Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals

Ethnopharmacological relevance

The rhizomes of Kaempferia parviflora Wall. ex Baker have been used in Thailand for treatment of gout, apthous ulcer, peptic ulcer and abscesses.

Aim of the study

In our previous study, the crude ethanol extract of Kaempferia parviflora and its compound (5, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone), was reported to show nitric oxide (NO) inhibition in RAW 264.7 cells. The present study is thus investigated the anti-inflammatory mechanism of Kaempferia parviflora extract and compound 5 against inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expressions.

Materials and methods

The extract of Kaempferia parviflora and its compound were tested against NO and prostaglandin E₂ (PGE₂) releases using RAW264.7 cells as well as studied on anti-inflammatory activity in carrageenan-induced rat paw edema and acute toxicity in mice.

Results

The results revealed that the ethanol extract of Kaempferia parviflora markedly inhibited PGE₂ release with an IC₅₀ value of 9.2 μg/ml. This plant extract and compound 5 also suppressed mRNA expression of iNOS in dose-dependent manners, whereas COX-2 mRNA expression was partly affected. According to the in vivo study, chloroform and hexane fractions greater decreased rat paw edema than ethanol, ethyl acetate and water fractions.

Conclusion

The mechanisms for anti-inflammatory activity of Kaempferia parviflora and compound 5 are mainly due to the inhibition of iNOS mRNA expression but partly through that of COX-2 mRNA.     

The constituents of Anisomeles indica and their anti-inflammatory activities

Ethnopharmacological relevance

Anisomeles indica (L.) Kuntze. (Labiatae), popularly known as ‘yu-chen-tsao’, has been traditionally used as anti-inflammatory agent.

Aim of the study

Investigate the chemical constituents from the whole plants of Anisomeles indica, and evaluate their in vitro anti-inflammatory activities.

Results

The combined MeOH extract was successively partitioned with CHCl₃ and n-butanol, then submitted to several column chromatographic, and HPLC purification procedures which led to the isolation of one cembrane-type diterpenoid (3), two benzenoids (4 and 5), five flavonoids (1, 2, 6, 7 and 14), and six phenyl propanoids (8–13). The compounds 1–14 were examined for their inhibitory effects on inflammatory mediator's enhanced production from LPS/IFN-γ-stimulated macrophages. Among these, ovatodiolide (3) exhibited potent inhibition on NO, TNF-α and IL-12 enhanced production at a concentration of 5 μM, followed by pedalitin (1), scutellarein 7-O-β-d-glucuronide methyl ester (6), and acteoside (12) at 40 μM (P < 0.05). Furthermore, 2 μM of 3, and 20 μM of 1 and 6 significantly (P < 0.05) arrested the cell cycle of Con A-stimulated spleen cells at the G0/G1 stage.

Conclusion

This is the first report on the presence of compounds 1 and 4–13 in this plant and of the potent anti-inflammatory activity of 1, 3, 6 and 12in vitro. These compounds may account for the use of Anisomeles indica in folk medicine to treat inflammation.     

4-Methylthio-butanyl derivatives from the seeds of Raphanus sativus and their biological evaluation on anti-inflammatory and antitumor activities

Ethnopharmacological relevance

Raphanus sativus seeds (Brassicaceae) known as Raphani Semen have long been used as anti-cancer and/or anti-inflammatory agents in Korean traditional medicine. This study was designed to isolate the bioactive constituents from the seed extracts of Raphanus sativus and evaluate their anti-inflammatory and antitumor activities.

Material and methods

Bioassay-guided fractionation and chemical investigation of a methanolic extract of the seeds of Raphanus sativus led to the isolation and identification of seven 4-methylthio-butanyl derivatives. Structural elucidation of the isolated compounds was carried out using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques (¹H, ¹³C, COSY, HMQC and HMBC experiments) and mass spectrometry.

Results

The isolated compounds were characterized as in the following: three new 4-methylthio-butanyl derivatives, sinapoyl desulfoglucoraphenin (1), (E)-5-(methylsulfinyl)pent-4-enoxylimidic acid methyl ester (2), and (S)-5-((methylsulfinyl)methyl)pyrrolidine-2-thione (3), together with four known compounds, 5-(methylsulfinyl)-4-pentenenitrile (4), 5-(methylsulfinyl)-pentanenitrile (5), sulforaphene (6), and sulforaphane (7). Full NMR data assignments of the three known compounds 4–6 were also reported for the first time. We evaluated the anti-neuroinflammatory effect of 1–7 in lipopolysaccharide-stimulated murine microglia BV2 cells. Compound 1 significantly inhibited nitrite oxide production with IC₅₀ values of 45.36 μM. Moreover, it also reduced the protein expression of inducible nitric oxide synthase. All isolates were also evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15), and all of them showed antiproliferative activity against the HCT-15 cell, with IC₅₀ values of 8.49–23.97 μM.

Conclusions

4-Methylthio-butanyl derivatives were one of the main compositions of Raphanus sativus seeds, and activities demonstrated by the isolated compounds support the ethnopharmacological use of Raphanus sativus seeds (Brassicaceae) as anti-cancer and/or anti-inflammatory agents.     

Phenolic derivatives from the rhizomes of Dioscorea nipponica and their anti-neuroinflammatory and neuroprotective activities

Ethnopharmacological relevance

Dioscorea nipponica (Dioscoreaceae) have been used as traditional medicines for diabetes, inflammatory and neurodegenerative diseases in Korea. The aim of the study was to isolate the bioactive components from the rhizomes of Dioscorea nipponica and to evaluate their anti-neuroinfalmmatory and neuroprotective activities.

Material and methods

The phytochemical investigation of 50% EtOH extract of Dioscorea nipponica using successive column chromatography over silica gel, Sephadex LH-20, and preparative high performance liquid chromatography (HPLC) resulted in the isolation and identification of 17 phenolic derivatives, including four new phenolic compounds (1–4). The structural elucidation of these compounds was based on spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques, mass spectrometry, and optical rotation. All isolated compounds were evaluated for their effects on nerve growth factor (NGF) secretion in a C6 rat glioma cell line and nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV2 cells. The neurite outgrowth of compound 16 was further evaluated by using mouse neuroblastoma N2a cell lines.

Results

Three new stilbene derivatives, diosniponol C (1), D (2) and diosniposide A (3) and one new phenanthrene glycoside, diosniposide B (4), together with 13 known compounds were isolated from the rhizomes of Dioscorea nipponica. Of the tested compounds (1–17), phenanthrene, 3,7-dihydroxy-2,4,6-trimethoxy-phenanthrene (16) was the most potent NGF inducer, with 162.35±16.18% stimulation, and strongly reduced NO levels with an IC₅₀ value of 19.56 μM in BV2 microglial cells. Also, it significantly increased neurite outgrowth in N2a cells.

Conclusions

This study supports the ethnopharmacological use of Dioscorea nipponica rhizomes as traditional medicine.     

Inhibitory effect of Physalis alkekengi L. var. franchetii extract and its chloroform fraction on LPS or LPS/IFN-γ-stimulated inflammatory response in peritoneal macrophages

Ethnopharmacological relevance

The aerial parts of the plant Physalis alkekengi L. var. franchetii (PA) are traditionally used in folk medicine to treat cough, middle ear infection, sore throat, abscesses, and urinary problems. However, the cellular mechanisms underlying PA's possible anti-inflammatory effects are unknown.

Materials and methods

We examined the effect of a methanol extract of PA on the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6. We also examined the extract's effect on the activity of IκBα, mitogen-activated protein kinases (MAPKs) and STAT1 in macrophages stimulated by lipopolysaccharide (LPS) or LPS/interferon-γ (IFN-γ). We further fractioned the extract into chloroform and water to investigate which fraction possessed anti-inflammatory activity.

Results

We found that the PA methanol extract significantly reduced the production of NO, iNOS, COX-2, TNF-α, and IL-6. The extract also inhibited LPS-induced IκBα degradation and MAPK activation as well as LPS/IFN-γ-induced STAT1 activation, effects observed at a higher concentration than that required to suppress iNOS. The chloroform fraction possessed the anti-inflammatory activity of PA by inhibiting the expression of iNOS, TNF-α and IL-6 through downregulation of IκBα degradation and MAPK activation.

Conclusion

Our findings indicate that the PA methanol extract contains different anti-inflammatory compounds, some of which suppressed iNOS expression and some of which inhibited IκBα degradation and MAPK activity. Further research is warranted to identify these anti-inflammatory components of PA and validate its use in animal studies.     

Selected compounds derived from Moutan Cortex stimulated glucose uptake and glycogen synthesis via AMPK activation in human HepG2 cells

Aim of the study

To evaluate the effect of selected compounds derived from Moutan Cortex on glucose uptake and glycogen synthesis associated with AMPK activation in insulin-resistant human HepG2 cell.

Materials and methods

The effect of isolated compounds (1-16) on glucose uptake and glycogen synthesis was performed using HepG2 cells. The western blot was used to determine the expression of AMPK and its downstream substrates, ACC, p-ACC, and p-GSK-3β.

Results

The effects of the 16 compounds from Moutan Cortex on glucose metabolism in HepG2 cells under high glucose conditions were evaluated. Compounds 2, 3, and 6 displayed highly potent effects on the stimulation of glucose uptake and glycogen synthesis in human HepG2 cells under high glucose conditions. Compounds 2, 3, and 6 phosphorylate AMPK (AMP-activated protein kinase), and resulted in increased phosphorylation of GSK-3β and suppression of lipogenic expression (ACC and FAS) in a dose-dependent manner. Compounds 2, 3, and 6 also demonstrated interesting, strong eNOS phosphorylation in human umbilical vein endothelial cells (HUVECs). Compounds 1, 4, 5-12, and 14 displayed considerable effects on hepatic glucose production, AMPK activation, and phosphorylation of GSK-3β in HepG2 cells under high glucose conditions.

Conclusions

These effects may indicate that the activation of AMPK by the active compounds from Moutan Cortex has considerable potential for reversing the metabolic abnormalities associated with type-2 diabetes.     

The constituents of Cibotium barometz and their permeability in the human Caco-2 monolayer cell model

Ethnopharmacological relevance

Cibotium barometz (L.) J. Sm. (Dicksoniaceae) has been traditionally used as anti-inflammatory and anodyne.

Aim of the study

To investigate the constituents in the rhizomes of Cibotium barometz, and evaluate their permeability in the human Caco-2 model.

Materials and methods

The rhizomes extracts of Cibotium barometz were isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods. The permeability of the main constituents was evaluated using human Caco-2 cell monolayer as a model system.

Results

Three unusual sesquiterpenes having 1-indanone nucleus (1, 3 and 4) and an unusual orthoester spiropyranosyl derivative of protocatechuic acid (2) were isolated from the rhizomes of Cibotium barometz. Among these, the bilateral permeation of 1, 3 and 4 in Caco-2 model was examined. The apparent permeability coefficients (P_app) of 1 was identical with those of propranolol, which is often used as reference standard of high permeability. The P_app values of 3 and 4 were in agreement with those of atenolol, which is often used as reference standard of poor permeability. The permeation rates of 1, 3 and 4 increased linearly as a function of time up to 180 min and with the concentration within the test range of 25–200 μM.

Conclusions

This is the first report on the presence of compounds 2 and 3 in this plant and 4 was a new compound. Compound 1 is assigned for a well-absorbed, and 2 and 3 are assigned for the poorly absorbed compounds in human intestine. A passive diffusion mechanism for transport of 1, 3 and 4 in Caco-2 model was proposed. The results provided some useful information for predicting the oral bioavailability of 1, 3 and 4.     

Sedative activity of hexane extract of Keampferia galanga L. and its active compounds

Ethnopharmacological relevance

It is well known that fragrance impacts behaviors and autonomic functions, and is increasingly used as relaxant, carminative, as well as sedative in aromatherapy. Kaempferia galanga L. is one of the popular traditional aromatic medicinal plants used in tropics and subtropics of Asia including China, Japan and Indochina.

Aim of the study

The aim of present study was to investigate sedative effects of hexane extract of Kaempferia galanga L.and 2 active aromatic compounds (compound 1: ethyl trans-p-methoxycinnamate and compound 2: ethyl cinnamate ) included in the extyract by means of inhalation in mice.

Materials and methods

Sedative activity was assessed by inhalation of the hexane extract and two major aromatic compounds.

Results

Inhalation of hexane extract at the doses of 1.5 and 10 mg showed significant reduction of locomotor activity, indicating considerable sedative and relaxant effects. Compound 1 and 2 were proved to possess sedative effects at 0.0014 mg and 0.0012 mg respectively.

Conclusions

These results suggest the promising application of Kaempferia galanga L. and its constituents in aromatherapy.     

Constituents isolated from Cordyceps militaris suppress enhanced inflammatory mediator's production and human cancer cell proliferation

Aim of the study

The purpose of this study is to isolate the pure compounds from the extracts of Cordyceps militaris obtained through solid-state cultivation process, and evaluate their anti-inflammatory and anticancer properties.

Materials and methods

Silica gel column chromatographic purification of Cordyceps militaris extracts resulted in the isolation of 10 pure compounds (1-10). The compounds 1-10 were examined for their growth inhibitory properties against nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-12 enhanced production from LPS/IFN-γ-stimulated macrophages. Additionally, the anti-proliferation effects of 1-10 on human cancer cell lines, colon (colon 205), prostate (PC-3), and hepatoma (HepG2) cells were also analyzed.

Results

Compound 8 displayed potent growth inhibition on NO, TNF-α and IL-12 production with an IC₅₀ value of 7.5, 6.3, and 7.6 μg/ml, respectively. A similar inhibitory trend on these inflammatory mediators was observed for 3, 7, 9 and 10 with an IC₅₀ values ranging from 10.8 to 17.2 μg/ml. On the other hand, compounds 3 and 8 were potent anti-proliferative agents with an IC₅₀ value of 35.6 and 32.6 μg/ml toward PC-3 and colon 205 cell lines, respectively. The compounds 1 and 2 showed potent anti-proliferation in PC-3 and colon 205 cells, while only 3 displayed such effect in HepG2 cells.

Conclusion

The present study provides scientific supporting information for the ethnopharmacological use of Cordyceps militaris as an anti-inflammatory and anticancer agent.     

Hypoglycemic evaluation of a new triterpene and other compounds isolated from Euclea undulata Thunb. var. myrtina (Ebenaceae) root bark

Aim of the study

Investigate the hypoglycaemic activity of the four isolated compounds from a crude acetone extract of the root bark of Euclea undulata var. myrtina, which is used by traditional healers in the Venda area, Limpopo Province in the treatment of diabetes.

Material and methods

The hypoglycaemic activity of the four compounds isolated from Euclea undulata was determined by in vitro screening of glucose utilization by C2C12 myocytes at a concentration of 25 μg/ml or 50 μg/ml. The inhibition of α-glucosidase was also tested at concentrations ranging from 0.02 to 200.00 μg/ml.

Results

Assay-guided isolation of the crude acetone extract of the root bark of Euclea undulata var. myrtina afforded a new triterpene, α-amyrin-3O-β-(5-hydroxy) ferulic acid (1), in addition to three known compounds; betulin (2), lupeol (3) and epicatechin (4). The in vitro results on C2C12 myocytes suggest that compound 4 may have some effect to lowers blood glucose levels, whereas compound 1 has the ability to inhibit α-glucosidase at a concentration of 200.0 μg/ml with an IC₅₀ value of 4.79 that correlates with that of the positive control acarbose IC₅₀ value 4.75.

Conclusion

The results suggest that 4 may have some ability to lower blood glucose levels, whereas 1 has the ability to inhibit α-glucosidase.

Ethnopharmacological relevance

These findings corroborate the ethnomedicinal use of Euclea undulata by traditional healers for the treatment of diabetes as two substances was isolated from the acetone plant extract that exhibit hypoglycaemic activity.     

Lipoxygenase inhibitory activity of crude bark extracts and isolated compounds from Commiphora berryi

Ethnopharmacological relevance

Commiphora berryi is traditionally used for the treatment of cold and fever as well as for wound healing in the southern parts of India.

Aim of study

The present study was designed to investigate in vitro soybean lipoxygenase inhibitory activity of crude extracts and compounds isolated from Commiphora berryi.

Materials and methods

The bark of Commiphora berryi was extracted with different organic solvents and subjected to chromatographic separation for isolation of bioactive compounds. Structures of isolated compounds were elucidated by spectroscopic methods. The anti-inflammatory activity of bark extracts and bioactive compounds were assessed by in vitro soybean lipoxygenase (SBL) assay.

Results

3β-Hydroxyglutin-5-ene (1), friedelin (2), cycloeucaneol (3) nimbiol (4), sugiol (5), surianol (6), daucosterol (7) and ursolic acid (8) were isolated from crude bark extracts of the Commiphora berryi. The structure of nimbiol (4) was also confirmed by single crystal X-ray analysis. The petroleum ether, methanol, chloroform and ethyl acetate extracts of bark of Commiphora berryi showed SBL inhibitory activity with the IC₅₀ values of 15.3, 54.2, 71.5 and 87.8 μg/ml respectively. Among all the isolates, friedelin (2) showed significant SBL inhibitory activity with IC₅₀ 35.8 μM.

Conclusion

The overall results provide evidence that the studied plant might be a potential source of anti-inflammatory agents.     

Immunomodulatory active compounds from Tinospora cordifolia

Ethnopharmacological relevance

Tinospora cordifolia mentioned as “Rasayana” is extensively used in various herbal preparations for the treatment of different ailments for its general tonic, antiperiodic, antispasmodic, antiinflammatory, antiarthritic, antiallergic and antidiabetic properties. It is extensively used in Ayurveda due to its potential in improving the immune system and the body resistance against infections.

Aim of the study

The aim of the study was to isolate and characterise the immunomodulatory active compounds of Tinospora cordifolia.

Materials and methods

The immunomodulatory activity of different extracts, fractions and isolated compounds in relation to phagocytosis and reactive oxygen species production in human neutrophil cells have been investigated using the PMN phagocytic function studies, NBT, NO and chemiluminescence assay.

Results

The results obtained indicate that ethyl acetate, water fractions and hot water extract exhibited significant immunomodulatory activity with an increase in percentage phagocyctosis. Chromatographic purification of these fraction led to the isolation of a mixture of two compounds 2, 3 isolated for the first time from natural source and five known compounds 1, 4–7 which were characterized as 11-hydroxymustakone (2), N-methyl-2-pyrrolidone (3), N-formylannonain (1), cordifolioside A (4), magnoflorine (5), tinocordiside (6), syringin (7) by nuclear magnetic resonance (NMR) and mass spectrometry (MS) and comparing the spectral data with reported one. Cordifolioside A and syringin have been reported to possess immunomodulatory activity. Other five compounds showed significant enhancement in phagocytic activity and increase in nitric oxide and reactive oxygen species generation at concentration 0.1–2.5 μg/ml.

Conclusions

Seven immunomodulatory active compounds belonging to different classes have been isolated and characterised indicating that the immunomodulatory activity of Tinospora cordifolia may be attributed to the synergistic effect of group of compounds.     

Hydrophobic constituents and their potential anticancer activities from Devil's Club (Oplopanax horridus Miq.)

Ethnopharmacological relevance

Devil's Club (Oplopanax horridus) is one of the most important spiritual and medicinal plants to many indigenous peoples of Alaska and the Pacific Northwest. It is widely used for external and internal infections as well as arthritis, respiratory ailments, digestive tract ailments, broken bones, fever, headaches, and cancer.

Aim of the study

To investigate hydrophobic constituents and their potential anticancer activity from Devil's Club, Oplopanax horridus.

Materials and methods

The root bark extract of Oplopanax horridus was isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods and comparison with published data. The anti-proliferation of isolated hydrophobic constituents in human breast cancer MCF-7 cells, human colon cancer SW-480 and HCT-116 cells were tested. The potential mechanism of anti-proliferation was also investigated using cell cycle and apoptosis assays.

Results and discussion

Six compounds were isolated and structurally identified as 9,17-octadecadiene-12,14-diyne-1,11,16-triol, 1-acetate (1), oplopandiol acetate (2), falcarindiol (3), oplopandiol (4), trans-nerolidol (5) and t-cadinol (6). These compounds showed potential anticancer activities on human breast cancer and colon cancer cells, of which compound 3 possesses the strongest activity. Further cell cycle and apoptosis tests by flow cytometry showed the polyacetylenes 1-4 induced HCT-116 cell arresting in G2/M phase and inhibited proliferation by the induction of apoptosis at both earlier and later stages.

Conclusion

These results provide promising baseline information for the potential use of Oplopanax horridus, as well as some of the isolated compounds in the treatment of cancer.