Safety Assessment of Ultra High Dilutions of Camphora officinarum: A Preclinical Investigation.

Background: Camphora officinarum (CO) is a commonly used homeopathic remedy for treating colds, collapse, and recurrent eruptive illnesses. Objective: Due to the non-availability of safety data on CO, the current study intended to determine the oral toxicity of CO in its ethanol-potentized dilutions 6C, 30C, and 200C in Wistar albino rats as per OECD guidelines. Materials and methods: A single oral dose of CO-6C, 30C, and 200C (2000 µl/kg) was administered, and the animals were monitored for behavior and mortality for up to 14 days in an acute toxicity study. In the subacute study, the effects of daily oral administration of CO-6C, 30C, and 200C (200 µl/kg) for 28 days were observed for clinical signs, change in body weight, and mortality. Hematological, biochemical, and histopathological analyses were assessed and organ weights were determined. Results: Results indicate no mortality of CO in its potencies in the acute toxicity study and was found to be safe at 2000 µl/kg dosage in the subacute toxicity study. CO (200 µl/kg/day) did not show any signs of toxicity in the hematological, biochemical, and histopathological analyses, along with organ weights. Conclusion: In conclusion, the findings suggest that CO in potencies of 6C, 30C, and 200C is safe up to a single oral dose of 2000 µl/kg body weight, and the No Observed Adverse Effect Level (NOAEL) was determined to be greater than 200 µl/kg/day.

Acute and Sub-acute Oral Toxicity Assessment of Ferrum phosphoricum in Rats.

BACKGROUND: Ferrum phosphoricum (FP) has been used by traditional medicine practitioners for various ailments since ancient times. However, scientific evidence on the safety of FP is still unavailable. Thus, the current study aimed to investigate the acute and sub-acute oral toxicity of homeopathic FP in experimental rats. METHODS: In an acute toxicity investigation, a single dose of 2,000 µL/kg of FP 6c, 30c and 200c was administered to female Wistar rats, which were monitored for up to 14 days according to the Organization for Economic Cooperation and Development (OECD) guideline 423. For a sub-acute toxicity study, FP 6c, 30c and 200c (200 µL/kg) were administered to male and female rats for 28 days as per the OECD guideline 407. All the animals were observed for mortality, clinical signs and body weight during the study. At the end of the experiment, hematological, biochemical and histopathological assessments were performed. RESULTS: During the acute toxicity study, no mortality was observed in rats administered with FP, and thus the median lethal dose (LD50) was identified as >2,000 µL/kg. In the sub-acute study, no mortality or adverse clinical signs were noticed with FP treatment. Moreover, weekly body weight gain was normal. Hematological and biochemical investigations revealed no abnormalities. Furthermore, histological analysis of FP-treated rats' vital organs revealed no pathological changes. CONCLUSION: Overall, our findings imply that FP 6c, 30c and 200c potencies are safe and do not cause toxicity when given orally to Wistar albino rats for an extended period at a dose of 200 µL/kg.

Aspirin 15cH has Different Effects on Morphology and Function of Lipopolysaccharide-Challenged RAW 264.7 Macrophages In Vitro Compared to a Pharmacological Dose of Aspirin.

INTRODUCTION: Aspirin is one of the most commonly used drugs worldwide. It is known to present antipyretic, anti-inflammatory and anti-thrombotic actions, making it extremely useful in a wide range of clinical contexts. Interestingly, homeopathically prepared Aspirin 15cH has been found to have a pro-thrombotic effect in rats, raising the hypothesis that Aspirin 15cH could also modulate the activity of inflammatory cells in different pathological processes. OBJECTIVE: Our objective was to assess what effect Aspirin 15cH has on RAW 264.7 macrophages in vitro. METHODS: The effects of Aspirin 15cH on biochemical and morphological activities of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were evaluated. These effects were compared with unchallenged macrophages (negative control), untreated LPS-stimulated macrophages, macrophages treated with succussed water (vehicle control), or aspirin 200 µg/mL (pharmacological inhibitor of LPS activity). Cell morphology (adhered cell area and cytoskeleton arrangements), cell viability, toll-like receptor-4 (TLR-4) expression, and the production of nitric oxide, cytokines and intracellular reactive oxygen species were assessed. RESULTS: Aspirin 15cH reduced the number of cells expressing TLR-4 on the surface (p = 0.03) and induced a "columnar" morphology of macrophage pseudopods, indicating changes in cytoskeleton arrangement. When cells were treated with both Aspirin 15cH and LPS, cell morphology became heterogeneous, suggesting that sub-populations of cells had differing sensitivities to LPS or Aspirin 15cH. Exposure of the cells to LPS alone, succussed water or aspirin 200 µg/mL produced effects consistent with the literature. CONCLUSION: Aspirin 15cH, aspirin 200 µg/mL, LPS and succussed water appear to act as independent stimuli able to induce different patterns of macrophage response. Aspirin 15cH induced changes suggestive of M2 polarization of the macrophages (i.e., toward a wound healing or tissue repair, rather than inflammatory, phenotype). These preliminary findings need to be confirmed in further specific studies.

The effects of tarantula cubensis extract on renal ischemia reperfusion injury in rats.

BACKGROUND: Renal ischemia-reperfusion (IR) related acute kidney injury (AKI) is an important health problem and has not yet been fully treated. Tarantula cubensis extract (TCE) is a homeopathic drug that has antiinflammatory and antioxidant effects. This study aimed to investigate the effects of TCE on renal ischemia-reperfusion injury in rats. METHODS: This study was carried out on 48 Spraque-Dawley male rats, which were divided into six groups. The first, second, and third groups were control, sham, and IR groups, respectively. Group four received IR and 0.2 mL of 96% ethanol. Group five and six received ischemia and reperfusion and TCE 0.01 and 0.1 mg per rat (which correspond to approximately 0.04 mg/kg, and 0.4 mg/kg), respectively. Tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1ß), total antioxidant status (TAS), and total oxidant status (TOS) levels in renal tissue were measured by enzyme-linked immunosorbent assay (ELISA). Oxidative stress index (OSI) was obtained by proportioning TAS and TOS. Superoxide dismutase (SOD), myeloperoxidase (MPO) activities, and malondialdehyde (MDA) levels were determined by manual spectrophotometric methods. The histopathological changes were evaluated via hematoxylineosin and immunohistochemical staining. RESULTS: In IR group, renal tissue TNF-α and IL-1ß levels were significantly higher than control group (p < 0.0001 for both), and low(p < 0.0001 for both) and high dose (p < 0.0001 for both) TCE administration decreased these markers. Low and high doses of TCE decreased OSI values compared with IR group (p = 0.04 and p = 0.001 respectively). Although TCE decreased MDA levels, it was not statistically significant. MPO levels significantly decreased. In addition, TCE has been found to prevent hemorrhage, cast formation, and dilatation caused by IR in renal tissues stained with hematoxylin-eosin. And also, the most intense nuclear factor kappa B (NFκB) and caspase-3 immunopositivity found in IR group was decreased in both of the TCE groups. DISCUSSION: Although TCE showed a protective effect by inhibiting inflammation against IR damage in renal tissues, there was no clear effect on oxidative stress. Larger and more detailed studies are needed to clarify the issue.

Dietary genistein and 17ß-estradiol implants differentially influence locomotor and cognitive functions following transient focal ischemia in middle-aged ovariectomized rats at different lengths of estrogen deprivation.

Genistein possesses estrogenic activity and has been considered a potential replacement for estrogen replacement therapy after menopause. In the current study, we investigated the neuroprotective effects of dietary genistein at varied lengths of estrogen deprivation in middle-aged ovariectomized Sprague-Dawley rats under ischemic conditions. Two weeks of treatment with dietary genistein at 42 mg/kg but not 17ß-estradiol implants improved cognitive flexibility (Morris water maze test) after short-term estrogen deprivation (2 weeks) but not long-term estrogen deprivation (12 weeks). 17ß-estradiol implants but not dietary genistein improved locomotor asymmetry (cylinder test) after long-term but not short-term estrogen deprivation. Dietary genistein but not 17ß-estradiol implant improved early phase motor learning (rotarod test) after long-term estrogen deprivation. Neither 17ß-estradiol implant nor dietary genistein reduced infarct size after either short-term or long-term estrogen deprivation. Genistein, however, reduced ionized calcium-binding adaptor molecule-1 (Iba1) expression, a marker of brain inflammation, at the ipsilateral side of stroke injury after short-term but not long-term estrogen deprivation. This study suggests that the neuroprotective effects of dietary genistein on motor and cognitive functions are distinctly influenced by the length of estrogen deprivation following focal ischemia. SIGNIFICANCE: There is an increasing postmenopausal population opting for homeopathic medicines for the management of menopausal symptoms due to the perceived distrust in estrogen use as hormone replacement. Basic and clinical studies support the notion that early, but not delayed, hormone replacement after menopause is beneficial. Furthermore, evidence suggests that delaying hormone replacement augments the detrimental, rather than the beneficial effects of estrogens. Because of the active consideration of soy isoflavones including genistein as alternatives to estrogen replacement, it is necessary to understand the ramifications of soy isoflavones use when their administration is begun at various times after menopause.

Opium Effect in Pregnancy on the Dynamics of Maternal Behavior: Testing a Neurochemical Model.

BACKGROUND: Investigations into neurochemical mechanisms of opioid addiction are difficult due to the complexity of behavior and multiplicity of involved neurotransmitter and hormonal systems. The aim of this study was to examine the benefits of structured analysis of these mechanisms using the framework of the neurochemical model Functional Ensemble of Temperament (FET) and the example of maternal behavior under the condition of opium consumption in pregnancy. The FET differentiates between (a) endurance, (b) speed of integration, and (c) emotionality aspects of behavior suggesting that these systems are differentially regulated by (a) serotonin-neuropeptides-brain-derived neurotrophic factor (BDNF), (b) dopamine-GABA, and (c) opioid receptor systems, correspondingly. The FET also suggests that mu-opioid receptors (MORs) binding the endorphines (including opium's ingredient morphine) have a stronger association with regulation endurance, whereas delta-OR have a stronger association with integration of behavior and kappa-OR - with the perceptual mobilization seen in anxiety. To test the predictions of this model, we compared the impact of massive MOR dysregulation on 3 behavioral aspects of behavior and on serotonin, BDNF, and corticosterone levels. METHODS: The study used 24 female white Wistar rats which were randomly divided into (1) control group: pregnant rats without any intervention; (2) opium-exposed group: animals that were exposed to opium during pregnancy and after the delivery until the end of the study. At the end of the study, the levels of BDNF, serotonin (5-HT) in the hippocampus of the mother's brain, and serum corticosterone, as well as 12 aspects of the maternal behavior were evaluated. The differences between control and experimental groups were assessed using the t test for independent samples. RESULTS: The BDNF and serotonin concentrations in the hippocampus of the mother rats which were exposed to opium were lower than in the control group; the mean corticosterone in exposed mothers was higher than in the control group. Behaviorally, opium-consuming mothers showed lower endurance in 4 distinct behavioral categories (nesting, feeding, grooming, and retrieval) than the mothers in the control group. Ease of integration of behavior was affected to a lesser degree, showing a significant effect only in 1 out of 5 applied measures. Self-grooming, seen as an emotionality-related aspect of behavior, was not affected. CONCLUSION: Opium exposure during pregnancy in our experiment primarily reduced the endurance of rat's maternal behavior, but the speed of integration of behavioral acts was less affected. This negative impact of opium on endurance was associated with a decrease of BDNF and serotonin levels in the hippocampus and an increase in corticosterone level in opium-consuming mothers. There is no effect of opium exposure on self-grooming behavior. This pattern supports the FET hypothesis about the role of 5-HT and BDNF in endurance, differential regulation of endurance, integrative and emotionality aspects of behavior, and differential association of the MOR system with endurance aspects, in comparison with kappa- and delta opioid receptors.

Comparison of the antidepressant like activity of homeopathic remedies (Argentum nitricum, Staphysagria and Ignatia amara) and their effect on the behavior of rodents.

The majority of the world population suffers from mental and behavioral disorder. It is the need of the time to find an alternate of presently available medicines in order to decrease the medical expense. Homeopathic remedies are available and prescribed by homeopaths for treatment of anxiety and depression. Unfortunately, no data are available that proves its potential to relieve mental illness. The current study is designed to assess neuro behavioral and antidepressant like effects of homeopathic remedies Staphysagria, Argentum nitricum and Ignatia amara in comparison with standard drug (escitalopram). Different neuro behavioral activities were analyzed. The animals were administered the doses of all homeopathic remedied (60 µl to the rats) and escitalopram (0.042 mg to rats) through the oral route. The activities were observed on day 30th and day 60th. Our result suggests that the swimming time in Staphysagria treated group were significantly improved (p<0.001) after day 60th and significance rise was observed (p<0.01) in Ignatia amara treated animals, whereas significant decline (p<0.05) in struggling time was observed in Argentum nitricum administered animals after the 60th day as compared to 30th day. The central square crossings were improved highly significantly (p<0.001) after the 30th day dosing, by all three remedies and peripheral squares crossing were found highly significantly increased (p<0.001) after chronic dosing in Staphysagria and Ignatia amara treated groups. It is concluded from the results that all three homeopathic remedies produce comparable effects like standard drug while among all three remedies Staphysagria possess a potent antidepressant activity. To the best of our knowledge the current study reports first time the anti-depressant potential of homeopathic remedies in rodents.

Oviductus Ranae protein hydrolyzate prevents menopausal osteoporosis by regulating TGFß/BMP2 signaling.

PURPOSE: It is known that menopausal osteoporosis (MOP) is the most typical form of osteoporosis, which is characterized by low bone mass and microstructure damage of the bone tissue, leading to increased bone fragility and risk of fracture. This study aimed to evaluate the protective effects of Oviductus Ranae protein hydrolyzate (ORPH) on the MOP in vivo. METHODS: Osteoporosis model was induced by ovariectomy, treated with ORPH 150 or 75 mg kg-1. Body weight and bone mineral density (BMD) of rats were measured at the beginning and the end of the experiment, and femoral maximum load was determined immediately after killing. The expression levels of alkaline phosphatase (ALP), Smad4, tartrate acid phosphatase (TRAP), BMP2, Runx2, CPB, ColI and osteocalcin were examined by RT-PCR or western-blotting. HE staining was used to observe the pathological changes in the femurs. Immunohistochemistry was used to detect the expression of ALP and BMP2. All data were analyzed by SPSS 13.0. RESULTS: The results revealed that ORPH had no effect on the weight of normal and osteoporotic rats. ORPH could significantly improve the femur BMD and increase the maximum load of the osteoporotic rats. ORPH could significantly upregulate the expression level of bone formation makers, ALP, osteocalcin, ColI, and Runx2, and downregulate the expression level of bone resorption marker, TRAP. In the ORPH group, the expression levels of BMP2, Smad4, and CPB of key proteins in the TGFß/BMP2 signaling pathway were significantly upregulated. In addition, immunohistochemistry showed that ALP and BMP2 expression in femurs of the ORPH group was stranger. H&E staining showed that ORPH (150 mg kg-1) significantly increased the thickness of trabeculae and decreased fracture risk. CONCLUSION: Collectively, ORPH plays a role in the prevention and treatment of osteoporosis, which may be a potential anti-osteoporosis drug.

Effects of Ultra-Low-Dose Aspirin in Thrombosis and Haemorrhage.

BACKGROUND: Aspirin is the oldest and possibly the most widely used pharmacologically active substance still used in allopathic medicine. Its effect on fever and inflammation has paved the way to its anti-thrombotic effect. Dilutions of aspirin have been tested for many years in the University of Bordeaux, in humans as well as in animal models. METHODS: This article is a review of the totality of articles published by the Laboratory of Hematology of the Faculty of Pharmacy of the University of Bordeaux, reporting different doses and dilutions of aspirin, different kinds of inhibitors, transgenic mice and animal models of disease such as portal hypertension and cirrhosis. RESULTS: Homeopathic dilutions of aspirin, notably 15 cH, have shown a pro-thrombotic effect in humans and in in-vivo animal studies. Longitudinal studies in rats have also shown an initial anti-thrombotic effect followed by a pro-thrombotic effect of aspirin several days after a single high-dose administration. This pro-thrombotic effect seems to act by inhibiting the cyclooxygenase (COX)-2 pathway in studies performed with COX selective inhibitors and in knock-out mice without COX-1 or COX-2. This effect may explain the thrombo-embolic complications described after aspirin withdrawal for the purposes of surgery or after non-compliance with anti-platelet therapy, and it may be beneficial in normalising primary haemostasis and decreasing haemorrhage in animal models of portal hypertension and cirrhosis. CONCLUSIONS: Aspirin 15 cH acts through the inhibition of the COX-2 pathway producing a clear pro-thrombotic effect. Further studies should clarify if the pro-thrombotic effect of aspirin withdrawal and the effect of aspirin 15 cH are related, as secondary effects of the same drug. Clarifying this last outcome may be of great significance to public health.

Gastroprotective effects of Kangfuxin against water-immersion and restraint stress-induced gastric ulcer in rats: roles of antioxidation, anti-inflammation, and pro-survival.

Context: Kangfuxin (KFX) is widely used for the treatment of gastric and duodenal ulcer; however, more research is needed to determine the protective mechanisms of KFX in ameliorating gastric ulcer.Objective: To investigate the efficacy and potential mechanism of Kangfuxin liquid (KFX) in water-immersion and restraint stress (WIRS)-induced gastric ulcer.Materials and methods: Seventy rats were randomly divided into seven groups (n = 10) as follows: the control group (normal saline, i.g.), the model group (normal saline, i.g.), the KFX groups (2.5, 5 and 10 mL/kg, i.g.), the omeprazole group (20 mg/kg, i.p.) and Sanjiuweitai Granules group (1850 mg/kg, i.g.). The WIRS model was applied to induce stress ulcers after 7 days of drug administration. Afterwards, rats were sacrificed at 10 h induced by WIRS.Results: Pre-treatment with KFX (5,10 mL/kg) could effectively reduce the area of gastric ulcers and improve the pathological changes of ulcerated tissue. Moreover, KFX (5,10 mL/kg) increased the prostaglandin E2 (52%) and cyclooxygenase-1 (30%) levels, and improved malondialdehyde (54%), superoxide dismutase (58%), catalase (39%), and nitric oxide (11%) and TNF-α (9%), IL-6 (11%), MMP-9 (54%) and MMP-2 (53%) of ulcer tissue. Furthermore, pre-treatment with KFX dramatically increased IGF-1, PTEN, and Akt protein expression.Conclusions: Our results suggest that KFX has protective effects on WIRS-induced gastric ulcer via inflammatory reactions, oxidative stress inhibition, and pro-survival action, which were the results of activating the IGF-1/PTEN/Akt signalling pathway. Our results provide evidence of KFX for treating gastric ulcer.

[Diuretic effect and material basis of Clematidis Armandii Caulis in rats].

To search for the active diuretic fractions of Clematidis Armandii Caulis( CAC) and determine its main active chemical components by using liquid chromatography-mass spectrometry( LC-MS) and diuretic activity evaluation. CAC 75% ethanol extracts and extracts from different polar solvents were orally administered to saline-loaded rats at different doses. 6 h urinary volume,p H and contents of electrolyte Na+,K+and Cl-were measured. The chemical components of the active fractions were separated and identified by ultra performance liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry( UPLC-ESI-Q-TOF-MS/MS) method. As compared with the control group,the urine volume was increased by 44%( P< 0. 01) and 34%( P < 0. 05) in CAC75% ethanol extract 57. 74 and 28. 8 mg·kg-1 groups respectively; the Na+excretion was increased by 52%( P< 0. 01) and 45%( P<0. 05),respectively; while the Cl-excretion was increased by 101%( P<0. 01) and 85%( P<0. 05),respectively. The urine volume,Na+excretion and Cl-excretion were increased by 50%( P< 0. 01),58%( P< 0. 05),and 65%( P< 0. 05) respectively in petroleum ether extract 70. 98 mg·kg-1 group as compared with the control group. While for the n-butanol extract 194. 18 mg·kg-1 group,the urine volume,Na+and Cl-excretion were increased by 42%( P<0. 01),41%( P<0. 05) and 97%( P<0. 01),respectively. The diuretic activity of other fractions was not obvious. There was no statistical difference in K+excretion in all groups. The results of LC-MS analysis showed that six compounds,including two sterols,one chromogen and three fatty acids,were identified from petroleum ether extract.Fourteen compounds,including six triterpenoid saponins,six lignin glycosides,one sterol glycoside and one phenolic glycoside,were identified from the n-butanol extract. All the results suggested that the ethanol extract of CAC had remarkable diuretic activity and its main effective components included sterol,triterpenoid saponin and lignin glycosides.

Phosphorus protects cardiac tissue by modifying the immune response in rats infected by Trypanosoma cruzi.

AIM: This study evaluates and correlates the number of myocarditis focuses and production of cytokines in Rattus norvegicus (Wistar lineage), experimentally infected with T. Cruzi and treated with Phosphorus. METHODS: In two blind, controlled and randomized trials, 53 45-day-old, male animals were allocated into groups Control (n=24): Control group infected and treated with 7% hydroalcoholic solution, the preparation vehicle of the test medication; and Phosphorus (n=24 on days 0, 5, 10 and 24 after infection): group infected and treated with Phosphorus 13cH, diluted 10-26 and dynamized (test medication). The animals were inoculated intraperitoneally with 5×106 blood trypomastigotes of T. cruzi-Y strain. The medication was administered overnight (16 consecutive hours), diluted in water (1mL/100mL) in amber water bottles. The animals were treated 2days before and 2, 4, and 6days after infection. Enumeration of inflammatory foci in cardiac tissue (Hematoxylin-Eosin) and dosage of cytokines TNF-α and IFN-γ in the serum were performed on days 0, 5, 10 and 24 after infection, using three animals/group. Mann-Whitney, Friedman ANOVA, Spearman correlation (p<0.05), and Statistica Single User Software version 13.2 were used for data analysis. RESULTS: The animals treated with Phosphorus 13cH had high concentration of INF-É£ on the 5th day of infection with significant decrease on the 10th and 24th days (p<0.05), and high concentration of TNF-α on the 5th and 10th days of infection with decrease on the 24th day (p<0.05). The treatment with Phosphorus caused a significant increase of INF-É£ and TNF-α on the 5th day of infection compared with the Control (p<0.05), with reestablishment on the 24th day, as well as in the Control group. The group treated with Phosphorus had 52.5% less number of myocarditis focuses in heart than Control group (p<0.05) on the 10th day of infection. The significant increase in cytokines on the5th day of infection in the Phosphorus group is related to a significant decrease in the number of inflammatory foci in cardiac tissue on the 10th day of infection in this group. DISCUSSION AND CONCLUSION: Treatment with Phosphorus 13cH promotes beneficial effects in T. cruzi infection in Wistar rats by modulating the secretion of IFN-γ and TNF-α with decreased inflammation in cardiac tissue. These results reinforce the importance of considering the use of homeopathy for establishing new therapeutic approaches in the management of patients with Chagas disease.

Beneficial immunomodulatory and neuro digestive effect in Trypanosoma cruzi infection after Lycopodium clavatum 13c treatment.

Studies show that highly diluted medications demonstrate benefits in treating infections, constituting an alternative for their treatment. The present study evaluated the effects of Lycopodium clavatum, dynamization 13c, in Wistar rats infected with T. cruzi. In this study 42 male rats were intraperitoneally inoculated with T. cruzi - Y strain and allocated into groups: IC (infected control group) and Ly (treated with L. clavatum 13c). The cytokines dosage (IFN-γ, IL-12, IL-10, IL-4), quantification and morphometry of myenteric neurons were evaluated. The treatment with L. clavatum modifies the immune response, with increase of IFN-γ on day 10 a.i. and IL-12 on day 24 a.i., decrease of IL-10 concentration on day 10 a.i. and subsequent increase of this cytokine and IL-4 on day 24 a.i., affording a bigger number of myenteric neurons compared to IC group. Thus, L. clavatum 13c promoted on rats infected with T. cruzi a beneficial immunomodulatory action reducing the pathogenic progression of digestive Chagas disease.

Induction of apoptosis by opium in some tumor cell lines.

The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

Pancuronium enhances isoflurane anesthesia in rats via inhibition of cerebral nicotinic acetylcholine receptors.

PURPOSE: This study was conducted to elucidate the mechanism of enhancement of volatile anesthetics by neuromuscular blocking agents in rats and to consider the relevance of this enhancement to clinical anesthesia. METHODS: Male Sprague-Dawley rats were used. After confirming a movement in response to tail clamping under 1.1 % isoflurane anesthesia, response was determined when the tail clamp was applied at several points after microinjection of pancuronium into the lateral ventricle. Arousal responses to microinjection of nicotine into the lateral ventricle were assessed with or without pretreatment with intraventricular pancuronium. The intravenous 50 % effective dose (ED50) and 95 % effective dose (ED95) for neuromuscular blockade with pancuronium administered in a cumulative fashion at 1.1 % isoflurane were calculated. RESULTS: Intraventricular pancuronium dose-dependently reduced the response to tail clamping, and the dose required to show immobilization of 50 % of rats (intraventricular ED50) was 1.62 µg/kg. Pretreatment with pancuronium at 6 µg/kg significantly reduced the effect of awakening by nicotine under isoflurane anesthesia (P = 0.044). The intravenous ED50 and ED95 for neuromuscular blockade were 63 µg/kg (90 % confidence interval [CI] 52-75 µg/kg) and 133 µg/kg (90 % CI 109-158 µg/kg), respectively. The ratio of intraventricular ED50 to intravenous ED50 was 0.026. CONCLUSION: Pancuronium microinjection into the lateral ventricle dose-dependently enhances the depth of isoflurane anesthesia, which might be caused by inhibition of neuronal nicotinic acetylcholine receptor transmission in the cerebrum. Intravenous injection of pancuronium at high doses might increase the cerebrospinal concentration to a level at which an effect can be observed.

Standardization of homeopathic mother tincture of Toxicodendron pubescens and correlation of its flavonoid markers with the biological activity.

BACKGROUND: Standardization and quality control of homeopathic drugs is very challenging. As mother tinctures are derived from complex natural resources, there is a need of systematic evaluation of chemical markers which correlate with the proposed biological activities of mother tinctures. METHODS: In present study, High-Performance Thin-Layer Chromatography (HPTLC) standardization method of homeopathic mother tinctures of Toxicodendron pubescens using quercitrin and rutin as chemical markers is validated and correlations of content of these markers with its anti-inflammatory effects are established. For HPTLC analysis, precoated silica gel plates were used as stationary phase. Two flavonoids, namely quercitrin and rutin were used as markers. Separation was achieved using methylene chloride:methanol:water:glacial acetic acid (15:1.5:1:8 v/v/v) as mobile phase. The developed plates were scanned at 365 nm. RESULTS: It was observed that quercitrin (Rf value 0.63) and Rutin (Rf value 0.41) are well resolved. The minimum detectable concentrations for quercitrin and rutin were 5 ng/spot. The linearity range was between 100 and 2000 ng/spot for both the markers. Subsequently, anti-inflammatory activity of these formulations was determined against carrageenan-induced paw edema in rats, pain threshold determined by electronic Von-Frey apparatus and paw withdrawal latency (PWL) on hot-plate. All the tested formulations of Rhus Tox showed anti-inflammatory and analgesic activity against carrageenan induced paw edema in rats. Quantitative correlation between the content of markers and anti-inflammatory activity of mother tinctures was established. RESULTS: Anti-inflammatory effect as well as effect on paw withdrawal and pain threshold, at third hour after carrageenan injection, correlated with quercitrin and rutin content in the respective formulations. CONCLUSIONS: This study validates a quantitative HPTLC method for standardization of homeopathic mother tincture of Rhus Tox and establishes quercitrin and rutin as markers corresponding its biological activity. Contents of quercitrin and rutin in T. pubescens mother tincture correlates with its anti-inflammatory and analgesic actions and the validated HPTLC method can be used in standardization of homeopathic mother tincture of T. pubescens.

Hepatoprotective effect of Lycopodium clavatum 30CH on experimental model of paracetamol-induced liver damage in rats.

INTRODUCTION: Homeopathic Lycopodium clavatum is indicated for disorders of the digestive system and its accessory organs, including atony of the liver and liver tissue failure. Tis suggests that it may have action on drug-induced hepatitis, as occurs in paracetamol overdose. PURPOSE: To evaluate the effectiveness of Lycopodium clavatum 30C (Lyc) as a hepatoprotector against liver damage experimentally induced by paracetamol (Pct) in Wistar rats. METHODOLOGY: Thirty animals subdivided into 6 groups were used. Animals from the treated groups were pretreated for 8 days with Lyc 30c (0.25 ml/day), receiving a dose of 3 g/kg of Pct on the 8th day. A positive control group received similar treatment, replacing Lyc 30c with 30% ethanol and a negative control received only 30% ethanol. After 24 and 72 h, the animals were sacrificed for tissue and blood sample collection. Subsequently, enzyme serum measurements indicative of liver damage (aspartate-aminotransferase (AST) and Alanine-aminotransferase (ALT)) and liver histological and morphometric analyses were performed. RESULTS: Pretreatment with Lyc 30c reduced hepatic lesions produced by Pct overdose as evidenced by a significant reduction (p < 0.05) in ALT levels in the LyP 24h-group (901.04 ± 92.05 U/l) compared to the respective control group (1866.28 ± 585.44 U/l), promoted a significant decrease in the number of acinar zone 1 affected by necrosis and inflammatory infiltration (15.46 ± 13.86 clr/cm(2) in LyP72 for 73.75 ± 16.60 clr/cm(2) in PC72), and inhibition of 1,2-glycol (glycogen) depletion in zone 3 (a significant reduction in Lyc 72 h group animals in comparison to the control group). Significant changes concerning the development of fibrosis or alterations in transaminase levels were not observed after 72 h. CONCLUSION: Lyc 30c exerted a moderate hepatoprotective effect on acute Pct-induced hepatitis, mainly shown by a histological decrease in necrosis and inflammatory foci, preserved glycogen and other 1,2-glycols in zone 3 and reduced serum levels of ALT and AST.

Effects of Tarantula cubensis D6 on aflatoxin-induced injury in biochemical parameters in rats.

INTRODUCTION: Aflatoxins are toxic fungal metabolites that have adverse effects on humans and animals. Tarantula cubensis D6 is used as a homeopathic medicine for different purposes. The present study investigates the effects of Tarantula cubensis D6 on the oxidant-antioxidant balance and some biochemical parameters against exposure to aflatoxin. METHODS: Thirty-two Sprague-Dawley female rats were used and evenly divided into four groups. Group 1 served as control. Groups 2, 3, and 4 received 200 µl/kg.bw/day Tarantula cubensis D6 (applied subcutaneously), 400 µg/kg.bw/day total aflatoxin (approximately 80% AF B1, 10% AF B2, 6 %AF G1, and 4% AF G2), and 200 µl/kg.bw/day Tarantula cubensis D6 plus 400 µg/kg.bw/day total aflatoxin, respectively, for 28 days. At the end of 28 days, blood samples and some organs (liver, kidney, brain, and spleen) were taken from all the animals. Oxidative stress markers (MDA, SOD, CAT, GSH-Px) and some biochemical parameters (glucose, triglyceride, cholesterol, BUN, creatinine, AST, ALT and ALP, total protein, albumin) were evaluated in blood samples and tissues. RESULTS: Aflatoxin caused negative changes in all oxidative stress parameters and some biochemical parameters (glucose, triglyceride, cholesterol, creatinine, AST, ALT, ALP, total protein, albumin). Administration of Tarantula cubensis D6 partly alleviated aflatoxin-induced negative changes. CONCLUSIONS: Our results indicated that Tarantula cubensis D6 partially neutralized the deleterious effects of aflatoxin.

Effect of homeopathic Lycopodium clavatum on memory functions and cerebral blood flow in memory-impaired rats.

BACKGROUND: Lycopodium clavatum (Lyc) is a widely used homeopathic medicine for the liver, urinary and digestive disorders. Recently, acetyl cholinesterase (AchE) inhibitory activity has been found in Lyc alkaloid extract, which could be beneficial in dementia disorder. However, the effect of Lyc has not yet been explored in animal model of memory impairment and on cerebral blood flow. AIM: The present study was planned to explore the effect of Lyc on learning and memory function and cerebral blood flow (CBF) in intracerebroventricularly (ICV) administered streptozotocin (STZ) induced memory impairment in rats. MATERIALS AND METHODS: Memory deficit was induced by ICV administration of STZ (3 mg/kg) in rats on 1st and 3rd day. Male SD rats were treated with Lyc Mother Tincture (MT) 30, 200 and 1000 for 17 days. Learning and memory was evaluated by Morris water maze test on 14th, 15th and 16th day. CBF was measured by Laser Doppler flow meter on 17th day. RESULTS: STZ (ICV) treated rats showed impairment in learning and memory along with reduced CBF. Lyc MT and 200 showed improvement in learning and memory. There was increased CBF in STZ (ICV) treated rats at all the potencies of Lyc studied. CONCLUSION: The above study suggests that Lyc may be used as a drug of choice in condition of memory impairment due to its beneficial effect on CBF.