Anti-ulcerogenic effects of Spartium junceum flowers on in vivo test models in rats

Journal of Ethnopharmacology 70 (2000) 219 – 226 www.elsevier.com/locate/jethpharm

Anti-ulcerogenic effects of Spartium junceum flowers on in vivo test models in rats Erdem Yes¸ilada a,*, Yoshihisa Takaishi b, Tetsuro Fujita c, Ekrem Sezik a a

Department of Pharmacognosy, Faculty of Pharmacy, Gazi Uni6ersity, Hipodrom 06330, Ankara, Turkey b Tokushima Uni6ersity, Faculty of Pharmaceutical Sciences, 770, Tokushima, Japan c Setsunan Uni6ersity, Faculty of Pharmacy, Osaka, Japan Received 18 December 1998; received in revised form 11 October 1999; accepted 11 October 1999

Abstract Flowers of Spartium junceum L. (Fabaceae) are used for the treatment of gastric ulcers in Turkish folk medicine. Through bioassay-guided fractionation using chemical and chromatographical means and water immersion and restraint-induced stress ulcer model in rats, a saponin fraction was determined as the potent anti-ulcerogenic ingredient. The active fraction was also highly effective in preventing ethanol- and pyloric ligation-induced gastric lesions as well as inhibiting gastric secretion volume, gastric pH and titratable acidity, but did not affect the hexosamine content of the gastric mucosa. A novel oleanen-type triterpenic saponin, named as spartitrioside, was isolated as the active principle by using chromatographical separation techniques. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Spartium junceum L.; Fabaceae; Anti-ulcerogenic activity; Water-immersion and restraint-induced stress ulcer; Ethanol-induced ulcer; Pyloric ligation-induced ulcer; Gastric secretion; Hexosamine level; Oleanene-type saponin; Spartitrioside

1. Introduction Flowers of Spartium junceum L. (Fabaceae) are known to have mild sedative and diuretic activities (Baytop, 1984). Our field surveys in southern Anatolia disclosed that an infusion prepared from the flowers of this plant is used for the treatment of gastric ulcers in folk medicine (Yes¸ilada et al., 1993a). Since the flowers are known to contain

* Corresponding author. Fax: +90-312-223-5018. E-mail address: [email protected] (E. Yes¸ilada)

cytisine-type alkaloids (0.56% of dry wt.)(Sadykov et al., 1981) which are reportedly toxic at a higher dose (Baytop, 1984), we first tested the anti-ulcerogenic activity of the flowers to prove the claimed usage by using water immersion and restraint-induced stress ulcer model in rats and determined a potent anti-ulcerogenic activity (Yes¸ilada et al., 1993b). The present study deals with the evaluation of anti-ulcerogenic activity of the fractions obtained from S. junceum flowers by using in vivo test models in rats and isolation of an active principle by bioassay-guided fractionation process.

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2. Experimental

2.1. Plant material Flowers of S. junceum were collected in Foc¸a (I: zmir) in May, 1992 – 1997, and dried under shade. A voucher specimen is deposited at the Herbarium of the Faculty of Pharmacy, Gazi University (Ankara) [No. 92A001].

2.2. Chemicals Amberlite XAD-2 (Organo), Sephadex LH-20 (Pharmacia Fine Chemicals), Dowex 50 (Bio-Rad Laboratories, CA), Kieselgel 60 (Merck). Solvent systems for tlc on precoated Kieselgel 60F254 plates (Merck, Art.5719): (a) n-BuOH-n-PrOHAcH-H2O (4:2:1:3); and (b) CHCl3-MeOH-H2O (7:4.2:1). Spots were visualized by spraying 5% H2SO4 in EtOH and then heating in an oven at 110°C.

2.3. Chemical experiments 2.3.1. Extraction Dried flowers (1.1 kg) were extracted with MeOH (21 l) at room temperature, then the sol-

Scheme 1. Extraction and fractionation chart of S. junceum flowers by solvent extraction.

vent was evaporated under reduced pressure to give 340 g extract. The material (residue of flowers) was further extracted with H2O on a waterbath (13 l) and the extract was evaporated to dryness to give 127.2 g aqueous extract (E-H2O fr.) (Scheme 1).

2.3.2. Fractionation of methanolic extract The methanolic extract was redissolved in 90% MeOH (2 l) and extracted with n-hexane (5× 500 ml). After the removal of MeOH, the remaining aqueous extract was extracted with EtOAc (4× 500 ml). Each of the combined extracts as well as the final aqueous layer was evaporated to dryness under reduced pressure to give a hexane extract (Hexane fr.) (30 g. viscous liquid), an ethylacetate extract (EtOAC fr.) (42.2 g), and the final H2O extract (Final-H2O fr./1 ) (265.2 g). The latter was further extracted with n-BuOH saturated with H2O (5 × 500 ml) and evaporated to dryness to give the n-butanol extract (n-BuOH fr.) (95.5 g) and the final H2O extract (Final-H2O fr./2 ) (167 g) (Scheme 1).

2.3.3. Fractionation of E-H2O extract The aqueous extract (E-H2O fr., 12.7 g) was submitted to chromatography on an Amberlite XAD-2 column (78× 8 mm, i.d.) and eluted with H2O (6 l) (Fr.1–21), H2O-MeOH (7:3) (4.5 l) (Fr.22–37), and MeOH (6 l) (Fr.38–66), successively. Fractions were combined into four groups on the basis of chromatographic behaviour on TLC (solvent system-a); XAD/Fr.1 – 2 (5.25 g), XAD/Fr.3 – 18 (1.47 g), XAD/Fr.19 – 37 (1.39 g), XAD/Fr.38 – 66 (1.27 g).

2.3.4. Fractionation of XAD/Fr.38 – 66 MeOH eluates from the Amberlite XAD-2 column was chromatographed on a Sephadex LH-20 column (1300×20 mm, i.d.), and eluted with MeOH. Fractions 2–9 (LH/Fr.2 – 9 ) and fractions 10–27 (LH/Fr.10 – 27 ) were combined into two separate groups on the basis of chromatographic behaviour on TLC (solvent systems a and b).

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2.3.5. Isolation of acti6e principle The detailed methodology for the isolation and structure elucidation of the saponin is reported previously (Yes¸ilada and Takaishi, 1999). Briefly, the n-butanol extract (n-BuOH fr.) was chromatographed in a MPLC column on silica gel (Kieselgel 0.040–0.063 mm, Merck) using CHCl3MeOH-H2O (10:3:0.3), (9:3:0.5) and (7:4.2:1) solvent systems, successively. Eluents which contain the main saponin were combined according to TLC control (solvent system-a and -b) and further purified by successive precipitation with MeOH. The structure determination of the isolated compound was carried out using the various techniques of NMR spectral analysis (1H, 13C, DEPT, C – H COSY, COLOC, NOESY, HMBC, HMQC), in conjuction with FAB- and EI-mass spectrometry. 2.4. Pharmacological experiments 2.4.1. General protocol Plant material was extracted with MeOH, and then fractionated by solvent extraction. Since the anti-ulcerogenic activity was detected in both the MeOH and aqueous extracts, in our previous study (Yes¸ilada et al., 1993b), polar as well as nonpolar solvent extracts were subjected to bioassay. The stress model for lesion production is considered to be the most useful methods for testing drugs against gastric ulcers (West, 1982). Accordingly, the water-immersion restraint-induced stress ulcer model was used for the bioassay-guided fractionation process. Male Wistar rats were left for 2 days for acclimatization to animal room conditions (24°C), exposed to 12 h of light and dark cycle and maintained on a standard pellet diet and water ad libitum. The food was, however, withdrawn 48 h before the experiment, but water was freely accessable. Coprophagy was prevented by fasting the animals in wire-bottomed cages. The control group animals received the same experimental handling as those of the test groups except that the drug was replaced with appropriate volumes of the dosing vehicle (0.5% carboxymethyl cellulose/distilled H2O). Test samples, unless otherwise stated, were administered to animals orally

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at a volume of 10 ml/kg (body weight) as a suspension in dosing vehicle by means of a gavage needle. For intraperitoneal (ip) administration, either physiological saline solution (0.9%) (for control group) or a suspension of a test sample in saline solution was injected at a volume of 3 ml/kg b.w. Famotidine was used as a reference drug at 20 mg/kg b.w. (oral) and 5 mg/kg b.w. (ip).

2.4.2. Water-immersion and restraint-induced stress ulcer A modification of the method described by Takagi and Okabe (1968) was employed. A group of six rats weighing 125–155 g was used for each treatment. The animals were placed individually in each compartment of a restraint cage (4.5× 4.5×18 cm) and immersed vertically up to the level of the xiphoid in a water-bath (19–22°C) to induce stress ulcer. Test sample was administered either orally or intraperitoneally to rats just before immobilization. After (7 h) immersion, rats were sacrificed by an overdose of ether. Their stomachs were removed, inflated with 10 ml of physiological saline solution and immersed in 1% formalin solution overnight to fix the outer layer of the stomach. Each stomach was opened along the greater curvature, rinsed with physiological saline to remove gastric contents and blood clots, and examined under a dissecting microscope (20×6.3× ) to assess the formation of ulcers. The sum of the length (mm) of all lesions for each stomach was used as the ulcer index (UI), and the inhibition percentage was calculated by the formula: ((UIcontrol − UIsample)/UIcontrol)× 100.

2.4.3. Ulceration induced by ethanol After 15 min the oral administration of a test sample to a group of six rats weighing 132–148 g, lesions were induced by an oral administration of 1 ml absolute ethanol by means of a gavage needle. Later (1 h), the animals were killed by an overdose of ether. The abdomen was immediately opened to remove the stomach and processed as described above to calculate the ulcer index.

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2.4.4. Pyloric ligation-induced ulcer Nine rats weighing 180 – 200 g were used in each group. Under ethereal anesthesia, the pyloric sphincter was ligated surgically, as described by Shay et al. (1945). Each test sample was administered at a volume of 3 ml/kg immediately following pylorus ligation. The animals were then placed in cages without food and water. After 4 h, the animals were sacrificed, their stomachs removed by clamping the esophagus at the cervical portion, and the inner surface of each stomach was examined for ulceration. The degree of ulceration was graded by scoring 0.5 for haemorrhages, erosions or each small ulcer less than 3 mm in length, one for each ulcer longer than 3 mm, and five for perforated ulcer. 2.4.5. Effects on gastric acid secretion in pylorus-ligated rats The gastric content of each stomach obtained from the pyloric ligation ulcer was drained into a centrifuge tube by filtering through glass wool, after an incision with a fine pair of scissors. After the centrifugation of the gastric content at 2500 rpm for 20 min at 4°C, the volume of the supernatant (ml) and the pH value (Horiba MD-8 pHmeter) were measured. The volume was expressed as ml per 100 g b.w. An aliquot (1 ml) of each sample was titrated against 0.01 N NaOH using the phenolphytalein reagent as an indicator. The acid concentration was expressed as m-equivalent/ l, and the acid output as m-equivalent per 4 h. The peptic activity was determined by using bovine serum albumin as a substrate and expressed in terms of the amount (mmoles) of liberated L-tyrosine according to the method described by Prino et al. (1971). A mixture of gastric juice (0.1 ml) and 0.5% bovine serum albumin (Sigma) in 0.01 N HCl (1 ml) was incubated at 37°C for 20 min and the reaction was stopped by adding 10% trichloroacetic acid (2 ml). After the denaturation of protein by heating in a boiling water bath for 5 min, the precipitate was removed by centrifugation (9000 rpm 10 min). A total of 1 ml of the supernatant was mixed with 0.4 ml of 2.5 N NaOH and 0.1 ml of Folin-Ciocalteu reagent (Nacalai Chemical) and the volume was adjusted to 10 ml with distilled water. The absorbence was

measured at 700 nm (Hitachi U-3210 spectrophotometer). A sample (gastric juice blank) containing no albumin was used as the control.

2.4.6. Measurement of gastric hexosamine content Male Wistar rats weighing 180–200 g were employed. Each test group consisted of seven rats, which had been fasted for 48 h on water prior to the experiment. Animals were exposed to waterimmersion stress ulcer as described earlier. The stomachs were then removed without inflating and cut open at the greater curvature and soaked in a mixture of EtOH-acetone (1:1 v/v) for 24 h to remove fat from tissues. The glandular stomach was then divided into the corpus and the antrum, each of which was dried in a desiccator under reduced pressure overnight and weighed. Accurately weighed portions of either corpus or antrum (35–45 mg) were digested with 1 ml of 2N HCl in a stoppered test tube in a sand bath (100°C) for 15 h. After cooling, the digested material was filtered through glass wool into a Dowex 50 column and 3 ml of the eluate was used to measure the content of hexosamine by Narumi’ and Kanno’s method (1972) spectrophotometrically at 530 nm (Hitachi U-3210 spectrophotometer). 2.5. Statistical analysis of data Data were expressed as mean 9S.E. The statistical significance of the difference between the mean ulcer index of the treated group and that of the control group was tested by Student’s t-test.

3. Results and discussion Fractions which were obtained from the MeOH extract of S. junceum flowers, by successive solvent extraction (Hexane fr., EtOAc fr. and Final H2O Fr./1) as well as E-H2O fr. were orally administered to rats and their effects were tested against water immersion and immobilization-induced stress ulcer model. As shown in Table 1, the anti-ulcerogenic activity of the E-H2O fr. was found to be very high; all the stomachs examined were completely free from any visible damage.

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Table 1 Effects of fractions from S. junceum flowers on water immersion and restraint-induced stress ulcer model in rats Ulcer index (mean 9S.E.)

Prevention from ulcerb

– 0/6 1/6 3/6

189 93 20

20.99 7.8 22.69 8.3 11.79 5.2 10.39 6.1 Toxic 0.09 0.0** 14.29 7.0 1.7 9 2.5** 1.99 3.3** 10.39 3.4 9.19 3.4 6.39 2.6 5.49 2.6 0.6 9 0.5** 3.79 1.5 0.29 0.2* 5.0 9 2.1 0.69 0.1*

Intraperitoneal administration Control [LH/Fr.2–9] 189 Famotidine 5

12.69 5.5 Toxic 2.69 3.0**

Fractions Per os administration Control Hexane Fr. EtOAc Fr. Final H2O Fr./1 E-H2O Fr. Control BuOH Fr. Final-H2O Fr./2 Control [XAD/Fr.1–2] [XAD/Fr.3–18] [XAD/Fr.19–37] [XAD/Fr.38–66] Control [LH/Fr.2–9] [LH/Fr.10–27] Famotidine

Dose (mg/kg)a

246 246 509 1099 750 566 530 754 209 150 136

6/6 – 2/6 3/6 – 1/6 1/6 1/6 3/6 – 5/6 0/6 0/6

Inhibition (%)

44.0 49.3 100.0 88.0 86.6 11.6 38.8 47.6 94.2 94.6 83.8

– 0/6

79.4

a

All drugs were administered to rats orally just prior to stress-loading. Number of rats whose stomachs were completely prevented from bleeding. * PB0.05; and **PB0.01.

b

The EtOAc fr. showed a weaker activity, however, even at twice as much higher dose. On the other hand, all animals died within 15 min by the administration of the Final-H2O fr./1, probably as a result of an accumulation of water soluble alkaloids in this fraction. Thus, we decided to administer this fraction in a half dose after further extraction with n-BuOH saturated with water. Both BuOH fr. and Final-H2O fr./2 showed significant anti-ulcerogenic activity without causing morbidity (Table 1). Since the activity of the E-H2O fr. was more pronounced and less toxic compared with the other fractions, further experiments were carried out on this fraction. E-H2O fraction was first fractionated by ion-exchange chromatography on Amberlite XAD-2 and then by molecular sieving on Sephadex LH20. Each fraction obtained by combining the eluents with similar TLC behavior, was tested using

the same ulcer model (Table 1). Fraction LH/ Fr.2–9 showed a prominent inhibitory effect on oral administration, but showed a high toxicity when injected intraperitoneally; animals died within 30 min after injection. Active fraction LH/Fr.2–9 also inhibited ethanol-induced (100%) (Table 2) and pylorus ligation-induced (83.9%) ulcerogenesis (Table 2) in rats when administered orally. This fraction also strongly inhibited gastric acid secretion in pylorus-ligated rats, showing a significant anti-peptic activity (60.8%) (Table 3). It inhibited gastric secretion volume (49.0%), as well as gastric pH (to pH 4.54), titratable acidity (47.7%) and acid output (82.7%), but did not affect the hexosamine content of the gastric mucosa (Table 4). The separation of the active components were carried out by using another chromatographical

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technique, medium pressure liquid chromatography and gradient elution technique. Due to the low yield of the components, only the main principle was isolated and the structure was elucidated by using 1D- and 2D-NMR as well as EI- and FAB-MS spectral techniques. It proved to be 3O-[a – L -rhamnopyranosyl-(1“ 2)-O-b- D -glucopyranosyl-(1“2)-O-b- D -glucuronopyranosyl]-3b,

16b,22b,24-tetrahydroxy-olean-12-ene and named as spartitrioside (Fig. 1). The detailed methodology for the isolation and structure elucidation of the saponin is reported elsewhere (Yes¸ilada and Takaishi, 1999). Because of the low yields of each saponin in the active fraction (LH/Fr.2–9), a study which is based on the structure-activity relationship of

Table 2 Effects of fractions from S. junceum flowers on various ulcer models Fractions

Dose (mg/kg)

Ethanol-induced ulcer Control [LH/Fr.2–9] 189 Spartitrioside 95 Famotidine 20 Pyloric ligation-induced ulcer Control [LH/Fr.2–9] 189 Famotidine 20

Ulcer index (mean 9S.E.)

Prevention from ulcera

Inhibition (%)

29.1 9 8.9 0.09 0.0** 0.59 0.5** 3.0 90.5*

– 6/6 4/6 0/6

100.0 98.3 89.7

4.69 1.8 0.79 0.3* 0.99 0.7*

2/9 2/9 0/6

83.9 80.4

a

Number of rats whose stomachs were completely prevented from bleeding. * PB0.05; and ** PB0.01. Table 3 Effect of active fraction (189 mg/kg) on gastric secretion in pylorus ligated rats Treatment

Gastric juice volume (ml/100 g/b.w.)

Gastric pH value Titratable acidity Titratable acid (mEq H+/ml) output (mEq/4 h)

Control [LH/Fr.2–9] Inhibitory ratio (%)

5.1 9 1.2 2.6 9 0.3* 49.0

2.369 0.3 4.549 0.4** 192.4

92.6 918.0 48.4 9 7.2* 47.7

696.9 9271.7 120.4 930.2* 82.7

Peptic activity (mmoles L-tyrosine/4 h) 15.04 911.5 5.89 92.2* 60.8

* PB0.05; and ** PB0.001. Table 4 Effects of the active fraction (189 mg/kg) on hexosamine content in the glandular stomach strips of rats exposed to water immersion and restraint-induced stress ulcer model Treatment

Control [LH/Fr.2–9] a

Mucosa weight (mg)a

135.89 5.5 131.69 5.4

Total weight of the dried glandular part of stomach.

Hexosamine content (mg/100 mg dry strip) Corpus

Antrum

561.0 9 19.6 605.6 9 108.5

1140.9 9165.2 1054.46 9278.9

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Fig. 1. Spartitrioside.

each component could not be established. But the characterized component, spartitrioside, showed a potent anti-ulcerogenic activity against ethanol-induced ulcerogenesis model in rats (Table 2). S. junceum flowers were found to be effective in preventing the gastric lesion formation induced by stress when administered orally. It has been reported that an increased acid output is closely associated with the occurrence of stress-induced lesions (Brodie et al., 1962). Typical antisecretory drugs, which are H2-receptor antagonists such as cimetidine, ranitidine, and famotidine, were markedly inhibitory on the stress ulcer model (Okabe et al., 1977; Yamamoto et al., 1984). Active fraction LH/Fr.2 – 9 also markedly inhibited the gastric secretory volume, reducing the total acid and pepsin output. Therefore, it is suggested that the inhibitory effect of S. junceum on stress-induced lesions might be mainly as a result of a decreased acid secretion. On the other hand, it is worth mentioning that S. junceum

flowers are claimed to have a mild sedative activity (Baytop, 1984) and Brodie et al. (1962) reported that this type of agents exert antisecretory effects through a central mechanism. In pyloric ligation ulcers, the volume and peptic activity of the gastric juice secreted are considered to be the main ulcerogenic factors, which may be controlled by antisecretory drugs or antiacids (Prino et al., 1971). S. junceum flowers also showed a statistically significant inhibition on this type of ulcer model, as might be expected. The active fraction was also highly effective in preventing ethanol-induced gastric lesions which might be regarded as a cytoprotective activity (Robert et al., 1979). The gastric mucosal barrier is thought to play an important role in protecting gastric mucosa from destructive effects of intraluminal acid (Davenport, 1968). Hexosamines are obligatory components of mucus and their quantitative determination has been used as a measure of mucus formation in the gastric mucosa (Lukie

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and Forstner, 1972). In the present study, the concentration of glucosamine in gastric mucosa was estimated by the method of Narumi and Kanno (1972). However, active fraction LH/ Fr.2 –9 was neither effective increasing the mucosa weight nor the hexosamine level when tested on stress-induced ulcerations. On the other hand, fractions obtained through solvent extration as well as active saponin fraction were studied for their possible effects against Helicobacter pylori and all were found ineffective against this organism (Yes¸ilada et al., 1999). Peptic ulcer formation is attributed to the imbalance appearing between aggressive factors (acid and pepsin) and defensive factors (mucosal resistance) (Chiu et al., 1984). Present experiments demonstrated that the active fraction of the plant was mainly effective in preventing the stomach from aggressive factors, but not in intensifying defensive factors. At present, it is yet unknown whether the active fraction has any effect on gastric blood flow or transmural potential activity or not. Further experiments on this subject remain to be performed.

Acknowledgements The first author, E.Y., acknowledges receiving grants from Fujii-Otsuka and Goho Life Sciences International Funds (Japan) to study in Japan.

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