Pharmacological

Research

Communications,

Vol. 8, No. 2, 1976

199

ANTI-INFLAMMATORY

AND ANTICONVULSANT PROPERTIES

OF SOME NATURAL PLANT TRITERPENOIDS'

A. K. Chaturvedi S. C. Bhatnagar**,

*+

** , S. S. Parmar +2 , S. K. Nigam , ** G. Misra , and B. V. R. Sastry *3

Department of Pharmacology University School of Medicine Nashville, TN 37232 and +Department of Pharmacology and Therapeutics King George's Mecical College Lucknow University Lucknow 226003, India and ** Utilization Research Laboratory National Botanic Gardens Lucknow 226001, India Vanderbilt
Received 27 October 1975

SUMMARY Some natural anticonvulsant a dose with the plant activities. against triterpenoids were evaluated afforded for antiinflammatory triterpenoids from which of all 9 to 48% were devoid and at

The protection carrageenin-induced caprylate Antiproteolytic inhibition

by these

of 40 mg/kg exception

edema ranged and friedelinoxime activity

of a-amyrin activity. by -- vitro in

of antiinflammatory was demonstrated

triterpenoids of

of trypsin-induced

hydrolysis

1) Supported in part by USPHS Grants No. RR-05424 and NS-04699, of Scientific and Industrial Research, New Delhi, India. 2) Senior Foreign The University Dakota 58201. 3) Inquiries should Scientist of North at the Dakota,

and Council

Department of Physiology and Pharmacology, School of Medicine, Grand Forks, North 8. V. R. Sastry at Vanderbilt University.

be directed

to Dr.

200 bovine trypsin sodium trypsin sis serum albumin

Pharmacological and casein. to be concentration used hydrolysis These as the of results reference bovine have serum Such

Research an inhibition dependent. drug, albumin

Communications, of the

Vol. 8, No. 2, 1976 activity of as well inhibition to the hydrolythe In as of

was found salicylate, during

The triterpenoids produced greater

as compared

of casein.

exhibited properties with 10 to All the

a good correlation of these exception plant of

between products. friedelinoxime pentylene-

antiinflammatory the present study,

and antiproteolytic all triterpenoids, provided

and acetylmethylursolate, tetrazol-induced except selectively DL-isocitrate, brain homogenates oxidation inhibition to their erythrodiol inhibited convulsions caprylate, the

40% protection natural plant

against

in mice. friedelinoxime NAD-dependent

triterpenoids,

and acetylmethylursolate, oxidation of pyruvate, citrate, by rat hand, NADstudy, however,

a-ketoglutarate, at a final of of

6-hydroxybutyrate, concentration remained of 2 mM.

and L-glutamate On the other

independent selective not related

succinate NAD-dependent

unaltered.

In the present
was,

oxidations antiinflammatory

by triterpenoids

anticonvulsant,

and antiproteolytic

properties.

INTRODUCTION
Recently, for anticonvulsant some natural plant coumarins and triterpenoids (Chaturvedi triterpenoids, of the al -et -' by "cotton prompted were et al --2
a-

evaluated 1974). and B-

and antiinflaaunatory properties as exhibited

activity plant

Anti-inflammatory amyrin acetates,

of two natural by the arthritis tissue protection

carrageenin-induced 1969) and the decrease

edema and formaldehyde-induced in the (Gupta natural protect convulsants produced formation et --' al plant against of 1971) granulation have

(Gupta induced This

wool" us to test

implantation some ability and as anticonvulsions to

been reported. as potential

triterpenoids the

anti-phlogistics in albino compounds

by their rats

edema induced the effect

by carrageenin of these mice.

by studying by pentylenetetrazol

on the

in albino

In addition,

the

role

of

Pharmacological proteolytic 1968) plant vitro activity mechanism Fig. 1.

Research enzymes

Communications,

Vol. 8, No. 2, 1976 process activity 1974) also (Barnhart of rat led et -' - al brain 1968; Bertelli, by the -in

201

in inflamatory of respiratory et --' al triterpenoids homogenates The structures

and inhibition products effects of rat

homogenates

(Chaturvedi of these brain

us to catabolism

investigate

on protein in an attempt of the

and on respiratory their studied biochemical are shown in

to elucidate

of action.

triterpenoids

MATERIALS AND METHODS General Commercial Chemical hederagenin and B-amyrin et -- al, acid 1969), (Nigam Company, methyl chemicals St. Louis, used in the present study were plant et --' al obtained triterpenoids unpublished), benzoate 1968), and Mitra, (Misra from Sigma were CL-

Missouri.

The natural (Nigam 1969),

ester

acetate, (Misra

hederagenin and Mitra, (Awasthi friedelinoxime

caprylates erythrodiol et --9 al

B-amyrin

caprylate

and Mitra, (Nigam

dihydrobetulic 1969) and

unpublished), (Misra

acetylmethylursolate Determination Carrageenin into the the foot right

and Mitra,

1968). activity in normal et --' al of saline) 1962) Buttle was injected and the et -al volume of

of anti-inflarrpnatory (0.05 hind

ml of a 1% suspension rats (Winter

paw of albino by the carrageenin

was measured hours after

micropipette injection. into the

method

(1957)

before,

and four were

The plant of five of rats

products rats

(40 mg/kg) before ml of a

injected

intraperitoneally injection, while

a group group

one hour 0.5

carrageenin

control

received

0.9% NaCl solution. induced edema,

The mean increase

in paw volume, drug,

due to carrageeninsalicylate, was calculated was used accord-

was measured.

The reference

sodium

at a dose ing to the

of 100 mg/kg. formula:

The anti-inflanmiatory

activity

% Anti-inflammatory

Activity

(1 - $

x 100

202

Pharmacological in the

Research average groups,

Communications,

Vol. 8, No. 2, 1976

where Vt and V, are increases

paw volume respectively.

due to carrageenin-

and control

(I) (II)

Hederagenin methyl R=H, R'=H Hederagenin;

ester

acetate;

(III)

a-Amyrin caprylate; R=C8H,60

R=CH$O;

R'=CH3

COOH

(IV) f+Amyrin
R'=CH3 (V) (VI) 8-Amyrin Erythrodiol R'=CH20H

caprylate; benzoate; caprylate;

R=C8H,60; R=C6H5CO; R'=CH3

(VII)

Dihydrobetulic

acic

R=C8H160,

(IX)

Acetylmethylursolate; R=CH$O, R'=CH3 tested

Figure

1.

Structure

of

triterpenoids

Pharmacological Determination Anticonvulsant seizures divided All

Research

Communications,

Vol. 8, No. 2, 1976 activity against either weights pentylenetetrazol-induced sex. The mice were

203

of anticonvulsant activity mice

was determined 25-30 the g, of group

in albino into groups

weighing

of ten, were

keeping

as equal

as possible. of anticomFour with produced 100% mortalduring alone. An episode a threshthe

triterpenoids activity,

suspended to give

in 5% aqueous

gum acacia, of 0.25% (w/v), a dose

devoid

convulsant pound hours

a concentration a group of s.c.). the of ten

The test of 100 mg/kg. injected

was injected after the

i.p.

into

mice

at the

administration (90 mg/kg,

compounds, This dose of

mice were

pentylenetetrazol convulsions ity within in all 24 hours. period were

pentylenetetrazol mice exhibited was observed test compounds

of

the

untreated other with for

mice, hand,

and the no mortality

On the treated

24 hour The mice of clonic old

in mice observed

100 mg/kg the

of the

60 minutes for

occurence five jerks seconds of

of seizures. was considered

spasm persisting Transient devoid of

at least

convulsion. Mice

intermittent threshold

trembulousness the

was not period group was was

counted. were

convulsions of

during

60 minute in each plant

considered and the

protected. anticonvulsant

The number activity

animals of these

protected natural then

recorded, represented and their

products for

as percent mortality

protection. was recorded.

The mice were

observed

24 hours

Determination The reaction of crystalline mM bovine volume of serum 1 ml.

of proteolytic mixture trypsin albumin All the ( consisted g sufficient

activity of 0.05 to M tris hydrolyze (substrates) plant compounds buffer (pH 8.2), casein), 0.075 0.035 mg

250 g of and water were

BSA) or casein test natural used

in a total in

dissolved

dimethylformamide 60 mg%. test addition

(DMF)

nd were amount of

at a final

concentration to the for control

of 40 mg% and tubes. prior All to the for 5 the

An equivalent were

DMF was added with trypsin

compounds of the

preincubated

10 minutes

substrate,

and the

reaction

mixture

was incubated

204 minutes addition products mined activity. Determination Male their sucrose ried out brains (1:9 at albino were w/v) of rats, after of 0.5 of by the the the addition

Pharmacological of the

Research

Communications,

Vol. 8, No. 2, 1976 by the soluble were of the deter-

substrate.

The reaction acid after (1951) solution.

was stopped The acid

ml of a 15% trichloroacetic protein catabolism, by Lowry obtained et -al

centrifugation, as an index

method

reported

enzyme

respiratory maintained

activity on an -~ libidum ad excised Elvehjem uptake technique pyruvate, and homogenized homogenizer. was measured with air citrate, and succinate solution at diet, were in ice All every as the sacrificed, cold 0.25 M were by the In the cr-ketoThe 6.7 carand

immediately in a Potter oxygen

incubations 10 minutes gas phase.

37OC and the Warburg

conventional present glutarate, reaction mM MgS04, brain study,

manometric

oxidation

of sodium

DL-isocitrate, was investigated. pH 7.4, cytochrome tissue.

B-hydroxybutyrate, mixture was carried

L-glutamate out in a buffer

containing c and rat The total for the

20 mM Na2HP04, equivalent It

1 mM AMP, 33 mM KCl, to

500 vg of of

homogenate ml.

100 mg of wet weight that well endogenous contained glycol

vol-

ume was 3.0 oxidative products solvent substrates RESULTS

was presumed The central in the

NAD was sufficient 0.2 ml of 20% KOH. An equal compounds volume

processes. were dissolved to used

The plant of the different

propylene

(100%). The test

was added were

control

vessels. concentration

and the

at a final

of 2 mM and 10 mM, respectively.

Anti-infldmmatory As is inflammatory caprylate while evident activity from

activity Table against 1, all the the triterpenoids exhibited edema, tested antiexcept a-amyrin of 40 mg/kg, 100 mg/kg.

carrageenin-induced were

and friedelinoxime. sodium salicylate, inhibition

The compounds as a reference drug,

at a dose at a dose of

was used with

The maximum

of 48.3%

was observed

hederagenin.

Table activity and anticonvulsant activity of triterpenoids

Anti-inflammatory

Compound Number aMean Increase in Paw Volume (ml) + S.E. Anti-inflammatory Activity % 'Anticonvulsant Activity %

Name

'Pentylenetetrazol Mortality 24 hr %

Sodium

Salicylate

(100

mg/kg)

0.58 + 0.01 0.59 + 0.02 32.3 48.3

33.3 20 30 30 40 20
10

I 0.45 2 0.01 0.88 + 0.03 0.54 5 0.02 0.79 + 0.03 0.67 + 0.02 0.47 t 0.01 0.89 _t 0.03 0.76 5 0.01 45.5
Nil 12.3 ml. of i.p. (100 mg/kg) 4 hr before 100 mg/kg. the

Hederagenin

methyl

ester

acetate

30 30 40 50 30 60 20
Nil Nil

II

Hederagenin

III 38.5 9.3 22.5

a-Amyrin

caprylate

IV

B-Amyrin

caprylate

B-Amyrin

benzoate

VI

Erythrodiol

caprylate

VII

Dihydrobetulic

acid

70
100 100

VIII

Friedelinoxime

IX

Acetylmethylursolate

a Mean increase in paw volume in control animals (normal saline 1 ml) was 0.87 5 0.01 b For procedure see text. Sodium salicylate was used as a reference drug at the dose were used at a dose of 40 mg/kg. were administered

Test administration

compounds of

For procedure pentylenetetrazol

see text. All (90 mg/kg,

compounds s.c.).

Table Antiproteolytic activitya of triterpenoids

% Inhibition BSAb Casein 60 mg %74.5 34.9 84.4 Nil 70.2 7.3 35.3 71.3 Nil 16.3 text. with t 0.7 was done in 8.3 triplicate 5 0.2 t 0.8 + 0.7 11.3 40.7 51.0 37.3 Nil 5 0.4 Nil 2 0.8 + 0.6 Nil -+ 0.4 and the values 12.3 are 20.4 52.3 45.3 + 2.1 48.2 + 0.7 + 1.0 15.3 5 0.9 -+.0.6 28.7 + 2.0 40 mg % 40 mg % 65.5 21.3 72.2 Nil 61.6 5.2 23.7 66.7 Nil 12.3 + 0.3 + 0.3 $- 0.8 + 0.4 t 0.8 + 1.0 2 0.7 t 0.9

of

the

hydrolysis

of

Compounds

60 mg % 32.8 20.7 68.3 t 0.9 + 0.8 t 0.5 Nil &, 0.8 Nil & 0.7 + 0.7 Nil t 0.5 the

Sodium

salicylate

Hederagenin methyl ester acetate

Hederagenin

a-Amyrin

caprylate

B-Amyrin

caprylate

B-Amyrin

benzoate

Erythrodiol

caprylate

Dihydrobetulic

acid

Friedelinoxime

Acetylmethylursolate

a Assay procedures are as indicated in the mean values of three separate experiments

Each experiment 5 S.E.

b Bovine

serum

albumin.

Pharmacological Anticonvulsant All sions ursolate. at doses the

Research

Communications,

Vol. 8, No. 2, 1976

207

activity were found to show protection except was from per cent friedelinoxime lo-40%. mortality against the the against the convul-

triterpenoids

produced

by pentylenetetrazol, of protection One hundred

and acetylmethylcompounds were with (Table used the 1).

The range of 100 mglkg. which

was observed convulsions

two compounds

exhibited activity effects hydrolysis except

no protection

Anti-proteolytic The inhibitory the the trypsin test induced compounds,

of these of a-amyrin activity. of with were

triterpenoids

and of sodium are recorded

salicylate 2. found

on All

BSA and casein caprylate The degree 72.2%

in Table were

and friedelinoxime, of inhibition

to show anti-proteolytic dependent. respectively, well as sodium than

was concentration

Maximum inhibitions were observed

and 84.4% All of

at 40 mg% and 60 mg% doses, of the new compounds with as

hederagenin. better inhibitors

salicylate with casein

trypsin

BSA as the

substrate

as the

substrate.

Respiratory Table vitro cellular

activity the effects of the of natural the rat plant brain triterpenoids homogenates. on the -in Inhibition a-ketoglutathese triterpenoids,

3 shows

respiratory oxidation

activity

of NAD-dependent rate, with

of pyruvate, and L-glutamate

citrate,

DL-isocitrate, with

8-hydroxybutyrate the exception which were of

was observed

erythrodiol of such oxidation

caprylate, inhibitory of

friedelinoxime effects at final

and acetylmethylconcentrations

ursolate of 2 mM.

devoid

NAD-independent

succinate

remained

unaltered.

DISCUSSION
In the between their present inhibition activity. hederagenin series of triterpenoids, of the trypsin induced there is a direct

relationship
antiby the activity,

hydrolysis activity

and their exhibited

inflammatory triterpenoid

Maximum anti-inflammatory also exhibits the

maximum antiproteolytic

Table on the Citrate 15.05.6 12.85.7 23.73.4 15.852.1 15.33.7 Nil 12.2+1.1 Nil Nil Nil Nil Nil Nil 13.35.4 9.95.8 12.95.2 Nil Nil Nil Nil Nil 16.35.5 10.951.5 11.45.4 16.151.4 18.3y.2 17.05.8 20.83.1 25.222.1 28.15.3 22.35.7 15.832.0 19.722.0 Nil 12.7y.8 Nil Nil 19.15.3 15.85.7 17.Oy.8 18.05.4 16.621.8 14.33.5 11.422.0 10.721.2 Nil Nil Nil Nil Nil Nil Nil Nil Nil DL-isocitrate B-Hydroxybutyrate L-glutamate Succinate % Inhibition a-Ketoglutarate respiratory activitya of rat brain homogenate

Effect

of

triterpenoids

Compound

Pyruvate

Hederagenin methyl ester acetate

12.321.8

Hederagenin

12.83.5

cr-Amyrin

caprylate

22.73.2

@-Amyrin

caprylate

17.3y.o

p-Amyrin

benzoate

20.922.0

Erythrodiol

caprylate

Nil

3 ? 3 h 0 0 rq . . 2 4 2 8 2 s f s 2' 2. 9 .T $ .Q

Dihydrobetulic

acid

11.23.7

Friedelinoxime

Nil

Acetylmethylursolate

Nil

a Different substrates were used at a final concentration of 10 mM. The oxygen uptake in ~1 during the oxidation of pyruvate, citrate, DL-isocitrate, a-ketoglutarate, 8-hydroxybutyrate, L-glutamate and succinate was 100 + 1.2, 90.7 + 1.2, 80.4 + 2.1, 120.3 + 2.1, 90.1 + 2.9, 89.3 ~1.2, 86.3 t 2.5 and 160.2 + 2.1, respectively. The final concentration of all these compounds-was 2 mM. Vessel contents and assay procedure are indicated in the text. Each experiment was done in duplicate. The percent inhibition was calculated from the decrease in the oxygen uptake/hr/lOO mg wet brain weight.

Pharmacological whereas of trypsin the

Research devoid

Communications,

Vol. 8, No. 2, 1976 activity also show no inhibition relationship was reported Chaudhari et -9 - al for

209

compounds induced

of anti-inflammatory of BSA and casein. and antiproteolytic et --' al 1969; here it than Gupta

hydrolysis

A similar activities et -- al, 1971; that

between the

anti-inflammatory acetates (Gupta results latter

amyrin

1974). caprylates higher acid this is

From the the activity the

reported

can be seen the former.

in a- and B-amyrin type of

was more active with

A similar

was observed second most potent

a- and B-amyrin after

acetates.

Dihydrobetulic in the active series. against than sodium In the

compound (II &VII) and the

hederagenin to be more

study

the

triterpenoids by carrageenin

were trypsin

found

edema-induced salicylate. Data

induced

hydrolysis

on the

anticonvulsant mortality from

activity did

of the not

triterpenoids

and the

24 hour between mortaloxidaof tested at a

pentylenetetrazol-induced increased ity tions of the by the protection experimental triterpenoids (Parmar affect the the There

indicate

an association pentylenetetrazol of inhibitory indicate rat brain that

convulsions animals. is

and decreased Selective inhibition with the

NAD-dependent effects the

in agreement 1965).

methaqualone compounds point chrome

and Seth,

Our results activity from of succinic respiratory

cellular passage cytochrome

respiratory of electrons system

homogenates

before b of

dehydrogenase chain the

to cytoand structure of failed

in the

of enzymes chemical inhibition have

coenzymes. of the respiratory to provide and/or ability

was no definite and their of the rat

relationship anticonvulsant brain

between effects

triterpenoids activity a definite

or the These

homogenates. anticonvulsant triterpenoids respiratory

results

correlation activities inhibit the

between of these

and anti-inflammatory as well activity as their of rat brain

antiproteolytic to selectively

cellular

homogenates.

210 REFERENCES Awasthi, Barnhart, Ann. Bertelli,

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Vol. 8, No. 2, 1976

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