Comparative analysis of biochemical, hormonal, and mineral compositions of preovulatory and cystic ovarian follicles in buffalo during the non-breeding season.

This study is a comparative analysis of the biochemical, hormonal, and mineral compositions of follicular fluid in preovulatory and cystic follicles of water buffalo (Bubalus bubalis). In total, reproductive tracts from 215 buffalo along with intact ovaries were collected randomly from an abattoir. The incidence of cystic conditions found in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances during the non-breeding season. Follicular fluid was aspirated from preovulatory follicles (12-15 mm diameter, oestrogen-active, follicular phase or stage IV corpus luteum on one of the two ovaries, n = 10) and cystic follicles (at least 20 mm diameter, no corpus luteum on any one of the two ovaries, n = 8). The follicular fluid samples were assayed for biochemical components (uric acid, creatinine, blood urea nitrogen, cholesterol, total protein, glucose, ascorbic acid, and alkaline phosphatase), hormones (progesterone, estradiol, and insulin), and minerals (calcium, magnesium, phosphorus, copper, zinc, and cobalt). Cystic follicles had greater (P < 0.05) concentrations of creatinine, blood urea nitrogen, cholesterol, progesterone, copper, zinc, and cobalt, and lesser (P < 0.05) concentrations of uric acid, glucose, ascorbic acid, estradiol, insulin, calcium, magnesium, and phosphorus compared with preovulatory follicles. These results indicated the marked differences in follicular fluid composition between preovulatory and cystic follicles in buffalo. Some of the changes were indicative of oxidative stress and disturbed steroidogenesis, two important mechanisms shown to be associated with cystic ovarian disease in various species. Further studies are warranted to investigate whether these differences are directly or indirectly involved in the formation of cystic follicles or are mere manifestations of the condition.

Response of anestrous heifers fed local grass or oak foliage-based diet with two different mineral mixtures.

The effect of incorporation of two different mineral mixtures and/or oak leaves was studied on nutrient utilization and reproductive performance in anestrous heifers. Twenty-one anestrous heifers (18.2 ± 1.45 months; 229 ± 14.2 kg body weight) were randomly distributed into three similar groups. Heifers in control (T1) and first treatment group (T2) were fed concentrate mixture incorporated with Bureau of Indian Standards (BIS) specific mineral mixture and a customized mineral mixture developed specially for Kumaon hills (MMKH), respectively, along with local green grass (Pennisetum orientale). In the second treatment group (T3), concentrate mixture was the same as that of T2, while the source of roughage was local oak (Quercus leucotricophora) leaves containing 3.35% condensed tannin. A digestibility trial was conducted after 120 days of study. The feed intake was similar among the groups. Digestibility coefficient of crude protein (CP) was lower in T3 than T2 and comparable to T1. Feeding oak leaves improved absorption of calcium as compared to grass-fed animals. Bioavailability of copper and zinc was higher (P < 0.05) in oak leaves and MMKH fed group (T3) as compared to T1, but similar to T2. Conversely, absorption of iron had the reverse trend and was reflected in serum Fe concentration. Hematological, biochemical, enzyme and hormonal profiles were not influenced by any of the treatments. The relative occurrence of estrus cyclicity and conception rate was more in groups T2 and T3, respectively, than other groups. It was concluded that feeding oak foliage-based diet containing 1.87% tannin along with customized mineral mixture developed for Kumaon hills improved certain nutrient utilization and reproductive performance as compared to local green grass supplemented with BIS-specific mineral mixture or MMKH.

Molecular analyses of genetic variability in the populations of Bergenia ciliata in Indian Himalayan Region (IHR).

Bergenia ciliata is an important medicinal plant species of Indian Himalayan Region (IHR). Genetic variability and population genetic structure of B. ciliata sampled from IHR was studied using two single primer amplification reaction (SPAR) methods (DAMD: Directed Amplification of Minisatellite region DNA; ISSR: Inter Simple Sequence Repeats). To provide a reasonable scientific basis for management and conservation of B. ciliata populations in IHR, genetic diversity analysis of 11 populations with 24 SPAR markers (15 ISSR and 9 DAMD) revealed significantly high level of (90.03%) polymorphism at species level. However, genetic variability was low at population level and KUL and BWS populations showed maximum while SHM population revealed least genetic diversity among the 11 populations. Analysis of molecular variance revealed highest percentage of variation (73%) within populations, followed by 17% among populations and least (10%) among the Himalayan regions. Clustering pattern obtained from UPGMA dendrogram was supported by STRUCTURE and principal coordinate analysis, segregating all the 11 natural populations of B. ciliata into two genetic clusters: Eastern and Western Himalayan populations. The clustering patterns of all the three statistical methods indicated that populations of B. ciliata have structured in response to the local micro-climates of the habitats in IHR, and therefore, it can be concluded that genetic variability is in congruence with the geographical diversity.

A pilot, randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of a novel Boswellia serrata extract in the management of osteoarthritis of the knee.

A double-blind, placebo-controlled human trial was conducted to evaluate the safety and efficacy of a standardized oral supplementation of Boswellin®, a novel extract of Boswellia serrata extract (BSE) containing 3-acetyl-11-keto-ß-boswellic acid (AKBBA) with ß-boswellic acid (BBA). A total of 48 patients with osteoarthritis (OA) of the knee were randomized and allocated to the BSE and placebo groups for intervention. Patients were administered BSE or placebo for a period of 120 days. The trial results revealed that BSE treatment significantly improved the physical function of the patients by reducing pain and stiffness compared with placebo. Radiographic assessments showed improved knee joint gap and reduced osteophytes (spur) confirming the efficacy of BSE treatment. BSE also significantly reduced the serum levels of high-sensitive C-reactive protein, a potential inflammatory marker associated with OA of the knee. No serious adverse events were reported. This is the first study with BSE conducted for a period of 120 days, longer than any other previous clinical trial on patients with OA of the knee. The findings provide evidence that biologically active constituents of BSE, namely, AKBBA and BBA, act synergistically to exert anti-inflammatory/anti-arthritic activity showing improvement in physical and functional ability and reducing the pain and stiffness.

Major bioactive phenolics in Bergenia species from the Indian Himalayan region: Method development, validation and quantitative estimation using UHPLC-QqQLIT-MS/MS.

Bergenia species are important medicinal plants used in indigenous systems of medicine for their antilithiatic and diuretic properties. An ultra-high performance liquid chromatography coupled to hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS) method has been developed and validated for the estimation of quantitative variation of eight major bioactive phenolics in the rhizomes (150 samples) of four species of this herb, Bergenia (B. ciliata, B. ligulata, B. purpurascens and B. stracheyi). Chromatographic separation was obtained on a Waters ACQUITY UPLCTM BEH (ethylene bridged hybrid) C18 column with a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile under a gradient elution manner. A hybrid linear ion trap triple quadrupole mass spectrometer was operated in negative electrospray ionization mode with multiple reactions monitoring for detection and quantification of the eight compounds. The validated method demonstrated good linearity (r2 ≥ 0.9991), precision (RSD ≤ 1.87%) and accuracy (95.16-102.11%, RSD ≤ 1.83%) for all reference analytes. The quantitative results revealed that B. ligulata contains the highest amount of the major active marker-bergenin. The results also suggest that sensitive UHPLC-QqQLIT-MS/MS method, a sensitive, accurate and convenient one, could be helpful in identification of potential accession(s), rapid quality control and establishing authenticity of Bergenia species as raw material for pharmaceutical industries.

Supplementation of Slow-Release Melatonin Improves Recovery of Ovarian Cyclicity and Conception in Summer Anoestrous Buffaloes (Bubalus bubalis).

The role of melatonin as a protective neurohormone against restoring cyclicity in summer anoestrous animals in photoperiod species has gained wider acceptance. This study was designed to uncover the evidence the slow-release melatonin (MLT) has on initiation of ovarian cyclicity and conception rate (CR) in summer anoestrous buffaloes. Thus, buffaloes diagnosed as summer anoestrous (absence of overt signs of oestrus, concurrent rectal examination and radioimmunoassay for serum progesterone at 10 days interval) were grouped as untreated (Group I, sterilized corn oil, n = 8) and treated (Group II, single subcutaneous injection of MLT @18 mg/50 kg bwt in sterilized corn oil, n = 20). Animals treated and detected in oestrus were artificially inseminated (AI) followed by division into Group III (second dose of MLT on 5th day post-AI, n = 8) and Group IV (no melatonin administration, n = 10). Blood samples were collected at 4 days interval for estimation of serum MLT, progesterone and oestrogen using radioimmunoassay kit. Mean oestrous induction rate (OIR), oestrous induction interval (OII), interoestrous interval (IOI) and CR were estimated. Compared to control, concentration of melatonin was significantly (p < 0.05) higher in treated group ranging from 14.34 ± 1.72 to 412.31 ± 14.47 pg/ml whereas other two hormones did not show any concentration difference. Melatonin-administered buffaloes showed significantly (p < 0.05) higher (90%) OIR with OII of 18.06 ± 1.57 days. Results showed improvement in conception rate in buffaloes administered with post-insemination melatonin. It can be concluded from the study that slow-release melatonin supplementation restored cyclicity in summer anoestrous animals resulting in improvement in conception rate in buffaloes.

Antitumor activity of melinjo (Gnetum gnemon L.) seed extract in human and murine tumor models in vitro and in a colon-26 tumor-bearing mouse model in vivo.

Melinjo (Gnetum gnemon L.) seed extract (MSE) and its active ingredient gnetin C (GC), a resveratrol dimer, have been shown to possess a broad spectrum of pharmacological activities. In this study, we investigated the antitumor activity of MSE and GC using human and murine tumor cell culture models in vitro. The antitumor activity of GC was compared with trans-resveratrol (tRV), a stilbenoid polyphenol. Our results show that MSE and GC at clinically achievable concentrations significantly inhibited the proliferation of pancreatic, prostate, breast, and colon cancer cell types (P < 0.05), without affecting normal cells. Interestingly, GC exerts enhanced antitumor activity than that of tRV (P < 0.05). MSE and GC significantly induced apoptosis in all the cancer cells, indicating MSE and GC inhibit tumor cell growth by inducing apoptosis (P < 0.001). Our findings provide evidence that MSE might induce apoptosis in cancer cells via caspase-3/7-dependent and -independent mechanisms. However, GC might trigger both early and late stage apoptosis in cancer cells, at least in part by activating caspase 3/7-dependent mechanisms. Furthermore, the antitumor efficacy of MSE observed in vitro was also validated in a widely used colon-26 tumor-bearing mouse model. Oral administration of MSE at 50 and 100 mg/kg per day significantly inhibited tumor growth, intratumoral angiogenesis, and liver metastases in BALB/c mice bearing colon-26 tumors (P < 0.05). In conclusion, our findings provide evidence that MSE and GC have potent antitumor activity. Most importantly, we provide the first evidence that MSE inhibits tumor growth, intratumoral angiogenesis, and liver metastasis in a colon-26 tumor-bearing mice.

The sirtuin 2 inhibitor AK-7 is neuroprotective in Huntington's disease mouse models.

Inhibition of sirtuin 2 (SIRT2) deacetylase mediates protective effects in cell and invertebrate models of Parkinson's disease and Huntington's disease (HD). Here we report the in vivo efficacy of a brain-permeable SIRT2 inhibitor in two genetic mouse models of HD. Compound treatment resulted in improved motor function, extended survival, and reduced brain atrophy and is associated with marked reduction of aggregated mutant huntingtin, a hallmark of HD pathology. Our results provide preclinical validation of SIRT2 inhibition as a potential therapeutic target for HD and support the further development of SIRT2 inhibitors for testing in humans.

Liposome encapsulation of curcumin and resveratrol in combination reduces prostate cancer incidence in PTEN knockout mice.

Increasing interest in the use of phytochemicals to reduce prostate cancer led us to investigate 2 potential agents, curcumin and resveratrol as preventive agents. However, there is concern about the bioavailability of these agents pertinent to the poor absorption and thereby limiting its clinical use. With the view to improve their bioavailability, we used the liposome encapsulated curcumin, and resveratrol individually and in combination in male B6C3F1/J mice. Further, we examined the chemopreventive effect of liposome encapsulated curcumin and resveratrol in combination in prostate-specific PTEN knockout mice. In vitro assays using PTEN-CaP8 cancer cells were performed to investigate the combined effects curcumin with resveratrol on (i) cell growth, apoptosis and cell cycle (ii) impact on activated p-Akt, cyclin D1, m-TOR and androgen receptor (AR) proteins involved in tumor progression. HPLC analysis of serum and prostate tissues showed a significant increase in curcumin level when liposome encapsulated curcumin coadministered with liposomal resveratrol (p < 0.001). Combination of liposomal forms of curcumin and resveratrol significantly decreased prostatic adenocarcinoma in vivo (p < 0.001). In vitro studies revealed that curcumin plus resveratrol effectively inhibit cell growth and induced apoptosis. Molecular targets activated due to the loss of phosphatase and tensin homolog (PTEN) including p-Akt, cyclin D1, mammalian target of rapamycin and AR were downregulated by these agents in combination. Findings from this study for the first time provide evidence on phytochemicals in combination to enhance chemopreventive efficacy in prostate cancer. These findings clearly suggest that phytochemicals in combination may reduce prostate cancer incidence due to the loss of the tumor suppressor gene PTEN.

Adenocarcina of the mouse prostate growth inhibition by celecoxib: downregulation of transcription factors involved in COX-2 inhibition.

BACKGROUND: Epidemiological studies have shown a decreased risk of prostate cancer among men who regularly take aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examined a dose-dependent effect of a cyclooxygenase-2 (COX-2) inhibitor, celecoxib against transgenic adenocarcinoma of the mouse prostate. METHODS: Efficacy of four different doses in parts per million of celecoxib, such as 200 ppm, 400 ppm, 600 ppm, and 1,000 ppm representing very low, moderate, and high doses, respectively were tested against adenocarcinoma of the mouse prostate using a transgenic adenocarcinoma of the mouse prostate (TRAMP) model assay. RESULTS: Dietary supplement of celecoxib at doses of 400 ppm, 600 ppm, and 1,000 ppm are most effective against mPIN (mouse prostatic intraepithelial neoplasia) and adenocarcinoma of the prostate. Tumor growth inhibition by celecoxib was associated with increased rate of apoptosis. At 1,000 ppm, a complete inhibition of the PIN lesions was extended to limit the growth of adenocarcinoma (from 85% to 15%) and metastasis of the mouse prostate. The chemopreventive effect was significant (P<0.01) at 400 ppm, 600 ppm, and 1,000 ppm doses compared to that at the lowest dose of 200 ppm and control. A dose-dependent effect on tumor growth inhibition was associated with reduced expression of NF-kappaBp65 and COX-2. CONCLUSIONS: Dietary supplementation of celecoxib at different doses provides evidence for the suppression of prostate adenocarcinoma tumor growth in a dose-dependent manner. Suppression of adenocarcinoma by celecoxib further limits the growth of metastatic prostate cancer.

A combination of docosahexaenoic acid and celecoxib prevents prostate cancer cell growth in vitro and is associated with modulation of nuclear factor-kappaB, and steroid hormone receptors.

Epidemiological studies have provided evidence that high intake of saturated fat and/or animal fat increases the risk of prostate cancer, but on the other hand, diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), present in fish oils were found to reduce the risk. There are indications of an increased expression of immunoreactive PPARgamma in prostatic intraepithelial neoplasia (PIN) and prostate cancer, suggesting that PPARgamma ligands may exert their own potent anti-proliferative effect against prostate cancer. The experimental evidence for the role of cyclooxygenase-2 (COX-2) in prostate carcinogenesis is well established through several investigations. It clearly suggests the need for development of strategies to inhibit COX-2 mediated prostate carcinogenesis. However, administration of high doses of COX-2 inhibitors, such as celecoxib, over longer periods may not be devoid of side effects. We assessed the efficacy of DHA and celecoxib individually and in combination at low doses in three prostate cancer cell lines (LNCaP, DU145 and PC-3) measuring cell growth inhibition and apoptosis, and on the levels of expression of COX-2, nuclear factor-kappaB (NF-kappaBp65), and nuclear receptors, such as PPARgamma and retinoid X receptors (RXR), all of which presumably participate in prostate carcinogenesis. A 48-h incubation of prostate cancer cells with 5 microM each of DHA or celecoxib induced cell growth inhibition and apoptosis, and altered the expression of the above molecular parameters. Interestingly, the modulation of these cellular and molecular parameters was more pronounced in cells treated with low doses of DHA and celecoxib (2.5 microM each) in combination than in cells treated with the higher doses of individual agents. In conclusion, the present study demonstrates for the first time that a combination of lower doses of the n-3 PUFA, and DHA with the selective COX-2 inhibitor celecoxib effectively modulates the above cellular and molecular parameters that are relevant to prostate cancer. This raises the intriguing prospect that the use of low doses of a COX-2 inhibitor in combination with an n-3 PUFA could be a highly promising strategy for prostate cancer chemoprevention while minimizing undesired side effects.

Regression of mouse prostatic intraepithelial neoplasia by nonsteroidal anti-inflammatory drugs in the transgenic adenocarcinoma mouse prostate model.

PURPOSE: Epidemiologic studies have revealed a decreased risk of colon cancer among people who have regularly taken cyclooxygenase (COX)-2 inhibitors such as aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs). Whereas the selective COX-2 inhibitor celecoxib and exisulind, a metabolic product of sulindac, have gained increasing attention as efficacious chemopreventive agents against colon and prostate cancer, not much is known about the underlying molecular targets and mechanisms. Moreover, the side effects of NSAIDs are a major obstacle for large-scale application to the prevention of cancer in humans; for example, in the United States in 1998, there were 16,550 deaths from NSAID-induced gastrointestinal complications. The toxicity associated with these compounds is raising concerns, and more needs to be known about their mode of action and molecular targets. EXPERIMENTAL DESIGN: We used the transgenic mouse prostate (TRAMP) model, which exhibits similarities with human prostate cancer, including epithelial origin, progression from the PIN stage to adenocarcinoma, and metastasis by a transgene that is hormonally regulated by androgens. In addition to histologically analyzing the PIN lesions of the dorsolateral prostate from TRAMP mice, we delineated the molecular targets and mechanisms of celecoxib and exisulind against mouse PIN lesions. We performed Western blot analysis of the total protein lysate from the tissues of mouse PIN lesions to measure the level of expression of androgen receptor, vascular endothelial growth factor, nuclear factor-kappaB p65, BclII, AKT (total and phosphorylated Ser473), p53, cyclin-dependent kinase inhibitor p21WAF1/CIP1, p27, BAX, and caspase-3 to demonstrate the COX-2-independent mechanism involved in the inhibition of PIN lesions of the dorsolateral prostate by both celecoxib and exisulind. RESULTS: We found for the first time that (a) both celecoxib and exisulind as dietary supplements induce strong inhibitory effects against prostate cancer at doses of 800 and 500 ppm, respectively, after 16 weeks; (b) the histologic analysis of the dorsolateral prostate after 2 weeks of treatment indicated a reduction of PIN lesions from 75% to 19% with celecoxib and to 16% with exisulind; (c) more importantly, those few PINs and adenocarcinomas in the groups treated with celecoxib or exisulind showed more apoptotic cells, lower levels of proliferating cell nuclear antigen, and a lower number of mitotic cells. To understand the molecular mechanisms involved in the inhibition of PIN lesions, first, we examined the expression of molecular targets involved in angiogenesis and inflammatory processes. It was clearly evident from Western blot analysis of the total protein lysate derived from the dorsolateral prostate tissues with PIN lesions that expression of androgen receptor, vascular endothelial growth factor, nuclear factor-kappaB p65, and BclII is down-regulated more effectively by celecoxib. Down-regulation of AKT protein (total and phosphorylated at Ser473) signaling by celecoxib clearly indicates an inhibition of the survival gene and the pathological process that could otherwise lead to adenocarcinoma. CONCLUSIONS: Overall, the findings from this study clearly show the effectiveness of celecoxib and exisulind in reducing the PIN lesions by modulating a cascade of molecular targets involved in COX-2-dependent and -independent mechanisms. Whereas these agents are already in clinical trial or in use as chemopreventive agents, findings from this study demonstrate the difference in their mode of action, thus helping us to understand the side effects.

Hyperbaric oxygen therapy and free radical production: an experimental study in doxorubicin (Adriamycin) extravasation injuries.

The role of hyperbaric oxygen (HBO) therapy in free radical-mediated tissue injury is not clear. HBO has been shown to enhance the antioxidative defense mechanisms in some animal studies, but HBO has also been reported to increase the production of oxygen free radicals. To investigate this controversy, we studied the effect of HBO in a doxorubicin (Adriamycin) extravasation model, because the cytotoxic activity of doxorubicin is partly related to its quinone structure, which leads to the formation of cytotoxic oxygen intermediates. Fifty-four Sprague-Dawley rats underwent injection of 0.3 ml doxorubicin solution (2 mg per milliliter) intradermally on both flanks as described by Rudolph and colleagues. Group I (N = 28) received HBO treatment (2 hours at 2 ATA) for 3 days prior to injection and 7 days postinjection. Group II (N = 26) received no HBO treatment. At 2, 3, and 5 weeks, the size of the ulcers and the surrounding area of alopecia in group I (+HBO) were significantly larger than in group II (-HBO): 112.2 mm2 vs. 42.8 mm2 (p < 0.01) and 1,132.2 mm2 vs. 364.8 mm2 (p < 0.005). Biochemical analysis of the biopsied skin ulcers, to measure the parameters of oxygen free radical production, indicated (similar) low levels of xanthine oxidase for both groups. However, significantly elevated levels of malonyldialdehyde (MDA), indirect evidence of free radical production, was observed in group I (+HBO) in comparison with group II (-HBO): 36.58 vs. 5.84 ng per minute per milligram protein (p < 0.001), which might indicate free radical-induced cellular injury. It is concluded that in this animal study the cytotoxicity of doxorubicin is potentiated by HBO therapy. The elevated levels of MDA suggest a direct additive cytotoxic effect by increased membrane lipid peroxidation. HBO therapy, therefore, might be deleterious in the early (preulcer) stage of doxorubicin extravasation.

The influence of the calcium channel blocker nimodipine on flap survival.

The calcium channel blocker nimodipine has been used successfully in neurological patients for the treatment of vasospasm after subarachnoid hemorrhage and stroke. In this study of experimental random pattern skin flaps in rats, an increase of nearly 50% in the survival rate to 94.33% (+/- 7.3% standard deviation) under nimodipine treatment versus 66.00% (+/- 16.5% standard deviation) in the controls could be demonstrated (p less than 0.001). Vital dye injections in a separate experiment indicated that the increased flap survival is mainly due to improved microcirculation. The distance traveled by the dye was 8.01 +/- 1.21 cm in the experimental versus 6.61 +/- 1.25 cm in the control group (p less than 0.05).

Influence of stage of lactation on the triacylglycerol composition of buffalo milk fat.

Milk fats obtained from colostrum and early, middle and late lactation samples of buffalo milk were analyzed for their triacylglycerol (TG) compositions. Each milk fat was first separated by thin layer chromatography (TLC) into high, medium and low molecular weight TG. The TG fractions thus obtained were further segregated by argentation TLC, according to their degree of unsaturation into saturated, trans-monoene, cis-monoene, diene and polyene species. With progressive lactation, the major changes from colostral fat were an increase in lower fatty acids and decline in oleic acid. This caused, in turn, marked variations in saturated TG and diene TG and, to a smaller extent, in polyene TG. Monoene TG, both cis and trans, remained practically constant throughout. These trends were largely reversed toward the end of lactation.