Currently, the most effective treatment for recurrent urinary tract infections in women is antibiotics. However, the limitation for this treatment is the duration and dosage of antibiotics and the resistance that bacteria develop after a long period of administration. With the aim of identifying mainly novel natural agents with antibacterial activity, the present study was undertaken to investigate the biological and phytochemical properties of extracts from the leaves
The increasing resistance of uropathogens to antibiotics and the recognition of the generally self-limiting nature of uncomplicated urinary tract infection (UTI) suggest that it is time to reconsider the empirical treatment of UTI by using antibiotics. Limitation for this treatment is the duration and dosage of antibiotics and the resistance that bacteria develop after a long period of administration [
Therefore, based on the above reasoning and observations, the aim of the study was to explore the phytochemical of n-hexane and ethyl acetate extracts of the
Results of the gas chromatography analysis of hexane and ethyl acetate extracts from leaves of
Main components of Hex extract.
No. | RT |
Main components | RI |
% area |
---|---|---|---|---|
1 | 14.74 | 2-Cyclohexen-1-ol | 892 | 1.81 |
2 | 16.47 | 2-Cyclohexen-1-one | 942 | 0.74 |
3 | 29.8 |
|
1372 | 0.29 |
4 | 34.01 | Brassicasterol acetate | 1538 | 0.25 |
5 | 35.61 | Diethyl phthalate | 1605 | 4.25 |
6 | 40.77 | 3,7,11,15-Tetramethyl-2-hexadecen-1-ol | 1841 | 13.08 |
7 | 40.91 | 4-Octadecenal | 1848 | 0.39 |
8 | 43.15 | n-Hexadecanoic acid | 1960 | 1.05 |
9 | 43.82 | Hexadecanoic acid, ethyl ester | 1994 | 0.41 |
10 | 46.13 | Phytol | 2118 | 3.48 |
11 | 47.13 | Ethyl 9,12,15-octadecatrienoate | 2174 | 1.49 |
12 | 71.44 | Lupeol | 3443 | 23.31 |
13 | 75.13 | Lupeol acetate | 3516 | 25.09 |
14 | 75.26 | Betulin | 3518 | 23.81 |
Main components of AcOEt extract.
No. | RT |
Main components | RI |
% area |
---|---|---|---|---|
1 | 13.64 | 1,2-Epoxycyclohexane | 865 | 0.19 |
2 | 14.72 | 2-Cyclohexen-1-ol | 892 | 0.90 |
3 | 16.44 | 2-Cyclohexen-1-one | 941 | 0.41 |
4 | 40.68 | Hexahydrofarnesol | 1837 | 0.76 |
5 | 40.79 | 3,7,11,15-Tetramethyl-2-hexadecen-1-ol | 1842 | 15.57 |
6 | 43.19 | n-hexadecanoic acid | 1962 | 0.83 |
7 | 43.83 | Hexadecanoic acid, ethyl ester | 1994 | 0.37 |
8 | 46.15 | Phytol | 2119 | 0.75 |
9 | 46.65 | Linolenic acid | 2147 | 0.67 |
10 | 47.01 | Linoleic acid ethyl ester | 2167 | 0.23 |
11 | 47.15 | Ethyl 9,12,15-octadecatrienoate | 2175 | 0.94 |
12 | 47.98 | 2-[(Z)-9-octadecenyloxyethanol] | 2222 | 0.48 |
13 | 51.17 | 3-Ethyl-3-hydroxy-5 |
2413 | 1.61 |
14 | 70.82 | (22Z)-Stigmasta-5,22-diene-3 |
3426 | 2.98 |
15 | 72.95 |
|
3483 | 4.55 |
16 | 73.43 |
|
3496 | 4.78 |
17 | 75.19 | Lupeol acetate | 3517 | 19.95 |
18 | 79.09 | Betulin | 3557 | 6.17 |
Forty components were identified in the hexane extract: 72.46% were triterpenoids, 16.56% terpenes, 4.25% phthalate ester, 1.46% fatty acids and derivatives, 1.42% aldehydes and ketones, 1.81% alcohols, and 0.55% unknown compounds. The hexane extract was mainly characterized by lupeol acetate (25.09%), betulin (23.81%), lupeol (23.31%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (13.08%), diethyl phthalate (4.25%), and phytol (3.48%). On the other hand, eighty components were identified in the ethyl acetate extract: 37.06% were triterpenoids, 17.08% terpenes, 3.04% fatty acids and derivatives, 1.50% ketones and alcohols, and 37.86% unknown compounds. The ethyl acetate extract was mainly characterized by lupeol acetate (19.95%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (15.57%), betulin (6.17%),
Most of the metabolites isolated belonging to the group of terpenes (lupeol, lupeol acetate,
Structures of main terpenes isolates of n-hexane and ethyl acetate extracts.
Recently, other authors have determined from ethanol extract three novel lupane-, bauerane-, and euphane-type triterpenoids and other known triterpenes present in dandelion roots, using spectroscopic analysis, which have potential anti-inflammatory activity [
It was determined that the total fatty acid content of the dandelion leaves, using the Soxhlet method, is
Fatty acids content in the
Fatty acid | mg/g dry weight of |
---|---|
C14 : 0 (myristic) | 0,1 |
C16 : 0 (palmitic) | 1,5 |
C18 : 0 (stearic) | 0,3 |
C18 : 1 n-9 (oleic) | 0,2 |
C18 : 2 n-6 (linoleic) | 0,7 |
C18 : 3 n-3 (linolenic) | 1,0 |
C20 : 0 (arachidonic) | 0,1 |
Fatty acid (% w/w of total fatty acids) composition in the leaves of
Figure
Manning 2001 [
The total fatty acid content in leaves, relative to dry plant weight, is 3,9 mg per gram and palmitic acid 1.5 mg per gram as the prevailing fatty acid in the sample. The next two most abundant fatty acids were 18 : 2n-6 and 18 : 3n-3 (Table
The antibacterial effects of the extracts of
Antibacterial activity of n-hexane and ethyl acetate extract of
Extracts | Percentage of growth inhibition (%)/MIC ( |
|||
---|---|---|---|---|
|
|
|
|
|
Hex | 72 ± 2.1 |
89 ± 3.3 |
52 ± 0.0 |
70 ± 0.8 |
EtOAc | 97 ± 0.9 |
0 ± 0.0 | - | - |
Chloramphenicol | 95 ± 0.0 |
- | 94 ± 0.0 |
89 ± 0.0 |
Streptomycin | - | 88 ± 1.1 |
- | - |
EtOAc extract showed low activity against
In addition, it was observed that as the extract concentration increased, so does the bacteria growth inhibition. This inhibitory effect may be due to the presence of phenolic compounds, terpenes, tannins, flavonoids, alkaloids, and/or proteins in the plant extracts. Such compounds had been reported to have an active effect on the bacterial cells membrane, which may destroy these microorganisms [
In studying the antipathogenic properties of genus
The leaves of
The extracts of
The n-hexane and EtOAc extracts were subjected to chromatography over a silica gel column (400 g) and eluted with mixtures of increasing polarity n-hexane and EtOAc. Fractions were combined based on TLC monitoring and purified by repeated CC on silica gel columns. Compounds were identified by chromatographic analysis and spectroscopic data, including 1H- and 13C-NMR, respectively, on a Bruker Avance 400 Digital NMR spectrometer operating at 400.1 MHz for 1H and 100.6 MHz for 13C, and comparisons with data reported in the literature were made.
The n-hexane and ethyl acetate extract was diluted with acetone, and analysis by gas chromatography (Hewlett Packard, Palo Alto, CA, USA) was carried out according to the method detailed elsewhere [
The gas chromatography analysis was performed on an Agilent Technologies 7890B gas chromatograph using a 30 m capillary column, Supelco Omega-Wax, 0.25 mm (Agilent Corp. CA). Hydrogen was used as a carrier gas at a flow rate of 1 mL/min. Detection was with flame-ionization detection and areal quantitation was made with an auxiliary automatic integrator. A temperature of 170°C was used at the beginning, which was maintained for two minutes, rising from 2,5°C/min to a final temperature of 240°C, which was maintained for 3 min. Injector and detector remained at 250°C and 270°C, respectively. Methyl esters were prepared according to our previously described procedure [
The uropathogenic bacteria were clinical isolates belonging to the Chemistry Department, Biological Tests Laboratory (Universidad Técnica Federico Santa María) collection. They comprised
The minimum inhibitory concentration (MIC) of the plant extract was determined using the broth serial dilution method with modifications, following Andrews 2001 [
Finally, the percentage of bacterial growth inhibition (GI) was calculated using the following formula:
Different secondary metabolites of the n-hexane and ethyl acetate extracts were isolated from
The authors declare no conflicts of interest.
Katy Díaz Peralta designed the research, performed extractions, separation, and purification of the compounds, performed the bioassays, and wrote the biological component of this document. Rolando Chamy Maggi contributed to research design, copywriting, and corrections. Luis Espinoza Catalán contributed to the structures determination by spectroscopic methods (1D, 2D NMR) and document writing. Alejandro Madrid Villegas contributed to the interpretation of the results (GC-M) and document writing. Leonardo Pizarro Dasso contributed to the determination of fatty acids by chromatographic methods. All authors read and approved the final document.
The authors thank FCR-CSB, CORFO-INNOVA project Grant no. 09CEII-6991, and the Department of Chemistry of Universidad Técnica Federico Santa María.