ANTI-ULCER ACTIVITY OF LEGUMINOSAE PLANTS

Atividade anti-úlcera de plantas leguminosas

Abstracts

Context

Ulcer is the most common gastrointestinal disturbance resulting from an inadequate gastric mucosal defense. Several drugs are available in the market to address the disease; however, these drugs are associated with unnecessary side effects.

Objectives

Previous research have confirmed the efficacy of plant extracts for possible treatment of the disease. This research aims to evaluate the anti-ulcer properties of medicinal plants.

Methods

Methanol extracts from the leaves of Intsia bijuga, Cynometra ramiflora, Tamarindus indica, Cassia javanica, Cassia fistula, Bauhini purpurea, Senna spectabilis, Senna siamea and Saraca thaipingensis were evaluated for their anti-ulcer activity using HCl-ethanol as ulcerogen.

Results

All extracts showed inhibitory activity with I. bijuga, T. indica, S. spectabilis and S. thaipingensis exhibiting more than 50% inhibition. S. thaipingensis showed the highest activity at 80%. S. spectabilis and S. thaipingensis were partitioned further into hexane, ethyl acetate and aqueous fractions. The aqueous and ethyl acetate fractions of S. spectabilis showed significant increased in its activity while the hexane and ethyl acetate fractions of S. thaipingensis gave higher activity than its aqueous portions.

Conclusions

We conclude that plant extracts are potential sources of new anti-ulcer agents.

Gastrointestinal diseases; Peptic ulcer; Leguminosae; Medicinal plants

Contexto

A úlcera é o distúrbio gastrointestinal mais comum que resulta de uma inadequada defesa da mucosa gástrica. Vários medicamentos estão disponíveis no mercado para tratar a doença, no entanto, estas drogas podem se associar a efeitos colaterais desnecessários.

Objetivos

Pesquisas anteriores confirmaram a eficácia de extratos de plantas como possível tratamento da doença. Esta pesquisa teve como objetivo avaliar as propriedades anti-úlcera de plantas medicinais.

Métodos

Extratos alcoólicos das folhas da Intsia bijuga, Cynometra ramiflora, Tamarindus indica, Cassia javanica, Cassia fistula, Bauhini purpurea, Senna spectabilis, Senna siamea e Saraca thaipingensis foram avaliados pela sua atividade anti-úlcera usando o HCl-etanol como ulcerogênico.

Resultados

Todos os extratos apresentaram atividade inibitória; I.bijuga, T. Índica, S. spectabilis e S. thaipingensis mostraram mais de 50% de inibição. A S. thaipingensis mostrou a maior atividade, atingindo 80%. S. spectabilis e S. thaipingensis foram divididos mais em hexano, acetato de etila e frações aquosas. As frações aquosas e acetato de etila de S. spectabilis mostraram aumento significativo em sua atividade, enquanto que as frações hexano e acetato de etila de S. thaipingensis resultaram em maior atividade do que em partes aquosas.

Conclusões

Pode-se concluir que os extratos vegetais são fontes potenciais de novos agentes anti-úlcera.

Gastroenteropatias; Úlcera péptica; Leguminosas; Plantas medicinais

INTRODUCTION

Peptic ulcer disease is the term used to describe a heterogeneous group of condition with ulcerations. It is characterized by the disruption of the mucosal integrity of the esophagus, stomach, or duodenum⁽¹⁶⁾. As the most common gastrointestinal disturbance, it affects 10%-15% of the population at any one time. Ulcers are primarily caused by an imbalance between some endogenous aggressive and protective factors in the stomach such as acid-pepsin secretion, integrity of the mucosal barrier, mucus secretion, blood flow, cellular regeneration, prostaglandins, and growth factors⁽¹²⁾. Several factors are also associated in the occurrence of peptic ulcer including stressful lifestyle, alcohol consumption, use of steroidal and non-steroidal anti-inflammatory drugs (NSAIDS), Helicobacter pylori infections, smoking, lower socio-economic status and family history⁽¹⁹⁾. Although ulcer is not a deadly disease, it can lead to more serious complications like gastrointestinal bleeding, perforations, penetration of ulcer into adjacent organs and gastric outlet obstruction⁽⁹⁾. Medications are used to relieve the pain, heal ulcerations and delay recurrence of ulcerations. These include antibiotics⁽³³⁾, antacids and proton pump inhibitors⁽³⁰⁾. Several drugs are available in the market for gastric ulcer therapy; however, most of these drugs are associated with unwanted side effects⁽²³⁾.

In this context, this research aims to evaluate the anti-ulcer properties of medicinal plants. Several researches have confirmed the efficacy of medicinal plants for the treatment of peptic ulcer disease. The observed activity is these plants is attributed with the presence of flavonoids, alkaloids, terpenoids, tannins, saponins, and phenolic acids^{(4, 10, 15, 21)}. Extracts of Wilbrandia ebracteata⁽⁶⁾, Eruca sativa⁽²⁾, Toona ciliata Roemer⁽¹⁸⁾, Calligonum somosum⁽¹⁷⁾, Voacanga Africana⁽²⁶⁾ and Pedalium murex⁽³⁾ have shown anti-ulcer activity. An alkaloid from the fruit of Voacanga africana and a protoberberine-type alkaloid from the bark of Enantia chlorantha were found to prevent ulcers^{(27, 28)}. An alkaloid extract and 2-phenylquinoline from Galipea longiflora Krause have also shown gastroprotective effects⁽³⁴⁾. Bauhinia purpurea, which belongs to the Leguminosae family, has been shown to inhibit aspirin-induced and ethanol-induced ulcers in mice⁽²⁹⁾. In this study, other species belonging to the same family were evaluated for their anti-ulcer activity.

METHODS

Plant Material

Fresh leaves of Intsia bijuga, Cynometra ramiflora, Tamarindus indica, Cassia javanica, Cassia fistula, Bauhini purpurea, Senna spectabilis, Senna siamea and Saraca thaipingensis were collected from the University of the Philippines, Diliman Campus and submitted to the Dr. Jose Vera Santos Herbarium, Institute of Biology, University of the Philippines, Diliman for authentication. Voucher specimen for each plant were also deposited.

Extraction and solvent partitioning

The plant samples were washed with running water and air-dried. The dried samples were homogenized for overnight soaking in methanol. The resulting extracts were filtered and concentrated in vacuo using a rotary evaporator at 40˚C. The methanol fractions were partitioned between hexane and water. The resulting aqueous layer was further extracted with ethyl acetate. The hexane and ethyl acetate portions were also concentrated in vacuo.

Phytochemical analysis

The phytochemical screening methods used were based on Harborne⁽¹³⁾ and Edeoga⁽⁸⁾. Qualitative test for terpenoids, saponins, tannins, flavonoids, steroids, phenolic compounds, alkaloids and cardiac glycosides were performed.

Bioassay

1) Animals

The mice used in the assay were 6-8 weeks old, Swiss Albino mice (ICR strain) purchased from the Food and Drug Administration (FDA) Philippines, Department of Health, Alabang, Muntinlupa City. The animals were acclimated for at least one week in standard cages. The mice were fed with commercial pellets with free access to purified drinking water ad libitum, standard conditions of 12h:12h light/dark cycle, and temperature (23˚C-25˚C). The protocol used for the anti-ulcerogenic assay was approved by the College of Science Animal Care and Use Committee (CSACUC) of the University of the Philippines Diliman with assigned protocol number IC 2011-06.

2) HCl/Ethanol-induced ulcer assay

The anti-ulcerogenic assay was adapted from the method of Schmeda-Hirschmann⁽²²⁾ with slight modifications. A total of 65 mice were randomly distributed into thirteen treatment groups with 5 mice for the initial assay. Mice weighing 26±5 g were deprived of food 24 hours prior to the experiment. Group 1 was given solvent solution with 5% Tween 80, 10% DMSO and 85% distilled water. Group 2 was given HCL/EtOH only. Group 3 was administered with Sucralfate. Groups 4-12 were treated with the plant samples. Group 13 did not receive any treatment. The plant samples, positive control, and solvent control were orally administered to the mice. The plant extracts were given at a dose of 1000 mg/ kg, 0.2mL/20 g body weight; Sucralfate at a dose of 200 mg/kg, 0.2 mL/20 g body weight; and 0.3 M HCl/60% EtOH.

After an hour, the mice were given 0.2 mL/20 g b.w. of 0.3 M HCl/60%EtOH solution to induce ulceration. The mice were sacrificed by cervical dislocation an hour after the induction of ulceration. The stomachs were excised and inflated by injecting with 0.9% normal saline solution. The excised stomachs were fixed with 10% phosphate buffered solution for at least 15 minutes, and opened along the greater curvature to expose the gastric mucosal layer. Hemorrhagic lesions in the mucosal membrane of the glandular region were observed under a dissecting microscope and were manually scored. Scoring of ulcerations was patterned after Adensawo et al.⁽¹⁾. Normal gastric mucosa was scored as 0, pinpoint ulcers were scored 0.5, one or two small hemorrhages were given 1.0 and ulcers with diameters greater than 3 mm or characterized by heavy bleeding were given a score 2.0.

Fifty mice were randomly distributed into ten treatment groups for the second assay. Groups 1-3 were given similar treatments as in the initial assay. Groups 4-9 were given plant extracts. Group 10 did not receive any treatment. Similar concentrations were used as in the first assay.

The ulcer index (UI) was obtained from the sum of the scores of all lesions for each stomach, and the mean ulcer index (UI_MEAN) was calculated for each group. Percent ulcer inhibition of the samples was determined using the following equation:

% ulcer inhibition =	(UIMEAN control- UIMEAN sample)	X 100%
	UIMEAN control

Data analysis

Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparison test using SPSS version 16.0 to determine statistical differences between the treated and the control group. The level of signficance was set at P<0.05

RESULTS

The methanol extracts of I. bijuga, C. ramiflora, T. indica, C. javanica, C. fistula, B. purpurea, S. spectabilis, S. siamea and S. thaipingensis were evaluated for their gastroprotective action against HCl-EtOH-induced ulcer. Figure 1 shows the ulcerations resulting from HCl-EtOH treatment. Varying gastroprotective activities of the extracts are shown in Table 1. The anti-ulcer activity of the extracts may be due to the phytochemicals they contain. The phytochemical profiles of all plant samples were determined and the results of the tests are shown in Table 2. The methanol extracts of S. spectabilis and S. thaipingensis were partitioned with hexane and ethyl acetate to further examine their high activity. The increase in gastroprotective activities of the hexane, ethyl acetate, aqueous extracts are shown in Table 3.

FIGURE 1.
Ulcerations caused by HCl/EtOH in mice

TABLE 1.
Gastroprotective activity of MeOH samples in HCl/EtOH-induced ulcer in mice

Treatment	Ulcer Index (mean±SEM)	Ulcer Inhibition(%)
Solvent	6.000±0.423
HCl/EtOH only	5.143±0.688
Sucralfate	1.000±0.244*	83.3
Intsia bijuga (Colebr.) O.K.	2.500±0.775*	58.3
Cynometra ramiflora L. Var. ramiflora	5.167±0.989	13.9
Tamarindus indicus L.	2.417±0.800*	59.7
Cassia javanica L. ssp. Nodosa (Buch.-Ham. Ex. Roxb)K. & S.S. Larsen	4.167±1.123	30.6
Cassia fistula L.	4.417±0.889	26.4
Bauhinia purpurea L.	3.500±1.084	41.7
Senna spectabilis (DC.) Irwin & Barneby	2.083±0.970*	65.3
Senna siamea (Lam.) Irwin & Barneby	3.917±0.908	34.7
Saraca thaipingensis Cantley ex Prain	1.200±0.464*	80.0
Normal (no treatment)	0.000±0.000*

• ^*
  Samples statisticaly different compared to the solvent control (P<0.05)

TABLE 2.
Phytochemical analysis of plant species in the Leguminosae family

Plant species	Flavonoids	Tannins	Terpenoid	Alkaloid	Cardiac Glycoside	Phenolics	Saponin	Steroids
Intsia bijuga (Colebr.) O.K.	+	+	+	+	+	+	+	+
Cynometra ramiflora L. Var. ramiflora	+	+	-	+	-	+	+	+
Tamarindus indicus L.	+	+	-	+	+	+	+	-
Cassia javanica L. ssp. Nodosa (Buch.-Ham. Ex. Roxb) K. & S.S. Larsen	+	-	+	+	+	+	+	-
Cassia fistula L.	+	-	+	-	-	-	+	-
Bauhinia purpurea L.	+	+	-	+	+	-	+	+
Senna spectabilis (DC.) Irwin & Barneby	+	+	-	+	-	+	+	+
Senna siamea (Lam.) Irwin & Barneby	+	+	-	+	+	+	+	+
Saraca thaipingensis Cantley ex Prain	+	+	+	-	+	+	+	+

TABLE 3.
Gastroprotective activity of S. spectabilis and S. thaipingensis fractions in HCl/EtOH-induced ulcer in mice

Treatment		Ulcer Index (mean±SEM)	Ulcer Inhibition (%)
Solvent		6.562±0.8097
HCl/EtOH only		5.000±0.8062
Sucralfate		3.143±0.7846*	52.1
S. spectabilis	Aqueous extract	1.357±0.7377*	79.3
	Ethyl acetate extract	1.857±1.0160*	71.7
	Hexane extract	5.833±0.7601	11.1
S. thaipingensis	Aqueous extract	5.000±0.5323	23.8
	Ethyl acetate extract	2.429±0.7975*	63.0
	Hexane extract	2.429±0.8690*	63.0
Normal (no treatment)		0.429±0.2974*

• ^*
  Samples statisticaly different compared to the solvent control (P<0.05)

DISCUSSION

All extracts were active and showed varying degrees of gastroprotection. It is possible that plants belonging to the Leguminosae family are able to inhibit ulcers. I. bijuga, T. indica, S. spectabilis and S. thaipingensis showed higher than 50% inhibition. S. thaipingensis showed the highest activity at 80% which is comparable with the activity of the positive control sucralfate at 83% inhibition. These plants showed significant anti-ulcer action against HCl-EtOH ulcerogen. Alcohol consumption is a contributor to gastric ulceration⁽¹¹⁾ and excessive consumption increases the risk for gastric mucosal damage. Ethanol causes gastric ulcers by lowering protective factors in the gastric mucosa⁽⁵⁾. Ethanol-induced ulcers in mice are characterized by heavy bleeding since it can cause immediate stasis in the blood flow⁽²⁰⁾. It is possible that the extracts contain compounds that can enhance protective factors and restore gastric blood circulation.

Phytochemicals refer to a wide-variety of compounds produced by plants with no nutritive value. They are promoted for their protective and disease-preventive properties according to the American Cancer Society website. Stilbenes and flavonoids were isolated from the heartwood of I. bijuga⁽¹⁴⁾. T. indica showed 59.6% ulcer inhibition. The results supported its used for gastrointestinal disorders in India^{(7, 25)}.

S. spectabilis activity showed significant increased inhibition for its ethyl acetate and aqueous fractions at 71.7% and 79.3%, respectively. Result showed that hexane and ethyl acetate extracts of S. thaipingensis exhibited significant bioactivity, both at 63 %. No significant activity was observed for the aqueous extract. The flowers of S. spectabilis previously yielded three new bioactive piperidine alkaloids⁽³¹⁾. Its leaves, roots and stems also gave different alkaloids^{(24, 32)}. Phytochemical screening of the methanolic extract of S. thaipingensis showed the presence of flavonoids, terpenoids, tannins, saponins, and phenolic acids which are known to have anti-ulcer activities^{(4, 19, 21)}. The bioactivity of the extracts could be attributed to these secondary metabolites.

CONCLUSION

The different plant extracts gave varying degrees of anti-ulcer activity and could be a potential source of new anti-ulcer agents. Further studies are underway to identify these compounds.

ACKNOWLEDGEMENT

This project was funded by the Natural Sciences Research Institute of the University of the Philippines Diliman.

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Publication Dates

• Publication in this collection
  Jan-Mar 2014

History

• Received
  23 Aug 2013
• Accepted
  17 Oct 2013