International Journal of Current Biotechnology

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1 Chinnaiah Alagarasan and Ganesan Vijaiyan Siva, Oral administration of ethanolic extract of Delphinium denudatum Wall, and its bio efficacy in Wister albino rats, Int.J.Curr.Biotechnol., 2016, 4(1):1-7. International Journal of Current Biotechnology ISSN: Journal Homepage : Oral administration of ethanolic extract of Delphinium denudatum Wall, and its bio efficacy in Wister albino rats Chinnaiah Alagarasan and Ganesan Vijaiyan Siva* Department of Biotechnology, University of Madras, Guindy Campus, Chennai Tamilnadu, India. A R T I C L E I N F O Article History: Received 05 January 2016 Received in revised form 14 January 2016 Accepted 22 January 2016 Available online 30 January 2016 Key words: Delphinium denudatum, oral toxicity, Animal modeling. A B S T R A C T Delphinium denudatum. Wall, (Dd) dried root sample were collected from authentic supplier and was further confirmed by taxonomist and extracted by absolute ethanol for oral toxicity studies. Based on this study, conclude that the extract from medicinal herb Delphinium denudatum can be administered at a dose of 1000 mg/kg/bw without any side effects. Since, the toxicity studies in experimental animals cannot always be totally extrapolated to humans, and a reasonable estimate of the self administered dose is difficult to make such as that applied during traditional use of this plant, additional clinical toxicological evaluations need to be performed to define a safe dose and protect the population from possible toxic effects of the plant. Introduction About 80% of the world population, mainly in the developing countries, depends on herbal medicine for primary health care. These medicines are more culturally acceptable, have better compatibility with the human body and lesser side effects (kamboj, 2000) 1. India has a rich diversity of medicinal plants and a number of plant extracts are used against diseases in various medicinal applications, e.g. Ayurveda, Unani, and Siddha. However, inspite of widespread use, only a few of these plants have been scientifically explored (Malaya, 2004) 2. Delphinium denudatum (Ranunculaceae) is a potent herbal medicine used in the Indian system of medicine, was known to possess diverse biological functions like antiseptic, anti diuretic, anti-inflammatory, antifungal (G.Vijaiyan Siva et al., , Rahman et al., 1997) 4, sedative, analgesic and as a nerve tonic. Silver nanoparticals synthesized fron Dd root extract exhibits antibacterial and mosquito larvicidal activity. (G.Vijaiyan Siva et al., 2014) 5 It is a great antidote to aconitum poisoning. Alkaloid delphinine is an antidote against muscarine and digitaline (Chatterji et al., 1997) 6. The root is used in Bashahr for toothache and also as an adulterant for aconite (Stewart). Root found beneficial in rheumatism, impotency and syphilis. (Rastogi et al., 1999) 7 Combined with other herbal stimulants, it is used as remedy in cardiac and cerebral diseases. Dried roots are a popular folk remedy for the treatment of epilepsy in the traditional *Corresponding author. address: gvs.bio@gmail.com Unani system in medicine (Kirthikar and Basu, 1999) 8. Active components of this extract include alkaloids, terpenoids. Any plant extract to be medically useful as therapeutic agent, it should be non-toxic or of low toxicity to human cells. Though Delphinium denudatum is widely used, limited information is available on the toxicity of this plant extract. This study is designed to determine the toxicity profile of the crude ethanolic extract of Delphinium denudatum Wall. (Jadwar) in Albino rats (Wistar strain). The acute oral toxicity study was conducted according to the OECD/OCDE guidelines 423/ , using various concentrations of Delphinium denudatum extract. Materials and Methods Chemicals: All the chemicals, solvents and reagents were of analytical reagent grade. Collection of plant samples The dried roots of Delphinium denudatum was procured from authentic suppliers from Hyderabad. The identity and confirmed by taxonomist, Center for advance studies in botany, University of Madras, Guindy campus, Chennai. Ethanolic Extraction Plant extracts were prepared from dried roots of Delphinium denudatum by using the procedure described by Phongpaichit et al., in The dried plant material was powdered by using mechanical grinder. 40 grams of dried Delphinium denudatum was packed well in the soxlets apparatus with the help of filter paper. 1 Int.J.Curr.Biotechnol. Volume 4; Issue 1; Jan, 2016

2 350 ml of absolute ethanol is taken as the extraction solvent. The extraction was carried out for 15 cycles at 55ºC, the crude ethanolic extract was further concentrated by evaporation at 55ºC using a solvent trap. Animals Male Wister albino Rats (6 8 weeks old) of body weight g was used for the study and given standard pellet diet and water ad labium. The control animals are treated with 0.9% saline, varied concentration of Delphinium denudatum extract using distilled water as the dose vehicle is administered for 21 days. All experiments performed as per the recommendations of Institution animal ethics committee with 6 animals in each group. Experimental design The experimental animals were divided into four groups, each group comprising of six animals. Group 1 served as normal control while those in group 2, 3 and 4 were fed orally with ethanolic extract of Delphinium denudatum at 100, 500 and 1000mg/kg BW, respectively, for 21 days. The body weight changes of control and experimental animals are being monitored every interval. At the end of the experimental period, the animals were anesthetized with diethyl ether followed by cervical decapitation, blood was taken by cardiac puncture, the liver and kidney were removed under ether anesthesia, washed thoroughly in ice-cold physiological saline [0.9% (w/v) NaCl], and weighed. Whole blood was separated in two tubes contain absence and presence of EDTA, for separation of serum and plasma, respectively, and the plasma and serum was stored frozen until further analysis. Hematology: The hematological parameters were analyzed using Horiba ABS 80 Diagnostics (ABX Pentra Montpellier, France). These parameters include red blood cells, white blood cells, neutrophils, monocytes, lymphocytes, eosinophils, basophils and platelets. Biochemical parameters Estimation of SGOT, SGPT and LDH The Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were estimated by UV kinetic method in which both SGOT and SGPT were assayed based on enzyme coupled system; where keto acid formed by the aminotransaminase reacts in a system using NADH. The coenzyme is oxidized to NAD and decrease in absorbance at 340 nm for SGOT malate dehydrogenase (MDH) reduces to malate with simultaneous oxidation of NADH to NAD. The rate of oxidation of NADH is measured, where as SGPT the pyruvate formed in the reaction is converted to lactate by lactate dehydrogenase (LDH). LDH was estimated in serum samples from each group of experimental animals. Estimation of ALP Alkaline phosphatase was assayed by the method of King et al., The incubation mixture of (final volume 3.2 ml) contained 1.5 ml of buffer, 1.5 ml of substrate, 0.1 ml MgCl 2 and 0.1 ml serum sample. The tubes were incubated at 37 C for 15 min and the reaction was arrested by the addition of Folin s phenol reagent. The content was then centrifuged. To the supernatant 1 ml of sodium carbonate was added and the tubes were incubated for a period of 10 min at 37 C. The colour developed was read against a reagent blank at 640 nm in Shimadzu UV spectrophotometer. The activity of ALP was expressed as ìmoles of phenol liberated/min/mg protein. Estimation of AST Aspartate transaminase was assayed by the method of King et al., ml of aspartate substrate was added to 0.1 ml of serum, incubated at 37 o C for 1 hr to which 1.0 ml of 2, 4-dinitrophenyl hydrazine reagent was added to arrest the reaction. To the blank tubes, 0.1 ml of double distilled water was added instead of serum and the tubes were incubated for 15 min, followed by addition of 10 ml of 0.4 N sodium hydroxide. The absorbance was read immediately at 520 nm in a Shimadzu UV spectrophotometer and the enzyme activity was expressed in µmoles of pyruvate liberated/min/mg protein. Estimation of ALT The activity of Alanine transaminase was assayed by the method of (King et al., 1965) ml of alanine substrate was added to 1ml of serum, incubated at 37 o C for 30 min, to which 1.0 ml of 2, 4-dinitrophenyl hydrazine reagent was added to arrest the reaction. To the blank tubes, 0.1 ml of double distilled water was added instead of serum and the tubes were incubated for 15 min, followed by addition of 10 ml of 0.4 N sodium hydroxide. The absorbance was read immediately at 520 nm in Shimadzu UV spectrophotometer and the enzyme activity was expressed in µmoles of pyruvate liberated/min/mg protein. Histopathological evaluation Histological evaluation was performed in the liver tissues and a portion of specimen was fixed in 10% formalin and embedded in paraffin wax. Sections were cut at 4 ìm in thickness, stained with hematoxylin and eosin (Booran et al., 1990) 12 and viewed under light microscope for histological changes. Statistical analysis All the grouped data were evaluated for statistical significance with SPSS v.10 software. Hypothesis testing methods included one way analysis of variance followed by least significant difference test. P values of less than 0.05 were considered to indicate statistical significance. All these results were expressed as mean ± S.D for six animals in each group. Results The body weight of the animals treated with the extract at the doses of 100, 500 and 1000 mg/kg BW and the control group were appreciably increased throughout the experimental period (Fig. 1) No significant changes were observed in the organs body weight ratio of the kidney, liver, lung, and heart as compared with control animals (Fig. 2). The hematological parameters The effect of oral administration of the ethanolic extract of Dd of the does investigated on hematological parameter and its function indicates in male Wister rats for 21 days. The extract did not significantly alter the level of RBC but efficiently reduced the level of WBC and its differentials, basophils, monocytes and as well as platelets (Table 1). The throughout the experimental period, while lymphocytes only reduced in Group 2 and Group 3 but significantly increased (p<0.05) at Group 4. The levels of eosinophils were not altered by the extract, whereas those neutrophils were markedly increased at specific dose. Volume 4; Issue 1; Jan, 2016 Int.J.Curr.Biotechnol. 2

3 Table 1: The effect of ethonoloic extracts of Delphinium denudatum on the haematological parameters of albino Wister rats. Delphinium denudatum ethanolic extracts (mg/kg body weight) Parameters Group 1 Group 2 Group 3 Group 4 RBC (X10 12 /l) 08.67± ± ± ±0.23 * WBC (X10 9 /l) 17.00± ± ± ±2.10 Neutrophils (%) 08.37± ± ± ±1.23 Monocytes (%) 16.53± ± ± ±2.64 Lymphocytes (%) 65.40± ± ± ±2.56 Eosinophils (%) 03.70± ± ± ±1.23 Basophils (%) 0.53± ± ± ±0.17 Platelets X ± ± ± ±12.46 Table 2: The effect of ethonoloic extracts of Delphinium denudatum on the SGOT, SGPT and creatine levels in control and experimental groups of animals. Parameters SGOT SGPT Creatine Group ± ± ±0.89 Group ± ± ±1.11 Group ± ± ±0.46 Group ± ± ±1.23 Figure 1: The effect of ethanolic extracts of Delphinium denudatum on the body weight of albino Wister rats.. G1: Control animals receiving only 0.9% saline water. G2: Animals fed with 100 mg/kg BW of extract, G3: animals fed with 500 mg/kg BW of extract, G4: animals fed with 1000 mg/kg BW of extract 3 Int.J.Curr.Biotechnol. Volume 4; Issue 1; Jan, 2016

4 Figure 2: The effect of ethanolic extracts of Delphinium denudatum on the organ body weight ratio of albino Wister rats.. G1: Control animals receiving only 0.9% saline water. G2: Animals fed with 100 mg/kg BW of extract, G3: animals fed with 500 mg/kg BW of extract, G4: animals fed with 1000 mg/kg BW of extract. No significant changes (p> 0.05) were observed in the organs body weight ration of the kidney, lung, liver and heart as compared with control group Figure 3: Serum markers of toxicological evaluation Figure 4: Serum LDH for toxicological evaluation Volume 4; Issue 1; Jan, 2016 Int.J.Curr.Biotechnol. 4

5 Figure 5: Histopathological observation of liver tissue viewed under light microscope. H & E staining (40 H & E) (A) Control animals showed normal architecture of the liver tissue. (B) Group mg/bw kg Dd treated animal cells (C) Group mg/bw kg Dd treated animal cells (D) Group mg/bw kg Dd treated animal cells showed normal architecture as that of control animals. Biochemical parameter The results indicate the non-toxic nature of the extract and between the two doses, mg/kg BW was found to be more effective. No Significant increases in AST, ALT and ALP were observed from Group 2 to Group 4 ( mg/kg/bw) as compared to normal control animals (Group 1) (Fig. 3). Serum AST, ALT and ALP, are the most sensitive markers employed in the diagnosis of hepatic damage because these are cytoplasmic in location and are released into the circulation after cellular damage (Sallie, 1991) 13. Ethanolic extracts of Dd at doses of mg/kg/bw (Group 2 to 4) did not show any effect on levels of AST, ALT and ALP concentrations as compared to normal control animals (Group 1). Lactate dehydrogenase Lactate dehydrogenase transfers hydrogen using NAD+ as hydrogen acceptor thus catalyzing the oxidation of L- lactate to pyruvate. LDH activity is present in all the cells of the body predominantly in cytoplasm of the cell. Thus tissue levels are 500 times greater than those in serum, thus even a small mass of damaged tissue causes leakage of enzyme and increasing its level in serum significantly. Ethanolic extracts of Dd at doses of mg/kg/bw (Group 2 to 4) did not show any effect on levels of serum LDH concentrations as compared to normal control animals (Group 1) (Fig. 4). Estimation of Serum SGOT, SGPT and creatine The enzyme SGOT and SGPT that is normally present in liver and heart cells. SGOT and SGPT are released into blood when the liver or heart is damaged. The blood SGOT levels are thus elevated with liver damage or with an insult to the heart. Serum SGOT, SGPT and creatine are the most sensitive markers employed in the diagnosis of hepatic damage and heart failure. Ethanolic extracts of Dd at doses of mg/kg/bw (Group 2 to 4) did not show any effect on levels of Serum SGOT, SGPT and creatine concentrations as compared to normal control animals (Group 1) (Table 2). 5 Int.J.Curr.Biotechnol. Volume 4; Issue 1; Jan, 2016

6 Histological examination The histological examination of liver section of control and experimental groups of animals related that, liver from control (Group 1) animals showed a normal architecture of cells with small uniform nuclei (Fig. 5A). Ethanolic extracts of Dd at doses of mg/body kg bearing animals (Fig. 5B, C, D) showed same architecture of the cells compared with control group animals exhibited no toxic effect of this experimental concentration of ethanolic extracts of Dd. Discussion Herbal medicines have attained greater importance as an alternative to conventional therapy. To optimize the safe use of a plant-based medicine, one should take into account their historical applications on humans and animals as well as toxicity evaluation of the medicinal herbs and their active components (Mukinda et al., 2007) 14. Many screening methods are employed to determine the safety and efficacy of these herbal medicines and also to establish the active component of the herbal products (Sim et al., 2010) 15. However, the scientific validation of its safety and efficacy has not been established so far. The present study gives detailed information on the toxicological profile of Delphinium denudatum oral toxicity studies in rats. A limit test was performed in oral toxicity study. According to the OECD test guideline 423/2001 when there is information in support of low or non-toxicity and immortality nature of the test material, then the limit test at the highest dose level (1000 mg/kg body weight) was conducted. There were no mortality and toxicity signs observed at 1000mg/ kg. Therefore, it can be concluded that Dd when administered at single dose is non-toxic and can be used safely in oral formulations. A 21-day oral toxicity study was performed followed OECD test guideline 423/2001 in Albino rats (Wistar strain). Since examination of clinical signs plays major role in toxicological testing (Stevens et al) 16. Dd did not produce any alterations in feed and water consumption and this reveals that it did not adversely affect the basic metabolic processes of the experimental animals. The haemopoietic system serves as important target for toxic chemicals and is a sensitive index for pathological conditions both in humans and animals (Adeneya et al.,) 17. In the present study, treatment with Dd did not produce any alteration in hematological parameters (i.e. RBC, WBC neutrophils, monocytes, lymphocytes, eosinophils, basophiles and platelets.), which indicate that Dd did not affect blood cells nor their production. Clinical biochemistry and hematological data holds significant role in determining the toxicity induced by drugs. Transaminases (SGOT and SGPT) are good indicators of liver function and biomarkers to predict the possible toxicity of drugs (Hilaly et al.,) 18. Any elevation pertaining to these enzymes indicate their outflow into the blood stream due to damage in liver parenchyma cells. There were no changes in the SGPT and SGOT levels which reveal that Dd did not affect liver function/or metabolism. In the present study, there were no treatment related abnormalities in renal function and other biochemical parameters suggesting that Dd is non-toxic. Histopathological studies provide supportive evidence for biochemical and hematological observations (Matsuzawa et al.,) 19. The relative organ weights were found to be nonsignificant between the control and Dd treated rats. No abnormality was recorded with respect to gross or histopathological examinations of liver examined. Since there were no signs of toxicity with respect to hematology, clinical chemistry, organ weight, gross and histopathological examinations noted in Dd it can be inferred that Dd will not produce delayed onset of toxicity. Based on these results, the No Observed Adverse Effect Level (NOAEL) of Delphinium denudatum is greater than 1000 mg/kg/day. Conclusion Based on this study, we conclude that the medicinal herb Delphinium denudatum can be administered at a dose range of 1000 mg/kg/bw without any side effects. Since, the toxicity studies in experimental animals cannot always be totally extrapolated to humans, and a reasonable estimate of the self administered dose is difficult to make such as that applied during traditional use of this plant, additional clinical toxicological evaluations need to be performed to define a safe dose and protect the population from possible toxic effects of the plant. References 1. Kamboj VP, Herbal Medicine. Current Science 1: Malaya G, Upal KM, Ramanathan SK, Thangavel S, Madgula L, Mohan V, Antitumor Activity and Antioxident Status of Caesalpinia Bonducella against Ehrlich Ascites Carcinoma in Swiss Albino Mice. J Pharmacol Sci. 94: Vijaiyan Siva.G, Sudha Revathy.S, Kaiser Rabee.U.Md (2006) Bioeficacy of the roots of delphinium denudatum (DD). Wall, against curvularia lunta. Journal of Drug Research in Ayurvedha and Siddha. Central council for research in ayurvedha and siddha.(ayush) Vol.XXVII, NO.1-2, pp Rahman A, Nasreen A, Akhtar F, Shekhani S, Clardy J, Parvez M and Choudhary I(1997) Antifungal diterpenoid alkaloids from delphinium denudatum J.Nat. Prod 60: Suresh G, Gunasekar PH, Kokila D, Prabu D, Dinesh D Ravichandran N Ramesh B, Koodalingam A, Vijaiyan Siva G(2014) Green synthesis of silver nanoparticales using Delphinium denudatum root extract exhibits antibacterial and mosquito larvicidal activities. Spectrochim Acta A Mol Biomol Spectrosc. 5;127: Chatterji A and Prakash C The treatise of Indian medicinal plant volume 1 (1997). 7. Rastogi P and Mehrotra N (1999) Compendium of Indian medicinal plants volume 1 8. Kirthikar and Basu, (1999) Indian medicinal plants volume 9. OECD: Guidelines for the Testing of Chemicals/Section 4: Health Effects Test No.423: Acute Oral toxicity - Acute Toxic Class Method.Paris,France:Organization for Economic Cooperation and Development; Phongpaichit.S, Pujenjob.N, Rukachaisirikul.V, and Ongsakul, Antimicrobial activities of the crude methanol extract of Acorus calamus Linn. Songklanakarin. Journal of Science and Technology 27: King J (1965) the transferase alanine and aspartate transaminase. In: Van.D. (Ed.). Practical clinical enzymology. Nostrand Co.Ltd, London, 1965a, pp Booran GA, Eustis SL, Elwell MR, Mont gory CA, Makenzie WF (1990) Pathology the fisher rat. Reference.D Atlas. Academic press, San Diego Newyork, London. Pp Sallie, R., R.S. 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7 14. Mukinda JT, Syce JA: Acute and chronic toxicity of the aqueous extract of Artemisiaa fra in rodents. J Ethnopharmacol 2007, 112: Sim KT, Sri Nurestri AM, Sinniah SK, Kim KH, Norhanom AW: Acute oral toxicity of Pereskia bleo and Pereskia grandiofolia in mice. Pharmacogn Mag 2010, 6: Stevens KR, Mylecraine L: Issues in chronic toxicology. In Principles and Methods of Toxicology. thirdth edition. Edited by Hayes AW. New York: Raven Press; 1994: Adeneyea AA, Ajagbonnab OP, Adelekec TI, Bellod SO: Preliminary toxicity and phytochemical studies of the stem bark aqueous extract of Musanga cecropioides in rats. J Ethnopharmacol 2006, 105: Hilaly JE, Israili ZH, Lyouss B: Acute and chronic toxicological studies of Ajuva Iva in experimental animals. J Ethnopharmacol 2004, 91: Matsuzawa T, Nomura M, Yonezawa H, Unno T: Selection of appropriate parameters, use of a quality control concept, and suitable statistical analyses for clinical pathology examination of animals in toxicity studies: results of a current survey by the Japanese Pharmaceutical Manufacturers Association. Comp Haematol Int 1995, 5: Int.J.Curr.Biotechnol. Volume 4; Issue 1; Jan, 2016