Phytochemical and Preliminary Study of the Antimicrobial Activity of Salvia Ort Amarissima

Salvia amarissima is a Mexican plant with special use in the traditional medicine that has showed biological activity against hypoglycemic, antiulcerant and antihelmintic diseases.

In this study we evaluated for the first time its antimicrobial activity. A hexanic, methanolic and aqueous extracts of the plant were tested against 8 strains of standard microorganisms by the techniques of Kirby-Bauer and the antimicrobial dilution in agar. There was not antimicrobial activity detected on different extracts. Finally, a preliminary spectroscopic analysis of the compounds in each extract was done.

Introduction
Medicinal plants have been used since ancient times to treat various illnesses. Therapeutic use has been systematically studied for two centuries under a scientific approach. With the discovery of penicillin, herbal step into the background and the development and use of antimicrobials is becoming widespread. Currently, there is renewed interest in natural and organic and has led to the emergence of natural extracts generating new lines of research.

Among the many therapeutic applications of plant antimicrobial action is included. An antimicrobial is a substance that low concentrations are able to inhibit or kill microorganisms.

The main effects of action of antimicrobials are:

Bacteriostatic: the minimum non-toxic concentration is reached in serum and tissues, which im-calls for the development and multiplication of microorganisms without destroying them, who may multiply again to disappear the antimicrobial agent. Bacteriostatic agents allow complete host mechanisms such as the synthesis of antibodies, complement or cytokines that allow the final destruction of the organism.
Bactericide: Its action is lethal on microorganisms, so these irreversibly lose their viability.

Antimicrobials are the fundamental basis of the treatment of infectious diseases, one of the most frequent problems and causing increased mortality in any specialty me certainty, such as pneumonia, myocarditis, hepatitis, tuberculosis, etc.

Unfortunately the irrational, given the inadequate medical prescription as well as poor use by patients has led to a search for new antimicrobial resistance due to ad-quirida by microorganisms, which now makes this therapeutic infectious. therefore has retaken the knowledge of traditional medicine, to detect new secondary metabolites in plants with antimicrobial potential.

The genus Salvia is the most diverse of Labiatae, with about 900 species widely distributed throughout the world, of these, about a quarter live in the mountains of Mexico. According to copies of ENCB Herbaria and MEXU, Salvia amarissima is distributed in the Valley of Mexico and in the states of Michoacan, Hidalgo, Guanajuato, San Luis Potosi, Oaxaca, Morelos, Chiapas, and grows wild in grasslands, shrublands, forests of pine and scrub oak xerophytic. Also grows widely in disturbed environments at an altitude of 1900-2460 meters.

Some species have been studied for their antimicrobial properties attributed to its essential oils such as Salvia sclarea, which have isolated four diterpenes abiatanos: salvipisona, aetiopinona, 1-ferru-ginol oxoaetiopinona and its roots, this diterpene last abiatano also was obtained from S. lavandulifolia and S. officinalis pinene, cienolo and camphor.

S. hispanica has been considered an important food and nutrition from the Aztecs which has been given a high amount of alpha-linolenic acid, a compound that decreases hypertriglyceridemia, while S. microphyla, S. leucantha and S. Mexico has been used to treat digestive problems. S. divinorum was used in certain rituals in the state of Oaxaca due to its hallucinogenic properties, produced by the compound Salvinorin A, which is an agonist opioid receptor is known in Latin America and the antimicrobial activity of Salvia rubescens, Colombian species – Biana known for its use against viral hepatitis and heart disease, among others.

Since essential oils S. amarissima the most studied species of salvias, was isolated amarisolido, a diterpene glycoside neoclerodano with antiinflammatory activity. This compound was also isolated in S. rubescens. and S. miltiorrhiza Bunge, which probably explains its use against heart disease. Despite efforts to isolate and characterize new secondary metabolites with potential therapeutic use are needed pharmacological and chemical studies of this genre. So in the present study evaluated the antimicrobial activity of three different extracts of S. amarissima because traditional medicine has been given to this genre antimicrobial, hypoglycemic and anthelmintic.

Objective
To evaluate the antimicrobial activity of the hexane extracts, methanolic and aqueous amarissima Salvia by agar diffusion method (Kirby-Bauer) and antimicrobial dilution technique in agar, to detect the compounds respon-sible for this by analyzing phytochemical to assist in the detection of bioactive compounds, which may constitute an alternative to the development of new antimicrobial therapeutic agents of natural origin.

Hexane Extract
40g were placed on the ground dried and crushed into a ball flask flat bottom and two liters of hexane added. After 24 h the solvent was removed by filtration, was collected in a flask ball and evaporated to dryness using a rotary evaporator. The procedure was repeated once more and produced about 14.9081g of extract, representing a yield of 37.3%.

Hexane crude extract was sampled for analysis in proton nuclear magnetic resonance (1 H NMR).

Methanol Extract
The residue of dried and crushed plant were added four liters of methanol for 48h (2L X 24 hours). The solvent was removed from the plant, filtered and collected in a flask ball, evaporated to dryness in a rotary evaporator thus obtaining about 20g corresponding to 50% yield.

Methanolic crude extract was sampled for subsequent 1 H NMR analysis.

Water Extract
Were placed 40 g of the dried plant and crushed into a flat-bottomed flask ball and they added two liters of water to make a decoction of the plant getting an infusion.

Science and Technology
volume is a portion of 100mL and lyophilized to concentrate the infusion; 5g lyophilisate was obtained corresponding to 12.5% ??yield. The other remaining volume was placed in a refractory and was added 50 mL of chloroform with a foil covering and concentrated to dryness at room temperature. Were obtained 25 g corresponding to 62.5%.

Spectroscopic analysis of nuclear magnetic resonance of hydrogen
The 1 H NMR spectra were performed on a 200 MHz spectrometer JEOL Eclipse-200 analytical. Using deuterated chloroform as solvent (CDCl3) and tetramethylsilane as internal reference. The chemical shifts (d) expressed in parts per million (ppm).

Determination of the antimicrobial activity of the hexane extract, methanol and aqueous amarissima Salvia
To demonstrate the presence of antimicrobial compounds present in the extracts hexane, methanol and aqueous bacterial strains were used in the area of ??culture collection of chemical-biological Sciences, Faculty of Science and Technology of USB.

To determine the possible microbial growth activity of the extracts of the plant Salvia loves-rissima took out two microbiological techniques developed under sterile conditions:

Antimicrobial diffusion technique in agar using filter paper discs (Kirby-Bauer method). This technique was used filter paper discs impregnated with the extract. The discs were placed on the surface of the plate in a solid medium seeded with massively test micro-organisms identified above.

The sensitivity was observed by the presence of a halo of growth inhibition around the disc. The halo size depends on the sensitivity of the organism and the concentration of the antimicrobial disk.

The inoculum of the reference strains was prepared from BHI plates were seeded with the strains by cross striations, which were taken from 3 to 5 colonies and these strains were adjusted to young nephelometer tube of 0.5 MacFarland corresponding at a concentration of 1.5 x 108 bacteria / mL.

After adjusting the bacterial culture was planted on agar plates massively Muller-Hinton Dibico ® (AMH) for a uniform inoculum. Then a known amount of aqueous and methanolic extracts were dissolved in 2.5 mL of sterile distilled water and the hexane extract was dissolved in 4.5 mL of dimethylsulfoxide. These two solutions were sterilized by filtration using a Millipore ® membrane of 0.45 microns in diameter.

And extract with 10 sterile serial dilutions were made with saline solution and impregnated with 10?L disks ® 40 Whatman filter paper with a diameter of 5mm sterile, by sticking on these plates inoculated with cultures AMH set of test organisms . Placing them in the direction of clockwise, using sterile forceps and pressing lightly on the agar to ensure complete contact with the surface that favors the diffusion of the compound to be tested. The plates were incubated at 37 ° C for 24 h. At the end of this period, was observed if plates showed an inhibition zone around the disk and if present diameter was measured.

Antimicrobial dilution technique in agar and determination of minimum inhibitory concentration (MIC). In sterile conditions and sterile extract and 10 serial dilutions were made with saline. 10 were labeled petri dishes and transferred to 2 mL of each dilution corresponding box and added 18ml of AMH subsequently cooled melt at a temperature close to 45 ° C. Antimicrobial homogenized in the culture medium by rotation of the plate on the table. Allowed to solidify. Sectors were drawn in each box with the antimicrobial and wrote on behalf of the reference strains. AMH was used as control without extract. Later take a roast each strain and was planted by estria open in the relevant sector. The plates were incubated at 37 ° C for 24h and treatment of the extract concentration observe possibly inhibit the growth of each strain.

Analysis of the 1H NMR spectrum of the hexane extract. The obtained spectrum shows signals corresponding to mainly aliphatic, signals at 0.5 – 1.5 ppm are indicative of hi-androgens for this type of compounds very likely internal double bonds exist due to the presence of signals complemented ad 4.8-5.4 ppm those observed ad 2.0 ppm. It shows the presence of other signals that may indicate the presence of some other group of compounds of pharmacological interest.

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