Isolation and Characterisation of a Proanthocyanidin With Antioxidative, Antibacterial and Anti-Cancer Properties from Fern Blechnum orientale.

BACKGROUND: Blechnum orientale Linn. (Blechnaceae), a fern, is traditionally used in the treatment of various ailments, such as skin diseases, stomach pain, urinary bladder complaints, and also as a female contraceptive. Previously, we reported a strong radical scavenging activity, antibacterial activity and cytotoxicity against HT29 colon cancer cells by aqueous extract of B. orientale. OBJECTIVE: In this study, we attempted to isolate and identify the active compound from the aqueous extract of B. orientale. MATERIALS AND METHODS: Aqueous extract of B. orientale was subjected to repeated MCI gel chromatography, Sephadex-LH-20, Chromatorex C18 and semi-preparative high performance liquid chromatography and was characterized using nuclear magnetic resonance and electrospray ionization mass-spectrometry spectroscopic methods. Antioxidant activity was determined using 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay. Antibacterial assays were conducted using disc diffusion whereas the minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined using the broth microdilution assay. Cytotoxicity was assessed using thiazolylblue tetrazoliumbromide. RESULTS: A polymeric proanthocyanidin consisting of 2-12 epicatechin extension units and epigallocathecin terminal units linked at C4-C8 was elucidated. Bioactivity studies showed strong radical scavenging activity (IC50 = 5.6 ± 0.1 µg/mL), antibacterial activity (MIC = 31.3-62.5 µg/mL) against five gram-positive bacteria and selective cytotoxicity against HT29 colon cancer cells (IC50 = 7.0 ± 0.3 µg/mL). CONCLUSION: According to our results, the proanthocyanidin of B. orientale demonstrated its potential as a natural source of antioxidant with antibacterial and anti-cancer properties. SUMMARY: A bioactive proanthocyanidin was isolated from the aqueous extract of medicinal fern Blechnum orientale Linn and the structure was elucidated using NMR and ESI-MS spectral studies.The proanthocyanidin compound possessed strong radical scavenging activity (IC50 5.6 ± 0.1 µg/mL)The proanthocyaniding compound showed bactericidal activity against five gram-positive bacteria inclusive of MRSA (minimum inhibitory concentration, MIC and minimum bactericidal concentration, MBC 31.3-62.5 µg/mL).The proanthocyanidin compound is strongly cytotoxic towards cancer cells HT29 (IC50 7.0 ± 0.3 µg/mL), HepG2 (IC50 16 µg/mL) and HCT116 (IC50 20 µg/mL) while weakly cytotoxic towards the non-malignant Chang cells (IC50 48 µg/mL). Abbreviation used: CC: Column chromatography, DP: degree of polymerization, DPPH: 2,2-diphenyl-1-picrylhydrazyl, ESI-MS: electronsprayionisation mass-spectrometry, MBC: Minimum bactericidal concentration, MIC: Minimum inhibitory concentration, MTT: Thiazolyl Blue Tetrazolium Bromide, MRSA: methicillin-resistant Staphylococcus aureus, NMR: nuclear magnetic resonance, TLC: thin layer chromatography, PD: prodelphinidin.

Anti-inflammatory potential of ellagic acid, gallic acid and punicalagin A&B isolated from Punica granatum.

BACKGROUND: Punica granatum (pomegranate), an edible fruit originating in the Middle East, has been used as a traditional medicine for treatment of pain and inflammatory conditions such as peptic ulcer. The numerous risks associated with nonsteroidal anti-inflammatory drugs (NSAIDs) for treatment of pain and inflammation give rise to using medicinal herbs as alternative therapies. This study aimed to evaluate the anti-inflammatory effect of isolated compounds from the ethyl acetate (EtOAc) fraction of P. granatum by determination of their inhibitory effects on lipopolysaccharide (LPS), stimulated nitric oxide (NO), prostaglandin E2 (PGE-2), interleukin-6 (IL-6) and cyclooxxgenase-2 (COX-2) release from RAW264.7 cells. METHODS: The compounds ellagic acid, gallic acid and punicalagin A&B were isolated from EtOAc by high performance liquid chromatography (HPLC) and further identified by mass spectrometry (MS). The inhibitory effect of ellagic acid, gallic acid and punicalagin A&B were evaluated on the production of LPS-induced NO by Griess reagent, PGE-2 and IL-6 by immunoassay kit and prostaglandin E2 competitive ELISA kit, and COX-2 by Western blotting. RESULTS: Ellagic acid, gallic acid and punicalagin A&B potentially inhibited LPS-induced NO, PGE-2 and IL-6 production. CONCLUSION: The results indicate that ellagic acid, gallic acid and punicalagin may be the compounds responsible for the anti-inflammatory potential of P. granatum.

An injectable poly(caprolactone trifumarate-gelatin microparticles) (PCLTF-GMPs) scaffold for irregular bone defects: Physical and mechanical characteristics.

Recently, a modified form of a three-dimension (3D) porous poly(caprolactone-trifumarate) (PCLTF) scaffold has been produced using a fabrication technique that involves gelatin microparticles porogen leaching. This poly(caprolactone trifumarate-gelatin microparticles) (PCLTF-GMPs) scaffold has been shown to be biocompatible, more flowable clinically, and has a shorter degradation time as compared to its existing predecessors. In this report, a detailed characterization of this new scaffold was performed by testing its cytocompatibility, analyzing the surface topography, and understanding its thermal, physical and mechanical properties. The result showed that the PCLTF-GMPs has no critical cytotoxic effect. To confirm improvement, the surface properties were compared against the older version of PCLTF fabricated using salt porogen leaching. This PCLTF-GMPs scaffold showed no significant difference (unpaired t-test; p>0.05) in mechanical properties before and after gelatin leaching. However, it is mechanically weaker when compared to its predecessors. It has a high biodegradability rate of 16weeks. The pore size produced ranges from 40 to 300µm, and the RMS roughness is 613.7±236.9nm. These characteristics are condusive for osteoblast in-growth, as observed by the extension of filopodia across the macropores. Overall, this newly produced material has good thermal, physical and mechanical properties that complements its biocompatibility and ease of use.

Pigmentation and dermal conservative effects of the astonishing algae Sargassum polycystum and Padina tenuis on guinea pigs, human epidermal melanocytes (HEM) and Chang cells.

BACKGROUND: The preference for a fairer skin-tone has become a common trend among both men and women around the world. In this study, seaweeds Sargassum polycystum and Padina tenuis were investigated for their in vitro and in vivo potentials in working as skin whitening agents. Seaweed has been used as a revolutionary skin repairing agent in both traditional and modern preparations. The high antioxidant content is one of the prime reasons for its potent action. It has been employed in traditional Chinese and Japanese medicine. For centuries, most medical practitioners in the Asian cultures have known seaweed as an organic source of vitamins, minerals, fatty acids like omega-3 and omega-6 and antioxidants. The present objective of the study was to evaluate the potent dermal protective effect of the two seaweeds Sargassum polycystum and Padina tenuis on human cell lines and guinea pigs. MATERIAL AND METHODS: Seaweeds were extracted with ethanol and further fractionated with hexane, ethyl acetate and water. The extracts were tested for mushroom tyrosinase inhibitory activity, cytotoxicity in human epidermal melanocyte (HEM), and Chang cells. Extracts with potent melanocytotoxicity were formulated into cosmetic cream and tested on guinea pigs in dermal irritation tests and de-pigmentation assessments. RESULTS: Both Sargassum polycystum and Padina tenuis seaweeds showed significant inhibitory effect on mushroom tyrosinase in the concentration tested. SPEt showed most potent cytotoxicity on HEM (IC50 of 36µg/ml), followed by SPHF (65µg/ml), and PTHF (78.5µg/ml). SPHF and SPEt reduced melanin content in skin of guinea pigs when assessed histologically. CONCLUSION: SPEt, SPHF and PTHF were able to inhibit HEM proliferation in vitro, with SPHF being most potent and did not cause any dermal irritation in guinea pigs. The results obtained indicate that SPHF is a promising pharmacological or cosmetic agent.

Alternanthera sessilis Red Ethyl Acetate Fraction Exhibits Antidiabetic Potential on Obese Type 2 Diabetic Rats.

The antidiabetic potential of Alternanthera sessilis Red was investigated using the obese type 2 diabetic rats induced by high fat diet and streptozotocin. Three fractions (hexane, ethyl acetate, and water) were obtained from the crude ethanol extract of Alternanthera sessilis Red. Alternanthera sessilis Red ethyl acetate fraction (ASEAF) was found to possess the most potent antihyperglycemic effect through oral glucose tolerance test. The ASEAF was subsequently given to the diabetic rats for two weeks. It was found that two-week administration of ASEAF reduces the fasting blood glucose level, triglyceride level, and free fatty acid level of the rats. ASEAF-treated diabetic rats showed higher pancreatic insulin content and pancreatic total superoxide dismutase activity compared to the untreated diabetic rats. Also, the insulin sensitivity indexes suggested that ASEAF ameliorates the insulin resistant state of the diabetic rats. In conclusion, ASEAF could be developed into a potential antidiabetic agent for the management of type 2 diabetes.

In vitro comparative study of white and dark polycaprolactone trifumarate in situ cross-linkable scaffolds seeded with rat bone marrow stromal cells.

OBJECTIVE: Dark poly(caprolactone) trifumarate is a successful candidate for use as a bone tissue engineering scaffold. Recently, a white polymeric scaffold was developed that shows a shorter synthesis time and is more convenient for tissue-staining work. This is an in vitro comparative study of both the white and dark scaffolds. METHODS: Both white and dark poly(caprolactone) trifumarate macromers were characterized via Fourier transform infrared spectroscopy before being chemically cross-linked and molded into disc-shaped scaffolds. Biodegradability was assessed by percentage weight loss on days 7, 14, 28, 42 and 56 (n = 5) after immersion in 10% serum-supplemented medium or distilled water. Static cell seeding was employed in which isolated and characterized rat bone marrow stromal cells were seeded directly onto the scaffold surface. Seeded scaffolds were subjected to a series of biochemical assays and scanning electron microscopy at specified time intervals for up to 28 days of incubation. RESULTS: The degradation of the white scaffold was significantly lower compared with the dark scaffold but was within the acceptable time range for bone-healing processes. The deoxyribonucleic acid and collagen contents increased up to day 28 with no significant difference between the two scaffolds, but the glycosaminoglycan content was slightly higher in the white scaffold throughout 14 days of incubation. Scanning electron microscopy at day 1 [corrected] revealed cellular growth and attachment. CONCLUSIONS: There was no cell growth advantage between the two forms, but the white scaffold had a slower biodegradability rate, suggesting that the newly synthesized poly(caprolactone) trifumarate is more suitable for use as a bone tissue engineering scaffold.

In vitro comparative study of white and dark polycaprolactone trifumarate in situ cross-linkable scaffolds seeded with rat bone marrow stromal cells

OBJECTIVE: Dark poly(caprolactone) trifumarate is a successful candidate for use as a bone tissue engineering scaffold. Recently, a white polymeric scaffold was developed that shows a shorter synthesis time and is more convenient for tissue-staining work. This is an in vitro comparative study of both the white and dark scaffolds. METHODS: Both white and dark poly(caprolactone) trifumarate macromers were characterized via Fourier transform infrared spectroscopy before being chemically cross-linked and molded into disc-shaped scaffolds. Biodegradability was assessed by percentage weight loss on days 7, 14, 28, 42 and 56 (n = 5) after immersion in 10% serum-supplemented medium or distilled water. Static cell seeding was employed in which isolated and characterized rat bone marrow stromal cells were seeded directly onto the scaffold surface. Seeded scaffolds were subjected to a series of biochemical assays and scanning electron microscopy at specified time intervals for up to 28 days of incubation. RESULTS: The degradation of the white scaffold was significantly lower compared with the dark scaffold but was within the acceptable time range for bone-healing processes. The deoxyribonucleic acid and collagen contents increased up to day 28 with no significant difference between the two scaffolds, but the glycosaminoglycan content was slightly higher in the white scaffold throughout 14 days of incubation. Scanning electron microscopy at days 1 and 14 revealed cellular growth and attachment. CONCLUSIONS: There was no cell growth advantage between the two forms, but the white scaffold had a slower biodegradability rate, suggesting that the newly synthesized poly(caprolactone) trifumarate is more suitable for use as a bone tissue engineering scaffold.

Potential dermal wound healing agent in Blechnum orientale Linn.

BACKGROUND: Blechnum orientale Linn. (Blechnaceae) is used ethnomedicinally to treat wounds, boils, blisters or abscesses and sores, stomach pain and urinary bladder complaints. The aim of the study was to validate the ethnotherapeutic claim and to evaluate the effects of B. orientale water extract on wound healing activity. METHODS: Water extract of B. orientale was used. Excision wound healing activity was examined on Sprague-Dawley rats, dressed with 1% and 2% of the water extract. Control groups were dressed with the base cream (vehicle group, negative control) and 10% povidone-iodine (positive control) respectively. Healing was assessed based on contraction of wound size, mean epithelisation time, hydroxyproline content and histopathological examinations. Statistical analyses were performed using one way ANOVA followed by Tukey HSD test. RESULTS: Wound healing study revealed significant reduction in wound size and mean epithelisation time, and higher collagen synthesis in the 2% extract-treated group compared to the vehicle group. These findings were supported by histolopathological examinations of healed wound sections which showed greater tissue regeneration, more fibroblasts and angiogenesis in the 2% extract-treated group. CONCLUSIONS: The ethnotherapeutic use of this fern is validated. The water extract of B. orientale is a potential candidate for the treatment of dermal wounds. Synergistic effects of both strong antioxidant and antibacterial activities in the extract are deduced to have accelerated the wound repair at the proliferative phase of the healing process.

Pharmacological, electrophysiological and toxicity studies of Limacia scanden Lour. (Menispermaceae).

Pharmacological studies showed that Limacia scanden Lour. extracts have sympathomimetic activities similar to noradrenaline (NA). A crude extract of Limacia scanden injected intravenously as a single bolus induced a dose-dependent increase in arterial blood pressure in anaesthetized rats and cats. Pretreatment with a non-specific alpha blocker phentolamine (10(-5) M) blocked this effect, whereas the beta blocker propanolol (10(-5) M) did not. The extract also reduced intestinal motility and this response could be blocked by pretreatment with phentolamine (10(-5) M) and specific alpha1-blocker, prazosin (10(-5) M). In superfused rabbit aorta preparations, it induced an increase in contractions. This effect was blocked by pretreatment with prazosin (10(-5) M), whereas the alpha2-blocker yohimbine (10(-5) M) had only a slight effect. The effects of NA on superfused aorta strip contraction were similar to extract. Toxic symptoms were manifested in less than 5 min when the mice were given 465 mg/kg of extract intraperitoneally. Physiological and behavioural changes observed in dying mice implicated serious malfunctioning of the autonomic nervous system and motor activity. Electrophysiological studies on the tonically autoactive neuron (TAN) of the snail Achantina fulica Férussac revealed that crude extract of Limacia scanden induced excitatory responses which were similar to those of serotonin (5-HT) stimulation. Studies with different ionic compositions of the bathing saline revealed that this excitatory effect of Limacia scanden could be attributed either to release of endogenous serotonin or inhibition of 5-HT reuptake in the CNS. This observation could tentatively be used to provide the framework towards elucidating the mechanism and rationale for the use of this plant in traditional medicine in the treatment of depression and affective disorders.