INTRODUCTION
Hyoscyamus, Datura, and Atropa species are well-known natural sources of tropane alkaloids, primarily scopolamine, atropine, and hyoscyamine. Due to their biological activity, these plants and substances are widely used in medicine as mydriatics, antiasthmatics, spasmolytics, etc.1,2 Furthermore, saponins, triterpenoids, phenolics, flavonoids, lignans, essential oils, sterols, and other compounds have been identified in the different organs of plants.2,3,4,5 Fatty acids are the main components of seed oils. The quality, consumption, industrial and medicinal uses of oils are mainly related to their fatty acids. According to various references, the percentage of oil differs from 15 to 35% in the seeds of these species.6,7
Fatty acids play an essential role in the metabolic processes of human organisms - in the storage of energy, as basic components of cell membranes, etc. Scientists have reported that the replacement of saturated fatty acids with monounsaturated and polyunsaturated fatty acids in the diet reduces the risk of cardiovascular disease by decreasing total and low-density lipoprotein cholesterol levels in the blood.7,8
In the human body, linoleic acid is converted into docosahexaenoic and eicosapentaenoic acids, which are responsible not only for the reduction of cholesterol but also for inflammation, enhancement of brain functions, and prevention of cancers and autoimmune conditions.9 Fatty acids are widely used in drug preparations as excipients and in the cosmetic industry to prepare soaps, fat emulsions, liposomes, etc. They are essential components in some drugs due to their biological activities.7,10 The seed oil of Datura stramonium is used as an analgesic in neurological practice, as well as for hair removal in cosmetology.11 Biodiesel was prepared with the fatty acids of some Datura species and suggested as an alternative fuel source.6,12,13 A scar cream is prepared with a Swiss recipe by using seed oil of Hyoscyamus niger under the brand name, i.e. Kelosoft®, and successfully sold in Europe.
In the present study, fatty acids of seed oils were analyzed in the accredited laboratory of the Food Safety Agency of the Republic of Azerbaijan.
MATERIALS AND METHODS
Plant collection
Plants were collected in the middle or end of vegetation periods, from various regions of Azerbaijan (Table 1). Species were authenticated at the Institute of Botany of the Azerbaijan National Academy of Sciences, Baku, where their voucher specimens were deposited (date: 01.09.2020, no: 01.04/1191).
Chemicals and solvents
Methanol, n-hexane and potassium hydroxide were purchased from Merck (Germany). 0.45 µm membrane filters were purchased from Isolab (Germany). A standard mix of fatty acid methyl esters (FAMEs) (Supelco 37 Component FAME mix, certified reference material, TraceCERT, in dichloromethane, ampule of 1 mL) was purchased from Sigma-Aldrich (Germany).
Extraction of oil
The seeds were dried and powdered. Extraction was carried out in a Soxhlet apparatus at 80°C for 6 h. 10 g of raw materials and 300 mL of n-hexane were used to obtain seed oils. After solvent evaporation, the percentage of oil was calculated by mass (w/w).
Preparation of fatty acid methyl esters
FAMEs for gas chromatography (GC) analysis. The oils (100 µL for each) and 5 mL of KOH-MeOH solution (0.5 M) were added to 10 mL-glass tubes. The mixtures were heated at 60°C for 15 min in the water bath under reflux, then, centrifuged in the closed tubes at 900 rpm for 8 min. After cooling to room temperature, 5 mL of n-hexane and 5 mL of distilled water were added and mixed thoroughly. The n-hexane phases were collected, filtered (membrane filter 0.45 µm), and applied to analysis.14
Gas chromatography conditions
Analysis of fatty acids was performed on an Aligent 7820A GC system equipped with a flame ionization detection (FID) detector and a capillary column HP-88 - 100 m × 0.25 mm × 0.25 µm. Helium was used as carrier gas with a flow rate of 1 mL/min. Oven temperature was initially programmed at 120°C, hold 1 min, and increased to 175°C with the rate of 10°C/min, hold 10 min. Temperature was elevated to 210°C, hold 5 min then 230°C and 5 min with a rate of 5°C/min. The total run time was 37.5 min. The injector and FID temperatures were set at 250°C and 280°C, respectively. The split ratio was 1:50. The injection volume was 1 µL.15,16
Standard sample
A standard mixture, which consists of 37 FAME components was applied. A 10 mg standard mixture was added to the volumetric flask, diluted to 10 mL with n-hexane. The injection volume was 1 µL.
Results calculation
The results were calculated compared with the retention times of standard samples. The concentrations of fatty acids were calculated using peak areas and expressed as percentages. Each analysis was repeated three times and mean values were reported. Statistical analysis has not been performed for evaluation of the results.
RESULTS
The percentages (w/w) of oils in the dried seeds are as follows: 22% in D. stramonium, 20% in D. stramonium var. tatula, 17% in H. reticulatus, 35% in H. niger, and 32% in A. caucasica. Results are presented individually for each fatty acid of five plant samples in Table 2.
Linoleic (55-79%), oleic (11-26%), palmitic (4-12%), and stearic (2-3%) acids compose 97% of total fatty acids. Percentage of unsaturated fatty acids are 83-91%. Changes of four main fatty acid concentrations are presented in Figure 1. Samples contained minor concentrations of trans-fatty acids.
Elaidic acid, a trans isomer of oleic acid (C18:1 t, <0.06%) and a trans isomer of linoleic acid (C18:2 t, <0.09%) are less than 1% of the total fatty acid mixtures. All other fatty acids are in the minor concentrations. The total number of fatty acids are varied - 28 in D. stramonium, 25 in D. stramonium var. tatula, 26 in H. reticulatus, 27 in H. niger, and 23 in A. caucasica.
The highest yield of oil was obtained from H. niger seeds. The maximum concentration of linoleic acid is observed in the seed oil of H. reticulatus (79%). D. stramonium var. tatula contains a higher amount of oleic acid (26%) than other samples. On the other hand, Datura species contain 20-22% oils as they are one of the optimal sources of plant oil, linoleic, and oleic acids because a whole plant produces at least 200-300 g seeds. 100 g of seeds were obtained from Hyoscyamus species, while approximately 40 g of seeds were gathered from A. caucasica. However, Datura species naturally grow in all regions of Azerbaijan. Hyoscyamus species were found in most parts of the country, but A. caucasica is rarely found in mountain forests.
DISCUSSION
The selected method, GC-FID is a useful and sensitive method for analyzing numerous compounds in a single run. Without wasting a long time, GC-FID method is successfully applied for routine analysis of fatty acids.17
Results of previous studies showed that 50-70% of polyunsaturated cis-linoleic acid (C18:2), 15-30% of monounsaturated oleic acid (C18:1) and 2-7% of saturated palmitic (C16:0) and stearic acids (C18:0) were found as major compounds in the seed oils of Hyoscyamus and Datura species.8,9,11,15,18 65% of monounsaturated fatty acids were reported by Koria and Nithya12 in the seed oil of D. stramonium grown in India. Contrary to other studies, polyunsaturated fatty acids (18%) were lower than monounsaturated fatty acids.11,12 Eicosanoic acid (C20:0) (34.55%), isomers of linoleic acid, (C18:2, n7) (4.56%) and (C18:2, n8) (3.61%), and eicosatrienoic acid (C20:3, n6) (4.39%), saturated - daturic acid C17H34O2 were reported in D. stramonium oil.11 Similar results were acquired compared with previous investigations. Total number of fatty acids was maximum in the present study.
CONCLUSION
The investigation evaluated the presence and concentrations of 37 fatty acids in five plant species from Azerbaijan. Datura, Hyoscyamus, and Atropa species are not harvested widely as oil and unsaturated fatty acid sources due to their toxicity. To the best of our knowledge, this is the first study on fatty acids in A. caucasica.
Depending on geographical and climate differences, components of the fatty acid mixture and their concentrations could be varied in the same species. An essential omega-6 fatty acid - linoleic acid dominates all investigated samples and gives them valuable nutritional and medicinal properties. Second major compound of oils is oleic acid. Another essential omega-3 fatty acid - linolenic acid, is found in trace amounts.
The results of the investigation might be useful for future researchers to evaluate these oils in medicinal, nutritional, cosmetic, fuel, and other industries.
ACKNOWLEDGEMENTS
The authors want to acknowledge the administration and laboratory members of the Food Safety Agency of the Republic of Azerbaijan.
Ethics
Ethics Committee Approval: Not applicable.
Informed Consent: Not applicable.
Peer-review: Externally peer-reviewed.
Authorship Contributions
Concept: A.V., Design: A.V., Data Collection or Processing: A.V., A.K., N.H., Analysis, or Interpretation: A.V., A.K., N.H., Literature Search: A.V., A.K., N.H., Writing: A.V., E.G.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.