Studies on antibacterial and antifungal of ethanol extract from wedelia chinensis in Viet Nam



This study examined the in vitro antibacterial and antifungal activities of ethanol extracts from Wedelia chinensis. The extracts were tested against two E. coli and Slamonella and two strains fungi A. flavus và F. solani (using disc diffusion method). The results showed that Ethanol extract has the ability to inhibit test strains, high resistance to E. coli (25 ± 0.67mm) and A. flavus (12 ± 0.84mm). The results obtained in the present study suggest that the extracts can be used in treating diseases caused by the test organisms.

Therefore, Wedelia chinensis can be used to develop new pharmaceutical for the treatment of bacterial and fungal diseases.

Keywords: Wedelia chinensis; extracts; Ethanol; antibacterial; antifungal.

1. Introduction

Human infections which involved microorganisms i. e., bacteria, fungi, viruses, nematodes cause serious damages in tropical and subtropical countries of the world.Keiji Fukuda, Ph.D., assistant WHO director-general, said that drug resistance is a major challenge in the treatment of infectious diseases today. The failure of chemotherapeutics and antibiotic resistance, forcing scientists to search for new antimicrobials from different types of sources

Therefore, the human demand need to discover new compounds with antimicrobial chemical structures and the diverse mechanisms of action to new infectious diseases are of current interest.

Medicinal plants are the richest source for strong antimicrobials. The medicinal value of these plants lies in some chemical active substances that produce a definite physiological action on the human body.

Wedelia chinensis Of the Asteraceae (Asteraceae). In Viet nam also known as «ngò núi», French Chrysanthemum flowers Ladle. This is a herbaceous plant, creeping, grow wild where moist cool.

Leaves almost no stalk, grows on, elongated oval bicuspid, both sides have hairy leaves, trees with yellow flowers.

According to herbal medicine, Wedelia chinensis sweet, sour, cool; there is heat effect, detoxification, holding cough, cooling blood; commonly used to treat flu, fever, sore throat, bronchitis, whooping cough, hypertension, hemorrhoids fistula, measles, pimples... Wedelia chinensis is considered curative herbs simple, effective and attracted the attention of many research scientists.

The purpose of this study evaluate antibacterial and antifungal of ethanol extract from Wedelia chinensis.

2. Materials and Methods

2.1.Material

Wedelia chinensis were collected in Da Nang city. The sample was determined by morphological comparison method according to Pham Hoang Ho's botanical classification [1]. The specimen template was preserved at Genetic Laboratory — Animal physiological anatomy, Department of Biology — Environment, University of Education, Da Nang.

Bacteria and Fungi:

Strains of 02 bacteria: Escherichia coli; Samonella sp and 02 strains of A. flavus fungus; F.solani were provided of lab microbiology — biochemical technology department of the Department of Biology — Environment, University of Education — Danang University.

2.2. Extraction method

W. chinensis collected in Da Nang city, was washed, shade dried, powdered leaf material was taken.

W. chinensis was extracted using the Ethanol. The extract drained was recovered by vacuum rotating with a temperature of 55ºC, 137atm pressure to create high extract.

2.3.Preliminary survey method of components in extract

Preliminary survey of chemical components (qualitative alkaloid, saponin, coumarin, tannin, flavonoid) applied the method of Romanian University of Pharmacy with improvement in accordance with the laboratory (Ministry of Health, 2006). The principle of the method is based on the solubility of the compounds in medicinal materials to separate compounds with different polarization solvents. Then, determine these compounds by specific reactions

2.3. Methods for determining antibacterial and antifungal activity

Ethanol extracts from W. chinensis soil is mixed in sterile DMSO solution at a concentration of 100; 200; 400; 800 mg / ml. The controlled substance in antibacterial is tetracylin (1mg / ml in DMSO) and in antifungal is 0.04mM nystatin with antifungal ability; Control evidence is sterile DMSO solution. Each concentration is repeated 3 times. The plates were incubated at 37⁰C for 16–20 hours

Antibiotic resistance of the extract was determined by the agar plate diffusion method of Hadacek et al. (2000) [6]. Accordingly, the tested antibacterial activity was assessed by measuring the microbial inhibitory ring diameter according to the formula.

2.4. Statistical analysis

Experimental data are statistically processed by biological statistical method, using data analysis tool of Microsoft excel. Experimental results are indicated by (M ± SD) & (M ± SE).

3. Results and discussion

3.1. Results of ingredients in Ethanol extracts from W. chinensis

The W. chinensis has been interested in research by Vietnamese scientists from the previous year. In order to determine the composition of substances in Ethanol extract from W. chinensis, we conduct qualitative analysis of some compounds in the sample as presented in Part 2. The results are shown in Table 1.

Table 1

Results of ingredients in ethanol extract from W. chinensis

Group of compounds	Reagents	Result
Flavonoid	NaOH 10 %	+
	FeCl3 5 %	+
Saponin	Foaming reaction	+
Ankaloid	Dragendorff	+
	Bouchardat	+
Coumarin	Open the lactone ring	_
Tannin	FeCl3 5 %	+
	Pb(CH3COO)2.3H2O.	+

It can be clealy seen that ethanol extracts from W. Chinensis gave a positive reaction to the substance class reagents: flavonoids, saponins, alkaloids and tannins. This result is similar to the study of Shanmugam Sureshkumar et al 2005 [9] in the chemical composition of W. Chinensis containing alkaloids, tannins, flavonoids, steroids, triterpenid...

3.2. Result of antimicrobial activity of Ethanol extract from W. Chinensis

Antimicrobial activity of Ethanol extract from W. Chinensis has been studied at different concentrations (100; 200; 400; 800 mg / ml) against 2 pathogenic strains of bacteria including two strains of Gram negative Salmonella sp, E. coli.

The antimicrobial capacity determined based on the ability to inhibit bacterial growth shown by the antibacterial diameter produced on Peptri dishes is presented in Table 2 and Figure 1.

Table 2

Antibacterial ring diameter of ethanol extract (mm)

Concentration(mg/ml)	Salmonella. sp	E.coli
100	8,78 ± 0,71	10,67 ± 0,50
200	11,55 ± 0,68	12,22 ± 0,62
400	13,89 ± 1,05	15,00 ± 0,21
800	21,4 ± 1,15	25 ± 0,67
tetracylin 1mg	24,67 ± 1,15	30 ± 1,27
DMSO 5 %	0,0	0,0

Salmonella. sp	E.coli

Figure 1. Antibacterial effect of W. chinensis against bacteria Salmonella.sp and E. coli

According to Table 2 and Figure 1, the results showed that the antimicrobial activity of the ethanol extract from W. Chinensis for Salmonella.sp and E. coli strains at the concentration of 800mg / ml showed the highest antibacterial activity against the two strains. (from 21.4 ± 1.15mm with Salmonella.sp and 25 ± 0.67 mm with E.coli), the lowest antibacterial activity at the concentration of 100mg / ml, the inhibitory zone is 8.78 ± 0.71 to 10.67 ± 0.50mm. However, a high concentration of 800mg / ml extracted W. Chinensis gives lower antibacterial effect of 1mg tetracycline.

Based on the experimental results with 5 % DMSO, negative controls did not appear antimicrobial rings, this was confirmed when most of the antibacterial experiments all use DMSO as a negative test. Therefore it can be concluded that DMSO dissolves most compounds and uses negative evidence with bacteria without affecting antibacterial results [5].

Thus, Ethanol extract from soil has good antibacterial ability even in low concentrations. The antimicrobial activity of high ethanol extract from W. Chinensis can be attributed to the effects of compounds of tannin and saponin class in extraction. According to the research results of the published works, extracts which was extracted from plants containing compounds of the class of saponins, tannins, flavonoids, alkaloids often have antibacterial activity [5,8,10]

− Antimicrobial activity of compounds of flavonoid class can be through the pathways such as inhibiting nucleic acid synthesis, inhibiting cytoplasmic function, inhibiting energy metabolism, inhibiting cohesion. and biofilm formation, porin suppression on cell membranes, changes in cell membrane permeability and impairment of pathogenicity [12].

− The antimicrobial activity of saponin may be through disturbance of the permeability of the outer membrane of bacteria. About 90 % of the surface of gram-negative biofilm membranes has no cholesterol but it is coated with lipopolysaccharide and protein. Saponins can interact with membrane lipids, thus promoting antibiotic absorption in bacteria [3].

− Tanin has the ability to inhibit bacterial enzymes and affect the metabolism of bacteria through inhibiting oxidative phosphorylation, so tannin has antibacterial activity. In addition, tannins can form complexes with many metal ions such as iron ions or copper ions... and through this activity, tannin prevents the growth of bacteria and thus has antibacterial activity [8].

− Alkaloid also exhibits anti-inflammatory activity by reducing the pathogenicity of bacteria by inhibiting the formation or dispersion of bacterial biofilm, inhibiting the production of calogen as well as excretion. Bacterial enzymes [5].

3.3. Result of mold resistance of Ethanol extract from W. Chinensis

Antifungal activity of Ethanol extract from W. Chinensis has been studied at different concentrations (100; 200; 400; 800 mg / ml) against pathogenic mold strains such as A. flavus and F. solani. 5 %. DMSO is a negative control that cannot resist mold-like bacteria. The antifungal ability is determined based on the ability to inhibit the growth of mold shown by the mold resistance diameter produced on petri dishes as shown in Table 3.

Table 3

Diameter of antifungal ring of Ethanol extract from W. Chinensis(mm)

Concentration(mg/ml)	A. flavus	F. solani
100	4,3 ± 0,41	0,0
200	6,83 ± 0,32	0,0
400	8,5 ± 0,84	0,0
800	12 ± 0,84	0,0
nystatin 0,04mM	25 ± 0,62	21,0 ± 1,73
DMSO 5 %	0,0	0,0

The results of the study showed that Ethanol extract from W. Chinensis does not have antifungal activity for F. solani strains but has a resistance to A. flavus strain and this activity is lower than the control nystatin.

A high concentration of 800 mg / ml found that the antibiotic ring was large, clear and there was a lot of difference with the result of 12 ± 0.84mm. A high concentration of 100 mg / ml of low fungal resistance only reaches a diameter of 4.3 ± 0.41mm.

The presence of compounds of flavonoid, tannin, etc. in ethanol extract from W. Chinensis can explain the antifungal activity of ethanol extract from soil. These compounds have antifungal properties through cell membrane breakdown, inhibition of cell wall formation, and mitochondrial inhibition. In addition, compounds of alkaloid class are also resistant to fungi through the breakdown of cell membranes by inhibiting ergosterol biosynthesis or complexing with ergorterol [7].

4. Conclusions

Based on these results, it can be concluded that Wedelia chinensis extracted from ethanol has great potential as an antibacterial and antifungal agent for the treatment of infectious diseases caused by pathogenic bacteria.

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