Anti-inflammatory and anti-arthritic activities ofsilymarin acting through inhibition of 5-lipoxygenase

O. P. Gupta, S. Sing, S. Bani, N . Sharma; S. Malhotra, B. D. Gupta, S. K. Banerjee , S. S. Handa

Regional Research Laboratory (CSIR) Jammu Tawi, India

Summary

Silymar in, a mixture of flavonolignans, comprised mainly of thre e isomers, silybin, silydia nin and sily­christi n isolated from the fru its of Silybum marianum , is currently in therapeutic use as a hepatop rot ec­tive agent. Silymarin on evaluation exhibited significant antiinflammatory and antiarthritic activities inthe papaya latex induced model of inflammation and mycobacterial adjuvant induced arthritis in rats.Results of the study indicate its action th rough inhibition of 5-lipoxygenase for anti inflammatory andanti arthritic activities.

Key words: Silymar in, anti-inflammatory, anti-arthritic, 5-lipoxygenase inh ibition.

Introduction

Silymarin, a mixture of flavonoligans comprised main­ly of three isomers silybin, silydianin and silychristinisolated from the fru its of Silybum marianum is report­ed to be hepatoprotective against a number of experi­mentally studied hepatotoxins (Antwei ler, 1976;Braatz, 1976; Rauen and Schriewer, 1971; Schriewer etal., 1973; William and Priestly, 1973; Hiroshi et al.,1984). Its hepatoprotective effect has been attri butedto membrane stabilizing and protein synthesis enhanc­ing effect coupled with antioxidant and free radicalsscavenging activities (Lonchampt, 1989; Chen, 1990).Recentl y reported gastric anti-ulcer activity of silymar­in has been att ributed to its inhibition of enzymaticperoxidation in the lipoxygenase pathway and therebyleukotriene synthesis (Alarcon et al., 1992). It has beenalso reported (Bauman et al., 198 0) that free radicalscavenging property affects various steps in the arachi­don ic acid (AA) cascade via cyclooxygenase and lipox­ygenase pathways.

In view of above reports on the free rad ical scaveng­ing and of 5-lipoxygenase activities of silymarin it wasthought desirable to evaluate silymarin for antiinflam­matory and ant iarthritic acti vities.

Materials and Methods

ChemicalsSilymarin employed in this study was isolated by treat­ing a defatt ed aceto ne extract of fruits of Silybum ma­rianum with an aqueous solution of sodium chloride inaccordance with the process (Soroceanu et al., 1991 )and acetyl salicylic acid was prepared by acetylatin gsalicylic acid with acetic anhydride using a smallamount of sulphuric acid as catalyst. On uv spectro­photometric determination silymarin was found tocontain not less than 80 % of flavonolignans taken assilybin [Amax (methano l): 288 nm (log E 4.33)] and itssilybin content was found to be not less than 40 % onHPLC analysis (Titte l and Wagner, 1978). The productwas found to be comparable with that from Aldrich inquality (uv and HP LC). Carrageenan and arachidonicacid (Sigma), mycobacteri al adjuvant (Difco Lab orato­ries, Michigan, USA) and papaya latex (collected by theinvestiga tors from the unripe fruit ) were used.

AnimalsMale Wistar rats of 160-175 g body weight reared andmaintained under controlled conditions of tempera­ture, air changes and light under hygienic conditions in

0944-7113/00/0 7/0 1-021 S 12.00/0

22 0. P. Gupta et aI.

the Institute's animal house were employed. Food andwater were given ad libitum.

Anti-inflammatory evaluation

Aqueous suspension of 1% each of carrageenan and0.25% of papaya latex in 0.05 M sodium acetate buf­fer of pH 4.5 were prepared . The se were injected in 0.1ml volume in the subplantar region of the left hind pawof the rat. The rat s were treated with test drugs p.o.1 hour before the injection of the inflammagen. Pawvolume was measured at a and 3 h after inflammageninject ion using volume differential meter, model 7101UGO, Basile. The agents employed for evaluation ofanti-inflammatory activity using the two inflammagensare carr ageenan (Winter et aI., 1962) and papaya latex(Gupta et aI., 1992) (see Table 1).

Arachidonic acid induced mouse ear edema

The edema was induced on the right ear of mice by top ­ical application of 2 mg/ear of AA in 20 ul acetone. Themice were first fasted for 5 hours and treated with testdru g 1 h before the application of AA. Thirty minutesafter arachidonic acid application, circular biopsies ofinflammed area of ear were taken out with a leatherpun ch of 8 mm diameter and weighed. Percentage inhi­bition was determined as compared to contro l group(see Table 2).

Table 1. Evaluation of anti- inflamm atory activity of Silymar in.

Anti-arthritic evaluation

The evaluation is in accordance with the method ofNewbould, 1963 and 1969. Fine suspension of myco­bacterial adjuvant in liquid paraffin, 4 mg/ml wa s pre­pared. 50 pl of this suspension was injected intrader­mally into plantar surface of right hind paw. Paw vol­ume was measured on day aand every alternate day till13th day in case of develop ing art hr itis and on 14thand every alternate day till 28th day in case of estab­lished arthritis. Paw volume was measured by volumedifferential meter model 7101 UGO , Basile. Drug treat­ment was started 1 day before the injection of adjuvantand continued till 14th day in case of developing ar­thritis and started from day 14 to 28 in case establishedarthritis.

Results and Discussion

Antiinflammatory activity: Table 1 covers the antiin­flammatory activity recorded with 25, 50 and 100mg/kg p.o. doses of silymarin aga inst carrageenan andpapaya latex induced models of inflammation. Thepercentage inhibitions record ed with 100 mg dose of si­lymarin are 14.97, 26.22%, against carrageenan andpapaya latex models of inflammation respectively. Theant iinflammatory activity of silymarin against arachi­don ic acid induced mouse ear edema using boswellic

Dru g Treatmentmg/kg p.o .

CARRAGEENANEdema (ml) %Mean :t SE inhibition

PAPAYA LATEXEdema (ml) %Mean :t SE inhibition

Contro lSy (25)Sy (50)Sy(100)

1.67:t 0.121.60 :t 0.061.47:t 0.041.42:t 0.11

4.1 911.9714. 97

1.22 :t 0.22 -0.97 :t 0.06 20.49 *0.95 :t 0.02 22.13 **0.90 :t 0.04 26 .22 **

N umber of observa tions = 12Sy- Silyma rin.* P-value < 0.0 1*" P-value < 0.001

Tabl e 2. Evalua tion of anti-inflammato ry activity of Silymarin (Sy) by AA indu ced mouse ear edema .

DrugTreatmentmg/kg p.o .

ControlSy (25)BA (200)

Weight of ear patch (mg)Mea n e S.E.

Unt reated

16.75:t 1.1816.50 :t 0.5016. 75 :t 0.75

AA treated

21.50 :t 1.0419.50 :t 0.2818.10 :t 0.60

Edema (ml)Mea n e S.E.

4.75 :t 0.4 73.00 :t 0.401.35 :t 0.25

%inhibition

36 .8471.57

Number of observations =8BA - Boswellic acids; positive control as 5-LO inhibitor.AA - Arachidonic acid; 2 mg in 20 pi acetone/ear.

Anti-inflammator y and anti-arthritic activ ities of silyma rin 23

Table 3. Evaluation of antiarthritic activity of Silymarin (Sy) in adjuvant induced Developing arthritis in rats monitoredthrough paw edema.

Drug Paw edema ml (Mean ± S.E.) % inhibition P-valueTreatment On Day on day on day(mg/kg p.o.) 3 5 7 9 11 13 13 13

Control 2.06 ± 0.09 3.54 ± 0.09 3.46 ± 0.08 3.22 ± 0.12 3.20 ± 0.13 3.16 ±0.17Sy (6.25) 2.00 ± 0.10 2.27 ± 0.17 2.97 ± 0.16 2.78 ± 0.26 2.72 ± 0.23 2.69 ± 0.23 14.87Sy (12.5) 1.84± 0.17 2.77 ± 0.31 2.68 ± 0.27 2.49 ± 0.27 2.46 ± 0.24 2.41 ± 0.29 23.73 < 0.05Sy (25) 1.80 ± 0.18 2.68 ± 0.18 2.54 ± 0.13 2.33 ± 0.18 2.32 ± 0.23 2.16 ± 0.23 31.64 < 0.01BA (200) 1.94 ± 0.14 2.62 ± 0.21 2.58 ± 0.18 2.40 ± 0.22 2.28 ± 0.17 2.12 ± 0.21 32.91 < 0.01

Number of observations: 10BA- Boswellic acids, Positive control as 5-LO inhibitor.Adjuvant injected on day O. Treatment from day 1 to day 13.

acids as positive control for 5-lipoxygenase inhibition(Safayhi et al. , 1992), is given in Tabl e 2. Silymarin inthe dose of 25 mg/kg p.o. and boswelli c acids in thedose of 200 mg/kg p.o. showed 36 .84 and 71.57% in­hibit ion s respectively.

Silymarin exhibited varying degr ees of antiinflamma­tory activity in the three models of inflammation em­ployed . The activity is less marked in ca rrageenan mod­el but significant in papaya latex and arachidonic acidmod els of inflammation. Further, the arachidonic acidinduced mou se ear edema has been held as selective forinh ibitors of enzyme 5-LO (Young et aI., 1989 ). In th ismodel , silyma rin in 25 mg/kg p.o. do se showed36. 84 % inhibition of oedema, wh ich ca n be attributedto inhibito ry action on th e for ma tio n of 5-LO prod­ucts, leukotrienes involved in inflammation. This find­ing is in tune with what has been reported earlier th atsilyma rin possesses anti-ulcer activity attributable toinhibition of enzymatic peroxid at ion in the lipoxyge­nas e pathway (Alarcon et aI., 1992) and that synthesisof pr oducts of lipoxygenase and cyclooxygenase path­ways of AA requires many free radi cals (Bauman et al.,1980 ).

Antiarthritic activity: Tested in 6.25, 12.5 and 25mg/kg p.o. doses, silymarin sho wed dose related inhibi­tion s of 14. 87, 23 .73 and 31.64 % as recorded on da y13 in th e ad juvant induced developing arthritis in rats.As co mpa red to th is, boswellic acids employed as posi­tive co nt rol showed 32 .9 1% inhibitio n (Ta ble 3). Fig. 1sho ws the effect of silymarin employed in 25 mg/kgp.o. dose on the established arthri t is. The effect record­ed in esta blished arthritis is less marked as compar ed tothat on the developing ar thritis . This difference in ac­tivity can be explained as fo llows. Silyma rin which hasfree rad ical scavenging activity cau ses inhibition of en­zymat ic peroxidation . Enzyma tic pero xidation is in­volved in arachido nic acid cascade leading to forma­tion of prosta glandins (PGs) and leuk otrienes (Lts)which are involved in developing arthritis and silyma r­in during this process is able to inh ibit the developmentof arthr itis more effect ively. It is in line with antihepa­tot oxic activity of silymarin whi ch has been reported tobe more effective when given pre as compared to posttr eatment of hepatotoxins (Braatz, 1976; Williams andPriestly, 1973 ). The role of free ra dica ls in hepatic inju-

6 .00 ,..------------------------,

III5.20co

...I:IIII::E 4.40

1EII 3.60§ 1 1'0>~ 2.80IIQ.

Fig. 1. Effect of Silymarin on established ar- 2.00thriris in rats (n = 6;~ control; -G-- Sily- 14 16 18 20 22 24 26 28 30marin 25 mg/kg) DAYS

24 O. P. Gupta et a I.

ry by hepatotoxins has been a lso reported (Lonchamp tet aI. , 1989; Chen, 1990 ).

From the above findi ngs it appea rs that silymarincould perhaps have cons idera ble ther apeutic potentialfor the treatment of rheumatoid arthritis.

References

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Address

O. P. Gupta , Regional Research Laboratory (CSIR),j ammei Tawi-1 80001-India .