Journal of Ethnopharmacology 157 (2014) 1–6
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Research Paper
Antiulcer activity of the ethanolic extract and ethyl acetate fraction of the leaves of Markhamia tomentosa in rats Margaret O. Sofidiya a,n, Foluso O. Agunbiade b, Neil A. Koorbanally b, Abimbola Sowemimo a, Dapo Soesan a, Titi Familusi a a b
Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos, Nigeria School of Chemistry, University of KwaZulu-Natal (Westville Campus), Durban, 4000, South Africa
art ic l e i nf o
a b s t r a c t
Article history: Received 5 May 2014 Received in revised form 21 August 2014 Accepted 8 September 2014 Available online 21 September 2014
Ethnopharmacological relevance: The leaves of Markhamia tomentosa (Benth.) K. Schum. Ex Engl. (Bignoniaceae) are used traditionally in the treatment of skin afflictions, sores, ulcers and inflammation. The aim of the study was to investigate the antiulcer activity of the crude ethanolic extract from the leaves of Markhamia tomentosa, determine the active fraction(s) of the extract and identify the chemical constituents in the active fraction by LC–MS. Materials and methods: The antiulcer activity of the crude extract (50, 100 and 150 mg/kg, p.o.) was evaluated in ethanol and indomethacin-induced models while the solvent fractions (150 mg/kg) were screened using ethanol-induced gastric lesions in rats. Furthermore, anti-ulcer activity of the active fraction (50, 100 and 150 mg/kg, p.o.) was performed using indomethacin and pylorus ligation models. Parameters such as gastric volume, pH and acidity were determined in the pylorus ligation model. LC– ESI–MS analysis was used to identify the components in the active fraction. Results: The extract at the dose of 50, 100 and 150 mg/kg caused a significant (po0.05) dose-dependent inhibition of ulcer in the ethanol and indomethacin-induced ulcer models, respectively. The ethyl acetate (EtOAc) fraction showed the most potent antiulcer activity from all the fractions tested. This fraction produced 72% and 92% inhibition of indomethacin and pylorus-induced ulcer at a dose of 150 mg/kg respectively. Acteoside, luteolin, luteolin-7-rutinoside, Luteolin-30 ,7-di-O-glucoside, carnosol, dilapachone, tormentic acid, oxo-pomolic acid and ajugol were detected in the EtOAc fraction. Conclusion: Our data provide a rational base for the folkloric use of Markhamia tormentosa in the treatment of ulcers. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Markhamia tomentosa Bignoniaceae Antiulcer LC–MS analysis
1. Introduction The focus on plant research has increased globally and experimental evidence has been provided to show the potential medicinal plants have in various traditional systems (Dahanuka et al., 2002). The use of medicinal plant in the treatment of symptoms related to gastric ulcer is also common in traditional medicine worldwide, with a large number of these plants shown to have gastroprotective effects (Borrelli and Izzo, 2000; Goel and Sairam, 2002;Falc~ao et al., 2008; Awaad et al., 2013). However, the potential of most of the
n
Corresponding author. Tel.: þ 234 8033356197. E-mail addresses: toyin_sofi[email protected] (M.O. Sofidiya), [email protected] (F.O. Agunbiade), [email protected] (N.A. Koorbanally), [email protected] (A. Sowemimo), [email protected] (D. Soesan), [email protected] (T. Familusi). http://dx.doi.org/10.1016/j.jep.2014.09.012 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
plants especially in developing countries as sources for new antiulcer drugs is still largely unexplored. Markhamia tomentosa (Benth.) K. Schum. Ex Engl. (Bignoniaceae) is one of the plants that has been unexplored. The plant is reputed for its medicinal use in Africa and is reported to be effective in treating skin-afflictions, sores, ulcers and inflammation in Nigeria and Cote d'Ivoire traditional folk medicine (Burkill, 1985). The pulped leaves are applied as a wet dressing under a bandage to sores (Bouquet and Debray, 1974). The leaf decoction is administered as diuretic medicine for edema of the legs, elephantiasis of the scrotum and for rheumatic pain (Burkill, 1985). The plant has been described for its antimicrobial (Aladesanmi et al., 2007; Ugbabe et al., 2010), and anti-inflammatory properties (Temdie et al., 2012; Sowemimo et al., 2013). Ibrahim et al. (2013) evaluated the cytotoxic effect and underlying mechanisms of the ethanolic extract of Markhamia tomentosa on HeLa and MCF-7 cancer cell lines and non-cancerous Vero cell lines. The plant has also been screened for acetylcholinesterase and butyrylcholinesterase inhibitory
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M.O. Sofidiya et al. / Journal of Ethnopharmacology 157 (2014) 1–6
activity (Elufioye et al., 2010) and for its effect against Aedes aegypti (Adebajo et al., 2012). The aim of this study was to investigate the antiulcer activity of the crude ethanolic extract of the leaves, determine the active fraction(s) of this extract and conduct LC–MS identification of the components in the active fraction.
2. Materials and methods 2.1. Plant collection and preparation of extract and fractions Fresh leaves of Markhamia tomentosa were collected from Ikire in Osun state, Nigeria in November, 2011. Identification and authentication were carried out in the Herbarium of the Department of Botany, University of Lagos where a voucher specimen was deposited (LUH 4593). The plant was dried in a hot air oven at 40 1C and pulverized in a mechanical grinder. The powdered material (1 kg) was macerated with absolute ethanol for 72 h at room temperature. The resulting extract was filtered and evaporated to dryness on a water bath at 40 1C to yield 15.4% (w/w). The extract was dissolved in just enough water to be loaded on to a column and fractionated by different solvents of increasing polarity using hexane, dichloromethane, ethyl acetate and butanol. The weight of the hexane (HEX), dichloromethane (DCM), ethyl acetate (EtOAc), butanol (BUT) and aqueous (AQU) fractions obtained were 19.48 g, 46.98 g, 20.66 g, 12.99 g, and 7.33 g, respectively. 2.2. Animals Male Albino Wistar rats (150–200 g) were obtained from the Laboratory Animal Centre of the National Agency for Food and Drugs Administration and Control, Yaba, Lagos, Nigeria. All animals were acclimatized for two weeks, under standard environmental conditions (23–25 1C, 12 h/12 h light/dark cycle). They were fed a rodent diet (Livestock Feeds PLC, Ibadan, Oyo State, Nigeria) and had free access to drinking water. The research protocol was approved by the Institutional Ethical Committee of the College of Medicine of the University of Lagos, Lagos, Nigeria (CM/COM/08/ VOL.XXV). 2.3. Evaluation of antiulcer activity 2.3.1. Ethanol induced gastric ulcer model The procedures for ethanol-induced ulcers were an adaptation of the method of Hollander et al. (1985). After a 24-h fast, rats were divided into 5 groups of 7 animals and treated orally with distilled water (10 ml/kg), misoprostol (0.1 mg/kg) and extract (50, 100 and 150 mg/kg). Sixty minutes after this procedure, every animal received absolute ethanol (1 ml/200 g). One hour later the rats were euthanized, stomach removed, opened along the greater curvature and ulcer score was determined using the Magistretti scoring scale (Magistretti et al., 1988). This procedure was also used for the screening of the solvent fractions at the dose of 150 mg/kg. 2.3.2. Indomethacin-induced gastric ulcer model The modified method of Kakub and Gulfraz (2007) was employed. Rats were fasted for 24 h and treated orally with vehicle (distilled water, 10 ml/kg), extract (50, 100 and 150 mg/kg), EtOAc fraction (50, 100 and 150 mg/kg) and omeprazole (100 mg/kg). One hour after the treatment, 60 mg/kg of indomethacin was administered orally to all the groups. Four hours later, the animals were sacrificed by cervical dislocation, stomachs removed, opened along the greater curvature and washed with normal saline. For
the determination of ulcer index, scores were attributed as described by Magistretti et al. (1988). 2.3.3. Pylorus ligation-induced gastric ulcer model Gastric antisecretory activity of the EtOAc fraction was evaluated using the method of de-Faria et al. (2012) with a few modifications. After 24 h of fasting, the animals were divided into groups (n¼ 5) and received the EtOAc fraction at a dose of 50, 100 and 150 mg/kg. The positive group was given cimetidine (100 mg/kg) and distilled water (10 ml/kg) was administered as negative control. All the samples were administered orally. After 1 h, the animals were anesthetized, abdomen incised and the pylorus ligated. Four hours later, the animals were sacrificed by cervical dislocation, the stomachs were removed and the gastric content collected and drained into a graduated centrifuge tube and centrifuged at 2000 rpm for 15 min. The supernatant volume was measured and the pH recorded with a digital pH meter (Elico, Hyderabad, India). Total acidity was determined by titrating with 0.01 N NaOH using phenolphthalein as indicator. 2.4. Statistical analysis The results were expressed as mean 7SEM. Statistical differences between control and test groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's or Tukey's test. Statistical calculations were carried out using GraphPad Prisms 5.0 (Graphpad Software, San Diego, USA). 2.5. Liquid chromatography-mass spectra (LC–MS) analysis of EtOAc fraction from Markhamia tomentosa LC–MS analysis was performed using an Agilent 1200 Series LC/ESI/MSD (Agilent Corp., Wilmington, DE) system equipped with a quaternary pump, diode array detector (DAD), thermostatted column compartment, degasser, auto-sampler Agilent 1100 series MSD ion trap with an electrospray ion source (ESI) and ChemStation software, Bruker Daltonics 4.2 software. The electrospray ion mass spectrometer (ESI–MS) was operated in the negative ion mode and an optimized collision energy level of 60% and scanned from m/z 50 to 2200 in an auto MS mode. ESI was conducted using a needle voltage of 3.5 kV. Nitrogen collision induced dissociation was achieved in a nebulizer set at 55 psi with the nitrogen dry gas at a flow rate of 10 L/min; capillary temperature was 350 1C. The ESI interface and mass spectrometer parameters were optimized to obtain maximum sensitivity. An Agilent C18 reverse phase column (150 4.6 mm, 5 mm) was used throughout this study and the column temperature was set at 35 1C. The mobile phase was a binary solvent system consisting of solvent A (Millipore water from an Elix MilliPore Water system with 0.1% formic acid) and solvent B (100% CH3CN, HPLC Chromasolvs grade). The gradient program used was as follows: 0–10 min, 10% B; 10–15 min, 30% B; 15–20 min, 60% B; 20–30 min, 90% B; 10% B for final washing and equilibration of the column for the next run. The flow rate was 0.5 ml/min and the injection volume was 10 ml. The identification of components present in the EtOAc fraction was based on comparison of the mass spectral data by computer matching with NIST Libraries, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature. Information from literature on the botanical family (Bignoniaceae) was found useful in the characterization process. 3. Results and discussion In order to demonstrate the potential antiulcer effect of the crude extract obtained from Markhamia tomentosa, ethanol and
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Table 1 Effect of the crude extract of Markhamia tomentosa on ethanol-induced ulcer in rats.
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Table 3 Effect of the crude extract and fractions (fr) of Markhamia tomentosa on ethanol induced ulcer.
Treatment
Dose (mg/kg)
Mean ulcer index 7S.E.M
Percentage ulcer inhibition
Treatment
Dose (mg/kg)
Mean ulcer score7 SEM
Ulcer index
% Protection
Control Extract
10 ml/kg 50 100 150 0.1
7.50 7 0.74 2.86 7 0.69nnn 1.217 0.66nnn 0.83 7 0.32nnn 0.337 0.25nnn
– 61.71 83.81 88.93 95.60
Control Crude AQU fr DCM fr BUT fr HEX fr EtOAc fr Misoprostol
10(ml/kg) 150 150 150 150 150 150 0.1
4.7 7 1.39 1.2 7 0.26n 4.8 7 0.58 2.2 7 0.96 1.7 7 1.02 4.17 0.95 0.6 7 0.19nn 1.3 7 0.41n
4.70 1.20 4.80 2.20 1.02 4.10 0.48 1.30
– 74.47 2.13 53.19 78.30 12.77 89.79 72.34
Misoprostol
Values are mean7 S.E.M, n¼ 7. nnn
p o 0.001 compared to control (One-way ANOVA followed by Tukey's test).
Values are expressed as mean 7 S.E.M. (n ¼5). Table 2 Effect of the crude extract of Markhamia tomentosa on indomethacin- induced ulcer in rats. Treatment
Dose (mg/kg)
Mean ulcer index7 S.E.M
Percentage ulcer inhibition
Control Extract
10 ml/kg 50 100 150 0.1
4.50 7 0.79 1.647 0.30nnn 1.29 7 0.46nnn 0.79 7 0.34nnn 0.36 7 0.18nnn
– 63.49 71.43 82.54 92.06
Omeprazole
Values are mean7 S.E.M, n¼ 7. nnn
p o 0.001 compared to control (One-way ANOVA) followed by Tukey's multiple comparison test.
n
p o 0.05. po 0.01 are different, when compared to the control group. (One-way ANOVA followed by Dunnett's test). nn
Table 4 Effect of the EtOAc fraction of Markhamia tomentosa on indomethacininduced ulcer. Treatment
Dose (mg/kg)
Mean ulcer score7SEM
Ulcer index
% Protection
Control EtOAc
10 (ml/kg) 50 100 150 100
6.2 7 1.11 4.5 7 1.29 2.2 7 1.13n 1.7 7 0.37n 0.0 7 0.0nn
6.20 3.60 1.76 1.70 0
– 41.94 71.61 72.58 100
Omeprazole
indomethacin-induced antiulcer models were employed. Both models have been widely used for the evaluation of gastroprotective activity. Ethanol is considered one of the agents that induce gastric ulcers. The effects of ethanol on the gastric mucosa may occur as a result of stasis in gastric blood flow, which contributes to the development of the hemorrhagic and necrotic aspects of tissue injury (Guth et al., 1984). Occurrence of these ulcers, which is predominant in the glandular part of the stomach, was reported to stimulate the formation of reactive oxygen species (ROS), resulting in damage to rat gastric mucosa (Gupta et al., 2013). The oral administration of absolute ethanol produced severe ulceration in the stomach of the control rats (Table 1). The extract at the dose of 50, 100 and 150 mg/kg exhibited a significant (p o0.001) reduction of gastric lesions with 61.71%, 83.81% and 88.93% inhibition, respectively when compared to the control group. This showed that the extract was effective in preventing ethanol-induced gastric lesion, which suggests its cytoprotective effect. Similar findings exist in the literature, where plant extracts have been shown to prevent gastric mucosal ulceration in rats using this model (Mahmood et al., 2011; Al-Rejaie et al., 2012; ). Moreover, the study by Rajasekaran, 2014 demonstrated that Kigelia pinnata, a species in the family Bignoniaceae significantly reduced the ulcer lesion index produced by ethanol in a dose dependent manner. The use of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and indomethacin is considered to be the major risk factor in gastric ulcers. The mechanisms suggested for the gastric damage caused by NSAIDs are inhibition of prostaglandin synthesis through cyclooxygenase-1 block and inhibition of epithelial cell proliferation in the ulcer margin, which is critical for the re-epithelization of the ulcer (Levi et al., 1990). Many taxa of medicinal plants have been investigated for their antiulcerogenic effect using this model. Bhattacharya et al. (2007) reported the healing effect of some Indian medicinal plants against indomethacin-induced gastric ulceration in rats. In this model, pretreatment of the extract (50, 100 and 150 mg/kg) offered significant (po0.001) protection to the gastric mucosa against ulceration (Table 2). The percentage of ulcer inhibition was 63.49%, 71.43%,
Values are mean 7 S.E.M. (n ¼5). n
p o 0.05. po 0.01 significantly different, when compared to the control group (Oneway ANOVA followed by Tukey's test). nn
82.54%, respectively compared to the control. The gastroprotective activity of the extract suggests a prostaglandin dependent effect. The view is supported by the fact that prostaglandins normally serve as protective function in stomach by maintaining gastric microcirculation and causes gastric secretion of bicarbonate and mucus (Nwafor et al., 2000). Based on these results, the solvent fractions from the crude extract were evaluated for antiulcer activity at the dose of 150 mg/ kg using ethanol-induced gastric ulcer model in rats. The results showed that the aqueous fraction did not exhibit anti-ulcerogenic effects (Table 3). The HEX, DCM, EtOAc and BUT fractions reduced ulcer lesion by 12.77%, 53.19%, 89.79% and 78.30% respectively. Among these, the EtOAc fraction was found to be the most active and thus tested in a dose-dependent evaluation using indomethacin and pylorus ligation models. In the indomethacin-induced assay, 50, 100 and 150 mg/kg doses of the EtOAc fraction were effective, exhibiting significant differences from the control group; the highest percentage of antiulcerogenic effect was at 150 mg/kg (72.58%) (Table 4). Oral administration of omeprazole at 100 mg/kg produced 100% protection. These data suggest that the EtOAc fraction produced gastroprotective effect, since it significantly reduced indomethacininduced ulcers. This further supports cytoprotective effects of the fraction, which may be mediated by prostaglandins. The activity of the EtOAc fraction was also evaluated by pyloric ligature-induced gastric ulcers in a rat model using parameters including ulcer index, gastric volume, gastric pH and total acidity. The causes of gastric ulcer by pyloric ligation are believed to be due to accumulation of gastric acid and pepsin leading to a breakdown of the gastric mucosal barrier (Gharate and Kasture, 2013). The ligation of the pyloric end of the stomach causes accumulation of gastric acid in the stomach. This increase in the
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Table 5 Effect of the EtOAc fraction of Markhamia tomentosa on pylorus ligation induced ulcer. Treatment
Dose (mg/kg)
Ulcer index 7 S.E.M
% Protection
Gastric volume (ml)
Gastric pH
Gastric acidity (mEq/L)
Control EtOAc fr
10(ml/kg) 50 100 150 100
2.007 0.62 1.08 70.20 0.727 0.25 0.167 0.25 0.48 70.37
– 45.83n 64.00n 92.00nn 76.00n
0.82 70.03 0.53 70.06n 0.32 70.09nnn 0.48 70.11n 0.3570.09nn
2.917 0.18 4.89 7 0.86 4.317 0.76 4.42 7 0.40 5.617 0.30n
10.40 71.04 9.50 70.95 14.80 71.48 8.80 70.88 9.00 70.90
Cimetidine
Values are mean7 S.E.M. (n¼ 5). n
po 0.05. p o0.01. nnn p o0.001 significantly different when compared to the control group. (One-way ANOVA followed by Dunnett's test). nn
Fig. 1. HPLC finger print of the ethylacetate fraction from Markhamia tomentosa. The chromato-grams were recorded at 280 nm.
Table 6 LC–MS/MS identification of compounds in ethylacetate fraction of Markhamia tomentosa. Peak no.
Rt (min)
Msn
Ms2
Identification
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
11.3 12.0 12.6 14.6 14.7 16.1 16.9 17.6 18.5 19.4 19.8 19.9 20 20.2 20.7 21.7 23 23.1 24 25.6
623 623 623 714 609 593 285 363 329 347 347 665 665 635 635 487 483 503.6 487.7 485.7
461(100); 462(17); 315(1.7) 461(100); 462(14); 315(1.7) 461(100); 462(14) 667(100) 285 (100); 447(35) 285(100); 447(35); 286 (14) 285 (100) 363 (100); 345(87); 301(8) 171(100); 311(48); 229(48); 293(38); 211(32) 347(100) 347(100) 633(100); 634(22); 631(12) 633(100); 634(82) No daughter ion 603(100); 604(33) 469(100); 423(34); 25(27); 470(22); 467(14) 241(100); 242(13); 221(9) 471(100); 472(20); 469(9) 469(100); 470(27) 467(100); 468(24)
Acteoside Acteoside Isoacteoside Not identified Luteolin-30 ,7-di-O-glucoside Luteolin-7-rutinoside Luteolin Dimethanol adduct of quercetin Carnosol Ajugol Ajugol Not identified Not identified Not identified Not identified Tormentic acid Dilapachone Pomolic acid derivative Tormentic acid isomer 2-oxo-pomolic acid
Li et al. (2005) Li et al. (2005) Li et al. (2005) – Dall'Acqua et al. (2011); Cvetkovikj et al. (2013) Abu-Reidah et al. (2013); Cvetkovikj et al. (2013) Cvetkovikj et al. (2013) Qiao et al. (2014) Zimmermann et al. (2011) Won et al. (2010) Won et al. (2010) – – – – Li et al. (2009); Gao et al. (2011) Jassbi et al. (2004) – – Gao et al. (2011)
gastric acid secretion causes ulcers in the stomach (Dhuley, 1999). The data in Table 5 showed that oral administration of the EtOAc fraction in the doses of 50, 100 and 150 mg/kg produced a significant reduction in the ulcer index, 1.08 70.20 (45.83%
protection), 0.72 70.25 (64.00% protection) and 0.16 70.25 (92.00% protection), respectively. The gastric volume was reduced significantly compared to the control. A non-dose dependent and non-significant increase in
M.O. Sofidiya et al. / Journal of Ethnopharmacology 157 (2014) 1–6
gastric pH was observed compared to the control. All tested doses of the fraction did not produce significant reduction in gastric acidity compared to the control. Cimetidine at 100 mg/kg significantly inhibited the formation of ulcers, reduced gastric juice volume and increased gastric pH. This data is similar to the results obtained with the ethanolic crude extract of Rosmarinus officinalis (Dias et al., 2000) and Casearia sylvestris (Esteves et al., 2005). Similarly, Santin et al., 2010 have reported that the parameters of gastric secretion (pH, volume, gastric acidity) showed no alteration in the different doses of Achyrocline satureoides administered to rats. Therefore, the protection offered by EtOAc fraction in the pylorus ligation experiment may not be due to an acid neutralizing effect. The chemical constituents in the active ethyl acetate fraction of Markhamia tomentosa were characterized by electrospray ionization mass spectrometry. The chromatogram is shown in Fig. 1, while Table 6 shows the retention times and spectral data in the negative ion mode. The identified compounds include some phenolic compounds (acteoside, luteolin, luteolin-7-rutinoside, luteolin-30 ,7-di-Oglucoside), terpenoids (carnosol, dilapachone, tormentic acid, oxopomolic acid) and iridiod, ajugol. Among these compounds, acteoside and ajugol appear to be the major components of this fraction. Peaks 4 and 12–15 could not be identified based on the available data. Further isolation and identification by NMR spectroscopy is required to determine the identity of these molecules. Three of the mentioned compounds have been reported previously in the genus Markhamia. Acteoside, also known as verbascoside was reported in Markhamia lutea (Kernan et al., 1998) while tormentic acid was reported from the stem bark of Markhamia tomentosa (Tantangmo et al., 2010) and from Malpighia obtusifolia (Nchu et al., 2010). The iridoid, ajugol has previously been isolated from related species, Markhamia stipulata (Kanchanapoom et al., 2002). A literature search for antiulcer molecules revealed that antiulcer activity is not confined to one class of compounds. Compounds identified in this fraction such as luteolin has been reported to have antiulcer property. Moreover, the anti-secretory potential of acteoside isolated from Tectona grandis has been demonstrated (Singh et al., 2010). The presence of these compounds, including others identified in the EtOAc fraction could be responsible for the observed antiulcer activity. These findings indicate that Markhamia tomentosa possesses an anti-ulcer effect, possibly through a cytoprotective mechanism, corroborating its use in traditional medicine and contributing to its pharmacological validation.
Acknowledgments The authors are thankful to the School of Chemistry and Physics, University of KwaZulu-Natal, South Africa for providing the necessary facilities for LC-MS data acquisition. The assistance of Dr. Seyi Aboyade, Mrs. Mutiat Ibrahim and Toyin Adetunde is acknowledged.
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Journal of Medicinal Plants Research 5 (23), 5577–5583. Nchu, F., Aderogba, M.A., Mdee, L.K., Eloff, J.N., 2010. Isolation of anti-Candida albicans compounds from Markhamia obtusifolia (Baker) Sprague (Bignoniaceae). South African Journal of Botany 76, 54–57. Nwafor, P.A., Okwuasaba, F.K., Binda, L.G., 2000. Antidiarrhoeal and antiulcerogenic effects of the methanolic extract of Asparagus pubescens root in rats. Journal of Ethnopharmacology 72, 421–427. Rajasekaran, S., 2014. Anti-ulcer activity of different extract of Kigelia pinnata in experimental animals. Asian Journal of Pharmaceutical and Clinical Research 7, 80–83. Santin, J.R., Lemos, M., Klein Junior, L.C., Niero, R., de Andrade, S.F., 2010. Antiulcer effects of Achyrocline satureoides (Lam.) DC (Asteraceae) (Marcela), a folk medicine plant, in different experimental models. Journal of Ethnopharmacology 130, 334–339. Singh, N., Shukl, N., Singh, P., Sharma, R., Rajendran, S.M., Maurya, R., Palit, G., 2010. Verbascoside isolated from Tectona grandis mediates gastric protection in rats via inhibiting proton pump activity. Fitoterapia 81 (7), 755–761.
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Sowemimo, A., Samuel, F., Fageyinbo, M.S., 2013. Anti-inflammatory activity of Markhamia tomentosa (Benth.) K. Schum. Ex Engl. ethanolic leaf extract. Journal of Ethnopharmacology 149, 191–194. Tantangmo, F., Lenta, B.N., Boyom, F.F., Ngouela, S., Kaiser, M., Tsamo, E., Weniger, B., Rosenthal, P.J., Vonthron-Sénécheau, C., 2010. Antiprotozoal activities of some constituents of Markhamia tomentosa (Bignoniaceae). Annals of Tropical Medicine and Parasitology 104 (5), 391–398.
Temdie, R.J.G., Fotio, L.A., Dimo, T., Beppe, J.G., Tsague, M., 2012. Analgesic and antiinflammatory effects of the leaves of Markhamia tomentosa (Benth.) K.Schum. (Bignoniaceae). Pharmacologia 3, 565–573. Ugbabe, G.E., Ayodele, A.E., Ajoku, G.A., Kunle, O.F., Kolo, I., Okogun, J.I., 2010. Preliminary phytochemical and antimicrobial analyses of the leaves of Nigerian Bignoniaceae Juss. Global Research Journals 1 (1), 001–005.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
Antiulcer activity of the ethanolic extract and ethyl acetate fraction of the leaves of Markhamia tomentosa in rats Margaret O. Sofidiya a,n, Foluso O. Agunbiade b, Neil A. Koorbanally b, Abimbola Sowemimo a, Dapo Soesan a, Titi Familusi a a b
Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos, Nigeria School of Chemistry, University of KwaZulu-Natal (Westville Campus), Durban, 4000, South Africa
art ic l e i nf o
a b s t r a c t
Article history: Received 5 May 2014 Received in revised form 21 August 2014 Accepted 8 September 2014 Available online 21 September 2014
Ethnopharmacological relevance: The leaves of Markhamia tomentosa (Benth.) K. Schum. Ex Engl. (Bignoniaceae) are used traditionally in the treatment of skin afflictions, sores, ulcers and inflammation. The aim of the study was to investigate the antiulcer activity of the crude ethanolic extract from the leaves of Markhamia tomentosa, determine the active fraction(s) of the extract and identify the chemical constituents in the active fraction by LC–MS. Materials and methods: The antiulcer activity of the crude extract (50, 100 and 150 mg/kg, p.o.) was evaluated in ethanol and indomethacin-induced models while the solvent fractions (150 mg/kg) were screened using ethanol-induced gastric lesions in rats. Furthermore, anti-ulcer activity of the active fraction (50, 100 and 150 mg/kg, p.o.) was performed using indomethacin and pylorus ligation models. Parameters such as gastric volume, pH and acidity were determined in the pylorus ligation model. LC– ESI–MS analysis was used to identify the components in the active fraction. Results: The extract at the dose of 50, 100 and 150 mg/kg caused a significant (po0.05) dose-dependent inhibition of ulcer in the ethanol and indomethacin-induced ulcer models, respectively. The ethyl acetate (EtOAc) fraction showed the most potent antiulcer activity from all the fractions tested. This fraction produced 72% and 92% inhibition of indomethacin and pylorus-induced ulcer at a dose of 150 mg/kg respectively. Acteoside, luteolin, luteolin-7-rutinoside, Luteolin-30 ,7-di-O-glucoside, carnosol, dilapachone, tormentic acid, oxo-pomolic acid and ajugol were detected in the EtOAc fraction. Conclusion: Our data provide a rational base for the folkloric use of Markhamia tormentosa in the treatment of ulcers. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Markhamia tomentosa Bignoniaceae Antiulcer LC–MS analysis
1. Introduction The focus on plant research has increased globally and experimental evidence has been provided to show the potential medicinal plants have in various traditional systems (Dahanuka et al., 2002). The use of medicinal plant in the treatment of symptoms related to gastric ulcer is also common in traditional medicine worldwide, with a large number of these plants shown to have gastroprotective effects (Borrelli and Izzo, 2000; Goel and Sairam, 2002;Falc~ao et al., 2008; Awaad et al., 2013). However, the potential of most of the
n
Corresponding author. Tel.: þ 234 8033356197. E-mail addresses: toyin_sofi[email protected] (M.O. Sofidiya), [email protected] (F.O. Agunbiade), [email protected] (N.A. Koorbanally), [email protected] (A. Sowemimo), [email protected] (D. Soesan), [email protected] (T. Familusi). http://dx.doi.org/10.1016/j.jep.2014.09.012 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
plants especially in developing countries as sources for new antiulcer drugs is still largely unexplored. Markhamia tomentosa (Benth.) K. Schum. Ex Engl. (Bignoniaceae) is one of the plants that has been unexplored. The plant is reputed for its medicinal use in Africa and is reported to be effective in treating skin-afflictions, sores, ulcers and inflammation in Nigeria and Cote d'Ivoire traditional folk medicine (Burkill, 1985). The pulped leaves are applied as a wet dressing under a bandage to sores (Bouquet and Debray, 1974). The leaf decoction is administered as diuretic medicine for edema of the legs, elephantiasis of the scrotum and for rheumatic pain (Burkill, 1985). The plant has been described for its antimicrobial (Aladesanmi et al., 2007; Ugbabe et al., 2010), and anti-inflammatory properties (Temdie et al., 2012; Sowemimo et al., 2013). Ibrahim et al. (2013) evaluated the cytotoxic effect and underlying mechanisms of the ethanolic extract of Markhamia tomentosa on HeLa and MCF-7 cancer cell lines and non-cancerous Vero cell lines. The plant has also been screened for acetylcholinesterase and butyrylcholinesterase inhibitory
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activity (Elufioye et al., 2010) and for its effect against Aedes aegypti (Adebajo et al., 2012). The aim of this study was to investigate the antiulcer activity of the crude ethanolic extract of the leaves, determine the active fraction(s) of this extract and conduct LC–MS identification of the components in the active fraction.
2. Materials and methods 2.1. Plant collection and preparation of extract and fractions Fresh leaves of Markhamia tomentosa were collected from Ikire in Osun state, Nigeria in November, 2011. Identification and authentication were carried out in the Herbarium of the Department of Botany, University of Lagos where a voucher specimen was deposited (LUH 4593). The plant was dried in a hot air oven at 40 1C and pulverized in a mechanical grinder. The powdered material (1 kg) was macerated with absolute ethanol for 72 h at room temperature. The resulting extract was filtered and evaporated to dryness on a water bath at 40 1C to yield 15.4% (w/w). The extract was dissolved in just enough water to be loaded on to a column and fractionated by different solvents of increasing polarity using hexane, dichloromethane, ethyl acetate and butanol. The weight of the hexane (HEX), dichloromethane (DCM), ethyl acetate (EtOAc), butanol (BUT) and aqueous (AQU) fractions obtained were 19.48 g, 46.98 g, 20.66 g, 12.99 g, and 7.33 g, respectively. 2.2. Animals Male Albino Wistar rats (150–200 g) were obtained from the Laboratory Animal Centre of the National Agency for Food and Drugs Administration and Control, Yaba, Lagos, Nigeria. All animals were acclimatized for two weeks, under standard environmental conditions (23–25 1C, 12 h/12 h light/dark cycle). They were fed a rodent diet (Livestock Feeds PLC, Ibadan, Oyo State, Nigeria) and had free access to drinking water. The research protocol was approved by the Institutional Ethical Committee of the College of Medicine of the University of Lagos, Lagos, Nigeria (CM/COM/08/ VOL.XXV). 2.3. Evaluation of antiulcer activity 2.3.1. Ethanol induced gastric ulcer model The procedures for ethanol-induced ulcers were an adaptation of the method of Hollander et al. (1985). After a 24-h fast, rats were divided into 5 groups of 7 animals and treated orally with distilled water (10 ml/kg), misoprostol (0.1 mg/kg) and extract (50, 100 and 150 mg/kg). Sixty minutes after this procedure, every animal received absolute ethanol (1 ml/200 g). One hour later the rats were euthanized, stomach removed, opened along the greater curvature and ulcer score was determined using the Magistretti scoring scale (Magistretti et al., 1988). This procedure was also used for the screening of the solvent fractions at the dose of 150 mg/kg. 2.3.2. Indomethacin-induced gastric ulcer model The modified method of Kakub and Gulfraz (2007) was employed. Rats were fasted for 24 h and treated orally with vehicle (distilled water, 10 ml/kg), extract (50, 100 and 150 mg/kg), EtOAc fraction (50, 100 and 150 mg/kg) and omeprazole (100 mg/kg). One hour after the treatment, 60 mg/kg of indomethacin was administered orally to all the groups. Four hours later, the animals were sacrificed by cervical dislocation, stomachs removed, opened along the greater curvature and washed with normal saline. For
the determination of ulcer index, scores were attributed as described by Magistretti et al. (1988). 2.3.3. Pylorus ligation-induced gastric ulcer model Gastric antisecretory activity of the EtOAc fraction was evaluated using the method of de-Faria et al. (2012) with a few modifications. After 24 h of fasting, the animals were divided into groups (n¼ 5) and received the EtOAc fraction at a dose of 50, 100 and 150 mg/kg. The positive group was given cimetidine (100 mg/kg) and distilled water (10 ml/kg) was administered as negative control. All the samples were administered orally. After 1 h, the animals were anesthetized, abdomen incised and the pylorus ligated. Four hours later, the animals were sacrificed by cervical dislocation, the stomachs were removed and the gastric content collected and drained into a graduated centrifuge tube and centrifuged at 2000 rpm for 15 min. The supernatant volume was measured and the pH recorded with a digital pH meter (Elico, Hyderabad, India). Total acidity was determined by titrating with 0.01 N NaOH using phenolphthalein as indicator. 2.4. Statistical analysis The results were expressed as mean 7SEM. Statistical differences between control and test groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's or Tukey's test. Statistical calculations were carried out using GraphPad Prisms 5.0 (Graphpad Software, San Diego, USA). 2.5. Liquid chromatography-mass spectra (LC–MS) analysis of EtOAc fraction from Markhamia tomentosa LC–MS analysis was performed using an Agilent 1200 Series LC/ESI/MSD (Agilent Corp., Wilmington, DE) system equipped with a quaternary pump, diode array detector (DAD), thermostatted column compartment, degasser, auto-sampler Agilent 1100 series MSD ion trap with an electrospray ion source (ESI) and ChemStation software, Bruker Daltonics 4.2 software. The electrospray ion mass spectrometer (ESI–MS) was operated in the negative ion mode and an optimized collision energy level of 60% and scanned from m/z 50 to 2200 in an auto MS mode. ESI was conducted using a needle voltage of 3.5 kV. Nitrogen collision induced dissociation was achieved in a nebulizer set at 55 psi with the nitrogen dry gas at a flow rate of 10 L/min; capillary temperature was 350 1C. The ESI interface and mass spectrometer parameters were optimized to obtain maximum sensitivity. An Agilent C18 reverse phase column (150 4.6 mm, 5 mm) was used throughout this study and the column temperature was set at 35 1C. The mobile phase was a binary solvent system consisting of solvent A (Millipore water from an Elix MilliPore Water system with 0.1% formic acid) and solvent B (100% CH3CN, HPLC Chromasolvs grade). The gradient program used was as follows: 0–10 min, 10% B; 10–15 min, 30% B; 15–20 min, 60% B; 20–30 min, 90% B; 10% B for final washing and equilibration of the column for the next run. The flow rate was 0.5 ml/min and the injection volume was 10 ml. The identification of components present in the EtOAc fraction was based on comparison of the mass spectral data by computer matching with NIST Libraries, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature. Information from literature on the botanical family (Bignoniaceae) was found useful in the characterization process. 3. Results and discussion In order to demonstrate the potential antiulcer effect of the crude extract obtained from Markhamia tomentosa, ethanol and
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Table 1 Effect of the crude extract of Markhamia tomentosa on ethanol-induced ulcer in rats.
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Table 3 Effect of the crude extract and fractions (fr) of Markhamia tomentosa on ethanol induced ulcer.
Treatment
Dose (mg/kg)
Mean ulcer index 7S.E.M
Percentage ulcer inhibition
Treatment
Dose (mg/kg)
Mean ulcer score7 SEM
Ulcer index
% Protection
Control Extract
10 ml/kg 50 100 150 0.1
7.50 7 0.74 2.86 7 0.69nnn 1.217 0.66nnn 0.83 7 0.32nnn 0.337 0.25nnn
– 61.71 83.81 88.93 95.60
Control Crude AQU fr DCM fr BUT fr HEX fr EtOAc fr Misoprostol
10(ml/kg) 150 150 150 150 150 150 0.1
4.7 7 1.39 1.2 7 0.26n 4.8 7 0.58 2.2 7 0.96 1.7 7 1.02 4.17 0.95 0.6 7 0.19nn 1.3 7 0.41n
4.70 1.20 4.80 2.20 1.02 4.10 0.48 1.30
– 74.47 2.13 53.19 78.30 12.77 89.79 72.34
Misoprostol
Values are mean7 S.E.M, n¼ 7. nnn
p o 0.001 compared to control (One-way ANOVA followed by Tukey's test).
Values are expressed as mean 7 S.E.M. (n ¼5). Table 2 Effect of the crude extract of Markhamia tomentosa on indomethacin- induced ulcer in rats. Treatment
Dose (mg/kg)
Mean ulcer index7 S.E.M
Percentage ulcer inhibition
Control Extract
10 ml/kg 50 100 150 0.1
4.50 7 0.79 1.647 0.30nnn 1.29 7 0.46nnn 0.79 7 0.34nnn 0.36 7 0.18nnn
– 63.49 71.43 82.54 92.06
Omeprazole
Values are mean7 S.E.M, n¼ 7. nnn
p o 0.001 compared to control (One-way ANOVA) followed by Tukey's multiple comparison test.
n
p o 0.05. po 0.01 are different, when compared to the control group. (One-way ANOVA followed by Dunnett's test). nn
Table 4 Effect of the EtOAc fraction of Markhamia tomentosa on indomethacininduced ulcer. Treatment
Dose (mg/kg)
Mean ulcer score7SEM
Ulcer index
% Protection
Control EtOAc
10 (ml/kg) 50 100 150 100
6.2 7 1.11 4.5 7 1.29 2.2 7 1.13n 1.7 7 0.37n 0.0 7 0.0nn
6.20 3.60 1.76 1.70 0
– 41.94 71.61 72.58 100
Omeprazole
indomethacin-induced antiulcer models were employed. Both models have been widely used for the evaluation of gastroprotective activity. Ethanol is considered one of the agents that induce gastric ulcers. The effects of ethanol on the gastric mucosa may occur as a result of stasis in gastric blood flow, which contributes to the development of the hemorrhagic and necrotic aspects of tissue injury (Guth et al., 1984). Occurrence of these ulcers, which is predominant in the glandular part of the stomach, was reported to stimulate the formation of reactive oxygen species (ROS), resulting in damage to rat gastric mucosa (Gupta et al., 2013). The oral administration of absolute ethanol produced severe ulceration in the stomach of the control rats (Table 1). The extract at the dose of 50, 100 and 150 mg/kg exhibited a significant (p o0.001) reduction of gastric lesions with 61.71%, 83.81% and 88.93% inhibition, respectively when compared to the control group. This showed that the extract was effective in preventing ethanol-induced gastric lesion, which suggests its cytoprotective effect. Similar findings exist in the literature, where plant extracts have been shown to prevent gastric mucosal ulceration in rats using this model (Mahmood et al., 2011; Al-Rejaie et al., 2012; ). Moreover, the study by Rajasekaran, 2014 demonstrated that Kigelia pinnata, a species in the family Bignoniaceae significantly reduced the ulcer lesion index produced by ethanol in a dose dependent manner. The use of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and indomethacin is considered to be the major risk factor in gastric ulcers. The mechanisms suggested for the gastric damage caused by NSAIDs are inhibition of prostaglandin synthesis through cyclooxygenase-1 block and inhibition of epithelial cell proliferation in the ulcer margin, which is critical for the re-epithelization of the ulcer (Levi et al., 1990). Many taxa of medicinal plants have been investigated for their antiulcerogenic effect using this model. Bhattacharya et al. (2007) reported the healing effect of some Indian medicinal plants against indomethacin-induced gastric ulceration in rats. In this model, pretreatment of the extract (50, 100 and 150 mg/kg) offered significant (po0.001) protection to the gastric mucosa against ulceration (Table 2). The percentage of ulcer inhibition was 63.49%, 71.43%,
Values are mean 7 S.E.M. (n ¼5). n
p o 0.05. po 0.01 significantly different, when compared to the control group (Oneway ANOVA followed by Tukey's test). nn
82.54%, respectively compared to the control. The gastroprotective activity of the extract suggests a prostaglandin dependent effect. The view is supported by the fact that prostaglandins normally serve as protective function in stomach by maintaining gastric microcirculation and causes gastric secretion of bicarbonate and mucus (Nwafor et al., 2000). Based on these results, the solvent fractions from the crude extract were evaluated for antiulcer activity at the dose of 150 mg/ kg using ethanol-induced gastric ulcer model in rats. The results showed that the aqueous fraction did not exhibit anti-ulcerogenic effects (Table 3). The HEX, DCM, EtOAc and BUT fractions reduced ulcer lesion by 12.77%, 53.19%, 89.79% and 78.30% respectively. Among these, the EtOAc fraction was found to be the most active and thus tested in a dose-dependent evaluation using indomethacin and pylorus ligation models. In the indomethacin-induced assay, 50, 100 and 150 mg/kg doses of the EtOAc fraction were effective, exhibiting significant differences from the control group; the highest percentage of antiulcerogenic effect was at 150 mg/kg (72.58%) (Table 4). Oral administration of omeprazole at 100 mg/kg produced 100% protection. These data suggest that the EtOAc fraction produced gastroprotective effect, since it significantly reduced indomethacininduced ulcers. This further supports cytoprotective effects of the fraction, which may be mediated by prostaglandins. The activity of the EtOAc fraction was also evaluated by pyloric ligature-induced gastric ulcers in a rat model using parameters including ulcer index, gastric volume, gastric pH and total acidity. The causes of gastric ulcer by pyloric ligation are believed to be due to accumulation of gastric acid and pepsin leading to a breakdown of the gastric mucosal barrier (Gharate and Kasture, 2013). The ligation of the pyloric end of the stomach causes accumulation of gastric acid in the stomach. This increase in the
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Table 5 Effect of the EtOAc fraction of Markhamia tomentosa on pylorus ligation induced ulcer. Treatment
Dose (mg/kg)
Ulcer index 7 S.E.M
% Protection
Gastric volume (ml)
Gastric pH
Gastric acidity (mEq/L)
Control EtOAc fr
10(ml/kg) 50 100 150 100
2.007 0.62 1.08 70.20 0.727 0.25 0.167 0.25 0.48 70.37
– 45.83n 64.00n 92.00nn 76.00n
0.82 70.03 0.53 70.06n 0.32 70.09nnn 0.48 70.11n 0.3570.09nn
2.917 0.18 4.89 7 0.86 4.317 0.76 4.42 7 0.40 5.617 0.30n
10.40 71.04 9.50 70.95 14.80 71.48 8.80 70.88 9.00 70.90
Cimetidine
Values are mean7 S.E.M. (n¼ 5). n
po 0.05. p o0.01. nnn p o0.001 significantly different when compared to the control group. (One-way ANOVA followed by Dunnett's test). nn
Fig. 1. HPLC finger print of the ethylacetate fraction from Markhamia tomentosa. The chromato-grams were recorded at 280 nm.
Table 6 LC–MS/MS identification of compounds in ethylacetate fraction of Markhamia tomentosa. Peak no.
Rt (min)
Msn
Ms2
Identification
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
11.3 12.0 12.6 14.6 14.7 16.1 16.9 17.6 18.5 19.4 19.8 19.9 20 20.2 20.7 21.7 23 23.1 24 25.6
623 623 623 714 609 593 285 363 329 347 347 665 665 635 635 487 483 503.6 487.7 485.7
461(100); 462(17); 315(1.7) 461(100); 462(14); 315(1.7) 461(100); 462(14) 667(100) 285 (100); 447(35) 285(100); 447(35); 286 (14) 285 (100) 363 (100); 345(87); 301(8) 171(100); 311(48); 229(48); 293(38); 211(32) 347(100) 347(100) 633(100); 634(22); 631(12) 633(100); 634(82) No daughter ion 603(100); 604(33) 469(100); 423(34); 25(27); 470(22); 467(14) 241(100); 242(13); 221(9) 471(100); 472(20); 469(9) 469(100); 470(27) 467(100); 468(24)
Acteoside Acteoside Isoacteoside Not identified Luteolin-30 ,7-di-O-glucoside Luteolin-7-rutinoside Luteolin Dimethanol adduct of quercetin Carnosol Ajugol Ajugol Not identified Not identified Not identified Not identified Tormentic acid Dilapachone Pomolic acid derivative Tormentic acid isomer 2-oxo-pomolic acid
Li et al. (2005) Li et al. (2005) Li et al. (2005) – Dall'Acqua et al. (2011); Cvetkovikj et al. (2013) Abu-Reidah et al. (2013); Cvetkovikj et al. (2013) Cvetkovikj et al. (2013) Qiao et al. (2014) Zimmermann et al. (2011) Won et al. (2010) Won et al. (2010) – – – – Li et al. (2009); Gao et al. (2011) Jassbi et al. (2004) – – Gao et al. (2011)
gastric acid secretion causes ulcers in the stomach (Dhuley, 1999). The data in Table 5 showed that oral administration of the EtOAc fraction in the doses of 50, 100 and 150 mg/kg produced a significant reduction in the ulcer index, 1.08 70.20 (45.83%
protection), 0.72 70.25 (64.00% protection) and 0.16 70.25 (92.00% protection), respectively. The gastric volume was reduced significantly compared to the control. A non-dose dependent and non-significant increase in
M.O. Sofidiya et al. / Journal of Ethnopharmacology 157 (2014) 1–6
gastric pH was observed compared to the control. All tested doses of the fraction did not produce significant reduction in gastric acidity compared to the control. Cimetidine at 100 mg/kg significantly inhibited the formation of ulcers, reduced gastric juice volume and increased gastric pH. This data is similar to the results obtained with the ethanolic crude extract of Rosmarinus officinalis (Dias et al., 2000) and Casearia sylvestris (Esteves et al., 2005). Similarly, Santin et al., 2010 have reported that the parameters of gastric secretion (pH, volume, gastric acidity) showed no alteration in the different doses of Achyrocline satureoides administered to rats. Therefore, the protection offered by EtOAc fraction in the pylorus ligation experiment may not be due to an acid neutralizing effect. The chemical constituents in the active ethyl acetate fraction of Markhamia tomentosa were characterized by electrospray ionization mass spectrometry. The chromatogram is shown in Fig. 1, while Table 6 shows the retention times and spectral data in the negative ion mode. The identified compounds include some phenolic compounds (acteoside, luteolin, luteolin-7-rutinoside, luteolin-30 ,7-di-Oglucoside), terpenoids (carnosol, dilapachone, tormentic acid, oxopomolic acid) and iridiod, ajugol. Among these compounds, acteoside and ajugol appear to be the major components of this fraction. Peaks 4 and 12–15 could not be identified based on the available data. Further isolation and identification by NMR spectroscopy is required to determine the identity of these molecules. Three of the mentioned compounds have been reported previously in the genus Markhamia. Acteoside, also known as verbascoside was reported in Markhamia lutea (Kernan et al., 1998) while tormentic acid was reported from the stem bark of Markhamia tomentosa (Tantangmo et al., 2010) and from Malpighia obtusifolia (Nchu et al., 2010). The iridoid, ajugol has previously been isolated from related species, Markhamia stipulata (Kanchanapoom et al., 2002). A literature search for antiulcer molecules revealed that antiulcer activity is not confined to one class of compounds. Compounds identified in this fraction such as luteolin has been reported to have antiulcer property. Moreover, the anti-secretory potential of acteoside isolated from Tectona grandis has been demonstrated (Singh et al., 2010). The presence of these compounds, including others identified in the EtOAc fraction could be responsible for the observed antiulcer activity. These findings indicate that Markhamia tomentosa possesses an anti-ulcer effect, possibly through a cytoprotective mechanism, corroborating its use in traditional medicine and contributing to its pharmacological validation.
Acknowledgments The authors are thankful to the School of Chemistry and Physics, University of KwaZulu-Natal, South Africa for providing the necessary facilities for LC-MS data acquisition. The assistance of Dr. Seyi Aboyade, Mrs. Mutiat Ibrahim and Toyin Adetunde is acknowledged.
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