Anti-ulcerogenic activity of some plants used in folk medicine of Pinarbasi (Kayseri, Turkey)

Journal of Ethnopharmacology 101 (2005) 313–318

Anti-ulcerogenic activity of some plants used in folk medicine of Pinarbasi (Kayseri, Turkey) b , Erdem Yesilada a , Osman Kutsal c ¨ Ilhan G¨urb¨uz a,∗ , Ayse Mine Ozkan b

a Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, Etiler 06330, Ankara Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Tandogan 06100, Ankara c Ankara University, Faculty of Veterinary Medicine, Department of Pathology, Diskapi, Ankara, Turkey

Received 29 June 2004; received in revised form 5 May 2005; accepted 21 May 2005 Available online 8 August 2005

Abstract Several ethnomedicinal plant remedies used in Pinarbasi, Kayseri, in particular those which are used for the treatment of several peptic ulcer symptoms such as stomach ache, heartburn, etc. were selected for evaluation of their anti-ulcerogenic potential. In order to confirm the claimed activities, decoctions were prepared from aerial parts of Malva neglecta Wallr. (Malvaceae), leaves of Potentilla reptans L. (Rosaceae), fruits of Rumex patientia L. (Polygonaceae), aerial parts of Sanguisorba minor Scop. ssp. muricata (Spach) Briq. (Rosaceae), aerial parts of Sideritis caesarea Duman, Aytac&Baser (Lamiaceae), and flowers of Verbascum cheiranthifolium Boiss var. cheiranthifolium (Scrophulariaceae) according to their described folkloric applications. Pharmacological experiments clearly demonstrated that the extracts of all plants given orally showed significant gastric protection against the ethanol-induced gastric ulcer model in rats. Furthermore, healing effects were also confirmed through histopathological examination. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Malva neglecta; Potentilla reptans; Rumex patientia; Sanguisorba minor ssp. muricata; Sideritis caesarea; Verbascum cheiranthifolium var. cheiranthifolium; Anti-ulcerogenic activity; Ethanol-induced ulcerogenesis; Histopathology; Peptic ulcer

1. Introduction During the past several decades, there has been a global trend for the revival of interest in the traditional system of medicine. Simultaneously the need for basic scientific investigation of medicinal plants using indigenous medical systems has become ever more interesting and relevant. A recent review of references indicates that the antiulcerogenic effects of many taxa of medicinal plants have been assessed worldwide (Yesilada and G¨urb¨uz, 2003). It is also reported that many plant species have been used to alleviate gastric symptoms such as gastric pain, etc. in Turkish folk medicine (Tabata et al., 1988; Sezik et al., 1992, 2001; Yesilada et al., 1995; Honda et al., 1996; Baytop, 1999). In the scope of an ethnobotanical survey conducted between 2000 and 2003 in Pinarbasi, a town in Kayseri ∗

Corresponding author. Tel.: +90 312 223 3274; fax: +90 312 223 5018. E-mail address: [email protected] (I. G¨urb¨uz).

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.05.015

province (Turkey), decoctions which were prepared from six different plants were found as reputed remedies for the treatment of stomach ache, heartburn and similar symptoms. These plants and their used parts were identified as, aerial parts or leaves of Malva neglecta Wallr. (Malvaceae), leaves of Potentilla reptans L. (Rosaceae), fruits of Rumex patientia L. (Polygonaceae), aerial parts of Sanguisorba minor Scop. ssp. muricata (Spach) Briq. (Rosaceae), aerial parts of Sideritis caesarea Duman, Aytac&Baser (Lamiaceae), and flowers of Verbascum cheiranthifolium Boiss var. cheiranthifolium (Scrophuloriaceae). To the best of our knowledge, a recent literature survey revealed that no experimental data was available on the anti-ulcerogenic activity of the above-mentioned traditional herbal remedies. In a continuous effort to evaluate the ethnobotanical information on Turkish medicinal plants (Yesilada et al., 1993; G¨urb¨uz et al., 2002, 2003), this study was undertaken to investigate the efficacy of the above-mentioned folk medici-

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nal plants in the treatment of gastric ulcers using the in vivo ethanol-induced ulcerogenesis model in rats.

2. Materials and methods 2.1. Plant materials Plants were collected from several locations in Pinarbasi, ¨ Kayseri and identified by Dr. A. M. Ozkan. Voucher specimens were deposited at the Herbarium of Ankara University, Faculty of Pharmacy (AEF). Local names of the plant parts which were used, the traditional preparation method, and the collection sites of the investigated plants along with herbarium numbers of voucher specimens are listed in Table 1, respectively. Experimental plant material was dried in the shade at room temperature and coarsely ground prior to extraction.

preparation of the remedy was not fully described, we used one spoonful of ground plant material which was estimated to be approximately 5 g for each material. A test sample of 5 g of each plant was extracted with H2 O (75 ml) and heated until boiling and kept boiling for a further 20 min. The resulting extracts were filtered and then lyophilised. The extract weight and % yields were found to be as follows: Malva neglecta aerial parts 1.24 g (24.8%); Potentilla reptans leaves 0.87 g (17.4%); Rumex patientia fruits 0.44 g (8.8%); Sanguisorba minor, ssp. muricata aerial parts 0.66 g (13.2%); Sideritis caesarea aerial parts 0.96 g (19.2%); Verbascum cheiranthifolium var. cheiranthifolium flowers 1.23 g (24.6%). Doses of the extracts were estimated as the amount of extract (mg) obtained from 5 g of plant material extracted with H2 O as described above and administered per kg of body weight (5 ml/kg) as a suspension of 0.5% (w/v) carboxymethylcellulose (CMC). 2.3. Animals and test samples

2.2. Extraction and preparation of test samples The extraction technique for each crude drug was adapted according to the traditional recipes or usage, if available. Although some plants were prescribed in different forms as listed in Table 1, decoction was a common form to prepare a remedy from all six plants. Therefore, H2 O extracts were employed for the activity assessment in this present study. The usual doses recommended by the informants were one or two glasses (50–100 ml) on an empty stomach before meals or in the morning if suggested to be administered only once a day. Since, the amount of the plant material used for the

Sprague-Dawley rats of either sex (120–200 g) purchased from G¨ulhane Military Academy of Medicine (Ankara) were used for biological tests. The animals were left at least 48 h for acclimatization to animal room conditions and were maintained with a standard pellet diet and tap water ad libitum. The food was withdrawn 24 h before the experiment, but free access to water was allowed. The water was withdrawn 1 h before the experiment. To avoid coprophagy the rats were fasted in wire-bottomed cages. For each group six rats were used. The test samples were administered to animals at a dose of 5 ml/kg body weight as a suspension in 0.5%

Table 1 Local name, parts used, traditional preparation reported, collection sites and the herbarium (AEF) numbers of traditional medicine Plant name

Local names

Parts used

Traditional preparation

Collection sites

AEF numbers

Malva neglecta

Ebeg¨umeci, cobanyatagi

Aerial parts

Boiled in water, after filtering drunk as tea on an empty stomach

22117

Potentilla reptans

Resatinotu

Leaves

Boiled in water, after filtering drunk as tea

Rumex patientia

Ebelik, kalmukcayi, yilki kulak

Fruits

Boiled in water, after filtering drunk as tea

Kazancik Village, gardens, 1800 m, 2 September 2001 Sariz Road, Uzunahmet Village, steppe, 1640 m, 13 June 2001 Egris¨og¨ut Village, gardens, 1740 m, 6 July 2000

Sanguisorba minor ssp. muricata

Yanikotu, mideotu

Aerial parts

Sideritis caesarea

Dagcayi

Aerial parts

Verbascum cheiranthifolium var. cheiranthifolium

Calba, yalangi

Flowers

Boiled in water, filtered, the solution is mixed with milk and boiled again, drunk as tea Boiled in water, after filtering drunk as tea Fresh herb is ingested Boiled in water, after filtering drunk as tea Boiled in water, after filtering drunk as tea

22284

21938

Sirvan Mountain, southern slopes, 1780 m, 24 May 2000

21219

Demircili Village, Kirecli Hill, pastures, 1770 m, 15 June 2001 Sirvan Mountain, steppe, 2000 m, 5 July 2000

22296

22395

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CMC/distilled water. The control group animals received the same experimental handling as those of the test groups except that the drug treatment was replaced by administration of an appropriate amount of the 0.5% CMC/distilled water. The cytoprotective drug misoprostol (400 ␮g/kg) was used as a reference substance. Test samples, reference drug and ethanol were administered to the animal orally with an intragastric gavage. All experimental protocols were approved by the ¨ Gazi University Ethical Council (G.U.ET-03.034).

sue (if present) was cut in half along the long diameter and two to five tissue samples were taken. If there were no visible lesions in gastric mucosa, several sections were removed from the different parts of the antrum and the corpus. After standard processing (Durry and Wellington, 1976), the cut tissue (Elliot Inc.) was embedded in paraffin and cut into sections (4 ␮m) in a rotary microtome (Reichert Inc.) and stained with haematoxylin–eosin (Merck) and then examined under microscope (Olympus FHY).

2.4. Effects on ethanol-induced ulcerogenesis

2.6. Statistical analysis of data

The method described by Robert et al. (1979) was employed with modifications. The test sample was administered orally 15 min before the oral application of ethanol 96% (1 ml) to a group of six rats. 60 min later, the animals were sacrificed with an over-dose of diethyl ether. The abdomen of the rat was dissected and the stomach was slightly taken out. Then the oesophagus was tied as a knot nearest the cardia by a surgical suture. From the duodenum side, 10 ml of 10% formalin solution was injected into the stomach. The distended stomach was immediately tied on the pyloric sphincter using another surgical suture to avoid leakage of the formalin solution. Finally, the distended stomach was removed from the abdomen, inflated with 10 ml of 10% formalin solution and immersed in the same solution to fix the outer layer of the stomach. Each stomach was then dissected along the greater curvature, rinsed with tap water 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 length (mm) of all lesions for each stomach was used as the ulcer index (UI), and the inhibition percentage was calculated by the following formula:

Results were expressed as mean ± S.E.M. The statistical difference between the mean ulcer index of each treated group versus control was calculated by using ANOVA and Student–Newman–Keuls multiple comparison test.

inhibition (%) = [(UI control − UI treated)/UI control] ×100. 2.5. Histopathological evaluation Stomachs were immersed in a 10% formalin solution for histopathological examination following the assessment of ulcer score. The central part of the damaged or ulcerated tis-

3. Results 3.1. Ethanol-induced ulcerogenesis The results of the in vivo experiments are shown in Table 2. The extracts of the six plants, which were claimed to be effective against ulcer symptoms in Pinarbasi (Kayseri), Turkish folk medicine, demonstrated significant anti-ulcerogenic activity in rats. The aqueous extract of Potentilla reptans showed to possess the highest protective effect with 99.4% inhibition against ethanol-induced ulcerogenesis. Stomachs of five out of six rats of the group treated with Potentilla reptans extract were completely protected from any visible damage. Even the weakest anti-ulcer ogenic effect observed for aerial parts of Sanguisorba minor ssp. muricata was found statistically potent (62.2%) and a stomach of one out of six rats of this group was further completely protected from any visible damage. Stomachs of two out of six rats of aerial parts of Malva neglecta and fruits of Rumex patientia, and three out of six rats of Sideritis caesarea aerial parts were completely protected from any visible damage. However, no rat was completely protected from any visible damage when the Verbascum cheiranthifolium var. cheiranthifolium extract was used.

Table 2 Effects of the plant extracts against gastric lesions induced by ethanol in rats Plant material

Dose (mg/kg)

Ulcer index (mean ± S.E.M.)

Control Malva neglecta Potentilla reptans Rumex patientia Sanguisorba minor ssp. muricata Sideritis caesarea Verbascum cheiranthifolium var. cheiranthifolium Misoprostol

– 1243 870 440 657 960 1227 0.4

155.4 28.1 0.9 27.0 58.7 6.6 27.1 0

*** a

P < 0.001 significant from control; S.E.M.: mean standard error. Number of stomachs completely prevented from any bleeding or lesion.

± ± ± ± ± ± ± ±

32.2 23.9*** 0.9*** 20.5*** 13.3*** 5.5*** 8.9*** 0***

Prevention from ulcera

Inhibition (%)

– 2/6 5/6 2/6 1/6 3/6 0/6 6/6

– 81.9 99.4 82.6 62.2 95.8 82.6 100.0

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3.2. Histopathological results In order to support and confirm the results of the observed pharmacological experiments as well as control and six experimental groups were also further examined by histopathological techniques. For the animal groups which were subjected to Malva neglecta extract, some regenerative improvements were observed in the lamina epithalis. However, some localized parenchymatic degenerations in gland epitheliums in lamina propria or scattered hyperemia in gastric vessels were also seen. In rare cases observed in this group, sheddings in lamina epithelialis and degenerative changes in the glands of lamina propria were noticed. For a group treated with Potentilla reptans extract, all of the stomachs were well protected. However, in two cases, the integrity of lamina epithalis was lost in small areas. There were some changes in lamina epithelialis, occasionally leading to necrosis. Mononuclear cell infiltration and edema were scattered. In lamina propria, vessels under the submucosa and subserosa were hyperemic; scattered microscopical haemorrhagic areas were also observed. Rumex patientia treated extract showed similar findings as in the Malva neglecta sample. Subserosal edema and hyperemia of the vessels were observed. But still, in some cases epithelial loss and degenerations were determined. In Sanguisorba minor ssp. minor extracts, coagulation necroses developed in some cases in lamina epithelialis as well as localized shedding of epithelium. Also, capillary vessels in lamina propria were hyperemic in some observations. In some cases only parenchymatic degeneration of the glands in lamina propria and hyperemia of the vessels were seen. The Sideritis caesarea extract treated group, albeit similar to the findings of the Malva group sample, hyperemia of the vessels and microscopic haemorrhagic areas were found. Finally in the Verbascum cheiranthifolium var cheiranthifolium extract, only a small number of stomachs, it was observed that the lamina epithelialis lost its integrity from place to place and that the lamina propria was opened. Changes from degeneration to necrosis at some of the glands of the lamina propria were observed. Both hyperemias of the vessels as well as microscopic haemorrhagic areas were found. Only a small amount of mononucleared cell infiltrations were considered in these questioned areas.

4. Discussion A literature survey revealed that the investigated plants are frequently employed for other purposes in folk medicine as well: Malva neglecta for the treatment of an abscess (Tabata et al., 1994), hemorrhoids (Yesilada et al., 1995), abdominal pain, common colds, wounds, infertility in women (Sezik et al., 1997), as choleretic and for bruises (Honda et al., 1996); Potentilla reptans as a tonic (Sayar et al., 1995) and an antidiarrheal (Vural et al., 1997); Rumex patientia is used as a digestive and to treat constipation (Baytop, 1999); San-

guisorba minor as a diuretic, as a digestive and appetite stimulant for children (Baytop, 1999); Verbascum species are used for bronchitis and itching (Yagmur, 1995), for pruritic conditions in urogenital organs, for abdominal pain, bronchitis as a desiccant for wounds (Sezik et al., 2001), expectorant, sudorific, sedative, diuretic and an antidiarrheal (Baytop, 1999); Sideritis caeserea was newly introduced to the literature, therefore no ethnobotanical data were obtained so far, but tea prepared from other Sideritis sp. are used commonly as stimulant, carminative and appetite stimulant in Anatolia (Baytop, 1999). A reference survey on the phytochemical composition and pharmacological effects of the titled plants evaluated in this study presented additional information to support the anti-ulcerogenic potential. For example; Sanguisorba minor was shown to possess potent elastase inhibitory activity due to its high tannin content (Lamaison et al., 1990). Several plants containing tannins have been reported to possess antiulcerogenic activity (Yesilada and G¨urb¨uz, 2003). Besides tannins, flavonoids (Villar et al., 1984; Alarcon et al., 1994) are also known as a naturally occurring compounds having gastroprotective effect. Several mechanisms have been proposed to explain their biological effects; including increase of mucosal prostaglandin content, decrease of histamine secretion from mast cells, inhibition of acid secretion and inhibition of Helicobacter pylori growth. In addition, flavonoids have been found to be free radical scavengers; free radicals play an important role in ulcerative and erosive lesions of the gastrointestinal tract (Borrelli and Izzo, 2000). Eventually, tannin and flavonoid content of Potentilla reptans could contribute to its gastroprotective effect (Gruenwald et al., 2000). Rumex patientia possesses laxative, diuretic, antipyretic, wound healing and anti-inflammatory properties according to Turkish folk medicine (Baytop, 1999). Recently, gastroprotective activity of the aqueous extract of Rumex patientia roots on the stress-induced ulcer model was investigated using rats. Results showed that, 500 mg/kg dose of the extract reduced the number of lesions 1.9 times compared to the control group, and decreased the size of lesion areas 1.1 times compared to the control group (S¨uleyman et al., 2002). In a more recent study, roots were found to be effective against indomethacine- and ethanol-induced lesions (S¨uleyman et al., 2004). However, weak activity was reported for Rumex obtusifolius L., a Bolivian anti-ulcer remedy, against ethanolinduced ulcerogenesis (Gonzales et al., 2000). In the present study, the fruit extract of Rumex patientia was investigated and shown to possess a significant anti-ulcer activity. On the other hand, aqueous extract from Rumex patientia roots was also shown to possess an anti-inflammatory effect (S¨uleyman et al., 1999). This might be of particular therapeutic importance as most of the anti-inflammatory drugs used in modern medicine are also ulcerogenic. Because of the colloidal character, mucilaginous plants have the property of covering and protecting the mucosa of the stomach and are used for the treatment of gastric ulcer

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as folk remedy. Mature okra fruit (Hibiscus esculentus L.) is known as a dietary meal, good for gastric irritations, due to its high mucilage content. In a previous report, the potent gastroprotective effect of okra fruits was reported against ethanol-induced ulcerogenesis in rats (G¨urb¨uz et al., 2003). Probably due to the high mucilage content of Malva neglecta leaves, they are used widely for the treatment of stomach ache in many parts of Turkey. Several plants containing high amounts of saponins have been shown to possess anti-ulcer activity in several experimental ulcer models (Yesilada and G¨urb¨uz, 2003). The protective activities of these saponins may be due to the activation of mucous membrane protective factors, inhibition of gastric secretion volume and acid secretion (Saito et al., 1989; Yesilada et al., 2000). Many phytochemical analyses led to the isolation of mucilages, flavonoids, phenylpropanoids and saponins from the inflorencence of some Verbascum species (Atas¨u, 1982; Klimek et al., 1992; Tatli et al., 2004). Consequently, data on the phytochemistry of Verbascum sp. suggests that the investigation of the anti-ulcerogenic activity of the flowers of Verbascum cheiranthifolium var. cheiranthifolium could be a promising approach. Generally, Sideritis sp. are widely used in folk medicine, especially for inflammatory and gastrointestinal disturbances. Their effectiveness was attributed mainly to their antioxidant properties originated from terpenoids and flavonoids in their composition (Triantaphyllou et al., 2001; Koleva et al., 2002). In addition, hypolaetin-8-glycoside was isolated as an anti-inflammatory and anti-ulcerogenic flavonoid from Sideritis mugronensis (Alcaraz and Tordea, 1988). The existence of this flavonoid has also been reported in several other species; Sideritis angustifolia, Sideritis saetabensis, Sideritis mugronensis, and Sideritis leucantha which are used in Spanish folk medicine for inflammatory and alimentary diseases (Barberan et al., 1987). The compound was found to be active on various gastric ulcer models in rats and mice (Alcaraz and Tordea, 1988). This group suggested a cytoprotective effect, which was mediated through endogenous prostaglandins and was involved in the possible mode of action for hypolaetin-8-glycoside. In another study conducted by Zarzuleo et al. (1993), four additional Sideritis species; Sideritis funkiana ssp. funkiana, and ssp. talaverana, Sideritis hirsuta, Sideritis incana var. virgata were studied using indomethacin- and cold plus restraint stress-induced gastric ulcer models and, except for Sideritis junkiana ssp. talaverana, all plants showed significant anti-ulcerogenic activity. Consequently, the potent gastroprotective effect of Sideritis caesarae extract on ethanol-induced ulcerogenesis might be attributed to the existence of similar components, which still needs further evaluation. The results obtained in the present study are mostly in accord with the information compiled by Dioscorides of Anazarbus (today’s Turkey). In a historical point of view, Dioscorides reported that, Rumex patientia, Sideritis sp., Sanguisorba sp., Potentilla reptans and Verbascum sp. have been used for their wound healing and gastroprotective proper-

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ties in folk medicine of Asia Minor for at least 2000 years (Gunther, 1959). The in vivo anti-ulcerogenic activities of five plant remedies (Potentilla reptans, Malva neglecta, Sanguisorba minor ssp. muricata, Sideritis caesarea, Verbascum cheiranthifolium var. cheiranthifolium) investigated in the present study are reported here for the first time to the best of our knowledge. Although, the fruits of Rumex patientia were investigated for the first time in this study, S¨uleyman et al. (2002, 2004) evaluated the gastroprotective activity of the roots. On the other hand, fresh leaves of Malva neglecta and Rumex patientia are used as vegetables in Anatolia widely, and this information gave a clue as to their safety. Our studies on the anti-ulcerogenic activity of the afore-mentioned plant remedies are still in progress for the isolation of active constituent(s) and to elucidate their mode of action. As a consequence of our in vivo anti-ulcerogenic activity screening studies carried out so far, it is noteworthy to point out that all remedies (23 remedies) which were reported to be used for the treatment of stomach ache or peptic ulcer in Turkish folk medicine have been shown to possess potent in vivo anti-ulcerogenic activity (Yesilada et al., 1993; G¨urb¨uz et al., 2002, 2003). This point is extremely important from the viewpoint of reliability of our folkloric records on Turkish folk remedies (Yesilada, 2002). The overall aim of these studies was to contribute to the global search for bioactive natural products as well as to the evaluation of rich tradition and flora in Turkey which is still ongoing with other medicinal plants which have traditional applications as well. Acknowledgement This project is financially supported by the Research Fund of Gazi University (No.: EF.02/2004-06).

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