Headnote
ABSTRACT
Objective: The synergistic or adjuvant effect on the antimicrobial effectiveness of pelargonium essential oil (PEO) in combination with eucalyptus essential oil (EEO) has been the objective of this work.
Theoretical Framework: The small amounts of oil in pelargonium (difficultly extractable by vapor drag) have motivated a liquid PEO-liquid EEO (L-L) extraction. EEO has been used as an extractant for PEO, giving a final proportion of 5:2.
Method: Both essential oils were obtained by steam stripping with water. Analysis by GC-FID chromatography gave 80.5% w/w of 1,8-cineole (eucalyptol), 3.05% w/w of limonene, 5.4% w/w of a-pinene, and antioxidant capacity of 76.0%. For the ME-ESP2 mixture, 56.4% of 1,8-cineole and 9.30% of citronellol were obtained from the essential oil of Pelargonium spp. with an antioxidant capacity of 78.6%. Due to the small amount of PEO (0.033% in the wet base of the plant) compared to EEO (5%), a direct soxhlet extraction of PEO with EEO as extractant was developed, until obtaining a proportion equivalent to EEO (5): PEO (2), so that volatility losses (typical of hydro distillation) were avoided. This mixture has been called the L-L EXTRACTION MIXTURE (ME-ESP2).
Results and Discussion: In vitro, the effectiveness of ME-ESP2 against Klebsiella pneumoniae had better effectiveness against K. pneumoniae ATCC 13883 with a minimum bacterial concentration (MBC) of 60.5 mg/mL, a minimum inhibitory concentration (MIC) of 130 mg /mL and an excellent antimicrobial sensitivity (AMS) between 15.0 to 29.0 mm compared to Imipenem and oxacillin. It was found that the combination of the essential oils of Eucalyptus globulus Labill and Pelargonium spp. in a ratio of 5:2 has better antimicrobial effectiveness against Klebsiella pneumoniae.
Research Implications: The practical and theoretical implications of this research are discussed, providing information on how the results can be applied or influence practices in the field of medicine and biology.
Originality/Value: The relevance and value of this research is evident in providing an efficient and natural alternative to the indiscriminate use of chemical antibiotics, avoiding microbial resistance.
Keywords: Effectiveness, Essential Oil, Eucalyptus, Pelargonium, Klebsiella pneumonieae.
RESUMO
Objetivo: O efeito sinérgico ou adjuvante na eficácia antimicrobiana do óleo essencial de pelargônio (PEO) em combinação com óleo essencial de eucalipto (AEE) foi o objetivo deste trabalho.
Referencial Teórico: As pequenas quantidades de óleo de Pelargonium (difícil de extrair por extração a vapor) motivaram uma extração de PEO líquido-EEO líquido (L-L). O EEO tem sido utilizado como extrator de PEO, dando uma proporção final de 5:2
Método: Ambos os óleos essenciais foram obtidos por decapagem a vapor. A análise por cromatografia GC-FID deu 80,5% p/p de 1,8-cineol (eucaliptol), 3,05% p/p de limoneno, 5,4% p/p de α-pineno e uma capacidade antioxidante de 76,0%. Para a mistura ME-E5P2 foram obtidos 56,4% de 1,8-cineol e 9,30% de citronelol a partir do óleo essencial de Pelargonium spp. com capacidade antioxidante de 78,6%. Devido à pequena quantidade de PEO (0,033% na base úmida da planta) em comparação com EEO (5%), foi desenvolvida uma extração direta de PEO em soxhlet com EEO como extratante, até obter uma proporção equivalente a EEO (5):PEO (2), para que fossem evitadas perdas de volatilidade (típicas da hidrodestilação). Esta mistura foi chamada de MISTURA DE EXTRAÇÃO L-L (ME-E5P2).
Resultados e Discussão: In vitro, a eficácia do ME-E5P2 contra Klebsiella pneumoniae teve melhor eficácia contra K. pneumoniae ATCC 13883 com concentração bacteriana mínima (MBC) de 60,5 mg/mL, concentração inibitória mínima (CIM) de 130 mg/mL e excelente sensibilidade antimicrobiana (AMS) entre 15,0 a 29,0 mm em comparação com Imipenem e Oxacilina. Verificou-se que a combinação dos óleos essenciais de Eucalyptus globulus Labill e Pelargonium spp. na proporção de 5:2 apresenta melhor eficácia antimicrobiana contra Klebsiella pneumoniae.
Implicações da Pesquisa: São discutidas as implicações práticas e teóricas desta pesquisa, fornecendo informações sobre como os resultados podem ser aplicados ou influenciar práticas no campo da medicina e da biologia.
Originalidade/Valor: A relevância e o valor desta pesquisa são evidentes ao fornecer uma alternativa eficiente e natural ao uso indiscriminado de antibióticos químicos, evitando a resistência microbiana.
Palavras-chave: Eficácia, Óleo Essencial, Eucalipto, Pelargonium, Klebsiella pneumonieae.
RESUMEN
Objetivo: El efecto sinérgico o coadyuvante sobre la eficacia antimicrobiana del aceite esencial de pelargonio (PEO) en combinación con el aceite esencial de eucalipto (AEE) fue el objetivo de este trabajo.
Marco Teórico: Las pequeñas cantidades de aceite del Pelargonio (difícilmente extraíble mediante arrastre de vapor) han motivado una extracción de PEO líquido-EEO líquido (L-L). Se ha utilizado EEO como extractante de PEO, dando una proporción final de 5:2
Método: Ambos aceites esenciales se obtuvieron mediante decapado con vapor de agua. El análisis mediante cromatografía GC-FID dio un 80,5 % p/p de 1,8-cineol (eucaliptol), un 3,05 % p/p de limoneno, un 5,4 % p/p de a-pineno y una capacidad antioxidante del 76,0 %. Para la mezcla ME-ESP2 se obtuvo 56,4% de 1,8-cineol y 9,30% de citronelol a partir del aceite esencial de Pelargonium spp. con una capacidad antioxidante del 78,6%. Debido a la pequeña cantidad de PEO (0.033% en la base hámeda de la planta) en comparación con EEO (5%), se desarrolló una extracción soxhlet directa de PEO con EEO como extractante, hasta obtener una proporción equivalente a EEO (5):PEO (2), de modo que se evitaron pérdidas de volatilidad (típicas de la hidrodestilación). Esta mezcla se ha denominado MEZCLA DE EXTRACCIÓN L-L (ME-E5P2).
Resultados y Discusión: In vitro, la efectividad de МЕ-Е5Р2 contra Klebsiella pneumoniae tuvo mejor efectividad contra K. pneumoniae ATCC 13883 con una concentración bacteriana mínima (MBC) de 60,5 mg/mL, una concentracion minima inhibitoria (CIM) de 130 mg/mL y una excelente sensibilidad antimicrobiana (AMS) entre 15,0 a 29,0 mm en comparación con Imipenem y Oxacilina. Se encontró que la combinación de los aceites esenciales de Eucalyptus globulus Labill y Pelargonium spp. en una proporción de 5:2 tiene mejor eficacia antimicrobiana contra Klebsiella pneumoniae.
Implicaciones de la Investigación: Se discuten las implicaciones prácticas y teóricas de esta investigación, proporcionando información sobre cómo los resultados pueden aplicarse o influir en las prácticas en el campo de la medicina y la biologia.
Originalidad/Valor: La relevancia y valor de esta investigación se evidencian en dar uma alternativa eficiente y natural frente al uso indiscriminado de antibióticos químicos, evitando la resisténcia microbiana.
Palabras clave: Efectividad, Aceite Esencial, Eucalipto, Pelargonium, Klebsiella pneumonieae.
1 INTRODUCTION
In the context of biodiversity in Peru and phytomedicines, some plant species' essential oils (EO) present antimicrobial compounds for treating infectious diseases of the respiratory tract. The EOs of species such as Eucalyptus (Eucalyptus globulus Labill) and Pelargonium (Pelargonium spp.) from the Cusco region present medicinal properties against acute respiratory infections with anti-cold, expectorant, antiseptic effects, pleasant aroma and probably with antibacterial and antiviral. It is currently used in industrial preparations to combat colds; analgesic and anti-inflammatory properties are attributed to pelargonium leaves (Bussmann & Sharon, 2015).
The interest of the present investigation was determining the in vitro antimicrobial effectiveness of Klebsiella pneumoniae of the mixture of the two essential oils by determining the MBC, MIC, and AMS. Due to the small amount of PEO (0.033% on the wet basis of the plant) compared to EEO (5%), a direct liquid-liquid (L-L) extraction in soxhlet of PEO with EEO as extractant was developed until an equivalent proportion was obtained to EEO (5): PEO (2) so that volatility losses (typical of hydro distillation) are avoided. This mixture has been called the L-L EXTRACTION MIXTURE (ME-E5P2)
2 THEORETICAL FRAMEWORKS
2.1 EUCALYPTUS GLOBULUS LABILL
The eucalyptus is a tall tree with more than 700 species; 300 contain essential oils in their leaves. Eucalyptus trees, sometimes exceeding 40 meters in height, have a straight and poorly branched trunk, usually letting off thin layers of bark, aromatic, simple, alternate, entire leaves. Flowers and fruits are relatively small and unremarkable. They belong to the botanical family Myrtaceae, a family of trees rich in essential oils; they are native to Australia and Tasmania. Many introductions of different species have been made in America, such as Eucalyptus globulus Labill. The various species of eucalyptus are used interchangeably as popula medicine or for uses as wood.
These trees have evergreen, grayish-green leaves, and evident foliar dimorphism. Those located on young branches or in grafts of old branches are opposite, sessile, with an oval and thin horizontal blade covered by a waxy layer. Those situated on old branches are alternate, with a twisted and wrinkled petiole (2-3 cm), entire, leathery, glabrous, with an elliptical or slightly falciform-shaped blade, relatively thick (up to 25 cm in length and 5 cm in width) , which hangs vertically. The central nerve is yellowish-green, and the lateral ones are thinner and anastomose at the edge, forming a continuous line. Both on the upper side and on the underside, there are small, irregularly distributed dark brown spots and glands secreting the essential oil. It has solitary, short-pedunculated flowers, with absent sepals and welded petals forming a hemispherical structure that, when detached, reveals numerous long stamens curved inwards. As fruits, it has globose capsules with four ribs, more acute at the base.
Eucalyptus essential oil contains 1,8-cineole (45.4%), limonene (17.8%), p-cymene (9.5%), y-terpinene (8.8%), a-pinene (4.2%) and a-terpineol (3.4%), while, in the vapor, 1,8cineole (34.6%), limonene (29.9%), p-cymene (10, 5%), y-terpinene (7.4%), a-pinene (4.0%) and a-phellandrene (2.4%) were identified (Tyagi & Malik, 2011). They are also finding two monoterpene glycosides, conjugated with gallic acid [globulusin A, B], along with four known compounds, cypellocarpin A, eucaglobulin, cuniloside and (1 5, 2 S, 4 R) - trans - 2-hydroxy1,8-cineole B- d-glucopyranoside, determined by spectroscopic analysis (Hasegawa et al., 2008).
The effectiveness of 1,8-cineole for acute bronchitis is also related to its ability to increase mucociliary clearance and its anti-inflammatory activity. The molecular mechanisms associated with plant extracts' antiviral effects may vary between viruses. However, the potential of the plant extract to boost the human body's inherent antiviral defense involving an intricate immune system could utilize common pathways. Thus, the immunostimulatory properties of plant extracts with antiviral properties have been explored. Mukhtar et al., 2008).
2.2 PELARGONIUM SPP.
An herbaceous, perennial plant, 30 - 50cm high. Erect stem with alternate, simple, aromatic leaves, lobular margin. Its veins indicate it is palmate, and its leaf base is cordate. The flowers are in an umbel; they can be simple or double; they are showy and aromatic; the calyx has five free sepals; the corolla has five free petals, 10-15 stamens (not all fertile), and a super ovary.
Pelargonium leaves are used in throat inflammation, diarrhea, bleeding, and toothache cases. Other uses: ornamental and as an insecticide to eliminate fleas. Its form of preparation is infusion; gargling is recommended for throat inflammations. In case of pain, apply the ground leaves to the affected area.
The essential oils of Pelargonium contain thirty-two compounds that constitute 99.23% of the essential oil identified; the main ones were citronellol (29.9%), trans-geraniol (18.0%), 10-epi-y- eudesmol (8.27%), isomanthone (5.44%), linalool (5.13%), geranyl acetate (4.52%), y-Cadinene (2.89%), geranyl butyrate (2. 53%), geranyl tiglate (2.50%) and gemacrene D (2.05%). Geranium essential oil is rich in oxygenated components and commercial rhodinol (linalool + citronellol + geraniol). С galloyl glycosidic flavones, non-galloyl flavones, phenolics (flavonoids and tannins), benzoic and cinnamic acid derivatives were reported in the aerial parts, and the diterpene (reniformin), which has a new diterpene skeleton linked to a unique poxyphenetanesulfonic acid in the roots of Pelargonium reniform-the new ellagitannins with a 1- C4. Glucopyranose core (pelargoniins) and 4-allyl-2,5dimethoxyphenol-1-b-glucoside were also found. Tartaric acid is a characteristic component of the genus Pelargonium.
The occurrence of coumarin sulfates (5,6-dimethoxycoumarin 7-sulfate and a mixture of 6- and 8-monosulfate 6,8-dihydroxy-5,7-dimethoxycoumarin), coumarin glycosides and proanthocyanidins was limited to P. Sidoides The presence of structurally related umckalin (coumarins) as an active ingredient in Umckaloabo (herbal extract derived from the plant Pelargonion Sidoides) decreases upper respiratory tract infection and flu, alleviates the severity of symptoms by improving the immune system. Antimicrobial activity of Pelargonium extracts and its constituents was reported on (Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Haemophilus influenzae), fungi (Microsporum canis, Microsporum gypseum, Aspergillus Jumigatus, Mucor racemosus, Rhizopus nigricans) and pathogens, as well as opportunistic yeasts such as Candida albicans, Candida glabrata, Candida krusei and Cryptococcus neoformans (J et al., 2011). Pelargonium odoratissimum (scented geranium), from the leaves and flowers of the species, presents flavonoids such as quercetin, kaempferol, and myricetin. The essential oil of its leaves is rich in methyl-eugenol, limonene, and fencone (Stashenko et al., 2007).
2.3 ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS
In various studies carried out, Correa et al. (2009) show that the antimicrobial activity presented by essential oils is due to terpenoids. Following in order of activity the terpenoids that contain alcohol groups, then those that have aldehydes, and finally those that have ketonic groups. In the same way, authors propose that oils with a high percentage of terpenoid compounds of the phenolic type have notable antimicrobial properties. The antimicrobial effects of essential oils are related to their composition of phenols and monoterpenes that they possess due to their direct interaction with the cytoplasm of the pathogen or due to their hydrophobicity that can be incorporated into the lipids of the bacterial cell membrane and cytotoxic effects, which cause damage to the cell membrane (Montero-Recalde et al., 2019b).
Essential oils are not pure compounds but mixtures of a multitude of substances (it can be more than 100 different chemical substances) that are found in different proportions and that together provide the essential oil with its characteristics. On the other hand, essential oil is the product obtained from the plant after physicochemical treatment. Among the components are terpenes, up to 75-90% of the total weight; they contribute little to the overall aroma. Those responsible for the aroma are usually substances that are found in a lower proportion, such as organic compounds with functional groups such as ketone, ester, alcohol, aldehyde, and ether, Which are often found in small quantities that make up the aromatic fingerprint of each essential oil (Sanchez, 2006).
Respiratory infections are high-frequency events in the population, defined as a condition that compromises one or more parts of the respiratory system for no more than 15 days. The leading causes are viral and bacterial origin; they are the first cause of antibiotic indication worldwide. Acute respiratory infections (ARI) are classified according to the anatomical site affected as high and low, with the epiglottis being the separation point. Among the high ones, we have rhinopharyngitis, pharyngotonsillitis, sinusitis, and acute otitis media, and the low ones include epiglottitis, laryngotracheobronchitis, bronchiolitis, and pneumonia. The incidence of mortality from ARI in developing countries is 14-24% in children under five years of age (Valero et al., 2009).
2.4 MINIMUM INHIBITORY CONCENTRATION (MIC) OR BACTERIOSTATIC EFFECT
The minimum concentration of antimicrobial in pg/ml inhibits the visible growth of a microorganism after 24 hours of incubation at 37° C. The MIC has been established as a "Gold Standard" compared to other methods that evaluate antimicrobial susceptibility, in addition to confirming unusual resistance, definitive responses when the result obtained by other methods is indeterminate (Horna et al., 2005).
2.5 MINIMUM BACTERICIDAL CONCENTRATION (MBC) OR BACTERICIDAL EFFECT
The minimum antimicrobial concentration eliminates more than 99.9% of viable microorganisms after a specific incubation time (generally 24 hours) (Horna et al., 2005).
2.6 ANTIMICROBIAL SENSITIVITY (AMS)
Sensitivity tests or antibiograms determine the susceptibility of a microorganism to antimicrobial drugs based on the exposure of a standardized concentration of the germ to these drugs. Sensitivity tests can be done for bacteria, fungi, or viruses (Herrera, 1999).
2.7 LIQUID-LIQUID (L-L) EXTRACTION OF ESSENTIAL OILS
The steam stripping method is frequently used for essential oil contents in green plants greater than 0.1%, as in the case of EEO. For plants with lower contents, solvent extraction is the most appropriate even though there are risks of incorporating contaminants.
The L-L extraction is appropriately adapted to Soxhlet extraction equipment, as shown in Figure 3. The chopped plant material packed in filter paper is introduced into the body, and the weight and volume of the solvent (EEO) is loaded into the balloon. After several cycles of evaporation-condensation-siphoning, the extraction is exhausted in the plant material and with new recharges until an increase in the volume of the solvent is obtained in the required proportion.
3 METHODOLOGY
3.1 MICROBIOLOGICAL MATERIAL:
* Klebsiella pneumoniae ATCC 13883 KWIK STIK Microbiology
Essential oils:
* Eucalyptus globulus Labill essential oil (50ml) - EEO
The fresh leaves were from the district of San Salvador, province of Calca, Department of Cusco, Coordinates 13°29'29" S / 71°46'30" W 3,118 т altitude. The sample selected for this investigation was taken by simple random steam extraction sampling.
* The leaves of Pelargonium (Pelargonium spp.) - PEO
Coming from the district of Wanchaq, province of Cusco, Department of Cusco, Adolfo Guevara Velasco National Hospital coordinates 13°31'33" $ / 71°57'31" W 3,340 т altitude. The sample selected for this research was taken by simple random sampling.
* L-L extraction mixture of PEO with EEO: obtained in Soxhlet extraction equipment, according to section 2 criteria, proportion EEO (5): PEO (2). Table 1 shows the laboratory instruments used for extraction, equipment, and culture media.
3.2 EXPERIMENTAL PLAN
The research used a completely randomized experimental design, with a 2-factor factorial arrangement (AxB) (Table 2), with two levels for factor A and four levels for factor B (Table 3), with three repetitions, making a total of 8 treatments, with 24 experimental units; a Tukey test of means at a significance level of 0.05, according to the following table.
3.2.1 Physical-chemical determinations and in vitro effectiveness of the essential oils of Eucalyptus globulus Labill and Pelargonium sp. envelope Klebsiella pneumoniae ATCC 13883
The physical-chemical analysis and the M.I.C., C.M.B., and S.A.M. tests of the Essential Oils of Eucalyptus globulus Labill and Pelargonium spp. were carried out in the "FRACTAL QUIMICOS E.LR.L." Laboratory.
3.2.2 Determination of the in vitro effectiveness of the essential oils of Eucalyptus globulus Labill and Pelargonium spp. Envelope Klebsiella pneumoniae ATCC 13883
Activation of A.T.C.C. strains. The strain of Klebsiella pneumoniae ATCC 13883 KWIK STIK Microbiology was activated according to the manufacturer's recommendations, for which the following steps were followed:
- The swab that comes with the ATCC strain was saturated in the hydrated material and transferred into selective culture media for each microorganism.
- For Klebsiella pneumoniae ATCC 13883 KWIK STIK Microbiologist on Mac Conkey agar.
- It was incubated at 35-37 °C for 24-48 hours.
3.2.3 Determination of the Minimum Inhibitory Concentration (M.I.C.) modified in this research (Gil et al., 2012)
After activation, the ATCC strains were pre-hydrated in B.H.I. broth and incubated for 24 hours at 37°C until reaching the standard turbidity of 0.5% of the Mac Farland scale equivalent to 1.5 x 108 cfu/ml.
For the dilutions, the macro dilution technique in tubes was used with some adaptations because it is a natural product different from common antibiotics, carried out by Gil et al. (2008) and proceeded as follows:
- For each ATCC bacteria used, a battery of 10 tubes of 2ml capacity was prepared with 20011 of B.H.I. broth to make 10-point dilutions, a negative control, and a positive control.
- Dilutions of the essential oil of Eucalyptus globulus Labill. They were prepared in proportions of %, 14, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256.
- In the same way, the essential oil of Eucalyptus globulus L. and Pelargonium spp., in a 5:2 proportion, proportions of 4, 4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256.
- Then, 10ul of the A.T.C.C. strain suspension at 0.5% of the Mac Farland Escla was added to each tube and incubated at 37°C for 24-48 hours.
- Turbidity observation was carried out in each tube compared to the controls.
- Subsequently, 101 of each tube was inoculated onto plates with the selective medium of Mac Conkey Agar for Klebsiella pneumoniae ATCC 13883
- It was incubated at 37°C for 24-48 hours.
- Colony counting was then performed to determine the Bacteriostatic effect (M.I.C.) using the following criteria:
+++ > 100,000 cfu/ml
++ 50,000 - 100,000 cfu/ml
+ <50,000 cfu/ml
- 0 cfu/ml
(ProQuest: ... denotes formula omitted.)(1)
3.2.4 Determination of the Minimum Bactericidal Concentration (M.B.C.), according to Gil (2012) modified in this research
- For each A.T.C.C. bacteria used, a battery of 10 tubes of 2ml capacity was prepared with 200u1 of B.H.I. broth to make 10-point dilutions, a negative control, and a positive control.
- Dilutions of the essential oil of Eucalyptus globulus Labill. They were prepared in proportions of %, Va, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256.
- In the same way, the essential oil of Eucalyptus globulus L. and Pelargonium spp., in a 5:2 proportion, proportions of %, Va, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256.
- Then 10pl of the A.T.C.C. strain suspension at 0.5% of the Mac Farland Scale was added to each tube and incubated at 37°C for 24-48 hours.
- Turbidity observation was carried out in each tube compared to the controls.
- Subsequently, 10ul of each tube was re-inoculated with 2ml of B.H.I. broth previously prepared in 10ml capacity tubes.
- It was incubated at 37°C for 24-48 hours.
Then, the turbidity reading was carried out to determine the bactericidal effect (C.M.B.) using the following criteria: + Apparent turbidity, - No turbidity
3.2.5 Antimicrobial Sensitivity (SAM), according to the dug-well method (Castresan et al., 2013) modified in the present research
Preparation of concentrations of Eucalyptus globulus Labill essential oil. Concentrations of 100%, 75%, 50%, 25%, and 12.5% diluted in absolute ethanol were prepared from the essential oil.
Preparation of concentrations of Eucalyptus globulus Labill and Pelargonium spp. essential oil in a 5:2 proportion. Concentrations of 100%, 75%, 62.5%, 50%, 37.5%, 25%, and 12.5% diluted in absolute ethanol were prepared from the mixture of essential oils in a 5:2 proportion.
After activating the ATCC strains in selective agar for each one.
A suspension was prepared with 3-5 colonies in 5ml of TSB broth incubated at 37°C for 2-5 hours compared to the Mac Farland scale of 0.5%.
Three 5.5mm diameter perforations were made in previously prepared Muller Hinton agar at 22mm from each well.
Each ATCC strain was shown homogeneously by spreading it on the surface with the help of a sterile swab.
50ul of each prepared concentration of the essential oils of Eucalyptus globulus and Eucalyptus globulus L. and Pelargonium sp. were applied. In a 5:2 ratio, in triplicate on plates with Muller Hinton agar.
They were incubated at 37°C for 24-72 hours.
4 RESULTS AND DISCUSSIONS
4.1 IDENTIFICATION OF BOTANICAL SPECIES
The Vargas Herbarium (CUZ) of the National University of San Antonio Abad of Cusco identified the botanical species under study in this research as:
Eucalyptus globulus Labill.
Pelargonium spp.
4.2 EXTRACTION OF ESSENTIAL OILS
The optimal results found in the essential oil extraction process by the steam drag method are presented below:
4.3 HPLC-DAD AND GC-FID CHEMICAL ANALYSIS OF AEE AND ME-E5P2 ESSENTIAL OILS
4.4 EFFECTIVENESS OF THE MIXTURE OF ESSENTIAL OILS OF EUCALYPTUS GLOBULUS LABILL AND PELARGONIUM SPP. IN PROPORTION 5:2 (МА-Е5Р2) ON KLEBSIELLA PNEUMONIEAE
4.4.1 Minimum Bactericidal Concentration (MBC)
The Minimum Bactericidal Concentration of the essential oil Eucalyptus-Pelargonium 5:2 on Klebsiella pneumoniae ATCC 13883 was found to be 1/16 (60.5mg/ml) at 24 hours, as shown in Table 7 and Figure 5.
4.5 MINIMUM INHIBITORY CONCENTRATION (MIC)
The Minimum Inhibitory Concentration of the essential oil of Eucalyptus-Pelargonium 5:2 on Klebsiella pneumoniae ATCC 13883 reached 1/8 (129.6 mg/ml) at 24 hours, as shown in Table 8 and Figure 6.
4.6 ANTIMICROBIAL SENSITIVITY halos of 5.5mm.
The results of the in vitro antimicrobial sensitivity carried out using inhibition zones of the essential oils of Eucalyptus-Pelargonium 5:2 is presented below: The in vitro antimicrobial activity of Eucalyptus-Pelargonium Essential Oil 5:2 against Klebsiella pneumoniae ATCC 13883 showed a more excellent significant inhibition zone of 15.01mm 29.04mm at 25% - 50% concentration at 50%, which is close to the control of Imipenem of 43.23mm and at a concentration of 62.5% or more it surpasses the control, the distilled water blank maintains halos of 5.5mm. In comparison, control Ethanol (Control Et-OH) used as diluent found a slight halo of 7.39mm, as shown in Table 9 and Figure 7.
5 CONCLUSIONS
The present works studied the synergistic or adjuvant effect on the antimicrobial effectiveness of pelargonium essential oil (PEO) in combination with eucalyptus essential oil (EEO). The small amounts of oil in pelargonium (difficultly extractable by vapor drag) have motivated a liquid PEO-liquidAEE (L-L) extraction. EEO has been used as an extractant for PEO, giving a final proportion of 5:2.
Analysis by CG-FID chromatography gave 80.5% w/w of 1,8-cineole (eucalyptol), 3.05% w/w of limonene, 5.4% w/w of a-pinene, and antioxidant capacity of 76.0%. For the ME-ESP2 mixture, 56.4% of 1,8-cineole and 9.30% of citronellol were obtained from the essential oil of Pelargonium spp. with an antioxidant capacity of 78.6%.
The in vitro effectiveness of Eucalyptus globulus Labill essential oil against Klebsiella pneumoniae ATCC 13883 demonstrated a Minimum Bactericidal Concentration (MBC) of 1/16 (60.50g/ml), Minimum Inhibitory Concentration (MIC) of 1/8 (125.58 mg /mL) and an antimicrobial sensitivity lower than the Imipenem standard (41.04mm), with an average inhibition zone of 13.47mm. Eucalyptus oil had better effectiveness against Staphylococcus aureus ATCC 25923 (Gram-positive) than Klebsiella pneumoniae ATCC 13883 (Gramnegative).
The essential oil of Eucalyptus globulus Labill and Pelargonium sp. 5:2 had better in vitro effectiveness against Klebsiella pneumoniae ATCC 13883 with an MBC of 1/16 (60.47mg/mL), a MIC of 1/18 (129.58mg/mL) and an excellent antimicrobial sensitivity between 25-37.5 % (15.01-29.04mm) compared to the Imipenem standard (43.23mm). Finally, it was shown that combining both oils in a 5:2 ratio was more effective than pure Eucalyptus essential oil.
ACKNOWLEDGEMENTS
The authors sincerely acknowledge to Andean University of Cusco through the vice-rector for Research.
References
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