Antinociceptive activities of Artocarpus lacucha Buch-ham (Moraceae) and its isolated phenolic compound, catechin, in mice.

BACKGROUND: The present study evaluated the antinociceptive effect of the bark of Artocarpus lacucha, which is used for the treatment of stomachache, headache and boils in the traditional system of medicine. METHODS: The antinociceptive activity was investigated by the tail immersion, hot plate, acetic acid- & formalin-induced nociception and carrageenan-induced paw edema tests using a hydro-methanolic extract of A. lacucha bark. The plant extract was found to contain a substantial amount of phenolic compounds according to the total phenolic and flavonoid content assay. A phenolic metabolite, (+)-catechin, has been isolated using different chromatographic techniques. The compound was characterized with 1D and 2D NMR spectroscopic data. (+)-catechin, isolated from A. lacucha was assessed for antinociceptive effects swiss albino mice. Furthermore, the possible involvement of opioid receptors and ATP-sensitive K+ channel for the effect of the plant extract and (+)-catechin has been justified using naloxone and glibenclamide, respectively. RESULTS: Oral administration (p.o) of the plant extract (50-200 mg/Kg b.w.) resulted in significant thermal pain protection in the hot plate and tail immersion tests. The action of the plant extract was significantly antagonized by naloxone, a non-selective opioid antagonist, in the hot plate and tail immersion tests, which supports the involvement of opioid receptors. Both the plant extract and (+)-catechin, (50-200 mg/Kg b.w., p.o.) significantly diminished the acetic acid- & formalin-induced nociception, and carrageenan-induced paw edema. Glibenclamide, an ATP-sensitive K+ channel blocker, significantly reversed their effect in the acetic acid-induced writhing test which indicates the participation of ATP-sensitive K+ channel system. CONCLUSIONS: The investigation revealed potential central and peripheral antinociceptive effects of A. lacucha bark supports its applications in the traditional system of medicine.

Antinociceptive effect of methanol extract of Celosia cristata Linn. in mice.

BACKGROUND: Celosia cristata Linn. (Amaranthaceae) is used in traditional medicine for the treatment of headache, sores, ulcers, eye inflammations, skin eruption, painful menstruation and carpal tunnel syndrome. This study was performed to evaluate the antinociceptive activity of methanol extract of the whole plant of C. cristata (MECC). METHODS: The evaluation of the antinociceptive effect of MECC was performed using thermal (hot plate, tail immersion test) and chemical (acetic acid, formalin, and glutamate-induced nociception test) pain models in mice at four different doses (50, 100, 200, 400 mg/kg; p.o.). Involvement of opioid receptors mediated central antinociceptive mechanism of MECC was evaluated using naloxone. Furthermore, the association of ATP-sensitive K+ channel and cGMP pathway were evaluated using glibenclamide and methylene blue respectively. RESULTS: Oral treatment of MECC produced significant, strong and dose-dependent central and peripheral antinociceptive effect in experimental pain models. MECC significantly increased the latency time of thermal threshold in both hot plate and tail immersion test. The inhibition of writhing syndrome by the extract in the acetic acid-induced writhing test was remarkable. MECC significantly reduced the formalin-induced neurogenic and inflammatory pain. In addition, the inhibition of glutamate-induced paw licking and edema by MECC was significant. The antinociceptive effect was significantly reversed by naloxone and glibenclamide, suggesting the association of opioid and ATP-sensitive K+ channel system respectively. In addition, MECC also demonstrated the involvement of cGMP pathway in the antinociceptive action. CONCLUSION: The study suggests that C. cristata possess significant antinociceptive effect which is associated with both central and peripheral mechanisms and provides a rationale for its extensive use at different painful conditions in traditional medicine.

Method for manufacture and cryopreservation of cartilage microtissues.

The financial viability of a cell and tissue-engineered therapy may depend on the compatibility of the therapy with mass production and cryopreservation. Herein, we developed a method for the mass production and cryopreservation of 3D cartilage microtissues. Cartilage microtissues were assembled from either 5000 human bone marrow-derived stromal cells (BMSC) or 5000 human articular chondrocytes (ACh) each using a customized microwell platform (the Microwell-mesh). Microtissues rapidly accumulate homogenous cartilage-like extracellular matrix (ECM), making them potentially useful building blocks for cartilage defect repair. Cartilage microtissues were cultured for 5 or 10 days and then cryopreserved in 90% serum plus 10% dimethylsulfoxide (DMSO) or commercial serum-free cryopreservation media. Cell viability was maximized during thawing by incremental dilution of serum to reduce oncotic shock, followed by washing and further culture in serum-free medium. When assessed with live/dead viability dyes, thawed microtissues demonstrated high viability but reduced immediate metabolic activity relative to unfrozen control microtissues. To further assess the functionality of the freeze-thawed microtissues, their capacity to amalgamate into a continuous tissue was assess over a 14 day culture. The amalgamation of microtissues cultured for 5 days was superior to those that had been cultured for 10 days. Critically, the capacity of cryopreserved microtissues to amalgamate into a continuous tissue in a subsequent 14-day culture was not compromised, suggesting that cryopreserved microtissues could amalgamate within a cartilage defect site. The quality ECM was superior when amalgamation was performed in a 2% O2 atmosphere than a 20% O2 atmosphere, suggesting that this process may benefit from the limited oxygen microenvironment within a joint. In summary, cryopreservation of cartilage microtissues is a viable option, and this manipulation can be performed without compromising tissue function.

Microtissue Culture Provides Clarity on the Relative Chondrogenic and Hypertrophic Response of Bone-Marrow-Derived Stromal Cells to TGF-ß1, BMP-2, and GDF-5.

Chondrogenic induction of bone-marrow-derived stromal cells (BMSCs) is typically accomplished with medium supplemented with growth factors (GF) from the transforming growth factor-beta (TGF-ß)/bone morphogenetic factor (BMP) superfamily. In a previous study, we demonstrated that brief (1-3 days) stimulation with TGF-ß1 was sufficient to drive chondrogenesis and hypertrophy using small-diameter microtissues generated from 5000 BMSC each. This biology is obfuscated in typical large-diameter pellet cultures, which suffer radial heterogeneity. Here, we investigated if brief stimulation (2 days) of BMSC microtissues with BMP-2 (100 ng/mL) or growth/differentiation factor (GDF-5, 100 ng/mL) was also sufficient to induce chondrogenic differentiation, in a manner comparable to TGF-ß1 (10 ng/mL). Like TGF-ß1, BMP-2 and GDF-5 are reported to stimulate chondrogenic differentiation of BMSCs, but the effects of transient or brief use in culture have not been explored. Hypertrophy is an unwanted outcome in BMSC chondrogenic differentiation that renders engineered tissues unsuitable for use in clinical cartilage repair. Using three BMSC donors, we observed that all GFs facilitated chondrogenesis, although the efficiency and the necessary duration of stimulation differed. Microtissues treated with 2 days or 14 days of TGF-ß1 were both superior at producing extracellular matrix and expression of chondrogenic gene markers compared to BMP-2 and GDF-5 with the same exposure times. Hypertrophic markers increased proportionally with chondrogenic differentiation, suggesting that these processes are intertwined for all three GFs. The rapid action, or "temporal potency", of these GFs to induce BMSC chondrogenesis was found to be as follows: TGF-ß1 > BMP-2 > GDF-5. Whether briefly or continuously supplied in culture, TGF-ß1 was the most potent GF for inducing chondrogenesis in BMSCs.

GC-MS analysis and pharmacological evaluations of Phoenix sylvestris (Roxb.) seeds provide new insights into the management of oxidative stress and hyperglycemia.

Phoenix sylvestris Roxb. (Arecaceae) seeds are used in the treatment of diabetes in the traditional system of medicine. The present study evaluated antihyperglycemic and antioxidant activities as well as the total phenolic and flavonoid content of the methanol extract of P. sylvestris seeds (MEPS). The constituents of the extract were identified by GC-MS analysis. MEPS demonstrated strong antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC50 = 162.70 ± 14.99 µg) and nitric oxide (NO) (IC50 = 101.56 ± 9.46 µg/ml) free radicals. It also possesses a substantial amount of phenolics and flavonoids. It significantly (p < .05) reduced blood glucose levels in glucose-loaded and alloxan-induced diabetic mice at the doses of 150 and 300 mg/kg b.w., respectively. A total of 46 compounds were detected and identified by gas chromatography-mass spectroscopy (GC-MS) analysis, among which 8-methylisoquinoline N-oxide (32.82%) was predominant. The phytochemical study by GC-MS revealed that the MEPS possesses compounds which could be related to its antidiabetic and antioxidant activities. To recapitulate, P. sylvestris seeds can be a very good option for antidiabetic and antioxidant activity though further studies are still recommended to figure out the responsible phytochemicals and establish their exact mechanism of action.

A tumour-spheroid manufacturing and cryopreservation process that yields a highly reproducible product ready for direct use in drug screening assays.

If it were possible to purchase tumour-spheroids as a standardised product, ready for direct use in assays, this may contribute to greater research reproducibility, potentially reducing costs and accelerating outcomes. Herein, we describe a workflow where uniformly sized cancer tumour-spheroids are mass-produced using microwell culture, cryopreserved with high viability, and then cultured in neutral buoyancy media for drug testing. C4-2B prostate cancer or MCF-7 breast cancer cells amalgamated into uniform tumour-spheroids after 48 h of culture. Tumour-spheroids formed from 100 cells each tolerated the cryopreservation process marginally better than tumour-spheroids formed from 200 or 400 cells. Post-thaw, tumour-spheroid metabolic activity was significantly reduced, suggesting mitochondrial damage. Metabolic function was rescued by thawing the tumour-spheroids into medium supplemented with 10 µM N-Acetyl-l-cysteine (NAC). Following thaw, the neutral buoyancy media, Happy Cell ASM, was used to maintain tumour-spheroids as discrete tissues during drug testing. Fresh and cryopreserved C4-2B or MCF-7 tumour-spheroids responded similarly to titrations of Docetaxel. This protocol will contribute to a future where tumour-spheroids may be available for purchase as reliable and reproducible products, allowing laboratories to efficiently replicate and build on published research, in many cases, making tumour-spheroids simply another cell culture reagent.

Collagenase treatment appears to improve cartilage tissue integration but damage to collagen networks is likely permanent.

When repairing cartilage defects a major challenge is achieving high-quality integration between the repair tissue and adjacent native cartilage. Matrix-rich cartilage is not easily remodeled, motivating several studies to trial enzyme treatment of the tissue interface to facilitate remodeling and integration. Studying and optimizing such processes is tedious, as well as potentially expensive, and thus simpler models are needed to evaluate the merits of enzyme treatment on cartilage tissue integration. Herein, we used engineered cartilage microtissues formed from bone marrow-derived stromal cells (BMSC) or expanded articular chondrocytes (ACh) to study the impact of enzyme treatment on cartilage tissue integration and matrix remodeling. A 5-min treatment with collagenase appeared to improve cartilage microtissue integration, while up to 48 h treatment with hyaluronidase did not. Alcian blue and anti-collagen II staining suggested that collagenase treatment did facilitate near seamless integration of cartilage microtissues. Microtissue sections were stained with Picrosirius red and characterized using polarized light microscopy, revealing that individual microtissues contained a collagen network organized in concentric shells. While collagenase treatment appeared to improve tissue integration, assessment of the collagen fibers with polarized light indicated that enzymatically damaged networks were not remodeled nor restored during subsequent culture. This model and these data paradoxically suggest that collagen network disruption is required to improve cartilage tissue integration, but that the disrupted collagen networks are unlikely to subsequently be restored. Future studies should attempt to limit collagen network disruption to the surface of the cartilage, and we recommend using Picrosirius red staining and polarized light to assess the quality of matrix remodeling and integration.

Polymethoxyflavones from Nicotiana plumbaginifolia (Solanaceae) Exert Antinociceptive and Neuropharmacological Effects in Mice.

Polymethoxylavones (PMFs) are known to exhibit significant anti-inflammatory and neuroprotective properties. Nicotiana plumbaginifolia, an annual Bangladeshi herb, is rich in polymethoxyflavones that possess significant analgesic and anxiolytic activities. The present study aimed to determine the antinociceptive and neuropharmacological activities of polyoxygenated flavonoids namely- 3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone (1), 3,3',4',5',5,6,7,8-octamethoxyflavone (exoticin) (2), 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone (3), and 3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone (4), isolated and identified from N. plumbaginifolia. Antinociceptive activity was assessed using the acetic-acid induced writhing, hot plate, tail immersion, formalin and carrageenan-induced paw edema tests, whereas neuropharmacological effects were evaluated in the hole cross, open field and elevated plus maze test. Oral treatment of compounds 1, 3, and 4 (12.5-25 mg/kg b.w.) exhibited dose-dependent and significant (p < 0.01) antinociceptive activity in the acetic-acid, formalin, carrageenan, and thermal (hot plate)-induced pain models. The association of ATP-sensitive K+ channel and opioid systems in their antinociceptive effect was obvious from the antagonist effect of glibenclamide and naloxone, respectively. These findings suggested central and peripheral antinociceptive activities of the compounds. Compound 1, 3, and 4 (12.5 mg/kg b.w.) demonstrated significant (p < 0.05) anxiolytic-like activity in the elevated plus-maze test, while the involvement of GABAA receptor in the action of compound 3 and 4 was evident from the reversal effects of flumazenil. In addition, compounds 1 and 4 (12.5-25 mg/kg b.w) exhibited anxiolytic activity without altering the locomotor responses. The present study suggested that the polymethoxyflavones (1-4) from N. Plumbaginifolia could be considered as suitable candidates for the development of analgesic and anxiolytic agents.

Antinociceptive and neuropharmacological activities of methanol extract of Phoenix sylvestris fruit pulp.

Fruits of Phoenix sylvestris Roxb. (Arecaceae) are used to treat back pain, toothache, headache, arthritis, nervous debility and as sedative. The aim of this study was to evaluate the antinociceptive and neuropharmacological activities of methanol extract of P. sylvestris fruit pulp (MEPS). The antinociceptive activity of MEPS was evaluated by heat-induced (hot plate, tail immersion test) and chemical-induced pain models (acetic acid-induced writhing, formalin-induced nociception, glutamate-induced nociception and paw edema test). The effect of MEPS on central nervous system (CNS) was studied using hole cross test, open field test, sodium thiopental-induced sleeping time and elevated plus maze test. MEPS showed strong, significant and dose-dependent antinociceptive activity in all heat-induced and chemical-induced pain models at all experimental doses. Involvement of opioid receptor mediated analgesia was evident from the reversal of analgesic effect by naloxone. MEPS also showed reduced locomotor activity in both hole cross and open field tests. The increase in sleeping time in sodium thiopental-induced sleeping test and anxiolytic activity in elevated plus maze test were also significant. So, it is evident that MEPS possesses strong central and peripheral antinociceptive activity as well as CNS depressant, sedative and anxiolytic activity. The results justify the ethnomedicinal use of P. sylvestris fruit in different painful conditions and CNS disorders.