Oxidative Stress and DNA Damage Effect of Dioscorea hispida Dennst. on Placental Tissues of Rats.

Dioscorea hispida Dennst. locally known as "ubi gadung" has been used as a traditional remedy and source of carbohydrate among Malaysians. To assess the effect of Dioscorea hispida aqueous extract (DHAE) on the production of reactive oxygen species (ROS) and their effects on DNA damage in Sprague Dawley rat's placental tissues, pregnant rats were randomly divided into four groups. The animals were orally treated with distilled water (negative control) and three different concentrations of DHAE (250, 500 and 1000 mg/kg body weight (BW)) from gestation day 6 until 20. The oxidative stress in placental tissues was evaluated at day 21 by measuring the level of ROS, superoxide dismutase (SOD) and lipid peroxidation biomarker, malondialdehyde (MDA) while comet assay was used for DNA damage. There was no significant production of ROS and SOD activities in all groups. Significant changes were observed in the MDA level at 1000 mg/kg BW DHAE. Comet assay revealed a significant increase (p < 0.05) of DNA damage on animals treated with 250 and 500 mg/kg BW DHAE but not at the highest concentration. It was postulated that the placental cells could have undergone necrosis which destroys all components including DNA. This occurrence simultaneously reduces the levels of DNA damage which can be represented by lower level of tail moments. This finding correlates with our histopathological examination where necrotic cells of spongiotrophoblast were observed in the basal zone of placental tissue. The high amount of hydrogen cyanide and other compounds in 1000 mg/kg BW DHAE could elevate the lipid peroxidation and directly induce cell necrosis which requires further investigation.

A 13-week subchronic toxicity study of a Dioscorea Rhizome water extract in rats.

Dioscorea Rhizome is widely used as a traditional herbal medicine to treat asthma, diarrhea, cough, bronchitis, spermatorrhea, leukorrhea, and rheumatoid arthritis. This study investigated the potential subchronic toxicity of a D. Rhizome water extract (DRWE) after repeated oral administration at 0, 800, 2000, and 5000 mg/kg/day in rats for 13 weeks. During the study period, clinical signs, mortality, body weight, food consumption, water consumption, urinalysis, ophthalmoscopy, hematology, serum biochemistry, gross pathology, organ weights, and histopathology were examined. The 13-week repeated oral administration of DRWE to rats resulted in an increased incidence of zona glomerulosa hypertrophy and hyperplasia in the adrenal gland at dose levels of ≥2000 mg/kg/day in both sexes. However, these findings are considered as non-adverse adaptive changes because of minimal histological changes in the lesions, which were not accompanied by any corresponding alterations in serum electrolytes and adrenal gland weight. No treatment-related adverse effects on clinical signs, body weight, food and water consumption, ophthalmic examination, urinalysis, hematology, serum biochemistry, necropsy findings, and organ weights were observed at any dose tested. Under the present experimental conditions, the no-observed-adverse-effect level of the DRWE was considered to be 5000 mg/kg/day in both sexes, and no target organs were identified.

The crucial role of metabolic regulation in differential hepatotoxicity induced by furanoids in Dioscorea bulbifera.

Diterpenoid lactones (DLs), a group of furan-containing compounds found in Dioscorea bulbifera L. (DB), have been reported to be associated with hepatotoxicity. Different hepatotoxicities of these DLs have been observed in vitro, but reasonable explanations for the differential hepatotoxicity have not been provided. Herein, the present study aimed to confirm the potential factors that contribute to varied hepatotoxicity of four representative DLs (diosbulbins A, B, C, F). In vitro toxic effects were evaluated in various cell models and the interactions between DLs and CYP3A4 at the atomic level were simulated by molecular docking. Results showed that DLs exhibited varied cytotoxicities, and that CYP3A4 played a modulatory role in this process. Moreover, structural variation may cause different affinities between DLs and CYP3A4, which was positively correlated with the observation of cytotoxicity. In addition, analysis of the glutathione (GSH) conjugates indicated that reactive intermediates were formed by metabolic oxidation that occurred on the furan moiety of DLs, whereas, GSH consumption analysis reflected the consistency between the reactive metabolites and the hepatotoxicity. Collectively, our findings illustrated that the metabolic regulation played a crucial role in generating the varied hepatotoxicity of DLs.

Serum microRNA-122-3p, microRNA-194-5p and microRNA-5099 are potential toxicological biomarkers for the hepatotoxicity induced by Airpotato yam.

Airpotato yam (the rhizome of Dioscorea bulbifera L.) is traditionally used to treat thyroid disease and various cancers in China. However, it was found to cause hepatotoxicity during clinical practice. This study aims to identify candidate serum microRNAs (miRNAs) as diagnostic biomarkers for the liver injury induced by Airpotato yam. The results of serum alanine/aspartate aminotransferase (ALT/AST) showed the remarkable hepatotoxicity induced by ethyl acetate fraction of Airpotato yam (EF) (450mg/kg) and diosbulbin B (DB) (300mg/kg) in mice. The results of miRNAs chip analysis showed that the expression of 28 and 37 serum miRNAs was obviously altered in EF- and DB-treated mice, respectively. Among these miRNAs, miRNA-122-3p, miR-194-3p and miR-5099 have passed the further validation in serum from both EF- and DB-treated mice. Moreover, the expression of miRNA-122-3p and miRNA-194-5p was significantly increased in EF (375mg/kg)-treated mice with no significant elevation of serum ALT/AST activity. Only the expression of serum miRNA-5099 was not altered in the liver injury induced by acetaminophen (APAP), monocrotaline (MCT) or toosendanin (TSN). In conclusion, this study demonstrated that miR-122-3p and miRNA-194-5p were two sensitive biomarkers, and miR-5099 might be a specific biomarker for reflecting the liver injury induced by Airpotato yam.

[Synergistic protective effect of Scutellariae Radix with Phellodendri Chinensis Cortex on Dioscoreae Bulibferae Rhizoma liver toxicity in rats].

Dioscoreae Bulibferae Rhizoma (RDB) is commonly used in clinical Chinese medicine. It has been used in many kinds of traditional Chinese medicine (TCM), but the toxicity of RDB, easily leads to hepatotoxicity. The objective of the present study is to investigate the synergistic protective effect of Scutellariae Radix (SR) with Phellodendri Chinensis Cortex (PCC) on RDB caused liver toxicity in rats. SD female rats were adopted to establish the hepatotoxicity models by RDB (9 g•kg⁻¹, ig) once daily for 28 consecutive days. After 28 days, liver histological changes were observed, and the activity of transaminase and antioxidant enzymes was evaluated. Morphological and biochemical indicators evaluation showed that, Dioscoreae Bulibferae Rhizoma-induced hepatotoxicity models were successful, and the liver cells were dissolved and swelling with fatty degeneration； inflammatory cells were present in gaps; local punctiformed or lamellar hydropic degeneration was found in liver tissues, with partial necrosis. Indexes of liver function (ALT, AST and ALP) were significantly increased (P<0.05 or P<0.01). The combination of SR and PCC has protective effect on RDB-induced hepatotoxicity in rats. SR+PCC exerted the strongest protective effects against RDB-induced hepatotoxicity. SR, PCC, and SB+CP were observed to exhibit hepatoprotective effect as demonstrated by significant decrease in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) (P<0.05 or P<0.01), and MAD level in liver tissue (P<0.001), significant increase in GSH content in liver tissue (P<0.001), and significant improvement in his to pathologic changes of liver tissues in rats. SR, PCC and their combinations could achieve liver protection effect by reducing ALT, ALP and AST level in serum, increasing GSH level and anti-oxidantability of liver tissues, and reducing hepatic tissue cells injury.

[Protection of Herba Scutellariae Barbatae against hepatotoxicity induced by Rhizoma Dioscoreae Bulbiferae and its mechanism].

This study was conducted to test the protective activity of ethanol extract of Herba Scutellariae Barbatae(SE) against hepatotoxicity induced by Rhizoma Dioscoreae Bulbiferae in mice and its mechanism. SE was orally given to mice at various doses, and ethyl acetate fraction of Rhizoma Dioscoreae Bulbiferae(EF, 450 mg·kg(-1)) was also orally given at the same time. After 11 days, serum levels of alanine/aspartate aminotransferase(ALT/AST), alkaline phosphatase(ALP), total protein(TP) and albumin(ALB) were measured, and liver histological examination was conducted. Liver glutathione(GSH) amount, myeloperoxidase(MPO) activity and serum tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interferon-γ(IFN-γ) levels were measured. The expression of heme oxygenase-1(HO-1), inhibitor of kappa B(IκB) and nuclear factor κB(NF-κB) p65 were determined by Western blot. The results showed that SE(200 mg·kg-1) reversed EF-induced changes of serum ALT, AST, ALP, TP and ALB. Liver histology also suggests the protection of SE against EF-induced liver injury. SE reduced the increased MPO activity in liver and TNF-α, IL-6, IFN-γ contents in serum, and blocked the decrease in IκB expression and subsequent increase in phosphorylation and nuclear translocation of p65 induced by EF. EF increased liver GSH amount and heme oxygenase-1(HO-1) protein expression in mice. SE increased liver GSH amount, but decreased the expression of HO-1. All those results suggest that SE alleviates liver injury induced by consecutive administration of EF by alleviating inflammatory injury via inhibiting NF-κB signaling pathway and elevating antioxidant capacity.

Renal and Hepatic Function in Hypercholesterolemic Rats Fed Jamaican Bitter Yam (Dioscorea polygonoides).

BACKGROUND: We reported that Jamaican bitter yam (Dioscorea polygonoides) has antilipemic potential in rats; however there is limited data on the toxicological profile of the yam. We therefore investigated the effects of bitter yam consumption for 6 or 12 weeks on renal and hepatic function in rats fed a high (4%) cholesterol diet. METHODS: Twenty four rats were divided into six groups (n = 4); three of which were used for each investigation (6 or 12 weeks). One group was administered 4% cholesterol diet, while the yam group had the cholesterol diet supplemented with 5% bitter yam. The control group was fed standard rat chow. Liver and kidney function tests were performed on serum, liver and kidney. Histological studies were conducted on liver samples. Acute toxicity tests were performed in rats and mice administered a single high dose of bitter yam (10 g/kg). RESULTS: Activities of liver and kidney AST and ALT differed (p ≤ .02) between control rats and those fed cholesterol with bitter yam for 12 weeks. Albumin to globulin ratio was reduced (p = .03) in rats fed cholesterol with bitter yam for 6 weeks as compared to the control group. Serum urea concentration was higher (p < .05) in rats fed bitter yam as compared to normal chow for 6 weeks. The cholesterol diet caused extensive fat deposition in liver cells; however this was inhibited by co-administration of bitter yam. CONCLUSION: Long-term administration of Jamaican bitter yam may induce slight changes in renal and hepatic functions.

Cytokines as potential biomarkers of liver toxicity induced by Dioscorea bulbifera L.

The present study is designed to search for the serum cytokine biomarker for liver injury induced by Dioscorea bulbifera L., which is a traditionally used herbal medicine in China. Mice were orally given various doses of ethyl acetate extract (EF) isolated from D. bulbifera for 12 days. The activity of serum alanine/aspartate transaminases (ALT/AST) was increased in EF (400 mg/kg)-treated mice. Histological assessment further confirmed EF (400 mg/kg)-induced liver injury. Results of a cytokine-antibody array demonstrated that there were 10 cytokines up-regulated and 1 cytokine down-regulated in EF (400 mg/kg)-treated mice. Enzyme-linked immunosorbent assay (ELISA) further confirmed the increased level of CD30 ligand (CD30L) and decreased level of interlukin-3 (IL-3) in EF-treated mice. In conclusion, our results demonstrate that the altered expression of CD30L and IL-3 may be potential biomarkers for hepatotoxicity induced by D. bulbifera.

[Protection of Grateloupia filicina polysaccharide against hepatotoxicity induced by Dioscorea bulbifera L].

The present study was designed to observe the protection of Grateloupia filicina polysaccharide (GFP) against hepatotoxicity induced by Dioscorea bulbifera L in mice and its underlying mechanism. GFP was intragastrically (ig) given to mice at various doses. After 6 days, the mice were treated with ethyl acetate extract of Dioscorea bulbifera L (EF, ig). Serum levels of alanine/aspartate aminotransferase (ALT/AST), alkaline phosphatase (ALP), total bilirubin (TB) were measured, and liver histological evaluation was conducted. Furthermore, reductions of liver glutathione (GSH) amount and glutamate cysteine ligase (GCL) activity were tested. The expressions of GCL-c, GCL-m, and HO-1 (heme oxygenase-1) in liver were observed by Western-blot. The results showed that GFP (600 mg x kg(-1)) decreased EF-induced the increase of serum ALT, AST and TB, and GFP (400, 600 mg x kg(-1)) inhibited EF-induced the increase of serum ALP. Liver histological evaluation showed that the liver injury induced by EF was relieved after treated with GFP. GFP further increased liver GSH amount and reversed EF-induced the decrease of GCL activity. The Western-blot result showed that GFP augmented EF-induced the increase of HO-1, and reversed EF-induced the decrease of GCL-c. In conclusion, GFP can act against the oxidative stress liver injury induced by Dioscorea bulbifera L in mice.

[Evaluation on hepatotoxicity caused by Dioscorea bulbifera based on analysis of bile acids].

Metabolic profile of bile acids was used to evaluate hepatotoxicity of mice caused by ethanol extraction of Dioscorea bulbifera L. (ethanol extraction, ET) and diosbulbin B (DB), separately. Ultra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) was applied to determine the contents of all kinds of endogenous bile acids including free bile acids, taurine conjugates and glycine conjugates. Obvious liver injuries could be observed in mice after administrated with ET and DB. Based on the analysis using principle components analysis (PCA), toxic groups could be distinguished from their control groups, which suggested that the variance of the contents of bile acids could evaluate hepatotoxicity caused by ET and DB. Meanwhile, ET and DB toxic groups were classified in the same trends comparing to control groups in the loading plot, and difference between the two toxic groups could also be observed. DB proved to be one of the toxic components in Dioscorea bulbifera L. Bile acids of tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), cholic acid (CA) and others proved to be important corresponds to ET and DB induced liver injury according to analysis of partial least square-discriminant analysis (PLS-DA) and the statistical analysis showed that there were significant differences between the control groups and toxic groups (P < 0.01). Furthermore, good correlation could be revealed between the foregoing bile acids and ALT, AST. It indicated that taurine conjugated bile acids as TUDCA, TCDCA, TCA and TDCA along with CA could be considered as sensitive biomarkers of ET and DB induced liver injury. This work can provide the base for the further research on the evaluation and mechanism of hepatotoxicity caused by Dioscorea bulbifera L.

Bitterness and toxicity in wild yam (Dioscorea spp.) tubers of Nepal.

Wild yams make a significant contribution to diets of tribal people in Nepal. However, these wild tubers are unpalatable, taste bitter, produce inflammation and show occasional toxicity. Four wild yam species, which are eaten after primary treatment by Nepali aborigines, were analyzed for bitter and toxic principles. Bitter components were identified as furanoid norditerpenes (diosbulbins A and B). Diosbulbins A and B were found in the range of 0.023-0.046 and 0.151-0.442 g kg(-1), respectively. Results demonstrated that diosbulbin B, with an average value of 0.314 g kg(-1), was the principal bitter compound as compared to diosbulbin A (0.037 g kg(-1)). The toxic alkaloid, dioscorine and histamine (an allergen) were not detected in these tubers, whereas cyanogens (as HCN equivalent) content were found ranging from 3.2 to 6.0 ppm. Our results revealed that Nepali wild yam tubers are not toxic varieties, as they do not contain either toxic dioscorine or histamine and cyanogens contents were satisfactorily below the safety limits. The inflammation and occasional toxicity observed could possibly be due to the presence of high level of oxalate in these tubers. Domestic cooking methods were found to be very efficient in removing bitterness, thus making the bitter yams palatable.

Hypoglycemic effects of steroidal sapogenins isolated from Jamaican bitter yam, Dioscorea polygonoides.

In this study, three steroidal sapogenins (Delta3 diosgenin, diosgenin, and pennogenin) and the phytosterols, stigmasterol and beta-sitosterol were isolated from Jamaican bitter yam, Dioscorea polygonoides. Their effects on fasting blood glucose and intestinal amylase and ATPases in streptozotocin-induced diabetic rats were studied. The diabetic rats (fed supplemented and unsupplemented diets) lost weight significantly compared to the normal group. There was a significant increase in the activity of alpha-amylase in the proximal region of the small intestinal mucosa of diabetic rats fed sapogenin extract or commercial diosgenin. However, this did not result in increased fasting blood glucose. Instead, supplementation of the diet with bitter yam sapogenin extract significantly decreased fasting blood glucose compared to the diabetic group. Supplementation of the diet with bitter yam sapogenin extract or commercial diosgenin significantly reduced Na+-K+-ATPase activity in all three regions compared to the diabetic control group. Commercial diosgenin supplementation resulted in a significant increase in Ca2+ ATPase activity in proximal region compared to the diabetic control and bitter yam sapogenin extract groups. The effect of bitter yam sapogenin extract or commercial diosgenin on intestinal Na+-K+-ATPase activity could account for their hypoglycemic properties. However, there was adverse effect on the body weight.

Final report of the amended safety assessment of Dioscorea Villosa (Wild Yam) root extract.

Dioscorea Villosa (Wild Yam) Root Extract is an extract of the rhizomes of the wild yam, D. villosa. A manufacturing process was described in which cut up and ground rhizomes are combined with an eluant (e.g., oleyl alcohol), the plant material precipitated with addition of a miscible solvent, washed, and redissolved in the original eluant. The extract contains glycoside and steroidal saponins (< or =0.4%), diosgenin (< or =3.5%), alkaloids, tannins, phytosterols, and starch. Levels of heavy metals, 1,4-dioxane, chloroform, methylene chloride, trichloroethylene, and benzene are reported to be below limits of detection. Although only one use was reported to the U.S. Food and Drug Administration (in a body and hand preparation), industry reported uses in body and hand creams, lotions, powders, and sprays at a concentration of 0.00001% (equivalent to 0.000002% plant solids), and in moisturizing creams, lotions, powders, and sprays at concentrations up to 15% (equivalent to 0.5% plant solids). Preparations fromD. villosaare used in herbal medicine for treatment of a variety of ailments and by the pharmaceutical industry in the preparation of steroids. Using Dioscorea Villosa (Wild Yam) Root Extract prepared via a specified process, it is possible to produce a stable extract with a narrow range of diosgenin content. The extract produced using this methodology was tested in acute and short-term toxicity tests, dermal irritation tests, a sensitization test, an ocular irritation test, a rat uterotropic assay, and genotoxicity tests. An acute oral toxicity test produced hypoactivity, piloerection, and dyspnea and a death in 1 of 10 rats at 2 g/kg using the specified extract, but no toxicity in rats given 0.5 g/kg. A dermal toxicity test using the specified extract demonstrated no acute toxicity in rats. Both a 7-day local tolerance test and a 28-day dermal toxicity test in rats produced no significant adverse effects at the maximum tested concentration of 10%. A single application of undiluted extract to the intact and abraded skin of rabbits produced sufficient irritation for the test material to be rated"irritant,"but a 10% dilution was not irritating. Undiluted extract was only mildly irritating to the conjuctiva of the rabbit eye; irritation in the iris and cornea was mild and transient. Undiluted extract was not irritating during the induction phase of a guinea pig sensitization study, nor did challenge with a 25% dilution elicit any sensitization. The specified extract at concentrations up to 500 mg/kg/day did not have any estrogenic activity in the juvenile rat uterotrophic assay. Genotoxicity assays in bacterial and mammalian systems were negative, except that Ames test strain TA 1537 was positive at one dose level using the plate incorporation method, but not using a preincubation method. Although the concentration at which the actual plant extract is used in cosmetic products is low, one of the primary safety concerns with this plant extract is the possible metabolic/endocrine activity, e.g., estrogen-like or progesterone-like activity as a result of the presence of small amounts of plant phytosterols such as diosgenin. Extracts prepared as described in this safety assessment, with an upper limit of 3.5% diosgenin, did not have any estrogenic activity, demonstrating that it is possible to produce material that does not present this specific safety concern. Although extracts from pesticide-free plants were not considered genotoxic and it was the view of the Cosmetic Ingredient Review (CIR) Expert Panel that there do not appear to be any components that could be carcinogenic, pesticide residues could raise this issue. It was urged that manufacturers limit pesticide residues to the limit previously used for lanolin of not more than 40 ppm (with not more than 10 ppm for any one residue). Based on these data, it was concluded that Dioscorea Villosa (Wild Yam) Root Extract is safe as used in cosmetic formulations. This conclusion regarding safety, however, is valid only for extracts prepared in a manner that produces a similar chemical profile as that described in this report, particularly as regards diosgenin. Extracts not prepared in a manner that produces a similar chemical profile would be considered safe if they have a similar safety test profile.

[Experimental pathological study of subacute intoxication by Dioscorea bulbifera L].

OBJECTIVE: To study the pathological change and the toxic mechanism of Dioscorea bulbifera L in mice. METHODS: Sixty ICR mice were randomly assigned to four groups poisoned respectively with 200% Dioscorea bulbifera L of 1/4 LD50, 1/10LD50, 1/30LD50 and a control group treated with distilled water by oral administration. All animals were pathologically examined with LM and some of them were examined with TEM when the mice died during the experiment or the survival mice were sacrificed after thirty days. RESULTS: The pathological changes showed fatty change and the increasing glycogen of liver cells; degeneration and necrosis of the epithelia of uriniferous tubules. The serum BUN and ALT of the experimental groups mice were higher than that of control group. Enzyme histochemical staining showed the decreasing activity of G-6-P and SDH in the liver cells in the experimental groups. CONCLUSION: The experiment suggests that the target organs were liver and kidney. The toxic mechanism of Dioscorea bublifera L was the damage of the mitochondrional and endoplasmic reticulum membrane directly. As a result, the activity of the SDH and G-6-P decreased, the metabolism was affected.

Experimental pathological study of subacute intoxication by Dioscorea bulbifera L / 法医学杂志

OBJECTIVE@#To study the pathological change and the toxic mechanism of Dioscorea bulbifera L in mice.@*METHODS@#Sixty ICR mice were randomly assigned to four groups poisoned respectively with 200% Dioscorea bulbifera L of 1/4 LD50, 1/10LD50, 1/30LD50 and a control group treated with distilled water by oral administration. All animals were pathologically examined with LM and some of them were examined with TEM when the mice died during the experiment or the survival mice were sacrificed after thirty days.@*RESULTS@#The pathological changes showed fatty change and the increasing glycogen of liver cells; degeneration and necrosis of the epithelia of uriniferous tubules. The serum BUN and ALT of the experimental groups mice were higher than that of control group. Enzyme histochemical staining showed the decreasing activity of G-6-P and SDH in the liver cells in the experimental groups.@*CONCLUSION@#The experiment suggests that the target organs were liver and kidney. The toxic mechanism of Dioscorea bublifera L was the damage of the mitochondrional and endoplasmic reticulum membrane directly. As a result, the activity of the SDH and G-6-P decreased, the metabolism was affected.