Characteristics of the main chromatographic techniques used for the study of natural extracts.
Abstract
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.
Keywords
- aromatic and medicinal plants
- Romanian
- traditional remedy
- scientific evidence
1. Introduction
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 [4]. 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 [25], impregnated in different supports [26–28] or used as intermediaries (for example for nanoparticle phytosynthesis) [29], 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 [30]. 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 [34]. 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) [43], gas chromatography (GC) [44], high-performance liquid chromatography and capillary electrophoresis (CE) [45]. 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 |
CE | Ionic | Simple | UV, MS |
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 [51].
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.
The
By determining the concentrations of carbon, hydrogen, nitrogen, sulphur or oxygen, the
2.3. In vitro protocols for the evaluation of natural extracts
The
Another very important group of
The
Unlike the previous presented assays, the
The
2.4. In vivo protocols for the evaluation of natural extracts
When exploring crude natural extracts, the use of
The
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.
Acknowledgments
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.
References
- 1.
Parvu C., Enciclopedia plantelor, Plante din flora Romanie ( Plants encyclopedia ,Plants from Romanian flora ). Bucharest: Ed. Tehnica; 2002. - 2.
Scarlat M.A., Tohanescu M. Mic tratat de fitomedicina ( Small treaty of phytomedicine ). Ploiesti: World Galaxy; 2003. - 3.
Tudor I., Minoiu M. Plantele medicinale miraculoase din flora Romaniei ( Miraculous medicinal plants from Romanian flora ). Bucharest: Artmed; 2004. - 4.
Olaru O.T., Nitulescu G.M., Ortan A., Dinu-Parvu C.E. Ethnomedicinal, phytochemical and pharmacological profile of Anthriscus sylvestris as an alternative source for anticancer lignans. Molecules. 2005;20 :15003–15022. - 5.
Bojor O., Popescu O. Fitoterapie traditionala si moderna ( Traditional and modern phytotherapy ). Bucharest: Fiat lux; 2001. - 6.
Milica C. Medicina naturista. Tainele tineretii, sanatatii si frumusetii ( Naturist medicine. Secrets of youth, health and beauty ) v.1 and 2. Iasi: Doxologia; 2013. - 7.
Ionescu D., Popescu M., Rizea G.D., Mihele D., Bulearca G., Ivopol M., et al. Polyphenols and minerals, antioxidants in the plants used in the natural treatment of hepatobilliary disorders. Rev. Chim. 2014; 65 (5):507–511. - 8.
Ionescu D., Predan G., Rizea G.D., Mihele D., Ivopol G., Ionita C. Antimicrobial activity of some hydroalcoholic extracts of artichoke ( Cynara scolymus ), burdock (Actium lappa ) and dandelion (Taraxacum officinale ). Bull. Transilvania Univ. Basov. 2013;6 (55):113–120. - 9.
Ionescu D. Studii privind influenta unor noi preparate de natura vegetala cu actiune hepato-colecistica asupra parametrilor biochimici la animale ( Studies on the influence of new preparations of plant acting on the liver and gallbladder biochemical parameters in animals ) [PhD thesis]. Bucharest:2014. - 10.
Handa S.S., An overview of extraction techniques for medicinal and aromatic plants. In: Handa S.S., Khanuja S.P.S., Longo G., Rakesh D.D., editors. Extraction technologies for medicinal and aromatic plants. Trieste: Int. Centre Sci. High Technol; 2008. - 11.
Deng C.H., Xu X.Q., Yao N., Li N., Zhang X.M. Rapid determination of essential oil compounds in Artemisia selengensis Turcz by gas chromatography–mass spectrometry with microwave distillation and simultaneous solid-phase microextraction. Anal. Chim. Acta. 2006;556 :289–294. - 12.
Chen L., Jin H., Ding L., Zhang H., Li J., Qu C., et al. Dynamic microwave assisted extraction of flavonoids from Herba epimedii . Sep. Purif. Technol. 2007;59 (1):50–57. - 13.
Schinor E.C., Salvador M.J., Turatti I.C.C., Zucchi O.L.A.D., Dias D.A. Comparison of classical and ultrasound-assisted extractions of steroids and triterpenoids from three Chresta spp. Ultrason. Sonochem. 2004;11 (6):415–421. - 14.
McHugh M., Krukonis V., Brenner H. Supercritical Fluid Extraction, Principles and Practice. 2nd ed. Amsterdam: Elsevier B.V.; 1994. - 15.
Paun-Roman G., Radu G.L. Obtinerea unor principii biologic active din plante in forma purificata si concentrate prin tehnologii avansate ( Obtaining biologic active principles from plants in a concentrated and purified form, using advanced technologies ). In: Cascaval D., Galaction A.I., editors. Biotehnologia, intre stiinta si arta. Iasi: Ed. Venus; 2007. p. 61–94. - 16.
Butler M.S. The role of natural product chemistry in drug discovery. J. Nat. Prod. 2004; 67 (12):2141–2153. - 17.
Fines-Neuschild S., Boucher E., de Vernal A., Gelinas Y., Leclerc P. Accelerated solvent extraction—An efficient tool to remove extractives from tree-rings. Dendrochronol. 2015; 36 :45–48. - 18.
Lawrence B.M. Commercial essential oils: Truths and consequences. In: Swift K.A.D., editor. Advances in flavours and fragrances: From the sensation to the synthesis. Cambridge: Royal Society of Chemistry; 2002. pp. 57–83. - 19.
Ortan A. Nanostructuri lipidice – vectori de distributie pentru principii active volatile ( Lipid nanostructures - distribution vectors for active volatile principles ). Bucharest, Romania: Ed. Printech; 2013. - 20.
Hui Y. H., editor. Handbook of food products manufacturing, Vol.1, Hoboken, New Jersey: Wiley; 2007. - 21.
Bhat S.V., Nagasampagi B.A., Sivakumar M. Chemistry of natural products. India: Springer Science & Business Media; 2005. 840 p. - 22.
Keville K., Green M. Aromatherapy: a complete guide to the healing art. 2nd ed. USA: Crossing Press; 2008. 256 p. - 23.
Garland S. The complete book of herb and spices. UK: Frances Lincoln; 2004. 288 p. - 24.
Paun G., Gheorghe O., Diaconu M. Curs procesare avansata a plantelor medicinale ( Course Advanced processing of medicinal plants ). 2011. Available from: http://medplanet.dbioro.eu/doc/Curs%20procesare%20avansata%20RO.pdf - 25.
Bucar F., Wube A., Schmid M. Natural product isolation – how to get from biological material to pure compounds. Nat. Prod. Rep. 2013; 30 :525–545. - 26.
Mustapa A.N., Martin A., Sanz-Moral L.M., Rueda M., Cocero M.J. Impregnation of medicinal plant phytochemical compounds into silica and alginate aerogels. J. Supercrit. Fluids. 2016; 116 :251–263. - 27.
Stoica R., Pop S.F., Ion R.M. Evaluation of natural polyphenols entrapped in calcium alginate beads prepared by the ionotropic gelation method. J. Optoel. Adv. Mat. 2013; 15 (7–8):893–898. - 28.
Lima S.S.P., Lucchese A.M., Araujo-Filho H.G., Menezes P.P., Araujo A.A.S., Quintans-Junior L.J., et al. Inclusion of terpenes in cyclodextrins: preparation, characterization and pharmacological approaches. Carbohydr. Polym. 2016; 151 :965–987. - 29.
Sutan N.A., Fierascu I., Fierascu R.C., Manolescu D.S., Soare L.C. Comparative analytical characterization and in vitro citogenotoxic activity evaluation of Asplenium scolopendrium L. leaves and rhizome extracts prior to and after Ag nanoparticles phytosynthesis. Ind. Crops Prod. 2016;83C :379–386. - 30.
Dhami N., Mishra A.D. Phytochemical variation: How to resolve the quality controversies of herbal medicinal products? J. Herb. Med. 2015; 5 (2):118–127. - 31.
Gutensohn M., Dudareva N. Tomato Fruits-A Platform for Metabolic Engineering of Terpenes. In: O'Connor S.E., editor. Methods in Enzymology. Synthetic Biology and Metabolic Engineering in Plants and Microbes Part B: Metabolism in Plants. 576th ed. Amsterdam: Elsevier B.V.; 2016. pp. 333–359. doi:10.1016/bs.mie.2016.03.012 - 32.
Istudor V. Farmacognozie, Fitochimie, Fitoterapie ( Pharmacognosy, Phytochemistry, Phytotherapy ). vol. 2 ed. Bucharest: Editura Medicala; 2001. - 33.
Dai J., Mumper R.J. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules. 2010; 15 :7313–7352. doi:10.3390/molecules15107313 - 34.
Misra B.B., Dey D. Phytochemical analyses and evaluation of antioxidant efficacy of in vitro Callus extract of East Indian sandalwood tree (Santalum album L.). J. Pharmacog. Phytochem. 2012;1 :7–16. - 35.
Jones C.G., Ghisalberti E.L., Plummer J.A., Barbour E.L. Quantitative co-occurrence of sesquiterpenes; a tool for elucidating their biosynthesis in Indian sandalwood, Santalum album . Phytochemistry. 2006;67 (22):2463–2468. doi:10.1016/j.phytochem.2006.09.013 - 36.
Doneva-Sapceska D., Dimitrovski A., Bojadziev T., Milanov G., Vojnovski B. Free and potentially volatile monoterpenes in grape varieties from the Republic of Macedonia. Bull. Chem. Technol. Macedonia. 2006; 25 (1):51–56 . - 37.
Singleton V., Rossi J. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am. J. Enol. Viticult. 1965; 16 :144–158. - 38.
ISO 14502–1:2005, Determination of substances characteristic of green and black tea-1: Content of total polyphenols in tea-Colorimetric method using Folin-Ciocalteu reagent. - 39.
Fierascu R.C., Padure I.M., Avramescu S.M., Ungureanu C., Bunghez R.I., Ortan A., et al. Preliminary assessment of the antioxidant, antifungal and germination inhibitory potential of Heracleum sphondylium L. (Apiaceae ). Farmacia. 2016;64 (3):403–408. - 40.
Porter L.J., Hrstich L.N., Chan B.G. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry. 1986; 25 :223–230. - 41.
Ortan A., Fierascu I., Ungureanu C., Fierascu R.C., Avramescu S.M., Dumitrescu O., et al. Innovative phytosynthesized silver nanoarchitectures with enhanced antifungal and antioxidant properties. Appl. Surf. Sci. 2015; 358 :540–548 - 42.
Arnous A., Makris D.P., Kefalas P. Effect of principal polyphenolic components in relation to antioxidant characteristics of aged red wines. J. Agricult. Food Chem. 2001; 49 (12):5736–5742. - 43.
Jesionek W., Majer-Dziedzic B., Choma I.M. Separation, identification and investigation of antioxidant ability of plant extract components using TLC, LC-MS and TLC-DPPH. J. Liq. Chromatogr. Relat. Technol. 2015; 38 (11):1147–1153. - 44.
Seenivasagan T., Paul A.V.N. Gas-chromatography and electroantennogram analysis of saturated hydrocarbons of cruciferous host plants and host larval body extracts of plutella xylostella for behavioural manipulation of cotesia plutellae. Ind. J. Exp. Biol. 2011;49 (5):375–386. - 45.
Rivasseau C., Boisson A.M., Mongelard G., Couram G., Bastien O., Bligny R. Rapid analysis of organic acids in plant extracts by capillary electrophoresis with indirect UV detection: directed metabolic analyses during metal stress. J. Chromatogr. A. 2006; 1129 (2):283–290. - 46.
Akhgari A., Laakso I., Seppanen-Laakso T., Yrjonen T., Vuorela H., Oksman-Caldentey K.M., et al. Determination of terpenoid indole alkaloids in hairy roots of Rhazya stricta (Apocynaceae ) by GC-MS. Phytochem. Anal. 2015;29 (5):331–338. - 47.
Qureshi M.N., Stecher G., Sultana T., Abel G., Popp M., Bonn G.K. Determination of carbohydrates in medicinal plants-comparison between TLC, mf-MELDI-MS and GC-MS. Phytochem. Anal. 2011; 22 (4):296–302. - 48.
Sripathi S.K., Poongothai G., Lalitha P. Identification of pinitol in plants extracts by HPTLC. J. Chem. Pharmaceut. Res. 2011; 3 (5):544–549. - 49.
Turkmen Z., Mercan S., Cengiz S. An HPTLC method for the determination of oleandrin in Nerium plant extracts and its application to forensic toxicology. J. Planar. Chromatogr. - Modern TLC. 2013; 26 (3):279–283. - 50.
Wang J., Yu J., Li L., Liu C.M., Zhang Y., Wang Q. Isolation of high purity ginsenosides from plant extract of panax ginseng by high performance centrifugal partition chromatography coupled with evaporative light scattering detection. J. Liq. Chromatogr. Relat. Technol. 2013; 36 (5):583–590. - 51.
Fong S.Y., Wong Y.C., Zuo Z. Development of a SPE-LC/MS/MS method for simultaneous quantification of baicalein, wogonin, oroxylin A and their glucuronides baicalin, wogonoside and oroxyloside in rats and its application to brain uptake and plasma pharmacokinetic studies. J. Pharm. Biomed. Anal. 2014; 97 :9–23. - 52.
Staerk D., Lambert M., Jaroszewski W.J. HPLC-NMR techniques for plant extract analysis. In: Kayser O., Quax W.J., editors. Medicinal plant biotechnology: from basic research to industrial applications. Weinheim: Wiley Verlag GmbH; 2006. pp. 29–48. - 53.
Bunghez F., Socaciu C., Zagrean F., Pop R.M., Ranga F., Romanciuc F. Characterisation of an aromatic plant-based formula using UV–Vis spectroscopy, LC–ESI(+)QTOF-MS and HPLC-DAD analysis. Bull. UASVM Food Sci. Technol. 2013; 70 (1):16–24. - 54.
Scoog D.A., Holler F.J., Hrouch S.R. Principles of instrumental analysis. 6th ed. USA: Cengage Learning; 2006. 1056 p. - 55.
Gonzales G.F., Miranda S., Nieto J., Fernandez G., Yucra S., Rubio J., et al. Red maca ( Lepidium meyenii ) reduced prostate size in rats. Reprod. Biol. Endocrinol. 2005;3 (6):1–9. - 56.
Ragavendran P., Sophia D., Raj C.A., Gopalakrishnan V.K. Functional group analysis of various extracts of Aerva lanata (L) by FTIR spectrum. Pharmacol. onLine 2011;1 :358–364. - 57.
Chaudhuri C., Pathak A.K., Sevanan M. Phytochemical screening and antimicrobial activity of extracts from leaves and stem of Ecbolium linneanum . Bangladesh J. Pharmacol. 2011;6 :84–91. - 58.
Edwards H.G.M., Farwell D.W., de Oliveira L.F.C., Alia J.M., Le Hyaric M., de Ameida M.V. FT-Raman spectroscopic studies of guarana and some extracts. Anal. Chim. Acta. 2005; 532 (2):177–186. - 59.
Chylinska M., Szymanska-Chargot M., Zdunek A. FT-IR and FT-Raman characterization of non-cellulosic polysaccharides fractions isolated from plant cell wall. Carbohydr. Polym. 2016; 154 :45–54. - 60.
Yan K.J., Chu Y., Huang J.H., Jiang M.M., Li W., Wang Y.F., et al. Qualitative and quantitative analyses of compound danshen extract based on 1H NMR method and its application for quality control. J. Pharm. Biomed. Anal. 2016; 131 :183-187. - 61.
Karioti A., Giocaliere E., Guccione C., Pieraccini G., Gallo E., Vannacci A., et al. Combined HPLC-DAD–MS, HPLC–MSn and NMR spectroscopy for quality control of plant extracts: The case of a commercial blend sold as dietary supplement. J. Pharm. Biomed. Anal. 2014; 88 :7–15. - 62.
Lopez-Garcia M., Garcia M.S.D., Vilarino J.M.L., Rodriguez M.V.G. MALDI-TOF to compare polysaccharide profiles from commercial health supplements of different mushroom species. Food Chem. 2016; 199 :597–604. - 63.
Martins F.T., dos Santos M.H., Coelho C.P., Barbosa L.C., Dias G.C., Fracca M.P., et al. A powder X-ray diffraction method for detection of polyprenylated benzophenones in plant extracts associated with HPLC for quantitative analysis. J. Pharm. Biomed. Anal. 2011; 54 (3):451–457. - 64.
Leonell F., Mostarda A., De Angelis L., Lamba D., Demitri N., La Bella A., et al. Proof of the structure of the Stemodia chilensis tetracyclic diterpenoid (+)-19-acetoxystemodan-12-ol by synthesis from (+)-podocarpic acid: X-ray structure determination of a key intermediate. J. Nat. Prod. 2016;79 (4):1155–1159. - 65.
Rodrigues D., Freitas A.C., Sousa S., Amorim M., Vasconcelos M.W., da Costa J.P., et al. Chemical and structural characterization of Pholiota nameko extracts with biological properties. Food Chem. 2017;216 :176–185. - 66.
Kostic D., Mitic S., Zarubica A., Mitic M., Velickovic J., Randjelovic S. Content of trace metals in medicinal plants and their extracts. Hem. Ind. 2011; 65 (2):165–170. - 67.
Kabata-Pendias A. Trace Elements in Soils and Plants. 4th ed. London: CRC Press; 2010. - 68.
Soare L.C., Visoiu E., Bejan C., Dobrescu C.M., Fierascu I., Iosub I., et al. Research on the in vitro bioaccumulation capacity of lead in some Pteridophyte species of the Romanian flora. Rev. Chim. 2015;12 :2017–2020. - 69.
Rice-Evans C.A., Miller N.M. Total antioxidant status in plasma and body fluids. Meth. Enzimol. 1994; 234 :279–293. - 70.
Alam M.N., Bristi N.J., M. Rafiquzzaman. Review on in vivo andin vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 2013;21 :143–152. - 71.
Sochor J., Ruttkay-Nedecky B., Babula P., Adam V., Hubalek J., Kizek R. Automation of methods for determination of lipid peroxidation. In: Catala A., editor. Lipid peroxidation. Rijeka: InTech; 2012. doi:10.5772/45945 - 72.
Tomer D.P., McLeman L.D., Ohmine S., Scherer P.M., Murray B.K., O'Neill K.L. Comparison of the total oxyradical scavenging capacity and oxygen radical absorbance capacity antioxidant assays. J. Med. Food. 2007; 10 :337–344. - 73.
Pisoschi A.M., Cimpeanu C., Predoi G. Electrochemical methods for total antioxidant capacity and its main contributors determination: a review. Open Chem. 2015; 13 :824–56. - 74.
Bauer A.W., Kirby W.M.M., Sherris J.C., Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 1966; 36 :493–496. - 75.
Balouiri M., Sadiki M., Ibnsouda S.K. Methods for in vitro evaluating antimicrobial activity: a review. J. Pharm. Anal. 2016;6 (2):71–79. - 76.
Cos P., Vlietinck A.J., Berghe D.V., Maes L. Anti-infective potential of natural products: how to develop a stronger in vitro proof-of-concept. J Ethnopharmacol. 2006;106 :290–302 - 77.
Ullah N., Parveen A., Bano R., Zulfiqar I., Maryam M., Jabeen S., et al. In vitro andin vivo protocols of antimicrobial bioassay of medicinal herbal extracts: a review. Asian Pac. J. Trop. Dis. 2016;6 (8):660–667. - 78.
Schmidt N.J., Dennis J., Lennette E.H. Plaque reduction neutralization test for human cytomegalovirus based upon enhanced uptake of neutral red by virus-infected cells. J. Clin. Microbiol. 1976; 4 (1):61–66. - 79.
Riss T.L., Moravec R.A. Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev. Technol. 2 (1):51–62. - 80.
Fan F., Wood K.V. Bioluminescent assays for high-throughput screening. Assay Drug Dev. Technol. 2007; 5 (1):127–136. - 81.
Ames B.N., Durston W.E., Yamasaki E., Lee F.D. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. 1973; 70 (8):2281–2285. - 82.
Stammberger I., Czich A., Braun K. Drug discovery and evaluation. In: Vogel H.G., Hock F.J., Maas J., Mayer D., editors. Genotoxicity. Heidelberg: Springer; pp. 829–840. - 83.
Kittakoop P. Anticancer drugs and potential anticancer leads inspired by natural products. Stud. Nat. Prod. Chem. 2015; 44 :251–307. - 84.
Attaway D.H., Zaborsky O.R., editors. Marine biotechnology, Vol. 1, Pharmaceutical and bioactive natural products. New York: Springer Science+Business Media; 1993. - 85.
Griffiths A.J.F., Miller J.H., Suzuki D.T., Lewontin R.C., Gelbart W.M. An introduction to genetic analysis. 7th ed. New York: W.H. Freeman; 2000. - 86.
Redei G., Acedo G., Andhu S. Sensitivity, specificity and accuracy of the Arabidopsis assay in the identification of carcinogens. U.S. Environ. Prot. Ag., Washington, D.C. 2002:EPA/600/D-84/105. - 87.
Misik M., Ma T.H., Nersesyan A., Monarca S., Kim J.K., Knasmueller S. Micronucleus assays with Tradescantia pollen tetrads: an update. Mutagenes. 2011;26 (1):215–221. - 88.
Teaf C.M., Middendorf P.J. Mutagenesis and genetic toxicology. In: Williams P.L., James R.C., Roberts S.M., editors. Principles of toxicology: environmental and industrial applications. 2nd ed. Hoboken: John Wiley & Sons; 2000. pp. 293–264. - 89.
Directive 2010/63/EU of the European parliament and of the Council on the protection of animals used for scientific purposes. 2010. - 90.
Ali R.B., Atangwho I.J., Kuar N., Ahmad M., Mahmud R., Asmawi M.Z. In vitro andin vivo effects of standardized extract and fractions ofPhaleria macrocarpa fruits pericarp on lead carbohydrate digesting enzymes. BMC Complement. Alt. Med. 2013:201313:39. - 91.
Lattanzio F., Greco E., Carretta D., Cervellati R., Govonic P., Speroni E. In vivo anti-inflammatory effect ofRosa canina L. extract. J. Ethnopharmacol. 2011;137 (1):880–885. - 92.
Al Batran R., Al-Bayaty F., Abdulla M.A., Al-Obaidi M.M., Hajrezaei M., Hassandarvish P., et al. Gastroprotective effects of Corchorus olitorius leaf extract against ethanol-induced gastric mucosal haemorrhagic lesions in rats. J. Gastroenterol. Hepatol. 2013;28 (8):1321–1329. - 93.
Itelima J.U., Agina S.E. In vivo antimicrobial activity of plant species onEscherichia coli O157:H7 inoculated into albino rats. World J. Microbiol. 2014;1 (1):002–009. - 94.
Betz U.A., Fischer R., Kleymann G., Hendrix M., Rubsamen-Waigmann H. Potent in vivo antiviral activity of the herpes simplex virus primase-helicase inhibitor BAY 57–1293. Antimicrob Agents Chemother. 2002;46 (6):1766–1772. - 95.
Robertson S., Narayanan N., Kapoor B.R. Antitumour activity of Prosopis cineraria (L.) Druce against Ehrlich ascites carcinoma-induced mice. Nat Prod Res. 2011;25 (8):857–862. - 96.
Bhowmick R., Sarwar S., Dewan S.M.R., Das A., Das B., Uddin M.M.N., et al. In vivo analgesic, antipyretic, and anti-inflammatory potential in Swiss albino mice andin vitro thrombolytic activity of hydroalcoholic extract fromLitsea glutinosa leaves. Biol. Res. 2014;47 (1):56. - 97.
http://www.genpharmtox.de/downloads/Toxicology.pdf - 98.
Ortan A., Ferdes M., Dinu Pirvu C. Comparative microbiological activity of volatile oils from Anethum graveolens species. Stud. Univ. V. Goldis, Life Sci. Ser. 2012;22 (4):531–535. - 99.
Ortan A., Popescu M.L., Gaita A.L., Dinu-Pirvu C., Campeanu G. Contributions to the pharmacognostical study on Anethum graveolens , dill (Apiaceae ). Rom. Biotechnol. Lett. 2009;14 (2):4342–4348. - 100.
Ortan A., Ferdes M., Rodino S., Pirvu C., Draganescu D. Topical delivery system of liposomally encapsulated volatile oil of Anethum graveolens . Farmacia. 2013;61 (2):361–370. - 101.
Ortan A., Dinu Parvu C., Ghica M.V., Popescu L.M., Ionita L. Rheological study of a liposomal hydrogel based on Carbopol. Rom. Biotechnol. Lett. 2011; 16 (S1):47–54. - 102.
Ortan A., Campeanu G., Dinu Pirvu C., Popescu L. Studies concerning the entrapment of Anethum graveolens essential oil in liposomes. Rom. Biotechnol. Lett. 2009;14 (3):4411–4417.