Antiproliferative and apoptotic effects of proteins from black seeds (Nigella sativa) on human breast MCF-7 cancer cell line.

BACKGROUND: Nigella sativa (NS), a member of family Ranunculaceae is commonly known as black seed or kalonji. It has been well studied for its therapeutic role in various diseases, particularly cancer. Literature is full of bioactive compounds from NS seed. However, fewer studies have been reported on the pharmacological activity of proteins. The current study was designed to evaluate the anticancer property of NS seed proteins on the MCF-7 cell line. METHODS: NS seed extract was prepared in phosphate-buffered saline (PBS), and proteins were precipitated using 80% ammonium sulfate. The crude seed proteins were partially purified using gel filtration chromatography, and peaks were resolved by SDS-PAGE. MTT assay was used to screen the crude proteins and peaks for their cytotoxic effects on MCF-7 cell line. Active Peaks (P1 and P4) were further studied for their role in modulating the expression of genes associated with apoptosis by real-time reverse transcription PCR. For protein identification, proteins were digested, separated, and analyzed with LC-MS/MS. Data analysis was performed using online Mascot, ExPASy ProtParam, and UniProt Knowledgebase (UniProtKB) gene ontology (GO) bioinformatics tools. RESULTS: Gel filtration chromatography separated seed proteins into seven peaks, and SDS-PAGE profile revealed the presence of multiple protein bands. Among all test samples, P1 and P4 depicted potent dose-dependent inhibitory effect on MCF-7 cells exhibiting IC50 values of 14.25 ± 0.84 and 8.05 ± 0.22 µg/ml, respectively. Gene expression analysis demonstrated apoptosis as a possible cell killing mechanism. A total of 11 and 24 proteins were identified in P1 and P4, respectively. The majority of the proteins identified are located in the cytosol, associate with biological metabolic processes, and their molecular functions are binding and catalysis. Hydropathicity values were mostly in the hydrophilic range. CONCLUSION: Our findings suggest NS seed proteins as a potential therapeutic agent for cancer. To our knowledge, it is the first study to report the anticancer property of NS seed proteins.

Quercetin modulates iron homeostasis and iNOS expression of splenic macrophages in a rat model of iron deficiency anemia.

Iron deficiency anemia is one of the most common micronutrient deficient conditions around the globe with various consequences, including the weakened immune system. Quercetin is widely distributed bioflavonoid; it has been debated for its dual roles in iron regulation. Quercetin-iron interaction in the body is a complex mechanism which has not been completely understood. The present study aimed to investigate the effect of quercetin on iron supplementation in iron deficiency anemia and on iNOS expression in splenic macrophages. The rat model of iron deficiency anemia was induced by feeding low iron diet to weanling rats for 20 days. The animals were then administered with ferrous sulfate, quercetin, and their combination for 30 days. Blood parameters, histopathological analysis, iron storage, CD68, iNOS and SLC40 expression in rat spleen were investigated. Our results showed that quercetin regulated iron absorption, despite SLC40 down-expression, indicating possible alternate route of iron transport, and that quercetin modulated iNOS production in splenic macrophages.

N-(2-Hydroxyphenyl)acetamide: a Novel Suppressor of RANK/RANKL Pathway in Collagen-Induced Arthritis Model in Rats.

RANKL and RANK are potential contributors of inflammatory cascade in human and animal model of arthritis. The current study aims to investigate the effect of N-(2-hydroxyphenyl)acetamide (NA-2) on regulation of RANKL pathway in collagen-induced arthritis (CIA) model in rats. CIA was induced using bovine type II collagen in female Wistar rats. The clinical parameters, level of pro-inflammatory and oxidative stress markers were measured to determine the progression of the disease. The mRNA level of RANKL and RANK and downstream mediators of inflammation i.e. c-fos, c-jun, NF-κB and Akt were analysed in spleen tissue using real-time PCR. Immunohistochemical analysis of iNOS, pAkt and c-Fos was also done in spleen tissue. Treatment with NA-2 and indomethacin showed increase in body weight and significant reduction in paw volume and arthritic score (p < 0.0001). Marked reduction in the level of oxidative stress markers, NO, PO and GSH (p < 0.0001), and pro-inflammatory markers, IL-1ß (p < 0.0001) and TNF-α (p < 0.01), was also observed. Likewise, NA-2 and indomethacin treatment also significantly suppressed the mRNA expression of RANKL, RANK, c-fos, c-jun, NF-κB (p < 0.0001) and Akt (p < 0.01) and protein expression of iNOS, pAkt and c-Fos (p < 0.0001) compared to the arthritic control group. Our findings suggest that NA-2 is an antiarthritic agent acting in a pleiotropic manner in CIA rats by not only reducing the clinical signs of arthritis, inflammatory cytokines and free radical production but also attenuating the RANK/RANKL signaling pathway.

Anti-TNF-α and anti-arthritic effect of patuletin: A rare flavonoid from Tagetes patula.

Rheumatoid arthritis (RA) poses a serious health problem as a chronic autoimmune joint disease with significant mortality and morbidity. Proinflammatory cytokines TNF-α and IL-1ß, reactive oxygen species (ROS), and activated CD4(+) T-cells play key roles in the progression of arthritis. The aim of the study is to evaluate the in vitro and in vivo immunomodulatory and anti-arthritic effect of flavonoid patuletin, isolated from Tagetes patula. ELISA was applied for quantification of TNF-α and IL-1ß. Intracellular and extracellular ROS production from phagocytes was measured by the chemiluminescence technique. Proliferation of T-cells was observed using a liquid scintillation counter. Cytotoxicity was assessed by a MTT assay. The serological and histological analysis studies were performed using a rodent model of adjuvant-induced arthritis (AIA). Expression of p38 and NF-κB after treatment of compound was observed by western blotting. Patuletin showed potent inhibitory effects on TNF-α in vitro as well as inhibited the production of both cytokines in vivo. It also showed potent suppression of proliferation of T-cells and significantly inhibited the extracellular and intracellular ROS production. Patuletin revealed significant anti-inflammatory and anti-arthritic activities in the rodent model of adjuvant-induced arthritis (AIA). Histologically, it causes mild bone destruction compared to the arthritic control group, thus representing its anti-arthritic potential. Based on these studies, patuletin could be considered as a potential immunosuppressive and anti-arthritic lead candidate.

The anti-arthritic and immune-modulatory effects of NHAG: a novel glucosamine analogue in adjuvant-induced arthritis.

Rheumatoid arthritis (RA) is potentially devastating condition which lacks good treatment options. Pro-inflammatory cytokines interleukin-1beta (IL-1 ß ), tumor necrosis factor-alpha (TNF- α ), and oxidative stress markers such as nitric oxide (NO) and peroxide (PO) are mediators of RA pathogenesis. In the present study N-[2,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-3-yl]acrylamide (NHAG), analogue of glucosamine, was evaluated in adjuvant-induced arthritic model of rats. The disease progression was monitored by analysing arthritis scoring, loss of body weight, paw oedema, and histological changes in joints. RA associated hyperalgesia was evaluated by gait analysis. The serum or plasma levels of NO, PO, glutathione (GSH) superoxide dismutase (SOD) IL-1 ß and TNF- α were analyzed to monitor the state of disease severity. The arthritic control animals exhibited significant increase in arthritic score (P < 0.003) and paw oedema (P < 0.001) with parallel loss in body weight (P < 0.04). The NHAG-treated arthritic animals exhibited refinement in the gait changes associated with arthritis. NHAG also significantly decreased the NO (P < 0.02) and PO (P < 0.03) with concurrent increased in GSH (P < 0.04) and SOD (P < 0.007). Both IL-1 ß (P < 0.001) and TNF- α (P < 0.001), were significantly decreased in NHAG-treated group. Thus NHAG might have a therapeutic potential for arthritis by exerting antioxidative and immunomodulatory effects.

Anti-arthritic effect of GN1, a novel synthetic analog of glucosamine, in the collagen-induced arthritis model in rats.

OBJECTIVE: Glucosamine is a naturally occurring amino monosaccharide that maintains the elasticity and strength of the cartilage tissues. It has been used to treat osteoarthritis in humans; however, in severe conditions of inflammation and pain, glucosamine alone is not enough, and it is important to improve its biological activity. Our research group has recently taken an interest in the synthetic manipulation of amino sugars to develop some efficient pharmacophores, e.g., ß-D-glucosamine, to combat rheumatoid arthritis, and tested its anti-arthritic effects in the collagen-induced arthritis (CIA) model in rats. METHODS: Arthritis was induced in female Sprague-Dawley rats by multiple intradermal injections of bovine type II collagen and challenged again with the same antigen preparation 7 days later. Arthritis was evaluated by arthritic score, body weight loss, paw volume measurement, and histological changes. RESULTS: The animals in the arthritic control group showed a gradual decrease in their body weight and concurrent increase in the paw volumes compared to the normal control group. In contrast, increased hind paw swelling was significantly suppressed with no further noticeable reduction in body weight in the glucosamine (p < 0.05) and GN1-treated (p < 0.02) arthritic animals. Histopathological evaluation of isolated knee joints by grading system and classification of the stages in arthritic lesion development revealed suppression of the inflammatory changes in the GN1-treated animals. Moreover, both the pro-inflammatory markers C-reactive protein (CRP) and low-density lipoprotein (LDL) levels were found to be significantly decreased in animals treated with GN1 (p < 0.03 for CRP and p < 0.05 for LDL) compared to the arthritic control group. CONCLUSION: These results suggest that GN1 has both anti-arthritic and anti-inflammatory properties. Its effects in the CIA model suggest that it could be useful in the treatment of rheumatoid arthritis.

Anticonvulsant activity of DNS II fraction in the acute seizure models.

AIM OF THE STUDY: Delphinium nordhagenii belongs to family Ranunculaceae, it is widely found in tropical areas of Pakistan. Other species of Delphinium are reported as anticonvulsant and are traditionally used in the treatment of epilepsy. Delphinium nordhagenii is used by local healer in Pakistan but never used for scientific investigation as anticonvulsant. Thus, Delphinium nordhagenii was subjected to bioassay-guided fractionation and the most active fraction, i.e. DNS II acetone was chosen for further testing in the acute seizure models of epilepsy to study the antiepileptic potential in male mice. MATERIALS AND METHODS: Different doses (60, 65 and 70mg/kg, i.p.) of DNS II acetone fraction of Delphinium nordhagenii was administered 30min prior the chemoconvulsant's injection in the male mice. Convulsive doses of chemoconvulsants (pentylenetetrazole 90mg/kg, s.c. and picrotoxin 3.15mg/kg, s.c.) were used. The mice were observed 45-90min for the presence of seizures. Moreover, four different doses of DNS II (60, 65, 70 and 100mg/kg, i.p.) were tested in the MES test. RESULTS: The DNS II acetone fraction of Delphinium nordhagenii has exhibited the anticonvulsant actions by preventing the seizures against PTZ- and picrotoxin-induced seizure as well as 100% seizure protection in MES test. The results are comparable with standard AEDs (diazepam 7.5mg/kg, i.p. and phenytoin 20mg/kg, i.p.). CONCLUSIONS: These findings suggest that the Delphinium nordhagenii possesses the anticonvulsant activity. Further analysis is needed to confirm the structure and target the extended activity profile.

Antinociceptive activity of natural honey in thermal-nociception models in mice.

Several honey samples of Pakistani origin have been analysed for their effect on nociception. Among the tested samples, Acacia honey showed most effective dose-dependent antinociceptive activity which was significantly different from the untreated group in tail-flick and paw-withdrawal tests (p < 0.01 and p < 0.003), respectively. The antinociceptive activity of honey reached a maximum at 60 min post-treatment and then gradually decreased, whereas the activity of indomethacin was persistent at 120 min post-treatment. The absence of antinociceptive activity in simulated honey (which contained fructose, glucose, maltose and sucrose representing the major constituents of honey) indicated that the active principle(s) might be present in minor constituents of honey.

Modulation of the gait deficit in arthritic rats by infusions of muscimol and bicuculline.

Gait analysis in the adjuvant-induced arthritic rat model of chronic pain was used to examine the role of GABA(A) receptors in the development of pain. Drug solutions were administered continuously at 5+/-0.75 microl/h for 14 days via Alzet osmotic pumps (2ML2) placed under the skin of the back. The GABA(A) receptor agonist, muscimol, produces a dose-dependent reversal of the gait deficits seen in arthritic rats without reducing the tibiotarsal joints inflammatory edema or the histological picture of joint erosion and inflammation. The higher infusion rate for muscimol, 20 microg/h, caused the gait for the arthritic rats to be indistinguishable from that of normal non-arthritic rats. In normal, non-arthritic rats, muscimol did not show any effect on gait. The GABA(A) receptor antagonist bicuculline showed small but significant exacerbation of stride length (P < 0.05) single and double stance time (P < 0.05) and swing time deficits (P < 0.05) in the arthritic rats, but no changes in measures of gait in the normal control rat. The results suggest that the development of arthritic pain is increased in the absence of GABA(A) receptor tone and that increasing GABA(A) receptor tone can reduce arthritic pain but does not affect the disease process.