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July - September 2015 1 Journal of Pharmacy and Chemistry • Vol.9 • Issue.3

J.Pharm.Chem CODEN: JPCOCM

Journal of Pharmacy and Chemistry(An International Research Journal of Pharmaceutical and Chemical Sciences)

Indexed in Chemical Abstract and Index Copernicus (IC Value 5.28)

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Editor-in-chief

Prof. K.N. JAYAVEERAJawaharlal Nehru Technological University Anantapur,

Anantapur, Andhra Pradesh -515001.

Associate EditorDr. K.V. Madhusudhan

Executive EditorDr. K. Balaji

Editorial Board

Dr. B.M. Vrushabendra Swamy Dr. A. Venkateshwar Reddy Dr. G. S. Kumar Dr. G. Madhu Dr. S. Subramanyam Dr. K. Yogananda Reddy

Dr. K. Bhaskar Reddy Dr. E. Sreedevi Dr. K.C. Chaluvaraju

Editorial Advisory Board

Prof. Nagarapu Lingaiah India Prof. G. Krishna Mohan India

Prof. T.R. Rao India Prof. M.L.N.Rao India

Prof. R.Nageshwara Rao India Prof. S. Srihari India

Prof. K.V.S.R.G. Prasad India Prof. Y. Rajendra Prasad India

Prof. K. Kannan India Prof. Yeoh Peng Nam IMU, Malaysia

Prof. D.R. Krishna U.S.A Prof. K.C.Naidu India

Prof. Jonathan R Dimmock Canada Prof. Ananth. P. Haridas India

Prof. Helton Max M. Santos Portugese Prof. Damaris Silveira Brazil

Prof. Mustafa Iraz Turkey Prof. Abdul Naser B Singab Egypt

Prof. Ali Asgarh hemmati Iran Prof. N. Devanna India

Prof. K.R.S. Sambasiva Rao India Prof. R. Shyam Sunder India

Dr. Nitin Mahukar India Prof. Arun Goyal India

Prof. Sarangapani India Prof. Sunil K. Khare India

Prof. Y. Narasimha Reddy India Dr. S. Narasimha Murthy U.S.A

Dr. Girish Gowda Saudi Arabia Dr. K. Suresh Babu India

Online : ISSN 2349-669X

Print : ISSN 0973-9874


ISSN 0973 – 9874 J.Pharm.Chem CODEN: JPCOCM

Journal of Pharmacy and Chemistry(An International Research Journal of Pharmaceutical and Chemical Sciences)

Volume 9 • Issue 3 • July – September 2015

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VIEWSThe views and opinions expressed in this journal are those of the contributors; Science-Tech Foundation does not necessarily concur with the same. All correspondence should be addressed to the Editor-In-Chief (Hon.), Journal of Pharmacy and Chemistry (Science-Tech Foundation), Plot No 22, Vidyut Nagar, Anantapur - 515 001, Andhra Pradesh, India. • e-mail:[email protected]. Send your queries at www.jpc.stfindia.com, www.stfindia.com

CONTENTS

Synthesis and Antimicrobial Evaluation of Some Novel Aryl Substituted Imino

Methylene Analogs of 2-Methyl-5, 6-Pentamethylene Thienopyrimidinones .............................................................. 2

RAMAMURTHY S AND JAYACHANDRAN E

Antidiabetic and Antioxidant Activities of Polyherbal Formulation in STZ Induced Diabetic Rats ................. 9

THANDAIAH KRISHNA TILAK, SHAIK ABDUL NABI, PARASARAM MURALI KRISHNA, MARELLA SARITHA AND CHIPPADA APPA RAO

Evaluation of Tephrosia Purpurea for Anti Hypelipidemic Activity in High Fat Induced Rats ........................... 20

RAGHAVENDRA HG, LAKSHMIKANTH G AND SAMATHA Y

Development of Dieteticbeverages by Using Aleo-Vera Juice and their Quality Evaluation ................................. 25

SANGITASOOD, SHIVANIWALIA AND MRINALINI

Design, Development and Characterization of The Dimenhydrinate Bilayered Caplets ........................................ 31

LAKSHMI NARAYANA REDDY G, DR. RAJNARAYANA K AND DR. JAYAVEERA KN

Alleviatory Effects of Brassica oleracea var. botrytis against Sodium Fluoride Induced Nephrotoxicity and Oxidative Stress on Male Wistar Rats .......................................................................................................................... 36

RAGHAVENDRA M, RAVINDRA REDDY K, AND JAYAVEERA KN

Instruction to Authors................................................................................................................................... Cover page 3




*Address for correspondence:[email protected]



Synthesis and Antimicrobial Evaluation of Some Novel Aryl Substituted Imino Methylene Analogs of 2-Methyl-5,

6-Pentamethylene Thienopyrimidinones

RAMAMURTHY S1* AND JAYACHANDRAN E2

1 Department of Pharmaceutical Chemistry, V.V.Pura institute of Pharmaceutical Sciences,B.S.K II Stage, Bangalore, Karnataka, India.

2 Department of Pharmaceutical Chemistry, S.C.S.College of Pharmacy, Harapanahalli, Karnataka,India.

ABSTRACT

In our present investigation 2 – Amino- 3–carbethoxy–4, 5 pentamethylene thiophene (compound-I)was prepared from ethyl methyl ketone and ethyl cyanoacetate. Reaction of this with acetic anhydride followed by treatment with hydrazine hydrate yielded the desired starting compound 3- N-amino -2-methyl-5,6- penta methylene thieno[2,3-d]- pyrimidin (3H)-4-one [III]. Further treatment of compound-III with various substituted aryl aldehydes resulted in a new series of aryl substituted imino-methylene analogs [IIIa-l]. These were characterized by physical data (m.p, TLC) and spectral data UV, IR, 1H NMR and Mass (representative compounds only). The antibacterial and antifungal activity of title compounds were evaluated by cup-plate agar diffusion method using Ampicillin and Miconazole nitrate as standard drugs respectively. Among the compounds tested, the compounds III-c, III-d, III-h and III-k showed good activity and compounds III-e, III-g, III-i and III-j exhibited moderate to mild activity compared to standard drugs used.

Key words: Synthesis, Thienopyrimidinone, Schiff bases, antimicrobial activities, Miconazole, ethyl cyanoacetate.

Introduction Although considerable advances have been achieved over the past few decades in the introduction of new structural prototypes as effective antimicrobials, current antimicrobial chemotherapy still suffers from major limitations like lack of selectivity of conventional drugs, their unwanted side effects, acquisition by the micro organism of multi drug resistance, uneconomical and time consuming synthetic routes for new potent drugs, etc. Nitrogen and sulphur containing heterocyclic compounds are indispensible structural units and provide considerable pharmacological and synthetic interest due to their extensive biological activities. This helped medicinal chemists to plan, organize and implement new approaches on discovery of more potent drugs and their molecular modifications. Thiophenes and pyrimidines form important class of heterocyclics reported to possess wide spectrum of biological activities such as antibacterial, antifungal, anti inflammatory, CNS depressant activity, analgesic, anti-proliferative, anti-ulcer and antitumor activities [1-10].

Thienopyrimidines are the fused ring systems involving thiophene and pyrimidine. Substituted thienopyrimidines are of great importance because of their remarkable biological activity for use as potential drugs. They proved to exhibit significant antibacterial, antifungal, anticonvulsant, angiotensin antagonistic, antimalarial and tyrosine kinase inhibitor activities. Up to now, there are many different structures containing thienopyrimidine nucleus have been synthesized and evaluated for biological activities [11-18].Despite the breadth of biological activities displayed by these agents, the antibacterial and antifungal activities of this class of compounds have shown only handful of results. Also various Schiff base analogs have been reported to possess a large number of biological activities and found to exhibit antimicrobial, anti-ulcer, anti HIV, anticonvulsant, and CNS depressant activities [19-23].

The above encouraging bioactive diversity of thienopyrimidines and Schiff bases prompted us to prepare a new series of structural variants of thieno[2, 3-d]-pyrimidinone derivatives in the present study. 2-Methyl 3-amino- 5, 6-pentamethylene thieno [2, 3-d] pyrimidin (3H)-4-one as key prototype structural unit was prepared and treated the active 3-amino group with substituted aryl


aldehydes to obtain a new series of Schiff bases (scheme 1). They were subjected for their possible antibacterial and antifungal investigations.

Materials and Methods The chemicals used in the present study were procured from Merck, S.D.Fine chem. Ltd, Mumbai and used directly. The melting points of the compounds were determined in an open capillary tube and are uncorrected. TLC was carried out using silica gel G with Benzene: Chloroform (1:1) as mobile phase to check the purity of synthesized compounds. The physical data of the compounds are tabulated (Table 1). 1H NMR spectra were recorded in CDCl3using tetra methyl silane (TMS) as internal standard on Bruker AMX 400MHz. IR spectra of the compounds were recorded using KBr pellet on Shimadzu FTIR-8700 spectrophotometer and frequencies were recorded in wave numbers. Mass spectra were recorded using Shimadzu LC MS-2010A at Quest Research Training Institute Ltd, Bangalore.

Synthesis of 2-Amino-3- carbethoxy-4, 5-pentamethylene thiophene [Compound-I]

To a mixture of the cycloheptanone (4.72 ml; 0.04mol), ethyl cyanoacetate (4.26 ml; 0.04 mol) and sulphur powder (1.28 g; 0.04 mol) in ethanol (40 ml), diethyl amine (4.0 ml) was added dropwise with stirring. The mixture was stirred further for 1h at 45-50°C, chilled overnight and the solid obtained was filtered, washed and recrystallized from ethanol. Mol.formula: C11H15O2NS; MW: 225; Yield: 50.87%; m.p:84°C; Rf : 0.60.

Synthesis of 2-Acetamido-3- carbethoxy-4, 5-penta-methylene thiophene[Compound-II]

A mixture of 2-Amino-3- Carbethoxy-4,5 penta-methylene thiophene [Compound-I] (2.25 g ;0.01mol),acetic anhydride (5.0ml, 0.05 mol)and zinc dust(0.30g; 0.01mol)was stirred and irradiated with microwave heating in Kenstar microwave oven(2450 MHz, 900 w) for 15 seconds and cooled, the resulting white solid was recrystallized from methanol. Mol.formula: C13H17O3NS; MW: 267; Yield: 78.25%; m.p:114°C; Rf :0.53.

Synthesis of 2-Methyl -3-N-amino-5, 6-pentamethylene thieno[2, 3-d]- pyrimidin (3H)–4-one [Compound-III]

A mixture of Compound-II (2.67 g, 0.01 mol), hydrazine hydrate ((15 ml; 0.03mol) and ethanol (20 ml) was irradiated for 20 seconds and cooled. A white crystalline product was obtained and was recrystallized from aqueous acetone (1:2). Mol.formula: C11H13ON3S; MW: 235; Yield: 60.42%; m.p:180°C; Rf :0.59; IR(KBr, cm-1): 3261.65(primary NH2), 3158.63 (Ar-CH-), 1680.18(C=O, aryl),1530.34(-N– C= O cyclic str.), 1634.12(NH bend), 821.90 (C-N),754.35(C-S); 1H NMR (δ ppm):2.6 (m, 4H of cycloheptane), 1.8m (4H of cycloheptane), 1.6 ( m, 2H of cycloheptane) , 1.0 (s, -CH3,3H at position 2), m/e: 239(100%), 193(68%),58(52%).

Synthesis of 2-Methyl-3-N-[(substituted aryl-methylene)-imino] -5, 6-pentamethylene thieno[2,3-d]- pyrimidin (3H)–4-ones: [Compound-IIIa-l]

A mixture of Compound-III (2.35g; 0.01 mol) and a substituted aryl aldehyde (0.01 mol) in isopropanol containing catalytic amount of glacial acetic acid (2 ml), was irradiated(2450 MHz, 900 w) for 20 seconds, then the mixture was cooled. The compounds III a- l obtained were purified from propanol/ethyl acetate.

Compound III a: 3-N-[(4’-dimethyl amino phenyl methylene)-imino]-2-methyl-5,6- penta methylene thieno[2,3-d]- pyrimidin (3H)–4-one IR (KBr, cm-1): 3162.68(Ar-CH), 1684.12(C=O, aryl), 1533.34 (-N– C= O cyclic str..), 1652.11(N=CH), 1612.12(NH bend), 1479.24(Ar C=C), 1290( C-N str.arom.amines), 823.80(C-N), 750.25(C-S);1H NMR (δ,ppm):8.0 (s,1H,–N=CH- ), 6.7 (d, 2H, Ar-H at 2’ and 6’), 7.7 (d, 2H, Ar-H at 3’ and 5’), 3.1(s, 6H, -N(CH3)2 at 4’), 2.4( d, 4H of cycloheptane), 1.9( m, 4H of cycloheptane), 1.8( m, 2H of cycloheptane), 1.1 (s, -CH3, 3H at 2).Compound IIIb: 3-N-[(4’-hyroxy phenylmethylene)- imino]-2-methyl-5,6- penta methylene thieno[2,3-d]- pyrimidin (3H)–4-one IR(KBr, cm-1): 3499.85( -OH), 3160.73 (Ar-CH), 1686.42(C=O, aryl), 1538.38 (-N– C= O cyclic str.), 1654.11(N=CH), 1614.18 (NH bend), 1487.97(Ar C=C), 823.72(C-N), 751.28(C-S); 1H NMR (δ, ppm):8.1 (s,1H,–N=CH- ), 6.8 (d, 2H, Ar-H at 2’ and 6’), 7.7 (d, 2H, Ar-H at 3’ and 5’), 5.2 (s, 1H, -OHat 4’),2.5 (d, 4H of cycloheptane), 1.8 (m, 4H of cycloheptane), 1.5 (m, 2H of cycloheptane) , 1.2 (s, -CH3, 3H at 2).

Compound IIIc: 3-N-[(2’-nitro phenylmethylene)-imino]-2-methyl-5,6- penta methylene thieno[2,3-d]- pyrimidin (3H)–4-one IR(KBr, cm-1): 3166.72(Ar-CH), 1686.74(C=O, aryl),1537.75(-N– C= O cyclic str.), 1650.88(-N=CH), 1620.20(NH bend), 1472.42(Ar C=C), 1236.72(Ar-O-C),1360.10 (N-O of NO2) 753.16(C-S), 1HNMR (δ ppm):8.2 (s,-N=CH- 1H), 7.1 (d, 2H, Ar-H at 2’ and 6’),7.5 (d, 2H, Ar-H at 3’ and 5’), 2.7 ( d, 4H of cycloheptane), 1.9 (m, 4H of cycloheptane), 1.5 (m, 2H of cycloheptane) , 1.0 (s, -CH3, 3H).Compound IIIe: 3-N-[(3’, 4’, 5’-trimethoxy phenyl methylene)-imino]- 2-methyl-5,6- penta methylene thieno[2,3-d]- pyrimidin (3H)–4-one IR(KBr, cm-1): 3160.73(Ar-CH), 1680.28(C=O, aryl),1535.38(-N– C=O cyclic str.), 1655.11(-N=CH), 1614.18(NH bend), 1479.56 (ArC=C), 1230.80 (Ar-O-C),1245.12 (O-CH3), 823.72(C-N), 751.28(C-S); 1HNMR (δ ppm):8.0 (s,–N=CH- 1H ), 6.9 (d, 2H, Ar-H at 2’ and 6’), 7.7 (d, 2H, Ar-H at 3’ and 5’), 3.4 (s, -OCH3, 9 H), 2.4 (d, 4H of cycloheptane), 1.8( m, 4H of cycloheptane), 1.6( m, 2H of cycloheptane) , 1.1 ( s, -CH3, 3H at 2).

Compound IIIg: 3-N-[(4’-hydroxy-3’-methoxy phenyl methylene)-imino]2-methyl-5,6-penta methylene


thieno[2,3-d]- pyrimidin (3H)–4-oneIR(KBr, cm-1): 3433.32(-OH), 3143.32 (Ar-CH), 1684.12 (C=O, aryl),1538.40(-N– C= O cyclic str.), 1658.32 (-N=CH), 1618.18(NH bend), 1482.58 (Ar C=C), 1235.62 (Ar-O C),1245.12( O-CH3),823.72(C-N), 751.28(C-S);1HNMR (δ ppm):8.2( s,–N=CH- 1H ), 7.6 (m.2H at 2’), 7.8 (m,2H at 5’), 7.1(m,2H at 6’), 5.3(s, 1H, -OHat 4’),2.5 (d, 4H of cycloheptane), 2.0(s.3H, 4’-CH3),1.9 (m, 4H of cycloheptane),1.4 (m, 2H of cycloheptane) , 1.2= ( s, -CH3, 3H at 2)).Compound III h: 3-N-[(4’-Chloro phenylmethylene)-imino] 2 –methyl-5,6- penta methylene thieno[2,3-d]- pyrimidin (3H)–4-one IR(KBr) cm-1: 3120.52(Ar-CH), 1678.28(C=O, aryl), 1535.44(-N– C= O cyclic str.), 1643.15(-N=CH), 1625.86 (NH bend), 1502.22(Ar C=C), 823.72(C-N), 751.28(C-S), 697.72(C-Cl), 1HNMR (δ,ppm):8.2 (s,-N=CH- 1H), 7.0 (d, 2H, Ar-H at 2’ and 6’), 7.6 (d, 2H, Ar-H at 3’ and 5’), 2.5 (d, 4H of cycloheptane), 1.9( m, 4H of cycloheptane), 1.6(m, 2H of cycloheptane) , 1.1 ( s, -CH3, 3H at 2)), m/e : 371(100%), 193( 58%),179(56%).

Compound III i : 3-N-[(4’-methoxy phenyl methylene)-imino]-2 –methyl-5,6-penta methylene thieno[2,3-d]- pyrimidin (3H)–4-one IR(KBr, cm-1): 3168.52 (Ar-CH), 1685.28(C=O, aryl),1539.64(-N– C= O cyclic str.), 1645.11(-N=CH), 1618.15(NH bend), 1475.12(Ar C=C), 1235.72(Ar-O-C),1252.24 (O-CH3),824.34(C-N), 753.16(C-S);1H NMR (δ ppm):8.2 (s,-N=CH- 1H), 7.0 (d, 2H, Ar-H at 2’ and 6’), 7.6 (d, 2H, Ar-H at 3’ and 5’),3.9 (s.3H,-OCH3),2.5 ( d, 4H of cycloheptane), 1.9 (m, 4H of cycloheptane), 1.6 (m, 2H of cycloheptane) , 1.1 (s, -CH3, 3H).

Compound III l: 3-N-[(4’-methyl phenyl methylene)-imino]-2 –methyl-5, 6- penta methylene thieno[2,3-d]- pyrimidin (3H)–4-one IR(KBr, cm-1): 3124.44(Ar-CH), 1684.64(C=O, aryl),1544.52(-N– C= O cyclic str.), 1650.11(N=CH), 1615.46 (NH bend),1508.72(Ar C=C),822.78(C-N), 754.52(C-S);1H NMR (δ ppm):8.2 (s,-N=CH- 1H), 7.0 (d, 2H, Ar-H at 2’ and 6’),7.6 (d, 2H, Ar-H at 3’ and 5’), 2.5 ( d, 4H of cycloheptane), 1.9( m, 4H of cycloheptane), 1.6 (m, 2H of cycloheptane) , 2.0(s.3H, 4’-CH3), 1.1 ( s, -CH3, 3H).

SCHEME 1:Synthetic protocol of title compounds


Antimicrobial Screening

The antimicrobial activity of the newly synthesized compounds were determined in vitro by cup-plate agar diffusion method [23-25] using DMSO as solvent. Antibacterial activity was determined against Gram positive bacteria Staphylococcus aureus(ATCC 11632)and Bacillus subtilis(ATCC 60711)and Gram negative bacteria Escherichia coli(ATCC 10536)and Klebsiella pneumoniae(ATCC 13883),while antifungal activity against Aspergillus niger(ATCC 1781), Candida albicans(ATCC 2501)and Cryptococcus neoformans(ATCC 32045) at

Where, III-a: 4’-Dimethyl amino III-e: 3’, 4’, 5’-Trimethoxy III-i: 4’-Methoxy III-b: 4’-Hydroxy III-f: 2’-Hydroxy III-j: 3’, 4’-Dimethoxy III-c: 2’-Nitro III-g: 4’-Hydroxy 3’methoxy III-k: 4’-Chloro III-d: 3’-Nitro III-h: 2’-Chloro III-l: 4’-Methyl

50 mcg/0.1 ml concentration. After 24 and 48 hours of incubation at 37±1oC, the antibacterial and antifungal activity respectively was determined by measuring zones of inhibitions in mm. Standard antibacterial Ampicillin and antifungal Miconazole nitrate were used under similar conditions for comparison. Control test with solvents were performed for every assay but showed no inhibition of microbial growth. The responses of organisms to the synthesized compounds were measured as mean of three values and compared with the standard response and also Standard deviation was also calculated (Table2&3).

Table - 1: Physical Data of Synthesized Compounds: [Compound-III a-l]

Code R Molecular Formula M.W % Yield M.P. (ºC) Rf ValueIII a Dimethyl amino-‘4 C21H23ON4S 370 52.2 203 0.47III b Hydroxy-‘4 C19H19O2N3S 353 54.5 223 0.50III c Nitro-‘2 C19H18O3N4S 382 52.2 205 0.34III d Nitro-‘3 C19H18O3N4S 382 63.5 209 0.38III e Trimethoxy-‘5,‘4,‘3 C22H25O4N3S 417 58.4 212 0.72III f Hydroxy-‘2 C19H19O2N3S 359 66.4 207 0.55III g Hydroxy 3’methoxy-‘4 C20H21O3N3S 383 60.8 172 0.57III h Chloro-‘2 C19H18ON3S 371 55.5 219 0.48III i Methoxy-‘4 C20H21O2N3S 367 59.4 185 0.65III j Dimethoxy-‘4,‘3 C20H21O2N3S 367 70.2 191 0.66III k Chloro-‘4 C19H18ON3S 371 65.6 189 0.45III l Methyl-‘4 C20H21ON3S 351 52.8 221 0.69

Table-2: Anti-bacterial activity data of title compounds

Code RZone of inhibition in mm.

S.aureus B.subtilis E.coli K.pneumoniaeIII a Dimethyl amino‘4 14.66±1.15 15.66±1.15 NA NAIII b Hydroxy-‘4 14.00±1.00 13.00±1.73 NA NAIII c Nitro-‘2 18.66±1.52 18.33±1.15 19.33±0.57 18.66±1.41III d Nitro-‘3 20.66±0.57 17.66±0.57 21.33±1.52 18.66±1.52III e Trimethoxy-‘5‘4‘3 13.33±0.57 12.66±1.15 14.66±1.52 13.66±0.57III f Hydroxy-‘2 15.00±1.00 13.33±0.57 NA NAIII g Hydroxy 3’-methoxy-‘4 13.66±0.57 14.66±1.52 14.66±1.52 13.66±0.57III h Chloro-‘2 20.66±0.57 18.00±1.73 19.66±0.57 18.66±1.52III i Methoxy-‘4 15.33±1.15 13.66±0.57 14.66±1.52 14.00±1.00III j Dimethoxy-‘5,‘3 15.00±1.00 15.33±1.15 14.66±1.52 14.66±1.52III k Chloro-‘4 20.66±0.57 17.33±0.57 20.66±0.57 18.33±1.52III l Methyl-‘4 13.66±0.57 13.33±0.57 NA NA

Ampicillin ...... 22.66±0.57 18.00±1.00 23.33±0.57 18.66±1.15

NA- No Activity


RESULTS AND DISCUSSION The starting compound III and the title compounds IIIa-l were synthesized and their structures were confirmed based on their spectral data.The UV absorption spectrum of the compound II exhibited λ max at 234 nm and the compound III at 214 nm and the title compound coded III a-l exhibited at 210-218 nm.This hypsochromic shifts with reduction in intensity clearly indicates the cyclic hetero aromatic cyclization seen in these products.

The compound III which was obtained by the cyclization of II showed no IR absorption band at 1620 cm-1 due to the absence of the CO group of the ester. It was also observed that compound III and its derivatives IIIa-l exhibited characteristic strong peaks at 1682 cm – 1 and 1676-1690 cm –1 respectively arising from C=O cyclic stretching due to the presence of pyrimidine ring (aromatic compound containing carbonyl system). There is one more strong peak at 1532-1645 cm – 1 in these compounds due to -N–C=O cyclic stretching vibration in pyrimidone ring.

In compound III there is an appearance of a specific peak at 3262 cm – 1 due to the presence of primary amino group. This peak is absent in the Schiff base derivatives III-a-l but there is an appearance of peak at 1650-1664 cm – 1 and suggests the absence of amino group and the formation of N-imino linkage in these compounds.

Among all the compounds screened for antibacterial activity, only the compounds bearing 2-nitro (III-c), 3-nitro (III-d), 2- chloro (III-h) and 4- chloro (III-k) substituents at R showed good activity against both gram positive and gram negative bacteria used in the study. Compounds bearing 3, 4, 5-trimethoxy(III-e),4 –hydroxy-3- methoxy (III-g), 4-methoxy (III-i) and 3, 4-dimethoxy(III-j) exhibited only

moderate activity. The other compounds showed very weak or no antibacterial activity compared to standard drugs tested.

Fungicidal screening data also revealed similar pattern of activity, but the results were not encouraging. Only a few compounds imparted considerable fungicidal activity. The compounds bearing R =2-nitro (III-c), 3-nitro (III-d), 2- chloro (III-h) and 4- chloro (III-k) substituents showed better antifungal activity, the remaining compounds exhibited very mild or no antifungal activity

Conclusion In the present study, convenient syntheses of a series of novel 2-methyl 3-N-substituted imino-5,6-penta-methylene thieno[2,3-d] pyrimidin-4-one analogs were described. Antibacterial activity was studied against Gram positive bacteria Staphylococcusaureus (ATCC 11632)and Bacillus subtilis (ATCC 60711)and Gram negative bacteria Escherichia coli (ATCC 10536)and Klebsiella pneumonia (ATCC 13883), while antifungal activity against Aspergilusniger (ATCC 1781), Candida albicans (ATCC 2501) and Cryptococcus neoformans(ATCC 32045).The results indicated that some of the target compounds bearing electron withdrawing groups like 2-nitro (III-c), 3-nitro (III-d), 2- chloro (III-h) and 4- chloro (III-k) substituents on the aldehydic phenyl ring, exhibited significant antibacterial activity compared to the compounds bearing electron donating groups like 3,4,5-trimethoxy(III-e),4–hydroxy-3- methoxy (III-g), 4-methoxy (III-i) and 3,4-dimethoxy (III-j). Only a few compounds coded III-d, III-j, III-f and III-l imparted comparable fungicidal activity. From our studies it can be concluded that suitable molecular modifications of these compounds may generate potent antimicrobial agents.

Table 3 : Antifungal activity data of title compounds

Code RZone of inhibition in mm.

A.niger C.albicans C.neoformansIII a Dimethyl amino‘4 NA NA NAIII b Hyroxy-‘4 10.33±1.52 10.33±1.15 NAIII c Nitro-‘2 14.66±1.52 15.66±1.15 14.66±0.57III d Nitro-‘3 14.00±1.00 14.66±1.52 13.00±1.73III e Trimethoxy-‘5‘4‘3 08.33±1.52 10.66±1.15 09.33±1.52III f Hydroxy-‘2 11.66±1.15 08.33±1.52 10.66±1.15III g Hyroxy 3’-methoxy-‘4 NA NA NAIII h Chloro-‘2 15.66±1.15 14.00±1.00 15.33±1.15III i Methoxy-‘4 NA NA NAIII j Dimethoxy-‘5,‘3 NA NA NAIII k Chloro-‘4 15.66±1.15 15.33±1.15 14.66±1.52III l Methyl-‘4 NA NA NA

Miconazole nitrate ... 27.33±1.52 25.33±1.15 23.33±1.52NA- No Activity


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*Address for correspondence: [email protected]

Antidiabetic and Antioxidant Activities of Polyherbal Formulation in STZ Induced Diabetic Rats

THANDAIAH KRISHNA TILAK1, SHAIK ABDUL NABI2, PARASARAM MURALI KRISHNA3, MARELLA SARITHA1 AND CHIPPADA APPA RAO*1

1Department of Biochemistry, Sri Venkateswara University, Tirupati,AP, India2Department of Biochemistry, University of Hyderabad, Hyderabad,TS,India

3Department of Panchakarma,Sri Venkateswara Ayurvedic College and Hospital,Tirupati,AP,India

ABSTRACT

This study was aimed to investigate the antidiabetic and antioxidant activities of aqueous suspension of polyherbal mixture in streptozotocin induced diabetic rats. Oral administration of aqueous suspension of polyherbal formulated (PHF) mixture at a dosage of 300 mg/kg body weight (600 mg/day ) for 35days showed significant decrease in fasting blood glucose (FBG),hepatic and renal thiobarbituric acid reactive substances (TBARS) and catalase (CAT) levels with concomitant improvement in the levels of enzymatic antioxidants like superoxide dismutase(SOD),glutathione peroxidise(GPx) and glutathione-s-transferase (GST) in liver and kidney of STZ induced diabetic rats when compared with diabetic untreated rats. These results clearly indicate the presence of antidiabetic and antioxidant activities of polyherbal formulated mixture in diabetic rats.

Keywords: Antidiabetic Activity, Poly Herbal Formulated (PHF) mixture, streptozotocin, Antioxidant Activity

Introduction Diabetes mellitus is a metabolic disorder in humans, worldwide with characteristic hyperglycemia associated with impairment in insulin secretion and / or insulin action with associated alteration in metabolism of carbohydrates, proteins and lipids .Several reports indicate the annual growth of DM will progress at alarming rate in future worldwide , especially in India . It has been proposed that approximately 57 million Indians will be affected by DM by the year 2025[1,2]. In diabetic stage, the condition of impaired glucose metabolism leads to an increase in free radical levels which is in correlation with increasing the levels of triglycerides and lipoproteins[3]. Oxygen free radical can initiate peroxidation of lipids which in turn stimulates glycation of protein further inactivating the antioxidant enzymes and triggering the long term complications of diabetes[4]. There are lots of chemical components available to control and treat diabetes by improving insulin sensitivity or by increasing insulin production or by making use of circulating glucose levels in blood. In addition to adverse effects, drug treatment are not always satisfactory in maintaining normal level of blood glucose and avoiding late stage diabetic consequences[5].The treatment with herbal products obtained from medicinal plants is good

because they are considered to be less toxic and free from side- effects compared to synthetic one[6]

Herbal drugs are gaining popularity both in developing and developed countries because of their natural origin and less side effects. Many traditional medicines in use are derived from medicinal plants, minerals and organic matter [7]. The medicines which are prepared from medicinal plants and their parts for treatment of diseases are called ayurvedic or herbal medicine. Ayurvedic medicines are composed of medicinal herbs mixture and their preparations. Different herbs have different modes of action in decreasing the blood glucose level. For example one herb is best in controlling the fasting blood glucose level , another herb is good at suppressing the levels of post prandial glucose and some other herb is good at peripheral glucose uptake. The selection of particular part of the medicinal herb or plant depends on the scientific data of that plant/herb, regarding its storage of its phytoconstitiuents in particular part like rhizome, leaves, fruit and seed. There are a number of medicinal plants with potential effect on blood glucose which were found to be Antidiabetic. Among these various therapeutic effects like antihyperglycemic, antihyperlipidemic and antioxidant activity may act in one or in group with their beneficial effect in preventing the complications of diabetes mellitus. Hence, in the preparation of polyherbal mixture different


plants are selected according to their phytoconstituents. Therefore, the current study was focussed in preparing the polyherbal drug and its long term effect on the control of diabetes and its associated effects.

Many polyherbal formulations such as Dihar, D-400, Trasina, Diasulin and Diamed have been shown for their antidiabetic and other effects i.e., antioxidant effect, antihyperlipidemic effect etc. Ayurvedic remedies for diabetes are usually mixed formulations containing blood sugar lowering herbs in combination with immune modulators, diuretics, antioxidants and detoxicants. Madhunorm is a one such polyherbal formulation composed of 4 medicinal plants. Which are known to posses antidiabetc, antihyperlipidemic and anti oxidant activity and have been used individually in indigenuous system of medicine to treat diabetes mellitus

Materials and Methods1. Collection and Preparation of Polyherbal

Formulated mixture: There are a number of polyherbal formulated mixtures, available in the market used to treat diabetes .Among these, “Madhunorm” is one of them which is being prepared and prescribed to diabetic patients in Sri Venkateswara Ayurvedic Hospital, Tirupati for the last 20 years. There is no scientific data regarding its effect in experimental rats. This mixture was prepared, standardized and used for evaluation of its anti diabetic activity in STZ induced diabetic rats.

It is composed of powders from seeds of Syzygium cumini, fruits of Philanthus emblica, Rhizome of Curcuma longa and leaves of Gymnema sylvestra. All of these ingredients were identified and stored as voucher specimen with herbarium accession No.897, 1311, 2094, & 997 by the Botanist of department of botany, S.V.University, Tirupati. They were collected, shade dried, powdered and mixed in desired proportions to get the herbal mixture.

2. Phytochemical analysis

Phytochemical analysis of the PHF was carried out using the standard methods of qualitative analysis.

3. Chemicals

Streptozotocin was obtained from Sisco Research laboratories pvt. Ltd., Mumbai .Blood glucose estimation was done by the glucose oxidase – peroxidise method using the Accu chek active of Roche diagnostics,germany.

4. Experimental animals

Male albino wistar rats of body weight 175–225 g (3-4 months aged), were procured from Sri Venkateswara Traders, Bangalore. Animals were acclimatized in clean, dry polypropylene cages with free access to standard pellet diet and water ad libitum, in well ventilated animal house with 12 hours of dark and light at uniform laboratory conditions as per the guidelines of Institute Animal Ethics committee.

5. Induction of diabetes:

A single intraperitoneal injection of freshly prepared STZ (55 mg/kg, dissolved in 0.1 M cold citrate buffer, pH 4.5, [8])was administered to overnight fasted rats to induce Type 2 diabetes mellitus. As a result of massive destruction of pancreatic β cells, a gradual output of insulin causes hypoglycaemia in rats after 6 hours of STZ administration. Therefore, the rats were fed with 15% glucose solution to prevent hypoglycaemia for next 24 hours.

Diabetes was identified by checking blood glucose levels after 3 days of induction and confirmed by polyuria ,polydypsia symptoms . Only rats with blood glucose levels >200–350 mg/dl were selected for the study.This study was approved by Institutional Animal Ethics Committee with Resolution No: 37/2012-2013/(i)/a/CPCSEA/IAEC/SVU/CAR-TKT/dt:01.07.2012.

Experimental ProcedureThe rats were divided into 5 groups that include 6 rats in each group

1. Group: Normal untreated rats.

2. Group: Normal rats treated with PHF suspension at 600 mg/kg b.wt./day divided into two doses (each 300mg/ kg b.Wt. morning and evening ) for 35 days.

3. Group: Diabetic untreated rats.

4. Group: Diabetic rats treated with PHF suspension at 600 mg/kg Bd wt divided into two doses (each 300mg/ kg b.Wt. morning and evening ) for 35 days.

5. Group: Diabetic rats treated with 20 mg of Glibenclamide/kg bw/day for 35 days.

Polyherbal mixture aqueous suspension was administered to the rats every day morning and evening for 35 days through force feeding by using gavage needle. Blood samples were collected for every week upto 5 weeks with one week interval (on final day of that particular week) to know the fasting blood glucose levels. All groups of rats were sacrificed on 35th day with overnight fasting by anesthetizing with anaesthetic ether and further by cervical dislocation. liver and kidney were collected and immediately stored at -200 c until the use.

Experimental methods for analysis:

Phytochemical analysis was carried out in the by different methods of phytochemical analysis [9].Estimation of blood glucose was carried out by glucose oxidase–peroxidase method[10]. The levels of TBARS in tissues were estimated by the method of Fraga et al .1988.[11]. CAT activity was assayed following the method of Sinha ., 1972 [12]. SOD activity was assessed according to the method of Kakkar et.al.,1984 [13]. GPx activity was measured as described by Rotruck et.al., 1973 [14]. GST activity was estimated according to the method of Habig et al. 1974[15].


Statistical analysis

All results were expressed as mean± standard deviation. The statistical analysis of results was carried out using one-way analysis (ANOVA) followed by DMRT.

Results:Phytochemical analysis of Polyherbal mixture Phytochemical analysis of the aqueous suspension of PHF has shown the presence of Saponins, alkaloids, carbohydrates, flavanoids, Tannins and glycosides. Table.1 shows effect of long term treatment with the PHF on fasting blood glucose and body weights of different experimental groups of rats.

On starting of experiment, the FBG of all diabetic groups were significantly higher than that of normals. But on 35th day of treatment , there was 72% reduction(P< 0.001) in the FBG levels of diabetic rats treated with PHF suspension. Only 45.7 % decrease in blood glucose levels in the diabetic rats treated with glibenclamide were observed .The untreated diabetic rats continued with the elevated blood glucose till the end of the experimental period . The normal rats treated with PHF showed the blood glucose levels similar to those of the normal untreated rats

The body weights of normal control rats were significantly increased by 44gr, while the Normal rats

treated with PHF showed only slight increase i.e., 2.5 gr. The diabetic untreated rats, diabetic treated with PHF and diabetic treated with glibenclamide showed decrease in their body weights (-44g, -17.5g and -40.8 g. respectively).

Table.2 and Table.3 show the Liver and Kidney (respectively) levels of TBARS, activities of SOD, CAT, GPx and GST in the Normal and Experimental Groups of rats.

The levels of TBARS and CAT activity were significantly increased whereas the activities of SOD, GPx and GST were decreased in both tissues of diabetic control rats.PHF treated diabetic rats showed decreased levels of TBARS, CAT activity and increased activities of SOD , GPx and GST in liver and kidney. Glibenclamide treated diabetic rats showed similar effects like that of PHF but with less magnitude when compared to those treated with PHF.

Discussion Streptozotocin Induced Diabetic Rats are widely accepted animal model and reported to resemble human hyperglycemic non ketotic diabetes mellitus[16]. Sometimes it exhibits the kidney hypertrophy which contribute to end stage renal damage, hepatotoxicity, oxidative stress and Hyper cholesterolemia[17,18].Due to beta cell damage in stz induced rats, hyperglycemia is sustained. The FBG in diabetic rats treated with PHF were maintained to normal

Table 1Effect of Long Term Treatment with PHF aqueous suspension on Blood Glucose and Body Weights of Normal,

Diabetic and Diabetic Treated Rats

Groups

Blood glucose(mg/dl) and change in body weights at different weeks during the experimental period with PHF

Initial Ist week IInd week IIIrd week IVth week Vth weekChange in body

weights (g)

N 98.16±7.8a 90.8± 4.6a 92.17 ±7.9a 89.17 ±8.2a 88.5 ±5.2a 86.67 ±9.42a +44

N T 96 ±9.38a 88.17 ±6.2a 91 ±6.6a 93 ±7.2a 90.7 ±9.4a 85.5 ±7.3a +2.5

DC 355±44.18b 378.3±40.3c 439±42c 461.2±41.2d 467.7±33d 449.2±43.6c -44

DT 357.16±42b 290 ±43.5b 231 ±43.7b 178 ±35.9b 130.5 ±16.1b 99.8 ±7.5a -17.5

DG 347.33±44b 302 ±42.6b 267 ±41.9b 235.2 ±43.3c 208.1 ±41.7c 163.7 ±27.2b -40.8

F value 101.409 97.553 112.479 139.733 236.091 254.438

Significance 0.000 0.000 0.000 0.000 0.000 0.000

Values are given as mean ± S.D from six rats in each group.Values not sharing a common superscript letter differ significantly at p < 0.01 (DMRT).


level indicating its antidiabetic potiental . This could be due to presence of phytoconstituents which control the FBG in different mechanistic pathways. The phytochemical analysis of Madhunorm revealed the presence of Saponins, Alkaloids, Carbohydrates, Flavanoids, Tannins and Glycosides.

Increased muscle wasting and loss of tissue proteins are the main reason behind the loss of body weight in STZ induced diabetic rat models [19,20]. The diabetic rats treated with PHF suspension were shown decreased body weights but the decrease was less than that of glibenclamide treated group. Very less increase in body weights in normal rats

treated with PHF indicates that the PHF prevent the increase in bodyweight in normal rats and loss of body weight in diabetic rats.

Diabetes induced in male albino wistar rats by streptozocin is via free radical generation which there by leads to DNA damage, protein degradation, lipid peroxidation and finally damaging various organs of the body like liver , kidney , brain and eyes. Hence, elevated levels of free radicals and decreased levels of antioxidant enzymes lead to cell disruption due to lipid peroxidation in the cell membranes [21]. In untreated diabetic rats, the

Table - 3Effect of Long Term Treatment with suspension of PHF on TBARS levels and Antioxidant Enzyme Activities in the

KIDNEY of Different Experimental Groups

GroupsLipid Peroxides

(nmoles MDA/mgprotein)

Catalase(U/mg protein)

Glutathione Peroxidase

(U/mg protein)

SuperoxideDismutase

(U/ mg protein)

Glutathione-S- Transferase

(U/mg protein)

N 0.20013±0.004 a 37.135±3.077 b 0.275±0.005 c 11.665±1.77 b 21.01±0.705 c

N T 0.1949±0.012 a 31.74±2.07 a 0.294±0.012 e 12.37±1.37 b 17.93±0.408 b

DC 0.3154±0.017 e 69.61±5.30e 0.1717±0.008 a 5.903±1.115 a 7.813±0.351 a

DT 0.2128±0.0045 b 35.363±4.6 b 0.265±0.018 c 10.6±1.052 b 18.56±1.057 b

DG 0.2249±0.0046 b 44.09±2.519 c 0.222±0.024 b 7.65±1.978a 18.59±0.385 b

F value 136 99.9 60 20 387

Significance 0.000 0.000 0.000 0.000 0.000


Table 2Effect of Long Term Treatment with the suspension of PHF on TBARS levels and Antioxidant Enzyme Activities in

the LIVER of Different Experimental Groups

GroupsLipid Peroxides

(nmoles MDA/mgprotein)

Catalase(U/mg Protein)

Glutathione Peroxidase

(U/mg protein)

SuperoxideDismutase

(U/mg protein)

Glutathione-S- Transferase

(U/ mg protein)

N 0.2801±0.005c 41.58±3.60a 0.313±0.007c 13.86±1.73b 22.77±0.86d

N T 0.264±0.004b 36.62±2.67a 0.330±0.016c 13.71±0.97b 23.73±0.82e

DC 0.3615±0.005e 94.23±11.5c 0.184±0.027a 6.97±1.71a 9.44±0.31a

DT 0.2321±0.007a 52.6±2.72b 0.240±0.011b 12.45±1.553b 19.55±0.55c

DG 0.2360±0.008a 59.48±6.07b 0.180±0.021a 8.05±0.93a 16.46±0.45b

F value 381 82 88 31 486

Significance 0.000 0.000 0.000 0.000 0.000



TBAR levels were increased, which are the end products of lipid peroxidation. The free radical scavenging effect of PHF is maximum with decreasing the levels of TBARS in the tissue like liver and kidney of diabetic treated with the PHF suspension. Implication of oxidative stress in the pathogenesis of diabetes mellitus is suggested not only by oxygen free radical generation but also due to non-enzymatic protein glycosylation, auto-oxidation of glucose, impaired antioxidant enzyme, and formation of peroxides [22,23]. The events of free radical formation and decrease in antioxidant enzymes conditions are common in diabetes with imbalance in the oxidant/antioxidant defence system [24]. The present data reveals that the levels of antioxidant enzymes like SOD, CAT, GPx and GST were altered. In our study, the activities of SOD, GPx and GST were decreased in the diabetic rats compared to normal rats, which could be due to free radical- induced inactivation and glycation of the enzymes in diabetic state [25].

In diabetic untreated rats, catalase activity in liver and kidney was increased, it could be a compensatory mechanism to prevent tissue damage by the increased levels of H2O2 and decreased levels of GPx. In diabetic condition , insulin levels were decreased, that increases the activity of fatty acyl coenzyme A oxidase, which initiates β- oxidation of fatty acids , resulting in increased levels of H2O2 .The PHF treated diabetic rats showed decrease in the catalase activity in both tissues and also simultaneous increase in the activities of SOD, GPx and GST indicating its anti oxidant effect by scavenging the free radicals generated during diabetic condition. The antidiabetic and antioxidant activity of (Madhunorm) poly herbal formulation could be due to different types of phytoconstituents from various plants which may have different mode of action in their mechanism. The combination of plants containing phytoconstituents, may be having additive effect at the same time. According to patel et al., 2012 [26] and Malviya et al,2010[27] the therapeutic effect of alkaloids,flavonoids,saponins and tannins were additive in collective action in controlling the blood glucose and maintaining normal levels of elevated lipids in blood . The herbal formulation is considered to be a, Safe therapy for long term and effective management of diabetes mellitus.

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[9] Harborne JB 2005; Phytochemical Methods. Analysis. A Guide to Modern Techniques of Plant, third ed. Springer International, India

[10] Kesari AN, Gupta RK, Singh SK, Diwakar S, Watal G. Hypoglycaemic and antihyperglycaemic activity of Aegle marmelos seed extract in normal and diabetic rats. J Ethnopharmacol 2006; 107: 374–379.

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[14] Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B. Selenium: Biochemical role as a component of Glutathione peroxidase. Science 1973; 179, 588-590.

[15] Habig WH, Pabst MJ, Jakoby WB. Glutathione transferase. The first enzymatic step in mercapturic acid formation. J Biol Chem 1974 ; 249, 7130-7139.

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Traditional plant treatment for diabetes: studies in normal and streptozotocin diabetic mice. Diabetologia 1990; 33: 462-464.

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[21] Yazdanparast R., Amin A, Jamshidi S. Experimental diabetes treated with Achillea santolina: effect on pancreatic oxidative parameters. J. Ethnopharmacol. 2007 ; 112, 13–18.

[22] Vincent AM, Russell JW, Low P, Feldman EL. Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev 2004; 25, 612–28.

[23] Pari L, Latha M. Antidiabetic effect of Scoparia dulcis: effect on lipid peroxidation in streptozotocin diabetes. Gen Physiol Biophys 2005; 24, 13–26.

[24] Nazirogilu M, Butterworth P. Protective effects of moderate exercise with dietary Vitamin C and E on blood antioxidative defense mechanism in rats with streptozotocin Induced diabetes. Can J Appl Physiol 2005; 30, 172–185

[25] Al-Azzawie HF, Alhamdani MS. Hypoglycaemic and antioxidant effect of oleuropein in alloxan-induced diabetic rabbits. Life Sci. 2006; 78(12), 1371-1377

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Synthesis, characterization and antimicrobial evaluation of novel compounds of 1-(2,5-difluorobenzoyl)-4-(2-(4-phenyl)hydrazono) -3-(5-(4-(trifluoro methyl) phenyl)-1H-tetrazol-1-yl)-1H-pyrazo l -

5(4H)-one

SWARNA KUMARI M1*, SUDHAKAR BABU K1, RAVINDHRANATH LK1, LATHA J^2, AND HARINAGAMADDAIH K 2

1Department of Chemistry, Sri Krishnadevaraya University, Anantapur, (AP) India.2Department of Bio-technology, SKUCET, Sri Krishnadevaraya University, Anantapur, (AP) India.

ABSTRACT

New novel derivatives of 3-chloro-6-(2,5-difluorobenzoyl)-1-(phenylamino)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl) -1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2a-g) were prepared by condensation of 1-(2,5-difluorobenzoyl)-4-(2-phenylhydrazono) -3 - (5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1H-pyrazol-5(4H)-one (1a-g). The newly synthesized compounds were characterized by IR, 1H-NMR, 13C-NMR, mass spectra & Elemental analysis. The newly synthesized compounds were screened for their Biological activity.

Key words:Tetrazol, monochloro acetyl chloride , Antibacterial and Antifungal activity,spectral data.

Introduction Tetrazoles are class of five member Heterocyclic compounds with four nitrozen and one carbon atom beside hydrozen. Terazoles and its derivatives are associated with a variety biological activities such as anti fungal[1-2], antinociceptive[3-4], anti-convulsant[5], antidiabetic [6], cyclo-oxygenase inhibitors [7], hypoglycaemic [8], antibacterial [9] and anti-inflammatory [10], activities. Tetrazoles are used as catalysts in the synthesis of phosphonates.

Materials & Methods All the chemicals used in the present investigation were purchased from Sigma-Aldrich Chemicals Company, Inc.USA. And used without further purification. TLC was performed on aluminum sheet of silica gel 60F254, E-Merk,Germany using iodine as visualizing agent. Melting point was determined in open capillary tubes on Mel-Tempapparatus and is uncorrected. Column chromatography was performed on silica gel with different solvent systemsas

Reagents & Reaction Conditions: monochloro acetyl chloride, dichloro methane ,stirred 8hr RT, (7:3)cyclohexane,ethyl acetate, left 3 days RT, vaccume RE,60-120 mesh , silica gel, crushed ice

R H- CH3- OCH3- Cl- Br NO2 CF3


eluents to afford the pure compound. The IR Spectra were recorded as KBr pellets on Perkin-Elmer 1000 units, instruments. All 1H and 13C-NMR spectra were recorded on a Varian XL-300 spectrometer operating at 400MHzfor 1H -NMR and 75 MHz for 13C-NMR were recorded on a Varian XL-spectrometer operating at161.89MHz. The compounds were dissolved in DMSO-d6 and Chemical shifts were referenced to TMS (1H and13C-NMR) .Mass spectral data were recorded on FAB-MS instrument at 70ev with direct inlet system. Elemental analysis wasrecorded on a Carlo Erba 1108 elemental Analyzer, Central Drug Research Institute, Lucknow, India.

RESULTS AND DISCUSSIONSynthesis of 1-(2,5-difluorobenzoyl)-4-(2-(4-substituted phenyl) hydrazono)-3-(5-(4-(trifluoro methyl ) phenyl)-1H-tetrazol-1-yl)-1H-pyrazol-5(4H)-one (1 a-g)

A mixture of 1-(2,5-difluorobenzoyl)-4-(2-(4–substituted phenyl) hydrazono)-3-((4-(trifluoro methyl )benzylidene) amino)-1H-pyrazol-5(4H)-one (1 a-g) (0.004 mol) and PCl5 (0.004 mol) was heated at 1000C for 1hour, when the evolution of fumes of HCl ceased, excess of PCl3 was removed under reduced pressure .The residual imidoyl chloride was treated with an ice-cold solution of sodium azide (0.0075 mol) and excess of sodium acetate (0.008 mol)in water (25 ml) and acetone (30 ml). The reaction mixture was stirred for 20 hours. The progress of the reaction was monitored by TLC using cyclohexane and ethyl acetate solvent mixture (7:3).After completion of the reaction, the acetone was removed under reduced pressure. The remaining reaction mixture was extracted with chloroform (3x20 ml). The chloroform extract was dried under reduced pressure and the residue purified by column chromatography using silica (60-120 mesh) and cyclo hexane and ethyl acetate solvent mixture(7:3) as an elutent.

Synthesis of 3-chloro-6-(2,5-difluorobenzoyl)-8-(5(4-trifluoromethyl-phenyl-1H-tetrazol-1-yl)-1-((4-substituted-phenyl-amino)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione 2a-g

Monochloro acetyl chloride 0.01 mole was added drop wise to 1-(2,5-difluorobenzoyl)-4-(2-(4-substitutedphenyl)hydrazono)-3-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1H-pyrazol-5(4H)-one (1a, 0.01 mole) and tri ethyl amine 0.02 mole in dioxane (25 ml) at room temperature. The reaction mixture was stirred for 8 hours and left at room temperature for three days. Poured the contents on crushed ice. The product 3-chloro-6-(2,5-difluorobenzoyl)-8-(5(4-trifluoromethyl-phenyl-1H-tetrazol-1-yl)-1-(phenyl-amino)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2a) thus formed was filtered and washed with absolute alcohol.

Physical, analytical and spectral data for the compounds:

3-chloro-6-(2,5-difluorobenzoyl)-1-(phenylamino)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2 a)

Yield 70%.

m p: 124-126OC. IR (KBr): 3225(stretching vibration of -NH), 1675 (stretching vibration of cyclic carbonyl in pyrazoline-5-one ring), 1656(Exo cyclic >C=O group), 1694(stretching vibration of >C=O group of azetidinone), 1157 and 2102(characteristic fundamental vibrations of tetrazole).

1H-NMR (400 MHZ DMSO-d6):) 5.3(s,1H,-CH of azetidinone group), 8.6(s,1H,Ar-NH-,attached to azaspiro ring),6.57-8.28(m,12H,C6H5,C6H4 and C6H3 group). 13CNMR(75MHz,DMSO-d6): 154.3, 113.5, 125.6, 126.9, 163.5, 56.5, 74.0 ,176.1, 155, 170.2, 126.7, 154.9, 118.7, 120.5, 158.3, 113.9, 163.5, 130.6, 127.5, 129.2, 131.1, 124.1 corresponding to C1,C2& C6,C3 & C5 , C4,C7, C8, C9, C10,C11,C12,C13,C14, C15,C16, C17, C18, C19,C20 , C21&C25,C22&C24,C23,C26 respectively.,Anal.Calcd. For C26H14ClF5N8O3 C 46.74% , H 2.25% and N14.39%. Found: C 46.34% , H 1.93% and N 14.02%.

3-chloro-6-(2,5-difluorobenzoyl)-1-((4-methoxyphenyl)amino)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2 b)

Yield 70%.m p: 120-123OC.

IR (KBr): 3233(stretching vibration of -NH), 1683 (stretching vibration of cyclic carbonyl in pyrazoline-5-one ring), 1664(Exo cyclic >C=O group), 1699(stretching vibration of >C=O group of azetidinone), 1154 and 2194(characteristic fundamental vibrations of tetrazole).

1H-NMR (400 MHZ DMSO-d6): ) 2.36 (s,3H, CH3 Group), 5.6(s,1H,-CH of azetidinone group), 8.9(s,1H,Ar-NH-,attached to azaspiro ring),6.59-8.29(m,12H,C6H5,C6H4 and C6H3 group). 13CNMR(75MHz,DMSO-d6): 154.3, 113.5, 125.6, 126.9, 163.5, 56.5, 74.0 ,176.1, 155, 170.2, 126.7, 154.9, 118.7, 120.5, 158.3, 113.9, 163.5, 130.6, 127.5, 129.2, 131.1, 124.1 corresponding to C1,C2& C6,C3 & C5 , C4,C7, C8, C9, C10,C11,C12,C13,C14, C15,C16, C17, C18, C19,C20 ,C21&C25,C22&C24,C23,C26 respectively.,Anal.Calcd. For C27H16ClF5N8O3 C 46.79% , H 2.29% and N14.39%. Found: C 46.39% , H 1.99% and N 14.09%.

3 - c h l o r o - 1 - ( ( 4 - c h l o r o p h e n y l ) a m i n o ) - 6 - ( 2 , 5 -difluorobenzoyl)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2c)Yield 70%.m p: 134-136OC. IR (KBr): 3240(stretching vibration of -NH), 1690 (stretching vibration of cyclic carbonyl in pyrazoline-5-one ring), 1661(Exo cyclic >C=O group), 1700(stretching vibration of >C=O group of azetidinone), 1159 and 2199(characteristic fundamental vibrations of tetrazole).

1H-NMR (400 MHZ DMSO-d6): ) 3.9 (s,3H, -OCH3 Group), 5.29(s,1H,-CH of azetidinone group), 8.59(s,1H,Ar-NH-,attached to azaspiro ring),7.19-7.49 (m,11H,C6H5,C6H4 and C6H3 group). 13CNMR(75MHz,DMSO-d6): 154.3,


113.5, 125.6, 126.9, 163.5, 56.5, 74.0 ,176.1, 155, 170.2, 126.7, 154.9, 118.7, 120.5, 158.3, 113.9, 163.5, 130.6, 127.5, 129.2, 131.1, 124.1 corresponding to C1,C2& C6,C3 & C5 , C4,C7, C8, C9, C10,C11,C12,C13,C14, C15,C16, C17, C18, C19,C20 ,C21&C25,C22&C24,C23,C26 respectively.,Anal.Calcd. For C27H16ClF5N8O 4 C 46.79% , H 2.29% and N15.39%. Found: C 46.34% , H 1.99% and N 15.02%.

1 - ( ( 4 - b r o m o p h e n y l ) a m i n o ) - 3 - c h l o r o - 6 - ( 2 , 5 -difluorobenzoyl)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2d)Yield 70%.m p: 129-134OC. IR (KBr): 32340 (stretching vibration of -NH), 1680 (stretching vibration of cyclic carbonyl in pyrazoline-5-one ring), 1664(Exo cyclic >C=O group), 1699(stretching vibration of >C=O group of azetidinone), 1154 and 2194(characteristic fundamental vibrations of tetrazole).

1H-NMR (400 MHZ DMSO-d6): ) 3.6 (s,3H, -OCH3 Group), 5.40(s,1H,-CH of azetidinone group), 8.54(s,1H,Ar-NH-,attached to azaspiro ring),7.14-7.44 (m,11H,C6H5,C6H4 and C6H3 group). 13CNMR(75MHz,DMSO-d6): 154.3, 113.5, 125.6, 126.9, 163.5, 56.5, 74.0 ,176.1, 155, 170.2, 126.7, 154.9, 118.7, 120.5, 158.3, 113.9, 163.5, 130.6, 127.5, 129.2, 131.1, 124.1 corresponding to C1,C2& C6,C3 & C5 , C4,C7, C8, C9, C10,C11,C12,C13,C14, C15,C16, C17, C18, C19,C20 ,C21&C25,C22&C24,C23,C26 respectively.,Anal.Calcd. For C26H13Cl2F5N8O3 C 46.74% , H 2.25% and N14.39%. Found: C 46.34% , H 1.93% and N 14.02%.

3-chloro-6-(2,5-difluorobenzoyl)-1-((4-nitrophenyl)amino)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2e)Yield 70%.m p: 123-129OC. IR (KBr): 3230(stretching vibration of -NH), 1680(stretching vibration of cyclic carbonyl in pyrazoline-5-one ring), 1661(Exo cyclic >C=O group), 1699(stretching vibration of >C=O group of azetidinone), 1151 and 2194(characteristic fundamental vibrations of tetrazole).

1H-NMR (400 MHZ DMSO-d6): ) 3.6 (s,3H, -OCH3 Group) , 5.20(s,1H,-CH of azetidinone group), 8.50(s,1H,Ar-NH-,attached to azaspiro ring),7.10-7.40 (m,11H,C6H5,C6H4 and C6H3 group). 13CNMR(75MHz,DMSO-d6): 154.3, 113.5, 125.6, 126.9, 163.5, 56.5, 74.0 ,176.1, 155, 170.2, 126.7, 154.9, 118.7, 120.5, 158.3, 113.9, 163.5, 130.6, 127.5, 129.2, 131.1, 124.1 corresponding to C1,C2& C6,C3 & C5, C4,C7, C8, C9, C10,C11,C12,C13,C14, C15,C16, C17, C18, C19,C20 ,C21&C25,C22&C24,C23,C26 respectively.,Anal.Calcd. For C26H13BrClF5N8O3 C 46.74% , H 2.25% and N14.39%. Found: C 46.34% , H 1.93% and N 14.02%.

3-chloro-6-(2,5-difluorobenzoyl)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1-((4-(trifluoromethyl)phenyl)amino)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione(2f)

Yield 70%.

m p: 127-129OC.IR (KBr): 3230 (stretching vibration of -NH), 1680 (stretching vibration of cyclic carbonyl in pyrazoline-5-one ring), 1661(Exo cyclic >C=O group), 1699(stretching vibration of >C=O group of azetidinone), 1151 and 2194(characteristic fundamental vibrations of tetrazole).

1H-NMR (400 MHZ DMSO-d6): ) 3.6 (s,3H, -OCH3 Group), 5.20(s,1H,-CH of azetidinone group), 8.50(s,1H,Ar-NH-,attached to azaspiro ring),7.10-7.40 (m,11H,C6H5,C6H4 and C6H3 group). 13CNMR(75MHz,DMSO-d6): 154.3, 113.5, 125.6, 126.9, 163.5, 56.5, 74.0 ,176.1, 155, 170.2, 126.7, 154.9, 118.7, 120.5, 158.3, 113.9, 163.5, 130.6, 127.5, 129.2, 131.1, 124.1 corresponding to C1,C2& C6,C3 & C5 , C4,C7, C8, C9, C10,C11,C12,C13,C14, C15,C16, C17, C18, C19,C20 ,C21&C25,C22&C24,C23,C26 respectively.,Anal.Calcd. For C26H13ClF5N9O5 C 46.74% , H 2.25% and N14.39%. Found: C 46.34% , H 1.93% and N 14.02%.

3-chloro-6-(2,5-difluorobenzoyl)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1-((4-(trifluoromethyl)phenyl)amino)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2g)

Yield 70%.

m p: 124-126OC.

IR (KBr): 3230 (stretching vibration of -NH), 1680 (stretching vibration of cyclic carbonyl in pyrazoline-5-one ring), 1661(Exo cyclic >C=O group), 1699(stretching vibration of >C=O group of azetidinone), 1151 and 2194(characteristic fundamental vibrations of tetrazole).

1H-NMR (400 MHZ DMSO-d6): ) 3.6 (s,3H, -OCH3 Group) , 5.20(s,1H,-CH of azetidinone group), 8.50(s,1H,Ar-NH-,attached to azaspiro ring),7.10-7.40 (m,11H,C6H5,C6H4 and C6H3 group). 13CNMR(75MHz,DMSO-d6): 154.3, 113.5, 125.6, 126.9, 163.5, 56.5, 74.0 ,176.1, 155, 170.2, 126.7, 154.9, 118.7, 120.5, 158.3, 113.9, 163.5, 130.6, 127.5, 129.2, 131.1, 124.1 corresponding to C1,C2& C6,C3 & C5 , C4,C7, C8, C9, C10,C11,C12,C13,C14, C15,C16, C17, C18, C19,C20 ,C21&C25,C22&C24,C23,C26 respectively.,Anal.Calcd. For C27H13ClF8N8O3 C 46.74% , H 2.25% and N14.39%. Found: C 46.34% , H 1.93% and N 14.02%.

Mass Spectra The electron impact mass spectrum of 3-chloro-6-(2,5-difluorobenzoyl)-8-(5-4-trifluoromethylphenyl-1H-tetrazol-1-yl)-1-((4-(trifluoromethyl)phenylamino)-1,6,7-triazaspiro [3.4]oct-7-ene-2,5-dione (2a) was recorded and interpreted. The mass spectral data of compound 6a showed the molecular ion [M+] ion peak at m/z= 616.08(100% ) and M+2 peak at m/z= 618.08(30.35%) the relative abundance of M7+ and M+2 were in there ratio of 3:1 indicate the presence of one chlorine atom. The even m/z value of molecular ion (M7+) indicates the presence of even number of nitrogen atoms in molecular ion.. The molecular ion signal obey Nitrogen rule, while the primary fragmented ions derived from molecular ion signal may or may not obey Nitrogen rule.


Table:Mass spectral primary fragmented data of 3-chloro-6-(2,5-difluorobenzoyl)-8-(5-(4-trifluoromethyl-phenyl-1H-

tetrazol-1-yl)-1-(phenylamino)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2a)

Molecular ion Lost radical Primary fragmented ion m/z values Relative abundance (R.A) (%)

C26H14ClF5N8O3 (M+)

m/z: 616.08 (100%)618.08(33.0%)

(6a)

C19H9ClF5N4O3• C7H5N4+(II) 145.05 9.0

C7H5N4• C19H9ClF5N4O3+(III) 471.03473.03

32.99.7

C19H9ClF5N7O3• C7H5F3N+(IV) 160.04 7.6

C7H5F3N• C19H9ClF5N7O3+(V) 456.04458.04

32.69.8

C7H3F2O• C19H11ClF3N8O2+(VI) 475.06477.06

32.69.8

C19H11ClF3N8O2• C7H3F2O+(VII) 141.02 7.6

C6H3F2• C20H11ClF3N8O3+(VIII) 114.02 6.5

C20H11ClF3N8O3• C6H3F2+(IX) 503.06505.06

33.29.4

C20H10ClF2N8O3• C6H5+(X) 113.02 7.6

C6H5• C20H10ClF2N8O3+(XI) 539.06541.04

32.069.8

Biological Sctivity

The antimicrobial activity [11-12] of these newly synthesized compounds was performed according to disc diffusion method, as recommended by the National

Committee for Clinical Laboratory [13]. The synthesized compounds Were used at the concentration of 250μg/ml DMF as a solvent [14].

The Atimicrobial profile of 3-chloro-6-(2,5-difluorobenzoyl)-1-(phenylamino)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2 a-g) Antibacterial activity (Diameter zone of Inhibition in mm) of Compounds of 2(a-g)

Entry

Bacteria FungiStaphylococcus

aureus NCCS2079

Bacillus cereus NCCS

2106

Escherichia coliNCCS2065

Aspergillus niger NCCS 1196

Candida albicans NCCS 2106

25 50 25 50 25 50 25 50 25 502a - 12 - 11 - 10 - 13 - 14 2b - 11 - 13 - 12 - 12 - 12 2c - 14 - 12 - 14 - 14 - 13 2d 13 17 13 18 12 16 11 16 10 15 2e 12 15 13 12 11 15 10 15 09 14 2f 19 19 16 15 15 18 14 19 15 18 2g 18 17 14 12 13 16 12 17 11 16

Chlorom-phenicol (5) - 25 - 26 - 22 - - - -

Ketocanazole(50) - - - - - - - 22 - 24

Each well contains 25 and 50 µg/mL of compounds ; Ch=Chloromphenicol 5 µg/mL, Ketocanazole 50 µg/mL


Result and Discussion The antibacterial activity of 3-chloro-6-(2,5-difluoro-benzoyl)-1-(phenylamino)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2a-g) was screened against the gram-positive bacteria Staphylococcus aureus NCCS 2079 and Bacillus cereus NCCS 2106 The gram negative bacteria used was Escherichia coli 2065.The antibacterial results reveals that most of the compounds exhibit good antibacterial activity against both bacteria at the concentration of 50µg/mL.The presence of nitro (2f), tri fluoro methyl(2g),Chloro (2d) and Bromo(2e) were showed more activity than other substituted compounds. Here Chloromphenicol was used as reference compound to Compare the activity.

The anti-fungal activity of 3-chloro-6-(2,5-difluoro-benzoyl)-1-(phenylamino)-8-(5-(4-(trifluoromethyl)phenyl)-1H-tetrazol-1-yl)-1,6,7-triazaspiro[3.4]oct-7-ene-2,5-dione (2 a-g) was screened against the Aspergillus niger NCCS1196 and Candida albicans NCCS 2106. The antifungal results reveal that most of the compounds exhibit good anti-fungal activity against both fungi at the concentration of 50µg/mL. The presence of nitro (2f), tri fluoro methyl(2g),Chloro (2d) and Bromo(2e) were showed more activity than other substituted compounds. Here Ketocanazole was used as reference compound to Compare the activity

The order of anti microbial activity ( 50µg/mL) 2f>2g >2d>2e>2c>2a>2b.

Acknowledgement The author is very thankful to UGC authorities, New Delhi for providing UGC BSR SAP fellowship to carry out present research work and also thankful to IICT, Hyderabad and CDRI to get spectral data. The author expresses sincere thanks to Dept. of Chemistry, Sri Krishna Deveraya University for carrying out research work.

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[2] Popat.B.Mohite, Vaidhun.H.Bhaskar; Orbital-The electronic Journal of Chemistry, 2(3), 2010, 311-315.

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Online : ISSN 2349-669X Print : ISSN 0973-9874


Evaluation of Tephrosia Purpurea for Anti Hypelipidemic Activity in High Fat Induced Rats

RAGHAVENDRA HG*, LAKSHMIKANTH G1 AND SAMATHA Y2

1Department of Pharmacology C.E.S College of Pharmacy, Kurnool2Department of Pharmacognosy J.N.T.U Hyderabad.

ABSTRACT

The anti-hyperlipidemic effect of methanolic extract of leaves of Tephrosia purpurea (T.purpurea) was tested in high fat diet induced hyperlipidemic rat models. Here, chronic hyperlipidemia was induced by feeding fat diet for 21 days. The hyperlipedemic rats are grouped and Treated with fenofibrate (5mg/kg), T.purpurea extract, (200 and 400 mg/kg, p.o). The plant extract treated groups are significantly reduced the hyperlipidemia i.e., decreased levels of serum Total Cholesterol, Trigly cerides, Low Density Lipoprotein Cholesterol (LDL-C), and increase of serum High Density Lipoprotein Cholesterol (HDL-C) whencompared to vehicle control and standard drug Fenofibate (5 mg/kg). The results demonstrated that methanolic extract of leaves of T.purpurea possessed significant antihyperlipidemic activity.

Introduction Hyperlipidemia is the term used to denote raised serum levels of one or more of total cholesterol, low density lipoprotein cholesterol, triglycerides or both total cholesterol and triglyceride (combined hyperlipidaemia). Dyslipidaemia is a wider term that also includes low levels of high-density lipoprotein cholesterol1.

Hyperlipidemia represents a major risk factor for the premature development of atherosclerosis and its cardiovascular complications. Hyperlipidemia is a disorder characterized by the increase in blood lipoprotein or cholesterol levels. Recent studies have shown that lipid associated disorders are not only attributed to the total serum cholesterol, but also to its distribution among different lipoproteins. The low density lipoproteins (LDL) are the major carriers of cholesterol towards tissues having atherogenic potential, while the high density lipoproteins (HDL) carry cholesterol from peripheral tissues to the liver. HDL thus give protection against many cardiac problems and obesity. Although genetic factors recline behind these lipid disorders2. Oxidative stress by oxidation of low density of low density lipoproteins plays a major role in hyperlipidemia.

The disorder of hyperlipidemia is considered to be a problem in patients with diabetes and is one of the major risk factor in the development of atherosclerotic heart diseases3.

Excess triglyceride accumulation and increased fatty acid oxidation in diabetic heart contribute to cardiac dysfunction. Lipid metabolism normally maintains an elegant balance between its synthesis and degradation when this balance is disrupted hyperlipidemia, hypertriglyceridemia, and hypercholesterolemia may develop, this can cause variety of diseases such as hypertension, diabetes, obesity, atherosclerosis4.

Plants have a significant role in maintaining human health and improving the quality of human life for thousands of years and served humans well as valuable components of medicine5. Ayurveda is an ancient form of Indian medicine which deals with plants products, this indigenous form of medicine uses the active ingredients present in plants for treating disease6 .Plant products are frequently considered to be less toxic and free from side effects than synthetic ones. The prophylactic and therapeutic effect of plant foods and extracts in reducing cardiovascular disease has been reviewed. A vast number of these plants are to receive attention in this regard and have been shown to lower plasma lipid levels, some examples are Gacinia cambogia, Zingiber officinale and Emblica officinalis7. The wide use of these herbal plants had now leads to carry out research in institutions and universities on the potential benefits of herbal drugs. Our present study is also an attempt towards this direction8.


Materials and Methodsi. Plant Collection

Tephrosia purpurea was collected around Kurnool, Creative Educational Society, JNTU University, Ananthapur. Leaves and stems were separated, washed in water, chopped into pieces and then shade dried. The coarsely powdered leaves were extracted with methanol using sox let apparatus for 3 hr. The cycle was repeated for three times. The extract was concentrated on rotary flash evaporator to semi solid consis-tence and then dried over a water bath (yield–30.6 g/kg).

ii. Animals

Male and Female albino rats (150-200gm) of body weight were obtained from Tirupati, and they were housed under standard husbandry conditions 25±5°C temperature, light/dark cycle with standard rat feed (Pravan agro Ltd. India) and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethical Committee of Sri Padmavathi School of Pharmacy (1016/a/06/CPCSEA/008/2009).

iii. Drugs and Chemicals Fenofibate c Epinephrine, DTNB (sigma), Thiobarbituric acid (TBA) and Trichloro acetic acid, hydrogen peroxide (SD fine chemicals Ltd). Sodium dihydrogen phosphate, potassium dihydrogen phosphate, tries buffer and all other reagents used were of analytical grade. Total cholesterol and HDL, Triglycerides and Total protein estimation kits were procured from Kamineni Life Sciences Pvt. Ltd. India.

iv. Instruments

Semi auto analyzer (mixpel)

v. Acute Toxicity Studies

Acute oral toxicity study was performed as per OECD – 423 guidelines (acute toxic class method). Wistar albino rats of either sex were selected by random sampling technique and divided into five groups (n = 6). The animals were fasted overnight and methanolic extract in doses of 500, 1000, 2000, 4000 mg/kg body weight, were administered orally to II – V groups. Group I which received vehicle (water) served as control. The animals were observed continuously for 2 hr, then intermittently for 6 hours at the end of 24 hours, the number of were noted deaths and to determine LD50 of extract9.Animals were also observed for behavioral, neurological and autonomic profiles simultaneously10.

vi. High Fat Diet (Fd) Induced Hyperlipidemic Model

Normal animal food pellets were crushed in mortar and pestle to crush into small pieces and then grinded into fine powder in mixer grinder. The other ingredients i.e. cholesterol 2% , Cholic acid 1% , sucrose 40% , and coconut oil 10% were added in the mixer grinder in an ascending order of their quantity and mixed well. This dried powder was then mixed with same quantity of water every time to make small balls of feed and later this was stored in self sealing plastic covers in refrigerator at 2°C to 8°C. The feed

for normal group was prepared similarly by grinding only the normal food pellets and then mixing with water without the other exepients. This preparation of feed was done once in three days for all the animals. Thirty Wister rats were randomly divided into five groups of six each. The chronic experimental hyperlipidemia was produced by feeding the above prepared food for 21 days. The rats are then given test plant extracts i.e., T.purpurea (200 and 400 mg/kg, p.o) and fenofibrate (5 mg/kg, p.o) once daily in the morning orally for 28 consecutive days. During these days, all the groups also received fat diet in the same dose as given earlier. The hyperlipidemic control i.e., group II animals received the hyperlipidemic diet and the vehicle. The control group animals received the normal laboratory diet and vehicle.

vii. Experimental Design

The experiment was conducted for 28 days, in which rats n=30 are randomly divided into five groups and fed with modified diet containing 20% ground nut oil, 0.5% cholesterol, 1% cholic acid and followed by treatment with standard, test doses of drugs and periodic blood samplings.

Treatment Schedule for Antihyperlipidemic activity

Groups Treatment (28Days) Purpose

I Vehicle(Distill water)

To serve as normalanimal

II High fat diet To serve as controlIII Fenofibate (5mg/kg) To serve as standard

IVTephrosia purpurea extract 200 mg/kg + fat diet

To assess the anti hyperlipidemic activity of Tephrosia purpurea

V Tephrosia purpurea extract 400 mg/kg+fat diet

To assess the anti hyperlipidemic activity of Tephrosia purpurea

viii. Collection Of Blood Samples

The blood samples were withdrawn on 28th day from the retrorbital venous plexus of rats without any coagulant for the separation of serum. After collecting the blood in eppendroff tubes kept for 1 hour at room temperature and serum was seperated by centrifugation at 2000 rpm for 15 min and stored analyzed for various bio chemical parameters.

ResultsAcute Toxicity

Administration of methanolic extract of leaves and stems of Tephrosia purpurea upto 4000 mg/kg body weight did not produce any mortality. The antihyperlipidemic activity of methanolic extract of Tephrosia purpurea was tested at 400mg/kg.

Effect On Serum Total Cholesterol

Animals treated with high fat diet (G-II) a significant increase in serum cholesterol was observed on 28th day, when compare to normal animals (G-I). This shows that


administration of high fat diet induces hyperlipidemia for the present study.

Group-III that received standard drug (Fenofibrate, 5mg/kg) showed a significant decrease of serum cholesterol on 28th day, when compare to high fat diet treated group (G-II).Group IV, V receiving T.purpurea extract 400 mg/kg and 200 mg/kg showed a significant reduction in serum cholesterol on 28th day.

These observations suggest that Tephrosia purpurea had a cholesteol lowering property.

Effect on Serum HDL Cholesterol

There was significant decreases in serum HDL cholesterol levels in control group (G-II), on 28th day when compared to normal group (G-I).

The group-III treated with fenofibrate showed a significant increase in serum HDL cholesterol levels, when compared to control group (G-II). The effect was significant from 28th day onwards.

The test group (G-IV and V) receiving BMLE in 400 mg/kg and 200 mg/kg also showed a significant increase in serum HDL levels when compared with control group (G-II) on 28th day.

Effect on Serum LDL Cholesterol

It was observed that there was significant increase in serum LDL cholesterol levels in rats treated with high fat diet group (G-II). The group III treated with standard drug fenofibrate 5mg/kg exhibits a reduction in LDL cholesterol levels on 28th day when compared to control group (G-II).

Group VI and V receiving BMLE 400 mg/kg and 200 mg/kg showed significant decrease in serum LDL cholesterol

levels on 28th day on comparison with control group (G-II).

Effect on Serum Triglycerides

Control group receiving high fat diet shows a significant increase in serum triglyceride levels on 28th day, when compare to normal group(G-I).

The group III treated with standard drug fenofibrate 5 mg/kg had significantly lowered triglyceride levels on 28th day, when compared with control group (G-III).

Decrease in serum triglyceride levels was observed in groups IV and V receiving BMLE 400 mg/kg and 200 mg/kg. This decrease was significant on 28th day.

Effect on Total Proteins

Administration of high fat diet in control group (G-II), a significant decrease in total proteins was observed on 14th day onwards when compared to normal group (G-I).

A significant increase in serum total proteins was found in animals treated with fenofibrate from 14th day onwards. Animals treated with BMLE 400 mg/kg and 200 mg/kg showed a significant increase in serum total proteins on 28th day

Effect on Body Weight

There was a significant increase in body weight of control group (G-II) administered with high fat diet, compared to normal group (G-I). Also it was found that group (III) receiving 5mg/kg of standard drug there was a significant reduction in body weight.

Group (G-IV and V) receiving the extract 400 mg/kg and 200 mg/kg, there was significant decrease in body weight compared to control group.

Effect of Methanolic extracts of T.purpurea on total cholesterol, HDL, LDL, triglycerides, Total proteins results on 28th day

Group Treatment Cholestirol HDL LDL Triglycerides Total Proteins

I Normal 237.3±14.64 61.52± 5.47 139.2±14.11 132.7±13.04 8.195±0.97

II High fat diet 380.0±15.06a 28.54±0.90a 306.±15.9a 290.8±14.23a 3.242.±0.67a

III Fenofibrate (5 mg/kg)+fat diet 269.2±13.87d 49.91±2.56b 189.2±11.32d 190.3±17.56d 6.102±0.84c

IV Fat diet +TPE (200 mg/kg) 280.0±14.83d 48.46±3.23b 198.1±16.31d 198.0±13.36c 5.580±0.96c

V Fat diet +TPE (400 mg/kg) 263.2±14.83d 54.58± 7.17c 192.0±13.90d 185.02±16.81d 6.624±0.93c

All the values are expressed as Mean±SEM (n=6)a = p <0.001 When compared to normal (G-I) b= p <0.01 When compared to control (G-II)c=p<0.001 When compared to control (G-II) d=p <0.001 When compared to control (G-II)


Effect of methanolic T.purpurea extract on Body weight

GROUPS TREATMENTBody Weight

0 Day 28th DayI Normal 180.0±12.71 183.3±14.47II High fat diet 181.7±16.41 290.0±18.80 aIII Fenofibrate (5 mg/kg)+fat diet 182.0±13.90 213.3±18.51bIV Fat diet +TPE (200Mmg/kg) 184.8±12.74 216.7±12.81bV TPE (400 mg/kg) +Fat diet 183.0±14.59 209.2±18.64b

All the values are expressed as Mean ± SEM (n=6)a= p <0.01 When compared to normal (G-I) b= p <0.05 When compared to control (G-II)

Effect of methanolic T.purpurea extract on Body weight at 28th day

All the values are expressed as Mean ± SEM (n=6)a= p <0.01 When compared to normal (G-I)b= p <0.05 When compared to control (G-II)

Discussion The experimental model selected for the present study is high fat diet induced hyperlipidemia in rats. These animal model of high fat diet in composition of 0.5% cholesterol mimics human hyperlipidemia, inducing fat deposition and damage to the cells of the organs and results in generation of free radicals showing the signs of oxidative stress.

In the present study administration of high fat diet to rats causes significant increases in serum cholesterol, LDL, triglycerides on 28th day, with a decrease in protective HDL total proteins. The effect on HDL was significant on 28thday when compared to normal group (G-I). Significant reduction of total cholesterol, LDL, triglycerides levels, with an increase in HDL, total protein levels were found in standard group (G-III) treated with fenofibrate 5 mg/kg.

The proposed mechanism of action for the improvement in serum lipid profile is, that fenofibrate is a lipid lowering agent comes under a class of Fibrates. Fibrates act on Peroxidase proliferated activated receptor (PPAR-α).Activation of this receptor leads to lowering of triglycerides,

VLDL and increase HDL level which clears the unused cholesterol from blood12.

It is also found from the present study that administration of Tephrosia purpurea extract 200 mg/kg and 400 mg/kg to high fat diet rats of test group (G-IVand V) showed a significant decrease in serum cholesterol. LDL, triglyceride and improvement in HDL, total proteins levels, when compared to control group (G-II).

A decrease in body weight of animals treated with 200 mg/kg and 400 mg/kg extract along with fenofibrate 5 mg/kg was found. This reduction in body weight may due to beneficial effects of oleic, linolenic, lenoleic acids on serum lipid profile13.

The effect could be due to Tephrosia purpurea enhancing hypolipidemic action of fibrates. Both polyunsaturated fatty acids and fibrates show similar actions enhancing endothelial nitric oxide synthesis and lower cholesterol levels prevent atherosclerosis, and are benefit in caronary heart diseases.


T.purpurea extract significantly protects against high fat diet and cholesterol (0.5%) induced oxidative stress in rats. Which was reflected by improved antioxidant parameters like increases in SOD, Catalase, GSH levels and decreased lipidperoxidation (LPO)? The protective effect of T.purpurea extract and combination groups may be due to anti oxidant action of butein of linoleic acid present in Tephrosia purpurea leaves14. The anti oxidant action depends on its participation in a series of reactions involving radicals15.

In conclusion methanolic extracts of Tephrosia purpurea offers significant protection against high fat diet induced hyperlipidemia. The study clearly reveals the antioxidant property of Tephrosia purpurea individually and in combination by decreasing lipid peroxidation and enhancing protective antioxidant enzyme levels. T.purpurea extract shows synergistic action with fenofibrate. Histopathological findings add an additional note for protective effect. Therefore it is a potential therapeutic agent for treating hyperlipidemia and can be further explored for future research studies.

Reference1. Sharma HL and KK Sharma.Principles of Pharmacology

first edition. Paras publishers 2007; 326.

2. Armugam Sattivel, Hanmanta Rao, Balaji, Raghavendran. Anti peroxidative and Anti hyperlipidemic nature of Ulva Lactuca crude polysaccharide on D-galactose amine induced hepatitis in rats. Food and chemical toxicology 2008;:46:3262-3267.

3. Paris. L, M.Amarnath Sateesh. Anti diabetic effect of Boerhavia diffusa on serum and tissue lipds in experimental diabetes. Journal of medicinal food 2004; 7(4): 472-476.

4. So Yeonkim, Hye-Jinkim, Z.Mikyung Lee, Seonmin,Gyeong-Min D, Eun-Young Lee Naringin time dependently lowers hepatic cholesterol biosynthesis and plasma cholesterol in rats fed with high fat and cholesterol. Journal of medicinal food 2006 9;(4):582-586.

5. Arul mozhi.S, Papiyamitra mazumder, Purnima Ashok, L.Sathiya narayana. Pharmacological activities of

Alstonia Scholaris (linn). Pharmacognosy Reviews 2007; 1:163-170.

6. Ashok D.B.Vaidya and Thomas P.A, Devasagaym . Current status of herbal drugs in India 2007; 41(1)1-11.

7. Hicham, Harnafi, Nouri-el-HoudaBounani, Hana Serghini Laid, Souliman Amrani The hypolipidemic activity of aqueous EricaMultiflora extract in triton WR-1339 iduced hyperlipidemic rats. Journal of ethanopharmacology 2007; 109:156-160.

8. Annien M.larson, Acute liver failure. Dismon 2008; 54: 457-485

9. Saroj Kothari, Anand K Jain, Swaroop C.Mehta, Srinivas D. Effect of fresh Triticum Aestivum grass juice on lipid profile of normal rats. Indian journal of pharmacology 2008;40(5):235-236.

10. Sehrawat.A, T.Khan, L.Prasad S.Sultana. Butea monosperma and chemo-modulation protective role against thiacetazone mediated hepatic alterations in wistar rats 2004; 13 (3) :157-163.

11. Seifollah Bahramika and Razieh Yazdan parast. Effect of hydroalcoholic extract of Nasturtium officinale leaves on lipid profile in high fat diet rats 2008; 115(1):116-121.

12. Ray SK, NN Rege. Atrovastatin in the management of hyperlipidemia. Journal of post graduate medicine 2000; 46(3):242-243.

13. Berrougui.H, A.Ettaib, M.D Herrere Gonzalez, M.A Luazez de Sotomayor, N.Bennani-Kabchi, M. Hmamouchi. Hypolipidemic and hypocholesterolemic effect of argan oil in meriones shawi rats. Journal of ethanopharmacology 2003; 89: 15-18

14. Jorge Limon –Pacheo, Maria E.Gonsebatt. The role of antioxidant and antioxidant related enzymes in protective response to environmentally induced oxidative stress. Journal of mutation research 2009; 674:137-147.

15. Reza Farhoosh. Antioxidant activity and mechanism of action of butein in linoleic acid. Journal of food chemistry 2005; 93:633-639.

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Online : ISSN 2349-669X Print : ISSN 0973-9874


Development of Dieteticbeverages by Using Aleo-Vera Juice and their Quality Evaluation

SANGITASOOD*, SHIVANIWALIA AND MRINALINIDepartment of Food Science, Nutrition and Technology, College of Home Science

Himachal Pradesh KrishiVishvavidyalaya, Palampur-176062, India

ABSTRACT

Many of the health benefits associated with Aloe -verahave been attributed to the polysaccharides contained in the gel of the leaves. It also contains more than two hundred tonic ingredients including essential amino acids, minerals, vitamins, enzymes and steroids. These biological activities include promotion of wound healing, antifungal activity, hypoglycemic or anti diabetic effects anti-inflammatory, anticancer, immune modulatory and gastro protective properties. The taste of the juice is a bit bitter which restricts its usage by people. To make it more palatable it was attempted to develop some of dietetic beverages viz.RTS,Squashes and Syrups by using sorbitol and steveosides as a source of sweetening agent instead of sugar. But the present processing techniques aims at producing best quality aloe products but end aloe products contain very little or virtually no active ingredients. Hence, appropriate processing techniques should be employed during processing in order to increase the use of aloe-vera gel.

Key words: Aloe-vera, Dietetic , Stevioside, Sorbitol, Syrup.

Introduction Aloe -vera is quite an incredible medicinal plant full of nutritional benefits. It is a succulent plant and parts of the lily family (Liliaceae), the same family that garlic and onions belong to. Different parts of the plant are used for different purposes and Aloe –verahas both internal and external applications. The plant has stiff grey - green lance - shaped leaves containing clear gel in a central mucilaginous pulp. Clinical evaluationsrevealed that the pharmacological active ingredients are concentrated in both the gel and rind of the Aloe –veraleaves [1]. Aloe vera is considered to be the most biologically active of the Aloe species. More than 75 potentially active constituents have been identified in the plant including vitamins, minerals, saccharides, amino acids, anthrax quinones, enzymes, lignin, saponins, and salicylic acids.The leaf exudate contains anthraquinones, particularly barbaloin), which appear to be responsible for its bitter taste and cathartic effect [2, 3]. Barbaloin and other products of the phenyl propanoid pathway are commonly referred to as polyphenolic compounds derived from the precursor phenolic acids, and they may act as antioxidants to inhibit free radical–mediated cytotoxicity and lipid peroxidation [4].

Potential use of aloe products often involves some type of processing e.g. heating, dehydration and grinding. Processing of Aloe vera gel derived from the leaf pulp of the plant has become the big industry worldwide due to the application in the food industry.It has been utilized as a resource of functional especially for preparation of health drinks. Aloe vera gel was not very popular due to their laxative effects and majority of them contained absolutely no active mucilaginous polysaccharides [5]. The production process of aloe products involves crushing, grinding and processing of the entire leaf of Aloe verato produce aloe-juice or gel. The system involves the colour change, which is totally unacceptable in some products. It therefore becomes imperative that a simple but effective gel extraction technique needs to be involves and to improve product quality, to preserve and maintain all the bioactive chemicals present inAloe veraplant. The extraction of juice is a tedious process. It is the need of the hour to develop some palatable products.

Aloe-verahas been attributed to the polysaccharides contained in the gel of the leaves. It also contains more than two hundred tonic ingredients including essential amino acids, minerals, vitamins, enzymes and steroids. These biological activities include promotion of wound healing, antifungal activity, hypoglycemic or antidiabetic effects


anti-inflammatory, anticancer, immune modulatory and gastroprotective properties.Aloe-vera exerts hypoglycemic effects and can be used as hypoglycemic agent for diabetic patients [6]. Its bitter taste restricts its usage.Keeping this in view, pure aloe-verabeverge, dietetic based aloe-vera beverage, RTS, syrup and squash were also prepared. The aloe-vera juice prepared for diabetic subjects as it provide better glycemic control along with the improvement in lipid profile as well as anthropometric measurements [7].

Materials of MethodsProcurement and Processing of selected raw ingredients

Aloe-vera was procured from local area of the Palampur at optimum maturity period .Then leaves were cleaned and washed and then lower part of leaf base or the white part attached to the stem of plant, and the tapering point of the top, and sharp spines located along the leaf margins were removed by a sharp knife. Then with the help

of knife mucilage layer was removed lies below the green top rind then similarly bottom rind.

Preparation of juice

This mucilaginous gel was again washed and gel was dipped in cold water to cut down the natural enzymatic reaction .The prepared pieces of aloe-vera gel were divided into four different lots and then subjected to following treatments. i) Untreated ii) Water blanched for 3 minutes iii) KMS (1% w/v) treated iv) Citric acid (1% w/v) treated

Then the treated pieces and extracted gel were crushed in a blender to extract the juice which stored still further use. The detailed process is shown in Figure 1


Beverages of Aloe-vera

Aloe-vera juice from aloe-vera gel was extracted by crushing in laboratory mixer cum grinder. The crushed material was strained through muslin cloth and again filtered through two folded muslin cloth. The prepared Juice of aloe-vera gel was used for chemical analysis.Development of Low Calorie Aloe-vera based RTS, Squash and Syrup Since the citric acid treated juice was adjudged best by the panel of judges, so it was used for the development of various products viz. RTS, squash and Syrup by using sorbitol and sugar. Zero calorie beverageswas also attempted by using stevioside in place of sugar as a sweetening agent. The detailed treatments, sub-treatments and interactions are given below:

Treatments Sub-treatments

T1-Control juice P1 S0-Sugar based RTS

T2-Water blanched P1 S1-Sorbitol based RTS

T3-KMS treated P2 S0-Sugar based Squash

T4-Citric acid treated P2 S1-Sorbitol based Squash

A1-Sugar based Beverage P3S0-Sugar based Syrup

A2-Stevioside based P3S0-Sorbitol based Syrup Beverage

P1-RTS A1-Sugar based Beverage

P2-Squash A2 - Stevioside based Beverage

P3 - Syrup

S0 - Sugar

S1 -Sorbitol

Nutritional evaluation

Aloe - vera juice as well all the value added products were analyzed for various chemical parameters.pH, TSS, Per cent acidity, Ascorbic acid, Minerals and Sugars were estimated by following the methods given [8]. Triplicates sample were analyzed and mean values are reported.

Organoleptic evaluation

The organoleptic evaluation was done as per method [9]. The sensory attributes like colour, flavor, taste, consistency and over all acceptability of the products were evaluated. A minimum of 10 judges were selected at random. The judges were required to record their preferences and acceptability of products on the evaluation sheets.

Results and Discussion Aloe-verajuice was prepared by giving different treatments and evaluated objectively as well as subjectively and the pertinent results are presented in respective Tables and discussed.

Nutritional evaluation of Aloe-vera Juice:

The juice was evaluated for pH,TSS,Acidity,Ascorbic acid and Sugars and the results are presented in Table 1.

Table 1Nutritional Composition of Aloe-vera Juice

Parameters T1 T2 T3 T4

pH 4.3 4.9 4.4 3.4TSS(°B) 1.2 0.90 0.89 0.90Acidity (%) 0.24 0.16 0.16 0.32Ascorbic acid (mg/100g) 1.20 0.56 0.72 0.88Reducing sugars (%) 0.82 0.94 0.48 0.36Total sugars(%) 1.12 1.98 0.78 0.72CD (P< 0.05) V x T = 0.04(NS)

Data in Table 1represents the chemical composition of aloe-vera juice. The pH value was higher in T2 (4.90), followed by T3 (4.40), T1 (4.30) and 3.40 in T4 juice. Regarding the values of TSS, which were 1.20, 0.90, 0.89 and 0.90 in T1,T2, T3 and T4 respectively. Per cent acidity

Flow sheet for preparation of Sugar and Dietetic Aloe-vera based Beverage, RTS, Squash and Syrup

Water + sugar/ dietetics

Preparation of syrup

Addition of pulp

Beverage RTS Syrup Squash

Adjusting TSS

Addition of KMS to a small portion andmixing to entire lot

Bottling and Corking

Pasteurization Cooling and Packaging


recorded higher in T4 (0.32%), followed by 0.24 in T1 and 0.16 per cent in T2 and T3. T2 showed the lowest value of ascorbic acid (0.56 mg/100g),which was followed by T3 ( 0.72 mg/100g), T4 (0.88mg/100g) and T1 (1.20mg/100g) as ascorbic acid sensitive to heat treatment.

The values of reducing and total sugar were found higher in T2 (0.94 and 1.98 %), followed by T1 (0.82 1nd 1.12%), T3 (0.48 and 0.78%) and (0.36 and 0.72 %) in T4 beverage. The lower value of per cent acidity was observed in water blanched beverage, which might be attributed to hydrolysis of polysaccharides and non-reducing sugars, where acid is utilized for the converting them to hexose sugar at blanching temperature. Researchers reported the (0.87 mg/100g) of ascorbic acid in aloe-vera juice [10]. A small variation could be due to the agro-climatic and varietal variations. Similar observations of aloe vera juice were reported [11].

Organoleptic evaluation of Aloe-vera juice

To get the best treatment the aloe-vera juice treated with different treatments were offered to a panel of judges to get the best one. The results are depicted in Fig. 1.

Fig. 1: Organoleptic Evaluation of Aloe-vera

The sensory score of aloe-vera juice as in Figure 1 revealed that the colour of T4 and T4 was more or less similar and high score than remaining samples but regarding the flavor T4 had a high score of 7 than the other counterparts. All the juices were par to each other regarding their consistency except T4 , which scored high value of 7. Data showed that highest score was evaluated by the panelists of T4 sample regarding their overall acceptability because the addition of citric acid enhance the colour of the beverage and also act as aciduant so gained high score in flavour also. Aloe-vera juice was organoleptically accepted by the diabetic subjects. Formulated aloe-vera gel (20%) based bread which attained golden brown crust, soft texture and creamish white colour [12].

Nutritional Composition of Sugar and Dietetic Aloe-vera based RTS, Squash and Syrup

It was attempted to prepare dietetic based RTS,

squash and syrup from aloe-vera juice. These products were also compared with sugar based RTS, squash and syrup. Perusal of data in Table 2 shows that higher value of pH was observed in P3S0 (4.20), P3S1 and P2S0 (4.10), P2S1 (4.00) and values of P1S0 and P1S1 were on a par with each other. The TSS values were observed high in P3S0 (66.90), P3S1(66.32), P2S0 (41.15), P2S1 (42.03), P1S0 (15.67) and P1S1 (15.16).

Table - 2Nutritional Composition of Sugar and Dietetics Aloe-

vera based RTS, Squash and Syrup

Para-meters

P1 P2 P3

S0 S1 S0 S1 S0 S1

pH 3.16 3.14 4.10 4.00 4.20 4.10TSS 15.67 15.16 41.15 42.03 66.90 66.32Acidity (%) 0.32 0.38 0.20 0.24 0.16 0.20

Ascorbic acid (mg/ 100g)

0.12 0.12 0.15 0.15 0.36 0.36

Reducing sugars (%)

3.68 0.19 18.63 2.46 24.71 5.21

Total sugars (%)

11.43 0.38 40.28 5.32 59.43 10.68

CD (P< 0.05) V x T = 0.054

The values of per cent acidity were found higher in P1S1 (0.38%), followed by P1S0 (0.32%),P2S1 (0.24%),P2S0 (0.20%), P3S1 (0.20%) and P3S0 (0.16%). The values of ascorbic acid were found as 0.12 mg/100g in P1S0 and P1S1,(0.15) in P2S0 and P2S1,(0.36mg/100g) in P3S1 and P3S0. The values of total and reducing sugars were found (11.43 and 3.68%) in P1S0, (0.38 and 0.19%) in P1S1, (40.28 and 18.63%) in P2S0, (5.32 and 2.46%) in P2S1, (59.43 and 24.71%) in P3S0 and (10.68 and 5.21%) in P3S1. Perusal of data indicates that ascorbic acid content remained same among all the treatments might be due to use of equal proportions of juice. Higher values of reducing and total sugar were observed in sugar based products, while sorbitol based beverage had a low values of total and reducing sugars as it provide 50 per cent less calories than sugar. Same observations were also reported [13], that the value of total and reducing sugars (32.34 & 18.70%) and (5.55 & 2.37%),respectively in sugar and sorbitol based plum squash.

Organoleptic evaluation of Sugar and Dietetic based Aloe-vera RTS, Squash and Syrup:

To assess consumers’ acceptability the beverages were evaluated organoleptically and the results are presented in Fig 2.


Fig. 2: Organoleptic evaluation of Sugar and Dietetic based Aloe-vera RTS, Squash and Syrup

Data on the Figure 2 revealed that the mean score differ significantly from each other. The high score of 6 for colour was given for P3S0, P3S1, P2S0 and P2S1. Regarding the flavor which was reported 6 in P3S0, P3S1 and P2S1 and 5 in P2S0, P1S1 and P1S0. The mean score of consistency was almost same in all the samples except P2S1 and P3S1 were scored 7. The data on overall acceptability shows that P2S1 and P3S1 samples were scored high as 7 by the panelists. Barwalet al. (13) also reported that sorbitol based plum squash contribute less calories to product and organoleptic score of the product was found to be acceptable.

Nutritional Composition of Stevioside and Sugar basedAloe- vera beverage

Simultaneously it was attempted to prepare zero calorie beverage by using stevioside and compare with sugar based beverage.

Table - 3Nutritional Composition of Dietetics and

Sugar based Aloe-vera beverage

Parameters A1 A2

pH 4.6 4.6TSS(°Brix) 0.00 10.2Acidity (%) 0.16 0.12Ascorbic acid (mg/100g) 1.12 1.08Reducing sugars (%) 0.82 2.65Total sugars (%) 1.62 9.85CD (P< 0.05) V x T 0.14

Stevioside and sugar aloe-vera based beverage was prepared and chemical composition is summerised in Table 3 The pH value was found to be 4.6, per cent acidity (0.16 & 0.12%), ascorbic acid (1.12 & 1.08mg/100g), reducing and total sugars were found as (0.82 & 2.65%) and (1.62 & 9.85%), respectively in A1 and A2 beverage. Considering the beneficial properties of stevia as it is 40 times sweeter than sucrose with no calories and also act as a hypoglycemic and cardio tonic and hence boon to the cardiac patients [14]. Few documented that stevia stimulate insulin secretion by

direct action on beta cells and have potential role in the treatment of diabetes mellitus [15].

Organoleptic Evaluation ofStevioside and Sugar Aloe-vera based beverage

The mean score of organoleptic evaluation is depicted in Figure 3. The samples when compared to each other regarding their colour, they both were adjusted well because of their score i.e. 7. The flavor of A2 was adjusted best scoring it maximum value of 8 over the score of 6 by A1. Both the samples retained their similar consistency.

The overall acceptability of T2 received 7 and T1 received 6 as its low rating for flavour.Researchers formulated stevia based tea and accepted as better taste, aroma and smooth texture [16].

Organoleptic Evaluation of Sugar and Dietetic based Aloe-vera RTS, Squash and Syrup

Fig. 3: Organoleptic Evaluation ofStevioside and Sugar Aloe-vera based beverage

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3. Boudreau M.D, Beland F.A. An evaluation of the biological and toxicological properties of Aloe barbadensis (Miller), Aloe vera. J Environ Sci Health C. 2006;24:103–54. [PubMed]

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Design, Development and Characterization of The Dimenhydrinate Bilayered Caplets

LAKSHMI NARAYANA REDDY G*1, DR. RAJNARAYANA K1 AND DR. JAYAVEERA KN2 1RA Chem Pharma Limited, Hyderabad, Telangana, 500 076

2Dept. of Chemistry, VEMU Institute of Technology, P.Kothakota (Near Pakala), Chittooor Dist., Andhra Pradesh.

ABSTRACT

The aim of the present investigation is to develop bilayer caplet (tablet) dosage form of Dimenhydrinate containing combination of immediate and sustained release layer for the treatment of nausea, vomiting and motion sickness. Dimenhydrinate bilayer caplets were prepared by wet granulation technique. Prepared granules as well as caplets were evaluated for various physical and chemical tests. Bilayer caplets were formulated using pre-gelatinized starch as a disintegrant in immediate release granules, shown 99.7% of release in 15 minutes and using hydrophilic polymer (Methocel K 4M) as release retardant which sustain the Dimenhydrinate release for the period of 12 hours. Optimized formulation was met the pre-determined specification. From this research work it is evident that the developed formulation was able to release drug in biphasic pattern.

Keywords: Dimenhydrinate, Hydrophilic polymers, Bilayer caplet

Introduction Bilayered caplet is suitable for combination therapy, i.e., for sequential release of two different drugs or the same drug or two incompatible substances and also for sustained release dosage form to provide two different release rates or biphasic release of a drug from a single dosage form in which one layer is formulated to obtain rapid action of the drug, with the aim of reaching a high plasma concentration in a short period of time while the second layer is designed as sustained released layer, which provides effective plasma concentration by a maintenance dose of drug for an extended period of time. The design of bilayered caplet dosage form holds many advantages over conventional dosage forms such as a reduction in frequency of drug administration, improved patient compliance, reduction in drug level fluctuation in blood and quantitative reduction in total drug usage when compared with conventional therapy [1].

Dimenhydrinate is an anti-emetic drug which prevents nausea, vomiting, or dizziness. It undergoes extensive first pass metabolism resulting in an oral bioavailability of 46 % and it shows variable absorption from GIT. Owing to these properties need for twice to three times administration which can also reduce patient compliance. Hence, to reduce the dosing frequency and improve the patient compliance, Dimenhydrinate was formulated as a bilayered caplet formulation [2, 3].

The objective of the present investigation is to prepare the dimenhydrinate bilayer sustained release matrix caplets (tablets) were prepared such that the dose distributed among the both immediate and sustain release layers as 1:3 ratio of the total dose and evaluate the various physico-chemical properties. Loading dose and maintenance dose are calculated based on the available literature using various kinetic equations.

Methods Dimenhydrinate was procured from Chiral drugs, Hydrophilic polymers like, Methocel K4M Prem CR, Dibasic calcium phosphate (Innophos) and Micro crystalline cellulose (FMC bio Polymers), Pre-gelatinized starch (Roquette) and Hydroxy propyl cellulose (Arihant), Plasdone (PVPK29/32) (ISP), Magnesium stearate (Ferro) and Talc (Luzenac), Aerosil (Evonik), Hypromellose (Pharmacoat 606) (Shin Etsu), Polyethylene glycol (PEG) 6000 (Clariant). All the other ingredients used throughout the study are of analytical grade.

Preparation of Dimenhydrinate Bilayered Caplets 100 mg [4, 5]

Dimenhydrinate Bilayered Caplets 100 mg were prepared by wet granulation method by optimizing the composition of both immediate release (Disintegrant concentration) and sustained release (Polymer & binder concentration) layers seperately. All the materials were sifted through #40mesh and allowed for dry mixing in RMG, then granulated with appropriate quantity of binder.


The wet mass was discharged and allowed for air drying followed by wet milling through 4mm screen at knife forward direction. The wet granules were dried at 50°C ± 5°C in Fluid bed drier (FBD) until Moisture content reaches less than 3.5%w/w. The dried granules were sifted through sieve no.25 (ASTM). These granules were lubricated with Magnesium Stearate (which was previously sifted through #60), in double cone blender for 2 minutes. The lubricated blend was evaluated for various physical properties. Finally both the layers were compressed into bilayered caplets and then subjected for coating. Both core and coated caplets were evaluated for various physic-chemical properties.

Table 1Composition of the Film Coated Dimenhydrinate

Blayered caplets 100mg

Composition of the Sustained release layer

F8 (Optimized formulation)

Dimenhydrinate 75.00Di basic calcium phosphate 266.05HPMC K4M 18.75PVPK-30 5.00Purified water QSTalc 7.60Aerosil 5.70Magnesium Stearate 2.00Composition of the Immediate release layerDimenhydrinate 25.00Di calcium phosphate Dihydrate 82.00

MCC PH 102 79.00PG Starch 22.00Iron oxide red 0.50PVPK-30 11.00Purified Water QSMagnesium Stearate 0.50Composition of the Film coatingHydroxy propyl methyl cellulose (Pharmacoat 606) 11.40

Polyethylene Glycol 6000 1.70Purified Water QS

Characterization of the Film coated Dimenhydrinate Caplets 100mg [6-12]:

i) Evaluation of Granular Properties:

Bulk density (BD), tapped density (TD), Compressibility Index (CI), Hausner ratio (HR) and the angle of repose of granules were determined. BD and TD were determined by USP method I using a Tapped density tester. Compressibility Index and hausner ratio were calculated using following formulae

Compressibility index = {(TD – BD) / TD} x 100 Hausner ratio = TD / BD; Angle of repose (θ) = tan -1 (h/r)

Where h and r are the height and radius of the powder cone, θ is the angle of repose.

ii) Evaluation of the Caplets/Tablets

IR and SR caplets were evaluated for physical properties like weight variation, hardness, thickness, diameter, friability, disintegration time and chemical properties such as assay, uniformity of dosage units and in vitro drug release studies (as described below).

a) Assay & Uniformity of dosage units (by Content uniformity)

Preparation of diluent: Prepare a mixture of Acetonitrile and water in the ratio of 18:82 V/V

Preparation of Mobile Phase A: Dissolve 10.0 g of Triethylamine in 950 mL of water and adjust pH to 2.5 with phosphoric acid and dilute to 1000 mL with water.

Preparation of Mobile phase B: Acetonitrile

Chromatographic conditions:

Column : Inertsil ODS 3V, 250 mm X 4.6 mm, 5µm or its equivalent

Wave length : 225 nm Injection volume : 10µLTemperature : 30°CRun time : 40 minutes

Standard and sample solutions were prepared according to the Standard testing procedure.

Assay of Dimenhydrinate:

RT WS 5 5 50 P = ––– x ––– x ––– x ––– x ––– x ––– x A= ––– mg/caplet RS 25 50 WT 5 100

Assay of Dimenhydrinate % of label amount = ––––––––––––––––––––––––– x 100 Label claim Where, RT = Peak area of diphenhydramine obtained from

the Sample Solution. RS = Average Peak area of diphenhydramine

obtained from the standard Solution WS = Weight of Dimenhydrinate working standard

taken in mg WT = Weight of sample taken in mg P = Assay of Dimenhydrinate working standard

used (on as is basis) A = Average mass of the caplets in mg.


Assay of 8-chlorotheophylline:

RT WS 5 50 50 P 214.61= –––x–––x–––x–––x–––x–––x A x––––– = ––mg/caplet RS 25 50 WT 5 100 469.96

Assay of 8-Chlorotheophylline % of label amount = ––––––––––––––––––––––––– x 100

Assay of DimenhydrinateWhere,

RT = Peak area of 8-chlorotheophylline obtained from the sample Solution.

RS = Average Peak area of 8-chlorotheophylline obtained from the standard solution

WS = Weight of Dimenhydrinate working standard taken in mg

WT = Weight of sample taken in mg P = Assay of Dimenhydrinate working

standard used (on as is basis) A = Average mass of the caplets in mg 214.61 = Molecular weight of 8-chlorotheophylline 469.96 = Molecular weight of diphenhydramine

b) In vitro drug release studies:

Dissolution Parameters Medium : Water; 900mL Apparatus : USP Type II (Paddle) RPM : 50 Temperature : 37 ± 0.5°C Time : 15 minutes (for IR); 1, 3, 6, 9 and 12 hours

Determine the amount of Dimenhydrinate dissolved by employing UV absorption at the wavelength of maximum absorbance at about 276 nm on filtered portions of the solutions under test and standard preparations by using dissolution medium as blank.

The % Dimenhydrinate dissolved calculated by following formulae:

AT WS 5 5 900 25 P 100 Dn ––– X––––X–––X–––X––––X–––X–––X–––––X––––X 10 AS 25 50 50 1 5 100 Labelclaim 900

Where AT = Absorbance count of Dimenhydrinate in

sample solution. AS = Average Absorbance counts of

Dimenhydrinate in standard solution WS = Weight of standard taken in mg P = Potency of Dimenhydrinate working

standard used (on as is basis)

c) Kinetic modeling of drug release [11-12]

The release pattern of the optimized formulation was evaluated to check the order of kinetics (Zero order or First order release kinetics) and mechanism of drug release (Higuchi’s and Korsmeyer– Peppas)

Drug release data were fitted according to the below mentioned Eq.

Mt/Ma = ktnWhere, Mt/Ma is the fractional drug released at time t; k is a constant and ‘n’ is a kinetic constant (used to characterize the transport mechanism).

If, n=0.45 for Case I or Fickian diffusion, n=0.89 for Case II transport, 0.45<n<0.89 for anomalous behavior or non-Fickian transport, and n>1.0 for Super Case II transport.

Results and Discussioni) Evaluation of granular properties

All the physical properties of both IR &SR layers are well within the acceptance limits.

ii) Characterization of IR Caplets:

Caplet thickness, hardness and diameter are well within the acceptance criteria. Physicochemical properties of the IR caplets computed in the below table (Table 2)

Table - 2Physicochemical properties of IR (layer) Caplets

Trial No.

% Fri-

ability

DT (min)

Assay (%)

Content uniformity(L1 value)

% drug dissolved

T1 0.049 9’30” 98.7 4.5 93.5T2 0.047 4’54” 99.0 4.7 97.5T3 0.046 2’57” 99.6 4.6 99.6T4 0.045 2’39” 99.7 4.4 99.5T5 0.048 3’01” 99.5 4.7 99.2T6 0.045 2’54” 100.1 4.5 99.7

Based on the obtained results, 8%w/w (T3) was selected as an optimum disintegrant concentration. Extra and intra granular addition of disintegrant (T5) doesn’t have tremendous impact on the disintegration time. Hence, 8%w/w of disintegrant is added intra-granularly in further studies.

iii) Characterization of Dimenhydrinate Bilayered Caplets100mg:

Caplet thickness, hardness and diameter are well within the acceptance criteria. Physicochemical properties of the Blayered caplets computed in the below table (Table 3).


Table - 3Physicochemical properties of bilayered caplets

F. Code

% Friability

DT (min)(IR layer)

Assay (%)


F1 0.11 3’14” 96.7 4.5

F2 0.09 3’04” 97.8 4.6

F3 0.08 2’57” 98.6 4.7

F4 0.08 2’49” 100.1 4.6

F5 0.06 2’44” 99.7 4.5

F6 0.07 2’54” 101.2 4.5

a) Study on impact of Polymer concentration on drug release

To study the impact of polymer concentration (with respect to drug), the formulations F1, F2 and F3 (1:1, 2:3 and 1:3 respectively) were prepared and evaluated for drug release studies along with the other physic-chemical properties. The dissolution results are presented below:

Table - 4Comparative dissolution data of formulations

executed with various concentrations of polymers

Time (Hr) Specification F1 F2 F3

0 0 0 0

1 20% to 45% 24.4 ± 10.7

30.1 ± 6.9

35.2 ± 5.7

3 40% to 60% 36.2 ± 7.9 43.2 ± 5.3

45.6 ± 4.1

6 60% to 85% 46.4 ± 5.4 53.5 ± 3.5

64.5 ± 3.2

9 Not less than 75% 56.3 ± 2.9 64.4 ±

2.875.1 ±

2.9

12 Not less than 80% 68.3 ± 1.9 74.8 ±

2.0 82 ± 1.4

From the obtained results, polymer to drug at a ratio of 1:3 (F3) is selected for further studies.

b) Study on impact of binder concentration on drug release

To study the effect of binder concentration, the formulations F3, F4, F5 and F6 (5, 4, 2.5 and 1.31%w/w respectively) were prepared and evaluated for drug release studies along with the other physic-chemical properties. The results are computed below:

Table - 5Comparative dissolution data of formulations

executed with various concentrations of binders

Time (Hr)

Specifica-tion F3 F4 F5 F6

0 0 0 0 0

1 20% to 45%

35.2 ± 5.7

36.9 ± 5.2

38.7 ± 5.1

39.2 ± 5.1

3 40% to 60%

45.6 ± 4.1

47.89 ± 3.6

52.4 ± 3.9

54.3 ± 4.0

6 60% to 85%

64.5 ± 3.2

65.8 ± 3.5

69.8 ± 2.4

72.1 ± 3.2

9 Not less than 75%

75.1 ± 2.9

77.7 ± 2.6

83.4 ± 1.9

85.6 ± 2.2

12 Not less than 80% 82 ± 1.4 83.5 ±

1.996.5 ±

0.996.9 ±

0.9

From the obtained results, 2.5%w/w (F5) was selected as a suitable binder concentration for further studies.

c) Study on impact of the concentration of the film coating polymer on drug release

The Optimized Caplet formulation (F5) was coated with various concentrations of the film coating polymer (Hypromellose) such as 2, 3 and 5%w/w weight gain to the uncoated caplet. The drug release from the film coated caplets mentioned below:

Table - 6Comparative dissolution profiles of Dimenhydrinate

film coated caplets coated with various concentrations of the film coating polymer

Time (Hr) Specification F7 F8 F9

0 0 0 0

1 20% to 45% 37.3 ± 4.1

36.5 ± 4.2

34.9 ± 4.7

3 40% to 60% 49.1 ± 3.6

48.3 ± 3.8

47.2 ± 3.6

6 60% to 85% 65.6 ± 2.1

64.7 ± 2.5

63.9 ± 2.2

9 Not less than 75%

81.9 ± 1.2

81.2 ± 1.6

81.0 ± 1.9

12 Not less than 80%

96.5 ± 0.9

96.8 ± 1.0

95.2 ± 1.1

From the obtained results, there is no impact of film coating polymer on drug release. Hence, 3%w/w is selected as an optimum by considering release as well as physical properties like elegance of the caplets.

F8 was evaluated for kinetic modelling, drug release followed the first order kinetics and as the n value is less


than 0.45; the mechanism of drug release is found to be Fickian diffusion/Case I transport.

Table - 7Physico-chemical properties of the Dimenhydrinate

Bilayered film coated caplets

F. Code DT (min)(IR layer)

Assay(%)


F7 3’39” 100.1 4.8F8 3’56” 99.8 4.7F9 5’20” 97.9 4.9

The optimized formulation was evaluated in different dissolution media (0.1N HCl, pH 4.5 acetate buffer, pH 6.8 phosphate buffer and water (QC release media)) and results are computed below.

Table - 8Comparative dissolution data of optimized

formulation (F8) in different dissolution media

Time (Hr)

Water media 0.1N HCl

4.5 Acetate buffer

6.8 phosphate

buffer0 0 0 0 0

1 36.5 ± 4.2 39.8 ± 2.9 36.2 ± 3.1 36.2 ± 3.5

3 48.3 ± 3.8 56.8 ± 2.2 49.8 ± 2.8 49.8 ± 3.1

6 64.7 ± 2.5 75.7 ± 1.8 64.8 ± 2.1 64.8 ± 2.2

9 81.2 ± 1.6 89.6 ± 1.5 75.1 ± 1.9 75.1 ± 1.8

12 96.8 ± 1.0 97.7 ± 1.1 85.7 ± 0.8 85.7 ± 0.9

Fig. 1: Comparative dissolution profiles of Optimized formulation (F8) in different dissolution media

From this research work it is evident that the developed formulation releases drug in the bi-phasic release pattern.

Acknowledgements The authors are thankful for the management of RA Chem Pharma Ltd, Hyderabad for providing necessary facilities to carry out this project.

References1. Nilawar, P.S., Wankhade, V.P., Badnag, D. B. An

emerging trend on bilayer caplets. Int J Pharm & Pha Scie Res 2013; 3(1): 15-21.

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3. Halpert AG, Olmstead MC, Beninger RJ, A Review: “Mechanisms and abuse liability of the anti-histamine dimenhydrinate” Pubmed, 2002; 26(1):61-67.

4. Lütfi Genç Hadi Bilaç, Erden Güler, Studies on controlled release dimenhydrinate from matrix caplet formulations,Pharmaceutica Acta Helvetiae, 1999;74 (1): 43–49

5. Colombo P, Swelling-controlled release in hydrogel matrices for oral route, Adv Drug Del Rev, 1993; 11: 37-57.

6. Aulton ME, Wells TI. Pharmaceutics: The science of dosage form design. London, England: Churchill Livingstone; 1998: 647-649.

7. Cooper J, Gunn C. Powder flow and compaction. In: Carter SJ, editor. Tutorial Pharmacy. New Delhi: CBS Publishers and Distributors, 1986: 211-233.

8. Leon Lachman et al, The Theory and Practice of Industrial pharmacy, Varghese Publication House, Bombay, 3rd edition, 1987.

9. Podczeck F., Drake K.R., Neton J.M., and Harian I., The strength of bi-layered caplet. European Journal of Pharmaceutical Sciences, 2008; 1-14.

10. Libermann HA, Lachman L, Schwartz JB; Pharmaceutical Dosage Forms: Caplets 2nd edition 1990: 274-278.

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12. Peppas NA. Analysis of Fickian and non-Fickian drug release from polymers. Pharm Acta Helv 1985;60:110:111.

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Alleviatory Effects of Brassica oleracea var. botrytis against Sodium Fluoride Induced Nephrotoxicity and Oxidative Stress on

Male Wistar Rats

RAGHAVENDRA MA*, RAVINDRA REDDY KB*, AND JAYAVEERA KNC,aDepartment of Pharmacology, CMR College of Pharmacy, Hyderabad, Telangana state, INDIA

bPrincipal, CES College of Pharmacy, Kurnool, Andhra Pradesh State, INDIAcDept. of Chemistry, VEMU Institute of Technology, P.Kothakota (Near Pakala), Chittooor Dt., Andhra Pradesh, INDIA

ABSTRACT

The present study was undertaken to investigate the alleviatory effects of hydroalcoholic extract of Brassica oleracea var. botrytis (BOB) leaves against sodium fluoride (NaF) induced nephrotoxicity. Thirty-six male wistar albino rats were divided into six groups of six animals each. Group I served as the normal. Group II served as toxic control. Group III, IV and V served as treatment groups received extract at three doses 100, 200, and 400 mg/kg (p.o) respectively. Group VI served as plant control received hydroalcoholic extract of BOB leaves 400 mg/kg (p.o). All groups except group I and group VI received NaF (100 ppm) through drinking water for 30 days. At end of the study of 30 days, serum profile (Urea, Uric acid, Creatinine, Blood urea nitrogen, Total protein and Albumin) and lipidperoxidation, reduced glutathione and catalase enzyme levels were measured in the homogenates of kidney. The results of the present study suggested that hydroalcoholic extract of BOB has alleviated sodium fluoride induced nephrotoxicity, probably via its antioxidant activity due to the presence of polyphenols.

Key words: Sodium fluoride, kidney biomarkers, Lipidperoxidation, Catalase, Reduced glutahione.

Introduction Over the last few decades there has been increasing global concern about the public health related to environmental pollution and their diseases are not easily to detect or diagnose early. One of the global spanning environmental pollutants is fluoride (F-). The high level of F- (> 1.5 ppm) is unfit for drinking purpose. The level of these toxic substance is mainly depends on geological, physical and chemical properties of soils, rocks and industrialization.

Excessive intake of fluoride might accumulate and predominately alters the structure and functions of skeletal systems and non skeletal systems [1-6]. Kidney is the major excretory organ for removal of most of the drug metabolites and toxic substances include fluoride (50-80%) from the body and is exposed to higher concentrations of fluoride than all other soft tissues (with the exception of the pineal gland), there is concern that excess fluoride exposure may contribute to kidney disease. Earlier studies revealed that kidney is the more sensitive organ in their histopathological responses to excessive amounts of fluoride showed that a significant dose-effect relationship between water fluoride level and damage to the kidney structure and functions of

kidney [7-11].

The whole plant of Brassica oleracea var. botrytis (BOB) is popularly called as cauliflower and the most commonly used edible plant for the preparation of curries and snacks. It is a rich source of polyphenols and ascorbic acid, which are known to control the lipidperoxidation and reduce the oxidative stress and also proved as antioxidant, antiobesity, anticancer and hepatoprotective substance [12-15]. Many research reports clearly disclosed that antioxidants and antioxidant rich food products are useful for management of fluoride toxicity [16-19]. Therefore, the purpose of present study is to find out the alleviatory effects of hydroalcoholic extract of BOB leaves against sodium fluoride (NaF) induced toxicity in kidney.

Material and MethodsCollection and authentication of plant material

The leaves of the BOB were collected from the local market of Kadapa, Kadapa district, Andhra Pradesh, India and were authenticated by Dr. Sunita Garg, Chief scientist, Raw Material Herbarium and Museum, Delhi (RHMD), CSIR-NISCAIR; Voucher specimen was stored in the department of Pharmacognosy, CMR college of Pharmacy, Hyderabad, Telangana state, India. The leaves were air-


dried, ground to powder and stored in an airtight container.

Preparation of Extract 100 g of the BOB powder was mixed with water-alcohol (30:70) in a round bottom flask and left for seven days at room temperature with occasional stirring. After seven days, the mixture was filtered with Whatman filter paper no.1. The filtrate was evaporated in vaccuo using rotary film evaporator. About 31.4 % of semi-solid yield was obtained. The extract was stored at 4 oC for further studies.

Experimental Animals Male wistar albino rats (36) weighing in between 220-250 g were procured from Sai Thirumala Enterprises, Hyderabad, India. The animals were acclimatized for 10 days before starting the experiment. Rat feed was provided with water ad libitum and maintained a photoperiod of 12 h light/ dark cycle. The study was completed as per the guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals, Government of India, after approval from Institutional Animal Ethics Committee (IAEC No:CPCSSEA/1657/IAEC/CMRCP/PhD-14/35).

Experimental Design

The dose of NaF was selected based on the previous studies [20]. After ten days of adaptation period, the experimental animals were divided into six groups of six animals each as follows:

Group I- Normal: The animals received drinking water.

Group II- Toxic control- Toxicity was induced by giving NaF (100 ppm) through drinking water for 30 days.

Group III- They were treated with a hydroalcoholic extract of BOB at an oral dose of 100 mg/kg b. wt/day for 30 days (p.o) and NaF was administered through drinking water.

Group IV- They were treated with a hydroalcoholic extract of BOB at an oral dose of 200 mg/kg b. wt/day for 30 days (p.o) and NaF was administered through

drinking water.

Group V- They were treated with a hydroalcoholic extract of BOB at an oral dose of 400 mg/kg b. wt/day for 30 days (p.o) and NaF was administered through drinking water.

Group VI- Plant control- They were treated with a hydroalcoholic extract of BOB at an oral dose of 400 mg/kg b. wt/day for 30 days (p.o) alone.

After the treatment schedule, animals were fasted overnight and blood was collected by puncturing the retro orbital plexus. Blood samples were allowed to clot for approximately 1 h at room temperature and centrifuged at 2500 rpm for 15 min to obtain the serum used for estimation of various biochemical parameters like Creatinine, Urea, Uric acid, Blood Urea Nitrogen (BUN) were estimated by using coral diagnostic kits, India. Kidney homogenate was used for the estimation of Lipidperoxidation, Reduced glutathione (GSH) and Catalase level [21-23].

Statistical Analysis

The values were expressed as Mean ± SEM, n=6 in each group. The statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Dunnett’s test using Graph pad prism version 5.0 Software. These values were significant at P < 0.05.

ResultsEffect on kidney biomarkers

Administration of sodium fluoride through drinking water for 30 days showed significant increase in the blood serum levels of Urea, Uric acid, Creatinine, BUN and decreased levels of Total protein and Albumin when compared to group I indicating its potential kidney dysfunction. Administration of hydroalcoholic extract of BOB leaves in group III, IV, V and VI showed a decrease in the serum level of Urea, Uric acid, Creatinine, BUN (Table 1) and increase in the levels of Total protein and Albumin of when compared to the Group II (Table 2).

Lipid peroxidation and antioxidant profile

Table - 1

Effects of hydroalcoholic extract of Brassica oleracea var. botrytis leaves on serum Urea, Uric acid, Creatinine and BUN in sodium fluoride intoxicated rats

GROUPS UREA (mg/dl) URIC ACID (mg/dl) CREATININE (mg/dl) BUN (mg/dl)

Group I 13.29±١٫٠٤ 1.01± 0.73 0.62±٠٫٠٥ 0.62±٠٫٤٩Group II 32.38±١٫٧٩ 1.99± 0.02 1.45±٠٫٠٦ 15.15±0.84Group III 24.37 ±1.57*** 1.89±٠٫٠٤ 1.02±0.04*** 11.38±0.73***Group IV 15.05 ±1.11*** 1.44±٠٫٠٤*** 0.77±0.05*** 7.03±0.52***Group V 9.77±٠٫٧٢*** 1.24±٠٫٠٢*** 0.70±0.03*** 4.56±0.34***Group VI 8.78±٠٫٥٠*** 0.74±0.04*** 0.57±0.04*** 4.10±0.23***

Where, ***p <0.001 when compared to Group II.


Prolonged intake of fluoride caused a significant increase in lipidperoxidation level and decrease in the levels of reduced glutathione and catalase when compared to the group I. Administration of hydroalcoholic extract of BOB leaves in group III, IV and V showed significant decrease in lipidperoxidation level and increase in the levels of reduced glutathione and catalase when compared to group II (Table 3).

Discussion Prolonged intake of fluoride causes adverse health or toxic effects collectively called as fluorosis. Hydrofluorosis is the major prevalent and wide spread entity in nature than industrial fluorosis [24, 25]. Thus, in the present study, fluoride was administered in rats through the drinking water.

Till date there are no drugs available which could effectively control the incidence, prevention or cure the renal damage caused by various agents such as drugs, industrial and environmental chemicals. Long term exposure of fluoride has an ability to accumulate in the soft tissues by simple diffusion mechanism. Numerous studies clearly disclosed that kidney has a close correlation between fluoride intake and renal injury [26].

The increase in serum levels of urea, uric acid, Creatinine and BUN and decrease in the levels of total protein and albumin indicate nephrotoxicity [27]. Data obtained in the present study revealed that disturbed kidney function in group II was reflected by increased serum levels of urea, uric acid, creatinine, BUN and decrease in the levels of total protein and albumin. These findings were coincided with the results of previous research reports [28, 29]. Treatment with hydroalcoholic extract of BOB leaves in group III, IV, V and VI significantly decreased in the serum levels of urea, uric acid, Creatinine, BUN and increase in the levels of total protein and albumin when compared to group II.

A relation between oxidative stress and nephrotoxicity has been demonstrated in many experimental animal studies [30]. The continuous exposure of fluoride significantly damages the kidney may be to the generation of free radicals and capacity to decrease in the level of antioxidant profile [28]. In the present study, the administration of NaF resulted in the increase of lipidperoxidation level and decrease in catalase and reduced glutathione levels of kidney. These findings were coincided with the results of Vasant & Narasimhacharya, 2011 [31]. Administration of hydroalcoholic extract of BOB leaves in group III, IV and V showed significant reduction in the lipidperoxidation

Table - 2

Effects of hydroalcoholic extract of Brassica oleracea var. botrytis leaves on serum Total protein, Albumin, Glucose and magnesium levels in sodium fluoride intoxicated rats

GROUPS TOTAL PROTEIN (mg/dl) ALBUMIN (mg/dl) Group I 7.92±0.37 2.46±0.21 Group II 6.39±0.41 2.09±0.18Group III 7.43±0.20** 2.93±0.10***Group IV 7.70±0.29 *** 3.02±0.15***Group V 7.94±0.32*** 3.41±0.27***Group VI 8.15±0.42*** 4.75±0.04***

Where, **p <0.01 and, ***p <0.001 when compared to Group II.

Table - 3

Effects of hydroalcoholic extract of Brassica oleracea var. botrytis leaves on Liver lipidperoxidation reduced Glutathione and Catalase levels in sodium fluoride intoxicated rats

GROUPS LIPIDPEROXIDATION(µMol/mg of tissue)

REDUCED GLUTATHIONE(µMol/g of tissue)

CATALASE(µMol/g of tissue)

Group I 4.22±0.38 10.59±0.56 1.11±0.05Group II 10.12±1.12 4.16±0.75 0.27±0.01Group III 8.06±0.56 8.15±0.49** 0.38±0.04Group IV 4.23±0.35*** 12.81±0.70*** 0.69±0.04*** Group V 3.78±0.34*** 17.28±0.81*** 0.76±0.04***Group VI 2.85±0.26*** 19.82±0.73*** 0.91±0.02***

Where, **p <0.01 and, ***p <0.001 when compared to Group II.


level and increase in catalase and reduced glutathione levels indicating its ability to decrease the lipidperoxidation and restore the antioxidative level.

Chronic exposure of NaF causes histological renal changes, intestinal edema, tubular destruction, inflammation, fibrosis, glomerular and medullary hyperemia [32]. Renal function especially glomerular filtration rate was very sensitive to fluoride exposure and has caused dental/skeletal fluorosis in the residents living in fluorosis areas [11].

In conclusion, the present study clearly observed that administration of hydroalcoholic extraction of BOB leaves alleviated the sodium fluoride induced nephrotoxicity probably by reducing the levels of free radical production and/or enhancing the enzymatic and non-enzymatic antioxidants due to the presence of polyphenols. The administration of BOB has effectively alleviated the toxic effects of fluoride in the above observed parameters in a dose dependent manner.

Acknowledgement The authors are special thanks to secretary cum Correspondent to Mr. Ch. Gopal Reddy, CMR group of institutions for his necessary support for successful completion of the project. The authors declare that there are no conflicting and competing financial interests among them.

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