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In vitro antibacterial activity of dried extract from Anredera vesicaria rhizomes

After the pharmaceutical industry was devoted exclusively to the manufacture of synthetic drugs leaving behind the old medicines based on plant extracts, today there is a qualitative change in the industrial programs dedicated to the search for new medicines of herbal origin.1 Plants are one of the largest reservoirs of medicinal wealth in nature and the benefits to health, as well as their economic value have motivated a growing attention in recent decades. Anredera vesicaria, known by the common name yucahiedra is a perennial herbaceous plant belonging to the Basellaceae family. The maceration in ethanol of its rhizomes is used in eastern Cuba for the treatment of wounds, blows, fractures and bruises.2 Polyphenols are related to the anti microbial and antioxidant activities of many plants,3–5 and the rhizomes of A. vesicaria have a high content of phenolic compounds and flavonoids,6 however, no reports of the antibacterial activity of this plant have been observed. In this work we describe a preliminary study of the antibacterial activity of the ethanolic extracts obtained from rhizomes of A. vesicaria.


Introduction
After the pharmaceutical industry was devoted exclusively to the manufacture of synthetic drugs leaving behind the old medicines based on plant extracts, today there is a qualitative change in the industrial programs dedicated to the search for new medicines of herbal origin. 1 Plants are one of the largest reservoirs of medicinal wealth in nature and the benefits to health, as well as their economic value have motivated a growing attention in recent decades. Anredera vesicaria, known by the common name yucahiedra is a perennial herbaceous plant belonging to the Basellaceae family. The maceration in ethanol of its rhizomes is used in eastern Cuba for the treatment of wounds, blows, fractures and bruises. 2 Polyphenols are related to the anti microbial and antioxidant activities of many plants, [3][4][5] and the rhizomes of A. vesicaria have a high content of phenolic compounds and flavonoids, 6 however, no reports of the antibacterial activity of this plant have been observed. In this work we describe a preliminary study of the antibacterial activity of the ethanolic extracts obtained from rhizomes of A. vesicaria.

Materials and methods
The plant material was collected on June 18, 2017 in Palmas Altas, community belonging to the municipality of Manzanillo, Granma province, Cuba (20° 19'54.588 "N -77° 4'19.128" E) at 8:10 a.m., at a temperature of 31.4 °C with a relative humidity of 73%. The experimental work was developed in the Natural Products Laboratory of the Center for Applied Chemistry Studies of the University of Granma. A sample of rhizomes of A. vesicaria was dried for a week in the shade on perforated cardboard sheets, stirring the material twice a day; this action was completed in a stove with air circulation at 40°C for 4h. 7 It was then pulverized until obtaining a particle size of 0.5mm in diameter, and it was subjected to an ultrasound extraction process using 70% ethanol as Solvent. 8 The extract obtained was concentrated in a rotary evaporator. The Phytochemical analysis of extractswas carried out by using following standard methods. [9][10][11] The reagents and solvents used were of "pure" or "analytical" quality from Merck. The antibacterial activity of the extract was evaluated by the method of diffusion method in agar by Bauer-Kirby 12 surface disc dissemination and by the method of micro dilution in broth 13 against a battery composed of strains of Pseudomonas aeruginosa (wild), Staphylococcus aureus (wild), Escherichia coli (wild) and Salmonella typhimurium (ATCC 14028). 4 treatments were applied with 3 replications in each applied method, using as a negative control dimethyl sulfoxide (DMSO) that also served as solvent for the dry extract, and as a positive control disks of the commercial antibiotic chloramphenicol of 30μg/disc (Sensi-DiscTM, France). 14 The variables measured were: a. Diameter of the inhibition halo (ID) (mm) whose results were expressed as the average diameter of the inhibition halos in each treatment; and b. Minimum inhibitory concentration (MIC), which was expressed as the minimum concentration of the extract that completely inhibited bacterial growth in each treatment with the naked eye in a range of concentration of the extract between 2-1500g/mL.

Results and discussion
Considering that the population uses the ethanolic extract of rhizomes of A. Vesicaria (Figure 1) for medicinal purposes, an extract was obtained in 70% ethanol that was treated until obtaining a dry extract which was preserved for the microbiological study.

Phytochemical analysis
A phytochemical analysis was performed from the ethanolic extract; 9-11 the results are shown in Table 1 The chemical composition of the ethanolic extract of rhizomes, matches with those reported by de la Cruz et al. 6 The presence of alkaloids and abundance of phenols and flavonoids give the plant antibacterial potential activity. 15 These results agree with those reported for other species fromanrederagenus. 16,17

Anti bacterial activity in vitro
Four bacterial strains of pathogenic and investigative interest were evaluated. The results obtained are shown in Table 2. Table 3 shows the MIC values obtained in the evaluation of the dry extract of the rhizomes of A. Vesicaria against a gram-positivestrain (S. aureus) and 3gram-negativestrains (E. coli, P. aeruginosa and S. typhimurium).

Minimum inhibitory concentration
The zone of inhibition in the experiment was conceived as the area formed around the antibiotic discs, here bacterial growth was completely in hibited ( Figure 1). The result of eachtrial (Table 1) was reported as a halo of inhibition. The ethanolic extract of rhizomes showed anti bacterial action against Gram-positive and Gramnegativestrains. In all cases, halos of inhibition greater than 9mm were observed, highlighting the effect against S. aureus and P. Aeruginosa with halos of inhibition of 15 and 18 mm respectively. These results are encouraging because it is not common to observe halos of inhibitions opronounced in plant extracts. 18,19 The MIC values for S. aureus and S. Triphymurium were very close to each other, however, in the E. Coli test a higher concentration of the extract (187.5) was needed to inhibit bacterial growth (Table 3). This could be due to the factthatE. coliis a gram-negative bacterium whose cell wall has two membranes in the form of lipoprotein matrices with intermediate inclusions of peptidoglycans that can hinder the diffusion of certain molecules. 20 A highly acylated lipopolysaccharide located in the external leaf let has been detected of the outer membrane of gram-negative bacteri at hatis essential to maintain the barrier function that prevents the passive diffusion of hydrophobic solutes such as antibiotics and detergents inside the cell. 21 The strain of E. Coli use diswild, there fore, It is more resistant to the action of antibacterials. 22 The result so btained constitute the first report of in vitro antibacterial activity of A. vesicaria and may contribute to justify the medicinal properties attributed to this plant.

Conclusion
The ethanolic extract of A. vesicaria rhizomes presented remarkable in vitro antibacterial activity against gram-positive and gram-negative strains. The metabolites responsible for the antibacterial activity could be the phenolic compounds, flavonoids and alkaloids.