Chinnaiah Alagarasan and Ganesan Vijaiyan Siva, Oral administration of ethanolic extract of denudatum Wall, and its bio efficacy in Wister albino rats, Int.J.Curr.Biotechnol., 2016, 4(1):1-7.

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Oral administration of ethanolic extract of Delphinium denudatum Wall, and its bio effi- cacy in Wister albino rats

Chinnaiah Alagarasan and Ganesan Vijaiyan Siva*

Department of Biotechnology, University of Madras, Guindy Campus, Chennai - 600 025. Tamilnadu, .

ARTICLE INF O ABSTRACT

Article History: Delphinium denudatum. Wall, (Dd) dried root sample were collected from authentic Received 05 January 2016 supplier and was further confirmed by taxonomist and extracted by absolute ethanol Received in revised form 14 January 2016 for oral toxicity studies. Based on this study, conclude that the extract from medicinal Accepted 22 January 2016 herb Delphinium denudatum can be administered at a dose of 1000 mg/kg/BW with- Available online 30 January 2016 out 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 Key words: dose is difficult to make such as that applied during traditional use of this , Delphinium denudatum, oral toxicity, additional clinical toxicological evaluations need to be performed to define a safe Animal modeling. dose and protect the population from possible toxic effects of the plant.

Introduction Unani system in medicine (Kirthikar and Basu, 1999)8. About 80% of the world population, mainly in the Active components of this extract include alkaloids, developing countries, depends on herbal medicine for terpenoids. Any plant extract to be medically useful as primary health care. These medicines are more culturally therapeutic agent, it should be non-toxic or of low toxicity acceptable, have better compatibility with the human to human cells. Though Delphinium denudatum is widely body and lesser side effects (kamboj, 2000)1. India has a used, limited information is available on the toxicity of rich diversity of medicinal and a number of plant this plant extract. This study is designed to determine extracts are used against diseases in various medicinal the toxicity profile of the crude ethanolic extract of applications, e.g. Ayurveda, Unani, and Siddha. However, Delphinium denudatum Wall. (Jadwar) in Albino rats inspite of widespread use, only a few of these plants (Wistar strain). The acute oral toxicity study was have been scientifically explored (Malaya, 2004)2. conducted according to the OECD/OCDE guidelines 423/ 20019, using various concentrations of Delphinium Delphinium denudatum () is a potent denudatum extract. herbal medicine used in the Indian system of medicine, was known to possess diverse biological functions like Materials and Methods antiseptic, anti diuretic, anti-inflammatory, antifungal Chemicals: All the chemicals, solvents and reagents were (G.Vijaiyan Siva et al., 20063, Rahman et al., 1997)4, of analytical reagent grade. sedative, analgesic and as a nerve tonic. Silver nanoparticals synthesized fron Dd root extract exhibits Collection of plant samples antibacterial and mosquito larvicidal activity. (G.Vijaiyan The dried roots of Delphinium denudatum was procured Siva et al., 2014)5 It is a great antidote to aconitum from authentic suppliers from Hyderabad. The identity poisoning. Alkaloid delphinine is an antidote against and confirmed by taxonomist, Center for advance studies muscarine and digitaline (Chatterji et al., 1997)6. The root in botany, University of Madras, Guindy campus, is used in Bashahr for toothache and also as an adulterant Chennai. for aconite (Stewart). Root found beneficial in rheumatism, impotency and syphilis. (Rastogi et al., 1999)7 Combined Ethanolic Extraction with other herbal stimulants, it is used as remedy in cardiac Plant extracts were prepared from dried roots of and cerebral diseases. Dried roots are a popular folk Delphinium denudatum by using the procedure remedy for the treatment of epilepsy in the traditional described by Phongpaichit et al., in 2005 10. The dried plant material was powdered by using mechanical grinder. *Corresponding author. 40 grams of dried Delphinium denudatum was packed Email address: [email protected] well in the soxlets apparatus with the help of filter paper.

1 Int.J.Curr.Biotechnol. Volume 4; Issue 1; Jan, 2016 350 ml of absolute ethanol is taken as the extraction Estimation of AST solvent. The extraction was carried out for 15 cycles at Aspartate transaminase was assayed by the method of 55ºC, the crude ethanolic extract was further concentrated King et al., 1965 11. 1.0 ml of aspartate substrate was by evaporation at 55ºC using a solvent trap. added to 0.1 ml of serum, incubated at 37oC for 1 hr to which 1.0 ml of 2, 4-dinitrophenyl hydrazine reagent was Animals added to arrest the reaction. To the blank tubes, 0.1 ml of Male Wister albino Rats (6–8 weeks old) of body weight double distilled water was added instead of serum and 100-150 g was used for the study and given standard the tubes were incubated for 15 min, followed by addition pellet diet and water ad labium. The control animals are of 10 ml of 0.4 N sodium hydroxide. The absorbance was treated with 0.9% saline, varied concentration of read immediately at 520 nm in a Shimadzu UV Delphinium denudatum extract using distilled water as spectrophotometer and the enzyme activity was the dose vehicle is administered for 21 days. All expressed in µmoles of pyruvate liberated/min/mg experiments performed as per the recommendations of protein. Institution animal ethics committee with 6 animals in each group. Estimation of ALT The activity of Alanine transaminase was assayed by Experimental design the method of (King et al., 1965)11. 1.0 ml of alanine The experimental animals were divided into four groups, substrate was added to 1ml of serum, incubated at 37oC each group comprising of six animals. Group 1 served as for 30 min, to which 1.0 ml of 2, 4-dinitrophenyl hydrazine normal control while those in group 2, 3 and 4 were fed reagent was added to arrest the reaction. To the blank orally with ethanolic extract of Delphinium denudatum tubes, 0.1 ml of double distilled water was added instead at 100, 500 and 1000mg/kg BW, respectively, for 21 days. of serum and the tubes were incubated for 15 min, The body weight changes of control and experimental followed by addition of 10 ml of 0.4 N sodium hydroxide. animals are being monitored every interval. At the end of The absorbance was read immediately at 520 nm in the experimental period, the animals were anesthetized Shimadzu UV spectrophotometer and the enzyme activity with diethyl ether followed by cervical decapitation, blood was expressed in µmoles of pyruvate liberated/min/mg was taken by cardiac puncture, the liver and kidney were protein. removed under ether anesthesia, washed thoroughly in ice-cold physiological saline [0.9% (w/v) NaCl], and Histopathological evaluation weighed. Whole blood was separated in two tubes Histological evaluation was performed in the liver tissues contain absence and presence of EDTA, for separation and a portion of specimen was fixed in 10% formalin and of serum and plasma, respectively, and the plasma and embedded in paraffin wax. Sections were cut at 4 ìm in serum was stored frozen until further analysis. thickness, stained with hematoxylin and eosin (Booran et al., 1990)12 and viewed under light microscope for Hematology: histological changes. The hematological parameters were analyzed using Horiba ABS 80 Diagnostics (ABX Pentra Montpellier, Statistical analysis France). These parameters include red blood cells, white All the grouped data were evaluated for statistical blood cells, neutrophils, monocytes, lymphocytes, significance with SPSS v.10 software. Hypothesis testing eosinophils, basophils and platelets. methods included one way analysis of variance followed by least significant difference test. P values of less than Biochemical parameters 0.05 were considered to indicate statistical significance. Estimation of SGOT, SGPT and LDH All these results were expressed as mean ± S.D for six The Serum glutamic oxaloacetic transaminase (SGOT) and animals in each group. serum glutamic pyruvic transaminase (SGPT) were estimated by UV kinetic method in which both SGOT and Results SGPT were assayed based on enzyme coupled system; The body weight of the animals treated with the extract where keto acid formed by the aminotransaminase reacts at the doses of 100, 500 and 1000 mg/kg BW and the in a system using NADH. The coenzyme is oxidized to control group were appreciably increased throughout the NAD and decrease in absorbance at 340 nm for SGOT experimental period (Fig. 1) No significant changes were malate dehydrogenase (MDH) reduces to malate with observed in the organs body weight ratio of the kidney, simultaneous oxidation of NADH to NAD. The rate of liver, lung, and heart as compared with control animals oxidation of NADH is measured, where as SGPT the (Fig. 2). pyruvate formed in the reaction is converted to lactate by lactate dehydrogenase (LDH). LDH was estimated in The hematological parameters serum samples from each group of experimental animals. The effect of oral administration of the ethanolic extract of Dd of the does investigated on hematological parameter Estimation of ALP and its function indicates in male Wister rats for 21 days. Alkaline phosphatase was assayed by the method of The extract did not significantly alter the level of RBC King et al., 1965 11. The incubation mixture of (final volume but efficiently reduced the level of WBC and its 3.2 ml) contained 1.5 ml of buffer, 1.5 ml of substrate, 0.1 differentials, basophils, monocytes and as well as ml MgCl2 and 0.1 ml serum sample. The tubes were platelets (Table 1). The throughout the experimental incubated at 37°C for 15 min and the reaction was arrested period, while lymphocytes only reduced in Group 2 and by the addition of Folin’s phenol reagent. The content Group 3 but significantly increased (p<0.05) at Group 4. was then centrifuged. To the supernatant 1 ml of sodium The levels of eosinophils were not altered by the extract, carbonate was added and the tubes were incubated for a whereas those neutrophils were markedly increased at period of 10 min at 37°C. The colour developed was read specific dose. 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.

Volume 4; Issue 1; Jan, 2016 Int.J.Curr.Biotechnol. 2 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

12 RBC (X10 /l) 08.67±0.63 08.17±0.23 08.37±0.13 08.77±0.23*

9 WBC (X10 /l) 17.00±4.63 09.88±1.21 10.78±1.11 12.14±2.10

Neutrophils (%) 08.37±0.45 12.32±1.65 17.46±5.46 07.56±1.23

Monocytes (%) 16.53±4.53 08.51±2.10 10.36±4.23 12.36±2.64

Lymphocytes (%) 65.40±6.58 54.07±4.89 59.46±4.75 65.49±2.56

Eosinophils (%) 03.70±0.45 03.20±0.2 03.40±0.56 02.53±1.23

Basophils (%) 0.53±0.61 0.45±0.16 0.40±0.12 0.40±0.17

9 Platelets X10 851.00±36.12 705.45±15.62 724.36±41.26 803.12±12.46

Each value is expressed as mean ± S.D for four determinations in each experimental group 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 1 125.67±12.63 43.17±2.23 04.86±0.89

Group 2 119.00±04.13 49.18±3.31 04.37±1.11

Group 3 121.07±06.25 47.87±9.05 04.58±0.46

Group 4 126.53±07.53 48.21±6.10 04.66±1.23

Each value is expressed as mean ± S.D for four determinations in each experimental group Figure – 1: The effect of ethanolic extracts of Delphinium denudatum on the body weight of albino Wister rats.

Each value is expressed as mean ± S.D for four determinations in each experimental group. 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 Figure – 2: The effect of ethanolic extracts of Delphinium denudatum on the organ body weight ratio of albino Wister rats.

Each value is expressed as mean ± S.D for four determinations in each experimental group. 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

Each value is expressed as mean ± S.D for four determinations in each experimental group Figure – 4: Serum LDH for toxicological evaluation

Each value is expressed as mean ± S.D for four determinations in each experimental group Volume 4; Issue 1; Jan, 2016 Int.J.Curr.Biotechnol. 4 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 2-100 mg/BW kg Dd treated animal cells (C) Group 3-500 mg/BW kg Dd treated animal cells (D) Group 4-1000 mg/BW kg Dd treated animal cells showed normal architecture as that of control animals.

Biochemical parameter serum, thus even a small mass of damaged tissue causes The results indicate the non-toxic nature of the extract leakage of enzyme and increasing its level in serum and between the two doses, 100 - 1000 mg/kg BW was significantly. Ethanolic extracts of Dd at doses of 100- found to be more effective. No Significant increases in 1000 mg/Kg/BW (Group 2 to 4) did not show any effect AST, ALT and ALP were observed from Group 2 to Group on levels of serum LDH concentrations as compared to 4 (100-1000 mg/kg/BW) as compared to normal control normal control animals (Group 1) (Fig. 4). animals (Group 1) (Fig. 3). Serum AST, ALT and ALP, are the most sensitive markers employed in the diagnosis of Estimation of Serum SGOT, SGPT and creatine hepatic damage because these are cytoplasmic in location The enzyme SGOT and SGPT that is normally present in and are released into the circulation after cellular damage liver and heart cells. SGOT and SGPT are released into (Sallie, 1991)13. Ethanolic extracts of Dd at doses of 100- blood when the liver or heart is damaged. The blood 1000 mg/Kg/BW (Group 2 to 4) did not show any effect SGOT levels are thus elevated with liver damage or with on levels of AST, ALT and ALP concentrations as an insult to the heart. Serum SGOT, SGPT and creatine compared to normal control animals (Group 1). are the most sensitive markers employed in the diagnosis of hepatic damage and heart failure. Ethanolic extracts of Lactate dehydrogenase Dd at doses of 100-1000 mg/Kg/BW (Group 2 to 4) did Lactate dehydrogenase transfers hydrogen using NAD+ not show any effect on levels of Serum SGOT, SGPT and as hydrogen acceptor thus catalyzing the oxidation of L- creatine concentrations as compared to normal control lactate to pyruvate. LDH activity is present in all the animals (Group 1) (Table 2). cells of the body predominantly in cytoplasm of the cell. Thus tissue levels are 500 times greater than those in

5 Int.J.Curr.Biotechnol. Volume 4; Issue 1; Jan, 2016 Histological examination respect to hematology, clinical chemistry, organ weight, The histological examination of liver section of control gross and histopathological examinations noted in Dd it and experimental groups of animals related that, liver from can be inferred that Dd will not produce delayed onset of control (Group 1) animals showed a normal architecture toxicity. Based on these results, the No Observed Adverse of cells with small uniform nuclei (Fig. 5A). Ethanolic Effect Level (NOAEL) of Delphinium denudatum is extracts of Dd at doses of 100 - 1000 mg/body kg bearing greater than 1000 mg/kg/day. animals (Fig. 5B, C, D) showed same architecture of the cells compared with control group animals exhibited no Conclusion toxic effect of this experimental concentration of ethanolic Based on this study, we conclude that the medicinal herb extracts of Dd. Delphinium denudatum can be administered at a dose range of 1000 mg/kg/BW without any side effects. Since, Discussion the toxicity studies in experimental animals cannot always Herbal medicines have attained greater importance as an be totally extrapolated to humans, and a reasonable alternative to conventional therapy. To optimize the safe estimate of the self administered dose is difficult to make use of a plant-based medicine, one should take into such as that applied during traditional use of this plant, account their historical applications on humans and additional clinical toxicological evaluations need to be animals as well as toxicity evaluation of the medicinal performed to define a safe dose and protect the herbs and their active components (Mukinda et al., population from possible toxic effects of the plant. 2007)14. Many screening methods are employed to determine the safety and efficacy of these herbal References medicines and also to establish the active component of 1. Kamboj VP, 2000. Herbal Medicine. Current Science 1: the herbal products (Sim et al., 2010)15. However, the 78 scientific validation of its safety and efficacy has not 2. Malaya G, Upal KM, Ramanathan SK, Thangavel S, been established so far. The present study gives detailed Madgula L, Mohan V, 2004. Antitumor Activity and information on the toxicological profile of Delphinium Antioxident Status of Caesalpinia Bonducella against denudatum oral toxicity studies in rats. A limit test was Ehrlich Ascites Carcinoma in Swiss Albino Mice. J performed in oral toxicity study. According to the OECD Pharmacol Sci. 94:177-184. test guideline 423/2001 when there is information in 3. Vijaiyan Siva.G, Sudha Revathy.S, Kaiser Rabee.U.Md support of low or non-toxicity and immortality nature of (2006) Bioeficacy of the roots of delphinium denudatum the test material, then the limit test at the highest dose (DD). Wall, against curvularia lunta. Journal of Drug level (1000 mg/kg body weight) was conducted. There Research in Ayurvedha and Siddha. Central council for were no mortality and toxicity signs observed at 1000mg/ research in ayurvedha and siddha.(AYUSH) Vol.XXVII, kg. Therefore, it can be concluded that Dd when NO.1-2, pp. 1-10. administered at single dose is non-toxic and can be used 4. Rahman A, Nasreen A, Akhtar F, Shekhani S, Clardy J, safely in oral formulations. A 21-day oral toxicity study Parvez M and Choudhary I(1997) Antifungal diterpenoid was performed followed OECD test guideline 423/2001 in alkaloids from delphinium denudatum J.Nat. Prod 60:472- Albino rats (Wistar strain). Since examination of clinical 474. signs plays major role in toxicological testing (Stevens 5. Suresh G, Gunasekar PH, Kokila D, Prabu D, Dinesh D et al) 16. Dd did not produce any alterations in feed and Ravichandran N Ramesh B, Koodalingam A, Vijaiyan Siva water consumption and this reveals that it did not G(2014) Green synthesis of silver nanoparticales using adversely affect the basic metabolic processes of the Delphinium denudatum root extract exhibits antibacterial experimental animals. The haemopoietic system serves and mosquito larvicidal activities. Spectrochim Acta A as important target for toxic chemicals and is a sensitive Mol Biomol Spectrosc. 5;127:61-6. index for pathological conditions both in humans and 6. Chatterji A and Prakash C The treatise of Indian animals (Adeneya et al.,) 17. medicinal plant volume 1 (1997) . 7. Rastogi P and Mehrotra N (1999) Compendium of Indian In the present study, treatment with Dd did not produce medicinal plants volume 1 any alteration in hematological parameters (i.e. RBC, WBC 8. Kirthikar and Basu, (1999) Indian medicinal plants neutrophils, monocytes, lymphocytes, eosinophils, volume basophiles and platelets.), which indicate that Dd did 9. OECD: Guidelines for the Testing of Chemicals/Section not affect blood cells nor their production. Clinical 4: Health Effects Test No.423: Acute Oral toxicity - Acute biochemistry and hematological data holds significant Toxic Class Method.Paris,France:Organization for role in determining the toxicity induced by drugs. Economic Cooperation and Development; 2001. Transaminases (SGOT and SGPT) are good indicators of 10. Phongpaichit.S, Pujenjob.N, Rukachaisirikul.V, and liver function and biomarkers to predict the possible Ongsakul, 2005. Antimicrobial activities of the crude toxicity of drugs (Hilaly et al.,) 18. Any elevation pertaining methanol extract of Acorus calamus Linn. to these enzymes indicate their outflow into the blood Songklanakarin. Journal of Science and Technology 27: stream due to damage in liver parenchyma cells. There 517-523. were no changes in the SGPT and SGOT levels which 11. King J (1965) the transferase alanine and aspartate reveal that Dd did not affect liver function/or metabolism. transaminase. In: Van.D. (Ed.). Practical clinical In the present study, there were no treatment related enzymology. Nostrand Co.Ltd, London, 1965a, pp.121- abnormalities in renal function and other biochemical 138. parameters suggesting that Dd is non-toxic. 12. Booran GA, Eustis SL, Elwell MR, Mont gory CA, Histopathological studies provide supportive evidence Makenzie WF (1990) Pathology the fisher rat. for biochemical and hematological observations Reference.D Atlas. Academic press, San Diego Newyork, (Matsuzawa et al.,) 19. The relative organ weights were London. Pp.9.30 found to be nonsignificant between the control and Dd 13. Sallie, R., R.S. Tredger and R. Williams, 1991. Drugs treated rats. No abnormality was recorded with respect and the liver. Biopharm. Drug to gross or histopathological examinations of liver Disposal, 12: 251-259. examined. Since there were no signs of toxicity with

Volume 4; Issue 1; Jan, 2016 Int.J.Curr.Biotechnol. 6 14. Mukinda JT, Syce JA: Acute and chronic toxicity of the aqueous extract of Artemisiaa fra in rodents. J Ethnopharmacol 2007, 112:138–144. 15. 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, 6:67–70. 16. Stevens KR, Mylecraine L: Issues in chronic toxicology. In Principles and Methods of Toxicology. thirdth edition. Edited by Hayes AW. New York: Raven Press; 1994:673. 17. 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, 105:374–379. 18. Hilaly JE, Israili ZH, Lyouss B: Acute and chronic toxicological studies of Ajuva Iva in experimental animals. J Ethnopharmacol 2004, 91:43–50. 19. 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, 5:196–200.

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