Characteristics of the main chromatographic techniques used for the study of natural extracts.
From ancient times, plants have been used by humans for food, fodder, fibre and medicinal purposes. Several plants were empirically considered as treatments for a large array of illness and medical conditions. Each community had specific natural remedies, based on the geographical area, environmental conditions and other factors. Thus, the use of plants can be considered as part of the intangible cultural heritage of each community. In the geographical area of today’s Romania, the ancient inhabitants, Dacians, had very good knowledge regarding the use of plants for medicinal purposes, as presented by several historical sources. The present work describes protocols for the extraction and purification of natural extracts, analytical characterisation, in vitro and in vivo evaluation of their potential applications as well as some practical examples of their application on selected Romanian native medicinal and aromatic plants. The presented results offer scientific support to their traditional use, suggesting in the same time some modern applications, for example in the nanotechnology field.
- aromatic and medicinal plants
- traditional remedy
- scientific evidence
1.1. Traditional use of medicinal and aromatic plants
The first human’s tries to treat diseases were aiming for the environmental plants, so natural products could be considered the main mean of diseases treatment across the globe until the advent of scientific medicine. During their evolution on Earth, plants have developed the ability to synthesize certain chemical compounds for protection in the fight against world’s predators, such as insects, fungi, herbivorous mammals, etc.
Although some of these compounds are toxic to predators, they proved to have beneficial effects in the treatment of human diseases. In the past centuries, the practice of empirical use of plants for therapeutic purposes was passed either in writing or orally, from generation to generation. Thus, the oldest way of treatment, phytotherapy, whose beginnings lie in Palaeolithic and whose traces are preserved in folk medicine up to our days, was developed on an empirical base, but also in the context of a magic vision of the world, where the symbol, the analogy and the correspondences principle have played and are playing an important role in the choice of useful plants [1–3].
People always appealed to nature, mainly to plants, for treating and curing various diseases; harvesting plants with everything they prove useful and their complete exploitation gave humans the opportunity to familiarize with their curative properties . The first more precise data about the use of plants for healing in what is today Romania were given by the Greek doctor Discorides (doctor in Nero’s army) in the five-volume treatise on plants “De Materia Medica”, the precursor of all modern Pharmacopoeia and one of the most important botanical atlases in history. He pointed out that in Dacia numerous plant species were used on a large scale, making a vague and incomplete description of them. Published in 77, the book was describing 600 species of medicinal plants, of which 40 species were specific to Dacia’s territory. Among these species, 27 plants have Daco-Thracian names, 8-Latin and 5-Greek, which is a confirmation of the age of phytotherapy in Romania [2, 3, 5, 6].
Over the time, the inhabitants of Romania’s lands kept the continuity of rich traditions in the use of plants from spontaneous flora, which proved to be effective in curing physical or psychological suffering (injuries, fractures, bleeding, poisoning, animal bites, sunstroke, frostbite, infectious diseases, etc.).
Due to its geographic position, with a varied landscape and a climate favourable for rich vegetation, Romania is the meeting place of the Eurasian and Mediterranean flora. Here, grow more than 3600 species of higher plants, of which over 700 have become medicines, thanks to the long experience of the Romanian people in their use to cure diseases. In the Romanian plant heritage, there are numerous wild and cultivated plants that have different uses [1–9].
1.2. Short presentation of plants selected for the study
Among the Romanian traditional plants,
2. Obtaining and characterisation of natural extracts with biomedical applications
2.1. Extraction and separation of active compounds
The concentration of biologically active compounds of natural extracts directly depends on a series of factors, such as genomic composition, biological value of the cultivar, maturity stage of the plant, climatic zone, environmental conditions, post-harvest and storage conditions, as well as
Specific biologically active compounds are obtained from the vegetal products (different parts of plants or various mixtures of aromatic and medicinal plants) using appropriate solvents. To obtain water-soluble active substance at a pH close to neutral one (such as acids, bases, salts, sugars, phenols and polyphenols, amino acids, glycosides, gums, tannins, enzymes) water is used as solvent. Given that water is not a good solvent for resins, alkaloid, and oils - type compounds, etc., for obtaining them, alkaline or acidified water can be used. By-products as volatile oils, pigments, lecithin, resins, etc., are obtained using alcohol as a solvent (alcoholic or hydroalcoholic extracts). To prepare extractive solutions, different concentrations are used, ensuring the best yield, but also different solvents, depending on the nature of the substance to be extracted or the nature of raw material. When preparing extracts, it must be taken into account the influence of the following factors:
At the basis of extraction of
2.2. Analytical characterisation of extracts/active compounds
The plants are considered in our days as established sources of pharmaceutical, aromatic and industrial compounds. Various biocompounds gives the colour, odour or therapeutic actions. Used as pure compounds , impregnated in different supports [26–28] or used as intermediaries (for example for nanoparticle phytosynthesis) , bioactive compounds offers a natural health source. The potential applications of medicinal plants are determined by their compositional profile and possible synergies between those compounds. Nevertheless, as previous stated, the composition of the natural extracts varies with a series of factors. So, a variation in the phytochemical profiles of extract of the same plant, harvested from different areas, in different seasons or using different techniques is inherent . In the following paragraphs, we will present the main methods used for the analytical characterisation of natural extracts.
2.2.1. Phytochemical assays
The phytochemical assays are currently used for the preliminary assessment of the extracts composition, following some major type of compounds, such as sesqui- and monoterpenoids, phenolics, anthocyanins, flavonoids, saponins, oligomeric proanthocyanidins, flavan-3-ols, tannins, o-quinone or other parameters, such as the polyphenol index or the potential browning.
The phytochemical assays usually involve a specific reaction, standard substances and spectrophotometric measurements at specific wavelengths . In the following paragraphs will be presented the most common photochemical assays; it must be mentioned that other particular recipes and standards are also used in the literature. The presented recipes could also be applied for the study of essential oils, with proper dilution in alcohols.
2.2.2. Chromatographic methods
The main objective of the analytical methods is to determine both quantitatively and qualitatively the target compounds from plant extract. It is a difficult task since generally an extract contain numerous compounds (some of them being highly labile) with a broad range of polarities, volatility, molecular weight and quantities. Therefore, it is unrealistic to believe that a complete evaluation of an extract can be performed using a single method. Several aspects must be taken into consideration for appropriate selection of analytical methods: what type of information we need from the sample, amount of sample available for analysis, relative quantities of different components present in the sample. Due to the extract complexity, the analytical tools fitted for this task fall mainly in the chromatographic methods area: thin-layer chromatography (TLC) , gas chromatography (GC) , high-performance liquid chromatography and capillary electrophoresis (CE) . These techniques show a good performance in plant extract analysis due to simple treatment required for samples and diversity of detectors corresponding to different molecules properties (Table 1).
|Methods||Target biomolecules||Sample preparation||Detector|
|GC||Non-polar, thermostable||Difficult, derivatisation||FID, TCD, NPD, MS, HePD|
|TLC||Large range||Simple||Colour reagents|
|HPLC||Polar||Simple||UV-VIS, RI, MS, ELSD, NMR, fluorescence|
Chromatographic methods represent a suitable choice for nearly all biomolecules that come across in plant extracts. These methods have the advantages of high specificity and also allow us to determine a large number of compounds in a single analysis and to benefit from a high dynamic range. Impact of gas chromatography in biomolecules analysis is somewhat hindered by low thermostability of some of these species which involve the necessity of time consuming and expensive pretreatment of samples, like derivatisation [46, 47].
A further improvement of analytical methods in the biological samples is represented by hyphenation techniques, which are a combination or coupling of two or more analytical techniques using an appropriate interface. Most common link between techniques is represented by separation methods (chromatography) with an online spectroscopic detection technology (mass spectrometry or NMR). Also hyphenation techniques can be extended in both ways: in the separation parts (two separation methods) or in the spectrometry zone (two or more spectrometry methods) such us: SPE-LC-MS, LC-PDA-MS, LC-MS-MS, LC-NMR-MS. Albeit these techniques are more expensive than former methods the advantages overcome the capital costs: fast analysis, better automation, large number of sample processed in a period of time, higher reproducibility, less contamination, etc.
Most common and established hyphenation technique is
LC-MS is another relatively frequently used method that became more and more used in biological samples analysis. LC‐MS advantages compared to GC‐MS are evident: higher sensitivity and specificity, easy sample preparation (aqueous matrix is frequent but forbidden in GC or GC-MS), co‐eluting compounds can be more easily separated, can be applied to detect non‐volatile, polar and thermally labile compounds, several mass analysers can be used: quadrupole ion traps, time of flight (TOF), time of flight reflection (TOFR) and ion cyclotron resonance (ICR). Among major disadvantages there are lack of mass spectral libraries, hindering of the analyses by matrix effect, high capital costs and the need for qualified operators .
A novel hyphenation technique is represented by
2.2.3. Other methods
Besides the above presented analytical methods, other techniques are currently applied for the characterisation of natural extracts.
By determining the concentrations of carbon, hydrogen, nitrogen, sulphur or oxygen, the
In vitro protocols for the evaluation of natural extracts
Another very important group of
Unlike the previous presented assays, the
In vivo protocols for the evaluation of natural extracts
When exploring crude natural extracts, the use of
3. Evaluation of some traditionally used plants
The following paragraphs will shortly present selected results published by the authors, regarding the obtaining, characterisation and application of natural extracts from selected medicinal and aromatic plants, presented in Section 1.2.
Recently, our group presented the preliminary evaluation of the crude hydroalcoholic extract (50% ethanol) obtained from the upper aerial part of
The essential oils extracted from various parts of
The hydroalcoholic extracts (40% alcohol) obtained from the leaves of
The present work aims not to exhaustively present the obtaining and characterisation methods of natural extracts, but to bring its contribution to the field of phytochemistry, by addressing the most common methods for obtaining/characterisation of natural products, supported by some examples from our previously published works.
This work was partially supported by the Romanian UEFISCDI—“Partnerships in priority areas” program, project number 176/01/07/2014 (PN-IIPT-PCCA-2013-4-0953). All the authors had an equal contribution to the present work.
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