SCREENING ANTIBACTERIAL ACTIVITY OF VIETNAMESE PLANT EXTRACTS AGAINST HUMAN PATHOGENIC BACTERIA

Objectives: Infectious diseases caused by bacteria are a leading cause of death worldwide. Hence, the objectives of the study are aimed to evaluate the antibacterial activity against five human pathogenic bacteria of methanolic extracts from 66 plants collected from Vietnam. 
Methods: The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of methanol extracts of 66 plant species against five bacterial strains. 
Results: In this study, all the plant extracts were active against at least one train with MIC values ranging from 24 to 2048 μg/mL. Twenty-five plant extracts were active against all three Gram-positive bacteria (Bacillus cereus, Bacillus subtilis, and Staphylococcus aureus). Of these, the extracts of Macaranga trichocarpa (Rchb. f. and Zoll.) Mull. Arg. (Euphorbiaceae), Calophyllum inophyllum L. (Clusiaceae) and Caryodaphnopsis baviensis (Lecomte) Airy Shaw (Lauraceae) exhibited the highest antibacterial activity (MIC =24–128 μg/mL), followed by extracts of Betula alnoides Buch.- Ham. e × . D. Don (Betulaceae), Acronychia pedunculata (L.) Miq. (Rutaceae), Croton alpinus A. Chev. ex Gagnep. (Euphorbiaceae) (MIC =64–256 μg/mL). Furthermore, the extract of Rhus chinensis Mill. (Anacardiaceae) and Annona reticulata L. (Annonaceae) exhibited potent antibacterial activity against the two Bacillus species (MIC =32–64 μg/mL). 
Conclusion: Results of this study reveal that plant extracts from Vietnam have highly antibacterial activity against Gram-positive bacteria. These results suggest that Vietnamese plant extracts may be a rich source of antibacterial drugs.


INTRODUCTION
Infectious diseases remain a major health concern, being the second leading cause of death worldwide, and remain a dominant feature of domestic and international public health considerations in the 21 st century [1,2]. Bacterial infections are prevalent in developing countries due to factors such as inadequate sanitation, poor hygiene, and overcrowded living conditions [3]. Antibiotics have proven to be powerful drugs for control of infectious diseases and remain one of the most important discoveries in modern medicine [4]. At present, the world is facing the widespread emergence of bacterial resistance to antibiotics [2]. Antibiotic resistance has been recognized by the World Health Organization as the greatest threat in the treatment of infectious diseases [4].
To combat antibiotic resistance, the development of new antibacterial agents that suppress bacterial resistance mechanisms is necessary. Plants have traditionally provided a source of new chemicals, and numerous clinical studies have demonstrated the therapeutic value of molecules of plant origin [4]. Mainstream medicine is increasingly receptive to the use of antimicrobial and other drugs derived from plants as traditional antibiotics [5]. Indeed, higher plant-derived products represent ~25% of drugs in current clinical use [4].
Considering the therapeutic potential of plants, the aim of the present study was to evaluate the in vitro antibacterial activity of Vietnamese plant extracts against five human pathogenic bacteria, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, and Staphylococcus aureus, which cause food poisoning and various infections in the community and in hospitals [6,7].  [8]. Briefly, air-dried and powdered aerial parts of the plant species (10 g) were extracted twice with 100 mL of methanol for 48 h at room temperature. Extracts were filtered and the filtrates were evaporated to dryness using a rotary evaporator, and then stored at −20°C until further use. Then, for the antibacterial activity assays, the extracts were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL and stored at 4°C as stock solutions.  25922 and P. aeruginosa ATCC 9027) strains, were purchased from the (ATCC; Manassas, VA, USA). Bacterial strains were cultured on nutrient agar plates and incubated aerobically at 37°C for 24 h. For in vitro antibacterial assays, the bacteria were cultured in nutrient broth (NB) and incubated aerobically at 37°C for 24 h, and the bacterial suspension was adjusted to 10 5 cfu/ml.

Antibacterial assay
The antibacterial activities of plant extracts were evaluated by broth microdilution assays and minimum inhibitory concentration (MIC) values were determined. Twofold serial dilutions of each plant extract were added to the wells of sterile 96-well plates containing 100 µl of inoculated NB medium. The final concentrations of the plant extracts were 15.6-2000 µg/ml and the final bacterial density was 10 5 cfu/ml. DMSO (2%) was used as a negative control and did not affect bacterial growth; streptomycin sulfate was used as a positive control. The plates were incubated at 37°C for 24 h. The MIC values were determined as the lowest concentration that completely inhibited the growth of bacteria.
For weakly soluble plant extracts, the MIC was determined following addition of 20 µl of 0.2 mg/ml iodonitrotetrazolium chloride (Sigma-Aldrich, Korea) to the test wells and incubation at 37°C for 3 h. Viable bacteria reduce this yellow dye to pink [8]. Experiments were conducted twice in triplicate for each extract against individual bacterial species at all test concentrations.

RESULTS AND DISCUSSION
A total of 68 plant extracts were evaluated for antibacterial activity against five human pathogenic bacteria using the broth microdilution method. The plant extracts exhibited considerable antibacterial activity against the Gram-positive bacteria. B. cereus exhibited the greatest susceptibility to all of the plant extracts, followed by B. subtilis and S. aureus. Twenty-four plant extracts had the antibacterial activity against all three Gram-positive bacteria. Of these, the extracts of Macaranga trichocarpa, Calophyllum inophyllum, and Caryodaphnopsis baviensis had the highest antibacterial activity (MICs 31.3-125 µg/ml). Extracts of Acronychia pedunculata, Betula alnoides, Croton alpinus, Garcinia cowa, and Annona reticulata showed slightly weaker antibacterial activity (MICs 62.5-250 µg/ml). To the best of our knowledge, this is the first report of antibacterial activity of C. alpinus. In contrast, antibacterial activities and phytochemistry have been described for A. pedunculata [9], B. alnoides [10], G. cowa [11][12][13], and A. reticulata [14,15]. Furthermore, the extract of Rhus chinensis exhibited potent antibacterial activity against Bacillus species with MIC values of 31.3 µg/ml (Table 1). Conversely, most of the test plant extracts were inactive against the two Gram-negative bacteria. Only the extract of C. inophyllum at a concentration of 2000 µg/ml completely inhibited the growth of E. coli (Table 1).
In the following sections, the plants showing the greatest activities against Gram-positive bacteria are discussed in terms of their known phytochemical components, together with biological and pharmacological activities.

C. inophyllum
All parts of C. inophyllum have considerable therapeutic properties, such as antiseptic, expectorant, diuretic, astringent, and purgative effects [16]. The fruit oil is beneficial in treatment of rheumatism, gonorrhea, and itching, the gum extracted from the stems is used to treat ulcers, and a decoction from the bark is used for hemorrhage and ulcers [17]. Oil of seeds and roots is used to treat wounds and scabies [16]. Phytochemical components of this plant have been reported previously [17]. Extracts from leaves and other parts exhibited antibacterial activities against various bacteria as described in the previous reports [16,18]. In our study, the methanol extract of leaves and branches of C. inophyllum had marked antibacterial activity against Gram-positive bacteria (MICs 31.3-62.5 µg/ml) and at 2000 µg/ml completely inhibited the growth of E. coli (Table 1).

C. baviensis
In this study, the methanol extract of C. baviensis exhibited significant antibacterial activity against Gram-positive bacteria (MICs 62.5-125 µg/ml) ( Table 1). At present, there is lack of scientific documentation on the pharmacological and biological activities, as well as the phytochemical components, of C. baviensis. To the best of our knowledge, isolation of compounds from this plant has been reported only by Anh et al. [19], and this is the first report of antibacterial activity of an extract of C. baviensis.

M. trichocarpa
The methanol extract of M. trichocarpa showed the strongest antibacterial activity against three Gram-positive bacteria among the 68 plant extracts tested in this study (MICs 31.3-62.5 µg/ml) ( Table 1). Leaves of some Macaranga species are used in folk medicine to treat swellings, cuts, sores, boils, and bruises. This genus is reported to be a rich source of isoprenylated, geranylated, and farnesylated flavonoids. Flavonoids and stilbenes are major constituents and most likely responsible for the activities of plants of this genus [20]. Flavanones and dihydrochalcones have been reported to show antibacterial activities against various bacterial species, including B. subtilis, S. aureus, E. coli, and P. aeruginosa [21]. However, the methanol extract of M. trichocarpa did not inhibit the growth of E. coli and P. aeruginosa at the maximum concentration tested (2000 µg/ml) ( Table 1).

R. chinensis
This plant has long been used by practitioners of folk medicine in Asia. R. chinensis plant parts, particularly the galls on its leaves, Galla chinensis, have preventative and therapeutic effects on diverse ailments, including diarrhea, dysentery, rectal and intestinal cancer, diabetes mellitus, sepsis, oral diseases, and inflammation. Phytochemical studies on R. chinensis have demonstrated that it contains high levels of two phenolic compounds, gallic acid and methyl gallate. Recent studies revealed that R. chinensis compounds possess strong antiviral, anticancer, hepatoprotective, and antioxidant activities. Extracts from G. chinensis inhibited several bacteria, including B. cereus, B. subtilis, S. aureus, E. coli, and others (MICs 0.5-8 mg/ml) [22]. In our study, the methanol extract of R. chinensis exhibited considerable antibacterial activity against the Gram-positive bacteria, especially against the two Bacillus species (MIC 31.3 µg/ml) ( Table 1).

CONCLUSION
The Vietnamese plant extracts investigated in this study significantly suppressed the growth of Gram-positive bacteria. Discovery of biological activities in Vietnamese plants is a new venture. Although the antibacterial activities of some highly antibacterial plant extracts have been reported, C. baviensis and C. alpinus extracts have not previously been reported to show potent antibacterial activities.
Our results provide important information on the antibacterial activities of Vietnamese plant extracts to medical plant consumers, pharmacologists and researchers. Some Vietnamese plant extracts have potential for application as natural antibacterial agents and can be used for the development of new antibacterial drugs.
To develop new plant-derived antibacterial agents, further studies are necessary to isolate and characterize the active components from the antibacterial plants. Furthermore, additional research on combinations of the antibacterial components or plants with other antimicrobial agents would be useful to enhance their antibacterial potency.