observed for G. calcicola, cytotoxicity against insect and human carcinoma cell lines was detected.

Acaricidal and cytotoxic activities of extracts from selected genera of Australian Lamiaceae.

Crude foliar extracts of 67 species from six subfamilies of Australian Lamiaceae were screened by whole organism contact toxicity on the polyphagous mite Tetranychus urticae Koch (Acari: Tetranychidae) by using a Potter precision spray tower. Cytotoxicity assessments against insect cell lines from Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) and Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) also were made. The Spodoptera cell line was more susceptible to extracts than the Drosophila cellline. No direct correlation was observed between the two screening methods, but several interesting relationships were identified. Extracts from subfamilies Ajugoideae, Scutellarioideae, Chloanthoideae, Viticoideae and Nepetoideae showed acaricidal activity, whereas only those from Ajugoideae and Nepetoideae displayed potent cytotoxic effects. A range of activities was observed for the 25 species of Plectranthus, 14 of which showed moderate-to-high contact toxicity against T. urticae. Overall, the lowest toxicity was observed for extracts from the plant subfamily Prostantheroideae, which showed little contact toxicity or cytotoxicity for the 18 extracts studied.

Modulation of macrophage immune responses by Echinacea.

Echinacea preparations are widely used herbal medicines for the prevention and treatment of colds and minor infections. There is little evidence for the individual components in Echinacea that contribute to immune regulatory activity. Activity of an ethanolic Echinacea extract and several constituents, including cichoric acid, have been examined using three in vitro measures of macrophage immune function - NF-kappaB, TNF- alpha and nitric oxide (NO). In cultured macrophages, all components except the monoene alkylamide (AA1) decreased lipopolysaccharide (LPS) stimulated NF-kappaB levels. 0.2 microg/ml cichoric acid and 2.0 microg/mL Echinacea Premium Liquid (EPL) and EPL alkylamide fraction (EPL AA) were found to significantly decrease TNF-alpha production under LPS stimulated conditions in macrophages. In macrophages, only the alkylamide mixture isolated from the ethanolic Echinacea extract decreased LPS stimulated NO production. In this study, the mixture of alkylamides in the Echinacea ethanolic liquid extract did not respond in the same manner in the assays as the individual alkylamides investigated. While cichoric acid has been shown to affect NF-kappaB, TNF-alpha and NO levels, it is unlikely to be relevant in the Echinacea alterations of the immune response in vivo due to its non- bioavailability - i.e. no demonstrated absorption across the intestinal barrier and no detectable levels in plasma. These results demonstrate that Echinacea is an effective modulator of macrophage immune responses in vitro.