Evaluation of the in vitro and in vivo anti-inflammatory activity of Gymnosporia montana (Roth). Benth leaves.

ETHNO-PHARMACOLOGICAL RELEVANCE: Gymnosporia montana (Roth) Benth an herbaceous shrub used in Indian traditional medicine their leaves decoction was used as mouthwash to get relieve from toothache, hence it is also known as Dantakashta in Sanskrit language which means the plant used for tooth problems. Traditionally the leaves juice used to alleviate inflammation and in some parts of India like Saurashtra in Gujarat, leaves were chewed as a folklore cure for Jaundice and in Bhandra region Karnataka, leaves extract mixed with cow milk used for jaundice. Hepatoprotective activity for G. montana leaves was well reported however, its use for inflammation and toothache are still not studied to investigate active phytoconstituents responsible for anti-inflammatory activity. AIM OF THE STUDY: The present study aimed at bioactivity guided isolation of G. montana leaves extracts using inhibition of pro-inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF-α), and interleukins (IL-1ß and IL-6) in RAW 264.7 cells in vitro assay to yield bioactive phytoconstituents. MATERIALS AND METHODS: The n-hexane, ethyl acetate and methanol extracts prepared from G. montana leaves were evaluated for cell viability using MTT assay. The effect of extracts to inhibit the pro-inflammatory mediators like NO, TNF-α, IL-1ß and IL-6 in RAW 264.7 macrophages was measured by enzyme-linked immunosorbent assay (ELISA). The quantitative analysis of the isolated phytoconstituents was performed using quantitative Nuclear Magnetic Resonance (qNMR). RESULTS: The n-hexane, ethyl acetate, and methanol extracts of G. montana leaves exhibited cell viability in the range of 97.43-84.88% at 50 µg/mL concentration in RAW 264.7 macrophages. In-vitro evaluation of extracts showed that n-hexane extract was most effective in inhibiting NO, TNF-α, IL-1ß and IL-6 inflammatory mediators at 50 µg/mL in lipopolysaccharides (LPS) stimulated RAW 264.7 cells. Further n-hexane extract, its fraction GMHA3 and ß-amyrin exhibited significant anti-inflammatory activity at 100, 50 and 30 mg/kg per oral, respectively in carrageenan-induced rat paw edema. The quantitative analysis by qNMR revealed ß-amyrin as a major compound in the n-hexane extract. CONCLUSIONS: In vitro and in vivo bioassay results suggested that G. montana n-hexane extract, its fraction GMHA3 and ß-amyrin exhibits significant anti-inflammatory activity proves the traditional uses of G. montana leaves. The reported activity of ß-amyrin for periodontitis provides evidence of profound the use of G. montana leaves for toothache and anti-inflammatory activity.

Design, Synthesis, Molecular Modelling, and Biological Evaluation of Oleanolic Acid-Arylidene Derivatives as Potential Anti-Inflammatory Agents.

INTRODUCTION: Oleanolic acid, a pentacyclic triterpenic acid, is widely distributed in medicinal plants and is the most commonly studied triterpene for various biological activities, including anti-allergic, anti-cancer, and anti-inflammatory. METHODS: The present study was carried out to synthesize arylidene derivatives of oleanolic acid at the C-2 position by Claisen Schmidt condensation to develop more effective anti-inflammatory agents. The derivatives were screened for anti-inflammatory activity by scrutinizing NO production inhibition in RAW 264.7 cells induced by LPS and their cytotoxicity. The potential candidates were further screened for inhibition of LPS-induced interleukin (IL-6) and tumour necrosis factor-alpha (TNF-α) production in RAW 264.7 cells. RESULTS: The results of in vitro studies revealed that derivatives 3d, 3e, 3L, and 3o are comparable to that of the oleanolic acid on the inhibition of TNF-α and IL-6 release. However, derivative 3L was identified as the most potent inhibitor of IL-6 (77.2%) and TNF-α (75.4%) when compared to parent compound, and compounds 3a (77.18%), 3d (71.5%), and 3e (68.8%) showed potent inhibition of NO than oleanolic acid (65.22%) at 10µM. Besides, from docking score and Cyscore analysis analogs (3e, 3L, 3n) showed greater affinity towards TNF-α and IL-1ß than dexamethasone. CONCLUSION: Herein, we report a series of 15 new arylidene derivatives of oleanolic acid by Claisen Schmidt condensation reaction. All the compounds synthesized were screened for their anti-inflammatory activity against NO, TNF-α and IL-6. From the data, it was evident that most of the compounds exhibited better anti-inflammatory activity.

Origanum vulgare L.: In vitro Assessment of Cytotoxicity, Molecular Docking Studies, Antioxidant and Anti-inflammatory Activity in LPS Stimulated RAW 264.7 Cells.

BACKGROUND: Inflammation involves a dynamic network that is highly regulated by signals that initiate the inflammation process as well as signals that downregulate it. However, an imbalance between the two leads to tissue damage. Throughout the world, inflammatory disease becomes common in the aging society. The drugs which are used clinically have serious side effects. Natural products or compounds derived from natural products show diversity in structure and play an important role in drug discovery and development. OBJECTIVE: Oreganum Vulgare is used in traditional medicine for various ailments including respiratory and rheumatic disorders, severe cold, suppression of tumors. The current study aims to evaluate the anti-inflammatory potential by evaluating various in vitro parameters. METHODS: Inflammation-induced in macrophages via LPS is the most accepted model for evaluating the antiinflammatory activity of various plant extracts and lead compounds. RESULTS: The extracts (OVEE, OVEAF) as well as the isolated compound(OVRA)of Oreganum Vulgare inhibit the pro-inflammatory cytokines (IL-6 and TNF-α) and NO without affecting cell viability. CONCLUSION: Our study established that the leaf extracts of Oreganum vulgare L. exhibit anti-inflammatory activity and thus confirm its importance in traditional medicine.

MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220.

MsrA, an efflux pump belonging to ATP-binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2-8-fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8-fold. The real-time fluorometry-based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post-antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.

Antioxidant and antiproliferative activity of indigocarpan, a pterocarpan from Indigofera aspalathoides.

OBJECTIVES: This study aimed to investigate the anticancer potential of indigocarpan (1), a pterocarpan isolated from Indigofera aspalathoides, a plant found in India which has been used in Ayurveda for centuries for the treatment of oedematous tumours. METHODS: The antiproliferative activity in a panel of four human cancer cell lines was studied. The mechanism of its antiproliferative activity in human colorectal adenocarcinoma LS174T cells was investigated in detail. KEY FINDINGS: Indigocarpan (1) showed antiproliferative activity in a panel of four human cancer cell lines with IC50 s ranging from 180 to 250 µm. Indigocarpan induces p53-dependent p21 upregulation and apoptosis in LS174T cells, upregulates p53 and p21(WAF1) protein levels, enhances cleavage of caspase-3 and downregulates cyclin D1, cyclin B1 and PCNA protein levels, indicating its role in modulating cell cycle progression. Indigocarpan also exhibited a strong antioxidative effect in LS174T cells. CONCLUSIONS: Along with the antiproliferative capacity, the strong antioxidative property of the compound makes it a promising candidate for further development as an anticancer and chemopreventive compound.

2'-Hydroxy flavanone derivatives as an inhibitors of pro-inflammatory mediators: Experimental and molecular docking studies.

2'-Hydroxy flavanone (1) was previously isolated from Mimosa pudica (L.) whole plant and was found to exhibit anti-inflammatory effects in vitro. There are also reports on anti-inflammatory properties of compounds bearing flavanone/chromone nucleus. Taking this into account, fourteen derivatives of 2'-hydroxy flavanone (1) were synthesized and evaluated against pro-inflammatory mediators (TNF-α, IL-1ß and NO) in in vitro and in vivo models. Results directed that among the synthesized compounds, four derivatives (11-14) showed profound inhibition of pro-inflammatory mediators as compared to the lead molecule. Further, 11-14 demonstrated comparable anti-inflammatory activity with ibuprofen in carrageenan-induced rat paw edema assay and appreciable inhibition of lipopolysaccharide (LPS) induced pro-inflammatory mediators (TNF-α and IL-1ß) in Sprague Dawley (SD) rats. The synthesized compounds were further subjected to molecular docking analysis and in silico prediction of pharmacokinetic properties.

2-Acetoxyverecynarmin C, a new briarane COX inhibitory diterpenoid from Pennatula aculeata.

A new briarane-type diterpenoid, named 2-acetoxyverecynarmin C, was isolated from the methanolic extract of an octocoral, Pennatula aculeata, that exhibited cyclooxygenase (COX) inhibitory activity. The structure of the compound was elucidated by ESI-HRMS, 1D and 2D NMR spectroscopy and comparison of the measured spectral data with those reported in the literature. The relative stereochemistry at chiral carbons was established from 2D NOESY experiments. 2-Acetoxyverecynarmin C is a tricyclic compound containing a furan ring at C-7,8 of a briarane skeleton. 2-Acetoxyverecynarmin C showed moderate inhibitory activity in in vitro COX-1 and COX-2 assays.

Phytochemical, therapeutic, and ethnopharmacological overview for a traditionally important herb: Boerhavia diffusa Linn.

UNLABELLED: Boerhavia diffusa (BD) is a plant of rasayana category as per ayurvedic claims. It is reported to possess antiaging, disease prevention, and life strengthening activities which hold enormous influence in disease burden and affordability/availability of healthcare in the world. Objective. This paper has been compiled to comment on the studies reported for BD to highlight its chemical and therapeutic potential along with its ethnopharmacological considerations. METHODS: In the present paper, a detailed account of chemical constituents and pharmacological activities has been presented. All the findings were correlated with modern pharmacological activities to appraise the value of BD. RESULTS: Chemical analysis of BD gives a wide variety of chemical constituents, namely, rotenoids, flavonoids, xanthones, purine nucleoside, lignans, and steroids. Various ethnopharmacological reports emphasize its role in disorders of reproductive system, gastrointestinal system, respiratory system, urinary system, hepatic system/jaundice, cardiovascular system, and cancer. CONCLUSIONS: The studies on the therapeutic activities of BD range from studies on crude extracts to isolated compounds; however some of the studies require sophistication and validated results. BD is a plant of enormous importance in the purview of its chemical and therapeutic properties.

Analysis of flavonoids and iridoids in Vitex negundo by HPLC-PDA and method validation.

The leaves of Vitex negundo have been reported to contain various bioactive constituents including iridoids and flavonoids. This is the first report on the simultaneous determination of iridoids and flavonoids by HPLC in three different samples of V. negundo leaves collected from three regions of India. Separation of iridoids and flavonoids was accomplished by HPLC and further elaborated for their quantification in V. negundo leaves using a C-18 column with detection at 254 and 330 nm, respectively. The developed HPLC method showed good linearity (r2 > or = 0.999), high precision (RSD < 5%) and a good recovery (99.3-103.0%) of the compounds. All the validation parameters of the developed HPLC were found to be within the permissible limits according to the ICH guidelines. The developed method was robust, accurate and reliable for the quality control of V. negundo leaves.

NorA efflux pump inhibitory activity of coumarins from Mesua ferrea.

The purpose of this investigation was to study the modulator and efflux pump inhibitor activity of coumarins isolated from Mesua ferrea against clinical strains as well as NorA-over expressed strain of Staphylococcus aureus 1199B. Seven coumarins were tested for modulator activity using ethidium bromide (EtBr) as a substrate. Compounds 1, 4-7 modulated the MIC of EtBr by ≥ 2 fold against wild type clinical strains of S. aureus 1199 and S. aureus 1199B, whereas compounds 4-7 modulated the MIC of EtBr by ≥ 16 fold against MRSA 831. Compounds 1, 4-7 also reduced the MIC of norfloxacin by ≥ 8 fold against S. aureus 1199B, and 4-6 reduced the MIC of norfloxacin by ≥ 8 fold against MRSA 831 at half of their MICs. Inhibition of EtBr efflux by NorA-overproducing S. aureus 1199B and MRSA 831 confirmed the role of compounds 4-6 as NorA efflux pump inhibitors (EPI). Dose-dependent activity at sub-inhibitory concentration (6.25 µg/mL) suggested that compounds 4 and 5 are promising EPI compared to verapamil against 1199B and MRSA 831 strains.

Analysis of homoisoflavonoids in Caesalpinia digyna by HPLC-ESI-MS, HPLC and method validation.

The roots of Caesalpinia digyna have been reported to contain gallic acid derivatives and minor homoisoflavonoids, but HPLC-ESI-MS and HPLC analyses of the homoisoflavonoids were challenging due to their low concentration in the roots. Separation and identification was accomplished by HPLC-ESI-MS and further elaborated for quantification using a C18 column with detection at 330 nm. A gradient mobile phase consisting of methanol and water (0.1% acetic acid) was used. The developed HPLC method showed good linearity (r2 > or = 0.998), high precision (RSD < 5%) and a good recovery (99.3-104.5%) of the compounds. The lowest detection limit was 0.75 ng and the method was found to be robust. All the validation parameters were found to be within the permissible limits and, therefore, the developed method is accurate and reliable for the quality control of C digyna and other Caesalpinia species. This is the first report of sample preparation on Diaion HP-20 resin and characterization of homoisoflavonoids by HPLC-ESI-MS, extended by extensive quantitative HPLC analysis of homoisoflavonoids in C. digyna roots and method validation.

Phenylpropanoids of Alpinia galanga as efflux pump inhibitors in Mycobacterium smegmatis mc² 155.

The first and second line drugs used for the treatment of tuberculosis are now becoming ineffective due to emergence of resistant strains. Efflux pump provokes resistance in mycobacterium and hence could be explored as a new target for the discovery of anti-TB agents. In search of efflux pump inhibitors, MIC and modulation factor of phenylpropanoids isolated from A. galanga rhizome were determined prior to the accumulation and efflux assay. Phenylpropanoid compounds viz. 1'-S-1'-acetoxychavicol acetate, trans-p-coumaryl diacetate and 1'-S-1'-acetoxyeugenol acetate were found to be potent modulators and decreased the MIC of ethidium bromide by 64 fold at the concentration of 2.5, 6.25 and 5.0 mg/L respectively. 1'-S-1'-acetoxyeugenol acetate enhanced the accumulation and inhibited the efflux of EtBr in Mycobacterium smegmatis mc² 155 cells.

Antiarthritic effects of Ajuga bracteosa Wall ex Benth. in acute and chronic models of arthritis in albino rats.

OBJECTIVE: To evaluate the antiarthritic activity of Ajuga bracteosa using albino rats. METHODS: The antiarthritic activity of 70% ethanolic extract of Ajuga bracteosa (EEAB) was evaluated against turpentine oil- and formaldehyde- induced acute non immunological and complete freund's adjuvant (CFA)-induced chronic immunological arthritis in albino rats. RESULTS: EEAB showed a significant (P<0.05) and dose dependent inhibitory effect against acute and chronic models of arthritis. EEAB exhibited better antiarthritic activity than the standard aspirin. CONCLUSIONS: EEAB exhibits a significant and promising antiarthritic activity against acute and chronic arthritis and supports the traditional use of Ajuga bracteosa for rheumatism and other inflammatory diseases.

Anti-inflammatory, cyclooxygenase inhibitory and antioxidant activities of standardized extracts of Tridax procumbens L.

The standardized EtOAc, MeOH and 70% EtOH extracts of Tridax procumbens aerial parts showed significant inhibition of rat paw edema at a medium dose of 200mg/kg and the EtOAC extract was the most active. These extracts were standardized by HPLC with the help of chemical markers. Further, the extracts were evaluated for COX-1 and COX-2 inhibitory activity and EtOAc extract exhibited the highest inhibition of COX-1 and COX-2 at 50 µg/mL. Cent aurein, centaureidin and bergenin were isolated as COX-1 and COX-2 inhibitory principles from the EtOAc extract. The extracts also exhibited antioxidant activity against DPPH and ABTS free radicals. The anti-inflammatory activity of T. procumbens aerial parts could be at least in part due to COX-1, COX-2 enzyme inhibition and free radical-scavenging activities which may be attributed to the presence of flavonoids and other polyphenols in the extracts.

Simultaneous determination of naphthalene and anthraquinone derivatives in Rumex nepalensis Spreng. roots by HPLC: comparison of different extraction methods and validation.

INTRODUCTION: Rumex nepalensis contains mainly anthraquinone and naphthalene derivatives. Although HPLC methods have been reported for the analysis of anthraquinones, neither a phytochemical analysis of Rumex species nor the simultaneous determination of anthraquinone and naphthalene derivatives in other samples has been reported so far. OBJECTIVE: To develop and validate a HPLC method for the simultaneous determination of anthraquinone and naphthalene derivatives in R. nepalensis roots. METHODOLOGY: Anthraquinones and naphthalenes were extracted from R. nepalensis roots by three methods (reflux, ultrasonication and pressurized liquid extraction) using methanol. Separation was achieved on an RP C18 column with a gradient mobile phase consisting of 0.05% orthophosphoric acid in water (solvent A) and methanol (solvent B) using a UV detector (254 nm). RESULTS: Small differences were observed in the contents of anthraquinone and naphthalene derivatives extracted by the three methods. Chrysophanol-8-O-ß-D-glucopyranoside and nepodin were detected as major constituents. The method showed a good linearity (r² > 0.9992), high precision (RSD < 5%) and a good recovery (97-105%) of the compounds. The lowest detection limit was found to be 0.97 ng and the method was found to be robust. CONCLUSION: Reflux and ultrasonication were found to be the best suited methods for the extraction of glycosides and aglycones, respectively. The developed and validated HPLC method is simple, precise and accurate; and can hence be recommended as the method of choice for the analysis of anthraquinones and naphthalenes in R. nepalensis and other Rumex species for both quality control as well as routine analytical purposes.

Anti-inflammatory effect of Ajuga bracteosa Wall Ex Benth. mediated through cyclooxygenase (COX) inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Ajuga bracteosa Wall Ex Benth. (Labiateae) is described in Ayurveda for the treatment of rheumatism, gout, palsy and amenorrhea. AIM OF THE STUDY: The aim of present investigation is to study anti-inflammatory activity of Ajuga bracteosa, to understand possible mechanism of action and to identify the constituents responsible for its activity. MATERIALS AND METHODS: The anti-inflammatory activity of 70% ethanolic extract was evaluated in TPA-induced mouse ear edema assay and in vitro cyclooxygenase (COX)-1 and COX-2 inhibitory activity was determined using EIA kits employing appropriate reference standards. Aajugarin I, lupulin A, withaferin A, reptoside and 6-deoxyharpagide were isolated from the 70% ethanolic extract by silica gel column chromatography. RESULTS: The 70% ethanol extract of whole plants of Ajuga bracteosa showed a significant (p<0.05) and dose-dependent anti-inflammatory activity in an acute inflammation model at the dose of 0.5 and 1.0 mg/ear. The extract also exhibited a strong in vitro COX-1 and COX-2 inhibitory activity at 25 and 50 µg/mL concentration. Among the isolated compounds 6-deoxyharpagide exhibited highest COX-2 inhibition while rest of the compounds exhibited weak to moderate COX-1 and COX-2 inhibition at 30 µM concentration. CONCLUSIONS: The results suggest that the 70% ethanol extract of Ajuga bracteosa possesses promising anti-inflammatory activity, which is possibly mediated through inhibition of COX-1 and COX-2 enzymes. The isolated constituents could be responsible in part for its anti-inflammatory and COX inhibitory activity. The study supports traditional use of Ajuga bracteosa for inflammatory diseases.

Determination of chromones in Dysophylla stellata by HPLC: method development, validation and comparison of different extraction methods.

An HPLC method with reflux as an efficient extraction method has been developed for quantification of chromones in Dysophylla stellata Benth. Separation was achieved on a C18 column with a mobile phase of 0.5% (v/v) acetic acid in water (A), and ACN (B) under gradient elution at 1 mL/min. Chromones (1 and 2) isolated from D. stellata were used as standards for method development and validation was achieved according to ICH guidelines. Extracts prepared by three different methods [reflux, ultrasonication and accelerated solvent extraction (ASE)] were used for analysis by the validated method. The proposed HPLC method is simple, accurate and selective for the separation and quantification of chromones in D. stellata.

Chemical changes during fermentation of Abhayarishta and its standardization by HPLC-DAD.

Abhayarishta is an Ayurvedic formulation prepared traditionally by the fermentation of the decoction of Terminalia chebula (pericarp), Vitis vinifera (fruits), Embelia ribes (fruits) and Madhuca indica (flowers). In the present communication, chemical changes occurring during fermentation in Abhayarishta have been studied for the purpose of its standardization. An HPLC-DAD method for quantitative estimation of selected marker constituents in the formulation has been developed and validated. A comparison of decoction and final processed formulation revealed that major polyphenolics (chebulagic and chebulinic acid) of T. chebula were hydrolyzed to their respective monomers and, consequently, there was an increase in the amount of chebulic acid, gallic acid, ellagic acid and ethyl gallate after fermentation. 5-Hydroxymethyl furfural (5-HMF) was also found in the formulation. Thus, emphasis is laid upon consideration of processing methods of formulation which has been lacking in the standardization of most of Ayurvedic formulations.

Anti-inflammatory, cyclooxygenase (COX)-2, COX-1 inhibitory, and free radical scavenging effects of Rumex nepalensis.

Evaluation of the topical anti-inflammatory activity of chloroform and ethyl acetate extracts of RUMEX NEPALENSIS roots in a TPA-induced acute inflammation mouse model demonstrated a significant reduction in ear edema. The extracts were further tested on purified enzymes for COX-1 and COX-2 inhibition to elucidate their mechanism of action, and a strong inhibition was observed. Six anthraquinones and two naphthalene derivatives were isolated from the ethyl acetate extract. Among the isolated compounds, emodin was found to be a potent inhibitor with slight selectivity towards COX-2, and nepodin exhibited selectivity towards COX-1. Emodin, endocrocin, and nepodin also exhibited significant topical anti-inflammatory activity in mice. Interestingly, nepodin showed better radical scavenging activity than trolox and ascorbic acid against DPPH and ABTS radicals. The strong radical scavenging activity of chloroform and ethyl acetate extracts could be explained by the presence of nepodin as well as by the high phenolic content of the ethyl acetate extract. Thus, the anti-inflammatory effect of R. NEPALENSIS roots was assumed to be mediated through COX inhibition by anthraquinones and naphthalene derivatives and through the radical scavenging activities of naphthalene derivatives.

Anti-inflammatory, cyclooxygenase (COX)-2, COX-1 inhibitory and antioxidant effects of Dysophylla stellata Benth.

The n-hexane and ethyl acetate extracts of whole plants of Dysophylla stellata significantly inhibited edema when applied topically at doses of 0.5 and 1mg/ear in TPA-induced ear edema assay in mice. Further, both the extracts were evaluated for COX-1 and COX-2 inhibitory activities and showed 85.42 and 57.38%; and 71.79 and 89.27% inhibition at 50 microg/ml, respectively. Chromones (1 and 2) present in these extracts could be responsible for their COX-1 and COX-2 inhibitory and anti-inflammatory activities. The ethyl acetate extract showed antioxidant activity in DPPH and ABTS radical scavenging assay where as n-hexane extract found to be inactive.