Joint research project

Qualitative and quantitative NMR study of saffron (Crocus sativus L.) extracts grown in Lebanon and comparison with saffron from different geographical origins

Project leaders
Donatella Capitani, Ramez Chahine
LIBANO - CNRS-L - National Council for Scientific Research of Lebanon
CNR/CNRS_L 2015-2016
Chemical sciences and materials technology
Thematic area
Chemical sciences and materials technology
Status of the project

Research proposal

Lebanon, previously one of the world's largest producers and exporters of hashish, in 1997 was removed from the international list of drug producing and exporting nations and became eligible to US aid for international loans from development institutions like the World Bank. Lebanese government negotiated a complete annihilation of cannabis fields of the Bekaa Valley. The fields were destroyed, which left some 70,000 families out of their yearly revenue. International aid programs were introduced to replace the plants with crops. In 2001 the government starts distributing saffron bulbs to farmers in the northern Bekaa under the supervision of Dr Hassane Makhlouf. Saffron apparently originated in the area of Iran, Turkey and Greece, but now it is successfully cultivated in many European and Asian countries as well as in Australia. While the world's total annual saffron production is estimated to be 205 tons, Iran produces about 80% of the total. Saffron is the most expensive food spice in the world obtained from the flower stigmas of Crocus sativus L. (Iridaceae family). It plays an important role in the culinary culture of different regions of the world, where it is used as a food additive, since it possesses powerful coloring and flavoring properties due to its glycosidic constituents. Since ancient times, saffron has been also used as a medicinal plant. The stigmas contain carbohydrates, minerals, vitamins and pigments such as carotenes, and flavonoids. The most important compounds in saffron are crocin, picocrocin and safranal, responsible for saffron color, flavor and aroma, respectively. High amounts of these main compounds are used to commercially indicate the quality of saffron. The international organization for standardization (ISO) has set a classification of saffron based on minimum requirements of each quality, ISO 3632. According to this classification, they established 4 categories (I-IV) on the basis of the relative quantity of Crocin, picocrocrocin and safranal by means of a spectrophotometric method using different wavelengths. For commercial purposes the quality of the colouring power of a saffron sample depends on the quantification of the crocin analogues by colorimetric and chromatographic techniques, such as GC-MS, and HPLC. The chemical composition of saffron samples indicated that the estimated amounts of constituents strongly depended on the methods employed for drying, extraction, separation and quantification. In 2009 the research team at the Lebanese University (Oxidative Stress and Antioxidants) decided to prepare different extracts from the "lebanese saffron" and to test both their antioxidant activity in vitro and their potential protective effect against doxorubicine cardiotoxicity in isolated rabbit heart model. Results obtained showed that extracts possess a high antioxidant scavenging activity against a broad variety of toxic free radicals [1 2], and also exhibit a high protective activity against ischemia reperfusion injury [3]. This finding makes the spice a promising candidate for being a functional food. A functional food is considered a natural or processed food that contains known biologically active metabolites in defined quali-quantitative amounts providing a clinically proven health benefit. However, a gap still needs to be filled. Actually, there are no studies concerning the quantification of the active constituents of the Lebanese saffron and this is mandatory to ensure the quality of the compound tested. A cooperation between the research team of Lebanese University and the Italian research team of IMC-CNR will help to achieve this aim. In fact the team from IMC-CNR has proposed the 1H NMR-based metabolic profiling of microwave-assisted saffron extracts of different geographical origins [4]. The assignment of some compounds was reported in more detail with respect to literature data after a simple and environmentally-friendly microwave-assisted extraction requiring only a minimal amount of these expensive dried stigmas with respect to the ISO specifications. Microwave-assisted extraction has well-known advantages (reduced solvent volume and extraction time, pressurized and sealed vials to reduce loss of volatile components, strict control of multiple parameters, such as temperature, pressure and irradiation power). This technique provides a better isolation of secondary metabolites due to the complete swelling of the subcellular structures under microwave irradiation which leads to a deeper penetration of the solvent and an easier release of intracellular metabolites. 1H high field NMR spectroscopy is one of the most suitable methodologies to obtain ''high-throughput'' spectroscopic/structural information on a wide range of compounds with a high analytical precision. It has been applied to the analysis of food (target analysis, metabolic profiling, metabolic fingerprinting) to obtain information on varieties, geographical origin, quality, adulteration, and so on [5].

[1] Makhlouf H et al Determination of antioxidant effect of saffron taken from the flower of Crocus sativus grown in Lebanon. Afr. J. Biotech. 2011, 10 (41): 8093-8100
[2] Chahine N et al Protective effect of saffron extract against doxorubicin cardiotoxicity in isolated rabbit heart. Pharm Biol 2013, 51: 1564-71
[3] Chahine N et al 2011. Protective effects of saffron extracts on ischemia/reperfusion inducing oxidative damage in isolated rabbit heart. Annual meeting on medicinal chemistry, Emerging Targets and Treatments: Opportunities and Challenges for Drug Design. 25 November 2011, Ghent, Belgique
[4] A. P. Sobolev, S. Carradori, D. Capitani, S.Vista, A. Trella, F. Marini, L. Mannina. Saffron samples of different origin: an NMR study of microwave-assisted extracts. Foods, 2014, 3, 403-419
[5] Mannina L, Sobolev AP, Capitani D. Applications of NMR metabolomics to the study of foodstuffs: truffle, kiwifruit, lettuce, and sea bass. Electrophoresis. 2012;33:2290

Research goals

This proposal joins two complementary teams, the Lebanese team that will promote the saffron cultivation by giving an ISO level to this compound and the Italian team helping in measurements and biological evaluations in vitro. The first objective is the quantification of major active components of saffron from the Bekaa and the comparison with the amounts of these compounds in saffron from other countries using NMR-metabolic fingerprint. Our choice of an alternative analytical technique with respect to that proposed by ISO guidelines arises from the fact that crocins have absorption at 257 and 330 nm (cis-crocin) in addition to their main peak at around 440 nm. So, safranal and picrocrocin value as direct measurement at 257 and 330 nm may be misleading. Because of the high variability of the content of the spice constituents, a proper analytical method is mandatory for monitoring the quality of the product. Metabolite profiling could be used both to standardize the production in order to obtain the same product every year and to optimize the crop production conditions. The second objective is to optimize the extraction procedure using microwave-assisted extraction. The third objective is to test the saffron extracts in vitro using cardiomyocyte in culture treated with doxorubicine and to assess other biological activities (monoamine oxidase and carbonic anhydrase inhibition) of Lebanese saffron in order to better characterize this product on the global market.

Last update: 27/11/2021