Some pharmacological, toxicological and phytochemical investigations on Centaurea phyllocephala

Journal of Ethnopharmacology, 9 (1983) 299-314 Elsevier Scientific Publishers Ireland Ltd.

299

SOME PHARMACOLOGICAL, TOXICOLOGICAL AND PHYTOCHEMICAL INVESTIGATIONS ON CENTA UREA PFIYLLOCEPHALA

HUSNI A.A. TWAIJ, AGNES KERY*

and NIRAN K. AL-KHAZRAJI

Department of Pharmacognosy and Pharmacology, P. 0. Box 4084, Baghdad (Iraq)

Biological Research Centre, Adhamiia,

(Accepted July 14, 1983)

Summary Centaurea phyllocephala Boiss. has been used in folkloric medicine as an antidiabetic agent. Present investigations on various extracts of C. phyllocephala revealed that basal plasma glucose concentration and plasma glucose response to glucose load were either elevated or unchanged by the i.v. administration of these extracts in anaesthetized rats or by i.p. or oral administration of the extracts in conscious rats. The predominant effect of C. phyllocephala is the toxicity in rats and mice and this indicates the presence of some toxic or active compounds which merit phytochemical isolation. Further, C. phyllocephala extracts also caused either an initial brief hypotension followed by a delayed hypertension or produced no changes when injected i.v. in the rats. The hypotensive effect was inhibited by atropine whereas the hypertensive effect was prevented by phentolamine or guanethidine but not by hexamethonium. The alcoholic extract also induced an initial brief negative inotropic effect, followed by delayed prolonged positive inotropic and negative chronotropic effects on the spontaneous inotropic and negative chronotropic effects on the spontaneous contractions of the guinea pig right atrium. The initial depressing effect and the delayed positive inotropic effect were inhibited by pretreatment with atropine. The extract of C. phyllocephala also produced a contractile activity on guinea pig ileum strips and this could be prevented by atropine. No significant diuretic effect was produced by the extract. Phytochemical screening revealed that C. phyllocephala contains tertiary and quaternary alkaloids, sesquiterpenic lactones, methylated flavones and their glycosides, as well as leuco- and proanthocyanidines. Further studies on *Permanent address: Semmelweis Medical University, Institute Budapest, Hungary.

of Pharmacognosy,

0 1983 Elsevier Scientific Publishers Ireland Ltd. 0378-8741/83/$05.70 Published and Printed in Ireland

sesquite~enic lactones and methylated flavones resulted in the isolation of lactones with a-methylene y-la&one and methylene side chain on the cyclopentyl ring as well as of four methylated flavones (hispidulin, nepetin, cirsiliol, jaceosidin) structurally closely related to the cytotoxic flavonoids of other Co,mpositae plants.

Introduction Centaurea (tribe Cynareae, family Compositae) is one of the most widely distributed plant genera in the world. The genus is attributed with various medicinal properties: hypoglycaemic (Aguilar and Rocasalono, 1944; Bigona et al., 1950; Villar and Paya, 1980), astringent (Leclerc, 1939), antimalarial, antibiotic and antifebrile activities (Ibn-El-Bitar, 1890; Dragendorff, 1898; Wehmer, 1962; Watt and Breyer-Brandwijk, 1962). The leaves, especially the tender ones of some species are commonly used as a vegetable and eaten as salad or used in preparing soup. Other species are used as food for animals (Uphof, 1968). At the same time some species are known to be poisonous to animals (Watt and Breyer-Brandwijk, 1962). Since the literature survey revealed that no work has been carried out so far on C. phyllocephalu Boiss., which is one of the most widely distributed Centuurea species in Iraq, it was deemed of interest to investigate this plant pharmacologically to determine whether the plant is toxic to laboratory animals. In addition we wanted to know what kind of subst~~es are present having potential toxicological and/or medicinal significance. Materials and methods Plant material The plant material used in this study was collected at the blooming stage around Baghdad. The samples were compared with authentic plant material and identified by the National Herbarium of Iraq, Botany Directorate of Abu-Graib. Voucher specimens are kept in the Herbarium of Biological Research Centre, Department of Pharmacognosy and Pharmacology, Baghdad. Preparation of extracts Toxicity test The dried plant material (500 g) was ground into a coarse powder and extracted at room temperature with 4 X 2000 ml of 80% ethanol. The solvent was removed in vacua at 40°C to produce a crude extract (73.0 g = 14.6%). Pharmacological studies Total alcoholic extract as for toxicity test: 1.4 g extract = 10 g of crude

drug. Doses used: 0.21 g/kg i.v., 0.42 g/kg orally and 0.5-4.2 mg/al of bath. Chloroform soluble part of the total extract: 1.4 g total extract was dissolved in chloroform by continuous solid-liquid extraction. The chloroform soluble part was filtered and evaporated and then dissolved in 10 ml of saline adding a few drops of ethanol (0.03 g of chloroform soluble part = 10 g of crude drug = 1.4 g of total extract). Dose used: 4.5 mg/kg i.v. Chloroform insoluble part of the total extract: 1.35 g (10 g crude drug = 1.4 g of total extract) of chloroform insoluble fraction from above was dissolved in 10 ml of saline. Dose used: 0.2 g/kg i.v. Alkaloid extract: 10 g crushed crude drug was extracted with 1 N hydrochloric acid (3 times). The combined acidic fraction was basified to pH 9 with NH,OH and extracted with CHCl, (3 times). The combined and dried chloroformic extracts were evaporated and then dissolved in 10 ml of saline f 10 drops of ethanol + 2 drops of 2 N HCl (pH 3.5). Blank solution: the vehicle used for the alkaloid extract was administered to control rats. Doses specified in millilitres which is equivalent to 10 g of crude extract. Aqueous extract: 100 g of the plant material was extracted with 50 ml of distilled water overnight. The suspension was filtered and the filtrate centrifuged. Dose: 1.5 ml of the filtrate/kg. Phytochemical tests Dried material of C. phyllocephala and crude ethanolic extract prepared for toxicity test were used, respectively. The extraction process for alkaloids, sesquiterpenic la&ones and flavonoids were as described (Kery et al., 1982a). Isolation, separation and structure elucidation of the flavonoid compounds were as described (Kery et al., 1982b). Parameters used for pharmacodynamic

studies

Effect on plasma glucose concentration Anaesthetised rat: anesthetised (pentobarbitone sodium 50 mg/kg i.p.) male, fed, white Wistar rats (300-350 g) were used. Extracts and their solvents were injected i.v. into the cannulated left femoral vein as a bolus in a volume of 0.2 ml unless otherwise stated, and washed in with 0.2 ml 0.9% sodium chloride solution. Blood samples (0.2 ml) were removed from the cannulated right femoral artery after discarding 0.2 ml to allow for the cannula dead-space. Plasma glucose was measured enzymatically (Beckmann Glucose Analyzer). Conscious rats: Rats were prepared 48 h before the experiment under ether anaesthesia, by inserting the cannula into the abdominal aorta (via the left femoral artery) for blood sampling. The cannula were exteriorized on the dorsal part of the neck and attached to 26 gauge needles, plugged with plastic closures. Post-operatively the animals were isolated in separate cages, allowed to recover, and then fed. Drugs were administered i.p. or orally. Plasma glucose was estimated as above. The purpose behind using conscious

302

rats was to give the extracts orally and to get precise and successive blood sampling. Cardiovascular activities Anaesthetised rats: anaesthetised rats (300-350 g) of either sex were used. Systemic blood pressure was recorded from the right femoral artery through a Bell & Howe1 pressure transducer coupled to MX412 Lectromed recorder. Heart rate was obtained by counting the number of systolic peaks occurring in 3 s when the chart speed was 25 mm/s. Drugs were administered through an in-dwe~ing polyethylene cannula in the left femoral vein. Isolated right atrium: guinea pigs (400-500 g) of either sex were stunned and the hearts were quickly removed. Right atria were dissected out and mounted in an organ bath containing 10 ml of Krebs solution at 37°C bubbled with carbogen (95% 0, + 5% C02). Spontaneous contractions of the right atria were isometrically recorded by using a Force transducer (UFI) coupled to Lectromed recorder. The results were confirmed at least 3 times in different experiments and different preparations. effect on s.mooth muscle Terminal strips of isolated guinea pig ileum (2-3 cm) from a stunned guinea pig were suspended in an isolated organ bath containing Krebs solution at 37°C and bubbled with carbogen (90% 0, + 5% CO,). Isometric contractions of the ileum were recorded by Force transducer (UFI) coupled to a Lectromed recorder. Diuretic effect Fifteen rats were placed in filter funnels. They were separated into three groups, each group received i.p. injection of either furosemide 35 mg/kg or saline or the total alcoholic extract of C. ~~yllocephala. Urine was collected in tubes over a period of 5 h following drug adm~istration. Behavioural effect and toxicity Gross behavioural effects (mice): these studies were carried out along with the toxicity studies. The animals were observed continuously for 1 h, intermittently for 4 h and then after 24 and 48 h (Turner, 1965). Determination of LDSO (Reed and Muench, 1938): graded doses of the total alcoholic extract of C. ~hyllocephula in 0.2 ml of saline were administered S.C.to two groups of six albino mice each (25-30 g). They were kept in transparent plastic cages at 24°C. Mort~ity was noted after 24 h. determination of chronic toxicity: (1) Mice. Groups of 6 fed male mice with an initial weight of 25-30 g were used for each dose level and the doses used were: Group 1: 3.5 mg/day (120 mg/kg body wt) in 0.125 ml of saline. Group 2: 7.0 mg/day (240 mg/kg body wt) in 0.25 ml of saline.

303

Group 3: 14.0 mg/day (480 mg/kg body wt) in 0.30 ml of saline. Group 4: 21.0 mg/day (720 mg/kg body wt) in 0.30 ml of saline. Group 5: (control group) 0.25 ml of saline/day (2) Rats. Groups of 6 fed male rats with an initial weight of 300-320 g were used for both subcutaneous and intraperitoneal injection of the crude extract dissolved in saline. Group 1: 140 mg/day S.C. (420 mg/kg body weight) in 1.00 ml of saline. Group 2: 140 mg/day i.p. (420 mg/kg body weight) in 1.00 ml of saline. Group 3: (control group) 1.00 ml of saline/day i.p. The animals were injected between 09:OO h and 10:00 h every day during 30 days and were observed continuously for 2 h and intermittently for each 24 h. Post mortem examinations were performed. Animals were killed after recording their behaviour. Small pieces of the liver, kidney, spleen and intestine were cut and fixed by immersion in 10% buffered formalin for 36 h. Embedding was performed in fibre wax at 56°C. Sections, 6-7 pm thick, were cut and stained with Mayer’s haematoxylin and counter-stained with aqueous eosin. Sections were examined microscopically. Drugs used Drugs used were obtained as follows: guanethidine sulphate (Ismilin, Ciba); phentolamine (Regitine, Ciba); hexamethonium chloride (Fluka); acetylcholine bromide (Fluka); L-isoprenaline HCI (Breon); atropine sulphate monohydrate (Fluka); diphenhydramine HCl (SDE); frusemide (E. Merck). Results Results are the mean of five or more experiments significance was assessed using Student’s t-test.

f S.E.M. Statistical

Effect on plasma glucose concentration The results (Table 1) show that the i.v. administration of either the total alcoholic extract (0.21 g/kg) or the chloroform insoluble part of the extract (0.2 g/kg) or the aqueous extract (1.5 ml/kg) caused elevation of plasma glucose concentrations at 5 and 15 min after their administration in anaesthetised, fed rats. However, the administration of the chloroform soluble part of the extract (45 mg/kg) or the alkaloid part of the extract did not induce a hyperglycaemic effect when compared with the effect of the extract solvent or saline. Additionally, the i.p. injection of the chloro-

iv. iv. i.v. i.v.

Saline Cl (0.21 g/kg) C2 (45 mg/kg) C3 (0.2 g/kg) C4 (1.5 ml/kg) Solvent of C4 C5 (1.5 ml/kg) C2 (45 w/kg)

Anaesthetised Anaesthetised Anaesthetised Anaesthetised Anaesthetised Anaesthetised Anaesthetised Conscious (5)

Animal (no. )

107 +_6 99 rt:8 96 + 4 103 dz8 92 zt5 10058 95+6 108 + 8 106 f4 130 + 5e 99+3 140 f 9c 95 -+7 101 f6 115 _+4a 120 +6

After 5 min

soluble extract; C3 = chloroform

(5) (6) (5) (5) (5) (5) (5)

Pretreatment value

109 +6 107 +4 z39+5 106 +3 99 2 9 107 +5 99 f 5 133 + 9

110 4 5 106+ 5 912 6 130 t 20 94f 7 105 +- 6 100 f 6 130 +- 9

After 60 min

IN ANAESTHE-

insoluble extract; C4 = alkaloid extract; C5 =

108k 5 136 zk 7c 952 4 135 * 1oe 1115 5 lOSz!z 8 125f 5b 126f 7

After 15 min

After 30 min

(C) ON PLASMA GLUCOSE CONCENTRATION

Plasma glucose mg/lOO ml (mean ?: S.E.)

OF C. PElYLLOCEPffALA

Cl = total alcoholic extract; C2 = chloroform aqueous extract. aP < 0.05. bP < 0.01. CP < 0.001.

i.v. i.v. i.p.

LV.

Route

Treatment

EFFECT OF VARIOUS EXTRACTS TISED AND CONSCIOUS RATS

TABLE 1

305

form soluble part of the extract (45 mg/kg) in conscious, fed rats caused a slight, but insignific~t increase in plasma glucose concentration. The i.v. administration of the total alcoholic extract of C, phylZ~ep~alu (0.21 g/kg) 1 min prior toglucose injection (0.5 g/kg) in anaesthetised, fed rats caused an increase in glucose elevation when compared to the effect of saline on glucose injection in control rats (Table 2). However, the oral administration of total alcoholic extract (0.42 g/kg) either alone or 5 min prior to glucose (2 g/kg orally) to conscious, fed rats produced only a small elevation in plasma glucose concentration at 30 min after its administration, when compared with the effect of oral saline alone or saline 5 min prior to glucose (2 g/kg) (Table 2).

C. phyllocephala produced a well-marked biphasic response, a sharp, brief (20 s) vasodepressor phase followed by a vasopressor phase of 5-10 min duration, in a dose range of 30-200 mg/kg i.v. (Table 3, Figs. 1 and 2). The hypotension was accompanied by bradycardia, while the hypertension was accompanied by tachycardia. The above effects were only produced by the total alcoholic, the chloroform insoluble and the aqueous extracts. The hypotensive response was completely abolished by pretreatment of the animal with atropine sulphate (2 mg/kg i.v.) given 10 min before the extract (Table 4, Fig, 3), while the hypertensive response was completely inhibited by pretreatment of the animal with phentolamine meth~os~pha~ (5 mg/ kg), or ~~ethidine (4 mg/kg) 5 min prior to the extract (Fig. 4). Pretreating the animal with hexamethonium (5 mg/kg i.v.) did not modify either response. There was no evidence of tachyphylaxis on repeating the dose. The C. phyllocephala total alcoholic extract (4.2 mg/ml of the bath) had an initial brief negative inotropic and a negative chronotropic effect on the spontaneous contraction of the right atrium. A dose of l-4.2 mg/ml produced a dose-dependent increase in the amplitude of contraction (29-105%) for 10 min. The increase in the amplitude of the contraction by the larger dose (4.2 mg) was accompanied by a negative chronotropic effect. The initial response as well as the delayed positive inotropic effect disappeared by previous treatment with atropine sulphate (0.6 pg/ml) whereas the delayed negative chronotropic effect was not modified. In the same set-up, isoprenaline produced a positive inotropic and chronotropic effect. Application of 4.2 mg of the total alcoholic extract 3 min after isoprenaline reversed the positive chronotropic response and gave an initial partial inhibition followed by a delayed potentiation on the positive inotropic effect of isoprenahne (Table 5 and Fig. 5). Effect on the isolated guinea pig ileum The application

of the total alcoholic

extract

of C. phyllocephala to ileum

TABLE

2

OF

3

OF

bf’ I’ 0 01. ‘P <10 001

alcoholic

i4.5r&kg) (0.21g/kg) (1.5ml/kg) (1.5ml/kg)

Cl = total aP < 0 02.

c2 C3 C4 C5

Cl 10.21 e/kg)

Solvent (control)

Treatment

+ S.E.)

extract;

5 14 7 8 6 6

C2

THE

chloroform

WHEN

C3

350?20 360+15 355+10 356210 365f20 370+21

extract:

75f8 78+33 76+7 73+6 75+44 86 f5

soluble

103+5 102+_3 10625 10224 105f4 118?6

insoluble

103*3 71+4c 101+5 66+-4c 101_+4 102+_5b

SBP

15 min

262 + 7b

222f6

after

extract:

phase

C4

=

g/kg

7 4

6

g/kg

C5

4

6

4

120+6 109 + 5

104f8

116+8

PRESSURE

5b 6

=

aqueous

350 f18 312f 6c 352+17 311+ 9 360+17 320 flgC

First phase

12a+5

phase

RATS

@BP).

121+4 110 +6

109f4

Nochange 420 f19= Nochange 410 *15= Nochange 419 f2W

Second

extract.

HR (beats/min)

ANAESTHETISED

BLOOD

117+

119%

1292

170f 132f

122+10

after

120 min

RESPONSES

90 min after

orally

GLUCOSE RATS

60 min after

THE

extract;

98+5b

87 f6a

9128"

DBP

IN

SYSTOLIC

120+ 121_+

130?

152 f 20 2102 6” 118? 5

i.v..

0.42

PLASMA

30 min after

alkaloid

Nochange 115f6a Nochange 116 +5b Nochange 132+_4

SBP

Second

INTRAVENOUSLY

(C) ON THE

f 4a

74+6 46 +3c 7126 39 +_3c 13 _+5 72+4b

DBP

INJECTED

First phase

DBP

330

315 * 9

5 min after

ON CONSCIOUS

f SE.)

0.21

OR

AND

ml (mean

of extract,

mg/lOO

Dose

PHYLLOCEPHALA

116 f4 Ill_+5

119_+5

91+7 96 c 5 115+5

treatment values

Pre-

SBP

=

(HR)

later.

ANAESTHETISED

CONCENTRATION

Plasma glucose

15 min

EITHER

BP (mmHg)

RATE

CENTAUREA

HR

HEART

(6)

(6) (5)

given

IN

GLUCOSE

Pretreatment

AND

OF

Conscious

Conscious Conscious

EXTRACTS

(DBP)

= chloroform

PRESSURE

OF

Orally

orally

TYPES

No. of rats

BLOOD

VARIOUS

DIASTOLIC

(MEAN

THE

ti:FFECT

TABLE

-Pi 0.05. “P < o.001.

Saline + glucose Extract + glucose

Extract

(5)

2 g/kg

Anaesthetised Conscious (5)

Orally OraIIy

(no.)

glucose.

(5)

Oral

Anaesthetised

later.

i.v. i.v.

5 min

Saline + glucose Extract + glucose Saline

given __

ORALLY

PLASMA

Animal

i.v.

ON GIVEN

Treatment

g/kg

EXTRACT

Route

0.5

OR

GLUCOSE

TOTAL

INJECTION

c. PHYLLoCWHALA

GLUCOSE

injection,

EITHER

Glucose

TO

EFFECT

-

307

Fig. 1. Effect of C. phyllocephala extract (C) on mean arterial blood pressure of anaesthetised rat. Fig. 2. Effect of C. phyllocephala extract (C) on mean arterial blood pressure of anaesthetised rat before and 5 min after atropine (AT) 2 mg/kg i.v.

strip preparations, caused a dose-dependent muscle contraction followed by a relaxation to base line tension. These contractile responses were inhibited by previous treatment with atropine sulphate (0.6 pg/ml of the bath) or the antihistamine diphenhydramine HCl (0.2 mg/ml of the bath). The alkaloid extract produced a relaxation rather than a contraction (Table 6 and Fig. 6). Diuretic effect As shown in Table 7 the administration of the total alcoholic extract of C. phyllocephala did not produce any significant changes in urine volume collected from the rats over a period of 5 h following its i.p. administration in comparison with the effect of frusemide and saline (Table 7). TABLE 4 EFFECT OF ATROPINE ON THE BLOOD PRESSURE RESPONSES TO C. PHYLLOCEPHALA (C), WHEN INJECTED IN ANAESTHETISED RATS Atropine administered 2 mg/kg i.v. 10 min before the extract. Treatment

No. of rats

Before atropine and extract

After atropine and extract 1st phase

SBP

C extract (9.21 g/kg) aP c: 0.05. bP < 0.01.

5

98+4

2nd phase

DBP

80+3

SBP

DBP

SBP

DBP

98 +4

80 +3

112 + 4b

89 f 5a

308

Fig. 3. Effect of C. phyilocephala extract (C) on mean arterial blood pressure of anaesthetised rat 5 min after phentolamine (P) 5 mg/kg i.v. Fig. 4. Effect of C. phyllocephala extract (C) on mean arterial blood pressure of anaesthetised rat before and after guanethidine (Q) 3 mg/kg i.v.

Behavioural

effects

and toxicity

C. phyllocephala had no significant effect on the spontaneous motor activity and rectal temperature. Higher doses reduced motor activity and reactivity. The method used for the determination of LDSOemploys cumulative values. The LD,, computed is 1510 mg/kg S.C.in mice (Table 8). After the S.C.and i.p. administration of different doses of C. phyllocephala extracts, some fatalities occurred within 3 days. Fifty percent of the first TABLE 5 RATIO OF TENSION IN THE RIGHT ATRIUM INDUCED BY VARIOUS CONCENTRATIONS OF C. PHYLLOCEPHALA TOTAL EXTRACTS (10 min periods) BEFORE AND AFTER ATROPINE (6 rg/ml of bath) AND DURING ISOPRENALINE (8 x 10‘‘M) Concentrations of extracts in 1 ml of bath (mg)

Saline 4.2 2.1 1 0.5 4.2 4.2

Extracts alone or atropine or isoprenalinea

Alone Alone Alone Alone Alone After atropine After isoprenalineb

First phase (2 min duration)

Second phase (10 min duration)

Rate change

Tension change

Rate change

Tension change

(beatslmin)

(g%)

(beatslmin)

(g%)

-

-

-

-

-33+8 -

-45 * -5f -

7 5

+105f +75+ +29* -

-40

+

5

-

-15f5

-90

+ 18

-10 -

+- 5

-80

? 28

*Five determinations were carried out for each treatment (mean 3~S.E.). bResults after isoprenaline were compared with the effect of isoprenaline

+30 f10

itself.

6 2 9

309

_/k_

---

c

i:

AT

42 mg

42mp

06Yg

Fig. 5. Effect of C. phyllocephala pig right atrium.

A

li

l0.g

1ous

extract (C) on spontaneous contraction

of the guinea

Fig. 6. Contractions of guinea pig ileum due to C. phyllocephala extract (C) (1, 2.1, 4.2 mg/ml), acetylcholine chloride (A) (10 kg/ml), histamine (H) (10 @g/ml), before and after atropine sulphate (AT) (0.6 pg/ml of the bath) applied for 10 min.

and second group of rats receiving 140 mg crude extract (= 1 g dried plant material) i.p. and s.c., respectively, died. The percent mortality increased with increasing dose as shown in Table 9. C. phyllocephala induced the following symptoms of poisoning on the rats in both groups, and on the mice in the third and fourth groups: buffness and abdominal oedema, bloat, apathy, skin fibrosis and in some cases tail necrosis (in S.C. injected mice), weakness, laboured respiration, blindness and death. Post mortem examination disclosed general cyanosis, marked hyperaemia, abdominal bloody fluid, hydrothorax, hydropericardium, hydroperitoneum, nodular and adhesive enlarged viscera, acute catarrhal gastroenteritis with numerous haemorrhages in the mucosa of the intestine. Liver enlargment and adhesion to the viscera and diaphragm occurred, Microscopical examination of the tissues revealed marked inflammatory cells, infiltration and organized exudate into collagen in the serosa; and in the liver dilatation of the sinusoids and early degeneration. In the kidney it showed a thickened capsule due to incidence of peritonitis (Figs. 7 and 8). TABLE 6 MUSCULAR CONTRACTIONS IN THE GUINEA PIG ILEUM, INDUCED BY TOTAL AND ALKALOID EXTRACTS OF C. PHYLLOCEPHALA The contractile effects of the total extract were inhibited by prior treatment with atropine (6 pg) and diphenhydramine (2 mg) added to the 10 ml of the organ bath solution. Treatment

Concentrations (in 1 ml of bath)

Total extract Total extract Total extract Alkaloid extract

4.2 2.1 1.0 0.5

mg mg mg ml

No.

Tension (g) (mean f SE.)

5 5 5 5

0.8 f 0.12 0.5 f 0.12 0.27 + 0.08 -0.5 + 0.1 (dilatation)

310 TABLE 7 EFFECTS OF C. PHYLLOCEPHALA ALCOHOLIC VOLUME OF NORMAL FED MALE RATS

EXTRACT

ON THE URINE

Agents and dose

Rats no.

Route

Urine volume (ml)

Solvent Frusemide (35 mg/kg) C. phyllocepholo extract (0.42 g/kg)

6 6 6

i.p. i.p. i.p.

5+1 16 + 2 521

TABLE 8 ACUTE TOXICITY OF TOTAL EXTRACT MUENCH (1938) METHOD

(mg/kg s.c.) IN MICE BY THE REED-

LD,, = 1510 mg/kg S.C. Group

1 2 3 4 5

---

Dose of C. phyllocephala extract (mg/kg s.c.)

Dead

3000 2000 1500 1000 500

6 4 3 0 0

Survival

Cumulative

0 2 3 6 6

% Survival

Dead

Survival

Total

6 10 13 13 13

11 11 9 6 -

17 21 22 19 -

54.7 48.1

TABLE 9 THE LETHAL EFFECT OF THE SUBCUTANEOUS AND/OR INTRAPERITONEAL INJECTION OF C. PHYLLOCEPHALA EXTRACT ON GROUPS OF FED MALE MICE AND RATS Animal group

No. of animals

Dose of extract mgiday

Mice Mice Mice Mice Mice Rats Rats Rats

1 2 3 4 5a 1 (s.c.) 2 (i.p.) 3b

6 6 6 6 6 6 6 6

3.5 7.0 14.0 21.0 0 140.0 140.0 0

mg/kg 120 240 480 720 0 420 420 0

Equivalent plant material

Mortality

mg/day

mg/kg

3 days

14 days

30 days

25 50 100 150 0 1000 1000 0

857 1715

1

-

-

3430 5145 0

1 2 -

2 2 -

_

3000 3000 0

3 3 -

1 3 -

-

%ontrol group, receiving 0.25 ml of saline/day. bControl group, receiving 1.0 ml of saline/day.

311

Fig. 7. Dilatation of the sinusoids and early hepatocytes

Phytochemical

degeneration (H&E,

x

25).

tests

The preliminary alkaloid tests were positive for both tertiary and quaternary alkaloids, according to Mayer’s and Dragendorff’s reagents. According to the intensity of the spots and reactions, the quaternary alkaloids are predominant. Screening for sesquiterpenic lactones, the IR spectra of selectively purified chloroform extracts showed distinct bands at 1763 cm-’ (carbonyl group of a y-lactone ring). Further studies revealed the presence of sesquiterpenic lactones with cr-methylene-y-lactone and methylene side chain on the cyclopentyl ring. Studies on the phenolic compounds showed the presence of

Fig. 8. Infiltration of the mononuclear cells and the organized exudate into collagen in the serosa (H&E, X 40).

312

HO

--OR

% 0 ,OH 2 OH ,OhkOH 3 OH.OH .OH 4 OH, H . OH R, R R,

1 OWOH

5 H 6Acyl R

Fig. 9. Chemical structures of the compounds Cirsiliol; 2, jaceosidin; 3, nepetin; 4, hispidulin; picrin.

identified in C. phyllocephala Boiss. 1, 5, desacyl-cynaropicrin; 6, acyl-cynaro-

methylated flavones and their glycosides as well as leuco- and procyanidines. Detailed isolation and structure elucidation work resulted in the identification of four methylated flavones, including the monomethoxylated 5,7,4’trihydroxy-6-methoxyflavone (hispidulin), 5,7,3’,4’-tetrahydroxy-6-methoxyflavone (nepetin) and the dimethoxylated 5,7,4’-trihydroxy-6,3’-dimethoxyflavone (jaceosidin); these were identified both in free and glycosidic form, while the 5,3’,4’-trihydroxy-6,7-dimethoxyflavone (cirsiliol) was detected only as the aglycone (Fig. 9) (Kery et al., 1982b). Discussion The main pharmacological effect of C. phyllocephala extracts was found to be its ability to increase the basal plasma glucose level in the anaesthetised rats, rather than to lower it, as was suspected from the folkloric use. When hyperglycaemia was prepared in the anaesthetised rats by the i.v. injection of glucose the total alcoholic extract was found to delay the disposal of this injectable glucose. Additionally, conscious rats were used in the present work to allow oral administration of the extract in the rat and to rule out the possibility of anaesthesia as the cause for hyperglycaemia, because anaesthesia was reported to increase the plasma glucose level by Green et al. (1973). It was very clear in the conscious rats that the total alcoholic extract of C. phyllocephalu did not modify the plasma glucose level of the orally given glucose. Thus the present results in both the anaesthetised and the conscious rats contradict the reported hypoglycaemic effects of other species of Centaurea (Aguilar and Rocasolano, 1944; Villar and Paya, 1980) and the folkloric use of C. phyllocephala. The predominant effect of C. phyllocephala was its toxicity in the laboratory animals (rats and mice). This is an interesting finding that indicates the presence of some active or toxic compounds. The toxicity of C. phyllocephala is in agreement with the results reported about the toxicity of other species of Centaurea (Watt and Breyer-Brandwijk, 1962).

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The other pharmacological effect of C. phyZlocephaZa was its effect on blood pressure and heart rate. The initial hypotensive response was abolished by atropine pretreatment, thereby suggesting the site of action to be the muscarinic receptor. This possibility was further supported by the contractile effect of C. phyllocephala total extract on guinea pig ileum and its inhibition by atropine pretreatment. However the delayed hypertensive phase was abolished or even reversed by prior treatment with phentolamine or guanethidine but not with hexamethonium. This would indicate that the Centaurea extract contains some catecholamine-releasing agent. Moreover, stimulation of the ganglia as the mechanism for this hypertension is ruled out, because hexamethonium failed to antagonise the vasopressor effect of Cen taurea.

Pretreatment of the right atrium with atropine prevented the initial depressing and the delayed positive inotropic effect of Cphyllocephala total alcoholic extract. This may indicate that its effect on the right atrium is possibly due to some histaminergic agent. However, the precise mechanism of the cardiovascular activities cannot be concluded from the data available. The family Compositae has been submitted to intensive investigations regarding its chemistry and taxonomy (Herz, 1971; Heywood et al., 1977), as well as biological activity (Mitscher, 1975). One of the most exciting findings has been the marked cytotoxicity and the cytostatic activity exhibited by a number of the characteristic compounds. As is widely accepted, therapeutic and toxic effects are equally interesting at the screening stage of investigations. From this point of view the toxicity found and the presence of sesquiterpenic lactones, 0-methylated flavonoids, tertiary and quaternary alkaloids as biologically active constituents are of interest. It is worth mentioning that the structure of sesquiterpenic lactones in Centaurea phyllocephala Boiss. can be characterised with the a-methyleney-lactone arrangement which is confirmed as a prerequisite for cytostatic activity (Gonzalez et al., 1980). Recently, the cytostatic activity of sesquiterpene lactones from C. hyssopipholia Vahl. and C. linifolia Vahl. has been reported (Gonzalez et al., 1980). From C. hyrcania Borum., C. melitensis L. and C. americana L. sesquiterpene lactones with cr-methylener-lactone structure are known (Eustratova, 1972; Gonzalez, 1975). The structures of the four flavonoids isolated from C. phyllocephala Boiss., especially jaceosidin and cirsiliol, are closely related to cytotoxic flavonoids (eupatorin, etc.) which were claimed to exhibit cytotoxicity against human carcinoma of the nasopharynx (Kupchan et al., 1969) and to the recently reported computer model of cytotoxic and anticancer flavonoid structure (Farkas, 1980). Acknowledgements This work has been supported by the Scientific Research Council of the Republic of Iraq, which is gratefully acknowledged. The authors thank

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the National Herbarium of Iraq for the identification of plant material and Dr. W.A. Al-Katib (College of Veterinary Sciences, University of Baghdad) for his work on the histopathological studies. References Aguilar, A. and Rocasolano, I. (1944) Monitor de la Farmacia y de la Terapeutica, 50, 1. Ahmed, Z.F., Hammouda, F.M., Rizk, A.M. and Ismail, S.I. (1969) Ahmed, Z.F., Hammouda, F.M., Rizk, A.M. and Ismail, S.I. (1969) Chemical studies on certain Egyptian desert plants. Thesis, Pharmaceutical Science Laboratory, NRC, Cairo, Dokki. Bigorra, I., Puig, A. Yufera, Y. (1950) Farmacognosia, 10,197. Dragendorff, G. (1898) Die Heilpflanzen der Verschiedenen Volker und Zeiten, Ferdinand Enke, Stuttgart. Eddy, N.B., Touchberry, C.F. and Lieberman, J.E. (1950) Journal of Pharmacology and Experimental Therapeutics, 98,121. Eustratova, R.J. (1972) The structure of sesquiterpene lactone repin in Centaurea hyrcania Borum. Chemistry of Natural Products, 8, 450-451. Farkas, G. (1980) Active principles of plants of traditional medicine as models of new drugs. Journal of Ethnopharmacology, 2,145-180. Garcia, A. (1950) Rev. Real Acad. Sci. Exact, Fis. Y. Nat. (Madrid) 44, 103. Gonzalez, A.G. Darias, V., Alonso, G. and Estevez, E. (1980) The cytostatic activity of the chlorohyssopifolins, chlorinated sesquiterpene lactones from Centaurea. Planta Medica, 40, 179-184. Gonzalez, A.G. (1975) Elemanolides from Centaurea melitensis L. Phytochemistry, 14, 2039-2041. Green, A.A., Biebuyck, J.F. and Alberti, KG. (1973) Diabetologia, 9, 274. Herz, W. (1971) Pharmacognosy and Phytochemistry, Springer Verlag, Berlin, p. 64. Heywood, V.H., Harborne, J.B. and Turner, B.L. (Eds.) (1977) The Biology and Chemistry of the Compositae, Academic Press, London, New York. Ibn-El Bitar (1890) Mofradat Al Adwiah Wa Al Agzia Azharia, Cairo, p. 148. Kery, A. Twaij, H.A.A. and Al-Khazraji, N.K. (1982a) Preliminary phytochemical screening of Centauren phyllocephala. Bulletin of Biological Research Centre, Baghdad, in press. Kery, A., Twaij, H.A.A. and Al-Khazraji, N.K. (198213) Methylated flavonoid constituents of Centaurea phyllocephala. Iraqi Journal of Pharmaceutical Sciences, in press. Kupchan, S.M., Udsyamustry, M.A., Sigel, C.W. Hemingway, R.J. and Knox, I.R. (1969) Tumor inhibitors XxX111. Tetrahedron, 25, 1603-1615. Leclerc, E. (1936) Presse Medicale, 44, 1216. Lobo, I. and Puig, A. (1953) Farmacognosia (Madrid), 13, 223. Lobo, I. and Puig, A. (1956) Anales de la Real Sociedad Espanola de Fisica y Quimica, 52B,583. Millar, R.A., Keener, E.B. and Benfey, B.G. (1959) British Journal of Pharmacology and Chemotherapy, 24,9. Mitscher, A. (1975) Recent Advances in Phytochemistry, Vol. 9., Plenum Press, New York, p. 243. Stern, D.N., Maling, H.M., Altland, P.D. and Brodie, B.B. (1964) Pharmacologist, 6,185. Uphof, J.C.Th. (1968) Dictionary of Economic Plants, 3301 Lehre Verlag I, Cramer, p. 118. Villar, A. and Paya, M. (1980) Study of the antihyperglycaemic activity of Centaurea seridis var. maritima. Planta Medica, 39, 248. Watt, J.M. and Breyer-Brandwijk, M.G. (1962) The Medicinal and Poisonous Plants of Southern and Eastern Africa. Livingstone, p. 210. Wehmer, C. (1962) Die Pflanzenstoffe, 2nd edn, Jena, Fischer, p. 210.