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 Table of Contents    
ORIGINAL ARTICLE  
Year : 2010  |  Volume : 4  |  Issue : 4  |  Page : 288-291
Antibacterial activity and qualitative phytochemical analysis of Vitex mollis fruit


1 Master of Science Program in Food Science and Technology, Faculty of Chemical and Biological Sciences (FCQB), Autonomous University of Sinaloa (UAS), Culiacan, Sinaloa;Regional Doctoral Program in Biotechnology, Faculty of Chemical and Biological Sciences (FCQB), Autonomous University of Sinaloa (UAS), Culiacan, Sinaloa, Mexico
2 Master of Science Program in Food Science and Technology, Faculty of Chemical and Biological Sciences (FCQB), Autonomous University of Sinaloa (UAS), Culiacan, Sinaloa, Mexico
3 Regional Doctoral Program in Biotechnology, Faculty of Chemical and Biological Sciences (FCQB), Autonomous University of Sinaloa (UAS), Culiacan, Sinaloa;Master of Science Program in Biomedical Sciences, Faculty of Chemical and Biological Sciences (FCQB), Autonomous University of Sinaloa (UAS), Culiacan, Sinaloa, Mexico
4 Master of Science Program in Biomedical Sciences, Faculty of Chemical and Biological Sciences (FCQB), Autonomous University of Sinaloa (UAS), Culiacan, Sinaloa, Mexico

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Date of Submission17-Sep-2010
Date of Acceptance26-Jun-2010
Date of Web Publication22-Dec-2010
 

   Abstract 

The pulp of the Vitex mollis fruit is edible and traditionally used to treat diarrhoea. The antibacterial activity of this fruit is reported here for the first time. The fruit pulp was extracted with methanol (ME) and the extract was fractionated with solvents. ME and their fractions [hexanic (HF), chloroformic (CF), ethyl acetate (EAF) and aqueous (AqF)] were assayed against human pathogenic bacteria (microdilution test) and their phytochemicals determined (qualitative chemical determinations). The samples (i.e., ME, HE, CF, EAF and AqF) showed antibacterial activity; EAF was the most active, showing such activity against Shigella dysenteriae [minimal inhibitory concentration (MIC)=2 mg/ml]. Phenolics were mainly found in ME and EAF; compounds of this chemical family are well known for their antidiarrhoeal and antimicrobial activities. The reported antibacterial activity and phenolics content of V. mollis fruit could be associated with its use in the treatment of diarrhoea.

Keywords: Antibacterial activity, diarrhoea, phytochemical analysis, Vitex mollis

How to cite this article:
Delgado-Vargas F, Félix-Favela F, Pío-León JF, López-Angulo G, López-Valenzuela JA, Díaz-Camacho SP, Uribe-Beltrán Md. Antibacterial activity and qualitative phytochemical analysis of Vitex mollis fruit. Int J Green Pharm 2010;4:288-91

How to cite this URL:
Delgado-Vargas F, Félix-Favela F, Pío-León JF, López-Angulo G, López-Valenzuela JA, Díaz-Camacho SP, Uribe-Beltrán Md. Antibacterial activity and qualitative phytochemical analysis of Vitex mollis fruit. Int J Green Pharm [serial online] 2010 [cited 2014 Oct 1];4:288-91. Available from: http://www.greenpharmacy.info/text.asp?2010/4/4/288/74140



   Introduction Top


Around the world, infectious diseases are among the main causes of morbidity and mortality. [1] In Mexico, gastrointestinal pathogens are important morbidity agents. [2] Nowadays, the treatment with commercial antibiotics is more complicated due to the resistance phenomena and new antibacterials are required. [1] Till date, plants are the main source of bioactive compounds for drug development by pharmaceutical companies, and mostly the only option for disease treatment in rural communities characterized by low per capita income. [3] Plants are commonly used to treat diarrhoea where they may act as antimicrobial, slowing the gastrointestinal transit or increasing water and electrolyte reabsorption by the colon. [4]

Vitex mollis HBK (Verbenaceae) is a native plant of Sinaloa, Mexico, where it is known as "uvalamo". [5] Its fruit (uvalama) is edible and rich in dietary fibre and minerals. [6] Fruit and leaves are traditionally used to treat diarrhoea, [7] but scientifically it has not been established. The leaves of V. mollis have been better studied than fruit, e.g., methanolic extract was spasmolytic in guinea pig intestines and the hexanic extract did not show in vitro antibacterial activity at doses up to 16 mg/ml. [8] On the other hand, an aqueous extract of Vitex doniana sweet fruits showed antidiarrhoeic effects. [9]

The aim of this research is to demonstrate the antibacterial activity of uvalama fruit extracts against human pathogens, and also their phytochemical composition. These results could give the scientific support for the traditional use of this fruit as an antidiarrhoeal and for the future development of nutraceutical products.


   Materials and Methods Top


Materials

Uvalama fruits were collected from the valley of Culiacan, Sinaloa, Mexico. The plant material's identity was confirmed by Dr. Rito Vega-Aviρa and a voucher specimen (VAR 2321) was deposited at the Herbarium of the Agronomy Faculty, Autonomous University of Sinaloa (UAS).

Bacterial strains were ATCC (DIFCO Laboratories, Detroit, MI, USA) and from diseased humans, kindly provided by the Instituto Nacional de Pediatrνa, Mιxico D.F. [Table 1].

Fruit Extract Preparation

Fruit was freeze-dried, milled in a waring blender (Waring commercial, Torrington, CT, USA), the meal was passed through a mesh 40 and stored at −20ºC until analysis.

Extraction was carried out as described by Wall et al. (2006) with minor modifications. [10] Briefly, uvalama meal (200 g) was thrice extracted with pure methanol (1:5 p/v), liquid phases were mixed and vacuum concentrated (40ºC) to obtain the methanolic extract (ME), the yield was 74.5% on a dry weight basis (d.w.). ME was dissolved in a minimal volume of 90% methanol solution and successively fractionated (1:1 v/v) with hexane, chloroform, ethyl acetate and water. Organic fractions were concentrated under vacuum (40ºC) and the aqueous extract was concentrated by freeze-drying to obtain the hexanic (HF) (1.11% d.w.), chloroformic (CF) (0.4% d.w.), ethyl acetate (EAF) (0.38% d.w.) and aqueous (AqF) (73% d.w.) fractions. Used solvents were of reagent grade (CTR Scientific, Monterrey, Nuevo Leon, Mexico).th

Antibacterial Activity

The minimal inhibitory concentration (MIC) was determined by broth microdilution. [11] This method was established in agreement with the recommendations of Cos y col., 2006. [12] Briefly, ME and AqF were dissolved in 10% dimethyl sulphoxide (DMSO) (v/v) and the organic fractions in 15% Tween 80 (v/v); these extracts were loaded in the wells of microplates at concentrations from 0.5 to 64 mg/ml and 5Χ10 5 UFC/ml of bacterial inoculum. Gentamicin was used as a positive control and dissolution solvents as negative controls. Microplates were incubated for 18 hours (35ºC) and the MIC values were determined as the concentration at which bacterial growth was not observed. Evaluations were done in triplicate.

Phytochemical Screening

ME, HF, CF, EAF and AqF samples were analysed using the following: the reagents of Dragendorff, Mayer and Wagner for alkaloids; Keller-Kiliani, Lieberman-Burchard and Salkowski for cardiotonics; 1.0% gelatin solution and quinine sulphate solution with FeCl 3 for tannins; the Shinoda test for flavonoids; yellow fluorescence by reaction with NaOH and KOH for coumarins; SbCl 3 and lather formation for saponins; NaOH for free anthracenes and the Salkowski and Lieberman-Burchard for terpenes. [13],[14],[15]


   Results and Discussion Top


The lowest antibacterial effect was observed for the ME (MIC≥32 mg/ml) and the AqF, CF and EAF were the most active fractions with MIC values in the range of 2-8 mg/ml. In general, the active samples were more effective against Gram positive bacteria. They also showed activity against enteropathogenic bacteria. The EAF antibacterial effect was remarkable against Shigella dysenteriae (2 mg/ml) [Table 1], the causal agent of dysentery. The activities of the CF and EAF were in the range of values reported for the aerial parts of other Vitex species (0.02-10 mg/ml). [16],[17],[18] The MIC values of ATCC strains to gentamicin were in the range of values previously reported [Table 1]. [11]

The evaluated samples showed a high content of phenolics (i.e. flavonoids and tannins), followed by terpenes [Table 2]. HF showed the highest content of terpenes but an intermediate antibacterial activity; thus, it is suggested that antibacterial activity of the most active fractions (CF and EAF) is mainly associated with phenolics [Table 1] and [Table 2]. Phenolics are the most common antibacterial agents isolated from plants, being mainly active against Gram positive bacteria; [19] they have been recommended as antidiarrhoeic agents. [4] The antibacterial effect of the ME could be considered low [Table 1]. Several studies have focused on antibacterial and antidiarrhoeal activity of Vitex spp. but not on V. mollis fruit. The methanolic extract of V. mollis leaves showed higher antispasmodic activity (37%) on guinea pig intestines than other Mexican plants, whereas the hexanic extract, evaluated up to 16 mg/ml, was not active against enteropathogenic bacteria. [8] Remarkably, aqueous extract of V. doniana fruit showed a significant dose-response antidiarrhoeic activity on mice (150-650 mg/kg) and it was not toxic; at the highest concentration, no significant difference was found with loperamide (5 mg/kg) (P<0.001). In addition, it was suggested that antidiarrhoeic activity could be associated with the content of phenolics. [9]
Table 1 :MIC of V. mollis fruit extracts against bacteria

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Table 2 :Phytochemical analysis of V. mollis fruit extracts

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   Conclusions Top


The antibacterial activity of the V. mollis fruit is reported for the first time and the fractions with highest activity (CF and EAF) have phenolics and terpenes as their constituents. Our results give scientific experimental support for the traditional use of this fruit as an antidiarrhoeic and suggest that this antibacterial activity is mainly associated with the phenolics content. Additional studies are required to characterise the chemical components of the active fractions.


   Acknowledgments Top


We thank the following institutions/persons: "Programa de Fomento y Apoyo a la Investigaciσn (PROFAPI)-UAS", "Consejo Estatal de Ciencia y Tecnologνa del Estado de Sinaloa (CECYT)" and "Consejo Nacional de Ciencia y Tecnologνa (CONACYT)" for the financial support; "CONACYT-Fondos Mixtos Sinaloa (Sin-2007-C01-69959)" for the scholarship to Pνo-Leσn JF; Dr. Rafael Garcνa-Gonzαlez, "Instituto Nacional de Peditarνa, Secretarνa de Salud, Mιxico, D.F.", for the bacterial strains of diseased patients; and Dr. Rito Vega-Aviρa, Agronomy Faculty - UAS, for the identification of plant material.

 
   References Top

1.Schmidt FR. The challenge of multidrug resistance: Actual strategies in the development of novel antibacterials [MINI-REVIEW]. Appl Microbiol Biotechnol 2003;63:335-43.  Back to cited text no. 1
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2.Boletín Epidemiología. México, D.F.: Dirección General de Epidemiología. Secretaría de Salud [last updated on 2009 Aug 5] Available from: http://www.dgepi.salud.gob.mx/boletin/boletin.htm [last cited on 2009 Sep 3].  Back to cited text no. 2
    
3.Fowler MW. Plants, medicines and man [Review]. J Sci Food Agric 2006;86:1797-804.  Back to cited text no. 3
    
4.Palombo EA. Phytochemicals from traditional medicinal plants used in the treatment of diarrhoea: Modes of action and effects on intestinal function. Phytother Res 2006;20:717-24.  Back to cited text no. 4
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5.Pennington TD, Sarukhán J. Tropical trees from Mexico. Manual for identification of the main species from Mexico: UNAM FCE; 2005.  Back to cited text no. 5
    
6.Montiel-Herrera M, Camacho-Hernandez IL, Rios-Morgan A, Delgado-Vargas F. Partial physicochemical and nutritional characterization of the fruit of Vitex mollis (Verbenaceae). J Food Comp Anal. 2004;17:205-15.  Back to cited text no. 6
    
7.Moldenke HN. Additional notes on the genus Vitex. XVII. Phytologia 1980;46:42.  Back to cited text no. 7
    
8.Osuna L, Tapia-Perez ME, Jiménez-Ferrer JE, Carrillo-Quiróz BA, Silva-Sanchez J. Screening of Alternanthera repens, Boerhavia coccinea, Flaveria trinervia, Tournefortia densiflora, and Vitex mollis extracts to evaluate their antibacterial activity and effect on smooth muscle. I. Pharm Biol 2005;43:749-53.  Back to cited text no. 8
    
9.Suleiman MM, Yusuf S. Antidiarrheal activity of the fruits of Vitex doniana in laboratory animals. Pharm Biol 2008;46:387-92.  Back to cited text no. 9
    
10.Wall ME, Wani MC, Brown DM, Fullas F, Oswaldo JB, Josephson FF, et al. Effects of tannins on screening of plants of plants extracts for enzyme inhibitory and techniques for their removal. Phytomedicine 1996;3:281-5.  Back to cited text no. 10
    
11.Lennette EH, Balows A, Hausler WL, Shadomy HJ. Susceptibility tests: Microdilution and macrodilution in broth. In: Lennette EH, Balows A, Hausler WL, Shadomy HJ, editors. Manual of Clinical Microbiology, Buenos Aires. Argentina: Editorial Medica Panamericana S.A.; 1987.   Back to cited text no. 11
    
12.Cos P, Vlietinck AJ, Berghe DV, Maes L. Anti-infective potential of natural products: How to develop a stronger in vitro ′proof-of-concept′. J Ethnopharmacol 2006;106:290-302.  Back to cited text no. 12
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13.Domínguez AX. Métodos de investigación fitoquímica. 4 th ed. México, D.F.: Limusa; 1988.  Back to cited text no. 13
    
14.Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian medicinal. Afr J Biotechnol 2005;4:685-8.  Back to cited text no. 14
    
15.Harborne JB. Phytochemical Methods (a Guide to Modern Techniques of Plant Analysis). London, UK: Chapman and Hall Ltd; 1980.  Back to cited text no. 15
    
16.Hernandez MM, Heraso C, Villarreal ML, Vargas-Arispuro I, Aranda E. Biological activities of crude plant extracts from Vitex trifolia L. (Verbenaceae). J Ethnopharmacol 1999;67:37-44.  Back to cited text no. 16
    
17.Kilani AM. Antibacterial assessment of whole stem bark of Vitex doniana against some enterobactriaceae. Afr J Biotechnol 2006;5:958-9.  Back to cited text no. 17
    
18.Nyiligira E, Viljoen AM, Van Heerden FR, Van Zyl RL, Van Vuuren SF, Steenkamp PA. Phytochemistry and in vitro pharmacological activities of South African Vitex (Verbenaceae) species. J Ethnopharmacol 2008;119:680-5.  Back to cited text no. 18
    
19.Rios JL, Recio MC. Medicinal plants and antimicrobial activity. J Ethnopharmacol 2005;100:80-4.  Back to cited text no. 19
    

Top
Correspondence Address:
Francisco Delgado-Vargas
Master of Science Program in Food Science and Technology, Faculty of Chemical and Biological Sciences, Autonomous University of Sinaloa, Ciudad Universitaria s/n C.P. 80010, Culiacan, Sinaloa
Mexico
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DOI: 10.4103/0973-8258.74140

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