Hydroxysafflor yellows alleviate thrombosis and acetaminophen-induced toxicity in vivo by enhancing blood circulation and poison excretion.

BACKGROUND: Hydroxysafflor yellow A (HSYA) from the flower of Carthamus tinctorius (Safflower) has been reported to have various pharmacological effects. However, little is known about the bioactivities of other chemical constituents in Safflower and the relationship between enhancement of blood circulation and hepatoprotection by HSYA. PURPOSE: The present research was to evaluate the antithrombotic and hepatoprotective activities of HSYA and C, examine their mechanisms of actions, including influence on the excretion velocity of acetaminophen, and the relationship between the antithrombotic, hepatoprotective, and other bioactivities. METHODS: The hepatoprotective activities were examined by acetaminophen (APAP)-induced zebrafish toxicity and carbon tetrachloride (CCl4)-induced mouse liver injury. The concentrations of APAP in zebrafish and APAP that was excreted to the culture media were quantified by UHPLC-MS. The anti-thrombosis effect of HSYA and C were examined by the phenylhydrazine (PHZ)-induced zebrafish thrombosis. RESULTS: HSYA and HSYC showed robust protection on APAP-induced toxicity and PHZ-induced thrombosis. The hepatoprotective effects of HSYA and C were more potent than that of the positive control, acetylcysteine (61.7% and 58.0%, respectively, vs. 56.9% at 100 µM) and their antithrombosis effects were more robust than aspirin (95.1% and 86.2% vs. 52.7% at 100 µM). HSYA and C enhanced blood circulation, rescued APAP-treated zebrafish from morphological abnormalities, and mitigated APAP-induced toxicity in liver development in liver-specific RFP-expressing transgenic zebrafish. HSYC attenuated CCl4-induced mouse liver injury and regulated the levels of HIF-1α, iNOS, TNF-α, α-SMA, and NFκB in liver tissues. HSYA was also protective in a dual thrombotic and liver toxicity zebrafish model. By UHPLC-MS, HSYA accelerated the excretion of APAP. CONCLUSION: HSYA and C are the bioactive constituents of Safflower that are responsible for the herbal drug's traditional use in promoting blood circulation to remove blood stasis. Safflower and its chalcone constituents may protect from damage due to exogenous or disease-induced endogenous toxins by enhancing the excretion velocity of toxins.

Protective Role of Selenium Against Hemolytic Anemia Is Mediated Through Redox Modulation.

Selenium (Se), an essential trace element and potent nutritional antioxidant, exerts its biological effects through incorporation into selenoproteins like glutathione peroxidase (GPx). Modest decrement in the levels of GPx could be partly responsible for peroxidation of RBCs, which results into hemolytic anemia. Therefore, it is hypothesized that dietary Se, as selenoproteins (GPx), can maintain the homeostasis in RBCs and regulate the erythropoiesis by preventing oxidative stress-mediated hemolysis. Se-deficient (0.01 ppm), Se-adequate (0.1 ppm sodium selenite), and Se-supplemented (0.5 ppm sodium selenite) status were created in Balb/c mice by feeding yeast-based diets for 8 weeks and established by measuring Se levels in plasma and activities, expressions of Se-dependent selenoproteins. Fifty percent of mice from each differential Se group were treated with phenylhydrazine (PHZ, 20 mg/kg, i.p.) to induce hemolytic anemia. Results indicated that PHZ-treated Se-deficient animals demonstrated increased hemolysis, abnormal RBC morphology, increase in Heinz bodies and reticulocytes, and denaturation of hemoglobin to globin precipitates and methemoglobin. Se supplementation protected against these hemolytic changes and makes RBCs less fragile. These findings were consistent with dietary Se concentration-dependent changes in activity and expression of GPx indicating that ROS-mediated oxidative stress is integral to hemolysis. Protective effects of Se supplementation against increased levels of ROS, protein carbonyls, and peroxide damage to membrane lipids and enzymatic antioxidants validated these observations. In conclusion, dietary Se supplementation protected the RBCs against hemolysis by mitigating ROS-mediated oxidative stress.

Chemical analysis, pharmacological activity and process optimization of the proportion of bilobalide and ginkgolides in Ginkgo biloba extract.

Variations on the efficacy of commercial Ginkgo biloba preparations have been reported, although all the products follow the same standards. Terpene trilactones (TTLs), including bilobalide (BB) and ginkgolides, are one of the main active components in G. biloba extract and have been received the most attention due to their chemical uniqueness and their importance for quality control. A plenty of studies demonstrated that BB and ginkgolides display differential activities on various biological processes. However, the influence of different ratios of BB and ginkgolides on the efficacy of TTLs has not been detected yet. The aims of this study were: (1) to test whether different ratios of BB and ginkgolides existed in commercial G. biloba preparations; (2) to detect the influence of different ratios of BB and ginkgolides on the in vivo efficacy of TTLs; and (3) to optimize the extraction process of G. biloba to approach the better BB and ginkgolides ratio with the maximum in vivo effects. First, the content and proportion of BB and ginkgolides in various G. biloba preparations were quantified by HPLC-MS analysis. As the results, an obvious fluctuation in the proportion of BB and ginkgolides was observed in the preparations from different commercial suppliers. The ratio was ranged from 0.3 to 0.8. Second, a zebrafish thrombosis model was used to evaluate the antithrombotic effects of different ratios of BB and ginkgolides. The result showed that the proportion of BB and ginkgolides at 1:2 produced the maximum antithrombotic effects. Third, the extraction process of G. biloba was optimized using a design space technique aiming to approach the best BB and ginkgolides ratio obtained from zebrafish experiment. The extraction process was modeled based on the results of Box-Behnken designed experiments. Design space was then calculated using a probability-based method. Within this design space, G. biloba extraction process can be guaranteed to achieve the better BB and ginkgolides ratio with high assurance. Normal operation space for G. biloba extraction process was recommended as ethanol concentration of 50% to 70%, liquid-to-solid ratio of 5.6 mL/g to 7.3 mL/g, and extraction time of 2.2 h to 3.0 h. This work not only suggest that the proportion of BB and ginkgolides should be used as a quality control index in ginkgo preparations besides the content of TTLs, but also provide a way to approach it with the extraction process parameters controlled in the normal operation ranges.

Production of Minor Ginenosides from Panax notoginseng by Microwave Processing Method and Evaluation of Their Blood-Enriching and Hemostatic Activity.

A green solvent extraction technology involving a microwave processing method was used to increase the content of minor ginsenosides from Panax notoginseng. This article aims to investigate the optimization of preparation of the minor ginsenosides by this microwave processing method using single-factor experiments and response surface methodology (RSM), and discuss the blood-enriching activity and hemostatic activity of the extract of microwave processed P. notoginseng (EMPN) The RSM for production of the minor ginsenosides was based on a three-factor and three-level Box-Behnken design. When the optimum conditions of microwave power, temperature and time were 495.03 W, 150.68 °C and 20.32 min, respectively, results predicted that the yield of total minor ginsenosides (Y9) would be 93.13%. The actual value of Y9 was very similar to the predicted value. In addition, the pharmacological results of EMPN in vivo showed that EMPN had the effect of enriching blood in N-acetylphenylhydrazine (APH) and cyclophosphamide (CTX)-induced blood deficient mice because of the increasing content of white blood cells (WBCs) and hemoglobin (HGB) in blood. Hemostatic activity in vitro of EMPN showed that it had significantly shortened the clotting time in PT testing (p < 0.05). The hemostatic effect of EMPN was mainly caused by its components of Rh4, 20(S)-Rg3 and 20(R)-Rg3. This microwave processing method is simple and suitable to mass-produce the minor ginsenosides from P. notoginseng.

Haematological and biochemical studies on Justicia carnea leaves extract in phenylhydrazine induced-anemia in albino rats.

BACKGROUND: Justicia carnea is a medicinal plant used widely in Nigeria which is reported to have diverse functions, including blood-boosting potential. Aim. The effect of the ethanol extract of Justicea carnea (JC) leaves in phenylhydrazine induced-anemia albino rats on haematological and lipid profile parameters was investigated. METHODS: The experimental animals were randomly grouped into five groups of six rats each – group 1 (non-anemic control), group 2 (anemic control), group 3 (500 mg/kg of JC extract), group 4 (1000 mg/kg of JC extract) and group 5 (DMSO control). Phenylhydrazine was administered once at a dose of 80 mg/kg b.w.  to induce hemolytic anemia. After 28 days of extract administration, they were humanely sacrificed and the serum collected was used for biochemical analysis. RESULTS: In the acute toxicity study, the LD50 was found to be above 5000 mg/kg body weight. Packed Cell Volume (PCV) values, Red Blood cell (RBC) and haemoglobin (Hb) concentrations decreased (p < 0.05) sig- nificantly after 48 hours of phenylhydrazine induction, but after 28 days of administering extracts of Justicia carnea, PCV values, RBC and Hb increased (p < 0.05) significantly. There were significant (p < 0.05) de- creases in cholesterol, triacylglycerol, and LDL cholesterol concentrations in the extract-administered groups (groups 3&4) relative to the anemic control. There was a significant (p < 0.05) increase in HDL-cholesterol concentrations in the extract groups (3&4) relative to the non-anemic control. CONCLUSIONS: Extracts of Justicia carnea not only reversed anemic conditions in the phenylhydrazine-induced rats, it also improved the lipid profile, and this may be attributed to its rich phytochemical, nutrient and vita- min composition. Therefore, the findings of the study suggest that J. carnea leaves could be used to manage lipid abnormalities associated with anemia.

Hematopoietic Effect of Amaranthus cruentus Extract on Phenylhydrazine-Induced Toxicity in Rats.

Amaranthus cruentus (Amaranthaceae) is one of the popularly grown leafy vegetables in the Indian subcontinent. Leaves of the plant are rich in polyphenols, tannins, flavonoids, steroids, terpenoids, saponins, and betalains. The plant also contains rich amounts of protein, calcium, iron, vitamins A, E, and C, and folic acid. The present work was undertaken to evaluate the antianemic effect of Amaranthus cruentus. Ethanol extract of Amaranthus cruentus was prepared. Acute oral toxicity of the extract was determined by Organization for Economic Cooperation and Development (OECD) Guideline 423. Doses of 200 and 400 mg/kg were used in the present study. Phenylhydrazine (60 mg/kg, intraperitoneal injection for three consecutive days) was used to induce anemia in rats. After anemia induction, animals were treated with standard preparation and extract. Amaranthus cruentus extract significantly aided in restoring the levels of red blood cells, white blood cells (WBCs), and hemoglobin. There was also an increase in hematocrit. Thus, it can be concluded that Amaranthus cruentus is a rich source of phytochemicals that are responsible for demonstrating hematopoietic effects. Isolation and structure elucidation of constituents, responsible for antianemic activity, is necessary to affirm the aforementioned effect.

Iron-rich drinking water and ascorbic acid supplementation improved hemolytic anemia in experimental Wistar rats.

Anemia is a frequent problem in both the primary and secondary health care programs. In contrast, most areas of northeast India are vulnerable to iron toxicity. In the present study, we documented the effect of administration of iron rich water on hemolytic anemia in a Wistar rats' animal model. Hemolytic anemia was induced by phenyl hydrazine through intraperitoneal route and diagnosed by the lowering of blood hemoglobin. After inducing the hemolytic anemia, 24 Wistar rats (n = 6 in four groups) were randomly assigned to 1 mg/l, 5 mg/l, and 10 mg/l ferric oxide iron along with 1 mg/ml ascorbic acid administered through drinking water; a control group was treated with iron-free water. The hematological and biochemical parameters, iron levels in liver, spleen, and kidney were estimated after 30 d of treatment. In the group treated with 5 mg/l iron and ascorbic acid, a significant increase of serum iron and ferritin, and a decrease of TIBC (total iron binding capacity) were observed without changes in other biochemical parameters and histopathological findings. However, in the group treated with 10 mg/l iron and ascorbic acid, hematological changes with significantly higher values for white blood cell count, serum glutamic phospho transaminase, serum glutamic oxaloacetic transaminase, alkaline phosphatase, glucose, splenic, and liver iron content, indicate potential toxicity at this supplementation level. Data suggest that the optimum concentration of iron (5 mg/l) and ascorbic acid solution may improve anemic conditions and may be therapeutically beneficial in the treatment of iron deficiency anemia without any negative impact, while 10 mg/l in drinking water seems to be the threshold for the initiation of toxicity.

Inhibitory effects of palm tocotrienol-rich fraction supplementation on bilirubin-metabolizing enzymes in hyperbilirubinemic adult rats.

BACKGROUND: Phenylhydrazine, a hemolytic agent, is widely used as a model of experimental hyperbilirubinemia. Palm tocotrienol-rich fraction (TRF) was shown to exert beneficial effects in hyperbilirubinemic rat neonates. AIM: To investigate the effects of palm TRF supplementation on hepatic bilirubin-metabolizing enzymes and oxidative stress status in rats administered phenylhydrazine. METHODS: Twenty-four male Wistar rats were divided into two groups; one group was intraperitoneally injected with palm TRF at the dose of 30 mg/kg/day, while another group was only given vehicle (control) (vitamin E-free palm oil) for 14 days. Twenty-four hours after the last dose, each group was further subdivided into another two groups. One group was administered phenylhydrazine (100 mg/kg, intraperitoneally) and another group was administered normal saline. Twenty-four hours later, blood and liver were collected for biochemical parameter measurements. RESULTS: Phenylhydrazine increased plasma total bilirubin level and oxidative stress in the erythrocytes as well as in the liver, which were reduced by the pretreatment of palm TRF. Palm TRF also prevented the increases in hepatic heme oxygenase, biliverdin reductase and UDP-glucuronyltransferase activities induced by phenylhydrazine. CONCLUSION: Palm tocotrienol-rich fraction was able to afford protection against phenylhydrazine-induced hyperbilirubinemia, possibly by reducing oxidative stress and inhibiting bilirubin-metabolizing enzymes in the liver.

Nutritional support in the treatment of aplastic anemia.

OBJECTIVE: Whether a specific nutritional support promotes healing of aplastic anemia (AA) patients is still unclear. Therefore, we explored the potential of a high-nucleotide, arginine, and micronutrient nutritional supplement on the nutritional rehabilitation of AA mice. METHODS: The BALB/c AA mice model was treated with hypodermic injections of acetylphenylhydrazine (100 mg/kg), x-ray (2.0 Gy), and intraperitoneal injections of a cyclophosphamide (80 mg/kg) combination. Then AA mice were fed with nutritional supplements in a dose-dependent manner (1445.55, 963.7, 674.59 mg/kg/d) for 7 wk. At the end of the experimental period, mice were autopsied. A full blood count was performed, and femoral marrow cell suspensions were prepared to assess the total femoral nucleated cell count and the number of committed hemopoietic progenitor cells (colony-forming units). The pathologic changes of liver and spleen were analyzed. RESULTS: The significant increases of nutrient mixture groups were evident in many peripheral blood parameters. The femoral nucleated cell count and colony-forming units of nutritional supplements groups were markedly increased, compared with the AA group. Transmission electron microscopy showed that the number of mitochondria in similar bone marrow cells was increased in nutritional supplements groups. The nutritional supplements also affected the recovery of livers and spleens of AA mice. CONCLUSION: Specific nutritional supplements accelerated rehabilitation of AA mice and can be used as nutritional support in the treatment of AA.

Effect of Tectona grandis on phenylhydrazine-induced anaemia in rats.

Traditional oral report indicates that Tectona grandis is used in the treatment of anaemia in Togo. For this purpose, the extract of T. grandis leaves is evaluated on anaemia model of rat induced by intraperitoneal injection of phenylhydrazine at 40 mg/kg for 2 days. Oral administration of T. grandis extract at 1 g/kg/day and 2 g/kg/day, to the rats previously treated with phenylhydrazine, increased the concentration of haemoglobin, red blood cells number, haematocrit and reticulocytes rate. Moreover, the extract of T. grandis enhanced the osmotic resistance of the red blood cells that confirm the important presence of young red blood cells. These results support partially the traditional use of T. grandis in the treatment of anaemia.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards.

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

The influence of Sacoglottis gabonensis stem bark extract and its isolate bergenin, Nigerian alcoholic beverage additives, on the metabolic and haematological side effects of 2,4-dinitrophenyl hydrazine-induced tissue damage.

This study was designed to study the influence of Sacoglottis gabonensis stem bark extract on the metabolic and cytotoxic side effects of 2,4-dinitrophenyl hydrazine (2,4-DNPH) on the brain and blood using male weaving rats as the experimental model. This was after the effect of the bark extract and bergenin, its isolate, on membrane lipid peroxidation and tissue natural antioxidant defences was reported. Lipid peroxidation was induced experimentally with a single intraperitoneal phenylhydrazine (2,4-DNPH) administration at the end of 3 days exposure to the bark extract or bergenin in drinking water. Three hours later, the brain, liver and red blood cells of the experimental animals were analysed for glucose level and the blood was analysed for selected key indices of oxidative stress: red blood cell (RBC) count haemoglobin (Hb), packed cell volume (PCV) and white blood cell (WBC) count (total and differential). The bark extract exhibited a protective action on brain glucose, significantly inhibiting the glucose-depleting action of both 2,4-DNPH and ethanol. It also inhibited the lowering action of DNPH and ethanol on PCV, RBC and Hb concentration of rat blood, but inhibited proliferation of white blood cells (total and differential). The data on the effect of bergenin, on the side effects of 2,4-DNPH experimental lipid peroxidation and on ethanol followed an essentially similar trend to those of the bark extract on brain glucose. Bergenin, similar to the bark extract, exerted a protective action on the brain tissue, though to a lesser extent, against the oxidants, 2,4-DNPH and ethanol. It is evident that aqueous ethanol extract of S. gabonensis stem bark has biological antioxidant properties against 2,4-DNPH and ethanol-induced tissue damage exerting its action on the haematological and metabolic side effects of the oxidants. By virtue of its essentially similar activity under the same conditions, bergenin appears to be the phytochemical constituent that is largely responsible for the observed action of the bark extract.

Antianaemic properties of ayurvedic drugs, raktavardhak, punarnavasav and navayas louh in albino rats during phenylhydrazine induced haemolytic anaemia.

Single injection of phenylhydrazine[PH] reduced the number of RBC and haemoglobin content; decreased myeloid; erythroid cell ratio in bone marrow and increased Cathepsin D activity in spleen of rats. Ayurvedic drugs raktavardhak, punarnavasav and navayas louh recovered the number of RBC and haemoglobin content and raised myeloid: erythroid cell ratio and normalised cathepsin D activities by counteracting the action phenyl hydrazine. The results confirm the claims of ayurveda that these drugs possess the potency to cure anaemia through protection of RBCs from haemolysis and simultaneously lowering cathepsin D activities from the spleen.

Inadequate treatment of excessive erythrocytosis / Tratamiento inadecuado en la eritrocitosis excesiva

Los pacientes con el mal de montaña crónico o eritrocitosis excesiva (EE) son residentes de la altura (3600 m), con mayor o igual 6.5 x 10 a la sexta glóbulos rojos (GR) que presentan cianosis.Esto ocasiona problemas estéticos y psicológicos en su vida ya que las demás personas creen que son alcohólicos. Cuando hay aumento de los GR, ellos buscan una cura milagrosa. De acuerdo a los conceptos evolutivos de la EE, los tratamientos han incluído; sanguijuelas, radioterapia de la médula ósea mediante administración de substancias radiactivas como el fósforo, y más recientemente, flebotomías, infusiuones de té, tabletas de ajo y la más peligrosa la administración de la fenilhidrazina, agente citotóxico prohibido. Encontramos que la mayoría de los pacientes con EE tienen placas radiográficas de tórax anormales. El concepto de los tratamientos es el de disminuir los GR. Sin embargo, la fenilhidrazina es tóxica para la médula ósea, el hígado y otros tejidos, cambiando el color de la piel de cianótica a icterica. Las conjuntivas se tornan ictéricas y la harina de café oscura. Una vez iniciado el tratamiento, la sangre de los pacientes es analizada periódicamente y el recuento de GR disminuyue, con lo que quedan satisfechos. Sin embargo, este medicamento tóxico puede producir la muerte. Al reducir los GR, el contenido arterial de oxígeno (CaO2) en la sangre disminuye. Las pruebas ergométricas en estos pacientes durante el tratamiento producen gran débito de oxígeno. En el paciente descrito, en el 4to nivel del protocolo de Bruce,m el dolor intenso de ambasd pantorrillas se hizo intolerable y requirió oxígeno post ejercicio. Al interrumpirse la fenilhidrazina, el, CaO2 retorna a niveles normales en aproximadamente 60 días, con una elevación de los GR por encima de los valores iniciales, y mejoría de la capacidad de ejercicio. Este y muchos otros casos nos llevan a creer que la EE es un mecanismo de compensación de la enfermedad pulmonar en la altura y que la cantidad de GR no debe ser disminuída.

[Quantities of agaritine in mushrooms (Agaricus bisporus) and the carcinogenicity of mushroom methanol extracts on the mouse bladder epithelium].

Consumption of the Agaricus species mushrooms has increased considerably in Japan as the Japanese have become accustomed to Western cooking. The Agaricus species mushroom contains hydrazine derivatives known as Agaritine. Bladder implantation was performed to test the carcinogenic potential of the Agaricus species mushroom which contains large quantities of Agaritine. The results are summarized as follows: 1) Agaritine was detected in fresh Agaricus bisporus mushrooms at the level of 228.2 micrograms/wet weight and in Shiitake (Agaricus edodes) mushrooms at 0.82 microgram/g wet weight, but was not detected in either canned Agaricus bisporus mushrooms or Matsutake (Armillaria edodes) mushrooms. 2) The observed rates of mouse urinary bladder carcinoma were 30.8% for fresh mushrooms (Agaricus bisporus), 23.5% for fresh Shiitake (Agaricus edodes), 9.8% for dry Shiitake, 50% for synthesized Agaritine and 5.4% for paraffin wax as a control. The methanol extract of fresh mushrooms (Agaricus bisporus) and synthesized Agaritine were found to be significantly carcinogenic on the mouse bladder epithelium by the bladder implantation test with a probability of less than 0.01. 3) A large quantity of Agaritine was detected in fresh mushrooms (Agaricus bisporus), but decreased after boiling the mushrooms in water at 100 degrees C for 10 min. 4) The methanol extract of fresh mushrooms (Agaricus bisporus) and synthesized Agaritine are suggested to be the agents for producing cancer in the bladder epithelium. 5) Adding heat to mushrooms containing Agaritine before cooking contributes to the prevention of any potential Agaritine hazard which may induce carcinogenic changes in the bladder epithelium.

Screening of toxic-haemolytic anaemia in laboratory rats: a model of phenylhydrazine-induced haemolysis.

An increase of the plasma haemoglobin level was found to be a reliable characteristic of toxic-haemolytic anaemia induced by a single dose of phenylhydrazine in laboratory rats. Very high doses of the substance increased the changes in many other characteristics to an extent that was up to now observed only for haemolytic events.