Corresponding author: Liliia Budniak ( stoyko_li@tdmu.edu.ua ) Academic editor: Plamen Peikov
© 2021 Svitlana Marchyshyn, Liudmyla Slobodianiuk, Liliia Budniak, Olha Skrynchuk .
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Marchyshyn S, Slobodianiuk L, Budniak L, Skrynchuk O (2021) Analysis of carboxylic acids of Crambe cordifolia Steven. Pharmacia 68(1): 15-21. https://doi.org/10.3897/pharmacia.68.e56715
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Crambe cordifolia Steven is a perennial herb and contains many biologically active substances, including amino acids, quercetin and glycosides of kaempferol. In continuation of the investigation of these plant compounds, it is advisable to study the qualitative composition and quantitative contents of carboxylic acids. Using a HPLC method the quantitative content of the following organic acids was identified and determined: pyruvic (40.66 mg/g), isocitric (12.88 mg/g), citric (8.71 mg/g), succinic (38.03 mg/g) and malic (0.75 mg/g). Among fatty acids the saturated and unsaturated acids were determined by the GC/MS method. The content of polyunsaturated fatty acids of the total fatty acids was 56.97%, saturated – 38.53% and monounsaturated – 4.50%. Linolenic and palmitic acids dominated among the determined 7 fatty acids, their content was 9.68 mg/g (47.87%) and 4.88 mg/g (24.14%). The results of the study show that Crambe cordifolia Steven leaves is a source of carboxylic acids.
Crambe cordifolia Steven, fatty acids, GC/MS, HPLC, organic acids
The importance of medicinal plants did not reduce the annual increase in the number of synthetic medicines, which often model biologically active substances of plants and are their chemical equivalents (
One of the most interesting plants of Brassicaceae are the genus Crambe L. species, that indicate the need for their widespread introduction and investigation due to promising properties such as food, decorative, medicinal etc. (
Genus Crambe L. is the largest genus in the family Brassicaceae. Much work has been done upon different species of Crambe in order to evaluate their industrial and pharmaceutical importance (
Nowadays the study with crambe seeds for bio diesel production has been carried out, mainly due to the high content of oil in it (
Crambe cordifolia Steven. is a perennial herb that may reach the height of 182 cm. It has lobed leaves with small white flowers. It contains many chemical compounds including amino acids, quercetin and glycosides of kaempferol (Dudkin et al. 1977;
The literature survey shows that there have not been many reported studies about some primary metabolites, such as organic and fatty acids in Crambe cordifolia Steven. This deficiency in experimental data motivated us to determine these compounds.
Leaves of the Crambe cordifolia Steven. were selected as the objects of the study. The raw materials were provided by the Department of cultural flora of M. Gryshko National Botanic Garden of the National Academy of Sciences of Ukraine. The leaves were collected in summer 2018. The leaves were dried using a conventional method and stored in paper bags in a dry place (
Standards of organic acids, including tartaric acid, pyruvic acid, isocitric acid, citric acid, succinic acid and malic acid, obtained from SigmaAldrich Chemical Company (St. Louis, MO, USA) were of analytical grade (≥95% purity) (Figs
Applied reagents (acetonitrile, undecanoic acid, methanol, heptane, toluene) were of the highest purity and purchased from the SigmaAldrich Chemical Company.
HPLC analysis of organic acids was performed using Agilent 1200 (Agilent Technologies, USA) (
Standard solutions. Standard solutions (1000 ppm) of tartaric, pyruvic, isocitric, citric, succinic and malic acids were prepared in the mobile phase consisting of 0.1% phosphoric acid solution. Stock solutions of every organic acid were made in the mobile phase by respective dilutions.
Extraction of organic acids from leaves of the Crambe cordifolia Steven. 700 mg of powdered raw material was placed in a vial and extraction in 10 ml of 0.1% solution of phosphoric acid. Extraction was performed in the ultrasonic bath at 80 °C for 4 h. 8.3 ml of the obtained extract was centrifuged at 3000 rpm/min for 30 minutes and filtered through Supelco Discovery DSC-18 filter and then concentrated to the residual volume of the extract of 20 μl.
Identification and quantification content of organic acids was performed using standard solutions of dicarboxylic compounds (tartaric, pyruvic, isocitric, citric, succinic and malic acids). The content of organic acids in µg/g was calculated by the following equation 1:
,
where:
C ‒ concentration, obtained from the chromatogram by calculating the reference solution and the test solution;
Vsolv. ‒ the volume of solvent for extraction;
Vinj. ‒ the volume of injection standard of organic acid;
Vactual inj. ‒ the volume of injection of extract;
m ‒ is a mass of the raw material (
GC/MS analysis of fatty acids was performed using gas chromatograph Agilent 6890N with mass detector 5973 inert (Agilent Technologies, USA) (
0.5 g (accurately mass) of the raw material was refluxed with a 3.3 ml mixture containing (methanol: toluene: sulfuric acid (44:20:2 v/v)) and 1.7 ml of internal standard solution (undecanoic acid in heptane solution). The sample was maintained in the ultrasonic water bath at 80 °C for 2 h. The resulting mixture was allowed to cool and centrifuged for 10 min at 5000 rpm. Then 0.5 ml of the upper heptane phase with containing methyl esters of fatty acids was selected.
The compositions of the product obtained were identified by comparison of their mass-spectrums with data obtained from National Institute Standard and Technology (NIST, 2008) database. The quantitative content of fatty acids was done using internal standard of undecanoic acid in heptane solution added to the sample.
The amount of fatty acids in mg/g was calculated according to the following equation 2:
,
where:
Sx – is a peak area of each fatty acid,
Minst – is a mass of the internal standard,
Sinst – is a peak area of the internal standard,
m – is a mass of a plant material (
In total, five organic acids were determined in the Crambe cordifolia Steven leaves, including pyruvic, isocitric, citric, succinic and malic acids by means of the HPLC method (Fig.
The results of the organic acids determination in Crambe cordifolia Steven leaves.
No. | Retention time | Common name of organic acid (IUPAC) | Molecular formula | Quantitative content (mg/g) x– ± Δ x– |
---|---|---|---|---|
1. | 2.262 | Pyruvic (2-oxopropanoic) acid | C3H4O3 | 40.66 ± 0.84 |
2. | 2.787 | Isocitric (1-hydroxypropane-1,2,3-tricarboxylic) acid | C6H8O7 | 12.88 ± 0.26 |
3. | 3.081 | Citric (2-hydroxypropane-1,2,3-tricarboxylic) acid | C6H8O7 | 8.71 ± 0.16 |
4. | 3.298 | Succinic (butanedioic) acid | C4H6O4 | 38.03 ± 0.83 |
5. | 4.813 | Malic ((2S)-2-hydroxybutanedioic) acid | C4H6O5 | 0.75 ± 0.02 |
As shown in Table
The quantitative content of fatty acids is present in Table
As shown in Fig.
The results of the determination of fatty acids in Crambe cordifolia Steven leaves.
No. | Retention time | Common name of fatty acid (IUPAC) | Chemical nomenclature | Quantitative content of methyl esters of fatty acids | |
---|---|---|---|---|---|
mg/g | % of the total | ||||
Saturated acids | |||||
1. | 3.59 | Undecylic (undecanoic) | С 11:0 | internal standard | |
2. | 14.31 | Palmitic (hexadecanoic) | С 16:0 | 4.88 | 24.14 |
3. | 18.88 | Stearic (octadecanoic) | С 18:0 | 0.92 | 4.55 |
4. | 30.76 | Lignoceric (tetracosanoic) | С 24:0 | 0.54 | 2.67 |
5. | 33.27 | Cerotic (hexacosanoic) | С 26:0 | 1.45 | 7.17 |
Monounsaturated acids (ω-9) | |||||
6. | 18.42 | Oleic (octadecenoic) | С 18:1 | 0.91 | 4.50 |
Polyunsaturated acids (ω-3 and ω-6) | |||||
7. | 18.12 | Linoleic (octadecadienic, ω-6) | С 18:2 | 1.84 | 9.10 |
8. | 18.30 | Linolenic (octadecatrienic, ω-3) | С 18:3 | 9.68 | 47.87 |
The amount of saturated fatty acids | 7.79 | 38.53 | |||
The amount of unsaturated fatty acids | 12.43 | 61.47 | |||
Total | 20.22 | 100 |
The biological role of saturated fatty acids is that they are a source of energy for the human body. They are also involved in the construction of cell membranes, hormone synthesis, transfer and absorption of vitamins and trace elements. Unsaturated fatty acids play an important role in the body’s vital functions.
The results of the study showed that the major components of raw material were linolenic acid (9.68 mg/g; 47.87%), palmitic acid (4.88 mg/g; 24.14%), and linoleic acid (1.84 mg/g; 9.10%). Crambe cordifolia Steven leaves is a source of essential fatty acids omega-3 (linolenic acid), omega-6 (linoleic acid) that must be supplied in the diet because the body needs them but cannot synthesize them. Obtained linolenic and linoleic acids are the starting point for the synthesis of a variety of other unsaturated fatty acids (
Linoleic acid performs a number of vitally important functions, such as the production of bile acids in the liver, the normalization of metabolic processes, the production of prostaglandins, the normalization of hormonal balance, and the improvement of digestive enzymes. Linoleic acid is converted in the body into γ-linolenic, which is the most active and turns into prostaglandin E1, which increases immunity. Prostaglandins also suppress inflammatory processes, regulate brain function, reduce the probability of vascular and cardiac diseases, normalize the nervous system, regulate metabolism and insulin levels. Linolenic acid provides the production of prostaglandins, normalizes blood pressure and blood cholesterol (
The organic and fatty acids profile plays an important role in the chemical properties therefore this is the useful information for the next researches.
The results of the research indicate that Crambe cordifolia Steven leaves are the source of carboxylic acids. Using the HPLC method, we determined the composition of organic acids in the raw material. The dominant organic acids in the studied Crambe cordifolia Steven were pyruvic and succinic acids, the content of which was 40659.83 µg/g and 38034.83 µg/g respectively. The qualitative composition and quantitative content of fatty acids were studied by GC/MS method. We determined 7 fatty acids in the Crambe cordifolia Steven leaves. The content of polyunsaturated fatty acids was 56.97%, monounsaturated was 4.50% and saturated was 38.53%. Linolenic and palmitic acids dominated among the fatty acids, their content was 9.68 mg/g and 4.88 mg/g that were 47.87% and 24.14% respectively of the total fatty acids. Thus, Crambe cordifolia Steven displayed particular composition of organic and fatty acids which could be of great interest for pharmaceutical industries, and this plant raw material can be used as a source for new medicines in the future.