Antibacterial and Antidiarrhoeal Activity of Alangium Salviifolium Wang Flowers

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Antibacterial and antidiarrhoeal activity of Alangium salviifolium

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Molecular & Clinical Pharmacology 2012, 2(1) 34-43

Antibacterial and antidiarrhoeal activity of Alangium salviifolium Wang flowers

Ronok Zahan1, M. Ashik Mosaddik1, Ranjan Kumar Barman 2, Mir Imam Ibne Wahed2, M. Ekramul Haque*,2.

1 Department of Pharmacy, BRAC University, Dhaka, Bangladesh 2 Department of Pharmacy, Rajshahi University, Bangladesh

*Corresponding Author: [email protected]

Abstract

The present study was designed to investigate antibacterial and antidiarrhoeal potential of the extract/fraction of Alangium salviifolium flowers. The methanol extract (MEAS) along with some organic soluble fractions of the flower of Alangium

salviifolium Wang were tested against six Gram-positive and six Gram-negative bacteria. Diethyl ether fraction (DEAS) was active against all tested microbial species and the highest activity was shown against Proteus sp with a zone of inhibition 12 ± 0.12 mm. The chloroform fraction (CAS) showed significant activity for all bacterial strains except Bacillus megatherium and Pseudomonas aeruginosa. The highest zone of inhibition was found against Escherichia coli (zone of inhibition 12±0.22 mm) for CAS. Among the all extract/fractions only pet ether fractions (PEAS) had no activity for all the test bacteria. CAS showed the maximum relative percentage inhibition against E. coli (34.21%) whereas; lowest relative percentage inhibition was found against Shigella flexneri (10.92%) for MEAS extract. The MEAS and CAS studied for antidiarrheal properties using castor oil and magnesium sulphate induced diarrheal model in mice. At the doses of 50 and 100 mg/kg body weight (MEAS) and 100 mg/kg b. wt. (CAS), the extract/fraction reduced the frequency and severity of diarrhea in test animals throughout the study period. Alt- ogether, these results suggest that the MEAS and CAS could be used as a potential antibacterial agent along with its antidiarrhoeal potentiality.

Keywords: Alangium salvifolium; antibacterial

Introduction

The frequency of life-threatening infections caused by pathogenic microorganisms has increased worldwide and become a prime factor of morbidity and mortality in immune compromised patients in developing countries and many infectious microorganisms are resistant to synthetic drugs and hence an alternative therapy is very much needed (Okeke et al.,

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2005). Medicinal plants represent a rich source of antimicrobial agents. Plants are used medi- cinally in different countries and are a source of many potent and powerful drugs (Srivastava et al., 1996). Plants are rich in a wide variety of secondary metabolites such as tannins terpenoids, alkaloids, flabonoids, etc, which showed wide range of in vitro antibacterial and antif- ungal activities (Dahanukar et al., 2000; Cowan., 1999) Traditional healers claim that their medicine is cheaper, more effective and impart least side effects as compaired to synthetic medicines. In developing countries, low income people such as farmers, people of small isol- ate villages and native communities use folk medicine for the treatment of common infections (Balandrin et al., 2006).

The incidence of food-borne illnesses is still a major problem, even in developed countries. It has been estimated that 6–81 million cases of illnesses and up to 9000 deaths annua- lly were attributed to food-borne pathogens in the USA alone (Mead et al., 1999). Five pathogens account for over 90% of the estimated food-related deaths: Salmonella (31%), Listeria (28%), Toxoplasma (21%), Norwalk-like viruses (7%), Campylobacter (5%) and Escherichia coli O157:H7 (3%). Oral rehydration therapy (ORT) remains the major treatment for diarrhoea, although it does not reduce the volume or duration of diarrhea (Subbotina et al., 2003). Other treatment options include antibiotics and gut motility suppressing agents, which aim to reverse dehydration, shorten the length of illness and reduce the period of time an individual is infectious (Allen et al., 2003). Treatment with pharmacological agents that are pathogen- specific or that suppress severe symptoms would be of benefit to patients suffering from prolonged diarrhoea (Takahashi et al., 2001; Oi et al., 2002).

Alangium salviifolium wang is a deciduous, rambling shrub or a tree belonging to the family Alangiaceae (Jubie et al., 2008). The different parts of this plant are used for a wide range of diseases. Root is used in diarrhoea, paralysis, piles and vomiting (Pandey et al., 2005). Root is also useful for external application in acute case of rheumatism, leprosy and inflammation (Anjaria et al., 2002). Antibacterial compound was isolated from the flower of Alangium salvifolium (Anjum et al., 2002). The plant has been reported for its anti-tubercula- r, anti-spasmodic and anti-cholinesterase activity (Warrier et al., 2005). Anti-Fertility activity of the stem Bark of Alangium salvifolium (Linn.F) Wang in Wistar female rats has also been reported (Murugan et al., 2000). Previous phytochemical investigation revealed that it is a rich source of alkaloids including ipecac alkaloid and benzopyridoquinolizidine alkaloids. It is also known to produce alangiside, a tetrahydroisoquinoline monoterpene glucoside (Itoh et al., 1992). Recent phytochemical studies of this plant resulted in the isolation of several flav- anoid, phenolic compound, irridoid glycosides and oxyoglucoside of some alcohol (Ramni et al., 2003). New alkaloid, ankorine was isolated from leaves (Jain et al 2002). Plant is also rich in tetrahydroisoquinoline monoterpene glycoside. e.g. alangiside-1 or ipecoside-2 whose structures are closely related to the ipecac alkaloid (Itoh et al 1994). In addition, considering the wide folk medicinal application of this plant, this work was set out in order to investigate the antimicrobial and antidiarrhoeal activity of extract and fractions of the flower of Alangiu- m salviifolium wang.

Materials and methods

Plant material

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The flowers of the Alangium salvifolium were collected from the adjoining area of Rajshahi University Campus, Bangladesh during February 2007 and were identified by Taxo- nomist, Department of Botany and University of Rajshahi, Bangladesh where a voucher spe- cimen number (Voucher No # 105) has been deposited.

Preparation of extracts

The flower material was shade-dried with occasional shifting and then powdered with a mechanical grinder, passing through sieve #40, and stored in a tight container. The powdered flower (850gm) was taken in large glass bottle and extracted with methanol for 7 days. The procedure was repeated twice using same solvent system for next 3 days. The extract was filtered through filter paper. The filtrate obtained by repeated maceration was evaporated under reduced pressure at 40°c using Rotary evaporator. The net weight of dry extract was 5.5 gm. The dry plant extract (5.5gm) was suspended in water and fractionated in a conical flask using diethyl ether, Petroleum ether and chloroform solvent system. Each fraction furth- er evaporates using Rotary evaporator and then air dried to solid mass 250mg, 230 and 300mg, respectively.

Preliminary Phytochemical Investigation

The extract/fraction was subjected to qualitative chemical investigation for the identification of different phytoconstituents like sterols, glycosides, saponins, carbohydrates, alkaloids, flavonoids, tannins, proteins and tri-terpenoids (Yarnalkar., 1991).

Test microorganisms

Strains bacteria (Gram positive and Gram negative) were obtained from International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B). Bacillus anthracis ATCC 14321, Bacillus cereus ATCC 14579, Bacillus megaterium ATCC 13578, Bacillus subtilis A- TCC 6059, Streptococcus agalactiae ATCC 6123, Staphylococcus aureus ATCC 6538, Esc- herichia coli ATCC 25922, Pseudomonus aeruginosa ATCC 27853, Shigella flexneri ATCC 9221, shigella boydii ATCC 9234, Shigella dysenteriae ATCC 9361, Proteus sp. ATCC 93- 41, were used as test microorganism. All these bacterial species are recommended by ATCC for their susceptibility assay. The strains are maintained and tested on Nutrient Agar media (NA) for bacteria.

Screening of antibacterial activity

The methanol extract (MEAS) and soluble fraction of chlororform (CAS), diethyle- ther (DEAS) and petroleum ether (PEAS) of the flower of Alangium salvifolium were tested for antibacterial activity by disc diffusion method (Olurinola., 1996). For MEAS, CAS, DE- AS and PEAS, each (20mg) was dissolved in respective solvent (1 ml) to get a concentration of 200g/disc. The test microorganisms were inoculated into respective medium by spread plate method with 24h cultured bacteria, grown in nutrient agar media. After solidification the filter paper disc (5mm in diameter) impregnated with sample, the standard antibiotic (Ka-

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namycin-30g/disc) as positive control and as negative control, a blank disc impregnated with 30µl respective solvent was used The spatial arrangement discs were such that the discs were not closer than 15 mm to the edge of the plate, which is to prevent overlapping the zone of inhibition. The plates were then kept in a refrigerator at 4°c for about 24 hours in order to provide sufficient time to diffuse the sample and standard from the discs to surrounding agar medium. Finally, the plates were invested and kept in an incubator at 37°C for 24 hours. After incubation the antibacterial activity of the test material was determined by measuring the diameter of the zone of inhibition in terms of millimeter (mm) with a transparent scale.

Determination of Relative Percentage Inhibition:

The relative percentage inhibition with respect to positive control was calculated by using the following formula (Ajay et al., 2002). Relative percentage inhibition of the test extract = [{100 x (a - b)}/(c - b)]. Where, a: total area of inhibition of the test extract; b: total area of inhibition of the solvent; c: total area of inhibition of the standard drug. The total area of the inhibition was calculated by using area = πr2; where, r = radius of the zone of inhibition.

In vivo antidiarrhoeal activity

Castor oil-induced diarrhoea

The experiment was performed according to the method described by Shoba & Tho- mas (2001). Briefly, mice fasted for 24 hr were randomly allocated to five groups of six animals each. The animals were all screened initially by giving 0.5 ml of castor oil. Only those showing diarrhea were selected for the final experiment. Group I received 1% Tween 80 in water (10 ml/kg, p.o), groups IV and V received p.o the methanol extract (100 and 50 mg/kg) whereas group III received p.o chloroform fraction (100 mg/kg). Group II was given antidiarrheal drug loperamide (3 mg/kg, p.o) in suspension. After 60 min, each animal was given 0.5 ml of castor oil, each animal was placed in an individual cage, the floor of which was lined with blotting paper which was changed every hour, observed for 4 hr and the character- istic diarrheal droppings were recorded.

Magnesium sulphate-induced diarrhoea

Diarrhea was induced by oral administration of magnesium sulfate at the dose of 2 g/kg to the animals 30 min after pre-treatment with vehicle (1% Tween 80 in water, 10 ml/ kg, p.o) to the control group, loperamide (3 mg/kg) to the positive control group, and the methanol extract at the doses of 50 and 100 mg/kg and chloroform fraction at 100 mg/kg body weight to the test groups (Doherty., 1981).

Statistical analysis

All assays were performed in triplicate under strict aseptic conditions to ensure consistency of all findings. Data of all experiments were statistically analyzed and expressed as the mean ± standard deviation of three replicate experiments. The analysis was performed

Molecular & Clinical Pharmacology 2012, 2(1) 34-43

Table 1. Result of chemical group tests of MEAS and CAS of Alangium salvifolim wang flower.

Extract Carbohydrate Tannin Flavonoid Saponin Phenol Steroid Alkaloid MEAS - ++ ++ - ++ + +++ CAS - - +++ - ++ ++ ++

MEAS: Methanol extract; CAS: Chloroform fraction; (+): Present; (-): Absent; (+ + +): Reaction intensity is high; (+ +): Reaction intensity is medium; (+): Reaction intensity is normal; by using ANOVA done in SPSS 15.0 followed by Dunnett’s test where *p0.001 were consi- dered to be statistically significant.

Results

Phytochemical Screening

Phytochemical screening of MEAS and CAS was represented in Table 1. Alkaloids, phenol, steroid, flavonoid and tannins were present.

Antibacterial activity

Tablet 2 expressed the antibacterial aqctivity (zone of inhibitions) of extract and fractions of the flower part of the A.salviifolium Wang. The DEAS showed significant activity against the entire tested microbial flora and among them proteus sp. was highly susceptible with the zone of inhibition 12 ± 0.12 mm. The MEAS was highly active against Staphylococ- cus aureus, and Bacillus cereus with the zone of inhibitions was 10 ± 0.32 and`

Table 2. Antibacterial activity of crude extract and its soluble fractions of Alangium salvifolim wang flower.

Diameter of zone of inhibition (mm) Bacterial strain bStd.K aCAS aDEAS aPEAS aMEAS

Gram (+) ve Staphylococcus 28 ± 0.12 10 ± 0.14 7 ± 0.32 NA 10 ± 0.32 aureus Streptococcus 22 ± 0.22 10 ± 0.23 7 ± 0.21 NA NA agalactiae Bacillus cereus 24 ± 0.31 10 ± 0.02 8 ± 0.28 NA 10 ± 0.52 Bacillus 26 ± 0.12 NA 7 ± 0.19 NA NA megatherium Bacillus subtilis 28 ± 0.42 8 ± 0.22 8 ± 0.13 NA NA Bacillus anthracis 25 ± 0.22 7 ± 0.32 8 ± 0.12 NA 7 ± 0.28 Gram(-) ve Pseudomonas 29 ± 0.21 NA 7 ± 0.16 NA NA aeruginosa Proteus sp. 25 ± 0.11 10 ± 0.12 12 ± 0.12 NA NA Shigella flexneri 27 ± 0.12 7 ± 0.33 8 ± 0.12 NA 7 ± 0.51 Shigella dysenteriae 28 ± 0.11 7 ± 0.62 8 ± 0.12 NA 8 ± 0.17 Shigella boydii 29 ± 0.14 7 ± 0.14 7 ± 0.14 NA NA Escherichia coli 30 ± 0.18 12 ± 0.22 7 ± 0.11 NA 7 ± 0.13 aValues of the observed diameter zone of inhibition (mm). Incubation conditions for bacteria – 24 hours at 370C. Assay was performed in triplicate and results are the mean of three values ± Standard Deviation. b Reference standard; NA- Zone of inhibition 5 mm consider as no activity.

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Figure 1. Relative percentage inhibition (%) of extract/fractions of Alangium salvifolim wang flower on various bacterial strains.

10±0.52 mm, respectively whereas Streptococcus agalactiae, Bacillus megatherium, Bacillus subtilis, Pseudomonas aeruginosa, Proteus sp. and Shigella boydii were resistant to MEAS. Among the all extract/fractions only PEAS had no activity for all the test bacteria. On the other hand, The CAS showed significant activity for all bacterial strains except Bacillus megatherium and Pseudomonas aeruginosa. The highest zone of inhibition was found against Escherichia coli (zone of inhibition 12 ± 0.22 mm), followed by Staphylococcus aureus, Streptococcus agalactiae, Bacillus cereus and Proteus sp. and the moderate activity was shown against Bacillus subtilis (zone of inhibition 8 ± 0.12). The weakest activity was shown against Bacillus anthracis, Shigella flexneri, Shigella dysenteriae and Shigella boydii.

Results of the relative percentage inhibition are reported in figure 1. MEAS showed the maximum relative percentage inhibition against B. cereus (27.10%) followed by S. aureus (26.81%) whereas, lowest relative percentage inhibition against Shigella flexneri (10.92 %). The highest relative percentage inhibition was found against Proteus sp. (27.10%) and lowest relative percentage inhibition against B. megatherium (11.21%) for DEAS. CAS showed the maximum relative percentage inhibition against E. coli (34.21%) whereas, lowest relative percentage inhibition against Shigella boydii (11.24%). PEAS had no relative percentage inhibition against the entire tested bacterial flora.

Antidiarrhoeal activity

Effect on castor oil-induced diarrhea

In the castor oil induced diarrheal mice, the methanolic extract and chloroform soluble fraction of the flower of A. salviifolium, at the dose of 50, 100 and 100 mg/kg, significantly (p 0.001) lessen the total number of faces as well as delayed the onset of diarrhea in a

Molecular & Clinical Pharmacology 2012, 2(1) 34-43

Table 3. Effect of A. salviifolium flower extract/fraction on castor oil-induced diarrhea in mice.

Onset of diarrhoea Animals with % inhibition of Treatment No. of faeces in 4 h (min) diarrhoea defaecation

1% Tween 80 in water (ml) (control) 0.4 56.45±5.31 5/5 16.68±0.45

Methanolic extract of Alangium salviifolium flowers (mg/kg body weight) (test) 100 155.48*±7.79 3/5 8.23*±0.25 66.01 50 90.28*±5.40 2/5 12.65*±0.28 39.48

Chloroform fraction of Alangium salviifolium flowers (mg/kg body weight) (test) 100 165.87*±6.45 2/5 5.36*±0.06 86.27

Loperamide (mg/kg body weight) (positive control) 10 180.15*±8.5 1/5 3.27*±0.21 93.52 Values are presented as mean ± SEM, (n=5); *p0.001, Dennett’s test compared to control dose dependent manner (Table 3).

Effect on magnesium sulphate-induced diarrhea

A. salviifolium flower extract/fraction exhibited significant antidiarrheal activity against magnesium sulphate-induced diarrhea (Table 4). The extract/fraction at both dose levels significantly (p 0.001) reduced the extent of diarrhea and also notably delayed the onset of diarrhea in a dose dependent manner.

Discussion

Preliminary phytochemical study indicated the presence of alkaloid, steroids, tannins, phenolic and flavonoid compounds in crude extract of Alangium salvifolium.wang flower. Phytoconstituents such as saponin, phenolic compounds, flavonoids and glycosides have

Table 4: Effect of A. salviifolium flower extract/fraction on magnesium sulphate-induced diarrhea in mice.

Onset of diarrhoea Animals with % inhibition of Treatment No. of faeces in 4 h (min) diarrhoea defaecation

1% Tween 80 in water (ml) (control) 0.4 48.42 ± 4.5 5/5 8.45 ± 0.07

Methanolic extract of Alangium salviifolium flowers (mg/kg body weight) (test) 100 137.46 ± 5.25* 3/5 3.34 ± 0.04* 69.51 50 80.39 ± 4.65* 2/5 6.12 ± 0.07* 35.51

Chloroform fraction of Alangium salviifolium flowers (mg/kg body weight) (test) 100 145.73± 7.3* 2/5 2.06± 0.07* 77.13

Loperamide (mg/kg body weight) (positive control) 10 160.54 ± 6.5** 1/5 2.23± 0.03* 86.73

Values are presented as mean ± SEM, (n=5); *p0.001, Dennett’s test compared to control

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been reported to inhibit bacterial growth and to be protective to plants against bacterial and fungal infections (Alam et al., 2010). Moreover, Adeeba et al. isolated various antibacterial alkaloids from the methanolic extract of A. salvifolium flower. So the antibacterial activity showed by the extract and its fractions may be due to the presence of alkaloidal compounds. Several mechanisms have been previously proposed to explain the diarrhoeal effect of castor oil including inhibition of intestinal Na+, K+-ATPase activity to reduce normal fluid absorption (Nell & Rummel., 1984) activation of adenylate cyclase or mucosal cAMP mediated active secretion28, stimulation of prostaglandin formation (Capasso et al., 1994), platelet activating factor and recently nitric oxide has been claimed to contribute to the diarrhoeal effect of castor oil (Mascolo et al., 1996). However, it is well evident that castor oil produces diarrhea due to its most active component recinoleic acid which causes irritation and inflammation of the intestinal mucosa, leading to release of prostaglandins, which results in stimulation of secretion (Gaginella et al., 1975). Since the extract/fraction of the flower of A. salviifolium successfully inhibited the castor oil-induced diarrhoea, the extract/fraction might have exerted its antidiarrheal action via antisecretory mechanism which was also evident from the reduction of total number of wet faeces (not shown separately) in the test groups in the experiment. Again, flavonoids present in the plant extract (Anjum et al., 2002) are reported to inhibit release of autacoids and prostaglandins, thereby inhibit motility and secretion induced by castor oil (Hasan et al., 2009).

On the other hand, magnesium sulphate has been reported to induce diarrhea by incre- asing the volume of intestinal content through prevention of reabsorption of water. It has also been reported that it promotes the liberation of cholecystokinin from the duodenal mucosa, which increases the secretion and motility of small intestine and thereby prevents the reabsorption of sodium chloride and water (Zavala et al., 1998). The methanol extract was found to improve the diarrheal condition in this model. The extract may increase the absorption of water and electrolyte from the gastrointestinal tract, since it delayed the gastrointestinal transit in mice as compared to the control. The delay in the gastrointestinal transit prompted by the extract/fraction might have contributed, at least to some extent, to their antidiarrheal activity by allowing a greater time for absorption.

In conclusion, the results of the present study, in agreement with other authors14 indicate that the extract/fraction of A. salvifolium flower exhibits interesting antimicrobial and antidiarrhoeal properties. These results of the investigation do not reveal that which chemical compound is responsible for aforementioned activity. Now our next aim is to explore the lead compound liable for aforementioned activity from this plant.

Acknowledgement

The authors are thankful to the Department of Pharmacy, BRAC University, Bangladesh for providing laboratory facilities to carry out this research work.

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