Int. J. Pharmacognosy, 30 (1992). No. 2, pp. 97-104 0925-1618/92/3002-07$3.00 0 Swets & Zeitlinger

Antiulcerogenic Effects of Ocirnurn basilicum Extracts, Vola- tile Oils and Flavonoid Glycosides in Albino Rats

Muhammad Shoaib AKHTAR', Abdul Hameed AKHTAR'** and Muhammad Afzal KHAN2

1. Department of Physiology and Pharmacology, University of Agriculture, Faisahbad, Pakistan 2. Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan

ABSTRACT

Antiulcerogenic effects of extracts, volatile oils and flavonoid glycosides of Ocimum basilicum leaves were studied in normal as well as aspirin-, acetic acid- and stress-induced ulcerated rats. Also, their effects on outputs of gastric acid, pepsin and hexosamines were recorded in normal and ulcerated rats. The aqueous, methanol and water-methanol extracts and flavonoid glycosides of 0. basilicum decreased the ulcer index, inhibited gastric acid and pepsin secretions and enhanced hexosamines in aspirin-treated rats. The aqueous extract increased glucosamine but did not affect the output of acid and pepsin. The methanol extract lowered acid and pepsin output but did not increase the glucosamine level. However, the water-methanol extract decreased acid output, increased glucosamine, and did not affect pepsin. The flavanoid glycosides of 0. basilicum decreased the ulcer index and the outputs of acid and pepsin, but increased glucosamines. Volatile oils of the plant were ineffective against stress-induced ulcers. However, high antiulcer activity was demonstrated by the aqueous extract in acetic acid-induced ulcerated rats. It therefore appears that anti ulcerogenic activity of 0. basilicwn is extractable into both water and methanol and its active principle(s) may include flavonoid glycosides. These substances may act by augmenting the gastric barrier.

INTRODUCTION

Although available antiulcer drugs including cimetidine and ranitidine have de- creased morbidity and mortality, adverse reactions like arrhythmias, impotence, gynaecomastia, and haematopoeitic changes have been observed, and recurrence rates are high (Ariyoshi et al . , 1986). Thus, there is still a need for safe and curative agents for gastro-intestinal ulcers. Recently, the dried aerial parts of Ocimum basilicum L . (Laminaceae) have been reported to decrease the ulcer index significantly in rats (Akhtar and Munir, 1989). It has been suggested that antiulcerogenic principles increase the gastric hexasamine level and that the plant drug acts by the enhancement of gastric mucosal strength. However, in- vestigations are required to isolate the active fractions and/or principle of the plant drug and to determine the mechanism of antiulcerogenic activity. Thus, the present studies were conducted to further evaluate the antiulcer activities of

Correspondence: Dr. Muhammad Shoaib Akhtar, Department of Physiology and Pharmacology, University of Agriculture, Faisalabad-38040, Pakistan.

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

01/0

8/14

For

pers

onal

use

onl

y.

98 M.S. AKHTAR ET AL

various extracts of 0. basilicum and its volatile oil and flavonoid glycosides, against aspirin-, acetic acid- and stress-induced gastric ulcers. In addition, the effect of these preparations on the outputs of gastric acid, pepsin and hexosamine were determined in normal (unulcerated) and ulcerated rats.

MATERIALS AND METHODS

Animals Male and female Sprague-Dawley rats (224k4g) were used in these experiments. Rats were kept in wire cages and given standard food (prepared by the Department of Animal Nutrition, University of Agriculture, Faisalabad) and water ad libitum.

Plant materials Leaves and flowering tops of Ocimum basilicum L. (Laminaceae) were obtained from the botanical gardens of the Agriculture University, Faisalabad. The plant was identified and authenticated by the Herbarium maintained by the Department of Botany, University of Agriculture, Faisalabad, where a voucher specimen is available, The plants were dried under shade and powdered with an electric grinder. To prepare methanol and water/methanol (1:l) extracts, powdered drug was extracted in a Soxhlet to exhaustion. The solvent was evaporated at 40'C under reduced pressure in rotavaporator. This material was then reconstituted in such a way to yield 1.0 g in 1 ml. An aqueous extract was prepared by maceration for two days with occasional shaking. This extract was then dried at low temperature and reconstituted as described above.

Extraction of volatile oils Following Brain and Turner (1975), 10 g of powdered Ocimum basilicum was placed in a closed vessel with 25 ml of petroleum ether, and maintained at room temperature for 1 hour with continuous shaking. After filtration, extraction was repeated with another 10 ml of petroleum ether. The filtrates were combined and the solvent was removed under reduced pressure to recover the volatile oil.

Extraction of flavonoid glycosides Crude drug was macerated with 70% ethyl alcohol for 2 hours, then filtered and concentrated. The concentrated extract was washed several times with petroleum ether, and the alcohol layer was then hydrolyzed with 2 M HCl (reflux, 3-4 hours). A precipitate was removed and the filtrate contained flavonoid glycosides (Harborne, 1984).

Preparation of test samples for administration Animals were treated with the aqueous, methanol and aqueous/methanol extracts as well as the volatile oil and flavonoid glycosides of 0. basilicum at a dose equivalent to 4 g/kg of the crude powder [i.e., maximum effective dose tested by Akhtar and Munir (1989)l. The amount of the test sample required for each rat was well triturated with 1% aqueous gum tragacanth solution, and the final volume was adjusted to 10 mlkg. Control rats received an equivalent amount of 1% aqueous gum tragacanth. The drug suspensions were administered to each animal orally using a feeding needle connected to a 20 ml syringe. Reference drugs were administered in the same manner.

Studies on normal rats Normal healthy albino rats of either sex were treated orally with various extracts equivalent (9) to 4.0 g of the 0. basilicum powder. After three consecutive days of treatment, all the groups underwent operative procedures on the fourth day. The rats were fasted for 24 hours with tap water ad libitum. The operative procedure adopted was that of Shay et 01. (1945) as modified by Takeuchi et al. (1976.

After pylorus ligation, drinking water was withheld and gastric juices were allowed to collect for a period of four hours. The rats were then killed by an overdose of chloroform and gastric contents were collected through the esophagus, centrifuged, and analyzed for acid output, peptic activity and glucosamine concentration.

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

01/0

8/14

For

pers

onal

use

onl

y.

ANTIULCEROGENIC EFFECTS OF OCIMUM EASIUCLIM EXTRACTS 99

Studies on aspirin treated rats A modification of the method of Geol er 01. (1985) was used for the production of experimental gastric ulceration in rats. Aspirin (0.2 g/kg) was administered suspended in 1% uagacanth solution at pH 1.3, (5 ml/kg). HCI was added to the aspirin suspension since pilot experiments showed that the rate of ulceration without exogenous acid was extremely variable, as previously reported by Foschi er al. (1984). The test drugs, various extracts and isolated fractions of 0. basilicum, were administered 3 hr before the aspirin preparation, and an equal dose was administered 3 hr later. This regimen was continued for three consecutive days and pylorus ligation was done on the fourth day. As described earlier, the gastric contents were collected and analyzed. The stomachs treated with 1% formalin were incised along the greater curvature and examined for lesions (Akhtar and Munir, 1989).

Studies on stress ulcerated rats Stress ulcers were induced by water immersion according to the method of Okabe er al. (1976). Un- der ether anaesthesia, the abdomen of each rat was opened and the pylorus was ligated. The incised abdomen was then closed and covered with collodion to prevent any leakage of water at the time of water immersion. A dose of the drugs was given after pylorus ligation. The temperature of the water bath was maintained at 25'C for 20 hours. After this stressing procedure, the animals were killed by a blow on head and the stomachs were removed. The gastric contents were collected, centrifuged and analyzed.

Studies on acetic acid-induced gastric ulcerous rats Acetic acid ulcer in rats was induced according to the method of Keijiro er al. (1969). The dosing of test drugs and various extracts of 0. basilicum were then started one day after operation for 10 con- secutive days, twice daily. Feed was withheld on the 1 lth day and the animals were sacrified on the 12th day. The stomach was removed and filled with 10 ml of 1% formalin solution for 5 min to fix the outer layer of the stomach. The stomachs were then cut open along the greater curvature, spread on a pad and examined macroscopically. The ulcer produced was round or oval in shape. The length and width of each lesion was determined under a dissecting microscope (lox, 4x) and an ulcer index was determined as described below. The curative ratio was determined by the formula:

Control Test Curative ratio = ulcer index - ulcer index x 100

Control ulcer index

Determination of acid output Acid output was determined by titration with 0.01N NaOH using phenolphthalein as an indicator as described by Harold (1980). Acid output was expressed in mmoles per hour.

Determination of peptic activity Peptic activity was measured according to the methods of Riggs and Stadic (1943) and expressed as velocity constant per ml of the gastric juice per min.

Determination of glucosamine concentration Hexosamines are obligatory components of mucus and their determination has been used to estimate the mucus content of gastric juice (John el al., 1973). The concentration of glucosamine in gastric juice was determined by the method of Elson and Morgan (1933).

Determination of ulcer index In aspirin treated and stress ulcerous rats, the ulcer index was determined by treating the stomachs with 10 ml of 1% formalin in solution for 5 min in order to fix the outer layer. The stomachs were then incised along the greater curvature and examined for lesions which had developed in the glandular portion. The length of each lesion was determined under a dissecting microscope (lox, 4x) and the sum of the lengths of all lesions in mm for each stomach was expressed as an ulcer index as suggested and used by Okabe er al. (1976).

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

01/0

8/14

For

pers

onal

use

onl

y.

100 M.S. AKHTAR ET AL.

RESULTS

Effect on acid output in normal rats Table 1 shows that no significant change in acid output was found with various extracts of the Ocimum basilicum. However, a significant reduction in acid out- put was found with the reference drug, cimetidine.

Effect on pepsin level in normal rats Table 1 shows that no extract caused any change in pepsin level in normal rats. Only the reference drug, gefarnate, was found to cause a significant decrease in pepsin level.

Effect on gastric hexosamine level in normal rats Table 1 shows that a significant increase in glucosamine level was observed with the aqueous extract of Ocimum basilicum. A reference drug, gefarnate, was also found to cause a significant increase in hexosamine concentration in these rats.

Effect on gastric acid output in aspirin ulcerated rats Table 2 shows that a significant decrease in acid output was observed with the methanol extract of 0. basilicum and its flavonoid glycosides. Cimetidine treat- ment did not show a significant change.

Table 1. Effect of placebo and test drugs on the acid output, peptic activity and glucosamine concen- tration in the gastric contenB of the normal rats.

Treatment Total oral Acid Output Peptic Activity Glucosamine dosage millirnoles/hr velocity constant/ Concentration

pg/100 g/4hrs ml of the gastric juice/min

193 f 32 Tragacanth (control) 10 ml/kg 0 . lOf .02Y 0.029 f .008 Gefarnate 7.5 mg/kg 0.078 f ,004 0.004 f .0009* 963 f 82** Cimetidine 15.0 mg/kg 0.065 f .005* 0.032 f .004 140 f 16 0. basilicum (A.E.) = 4.0 g/kg 0.219 f .062 0.034 f .019 468 f 36** 0. basilicum (M.E.) = 4.0 g/kg 0.099 k .014 0.021 f .005 301 f 71 0. basilicum (A/M.E) +i 4.0 g/kg 0.1 10 f .COY 0.027 f .008 2 5 1 f 61

Significant from control * P<0.05

N = 6/group except for tragacanth control (N=10).

A.E. = Aqueous extract M.E. = Methanol extract A/M.E. = Aqueoushlethanol extract

** P<O.OOI

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

01/0

8/14

For

pers

onal

use

onl

y.

ANTIULCEROGENIC EFFECTS OF OCIMUM EASIUCUM EXTRACTS 101

Table 2. Effect of placebo and test drugs on the acid output, peptic activity and glucosamine in aspirin ulcerated rats.

Treatment Total oral Acid Output Peptic Activity Glucosamine dosage millimoles/hr velocity constantl Concentration

pg/100 g/4hrs ml of the gastric juice/min

Tragacanth (control) Gefarnate Cimetidine 0. basilicum (A.E.) 0. basilicum (M.E.) 0. basilic urn ( A N . E) Volatile oil of 0. basilicum Flavonoid glycosides of 0. basilicum

10 ml/kg 7.5 mgkg 15 mgAg = 4.0 g k g = 4.0 gkg = 4.0 g/kg

= 4.0 gkg

= 4.0 g/kg

0.965 f 0.14 0.576 f 0.14 0.774 f 0.05 0.899 f .07 0.138 * .024** 0.51 1 ? .09*

0.60 * .05

0.1 1 ? .02**

0.026 f .OM 0.007 f .002* 0.004 f .0009** 0.014 f ,006 0.011 f .007* 0.027 f 0.005

0.020 f 0.005

0.008 f .003**

637 k 54 1288 f 21** 875 f 118

1451 f 34** 789 f 15 882 f 55**

774 f 16

1083 k 31**

Significant from control * P<0.05

N = 6/group except for tragacanth control (N=10).

A.E. = Aqueous extract M.E. = Methanol extract A/M.E. = AqueousNethanol extract

** P<O.OOl

Effect on pepsin level in aspirin ulcerated rats Table 2 shows that a significant decrease in pepsin level was observed with both reference drugs. Significant decreases in pepsin levels were also found with the methanol extract of 0. basilicum and with the flavonoid glycosides.

Effect on gastric hexosamine level in aspirin ulcerated rats Table 2 shows that a significant increase in glucosamine concentration was observed with the water/methanol extract of 0. basilicum and reference drug, gefarnate. A significant change was also found with the water extract and the flavonoid glycosides.

Effect on gastric acid output in stress ulcerated rats Table 3 shows that a significant decrease in acid output was observed only with gefamate.

Effect on pepsin level in stress ulcerated rats Table 3 shows that no change in pepsin level was found with any of the reference drugs or extracts of 0. basilicum.

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

01/0

8/14

For

pers

onal

use

onl

y.

102 M.S. AKHTAR ET AL.

Table 3. Effect of placebo and test drugs on the acid output, peptic activity and glucosamine concen- tration in stress ulcerated rats (cold water immersion stress).

Treatment Total oral Acid Output Peptic Activity Glucosamine dosage millimoles/hr velocity constant/ Concentration

u 1 0 0 g/4hrs ml of the gastric juice/min

Tragacanth (control) 10 ml/kg 0.12 f .005 0.045 f ,007 265 f 48 Gefarnate (reference) 7.5 mgkg 0.02 f .004* 0.032 f .009 913 f 53** Cimetidine (reference) 15.0 mgkg 0.19 k .029 0.049 f .007 608 f 98** 0. basilicum (A.E.) = 4.0 g/kg 0.17 It: .05 0.042 f .004 676 f 34** 0. basilicum (M.E.) = 4.0 gkg 0.06 f .01 0.043 f .006 678 f 60** 0. basilicum (A/M.E) = 4.0 g/kg 0.13 k .007 0.040 f .004 676 f 41**

Significant from control * P<0.05 ** P<O.OOI

N = 6/group except for tragacanth control (N=10).

A.E. = Aqueous extract M.E. = Methanolic extract A/M.E. = Aqueoushtethanolic extract

Effect on gastric hexosamine level in stress ulcerated rats Table 3 shows that a significant increase in glucosamine concentration was ob- served with all the extracts of 0. basilicum and reference drugs.

Effect on ulcer index in aspirin ulcerated rats Table 4 shows that a significant decrease in the ulcer index was observed with all the extracts of 0. basilicum and flavonoid glycosides.

Effect on ulcer index in stress ulcerated rats Table 4 shows that no change in the ulcer index was observed with any extract of 0. basilicum. Only the reference drug, gefarnate, was found to cause a significant decrease in the ulcer index in these rats.

Effect on ulcer index in acetic acid induced ulcerated rats Table 4 shows that a significant decrease in ulcer index was found with the aqueous and the aqueouslmethanol extracts of 0. basilicum. Reference drug, gefarnate, also showed a significant decrease in the ulcer index in these rats.

DISCUSSION

Various extracts of 0. basilicum have been found to have no effect on the outputs of acid and pepsin in normal (non-ulcerated) rats. However, a significant increase

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

01/0

8/14

For

pers

onal

use

onl

y.

ANTIULCEROGENIC EFFECTS OF OCIMUM BASILICUM EXTRACTS 103

Table 4. Ulcer indices (mean + S.E.M.) after treatment with placebo or test drugs in aspirin ulcerated, stress ulcerous, and acetic acid - induced ulcerous rats.

Experimental hccedure Treatment

Total oral % dosage Ulcer index change

Aspirin Tragacenth (control) 10 ml/kg 4 3 f 6 control ulcerated Gefarnate 7.5 mgkg 3 2 f l 25

Cimetidine 15.0 mg/kg 24+ 1 1 42

0. basilicum (M.E) = 4.0 g/kg 25 f 4* 41 0. basilicum (A.E) = 4.0 g/kg 20+ 3* 53

0. basilicum ( A m ) = 4.0 g/kg 2 4 f 11, 44

Stress Tragacanth (control) ulcerous Gefarnate rats Cimetidine

0. basilicum (A.E.) 0. basilicum (M.E) 0. basilicum A/M.E) Volatile oils of 0. basilicum Flavonoid glycosides of 0. basilicum

10 rnl/kg 7.5 mgkg 15.0 mg/kg = 4.0 g/kg = 4.0 g/kg = 4.0 g/kg

= 4.0 g k g

= 4.0 g/kg

15f5 control 0.77 f 53** 21

l l f 6 95 18+5 120 22 f I 166 23 f I 156

24-t 1 1 142

24 f 4 40

Acetic Tragacanth (control) 10 ml/kg 3 1 f l control

induced- Cimetidine 15.0 mg/kg 21 f 13 31

rats 0. basilicum (M.E) = 4.0 g/kg 2 6 f 5 16

acid Gefarnate 7.5 mgkg 15 f 5** 51

ulcerous 0. basilicum (A.E) = 4.0 g/kg 4 f I * * 88

0. basilicum (A/M.E) = 4.0 g/kg 1 1 f 5** 45

Significant from control * Pc0.05

N = 6/group except for tragacanth control (N=10).

A.E. = Aqueous extract M.E. = Methanol extract A/M.E. = Aqueous/Methanol extract

** P<0.001

in glucosamine concentration was observed with an aqueous extract, demonstrat- ing that active constituents of the drug may be extracted into water. All of the extracts of 0. basilicum were not able to reduce basal gastric secretions. Signifi- cant decreases in acid output and peptic activities were observed with a methanol extract in aspirin-treated rats, and a significant decrease in acid output and a significant increase in glucosamine concentration were observed with a water/ methanol extract of 0. basilicum. From these results, it may be speculated that active constituents responsible for antiulcer activity may be extracted in both water and methanol solvents, and a dual action is exerted, viz., inhibition of gastric acid output and strengthening of the mucosal barrier. Flavonoids ex- tracted from 0. basilicum also significantly increased the glucosamine concen-

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

01/0

8/14

For

pers

onal

use

onl

y.

104 M.S. AKHTAR ET AL.

tration and decreased acid output and peptic activity. Therefore, it is conceivable that antiulcer activity could be due these flavonoids which possess a dual action, i.e., inhibition of harmful factors like acids and pepsin and increasing protective factors like mucus.

Neither of the extracts of the drug were effective for the stress lesions induced by water immersion. Stress induced gastric lesions in rats produced by water immersion is a suitable model for drugs with inhibitory action (West et al., 1982). Gefarnate demonstrated a significant decrease in the ulcer index in the present studies.

A high curing rate (88%) was observed with a water extract in acetic acid- induced gastric ulcer. Antiulcer principles responsible for stimulating the epithelization or granulation of the tissues and thus accelerating the repair proc- esses may be responsible for this activity. However, prevention of ulcer formation by entirely different mechanisms is also possible and more work is required to establish the mechanism of action of the extracts and isolated entities of the plant drug 0. basilicum.

REFERENCES

ARIYOSHI, I., TOSHIHARA, A., SUGIMURA, F., ABE, M., MATSUO, Y. and HONDA, T. (1986).

AKHTAR, M.S. and MUNIR, M. (1989). J. Ethanopharmacol. 27: 163-176. BRAIN, K.R. and TURNER, T.D. Practical Evaluation of Pharmaceuticals (1975) 1st Ed. Wright

ELSON, L.A., MORGAN, W.T.T. (1933). Biochem. J. 27: 962-963. FOSCHI, D., and IMBIMBO, B., DANIOTI, S. and SOLDSATO, P.D. (1984). Symposium on

Pirenzepine: New Aspects in Research and Therapy. Excerpta Medica, Amsterdam, pp. 61- 68.

GEOL, R.K., CHAKRABARTI, A. and SANYAL, A.K. (1985). Planta Medica 51: 85-89. HAROLD, V., (1980). Practical Clinical Biochemistry. Vol. 5, p. 1000. William Heinemann Medical

HARBORNE, J.B., Phytochemical Methods (1984) 2nd ed. Chapman and Hall 733 3rd Avenue., New

JOHN, D.J.B., YEOMANS, N.D., McDERMOTT, F.T. and BOER, W.G.R.M. (1973). Am. J. Dig.

KEIJIRO, T., OKABE, S., SAZIKI, R. (1969). Jpn. J. Pharmacol. 19: 418-426. OKABE, S., TAKATA, K., TAKEUCHI, K., NAGANUMA. T. and TAKAGI, K. (1976). Am. J. Dig.

RIGGS, B.C., and STADIE, W.C. (1943). J. Biol. Chem. 150 463-470. SHAY, H., KOMAROV, S.A., FELS, S.S., MERANZE, D., GRUENSTEIN, M. and SIPLET, H.

(1945). Gastroenterol. 5: 43-61. TAKEUCHI, K., OKABE, S. and TAKAGI, K., (1976). Am. J. Dig. Dis. 21: 782-788. WEST, G.B., (1982). J. Pharmacol. Meth. 8: 33-37.

Accepted October 21, 1991

Nihon University Journal of Medicine 28: 69-74.

Scientechnica, Bristol.

Book Ltd., Londen.

York. N.Y. 10017.

Dis. 18: 881-886.

Dis. 21: 618-625.

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

01/0

8/14

For

pers

onal

use

onl

y.