Indi an Journal of Experimental Biology Vol. 38, July 2000, pp. 675-680

Anticonvulsant and behavioral actions of triterpene isolated from Rubia cordifolia Linn.

Veena S. Kasture*, V.K.Deshmukh" & C.T.Chopde"

*M.Y .P.Samaj ' s College of Pharmacy, Gangapur Road, Nashik 422 002, Indi a

aDepartment of Pharmaceutical Sciences, Nagpur University, Nagpur, 440 010, India.

Received: I Apri/1999; revised 9 March 2000

Effect of a triterpene isolated from the acetone soluble part of petroleum ether extract of R. cordifolia was studi ed on convul sions induced by maximum electro shock (MES), electrical kindling and various chemoconvulsants in rats and mice. The effect of triterpene was also investigated on behavior and gamma-aminobutyric acid (GABA) and serotonin (5-HT) content in mouse brain . Triterpene inhibited seizures induced by MES, electrical kindling, pentylenetetrazol (PTZ), and lithium-pilocarpine. However, seizures induced by strychnine were not inhibited. Triterpene reduced locomotion as well as rearing. Pentobarbi tone induced sleep was potentiated and amphetamine induced stereotypy was inhibited. The triterpene was found to possess anxiogenic activity. Brain GABA and 5-HT contents were raised by the compound. The study suggests that the triterpene isolated from R.cordifolia bear a potential fo r further study.

Rubia cordifolia Linn (Family: Rubiaceace) is a climbing plant growing in north-west Himalaya, and other hilly districts of India. The methanolic ex tract of roots has anticancer activity' . Dried roots are used as astringent and diuretic. They are used in folklore medicine for treatment of dropsy, paralysis, jaundice amenorrhoea, and visceral obstructions2

. Ethanolic extract of the aerial parts of the plant shows hypoglycemic activity in albino rats' . Tripathi and her associates have reported the lipoxygenase activity of ethanolic extract of roots and ethyl acetate fraction of this extract is the most active lipoxygenase inhibitor4

•

During our preliminary studies, the acetone soluble fraction of petroleum ether extract, which contained a triterpene, was found to possess CNS depressant activity. Therefore, we investigated the effects of the triterpene isolated from the acetone soluble part of the pet.ether extract on chemically induced and max imum electro shock- induced convulsions, and electrically kindled seizures. Its effect on gross behavior, ambulation and rearing, pentobarbitone-induced sleep, and amphetamine-induced stereotyped behavior was also studied .

Materials and Methods Extraction- The dried roots and rhizomes of R.

cordifolia (500 g), collected in March, after identification and authentification by Dr.V .K. Deshmukh, Ex-Prof. of Pharmacognosy, Department of Pharmaceutical Sciences, Nagpur University, Nagpur, were extracted with petroleum ether (60°-

80°C). The extract was concentrated under vacuum and was then partitioned into acetone soluble and acetone insoluble parts . The yields of pet.ether extract, acetone soluble and insoluble parts were 0.5%, 0 .2%, and 0.3% w/w respectively. The acetone soluble part was then separated into four fractions using column chromatography, with neutral alumina as a stationary phase and solvents of varying polari ty like benzene, benzene:ethyl acetate (5:5), ethyl acetate and methanol as mobile phase. The benzene:ethyl acetate (5:5) fraction was obtained in sufficient quantity ( yield 750 mg) . The fraction was thoroughly washed with acetone to obtained white needle shaped crystals, which were triterpene chemjcall/. The crystals of triterpene, (RCT), were suspended in 0.5% (w/v) carboxy-methylcellulose and administered orally.

Animals-Albino mice (NIN strain) of either sex weighing 20-22 g and albino rats (NIN strain) of e ither sex, weighing 150-175 g were housed into groups of 8 in standard laboratory conditions. The experiments were performed during 900-1600 hrs in the laboratory, which was maintained at 23° ± 1 °C.

Drugs-The following drugs were used : pentobarbitone sodium, d-amphetamine (Sigma, USA), lithium sulphate (Gienmark, India), pilocarpine (FDC, India) , diazepam (Ranbaxy, India). All drug solutions were prepared in distilled water immediately before the experiment and administered in a constant volume (5mllkg in mice and I ml/kg in rats) .

676 INDIAN J EXP BIOL, JULY 2000

Acute toxicity - The RCT was administered in doses of I 00, 200, 400, and I 000 mg/kg orall y and percentage mortality was observed after 24 hr6

.

Behavioral studies - To investigate the central acti ons of RCT, ( I 00-200 mg/kg, p.o.) , the method described by Irwin7 was employed . Briefly , an imals were observed fo r 60 min after oral admini strati on of RCT. The procedure involved an initial phase of undisturbed observati ons and a later manipul ati ve phase during which animal s were subjected to the least provocating stimuli . In the initial phase the animal was observed for body pos ition, locomoti on, rearing, respirati on, tremors; staggering gait , and in the later phase, the effect on grip strength , pass ivity, righting refl ex, pa in response and lac rimati on was also observed.

Convulsions - Mice (n = 6/8) were pretreated with e ither RCT (25-200 mg/kg p.o.) or vehicle or diazepam (2mg/kg i.p.), 30 min before the admini strati on of e ither pentylenetet razo l (80 mg/kg s.c.), strychnine ( I mg/kg i.p.), or an electri c shock ( 42mA fo r 0 .2 sec us ing corneal electrodes) . The animals were pl aced in isolated cages and observed fo r the presence or absence of seizures. The onset of convul sions and the inc idence of seizures were recorded in case of chemoconvu lsants, whereas the duration of toni c hind leg ex tension and inc idence of convul sions were observed in case of MES induced convul sions8

.

Lithium-pilocarpine-induced seizures- The rats (n = 8) received lithium sulphate (3 meg/kg i.p. ) 24 hr before pilocarpine (30 mg/kg i.p.) . The animals received vehicle or RCT ( I 00-200 mg/kg) orally 30 min before pilocarpine. The. severity of convul sions was assessed for 90 min using the fo llowing scoring sys tem suggested by Pate l et at No response = 0, fi cti ve scratching = I , tremors = 2, head noddin g =3, fo re limb clonus = 4, reari ng, falling and clonus = 5.

Electrically kindled seizures - Rats (n = 6) received electric shock dail y, (70 mA, for 0 . 1 sec, twice a day, 3 hr apart using Techno Electroconvulsiometer) till all the animals ex hibited full blown seizures . A ll animals received equal number of shocks ( II shocks), although some ex hibited complete deve lopmen t of kindling with less number of shocks. T he vehic le, diazepam (2mg/kg i.p.) or RCT ( I 00 mg/kg p.o.) was admini stered 30 min before the electric shock and the severity of seizures was recorded as score I =fac ial movements, 2 =head nodding, 3 =forelimb clonus, 4 = rearing with forelimb c lonus, 5 =clonic seizures 10

.

The scores representing the intensity of pilocarpine induced behavior in lithium pretreated rats were noted every I 0 min fo r 90 min and the latency to first fore limb c lonus was noted .

Acute saf ety test- The in verted screen procedure was used to study the acute safety of RCT, as described by Coughenour and hi s assoc iates II_ The apparatus consisted of a 12.6cm square pl atform of 0 .6cm wire mesh supported by metal bars and mounted on a steel rod . Mice were pretested by pl acing them on the platform and gently rotating it upside down and observing the an imals fo r their ability to c limb on the pl atform with in I min . The animals failing the task were not used fo r the test on the nex t day . For the test, animals (n = 6) were admini stered vehicle or RCT ( I 00 mg/kg) orally or di azepam ( I mg/kg i.p .) and were p laced on the platform and the rod rotated through an arc of 180°C. Mice unable to c limb to an upri ght pos iti on within I min were considered to be neurall y impaired. Six animals were used in each group .

Activity on rotating rod - Mice were prev iously trained to remain on the rod (2.54 em di ameter) rotating at a speed of 20 rev/min fo r a pe ri od of 5 min . On the next day the animals were randoml y di vided into groups of 5 each. The RCT ( I 00 or 200 mg/kg p.o.) or diazepam ( I mg/kg i.p.) or vehic le (5 ml/kg, p.o .) was admini stered 30 min before the test and the time required to fall off the rotating rod was noted fo r each anima!I 2

.

Locomotion and rearing - The effect of RCT was observed on locomotion and rearing us in g open fi e ld test I3

. The open fi e ld test apparatus (24x24x 15") made up of plywood and the fl oor d ivided in 16 squares of same s ize. Mice (n = 5) were treated with vehicle or RCT ( 100 mg/kg) ora ll y or di azepam ( I mg/kg i.p.) and 30 min later the animals were pl aced (individually) gently in one corner of the apparatus and the number of squares traversed and rearings made in 5 min were noted.

Pentobarbitone-induced sleep-Female mice in groups of 5 each were treated wi th veh icle or RCT ( I 00/200 mg/kg) orally 30 min before pentobarbitone (40 mg/kg i.p.) and the duration of loss of righting refl ex was observed in each group. The righting refl ex was considered to be lost when the animal placed on its back, failed to regain its normal posture within I 0 sec i4 .

Amphetamine antagonism - Mice were di vided in groups of fi ve each. Mice were pretreated with vehicle or RCT (200 mg/kg) p.o. or chlorpromazine ( I 0 mg/kg i.p.) and 30 min later, d-amphetam ine

KAST URE et a/. ANTI CONV ULSANT ACTIVITY OF R. CORD/FOLIA 677

( I mg/kg i.p. ) 15 was admini stered. The animals were observed for the onset of tremors and biting.

Effect on haloperidol-induced catalepsy - Catalepsy was measured using "Bar tes t" 16

• In brief, mice (n = 5) were treated with vehicle or RCT ( I 00 mg/kg) p.o. 30 min before haloperidol ( I mg/kg i.p.) and the ir forepaws were pl aced on a bar ( I em di a. pl aced 2.5 em above the table) and the durati on of catalepsy was measured at 5, 15 , 30, 60 and 90 min interva ls.

Assessment of anxiolytic activity - Elevated plus maze was used as described earlier17

• In brief, mi ce (n = 5) were pl aced indi vidually in the centre of elevated plus maze (35x6 em, open arm and 35 x 6 x 15 em, c losed arm, e levated to the height of 25 em) facing an enclosed arm. The time spent by the mice in open and c losed arm was noted. The mice were treated with vehic le or RCT ( I 00 mg/kg) p.o . or diazepam ( I mg/kg i.p.) 30 m_in before the tes t.

Assessment of analgesic activity- The ana lgesic acti vity was studied using the hot plate analges iometer1

R. Mice (n = 5) were treated with vehicle or RCT ( I 00 mg/kg) p.o. or pentazocine ( I 0 mg/kg i.p.) and they were placed indi viduall y on the hot plate maintained at 55°± I 0 C. The time required to lick paws was noted .

Estimation of gamma-aminobutyric acid and serotonin in brain - Whole brain contents of GAB A and 5-HT were measured 30 min after vehic le or RCT ( 100 mg/kg) p.o. by the method of Klingman and Mennet19 and Curzon and Green 20 respecti ve ly.

Phytochemical study - Chemical tests were carried out to determine the chemical nature of RCT. Its me lting point was determined using conventi onal method. Thi s was followed by UV, IR, Mass, PMR and 13C NMR spectra and e lemental analys is.

Statistical analysis - The data obtained were analysed using one way analys is of vari ance (ANOVA) fo ll owed by Dunnett 's test or Student 's t test. Mann-Whitney U tes t was used fo r non­parametric data and the complete e ffec t or lack of e ffec t was analysed using Fisher exact tes t.

Results Acute toxicity- The approx imate LD50 of the

triterpene, RCT, was found to be 400 mg/kg ora lly. The signs preceding death usuall y inc luded prostration, atax ia and convul sions. Convu lsions were observed before death. They were not observed in the surviving animals.

Behavioral studies - The RCT had no effect on body position and respirati on, grip strength , ri ghting refl ex , and pass ivity at the doses used ( 100 /200

mg/kg, p.o.), however locomoti on, rearing, and reaction to pain ful stimuli decreased noticeably. Tremors, staggering gait , and lac ri mation were not observed .

Convulsions - The RCT dose dependentl y inhibited the incidence of convulsions and significantly de layed the onset of spasm and c lonus in the PTZ treated mice . However, it fa iled to inhibi t strychnine-induced seizures (Tables I and 2).

In the MES test, the durati on of tonic ex tensor phase reduced dose dependentl y. RCT at the dose of 200 mg/kg, p.o. prevented the toni c extensor phase completely. Diazepam (2 mg/kg, i.p.) affo rded complete protection to mice (Table 3). The severity of electrically kindled seizures was reduced and latency to attain forelimb c lonus in lithium pilocarpine treated rats was delayed s ignificantl y (P < 0.05, Fig I and 2).

Acute safety study- All the mice treated with RCT ( I 00-200 mg/kg, p.o.) could c limb the screen within I min . However, animals treated with diazepam ( I mg/kg, i.p.) failed to climb the screen within I mi n.

Activity on rotating rod- Mice treated with ve hic le could remain on the rotating rod for 300 sec and the mice treated with RCT ( I 00-200 mg/kg, p.o .) remained on the rotating rod for 264 .4 ± 30.7 and 248.4±34.9 sec respective ly. Thi s difference mi ssed the stati stical significance. Animals treated with

Tab le I -Effect of a tri terpene (RCT) iso lated fro m pet ether of Rubia cordifolia on pentylenetetrazo l induced seizures in mice.

[V alues are mean± SE o r 8 animals in eac h group]

Treatment (Dose: mg/k g)

Vehi cle RCT (25)

(50) ( 100) (200)

Diazepam ( I)

Latency to Latency to Incidence of spasm in sec convul sions in sec seizures

58.0±5.4 I 08.4±7.5 8/8

68.7±5.9 139.5±8.9 6/8

104.7±8.2** 164.6±12.4* 5/8

135 .7±7.2*** 189.3±11.4** 4/8t 0/8t

178.7±7.2# 2 10.5±16.3# 0/8t

Pentylenetetrazol was ad mini stered s.c. in a dose of 80 mg/kg. P values : *< 0.05 , **< 0.01. ***< 0.00 1, #< 0.000 1 (S tudent's r test) . 1 P < 0.05 , Fi sher exact test.

Table 2- Effect of a tri tcrpene (RCT) isolated from pet ether extrac t o r Rubia cordifolia on stryc hnine induced seizures in mice

[V alues arc mean±SE or 8 animals in each group]

Treatmen t Latency to Latency to Incidence or (Dose: mg/kg) spasm in sec convul sions in sec seizures

Vehicle 124.6±9.7 168 .2±7.8 8/8 RCT ( 100) 132.9±6.1 154.3±6.2 8/8

(200) 14 1.2±7.8 16 1.3±7.3 8/8

St rychni ne was administered in a dose of 1.2 mg/kg i.p.

678 INDIAN J EXP BIOL, JULY 2000

diazepam (I mg/kg i.p.) cou ld remain on the rotating rod for 46.4 ± 5.4 sec only (P < 0.00 I).

Locomotion and rearing- Locomotion was significantly reduced by RCT ( I 00 mg/kg p.o.). Whereas rearing was completely suppressed . RCT was more effective than diazepam (I mg/kg i.p.) in suppressing locomotion and rearing (Table 4).

Pentobarbitone-induced sleep- The pretreatment with RCT (I 00 and 200 mg/kg) orally prolonged sleeping time from 69 .6±5.4 min to 98.4±3.7 and II 0.4 ± 13.4 min respectively (P < 0.05).

Amphetamine-induced stereotypy- Prior treatment with RCT (200 mg/kg p.o.) de layed the onset of

Table 3- Effect of a triterpene (RCT) isolated from pet ether extract of Rubia cordifo/ia on maximum electroshock-induced

seizures in mice

[Values are mean ±SE of 8 animals in each group]

Treatment (Dose: mg/kg)

Vehicle RCT

Diazepam (2)

(25) (50) ( 100) ( 150) (200)

Durat ion of Incidence of Percentage tonic convulsions mortality

hindlimb extension

in sec

14.9±5.4 10.4±3.2 7.8 ±2.5* 5.4±2.2* 3.2±0.2*

8/8 100 6/8 75 5/8 50

4/8** 25 2/8** 00 0/8** 00 0/8** 00

*?<0.05, Student's 1 test. **?<0.05, Fisher exact test.

4.5

4

3.5

Cl) ... 3 0 u en

2.5 iij ... 0

2 ·;

"' ~ Cl) 1.5 III

0.5

0 +-~---L--+-~---L--+-~---L~

Vehicle RCT 1 00 Diazepam2

Treatment ( mg/kg)

Fig. I -Effect of RCT on severity of electri cally induced kindling seizures in rats. *?<0.05 (Student's t test).

tremors from 36.4±5.8 sec to 119.7± 8.9 sec and the onset of biting from 79.6 ± 8.4 sec to 320.6 ± 31.5 sec (P < 0.05).

Effect on haloperidol-induced catalepsy- The triterpene RCT, as such was devoid of cataleptic activity but potentiated and abbreviated the haloperidol-induced catalepsy significantly (Fig. 3).

Assessment of anxiolytic activity- The animal s treated with RCT remained for most of the time in the closed arm, whereas animals treated with diazepam spent more time in the open arm (P < 0.05, Table 5).

Assessment of analgesic activity- Animals treated with RCT exhibited increase in reaction time when placed on hot surface (P < 0.05). The activi ty was comparable with that of pentazocine, I Omg/kg i.p. (Table 6).

Estimation of brain GABA and 5-HT- The RCT in a dose of I 00 mg/kg p.o . increased the brain content of GABA from 19.6 ± 0.3 to 33 .59 ± 2.1 J..Lg/g and brain content of 5-HT was raised from 492.0 ± 43.1 to 1180.8 ± 23.4 ng/g. The increase in

60

70

~ CD 60 ... 0 0 fl)

r:::: 50

~ .s 40 r::::

E c 30 >o 0 r::::

~ 20

10

•

Vehicle RCT 100 Diazepam2

Treatment (Dose: mg/kg)

Fig. 2- Effect of RCT on severity of lithium-p ilocarpine induced seizures. * P < 0.05 (Student 's t test)

KASTURE el a/. ANTICONVULSANT ACTIVITY OF R. CORD/FOLIA 679

brain contents of both the neurotransmitters was significant at P < 0.0 I.

Phytochemical study of RCT- The Liebermann­Borchard test indicated triterpene nature of the compound. Its melting point was II 0°C. UV spectrum (A. max) at 217,250, 255 and 316 nm. IR (KBr) spectra-3450, 3370, 2900, 2800, 1660, 1640, 1480, 1320, 1060, 1010, and 780 cm·1

• Mass spectra indicated molecular ion peak at 478 . Other peaks were observed at 430, 426, 412, 396, 379, 271, 202, 148.

Elemental analysis : % C = 78.91 , % H = 11 .03, %0 = 10.06.13C NMR (200 Hz, CDCI3): Peaks were observed at 130.82, 128.8, 121 .62, 77 .31, 77 .20, 77.00, 76.6, 71.71' 56.7, 56.08, 55.2, 45 .09, 42.03, 37.27, 36.1, 34.0, 31.87, 29.62, 29.26, 29.08, 27.93, 27.70, 26.24, 24.74, 23.11, 22.61, 19.74, 19.34, 19 .05, 14.01, 11.94, 11.83 (Total 32 carbon atoms) . PMR (300MHz): 8 0.69, 0.8, 0.86, 1.0, 1.29, 1.5, 1.54, 1.66, 1.82, 1.89, 2.0, 2.4, 3.54, 4.9,'5.05, 5.15, 5.2, 5.35 .

From the elemental analysis and other spectral data, the empirical formula was determined as CJ2H460 3. The compound has not been reported in the literature.

Discussion The results of behavioral assessment indicate that

the triterpene RCT, isolated from the acetone soluble

Table 4- Effect of the triterpene (RCT) isol ated from pet ether extract of Rubia cordifolia on locomotion and

rearing in mice

[Values are mean±SE of 5 animals in each group]

Treatment Locomotion Rearing (Dose:mg/kg) Vehicle 46.2±6.68 7.0±1.08 RCT (100) 3.25 ±2.3* 0* Diazepam (I) 24.5±4.5" 4.5 ±2.25"

*P<0.05 compared to vehicle (Student's 1 test) . "P<0.05 compared to diazepam treated group (Student's 1 test) .

Table 5-Effect of the triterpene (Rcn isolated from pet ether extract of Rubia cordifolia on the time spent in open and closed

arm of the elevated plus maze in mice

[Values are mean±SE of 5 animals in each group]

Treatment

(Dose:mg/kg) Treatment RCT (100) Diazepam ( I)

Time spent in closed Time spent in open arm

in sec 216.5±1 0.24

280.75±12.06* II 0±4.58***/ a

arm in sec

82.25±1 0.24 19.25±12.06**

190.0±4.58***1"

*?<0.05, **P<O.O I, ***P<O.OO I compared to vehicle treated group (Student's 1 test). " P<O.OO I compared to diazepam treated group Student's 1 test).

part of petroleum ether extract of R.cordifolia produced a depressant effect on the central nervous system. The depression which occurred did not resemble the sedative effect of benzodiazepines, as the animals were still responsive and did not show prominent muscle relaxation. The compound inhibited MES and PTZ-induced convulsions but not strychnine-induced convulsions. These observations indicate that the anticonvulsant effect of the triterpene is possibly mediated by chloride channels of GABA/benzodiazepine receptor complex but not by chloride channel of glycine receptors 21

•

The triterpene, RCT, inhibited electrical kindling as well as lithium-pilocarpine-induced seizures . Lithium alone does not have general proconvulsant effect in rats22

. However, rats pretreated with lithium have limbic seizures, following subconvulsive doses of pilocarpine23

. The combined treatment with lithium and pilocarpine results in decrease in cortical inositol and accumulation of inositol monophosphate that are

Table 6-Effect of the triterpene (RCT) isolated from pet ether extract of Rubia cordifolia on nociception in mice.

Treatment (Dose:mg/kg) n=5

Vehicle RCT (100) Penta (10)

Latency to lick paws in sec (mean±SE) at 15 30 60 90 min

4 .0±0.31 4.2±0.6 12.3± 3.2 14.4±2.4 10.2±0.4 12.3±2.7

3.9±0.6 4.2±0.7 15.7±4.1 23 .2±2.1 I 0.1 ± 2.3 8.3± 1.4

Penta= pentazocine. All differences were signiticant at P<0.05 (Student's t test) when compared with vehicle treated group.

7 -Vehicle

~6 Q. QJ s 5

- x- RCT ftS u .... 4 0 c:: 0 3 -ftS '-::J 2 , .... 0 c:: 1

0 0 5 15 30 60 90

Time in min

Fig. 3-Effect of RCT on haloperiod-induced catalepsy. *P<0.05 (Student's t test). It is the logari thm to the base n.

680 INDIAN 1 EXP BIOL, JULY 2000

about I 0 times greater than the effects obtained with either drug alone24

. These seizures are prevented by diazepam25

. It is reported that GABA level in the brain decreases in both the electrical kindling and lithium-pi locarpine induced seizures26

. The RCT increased both GABA and 5-HT concentration in the brain . The 5-HT, like GABA is an endogenous an ticonvulsant and elevation of 5-HT is responsible for the anxiogenic effect observed in the elevated plus

27 ?8 ° maze ·- paradtgm. In the elevated plus maze test, reduction in the time spent in the open arm provides a measure of fear induced inhibition of exploratory activity which is increased by anxiogenic agents 29

.

A reciprocal relationship has been observed in the concentration of 5-HT and dopamine (OA) 30

. The potentiation of haloperidol induced catalepsy is indicative of decrease in the DA transmission in the substanti a ni gra. This observation has also been reflected in the amphetamine antagonism as observed from the delay in onset of tre~nors and biting.

The triterpene reduced locomotion and rearing. Although suppression of exploratory rearing has some face value as an animal model of mania, thi s behavior has a large variance and is not always replicable31

• It has been reported that the general depressants of nervous system decrease the locomotion and rearing 32

.

The potentiati on of pentobarbitone sleepin g time and the increase in the reaction time on hot plate are also suggestive of central depressant action of the RCT.

Thus function al alterations of GABA and 5-HT systems may be responsible for anticonvul sant , anxiogenic, and other behavioral actions of the RCT isolated from Rubia cordifolia.

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