Imlial Journal of Expcrimcntal Biology Vol. 4 1, March 2003, pp. 242-247

A comparative study of allitin and garlic on lipid turnover in a teleost, Anabas testudineus (Bloch)

Cyril Johnson I, A Banelji2 & OOITImen V OomITIen*1

I Dcpartlllcnt of Zoology, Uni vcrsity of Kcrala, Thiruviln

JOHNSON el at : ALLITI N & GARLIC ON LIPID TURNOVER IN ANABAS 243

Materials and Methods

Section I:

Anabas testudin eus (Bloch) captured from the Kananmula canal running through the outskirts of Thiru vanthapuram City (Kerala State) were captured using a net and transferred to cement tank s with proper aerat ion and natural li ght. Healthy ad ult fish (60±5 g) after accl imat izati on for a month were sepa-rated into six groups of to each in similar sized glass tanks, three each serving as cont ro l and experimental. The tanks were placed in the laboratory at suitable locations in order to get sufficient light and aerators were used for air suppl y. Feed (hi gh fat feed) pre-pared by add ing 20 g beef fat to 80 g the standard 40% protein feed (prepared as per Hardy' I) pelletized and sundri ed was given ad libitum. Water in the tanks was changed da il y to avoid turbidity.

Allitin used was prepared by the alkaline hydroly-sis of S-allyl-thiouronium chl oride under inert (nitro-gen) atmosphere, whi ch yielded all yl thi ol. Oxidati on of thi s thi ol wi th iodine gave crude di all yl di sulphide, which was purified by column chromatography (S i02; elution with petroleum ether, 40°-60°C) and vacuum dist ill ati on (b .p. 82°C/13 mm). The co lourl ess product obtained con tained 98% diallyl di sulphide and ap-prox imately 2% diallyl trisulphide as estimated by GLC and hi gh pressure liquid chromatography (Micro Bonda Pack 75 % aq ueous meth anoI12). This was the sample, given the tri vial name allitin , used in the pre-sent studi es .

The experimental animals were ad mini stered in-traperitoneall y with allitin at a dose of 10 mg/kg body weight fo r a period of 10, 20 and 30 days using physiological saline as the vehicle. The control s were given vehicle onl y. At the end of the stipul ated period and overnight fas ting, blood was collected in heparini zed tubes by puncturing the heart. The plasma separated by centrifugation was collected in eppen-dorf tubes and kept at -4°C for total cholestero l, HDL and LDL determination l3, employ ing chod/pod-phosphotungstate method. The fish were then sacri-ficed by spi nal transection and the li ver was sepa-rated, washed in sa line, blotted and used immedi ately fo r lipogeni c' enzymes assay th at included malic cn-zy me (Ec. 1.1.I .40)14, glucose -6- phosphate dehydro-genase (EC. 1.1.1.49) 15, and isoc itrate dehydrogenase (EC. I. I. 1.42)1 6. Total lipid l 7 and its profile [ti ssue choles terol18, triglycerides (mod ifi ed) 19, free fa tty ac-ids 20 and phospholipids 21] were also es timated.

Section II: After acclimatization similar to secti on I, the fish

(60±5 g) were segregated into four tank s of 10 each. While those in tank one served as the contro l, the re-maining three as ex perimental groups. All phys ical parameters were the same as in sec ti on I. Feeding and change of water were done every morning at 10 hI'S. While the control group was given food as in section I , the ex perimental groups were provided with the same type of food having additionally crushed raw garli c added to it in concentrations equi valent to hu-man consumption of 10, 20 and 40 g/day. The amount of garlic required to equali ze the stipul ated hu man consumption through food intake was calcul ated after assess ing the amount of food consumed by the fish. One gram of feed co nsumed/fish/day (establ ished af-ter study) contained 0.017 g garli c to equali ze 20 g garlic consumed by a person weighing 70 kg. Both control and experimental groups consumed the same quantity and were given food ad libitum. The time of sacrifi ce was so adju sted th at the duration of the ex-periment was 45 days. Blood and various tissues were coll ected for di ffe rent estimat ions after overnight fast-ing. Blood collected in heparini zed tubes by direct puncture into the heart was centrifuged and the plasma separated was transferred in to eppendorf tubes and kept at - 4°C for further study of choles terol, HDL, LDL, and tri glycerides. The liver and musc le ti ssue excised immedi ately after spinal transec tion were washed in saline, blotted and frozen for the study of total lipid.

Chemicals used in both sections were of the ana-lyti cal grade procured from SRL, Bombay. The data were analysed using Student 's t test fo r the first sec-tion and Duncan's mUltiple range tes t for the second section.

Results and Discussion The results are presented in Tables 1 and 2 (sec ti on

I) and Table 3 (section II). An overall evaluation of the results confirm the

lipid lowering properties of garli c and/or its deriva-tives, espec ially diallyl disulphide (DADS), the major constituent of allitin , for the firs ~ time in a teleos t. AI-litin and raw garlic are undoubted ly very effecti ve agents in lowering tissue (hepatic and muscle) and plasma lipids in higher animals and it has been dem-onstrated in many experimental animals22 . An analy-sis of the effect of garlic showed a signi ficant red uc-ti on of serum total choles terol7 . Li ver being the most important drug-metabolizing organ was used to estimate

244 I DIAN J EXP BIOl, MARCH 2003

the various lipid parameters23 . The results obtained from thi s study confirm a decrease in the act ivity of lipogenic enzymes after allitin adm ini stration. Vari-ous other reports confirm the decreased activity of lipogeni c enzymes after allitin administration, which in tLirn plays a very important role in lowering the lipid turnover in animals. Studies conducted in rats have ascertained that allitin ( 100 mg/kg. body weight) reduced plasma cholesterol signifi cantly 30 min and I hr after trea tment without any change in hepatic cho-lesterol 24 . Garlic when given through diet reduced the choles terol levels in present experimental fish . The consumption of garli c tends to show a decrease in the plasma cholesterol and LDL level with the maximum effect at the 40 g dose per day. Another finding with allitin was the reduction in both HDL and LDL levels, however there was a marked increase in HDL (65% and 64%) and decrease in LDL (35 % and 36%) at 10 and 20 day doses when co mpared to their respect ive

control s (55 and 61 % for H DL and 4.5 and 39% for LDL). With the suppl ementat ion of raw garli c, HDL level showed a significant increase with a corre-spond ing decrease in LDL. The 40 g garlic admini-stration dose showed a promising result with low plasma cholesterol and hi gh HDL leve ls. The prob-able ex planati on for the red uction in the hepatic total lipid along with cholesterol in fish adm inistered with allitin at all the three doses (10 mg/kg . body weight/day for 10,20 and 30 days respectively) may be due to the deposition of lipids in other ti ssues or the conversion to other sources of stored forms in the animal body. The reduction in the lipi d profiles sub-stantiated the decrease in the total hepatic lipid level. The total lipid content in garl ic fed fi sh confirmed an in verse relationship between the I i vc r and muscle. Contradictory to the influence of allitin where there was a direct relationship between the li ve r lipid and blood cholesterol, feeding the fish with garlic

Table I - Inhi bition or lipogc nic cnzymcs in livcr of A. l esllldill ellS ro ll owi ng 10 mg/kg body wc ight/clay alli tin ad mini st ration 1'0 1' vari-ous clumtions

I Valucs arc mcan ± SI: o r 6 anillla is in cac h group. Figures in parenthcscs nrc % dccreasc ovcr con tmll

10 cl ays 20 cla~s Control Experimcnt Contro l Experiment Control

ME 0.95 ±O .04 0.37±0.01 < 0.88 ±0.04 0.43 ± 0.04< 0.~9 ± 0.05 (38 %) (48 %)

G6PDH 1.20±0.4 0.80±0.20h 1.20 ±0. 10 1.0± 0.10 NS 1.1 ± 0. 20 (66%) (88 %)

ICD H 0 .90±0. 1 0 .70±0.20" 1.20±0.30 I.O ± 0 .30 NS 0.90±0.100 (77% ) (83 %)

ME= Malic cnl-yme; Gr. PDH= Glucosc-6-phosphatc clchydmgcnasc; ICDH= Isoci tratc dchydmgcnasc Un it s (ME, G6 PDH ancl lCDH) = IU/mg protcin/m in P va lucs: "< 0.05; b< 0.0 I; ,. P < 0.00 I

30 clays Ex periment 0.58 ± 0.06'

(65 %)

0.90±0 30" (8 1%)

0.70±0.20b (78 %)

Tablc 2 - lnhi bition of li ve r and serum lipicl sy nthcsi s in A. II:'SlIIdill l:' lIS fo llowing 10 mg/kg body wc ight/clay alli tin ad ministration for various dura ti ons

I Va lues arc mcan ± SE or 6 animals in cac h gmu p. Figurcs in parcnthcscs arc % dccrcasc over con tro l I

10 days

Total lipicl '" (l ) Phosphol ipid* (l)

Triglyccride* (l)

Choles tero l'" (l) Free ratty acid* (l)

Cholestem lt (1') HDlt (1')

lDlt (P)

Con trol

60.19 ± 1.73

34 ± 1.0 0.70 ±0.01 8. 14 ±0. 15 2.45 ± 0.04

188.S9 ± 1.50 103.97± 1.22

(55 %)

84 .92 ±2.20 (45 %)

P val ues: "< 0.05; hp < 0 .0 I; '< 0.00 I . 0= Liver ti ssuc; P - Plasma Units: *: IIlg/g; t : Illg/dl

Experi me nt

52.42 ± I.II <

9.33 ±0.2 1<

0.25 ±0.0 1<

6.67 ±0. 14b

2. 17 ± 0.04"

135.45 ± 1.79' 89.29 ± 1.0<

(65 %)

46. 16 ± I. 97' (35%)

20 days Contml Expcrimcnt

70.33 ±0. 17 52.78 ± 0.15<

37.33 ±0.52 10.40 ±O. IOc

0.70 ±0.02 0.36 ±005e

7.67 ±0.07 6.93 ±0.07b

1.83 ± 0 .06 1.58 ±0.02e

202 .3 ±2.4 142.5 ± 2.58<

125±3.75 92 ± 1.6< (6 1%) (64%)

76.5 ± 1.94 50 ± 2.40< (39%) (36%)

30 days Contml Experiment

65.92 ±1 .49 54 .86 ± 1.22"

40 ± 1.0 11 .2 1 ±0.22<

0.70 ±0.003 0.25 ±0.009'

7.67 ±0.05 7.05 ±O.OS"

1.29 ±0.0 1 2.47 ± 0.03' 234.931 2.09 179.89±2.73' 128.57 ± 2.80 9S.28 ± 1. ~6h

155%) (55 %)

106.35±2.11 ~ 1.61 ±2.63h

(45 %) (45 %)

JOHNSON el 01: ALLITI N & GA RLI C ON LIPID TU RNOVE R IN ANABAS 245

Table 3 - Innuence of dietary garlic on tissue and plas ma li pid and its profiles in A. leswdill ells.

rValues are mean ± SE of 6 anima ls in each group. Mean values of groups wi th different superscript letters in a given row are significantly difle rent (P

246 INDIAN J EX P BIOl, MARCH 2003

alliti n admini strat ion produced a hypocholes terolemic effect as ev idenced by changes in the activity of lipo-genic enzymes and lipid profil e. The decrease in LDL levels and simultaneous increase of HDL when given raw garli c provides ev idence for thi s compound to be Ll sed as a therapeutic agent. It w ill be of interest to include garli c products in aquaculture fish feed and to have fi sh wi th low LDL and choles terol and high HDL, fo r human consumption.

Acknowledgement One of the authors (eJ) ack now ledges the FIP

(Faculty improvement programme) of the UGe, the Uni versity of Kerala {'or the laboratory facil iti es and the Government of Kerala fo r the deputation granted. AS acknowledges the finan cial SLipport from the eSIR.

References I Mi l ner J A, Garli c: I ts an ticarcinogenie and anti tumour

properties, Nll lr ReI!, 54 ( 1996) 583. 2 Srivastava K C. Bordia A & Vcrma S K , Garl ic (A ll ium sati -

VU Ill) for discase prevention. S Aj;·.J. Sci , 9 1 ( 1995) 68. 3 Block E, The organosulfur chemistry of the genus Al l ium -

Impli ca tions for the organic chcllli stry of sul fur, Allgew Chelll 1111 ErllI EllgI, 3 1 ( 1992) 11 35.

JOHNSON el 01: ALLITI N & GA RLI C ON LIPID TURNOV ER IN ANABAS 247

34 Aga rwa l K C, Thcrapcutic actions of garli c constitucnts, Med Res Rev, 16 ( 1996) III.

35 Ali M, Bord ia T & Mustafa T, Effect of raw versus boil cd aqueous cx trac t of garli c and oni on on platelet aggregati on, Prostoglolldills Leukot Essell l FOlly Acids, 60( I ) ( 1999) 43.

36 Arora R C, Arora S & Gupta R K, The long-term use of gar-li c in ischcmic heart diseasc - An appraisal, Atherosclerosis. 40(2) ( 198 1) 15.

37 Al i M & Thomson M, Consumpti on of garlic c10vc a day could be bcneficial in prcvcnt ing thrombosis, Prostaglolldills Leukot Essellt Fa lly Acids. 53(3) ( 1995) 2 11.

38 Siege l G, Walter A, Enge l S, Walpcr A & Mi chcl F, Plicotropic effect s of garli c, Wien Med Wochenschr. 149(8-10) ( 1999) 2 17.

39 Ernst E, (Garlic thcrapy? Thcorics of a fo lk remedy (authors transl»), MMW MUllch Med Wochem chr. 123(4 1) ( 198 1) 1537.

40 Myung S C, Eunsook T K & Stcwart T J, Effcc ts of garlic on lipid metaboli sm in rats fed cholesterol or lard , J NUl,.. 11 2 ( 1982)24 1.

4 1 Sk lan D, Berner Y N & Rabinowitch H D, Thc effec t or dic-tary onion and garli c on hepati c lipid concent rations and ac-ti vit y of anti ox ida ti vc cnzy mes in ch icks. J NII/r Biochelll. 3 ( 1992) 322.

42 Clare Stevinson, Max H, Pittlcr & Edzard Ernst, Garlic fo r trcat-ing hyperholcsterolcmia, A IIIl Intern Med, 133 (2000) 420.

43 Augusti K T, Aswathy Narayanan , Lekha S Pill ai, Razia . S Ebrahim, Reshma Sivaciasan, Sidhu K R, Subha I. Sunitha Abdecn & Sunitha S Nair, Beneficial effects of garli c (AlliulII sativlIIlI ) on rats fcd with diets cont aining cholestcro l and ei-thcr of the oil secds, coconuts or groundnut s, Ill dioll J Exp Bioi. 39 (200 I ) 660.