Ahmed et al. SpringerPlus (2015) 4:315 DOI 10.1186/s40064-015-1059-7

RESEARCH Open Access Antidiabetic, antioxidant, antihyperlipidemic effect of extract of Euryale ferox salisb. with enhanced histopathology of pancreas, liver and kidney in streptozotocin induced diabetic rats Danish Ahmed1*, Vikas Kumar1, Amita Verma1, Girija Shankar Shukla3 and Manju Sharma2,4*

Abstract Background: Ethanolic extract of Euryale ferox salisb. (EFx) may have an effect on the activity of hepatic antioxidant enzymes, glycemic control and lipid profile and histopathology of pancreas, liver and kidney of streptozotocin (STZ)— induced diabetic wistar rats. Methods: Wistar albino rats were divided into eight groups viz. non-diabetic (normal control), diabetic control (STZ- induced), diabetic treated (infused with different doses of Euryale ferox. Salisb. ethanolic extract) and diabetic conven- tional treated (treated with Glibenclamide). Diabetes was induced by administering streptozotocin (60 mg/kg body weight) intraperitoneal (i.p). The ethanolic extract was supplemented in different doses through oral route. Biochemi- cal investigations were carried out according to previously reported methods. Histopathological examinations were done accordingly. Results: The EFx supplemented diabetic rats significantly (p < 0.001) decreased the blood glucose level in a dose dependent manner. Plasma insulin level was significantly increased in EFx treated rats. The hepatic gluconeogenic enzymes activities were restored to normal in EFx treated rats. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH) were significantly increased (p < 0.001) among EFx treated rats. Lipid profile was reinstated to nearly normal level among EFx treated rats. Histopathological investi- gations revealed that microscopic architecture of pancreatic, hepatic and renal cells improvised in EFx treated diabetic rats. Conclusion: EFx supplement could improve the glycemic control as well as lipid profile in diabetic rats along with improvised antioxidant enzymes which has beneficial effect in preventing the diabetic complications by scavenging the free radicals in diabetic rats. Keywords: Euryale ferox salisb. diabetes, Hyperlipidemia, Oxidative stress

*Correspondence: [email protected]; [email protected] 1 Department of Pharmaceutical Sciences, Faculty of Health Sciences, Sam Higginbottom Institute of Agriculture, Technology and Sciences (SHIATS)-Deemed University, Allahabad, India 2 Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India Full list of author information is available at the end of the article

© 2015 Ahmed et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Ahmed et al. SpringerPlus (2015) 4:315 Page 2 of 17

Background (Moncada et al. 1991). It is well known that the activity There are several evidences that demonstrate that dia- of free radical scavenging compounds i.e. antioxidant betic complications are closely associated with oxidative enzymes is very low in the islet of langerhans of pancreas stress persuaded by free radicals generation (Pitkänen as compared to the other tissues (Gandy et al. 1981). On et al. 1992) Type-2 diabetes mellitus (T2DM) is a multi- the basis of antioxidant activity of the E. Ferox salisb. functional disease which is associated with hypergly- seeds, we aimed to investigate the plausible role of EFx cemia and lipoprotein abnormalities (Ugochukwu and on STZ-induced diabetic rats. Babady 2002). Damage to the cell, in turn results in the Thus, the present study was carried out to investigate enhancement of reactive oxygen species (ROS) produc- the effect of ethanolic extract of E. Ferox salisb. on the tion. Abnormally high level of ROS has been found to carbohydrate metabolism, glycemic control, insulin level, play an important role in causation of the development hepatic gluconeogenic enzymes, renal function param- and pathogenesis of T2DM. Free radicals easily formed eters and antioxidant enzymes level in conjunction with during diabetic state, serves an important role in the gen- the pathological corrections in architecture of pancreas, esis of diabetic complications (Ahmed et al. 2014a). liver and kidney. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and Methods reduced glutathione (GSH) provides an important assis- Experimental animals tance in scavenging the free radicals by breaking down Healthy adult male albino Wistar rats, bred and reared the ROS. Prior researches have reported that a defect at Indian Institute of Toxicological Research (IITR), in the ROS scavenging machinery in T2DM patients as Lucknow were used for purpose of the experimenta- compared to the normal control (Ahmed et al. 2013). tion. Weight-matched animals (140–160 g) were selected Disturbances in the lipid profile is so high that diabe- and housed in polypropylene cages layered with husk tes has been called “more a disease of lipid than carbo- and kept in a semi-natural light/dark condition (12 h hydrate metabolism” (Rawi et al. 1998). Levels of total light/12 h dark). The animals were allowed free access cholesterol (TC), triglycerides (TG), low density lipopro- to water and standard pellet diet (Amrut Feeds, Pune, tein cholesterol (LDL-c) were increased to a momentous India). level in T2DM. In contrast the level of high density lipo- protein cholesterol (HDL-c) was significantly reduced in Chemicals, reagents and biochemical estimation kits T2DM (Ahmed et al. 2014b; Kumar et al. 2013a, b). Hexokinase, glucose-6-phosphate dehydrogenase, glu- Due to the competence of antioxidants to assuage oxi- cose-6-phosphatase, fructose-1-6-bisphosphate were dative stress in cells and its contribution in helping to purchased from Sigma Aldrich (India). Blood glucose, prevent diabetic complications, the chase for antioxi- lipid profile, and antioxidant enzymes kits were pur- dants in the past decades has attracted much interest and chased from Span Diagnostics, Surat (India). Streptozo- many plants have been discovered to have great antioxi- tocin was purchased from Sigma Aldrich (India). Other dant potential. Euryale ferox salisb. is an aquatic plant needed chemicals used in the study were procured either that belongs to family nymphaeaceae. It is also known from Sigma Aldrich (India) or CDH Mumbai (India). as fox nut or gorgon nut. It is mainly distributed in East Asia to China to North and North East India. Eurylae Induction of diabetes ferox salisb. produces starchy white seeds which are edi- Wistar rats were injected intraperitoneally with STZ dis- ble. In India, the processed white seeds of Euryale ferox solved in 0.1 M citrate buffer (pH = 6.5) at 60 mg/kg. salisb. is known as Makhana. Leaves of E.Ferox salisb. Animals of control group received equal volume of vehi- are used in condition of difficult parturition (Duke and cle. After 48 h of STZ injection, blood glucose level of the Ayensu 1985). Further, E.Ferox salisb. has also been used diabetes induced rats was estimated. The rats depicting as traditional oriental medicine to treat various ailments values ranging FBG ≥ 230 mg/dL, were considered to be including kidney failure, diarrhea and spleen dysfunc- diabetic. tion. Recent research studies indicate that E. Ferox salisb, seeds could reduce the myocardial ischemic reperfusion Preparation of extracts injury (Das et al. 2006). One of the recent study explores Extract of Euryale ferox salisb. seeds was prepared the antioxidant activities of the isolated compounds from according to guideline of Lee et al. (Lee et al. 2002). Dried E. Ferox Salisb. seeds (Song et al. 2011). The mechanism powder (100 g) of E. Ferox salisb. seeds was placed in a of action of STZ to induce diabetes is its selective toxicity conical flask with 70% ethanol and extracted at 80°C for to pancreatic β-cells via generation of NO free radicals. 3 h. The filtration of the extract was performed along Ahmed et al. SpringerPlus (2015) 4:315 Page 3 of 17

with the concentration of the extract by rotary evaporator for 15 days, respectively. Group 7 was treated with insulin (Buchi, India) under low pressure. The resultant residue (3-IU/kg) for 15 days. Blood samples were collected peri- was freeze dried and stored at −70°C. After freeze dry- odically. At the end of the repeated administration of the ing the whole extract was eluted with n-hexane, dichlo- drug, the collected blood samples were analyzed for any romethane, ethyl acetate and n-butanol in a stepwise toxic effect. Different doses of extract did not show any manner. After the sequential screening of the extract, the significant difference in the food consumption and body resultant fractions were collected and the remaining sol- weight when compared to the normal rats. There was no vents were removed by rotary evaporation. The powder lethality and toxicity found with any dose till the comple- (dried) was dissolved in dimethyl sulfoxide (DMSO) and tion of the experiment. Therefore, the conclusion was that diluted with phosphate buffered saline (pH = 7.4). The 400 mg/kg body weight was safe to use. percentage yield of the crude extract was calculated to be 3.17%. Experimental design A total of 35 male albino wistar rats were utilized and Administration of plant extracts were randomly divided into 7 groups of 5 animals in each The extract of the parts of Euryale Ferox Salisb. (EFx) was group: administered to the rats through force-feeding gastric gavage route. This was executed by inserting an infant Group I—Normal rats (untreated with dimethylsulfox- oral feeding tube, which was connected to a syringe con- ide, [DMSO]. taining the extract, into the gastric region of the rat. The Group-II—Diabetic control (administered with Strep- animals were fasted 30 min before and after the treat- tozotocin (STZ). ment to ensure maximum bioavailability (Sridhar et al. Group-III—Diabetic control + EFx seed extract (EFx) 2005). (100 mg/kg body weight). Group-IV—Diabetic control + EFx seed extract (EFx) Preliminary study—determining the dosage for crude drug (200 mg/kg body weight). One week after the induction of diabetes, rats with blood Group-V—Diabetic control + EFx seed extract (EFx) glucose level >250 mg/dL, were subjected to fasting for (300 mg/kg body weight). 16 h. They were divided into different groups, with five Group-VI—Diabetic control + EFx seed extract (EFx) rats in each group. N-hexane, di-chloromethane, ethyl (400 mg/kg body weight). acetate, n-butanol and aqueous extract ranging from 100 Group-VII—Diabetic control + Glibenclamide (1 mg/ to 800 mg/kg body weight, were administered to the ani- kg body weight). mals and blood glucose was determined at the end of 5 h after the oral administration of the extracts (Karunanay- The extract was administered to the respective groups ake et al. 1984). The lowest dose that brought about the through oral route using intragastric tube for 45 days. maximum antihyperglycemic effect for each plant extract was given through oral intubations for the repeated Biochemical evaluation administration. The effective dose of all the extracts was Rats of the different groups were kept on fasting over- found to be 400 mg/kg body weight. The selected doses night and the blood was withdrawn by retro orbital punc- of the plant extracts were given on everyday till comple- ture under light anesthesia. Blood was withdrawn from tion of the experiment (i.e., 45 days). the rats on the 1st, 22nd and 45th day after the induction of diabetes to assess the blood glucose and plasma insulin Repeated administration of E. Ferox salisb. seeds extract level by glucose oxidase method (Kesari et al. 2005) and (EFx) modified method of Herbert (Herbert et al. 1965) respec- After a week of induction of diabetes in albino rats, fasting tively. The alteration in the body weight was observed blood glucose levels of fasted rats was measured (Ahmed throughout the therapy in the experimental animals. et al. 2014a). Rats with plasma glucose level more than Oral glucose tolerance test (OGTT) was performed >250 mg/dL were included in the study. Rats were divided according to the method described by Kumar et al. into seven groups of five each. Groups 1 and 2, viz, nor- (2013). OGTT was evaluated to determine the effect of mal and diabetic control rats received vehicle alone [0.1% EFx on peripheral utilization of glucose in normal rats. dimethyl sulfoxide (DMSO) 1 ml/kg body weight]. Groups Wistar-albino rats were grouped into 7 groups (n = 5). 3–6 were treated with the effective dose of n-hexane, All the rats were administered with different doses of di-chloromethane, ethyl acetate, n-butanol and water EFx and Glibenclamide for 30 min. On completion of extracts dissolved in vehicle solution through oral route 30 min all the groups received 2 g/kg of glucose by oral Ahmed et al. SpringerPlus (2015) 4:315 Page 4 of 17

route. The blood samples were collected at regular time were added. The blue color developed after 20 min and period of 0, 0.5, 1.0, 1.5 and 2.0 h to estimate the oral glu- measurement of the absorbance at 680 nm was done. The cose tolerance. At the termination of treatment i.e. after activity of the fructose-1,6-bisphosphatase measured in 45 days, animals were deprived of food for overnight. units/min/mg of protein. Activities of hepatic hexokinase, glucose-6-phsophatase, The lipid parameters viz. total cholesterol, HDL cho- fructose-1-6-bisphosphatase, glucose-6-phosphate lesterol and triglycerides were evaluated according the dehydrogenase were assayed according to the method methods of Zlatkis (1953), Burnstein (1970) and Foster of Branstrup (1957), method of Koide and Oda (1959), and Dunn (1973), respectively. Level of serum LDL cho- method of Gancedo (1971) and method of Robert (1966), lesterol and VLDL cholesterol were estimated accord- respectively. ing to the Friedewald formula (Friedewald et al. 1972). The level of the hexokinase was determined by the Hepatic glycogen level was assessed by the method given using reported method with minor modification (Bran- by Kemp (1954). The levels of lipid peroxidation (LPO) strup et al. 1957). The liver tissue was thawed, weighed in the tissues were evaluated by the method of Ohkawa and homogenized in ice-cold 0.1 M phosphate-buffered (1979). Level of superoxide dismutase (SOD) was assayed saline (pH 7.4) to a concentration of 50 mg/ml. The by the method of Kakkar (1984). The level of catalase assay mixture contains the 7.5 mM MgCl2, 3.7 mM (CAT) enzyme was evaluated by the method given by glucose, 11 mM thioglycerol and 45 mM HEPES Sinha (1972). Glutathione Peroxidase (GSH-Px) was (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) assayed by the method given by Rotruck (1973). Level of buffer. Assay mixture 0.9 ml added into the culvert and reduced glutathione (GSH) was assessed by the method 0.3 ml of 0.22 M ATP added and mixed well. After that, of Ellman (1959). 0.1 ml of the tissue supernatant was added into the cul- vert and determinations of the absorbance at 340 nm the Production of liver and kidney homogenate hexokinase activity as units/min/mg of protein of tissue For the estimation of the antioxidant level, the rats of was recorded. The level of the glucose-6-phosphatase the respective groups were kept on fasting overnight. All activity was measured according reported method of the rats were decapitated and an abdominal incision was Koide et al. with minor modification (Koide and Oda performed, in order to harvest liver and pancreas. The 1959). Liver tissue was homogenate in ice-cold 0.1 M whole organs were thoroughly cleaned with chilled nor- phosphate buffer saline (pH 7.4) to a concentration of mal saline on ice. A 10% (w/v) homogenate of the liver 50 mg/ml. 0.1 ml of 0.1 M glucose-6-phosphatase solu- and pancreas (0.03 M sodium phosphate buffer, pH-7.4) tion and 0.3 ml of 0.5 M maleic acid buffer (pH 6.5) was was prepared with the help of Ultra-Turrax homogenizer mixed in a test tube and incubated at 370°C for 15 min. maintaining the speed at 9500 rpm. The reaction was stopped by adding 1 ml of 10% trichlo- roacetic acid, followed by chilling in ice, after stopping Observation of general condition of rats reaction the mixture was centrifuged at 3000 rpm for The overall general condition of rats such as psychologi- 10 min. The absorbance was measured at the 340 nm cal activity, food intake, water intake, urine output, gen- and expressed of the glucose-6-phosphatase at the lib- eral locomotion, and skin was observed every day. The eration of the inorganic phosphate units/min/mg of parameters such as body weight and food intake were protein of tissue. The level of the fructose-1,6-bispho- determined for every week. sphatase was estimated by using the reported method with minor modification (Gancedo and Gancedo 1971). Histological assessment of liver, kidney, pancreas Liver tissue was homogenized in ice-cold 0.1 M phos- and heart sample by Heamatoxylin Eosin (H/E) staining phate buffer saline (pH 7.4). The assay mixture containing At the end of the therapy, all the rats of different groups the 1.2 ml of trise HCl buffer (0.1 M, pH 7.0), 0.1 ml of were sacrificed using mild anesthesia. After collection of substrate (0.05 M), 0.25 ml of MgCl2 (0.1 M), 0.1 ml of the blood samples, the liver, kidney, pancreas and heart KCl (0.1 M), 0.25 ml of ethylenediaminetetraacetic acid tissues were fixed in neutral formalin solution for 48 h, (0.001 M) solution and 0.1 ml of enzyme homogenate dehydrated by passing through graded series of alcohol and maintained the final sample volume 2 ml. The mix- embedded in paraffin blocks. 4 µm thick sections were ture was incubated for 15 min at 370°C. The 10% solution prepared using a semi-automated rotator microtome. of the trichloroethanoic acid was used for the termina- tion of the reaction. The reaction mixture was centri- Statistical analysis fuged and the supernatant was used for the phosphorous Statistical analysis was performed using GRAPH PAD estimation). In the supernatant, the 1 ml of ammonium Prism software package, Version 5.0. All the data were molybdate and 0.4 ml of amino naphthol sulfonic acid expressed as mean ± standard error mean (SEM). The Ahmed et al. SpringerPlus (2015) 4:315 Page 5 of 17

comparisons within groups was evaluated utilizing inde- and the level of plasma insulin was further decreased in pendent student T test and one way analysis of variance the untreated diabetic rats at the end of the study i.e. after (ANOVA). The value of p < 0.05 or p < 0.01 was consid- 45 days. Treatment with the methanol/dichlorometh- ered to be statistically significant. ane extract of EFx in a dose dependent manner. Treat- ment with 400 mg/kg body weight of EFx was shown to Results produce most significant (p < 0.05) effect on the level of Effect of EFx on blood glucose level in normal and STZ plasma insulin. It amplified the level of plasma insulin induced diabetes treated rats nearly to the normal as compared to the other doses of The biochemical parameters of glycemic control in the EFx at the end of research exertion (Table 2; Figure 2). animals has been summarized in Table 1 (Figure 1). The intraperitoneal administration of streptozotocin (STZ) Effect of EFx on OGTT in normal and STZ induced (treated) resulted in nearly fourfold increase of the fasting blood diabetic rats during 120 min (2 h) glucose levels in the male/female diabetic Wistar rats. The results from the research exertion clearly indicate The blood glucose level was measured at different time that the of methanol/dichloromethane extract of Albizzia intervals during the research exertion viz. on the very Lebbeck Benth. stem bark (EFx) (400 mg/kg body weight) first day of induction of diabetes, at the middle of the and Glibenclamide (1 mg/kg) reduced the blood glucose study i.e. on 21st day and at the end of the experiment level (significant hyperglycemia due to administration of i.e. on 45th day. It was observed that with the gradual glucose load of 2 g/kg p.o to a significant level (p < 0.05) increase in dose of the EFx, the blood glucose level was after 2 h of oral administration as compared to the dia- improvised. At the end of 45 days period, EFx treated betic control group (Table 3; Figure 3). diabetic animals showed a significant reduction of blood glucose level nearly to the normal level compared with Effect of EFx weight variation (grams) in normal and STZ the diabetic animals (p < 0.05). induced diabetic (treated) rats The body weight variation of the rats was observed at Effect of EFx on plasma insulin level in normal and STZ the beginning and end of the research exertion. As it is induced diabetes treated rats obvious from the table (Table 4; Figure 4), that weight of The level of plasma insulin was measured at differ- the diabetic (untreated) rats was reduced to a significant ent periods during the experimentation. A significant level. Weight of the EFx treated rats was increased to a decrease in the level of plasma insulin was observed in momentous level (p < 0.05) as compared to the weight of the untreated diabetic rats compared to the normal rats normal rats.

Table 1 Effect of Euryale ferox salisb. seeds extract (EFx) on blood glucose level in normal and STZ induced diabetic treated rats

Groups Blood glucose level in mg/dL at different time intervals during experimentation At start (on 1st day) On 21st day On 45th day

Normal rats [untreated with dimethylsulfoxide, (DMSO)] Group 1 82.38 0.6308a 82.25 0.2649a 81.80 0.4055a ± ± ± Diabetic control [administered with streptozotocin (STZ)] Group 2 303.8 0.9011b 310.5 0.2231b 312.5 0.4212b ± ± ± Diabetic (EFx) (100 mg/kg body weight) Group 3 301.0 0.2698 264.1 1.152 201.7 0.3738 + ± ± ± Diabetic (EFx) (200 mg/kg body weight) Group 4 297.8 0.3096 235.4 0.8799 173.1 0.8970 + ± ± ± Diabetic (EFx) (300 mg/kg body weight) Group 5 295.4 0.3857 184.0 0.6072 107.6 1.422** + ± ± ± Diabetic (EFx) (400 mg/kg body weight) Group 6 293.6 0.3771 146.7 0.5004 91.08 0.5402*** + ± ± ± Diabetic glibenclamide (1 mg/kg body weight) Group 7 290.9 0.2182 117.4 1.004** 83.14 0.6532*** + ± ± ± The data are expressed as mean SEM (n number of animals in each group 5). The comparisons has been made by one way ANOVA followed by Dunnent’s test. ± = = ns non-significant, STZ Streptozotocin. * p < 0.05 is considered as significant when compared to the control group (0 h). ** p < 0.001 is considered as very significant when compared to the control group (0 h). *** p < 0.001 is considered as extremely significant when compared to the control group (0 h). a Compared to diabetic control. b Compared to normal control. Ahmed et al. SpringerPlus (2015) 4:315 Page 6 of 17

Figure 1 Effect of EFx on blood glucose level at different time interval of therapy, Group 1: normal control; Group 2: STZ (60 mg/kg i.p.); Group 3: diabetic control (EFx) (100 mg/kg body weight); Group 4: diabetic control (EFx) (200 mg/kg body weight) and continue for 45 days; Group5: + + diabetic diabetic control (EFx) (300 mg/kg body weight) and continue for 45 days; Group 6: diabetic control (EFx) (400 mg/kg body weight) + + + and continue for 45 days; Group 7: diabetic control glibenclamide (1 mg/kg body weight) and continue for 45 days. +

Table 2 Effect of Euryale ferox salisb. seed extract (EFx) on plasma insulin level in normal and STZ induced and treated diabetic rats

Groups Plasma insulin level in µM at different time interval of experimentation At start (on 1st day) On 21st day On 45th day

Normal rats [untreated with dimethylsulfoxide, (DMSO)] Group 1 12.22 0.2028a 12.37 0.1772a 12.39 0.1154a ± ± ± Diabetic control [administered with Streptozotocin (STZ)] Group 2 3.710 0.1482b 3.136 0.01208b 3.034 0.02482b ± ± ± Diabetic (EFx) (100 mg/kg body weight) Group 3 4.260 0.1040 5.812 0.03929 6.822 0.02596 + ± ± ± Diabetic (EFx) (200 mg/kg body weight) Group 4 4.250 0.02168 6.028 0.005831 7.936 0.02542 + ± ± ± Diabetic (EFx) (300 mg/kg body weight) Group 5 4.806 0.03655 7.058 0.03277 9.496 0.1394** + ± ± ± Diabetic (EFx) (400 mg/kg body weight) Group 6 4.896 0.02786 9.136 0.04854** 11.66 0.1689*** + ± ± ± Diabetic glibenclamide (1 mg/kg body weight) Group 7 5.058 0.03382 9.734 0.09304** 12.34 0.1080*** + ± ± ± The data are expressed as mean SEM (n number of animals in each group 5). The comparisons has been made by one way ANOVA followed by Dunnent’s test. ± = = ns non-significant, STZ Streptozotocin. * p < 0.05 is considered as significant when compared to the control group (0 h). ** p < 0.001 is considered as very significant when compared to the control group (0 h). *** p < 0.001 is considered as extremely significant when compared to the control group (0 h). a Compared to diabetic control. b Compared to normal control. Ahmed et al. SpringerPlus (2015) 4:315 Page 7 of 17

Figure 2 Effect of EFx on plasma insulin level at different time interval of therapy, Group 1: normal control; Group 2: STZ (60 mg/kg i.p.); Group 3: diabetic control (EFx) (100 mg/kg body weight); Group 4: diabetic control (EFx) (200 mg/kg body weight) and continue for 45 days; Group5: + + diabetic diabetic control (EFx) (300 mg/kg body weight) and continue for 45 days; Group 6: diabetic control (EFx) (400 mg/kg body weight) + + + and continue for 45 days; Group 7: diabetic control glibenclamide (1 mg/kg body weight) and continue for 45 days. +

Table 3 Effect of Euryale ferox salisb. seeds extract (EFx) during 120 min (2 h) on OGTT in normal and STZ induced dia- betic (treated) rats

Groups Time (h) 0 h 0.5 h 1 h 1.5 h 2 h

Normal rats [untreated with dimethylsulfoxide, (DMSO)] 82.91 0.3829a 95.60 0.2651a 108.62 0.3387a 117.67 0.9812a 138.15 0.4991a Group 1 ± ± ± ± ± Diabetic control (administered with Streptozotocin (STZ) 265.9 0.9826b 278.2 0.8519b 284.7 0.2911b 279.1 0.8171b 274.9 0.3012b Group 2 ± ± ± ± ± Diabetic (EFx) (100 mg/kg body weight) Group 3 235.0 1.086 225.0 0.8882 221.0 0.2465 203.9 0.8683 182.4 0.2997 + ± ± ± ± ± Diabetic (EFx) (200 mg/kg body weight) Group 4 225.2 0.9767 214.8 0.8538 212.4 0.2989 198.0 0.6565 171.6 0.4256 + ± ± ± ± ± Diabetic (EFx) (300 mg/kg body weight) Group 5 214.2 1.263 211.5 0.6072 202.8 0.4556 185.7 1.784 142.8 0.7964** + ± ± ± ± ± Diabetic (EFx) (400 mg/kg body weight) Group 6 203.1 1.652 192.3 0.5702 182.8 0.3597 162.4 0.4513* 88.45 0.7672*** + ± ± ± ± ± Diabetic glibenclamide (1 mg/kg body weight) Group 7 198.3 0.5980 182.4 0.5737 170.9 0.3304 141.7 0.5050** 82.80 0.5378*** + ± ± ± ± ± The data are expressed as mean SEM (n number of animals in each group 5). The comparisons has been made by one way ANOVA followed by Dunnent’s test. ± = = ns non-significant, STZ Streptozotocin. * p < 0.05 is considered as significant when compared to the control group (0 h). ** p < 0.001 is considered as very significant when compared to the control group (0 h). *** p < 0.001 is considered as extremely significant when compared to the control group (0 h). a Compared to diabetic control. b Compared to normal control. Ahmed et al. SpringerPlus (2015) 4:315 Page 8 of 17

Figure 3 Effect of EFx on oral glucose tolerance test (OGTT) during 120 min (2 h) of therapy, Group 1: normal control; Group 2: STZ (60 mg/kg i.p.); Group 3: diabetic control (EFx) (100 mg/kg body weight); Group 4: diabetic control (EFx) (200 mg/kg body weight) and continue for 45 days; + + Group 5: diabetic diabetic control (EFx) (300 mg/kg body weight) and continue for 45 days; Group 6: diabetic control (EFx) (400 mg/kg body + + + weight) and continue for 45 days; Group 7: diabetic control glibenclamide (1 mg/kg body weight) and continue for 45 days. +

Table 4 Effect of Euryale ferox salisb. seeds extract (EFx) on body weight variation (grams) in normal and STZ induced diabetic treated rats

Groups Time (h) 1st day 45th day

Normal rats [untreated with dimethylsulfoxide, (DMSO)] Group 1 263.6 0.8124a 265.0 1.673a ± ± Diabetic control [(administered with Streptozotocin (STZ)] Group 2 213.8 1.594b 210.8 0.4899b ± ± Diabetic (EFx) (100 mg/kg body weight) Group 3 217.0 1.140 222.8 1.655 + ± ± Diabetic (EFx) (200 mg/kg body weight) Group 4 220.6 0.2449 234.2 1.463 + ± ± Diabetic (EFx) (300 mg/kg body weight) Group 5 222.6 0.5099 237.6 0.5099 + ± ± Diabetic (EFx) (400 mg/kg body weight) Group 6 226.2 0.8000 252.6 1.631** + ± ± Diabetic glibenclamide (1 mg/kg body weight) Group 7 227.0 0.9487 262.0 0.7071*** + ± ± The data are expressed as mean SEM (n number of animals in each group 5). The comparisons has been made by one way ANOVA followed by Dunnent’s test. ± = = ns non-significant, STZ Streptozotocin. * p < 0.05 is considered as significant when compared to the control group (0 h). ** p < 0.001 is considered as very significant when compared to the control group (0 h). *** p < 0.001 is considered as extremely significant when compared to the control group (0 h). a Compared to diabetic control. b Compared to normal control. Ahmed et al. SpringerPlus (2015) 4:315 Page 9 of 17

Figure 4 Effect of EFx on body weight variation at different time interval of therapy, Group 1: normal control; Group 2: STZ (60 mg/kg i.p.); Group 3: diabetic control (EFx) (100 mg/kg body weight); Group 4: diabetic control (EFx) (200 mg/kg body weight) and continue for 45 days; Group 5: + + diabetic diabetic control (EFx) (300 mg/kg body weight) and continue for 45 days; Group 6: diabetic control (EFx) (400 mg/kg body weight) + + + and continue for 45 days; Group 7: diabetic control glibenclamide (1 mg/kg body weight) and continue for 45 days. +

Table 5 Effect of Euryale ferox salisb. seeds extract (EFx) on hepatic enzymes in normal and STZ induced diabetic treated rats

Groups Biochemical parameters of hepatic enzymes Hepatic hexokinase Gluocse-6-phosphatase Fructose 1-6-biphos- Glucose-6-phosphate (units/min/mg (units/min/mg of protein) phatase (units/min/mg dehydrogenase of protein) of protein) (units/min/mg of protein)

Normal rats [untreated with 0.2360 0.007483a 0.0356 0.0002449a 0.0320 0.0004472a 0.1620 0.003742a dimethylsulfoxide, (DMSO)] ± ± ± ± Group 1 Diabetic control (administered with 0.0880 0.003742b 0.0646 0.002249b 0.0664 0.0009274b 0.0714 0.0006782b Streptozotocin (STZ) Group 2 ± ± ± ± Diabetic (EFx) (100 mg/kg body 0.1080 0.003742 0.0602 0.0004899 0.0584 0.0007483 0.0754 0.0012080 weight)+ Group 3 ± ± ± ± Diabetic (EFx) (200 mg/kg body 0.1400 0.003162 0.0548 0.0005831 0.0526 0.0014 0.0822 0.00080000 weight)+ Group 4 ± ± ± ± Diabetic (EFx) (300 mg/kg body 0.1800 0.003162 0.0464 0.0009274 0.0466 0.001030 0.08808 0.01949 weight)+ Group 5 ± ± ± ± Diabetic (EFx) (400 mg/kg body 0.2024 0.0006782** 0.0408 0.0003742** 0.0376 0.0005099*** 0.1340 0.0050990** weight)+ Group 6 ± ± ± ± Diabetic glibenclamide (1 mg/kg 0.2220 0.003742*** 0.0532 0.001497** 0.0454 0.002315** 0.1144 0.003919** body weight)+ Group 7 ± ± ± ±

The data are expressed as mean SEM. (n number of animals in each group 5). The comparisons has been made by one way ANOVA followed by Dunnent’s test ± = = ns non-significant, STZ Streptozotocin. * p < 0.05 is considered as significant when compared to the control group (0 h). ** p < 0.001 is considered as very significant when compared to the control group (0 h). *** p < 0.001 is considered as extremely significant when compared to the control group (0 h). a Compared to diabetic control. b Compared to normal control.

Effect of EFx on hepatic enzymes in normal and STZ animals. The activities of hepatic hexokinase and glu- induced diabetic treated rats cose-6-phosphate dehydrogenase (G6PD) were found Table 5 (Figure 5) portrays the alteration in the activi- to be decreased. On the other hand, the level of glu- ties of carbohydrate metabolizing enzymes in the liver of coneogenic enzymes viz. glucose-6-phosphatase and diabetic control group and other groups of experimental fructose-6-phosphatase were significantly increased in Ahmed et al. SpringerPlus (2015) 4:315 Page 10 of 17

Figure 5 Effect of EFx on various hepatic enzymes at the end of therapy, Group 1: normal control; Group 2: STZ (60 mg/kg i.p.); Group 3: diabetic control (EFx) (100 mg/kg body weight); Group 4: diabetic control (EFx) (200 mg/kg body weight) and continue for 45 days; Group 5: dia- + + betic diabetic control (EFx) (300 mg/kg body weight) and continue for 45 days; Group 6: diabetic control (EFx) (400 mg/kg body weight) and + + + continue for 45 days; Group 7: diabetic control glibenclamide (1 mg/kg body weight) and continue for 45 days. +

the diabetic animals compared to those in normal rats. Effect of EFx on oxidative stress parameters in normal Administration of different doses of EFx among diabetic and STZ induced diabetic (treated) rats rats reversed the alterations in the hepatic enzymes such Table 7 clearly illustrates the effect of EFx on the anti- that animals received 400 mg/kg body weight showed oxidant enzymes. A marked reduction was noted in the the significant improvement (p < 0.05) in all the hepatic level of superoxide dismutase (SOD), catalase (CAT), enzymes alterations as compared to the other doses. Glutathione Peroxidase (GSH-Px), and reduced glu- tathione (GSH) in the STZ induced diabetic rats. Admin- Effect of EFx on serum lipid profile in normal and STZ istration of EFx at different doses for the 45 days to STZ induced among diabetic (treated) rats induced diabetic rats significantly (p < 0.05) increased As Evident from the Table 6 that diabetic rats exhibited SOD, CAT, GSH-Px levels with maximum effect seen at significant increase in serum total cholesterol, VLDL 400 mg/kg body weight. The enhanced level of TBARS cholesterol, LDL cholesterol, triglycerides levels and was reversed to near normal after administration of EFx decreased level of HDL cholesterol and hepatic glycogen. after administering 400 mg/kg body weight of EFx. It is Lipid profile of the EFx treated diabetic rats was signifi- pertinent to note that the EFx was found to be equipped cantly improved (p < 0.05) as compared to the untreated with the antioxidant effect in a dose dependent manner diabetic rats (Figure 6). (Figure 7). Ahmed et al. SpringerPlus (2015) 4:315 Page 11 of 17

Effect of EFx on histopathology of pancreas, kidney, liver deposition on the glomeruli along with destructed glo- and heart meruli and infiltration of red blood cells (Figure 8). Pancreas Groups received the EFx demonstrated reversal of these Normal control rats exhibited normal histological archi- pathological destructions as apparent by the cells regen- tecture. Many rounded normal proportions of islet of eration and removal of crystals deposition. langerhans were found all around the pancreatic acini. Prominent nuclei with well arranged lobules with sur- Liver rounding islet cells, were found among normal control The liver cells of normal control groups showed eminent rats (Figure 8). Groups received STZ, demonstrated cel- hepatocytes with central vein along with portal triad lular damage to the pancreatic acini and islets, which (Figure 10). The damage to the liver cells in the form of showed pancreatic β-cell damage and degeneration with damaged central vein, hepatocytes and portal trial could asymmetrical vacuoles. EFx treated STZ induced-DM be clearly seen in the group received STZ. The dam- rats showed marked improvement of the cellular injure as age to the liver cells were reversed in all the EFx treated (Figure 9), as evident from the partial restoration of islet groups. cells, reduced β-cell damage, more symmetrical vacuoles and an increase in number of islet cells. Discussion Acute toxicity study of the methanolic extract dem- Kidney onstrated that different doses of Euryale ferox salisb. Morphological features of kidney remained normal in were non-toxic throughout the experiment. The lethal- the control group like prominent glomeruli, collect- ity was found to be zero in the groups received the dif- ing ducts, tubules and ascending and descending loops. ferent doses of Euryale ferox salisb. seeds extract. Oral STZ-induced DM group showed presence of crystal glucose tolerance test studies showed EFx significantly

Table 6 Effect of Euryale ferox salisb. seeds extract (EFx) on lipid profile in normal and STZ induced and diabetic (treated) rats

Groups Serum lipid profile Total cholesterol HDL cholesterol LDL cholesterol Triglycerides Hepatic glycogen (mg (TC) (mg/dL) (HDL-c) (mg/dL) (LDL-c) (TG) (mg/dL) glucose equivalents/ mg wet tissue)

Normal rats [untreated with dimethyl- 137.7 1.315a 63.19 0.4878a 23.54 0.5120a 67.57 0.5943a 55.70 1.523a sulfoxide, (DMSO)] Group 1 ± ± ± ± ± Diabetic control [(administered with 283.0 0.7544b 16.65 0.1162b 114.6 0.5353b 203.7 0.8567b 15.61 0.1631b Streptozotocin (STZ)] Group 2 ± ± ± ± ± Diabetic (EFx) (100 mg/kg body 231.8 0.3189 24.83 0.4556 92.14 0.2920 184.7 0.6979 20.97 0.2149 weight)+ Group 3 ± ± ± ± ± Diabetic (EFx) (200 mg/kg body 198.1 0.4720 36.27 0.5386 71.43 0.4485 141.6 0.5797 34.01 0.7427 weight)+ Group 4 ± ± ± ± ± Diabetic (EFx) (300 mg/kg body 172.8 0.3100 42.49 0.7798 53.04 0.2755 111.3 0.4677 42.61 0.4618 weight)+ Group 5 ± ± ± ± ± Diabetic (EFx) (400 mg/kg body 151.4 0.4808*** 58.34 0.5236*** 32.03 0.5791** 74.77 1.225** 51.68 0.3041*** weight)+ Group 6 ± ± ± ± ± Diabetic glibenclamide (1 mg/kg 165.7 0.5246** 41.62 0.2302** 47.58 0.4332** 91.99 0.5388** 55.79 0.9709** body weight)+ Group 7 ± ± ± ± ±

The data are expressed as mean SEM (n number of animals in each group 5). The comparisons has been made by one way ANOVA followed by Dunnent’s test. ± = = ns non-significant, STZ Streptozotocin. * p < 0.05 is considered as significant when compared to the control group (0 h). ** p < 0.001 is considered as very significant when compared to the control group (0 h). *** p < 0.001 is considered as extremely significant when compared to the control group (0 h). a Compared to diabetic control. b Compared to normal control. Ahmed et al. SpringerPlus (2015) 4:315 Page 12 of 17

Figure 6 Effect of EFx on lipid profiles at the end of therapy, Group 1: normal control; Group 2: STZ (60 mg/kg i.p.); Group 3: diabetic control (EFx) + (100 mg/kg body weight); Group 4: diabetic control (EFx) (200 mg/kg body weight) and continue for 45 days; Group 5: diabetic diabetic + + control (EFx) (300 mg/kg body weight) and continue for 45 days; Group 6: diabetic control (EFx) (400 mg/kg body weight) and continue for + + 45 days; Group 7: diabetic control glibenclamide (1 mg/kg body weight) and continue for 45 days. +

Table 7 Effect of Euryale ferox salisb. seeds extract (EFx) on oxidative stress parameters in liver of normal and STZ induced diabetic treated rats

Groups Oxidative stress SOD (units/mg CAT (µ mol/min/mg GSH-px ((µ mol/min/mg GSH (mM/100 g protein) protein) protein) tissue)

Normal rats [untreated with dimethylsulfoxide, 12.30 0.1642a 82.84 0.6566a 13.71 0.1077a 53.63 0.1813a (DMSO)] Group 1 ± ± ± ± Diabetic control [administered with Streptozotocin 3.670 0.07218b 22.28 0.2578b 5.274 0.1402b 21.98 0.2745b (STZ)] Group 2 ± ± ± ± Diabetic (EFx) (100 mg/kg body weight) group 3 5.726 0.1197 36.09 0.2578 7.628 0.08243 35.30 0.3952 + ± ± ± ± Diabetic (EFx) (200 mg/kg body weight) Group 4 8.366 0.01965 48.08 0.4018 8.820 0.01304 39.09 0.2614 + ± ± ± ± Diabetic (EFx) (300 mg/kg body weight) Group 5 9.440 0.1626 68.61 0.2086 10.29 0.2236 43.76 0.4200 + ± ± ± ± Diabetic (EFx) (400 mg/kg body weight) Group 6 11.47 0.1209*** 76.68 0.8596** 12.64 0.04104*** 50.91 0.3273*** + ± ± ± ± Diabetic Glibenclamide (1 mg/kg body weight) 9.608 0.1329** 71.70 0.3430** 10.53 0.1955** 34.52 0.3057** Group 7+ ± ± ± ±

The data are expressed as mean SEM (n number of animals in each group 5). The comparisons has been made by one way ANOVA followed by Dunnent’s test. ± = = ns non-significant,STZ Streptozotocin. * p < 0.05 is considered as significant when compared to the control group (0 h). ** p < 0.001 is considered as very significant when compared to the control group (0 h). *** p < 0.001 is considered as extremely significant when compared to the control group (0 h). a Compared to diabetic control. b Compared to normal control. Ahmed et al. SpringerPlus (2015) 4:315 Page 13 of 17

Figure 7 Effect of EFx on various antioxidant marker enzymes at the end of therapy, Group 1: normal control; Group 2: STZ (60 mg/kg i.p.); Group 3: diabetic control (EFx) (100 mg/kg body weight); Group 4: diabetic control (EFx) (200 mg/kg body weight) and continue for 45 days; Group5: + + diabetic diabetic control (EFx) (300 mg/kg body weight) and continue for 45 days; Group 6: diabetic control (EFx) (400 mg/kg body weight) + + + and continue for 45 days; Group 7: diabetic control glibenclamide (1 mg/kg body weight) and continue for 45 days. +

to nearly normal in STZ-induced diabetic treated rats (88.45 ± 0.7672) declined the level of the blood sugar after 45 days of therapy. Distorted central vein and when compared to the glibenclamide (82.80 ± 0.5378). Glibenclamide is often used as insulin stimulant in hepatocytes were reinstated to normal in liver of STZ- many studies and also as standard antidiabetic drug induced diabetic rats treated with various doses of EFx in STZ-induced moderate diabetes to compare the when compared to the toxic control. Ascending and antidiabetic potential of various hypoglycemic agents descending loops were recovered almost to its normal (Andrade et al. 2000). Furthermore, the blood glucose morphological features. All histological changes were level of STZ-induced diabetic rats was increased to a well supported with the restored levels of insulin, glu- significant level. EFx treated STZ-induced diabetic cose, glycosylated hemoglobin. Glucose-6-phosphate rats, lowers the increased blood glucose level to a sig- dehydrogenase (G6PD) is the key enzyme to maintain the blood glucose level (Mayes 2000). In streptozo- nificant (91.08 ± 0.5402) extent. EFx treated group rats showed improved level of the insulin as compared to tocin-induced diabetic rats, the reduction of G-6-PDH the glibenclamide. It has been stated that most of the activity in liver which obstruct glucose utilisation diabetic complications are mediated through oxida- through pentose phosphate pathway as this enzyme tive stress (Matkovics et al. 1997; Kavalali et al. 2003). activity is controlled by insulin (Ugochukwu and Studies also suggest involvement of free radicals in Babady 2003). The plant extract significantly improves pancreatic cell destruction (Lenzen 2008). Histopatho- this enzyme activity in hepatic tissue which enlighten logical studies of the sections of pancreas, liver and its another possible way of antidiabetogenic activ- kidney revealed the disturbed morphological features. ity. Glucose-6-phosphatase and fructose-1,6-bispho- Islets of langerhans containing β-cells were restored phatase play an important role in glucose homeostasis Ahmed et al. SpringerPlus (2015) 4:315 Page 14 of 17

Figure 8 Effect of Euryale ferox salisb seeds extract (EFx) on histological profile of liver in normal, STZ-induced diabetic untreated and STZ-induced diabetic treated wistar rats (original magnification 40, DXIT 1200, Nikon, Japan). (1) NLIV: heamatoxylin and eosin (H/E) stained sections of liver × of normal control rats showing normal portal triad along with normal hepatocytes with central vein (yellow arrows). (2) LSTZ: liver section of rats received streptozotocin depicting destructed portal trial, disarranged hepatocytes and central vein (red arrows). (3) LEFx100: section of liver sup- plemented with 100 mg/kg body weight of EFx portraying improvement in structure of portal triad (green arrows). (4) LEFx200: liver section of rats received 200 mg/kg body weight of EFx showing arranged hepatocytes (green arrows). (5) LEFx300: section of liver or diabetic rats treated with 300 mg/kg body weight. of EFx depicting arranged central vein (yellow arrows). (6) LEFx400: liver of diabetic rat showing normal portal traid, central vein and hepatocytes (green arrows). (7) LGLIM: liver section of rat administered with Glibenclamide showing normal microvasculature along with normal hepatocytes (dark yellow arrows).

(Berg et al. 2001). Insulin deficiency results in the acti- seeds extract (EFx) and Glibenclamide administration vation of gluconeogenic enzymes during diabetes. EFx controlled loss of body weight. Hexokinase plays an administration significantly decreased the activity of important role in maintaining the blood glucose home- gluconeogenic enzymes in diabetic rats. The level of ostasis (Saxena et al. 1992). The activity of hepatic plasma insulin was found to increase significantly in hexokinase was significantly decreased in the STZ- diabetic rats treated with EFx, which might be a pos- induced diabetic rats. Treatment with different doses sible reason for significant reduction in the level of of EFx significantly reversed the activity of hexokinase. gluconeogenic enzymes. STZ-induced diabetes is Increased activity of hexokinase resulted in increased characterized by loss in body weight (Al-Shamaony glycolysis which inturn causes improvement in glu- et al. 1994) and this was also observed in the present cose utilization and glycolysis for the production of research exertion. The characteristic loss of body energy. Oxidative stress has been linked with obesity weight is mainly due to increased muscle wasting in and insulin resistance, which is recognized by exces- diabetes (Swanston-Flatt et al. 1990). E. ferox salisb. sive generation of reactive oxygen species (ROS). At Ahmed et al. SpringerPlus (2015) 4:315 Page 15 of 17

Figure 9 Effect of Euryale ferox salisb seeds extract (EFx) on histological profile of pancreas in normal, STZ-induced diabetic untreated and STZ- induced diabetic treated wistar rats (original magnification 10, DXIT 1200, Nikon, Japan). (1) NPAN: heamatoxylin and eosin (H/E) stained sections × of pancreas of normal control rat portraying normal islet of langerhans shown by yellow arrows. (2) PSTZ: pancreatic section of streptozotocin induced diabetic rat showing no/destroyed islet of langerhans and beta cells depicted by red arrows. (3) PEFx-100: pancreatic section of STZ- induced diabetic rats treated with EFx at 100 mg/kg body weight showing small number of islet of langerhans (green arrows). (4) PEFx-200: section of pancreas of STZ-induced diabetic rats treated with EFx at 200 mg/kg body weight portraying increased number of islet of langerhans with small proportions of beta cells (green arrows). (5) PEFx-300: pancreas of diabetic rats treated with 300 mg/kg body weight. EFx depicting nearly normal islet of langerhans (green arrows). (6) PEFx-400: sections of pancreas of diabetic treated rats with 400 mg/kg body weight. EFx showing normal islet of langerhans with numerous beta cells (green arrows). (7) PGLIM: pancreatic section of diabetic rats treated with Glibenclamide showing normal pancreatic islet of langerhans with enhancement in the number of beta cells (dark yellow arrow). present the indicators for evaluation of ROS are the profile was observed with significantly increased con- endogenous enzymes such as superoxide dismutase centration of TC, TGs, LDL and HDL and decreased (SOD), catalase (CAT), glutathione Peroxidase (GPx) concentration of HDL among diabetic rats (DC) than and glutathione (GSH). Administration of EFx to STZ- their normal counterparts. Administration of EFx for induced diabetic rats significantly restored the altered 45 days significantly improvised the altered level of level of antioxidant enzymes in a dose dependent man- TC, TGs. LDL and HDL in a dose dependent man- ner. Glycosylation of proteins, oxidative stress and ner. Therefore, the plausible mechanism of action of chronic hyperglycemia potentiate the increased risk of EFx in controlling the blood glucose level might be the cardiovascular diseases through the altered lipid pro- enhancement of secretion of insulin from pancreatic file. As diabetes is a metabolic disorder, it is character- β-cells. Furthermore, the mechanism of correction of ized not only by increased glucose concentrations but lipid profile might be due the improved glycogenesis in also by altered level of lipid profile. An altered lipid the liver. Ahmed et al. SpringerPlus (2015) 4:315 Page 16 of 17

Figure 10 Effect of Euryale ferox salisb seeds extract (EFx on histological profile of kidney in normal, STZ-induced diabetic untreated and STZ- induced diabetic treated wistar rats (original magnification 40, DXIT 1200, Nikon, Japan). (1) NKID: heamatoxylin and eosin (H/E) stained sections × of kidney of normal control rats showing normal glomeruli with normal baseline and tubules (yellow arrow). (2) KSTZ: section of kidney of STZ- induced diabetic rats depicting destroyed glomeruli with fat deposition on baseline along with infiltration of lymphocytes (red arrows). (3) KEFx100: kidney section of diabetic rats treated with EFx at dose of 100 mg/kg body weight portraying improved vasculature and glomeruli (green arrows). (4) KEFx200: section of kidney of diabetic rats treated with 200 mg/kg body weight of EFx showing normal tubules along with virtually improved struc- ture of glomeruli (green arrows). (5) KEFx300: kidney section of diabetic treated rats with 300 mg/kg body weight of EFx depicting nearly normal glomeruli (green arrows). (6) KEFx400: section of kidney of diabetic rat received 400 mg/kg body weight of EFx showing normal glomeruli with no infiltration of lymphocytes (green arrows). (7) KGLIM: section of kidney of the rat supplemented with Glibenclamide showing normal glomeruli with improved structure of tubules (dark yellow arrows).

Conclusion Authors’ contributions DA and MS designed the research exertion and performed the analysis and The above results indicated that E. Ferox salisb. seeds interpretation of the data. GSS has done the proofreading of the manuscript. extract protect β-cells against ROS-mediated destruc- VK and AV have been involved in drafting and revision of the manuscript. DA and VK have done the histopathological analysis of the different sections. All tion by improvising the levels of antioxidant enzymes authors read and approved the final manuscript. and minimizing hyperglycemia which could be due to release of insulin from remnant and recovered β-cells in Author details 1 Department of Pharmaceutical Sciences, Faculty of Health Sciences, Sam pancreas in STZ-induced diabetic rats as confirmed by Higginbottom Institute of Agriculture, Technology and Sciences (SHIATS)- the ultrastructural, histopathological studies. The above Deemed University, Allahabad, India. 2 Department of Pharmacology, Faculty research exertion clearly indicates that ethanolic extract of Pharmacy, Jamia Hamdard, New Delhi, India. 3 Faculty of Health Sci- ences, Sam Higginbottom Institute of Agriculture, Technology and Sciences of E. Ferox salisb. may be utilized as important source of (SHIATS)-Deemed University, Allahabad, India. 4 Department of Pharmacology, natural antioxidants with antidiabetic and antihyperlipi- Hamdard Institute of Medical Sciences and Research (HIMSR), Jamia Hamdard, demic potential and can be used as plausible food addi- New Delhi, India. tives or as a functional food in future. Ahmed et al. SpringerPlus (2015) 4:315 Page 17 of 17

Acknowledgements Kakkar P, Das B, Viswanathan P (1984) A modified method for assay of superox- Authors wish to thank the authorities of Sam Higginbottom Institute of ide dismutase. Ind J Biochem Biophys 21:131–132 Agriculture, Tehcnology & Sciences (SHIATS)-Deemed University for providing Karunanayake EH, Welihinda J, Sirimanne SR, Sinnadoria H (1984) Oral hypogly- necessary facilities. cemic activity of some medicinal plants of Sri Lanka. J Ethnopharmacol 1(2):223–231 Compliance with ethical guidelines Kavalali G, Tuncel H, Goksel S, Hatemi HH (2003) Hypoglycemic activity of Urtica pilulifera in streptozotocin-diabetic rats. J Ethnopharmacol Competing interests 84:241–245 The authors declare that they have no competing interests. Kemp A, Kits van Heijningen AJM (1954) A colorimetric micromethod for the determination of glycogen in tissues. Biochem J 56:646–648 Consent for publication Kesari AN, Gupta RK, Watal G (2005) Hypoglycemic effects of Murraya koenigii Animal handling and experimental procedures were approved by the Institu- on normal and alloxan-diabetic rabbits. J Ethnopharmacol 97:247–251 tional Animal Ethical Committee of Adina Institute of Pharmaceutical Sciences, Koide H, Oda T (1959) Pathological occurrence of glucose-6-phosphatase in Sagar, MP (IAEC Reg. no. 1546/PO/a/11/CPCSEA). liver disease. Clin Chim Acta 4:554–561 Kumar V, Ahmed D, Anwar F, Ali M, Mujeeb M (2013a) Enhanced glycemic Received: 8 October 2014 Accepted: 26 May 2015 control, pancreas protective, antioxidant and hepatoprotective effects by umbelliferon-α-D-glucopyranosyl-(2I 1II)-α-D-glucopyranoside in streptozotocin induced diabetic rats. SpringerPlus→ 2:639 Kumar V, Ahmed D, Gupta PS, Anwar F, Mujeeb M (2013b) Anti-diabetic, anti- oxidant and anti-hyperlipidemic activities of Melastoma malabathricum Linn leaves in streptozotocin induced diabetic rats. BMC Complement References Altern Med 13:222 Ahmed D, Sharma M, Mukerjee A, Ramteke PW, Kumar V (2013) Improved Lee SE, Ju EM, Kim JH (2002) Antioxidant activity of extracts from Euryale ferox glycemic control, pancreas protective and hepatoprotective effect seed. Exp Mol Med 34:100–106 by traditional poly-herbal formulation “Qurs Tabasheer” in strepto- Lenzen S (2008) Oxidative stress: the vulnerable beta-cell. Biochem Soc Trans zotocin induced diabetic rats. BMC Complement Altern Med 13:10. 36:343–347 doi:10.1186/1472-6882-13-10 Matkovics B, Kotorman M, Varga IS, Hai DQ, Varga C (1997) Oxidative stress in Ahmed D, Sharma M, Kumar V, Bajaj HK, Verma A (2014a) 2β-hydroxybetulinic experimental diabetes induced by streptozotocin. Acta Physiol Hung acid 3β-caprylate: an active principle from Euryale Ferox Salisb. seeds 85:29–38 with antidiabetic, antioxidant, pancreas & hepatoprotective potential in Mayes PA (2000) The pentose phosphate pathway and other pathway of streptozotocin induced diabetic rats. J Food Sci Technol. doi:10.1007/ hexose metabolism. In: Murray RK, Granner DK, Mayes VW (eds) Herper’s s13197-014-1676-0 Biochemistry. McGraw-Hill, USA, pp 219–237 Ahmed D, Kumar V, Verma A, Gupta P S, Kumar H, Dhingra V et al (2014b) Moncada S, Palmer R, Higgs E (1991) Nitric oxide: physiology, pathophysiology Antidiabetic, renal/hepatic/pancreas/cardiac protective and antioxidant and pharmacology. Pharm Rev 43(2):109–142 potential of methanol/dichloromethane extract of Albizzia Lebbeck Benth. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxidation in animal tissues stem bark (ALEx) on streptozotocin induced diabetic rats. BMC Comple- by thiobarbituric acid reaction. Ann Biochem 95:351–358 ment Altern Med 14(1):243. doi:10.1186/1472-6882-14-243 Pitkänen OM, Martin JM, Hallman M, Åkerblom HK, Sariola H, Andersson SM Al-Shamaony L, al-Khazraji SM, Twaij HA (1994) Hypoglycaemic effect of (1992) Free radical activity during development of insulin-dependent Artemisia herba alba. II. Effect of a valuable extract on some blood diabetes mellitus in the rat. Life Sci 50(5):335–339 parameters in diabetic animals. J Ethnopharmacol 43:167–171 Rawi S, Abdel Moneim A, Ahmed OM (1998) Studies on the effect of garlic oil Andrade Cetto A, Wiedenfeld H, Revilla MC, Sergio IA (2000) Hypoglycemic and glibenclamide on alloxan diabetic rats. Egypt J Zool 30:211–228 effect of Equisetum myriochaetum aerial parts on streptozotocin diabetic Robert Langdon G (1966) Glucose-6-phosphate dehydrogenase from erythro- rats. J Ethnopharmacol 72:129–133 cytes. In: Willis A, Woo (eds) Methods in enzymology IX. Academic Press, Berg JM, Tymoczko JL, Stryer L (2001) Glycolysis and glyconeogensis. In: Berg New York JM, Tymoczko JL, Stryer L (eds) Biochemistry. WH Freeman and Company, Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG New York, pp 425–464 (1973) Selenium: biochemical role as a component of glutathione peroxi- Branstrup N, Krik JE, Bruni C (1957) The hexokinase and phosphogluco dase. Science 179:588 isomerase activities of aorta and pulmonary artery tissue in individuals of Saxena AK, Srivastava P, Baquer NZ (1992) Effects of vanadate on glycolytic various ages. J Gerontol B-Psychol 12:166–171 enzymes and malic enzyme in insulin-dependent and -independent Burnstein M, Scholnic HR, Morfin R (1970) Rapid method of isolation of tissues of diabetic rats. Eur J Pharmacol 1216:123–126 lipoproteins from human serum by precipitation with polyanions. J Lipid Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47:389–394 Res 11:583–587 Song CW, Wang SM, Zhou LL, Hou FF, Wang KJ, Han QB et al (2011) Isolation Das S, Der P, Raychaudhuri U, Maulik N, Das DK (2006) The effect of Euryale and identification of compounds responsible for antioxidant capacity of ferox (Makhana), an herb of aquatic origin, on myocardial ischemic reper- Euryale ferox seeds. J Agric Food Chem 59(4):1199–1204 fusion injury. Mol Cell Biochem 289(1–2):55–63 Sridhar SB, Sheetal UD, Pai MRSM (2005) Preclinical evaluation of the anti- Duke JA, Ayensu ES (1985) Medicinal plants of China. 2 Vols. 705 S., 1300 diabetic effect of Eugenia jambolana seed powder in streptozotocin- Strichzeichnungen, Reference Publ., Inc. Algonac. Michigan diabetic rats. Braz J Med Biol Res 38(3):463–468 Ellman G (1959) Tissue sulphydryl groups. Arch Biochem Biophys 32:70–77 Swanston-Flatt SK, Day C, Bailey CJ, Flatt PR (1990) Traditional plant treat- Foster LB, Dunn RT (1973) Stable reagents for determination of serum triglyc- ments for diabetes. Studies in normal and streptozotocin diabetic mice. erides by a colorimetric Hantzsch condensation method. Clin Chem Diabetologia 33:462–464 19:338–340 Ugochukwu NH, Babady NE (2002) Antioxidant effects of Gongronema lati- Friedewald WT, Levy RI, Fredrickson DS (1972) Estimation of the concentra- folium in hepatocytes of rat models of non-insulin dependent diabetes tion of low-density lipoprotein cholesterol in plasma, without use of the mellitus. Fitoterapia 73:612–618 preparative ultracentrifuge. Clin Chem 18:499–502 Ugochukwu NH, Babady NE (2003) Antihyperglycemic effect of aqueous and Gancedo JM, Gancedo C (1971) Fructose 1, 6-bisphophatase, phosphofruc- ethanolic extract of Gongronema latifolium leaves on glucose and glyco- tokinase and glucose 6-phosphate dehydrogenase from fermenting gen metabolism in liver of normal and streptozotocin-induced diabetic yeast. Arch Microbiol 76:132–138 rats. Life Sci 73:1925–1938 Gandy SE, Galbrath RA, Crouch RK, Buse MG, Galbraich GM (1981) Superoxide Zlatkis A, Zak B, Boyle AJ (1953) A new method for the direct determination of dismutase in human islets of Langerhans. N Engl J Med 304:1547–1553 serum cholesterol. J Lab Clin Med 41:486–492 Herbert V, Lau KS, Gottlieb CW, Bleicher SJ (1965) Coated charcoal immunoas- say of insulin. J Clin Endocrinol Metab 25:1375–1384 570 Biochemistry: Harms-Ringdahl et al. Proc. Nat. Acad. Sci. USA 70 (1973)

D10 600 SMG solution to yield a standard bacterial cell suspension of 0 3.0 X 107 celis per ml. .2. In order to measure the stimulation of RNA, protein, or 0 i DNA synthesis, the cells were incubated with the factor to 5, 8 400 W! be assayed at 350 in the presence of a suitable reaction mix- 04 ture containing ['HJuridine, or ['H phenylalanine, or ['HI- >, 2. thymidine; incorporation of these precursors was measured. E 200 The reaction mixture, derived from the one generally used in ., cell-free protein-synthetizing systems, was freshly prepared E every day. The composition of the reaction mixture was as follows: Tris HOl buffer (112 mM) (pH 7.8), NH4Cl (0.1 M), O " 0.4 2 10 50 160 KCl (3.7 mM), Mg acetate (2.6 mM), ATP (Na salt) (2.5 skI PHA-P/rrg test mM), GTP (Na salt) (0.6 mM), creatine phosphate (Na salt) (26 mM), creatine phosphokinase (0.13 mg/ml), sucrose FIG. 1. Effect of PHA-P (Difco) on RNA synthesis in chicken- (0.34 M), uridine (15 uM), each of 19 amino acids (except spleen lymphocytes and in plasmolyzed E. coli Q 13 cells. Incuba- phenylalanine) (70 uM), phenylalanine (15 MM), thymidine tion time was 150 mini for E. coli Q 13 and 40 hr for chicken-spleen (15 MM), 2-mercaptoethanol (10 mM), and ['H]uridine (7.5 lymphocytes. (The content of one vial of PHA-P, Difco, was dissolved in 5 ml of distilled water.) MCi/ml; 24 Ci/mmol) or ['H]phenylalanine (7.5 MCi/ml; 18 Ci/mmol) or ['H ]thymidine (7.5 MuCi/ml; 14.1 Ci/mmol). The incubation mixture for one experiment consisted of 134 of reaction mixture to which was added the to be mM uridine, and 0.44 ml of [3H containing 100 ,ul sample ]uridine MACi/ml tested dissolved in 66 Mul of distilled water. The incubation (specific activity 24 Ci/mmol). Medium T was the same, but was started by addition of 50 Mul of the standard bacterial cell contained 0.22 ml of 0.4 mM thymidine instead of uridine and suspension and placement of the tube in a 350 water bath. 0.44 ml of ['H ]thymidine (100 MCi/ml, 14.1 Ci/mmol) in- After an appropriate time, an aliquot of the incubated mixture stead of ['H ]uridine. was mixed with an equal volume of 10% sodium dodecyl sul- At the appropriate time (usually after 40-45 hr), the tubes fate, and after 15 min incubation at 350, when the cells were were chilled in ice. For determination of the amount of in- lysed, 50 Mul was transferred to the middle of a glass-fiber filter corporated uridine or thymidine, the assay suspension was (Whatman GF/C, 2.5 cm). The filter was dried for 1 min at treated as described by Goldberg et al. (8). The radioactivity room temperature and immersed in ice-cold 10% trichloro- was measured in a Nuclear Chicago Liquid Scintillation acetic acid. For investigation of aminoacid incorporation, the Counter model 6850 with an efficiency of 16%. The results filters were further processed to hydrolyze aminoacyl-tRNA obtained were expressed as pmol of uridine or thymidine and to eliminate unincorporated radioactivity (13). When in- incorporated in 106 cells. corporation of uridine or thymidine was measured, hydrolysis Assay for Stimulation of RNA, Protein, or DNA Synthesis with hot trichloroacetic acid was omitted, and the filters were in Bacteria. The bacterial system developed for study of cell washed several times with 5% ice-cold trichloroacetic acid, stimulation consists of E. coli cells subjected to plasmolysis. followed by washing with alcohol and ether. Finally the filters The treatment of the cells is related to the procedure applied were dried, placed in scintillation vials, and incubated at by Heppel in order to increase permeability of the bacterial 550 for 30 min with 0.5 ml of Soluene (TM 100, Packard). cell wall (11, 12). E. coli Q-13 (kindly provided by Dr. J. T. Then 10 ml of scintillation fluid (0.5% PPO and 0.005% August, Albert Einstein College, Bronx) was grown in a POPOP in toluene) was added, and radioactivity was mea- medium containing Na2HPO4* 2H20 (8.8 g), KH2PO4 (3.0 g), sured in a Nuclear Chicago Liquid Scintillation Counter model NaCl (0.5 g), NH4Cl (1.0 g), glycerol (3.0 g), casamino acids 6850 with an efficiency of 19%. The data obtained were ex- (technical) (6.0 g), and MgSO4 * 7H20 (1.25 g) per liter. The pressed as pmol of uridine (or thymidine or amino acid) medium was sterilized by filtration through a Sartorius mem- incorporated in 106 cells. brane filter, pore size 0.2 Mm. The culture was aerated by Quantitative Presentation of Activity of Stimulators. In the shaking at 370 until the bacteria reached a density of 10' figures and tables, the amount of stimulator present in 1 ml cells per ml. It was then cooled rapidly to 00, and the bacteria of incubation mixture is given. In calculations of yields, one were collected by centrifugation at 20 for 15 min at 12,000 X bacterial unit (B.U.) and one lymphocyte unit (L.U.) is taken g. The cells were suspended in the original volume of 30 mM to mean the amount of stimulator that has to be added to 1 Tris* HCl buffer (pH 8.1) at 00. After they were slowly shaken ml of the bacterial or lymphocyte test suspension, respec- for 5 min at 00, the centrifugation was repeated. The collected tively, in order to induce 50% of the maximum (saturation) cells were then suspended at 00 in SMG solution (containing stimulation with regard to uridine incorporation during 150 10% sucrose, 0.1% MgSO4 7H20, and 0.1% glucose) to min of incubation for E. coli Q 13 and 40 hr for chicken spleen yield a cell suspension of 6.0 X 108 cells per ml. The cell sus- lymphocytes (compare ref. 14). pension was stored at 20 and was used for experiments after RESULTS 12 hr and up to 1 week. During storage for 1 week, the rate of incorporation of uridine usually drops by a factor of 2, al- Effect of Commercial PHA in Lymphocytes and E. coli. though the ability to become stimulated relative to the controls Fig. 1 demonstrates the RNA synthesis stimulating activity remains practically unchanged. Before use, an aliquot of the of commercial PHA in E. coli cells, as well as in chicken- suspension was centrifuged as above, the supernatant was spleen lymphocytes. The same batch of PHA-P (Difco) was discarded, and the cell pellet was suspended at 00 in a new used in both systems. 1 L.U. corresponds to 2.7 Ml of PHA-P Proc. Nat. Acad. Sci. USA 70 (1973) Substances That Stimulate Nucleic Acid Synthesis 571

solution, and 1 B.U. to 9 /l of PHA-P solution. The usual inhibitory action of a high concentration of PHA appears in the lymphocyte system.

Extraction and Purification of Active Fractions from Beans. N ID PHA-P (Difco) is a rather complex extract of red kidney beans containing about 17 different proteins, as revealed by polyacrylamide gel electrophoresis (14). In order to isolate from the extract the substances that stimulate RNA synthe- sis responsible for the effects shown in Fig. 1, the following purification procedure was used: Red kidney beans (Phaseo- lus vulgaris) were extracted for preparation of "dry powder" (15). Fraction I (F I) was prepared by extraction of 6.0 g of "dry powder" (derived from 500 g of beans) with 100 ml of PM buffer (8). The cloudy suspension was centrifuged, the precipitate was discarded, and the supernatant was filtered through a Sartorius membrane filter, pore size 0.22 um. Unless otherwise indicated, all centrifugations were at 20 for 5 RF 1.0 + 20 min at 30,000 X g. To each ml of F I, 0.16 ml of 11.5 M HCl04 was added at 1.0 (c) 00, as described (8). After 40 min at O°, the suspension was 0.0 R centrifuged. The precipitate was discarded; the supernatant was neutralized at 00 by addition of about 0.2 ml of 8 M KOH to each ml of supernatant until pH 7.6 was reached. After 20 min at 00, the suspension was centrifuged and the precipi- C tate was discarded. To each 1 ml of supernatant, 0.43 ml of cold absolute ethanol was added at 00. After 50 min, the suspension was centrifuged, and both the precipitate (p3) _0 0.5 R. 10 + and supernatant (s3) were kept. The p3 was washed twice with 25 ml of ice-cold ethanol (30%) and was extracted at 00 FIG. 3. Polyacrylamide gel electrophoresis of (a) fraction F with 50 ml of 5 mM potassium phosphate buffer (pH 7.5). III, sample size 40 pl; (b) fraction F III p, sample size 60 Al; The turbid suspension was centrifuged, the precipitate was (c) fraction F V, sample size 30 Il. discarded, and the supernatant was kept and called F III. Besides protein (about 0.5-0.7 mg of N per ml), F III con- rated from the polysaccharide by precipitation at pH 2.5- tains a large amount of some polysaccharide containing about 3.0. This principle was used for the further purification of F 6.0 usmol of ribose-equivalent pentose per ml, measured by III: ml of F III was mixed with 1.0 ml of concentrated the orcinol reaction (16). Most of the lymphocyte-stimulating 50 acetic acid at 00, and after 30 min at 00 the suspension was activity present in the bean extract is associated with the centrifuged. The supernatant was freeze-dried, dissolved in protein fraction of F III. The protein fraction can be sepa- 50 ml of distilled water with a drop of diluted NaOH (to pH 7.8), and was called F III s. The precipitate was washed with 0 0.5 1.0 10 ml of ice-cold 1 M acetic acid, then freeze-dried, dissolved ATM E in 50 ml of distilled water with a drop of diluted NaOH (to was F III E pH 7.8), and called p. .i- Most of the bacteria-stimulating activity remained in s3, I' ',N was with an CL .0 which was further purified as follows: s3 mixed ,I I E 40 0.4 equal volume of 95% ethanol, and the mixture was boiled E 0 m (about 800) for 5 min under reflux and in N2 atmosphere. 4 *. 1 I , 2 2.0 I++.++ Ig1~ - 0.2 The warm suspension was centrifuged (20,000 X g, room + o2 ,I temperature, 10 min), the precipitate was discarded, and the 2832 3640 4448 2 56 60 64 6872 supernatant was evaporated at 500 under reduced pressure to Froction number a final volume of 25 ml. The solution was centrifuged (20,000 FIG. 2. Gel filtration of fraction F IV on Sephadex G-50 fine. X g, room temperature, 10 min), and the resulting 24-ml Bed diameter: 3.2 cm; bed volume: 320 ml; flow rate: 10 ml/hr; supernatant was mixed with 1.2 ml of Pronase solution con- eluant: 1 mM phosphate buffer (pH 7.6). Sample size was 4 ml taining 10 mg of Pronase per ml in 0.1 M NaCl-0.05 M Tris- of F IV. Fraction size was 5 ml. Temperature 5°. For calibration HCl buffer (pH 7.8). After incubation at 40° for 4 hr the solu- of the Dextran 2000 and tritiated water were used. column, Blue tion was mixed with 2 volumes of 95% ethanol, boiled for 5 K., is calculated from the formula K,<", = (ve - vo)/(vt - vo), min in N2 atmosphere and reflux, and centrifuged (20,000 X where ve is the elution volume, vo the void volume, and vt the total g, room temperature, 10 min). The supernatant was concen- volume. RNA synthesis-stimulating activity in E. coli is ex- to a final volume of 6-7 pressed in B.U./ml. For definition of B.U. see Methods, and com- trated at 500 under reduced pressure inter- pare Table 1. Pentose was determined by the orcinol reaction ml, and was then dialyzed against three changes at 4-hr (16). Pentose (+++); absorbancy ( ); bacterial units vals of 1 liter of distilled water at 20. The dialyzed solu- tion was centrifuged; the final volume of the supernatant was 572 Biochemistry: Harms-Ringdahl et al. Proc. Nat. Acad. Sci. USA 70 (1973)

especially of RNA synthesis in the bacteria, but its action in the lymphocyte system is limited to a although signifi- 450 small, 0 A~~~ cant, increase of nucleic-acid synthesis, probably a conse- 300- quence of a delayed dying of the cells. The mechanism of action of the bacteria-stimulating frac- cs tion, F V, was studied with rifampicin and chloramphenicol, E inhibitors of RNA and protein synthesis, respectively. Table 3 shows that F V increased RNA synthesis in rifampicin- 0.5 1.0 1.5 blocked cells. Incorporation of phenylalanine was also in- log o g N of F 7mI test creased by F V in rifampicin-blocked cells, whereas, in the presence of chloramphenicol, only a minute effect of F V on FIG. 4. Effect of rifampicin per ml test. (0) Control; (U) 0.5 incorporation of phenylalanine could be observed. The RNA Mg; (A) 2.0 lg; (*) 4.0 ug. Incubation time, 150 min. synthesis of chloramphenicol-blocked cells could be stimulated proportionally to that of the uninhibited control. The de- adjusted to 10 ml and was called F IV. F IV was further puri- creased incorporation of uridine in chloramphenicol-treated fied by gel filtration on Sephadex G-50. A typical pattern of cells shows that the RNA synthesis was promoted by products gel filtration of 4 ml of F IV is shown in Fig. 2. Fractions no. of protein synthesis. The dose-response curves of F V in the 40-52 were combined (48 ml), freeze-dried, dissolved in 4 ml bacterial system at different rifampicin concentrations (Fig. of distilled water, and called F V. 4) suggest that F V in some way counteracted the RNA syn- thesis-inhibiting effect of rifampicin. Biological Activity of Fractions from Bean Extract. Tests for the ability of different fractions to stimulate RNA syn- DISCUSSION thesis in the two biological systems are presented in Table 1. Using chicken-spleen lymphocytes and plasmolyzed E. coli The lymphocyte-stimulating activity of F I was recovered cells, we isolated RNA synthesis stimulating factors, here with a 93% yield in F III from which F III p was prepared by called F III p and F V from an extract of red kidney beans, acid precipitation without further loss of lymphocyte-stimu- the source of commercial phytohemagglutinins. The lympho- lating activity. The orcinol-positive fraction F III s had neither cyte-stimulating component of the bean extract was found in a lymphocyte- nor bacteria-stimulating activity. Bacteria- fraction, here called F III, that consists mainly of two proteins stimulating activity was found in F V, which was obtained and carbohydrates. The biologically active proteins, F III p, from F I with 12% yield. which appear to be the well-known mitogens for lymphocytes, Electropherograms of the biologically active fractions (Fig. could be separated from contaminating carbohydrates by 3) indicate that the lymphocyte-stimulating fraction, F III acid precipitation. The property of stimulation of lymphocytes p, consists of two main proteins. The bacteria-stimulating and the preliminary determination of the aminoacid composi- fraction, F V, contains one protein. Typical experiments demonstrating the lymphocyte- and bacteria-stimulating TABLE 2. Effect of F III p and F V on RNA and DNA properties of F III p and F V are shown in Table 2. At a synthesis in lymphocytes and in bacteria* concentration of 2.7 ,ug N/ml, F III p gives the strong stimulation of lymphocytes characteristic of phytohemag- F III F III p glutinins. This concentration of F III p is without effect in the p + F V bacterial system, and even at a 50-times higher concentration, (140 F V (2.7 Ag only a weak effect is obtained. F V gives a strong stimulation, F III p ,Ag (8.3 N/ml + Con- (2.7 Ag N! Mg 8.3 Ag Hr trol N/ml). ml) N/ml) N/ml) TABLE 1. Biological activity and content of nitrogen and pentose* in different fractions of the bean extract Lymphocytes 18 0.85 6.70 - 1.18 6.71 Nitrogen RNA syn- (100)t (789) (139) (790) Activity in per unit thesis 42 0.29 19.1 0.53 19.5 (100) (6,660) (187) (6,800) Lym- E. Lym- - 2.48 Pen- pho- coli pho- 18 0.14 2.02 0.26 DNA syn- (100) (1,470) (188) (1,610) tose E. coli cytes (Mg cytes 0.051 6.25 Fraction N (Mmol/ (B.U./ (L.U./ N/ (Mg thesis 42 0.028 6.50 (ml) (mg/ml) ml) ml) ml) B.U.) N/L.U.) (100) (23,400) (183) (22,400) F I (100) 2.3 28.0 230 3600 10.0 0.64 Bacteria F III (50) 0.60 5.4 4 6700 (150) 0.09 RNA syn- 2.5 88 89 201 448 440 F III s (50) <0.05 4.8 0 0 thesis (100) (100) (228) (510) (500) III p (50) 0.54 <0.2 2.5 6700 (220) 0.08 DNA syn- 2.5 1.40 1.35 1.52 2.82 2.89 F V (10) 0.67 <0.2 270 t 2.5 -t thesis (100) (96) (113) (202) (206) For compari- son: PHA-P 1.30 1.2 100 500 13.0 2.6 * Given as pmol of uridine and thymidine incorporated, re- spectively, per 106 cells. * Ribose equivalents were determined with orcinol (16). t Incorporation in % of the respective control is given in t A slight activity was observed; see text and Table 3. parenthesis. Proc. Nat. Acad. Sci. USA 70 (1973) Substances That Stimulate Nucleic Acid Synthesis 573

TABLE 3. RNA, DNA, and protein synthesis and effects of glutinin, is here stated. Pokeweed mitogen is the only plant rifampicin and chloramphenicol on incorporation of protein that contains a similarly high amount of cysteine (17); [3H]uridine and [3H]phenylalanine in E. coli. this protein activates lymphocytes to cell division as well as to large-scale production of immunoglobulins (18). Under the Uridine Phenylalanine Thymidine conditions in the present study, F V had only a weak RNA Control F V Control F V Control F V synthesis-stimulating effect on lymphocytes. This effect was not comparable to the characteristic activating effect in- Control 81(100) (490)398 15.4(100) (320)48 1.32(100) 2.75(208) duced by F III p. The particular stimulating effect of F V on RNA synthesis Rifampicin 2.47 53 0.83 7.7 (9 ug/ml) (100) (2150) (100) (930) in the bacterial system is not yet understood. The antagonism Chloram- of F V and rifampicin (see Fig. 4) indicates that F V might act 17.0 95 1.82 2.48 on the level of DNA-dependent RNA polymerase. However, phencol) (100) (633) (100) (136) this hypothesis was not supported by in vitro studies [done ac- cording to Sippel et al. (19), kindly performed by Dr. G. R. Inhibitors were added before the start of the test, the duration Hartmann, Institute of Biochemistry, University of Wurz- of which was 150 min. F V was added to a final concentration of burg, Wurzburg] of the action of F V on the purified enzyme 6 ,gg N per ml test. Incorporation of precursors is given in pmol in the presence and absence of rifampicin. This result leads to per 106 cells, and, in parenthesis, as % of the respective control. the assumption that F V modifies the rate of RNA synthesis on some other level. tion of F III p show that it is identical or similar to the pooled We thank Mrs. Marie-Louise Hanngren for skillful assistance L-PHAP + H-PHAP, described et al. and AB KABI, Stockholm, for preparation of alcohol-ether dried proteins, by Allen (14). bean powder. The work was done within the frame of collabora- The orcinol-positive fraction, F III s, was inactive in the tion of the Hungarian Academy of Sciences and the Department tests applied. This fraction contains no protein. By comparing of Radiobiology, University of Stockholm; it was supported the electropherograms of F III and F III p (Fig. 3), one can financially by the Swedish Board for Technical Development and see that removal of this component did not influence the elec- by the Swedish Natural Science Research Council. trophoretic patterns. 1. Cooper, H. L. & Rubin, A. D. (1965) Blood 25, 1014- The bacteria-stimulating component of the bean extract *1027. was found in a fraction, called F V, which gives one band in 2. Pogo, B. G. T., Allfrey, V. G. & Mirsky, A. E. (1966) Proc. Nat. Acad. Sci. USA 55, 805-812. polyacrylamide gel electrophoresis. 3. Mueller, G. C. & Le Mahieu, M. (1966) Biochim. Biophys. RNA synthesis in the bacterial system cannot be stimulated Acta 114, 100-107. by F III p, the slight effect observed being assignable to the 4. Kay, J. E. (1968) Eur. J. Biochem. 4, 225-232. small contaminating amount of F V revealed by polyacryl- 5. Epstein, L. B. & Stohlman, F. Jr. (1964) Blood 24, 69-75. amide the other F V a 6. Cooper, H. L. (1970) in Proc. Vth Leukocyte Culture Conf., gel electrophoresis. On hand, gives only ed. Harris, J. E. (Academic Press, New York), pp. 15- weak stimulation of RNA synthesis in the lymphocyte sys- 30. tem. The slight increase in uridine incorporation and the 7. Rivera, A. & Mueller, G. C. (1966) Nature 212, 1207-1210. better survival of lymphocytes caused by F V is not compa- 8. Goldberg, M. L., Rosenau, W. & Burke, G. C. (1969) rable to the characteristic lymphocyte-stimulating effect of F Proc. Nat. Acad. Sci. USA 64, 283-289. 9. Fromageot, H. P. M. & Zinder, N. D. (1968) Proc. Nat. III p. Acad. Sci. USA 61, 184-191. These observations lead us to conclude that the lympho- 10. Weber, W. T. (1970) J. Reticuloendothel. Soc. 8, 37-54. cyte-stimulating substance (F III p) and the bacteria- 11. Heppel, L. A. (1967) Science 156, 1451-1455. stimulating substance (F V) in the bean extract are different 12. Heppel, L. A. (1968) in Methods in Enzymology, "Nucleic entities that exert their actions by different mechanisms. Acids," eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. XII, Part B, pp. 841-846. Preliminary data indicate that the aminoacid composition of 13. Mans, R. J. & Novelli, G. 1). (1961) Arch. Biochem. Biophys. F V is different from that of F III p. F V contains about 20 94, 48-53. mol % cysteine, and the molecular weight seems to be 9,500- 14. Allen, L. W., Svenson, R. H. & Yachnin, S. (1969) Proc. 10,000, as measured by ultracentrifugation or gel filtration (to Nat. Acad. Sci. USA 63, 334-341. 15. B6rjeson, J., Bouveng, R., Gardell, S., Norden, A. & Thunell, be published). F V and the components of F III p agree, how- S. (1964) Biochim. Biophys. Acta 82, 158-161. ever, with respect to the N-terminal amino acid, only serine 16. Ceriotti, G. (1955) J. Biol. Chem. 214, 59-70. being found in this position. 17. Reisfeld, R. A., B6rjeson, J., Chessin, L. N. & Small, P. A., The uniqueness of a protein, here called F V, with regard to Jr. (1967) Proc. Nat. Acad. Sci. USA 58, 2020-2027. its specificity to stimulate RNA synthesis in the bacterial 18. Parkhouse, R. M. E., Janossy, G. & Greaves, M. F. (1972) Nature New Biol. 235, 21-23. system, unusually high cysteine content, low molecular weight, 19. Sippel, A. E. & Hartmann, G. R. (1970) Eur. J. Biochem. and presence in red kidney beans, the source of phytohemag- 16, 152-157.