Anthemis tinctoria L. belongs to Asteraceae family and it is used for a long time by the Romanian people for its tinctorial and medicinal properties. The pharmacognostical study of this species is important, the knowledge about Anthemis tinctoria L. being poor. The carotenoids are important compounds present in Anthemis tinctoria L. flowers. They are mentioned for the first time in this paper. Total carotenoids were extracted with a solvent mixture, saponified, separated on alumina open column and analyzed by High Performance Liquid Chromatography with Photodiode Array Detector (AHPLC-PDA). The fresh flowers contain 46,86 mg carotenoid /100 g and the dried flowers contain 6,25 mg carotenoids /100 g. There were identified carotenoids from carotene series (β-carotene), monoxantophyll series (α- and β-criptoxantine), dixantophyll series (luteine) and epoxidic series (neoxanthine and violaxanthine). The drug is a good source of xanthophylls (the concentration of lutein is 46,8%), which has important therapeutical properties. The results show that it is possible to obtain some standardized extracts rich in carotenoids, especially in xantophylls.
Anthemis tinctoria L. este o plantă din familia Asteraceae, utilizată de multă vreme pentru proprietăţile tinctoriale şi în medicina populară românească. Cercetarea farmacognostică a speciei devine importantă în condiţiile în care ea este puţin studiată. Carotenoidele, compuşi importanţi din florile speciei, sunt semnalate pentru prima dată în această lucrare. Acestea au fost extrase cu un amestec de solvenţi, extractul obţinut a fost supus saponificării, separării pe coloană deschisă de oxid de aluminiu şi analizei prin cromatografie de lichide de înaltă performanţă, utilizând detector cu şir de diode (HPLC-PDA). Florile proaspete conţin 46,86 mg carotenoide /100 g, iar cele uscate conţin 6,25 mg carotenoide/100 g. Au fost identificate carotenoide din seria carotenelor (β-caroten), seria monoxantofilelor (α- şi β-criptoxantina), seria dixantofilelor (luteina) şi epoxizilor (neoxantina şi violaxantina). Produsul vegetal reprezintă o importantă sursă de xantofile (46,8% luteină), care au importante proprietăţi terapeutice. Rezultatele dovedesc posibilitatea obţinerii de extracte standardizate îmbogăţite în carotenoide, în special în xantofile.
Carotenoids are natural pigments produced by plants, also found in animals. In plants they are involved in photosynthesis and photoprotection. In animals, besides the role of provitamin A, they act as antioxidants and tumor-preventing agents. In consequence, carotenoids are largely used in food industries, cosmetics and in medicine.
Anthemis tinctoria L. belongs to Asteraceae family and its flowers (Anthemidis flos) have been used for a long time by Romanian people for its tinctorial and medicinal properties . By phytochemical studies we identified in Anthemidis flos, the polyphenolcarboxilic derivatives (2.066%), flavonoides (1.34%), cumarines (0.043%), saponins (2.97%), sugars (3.39%), fatty acids, acetilene derivatives. The identified polyphenolcarboxilic derivatives are caffeic acid and chlorogenic acid. From flavonoid class we identified the rutosid, iso-cvercetrine and luteolin-7-glucoside. From the class of cumarines (0.043%) we identified the scopoletole and the umbeliferone. The saponines are triterpenoid type. The sugars are osuronic type and the identified monomers are galacturonic acid, galactose, arabinose, xilose and ramnose. The identified fatty acids are miristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arahic acid, alfa-linolenic acid [1, 5- 8].
There isn’t any data about the content in carotenoids in Anthemis tinctoria L. (Asteraceae). The aim of this study was the separation and identification of the main carotenoids from Anthemidis flos. Total carotenoids were extracted with a solvent mixture. The carotenoid extract was saponified and separated on open alumina column. Three fractions were colected: carotenes, monoxantophylls and dixantophylls. Each fraction was quantified by relating the absorbance reading (λ=450 nm) to the specific absorbtion of coloured carotenoids (A1%1cm=2500). The most important quantity was the third fraction – around 85% from the total extract (dried flowers). All fractions were separated on TLC Silica plates and HPLC on reversed-phase column, in gradient elution system. Carotenoids were identified by comparing the retention factor (Rf) values, retention times and wavelengths with those of the standards.
MATERIALS AND METHODS
Two experiments were made: the first using dried flowers of Anthemis tinctoria L. and the second using fresh flowers separated in ligulate and tubular.
Extraction and saponification
The material was ground with acetone in the dark, overnight, and further with petroleum ether for 2 hours. BHT (butyl hidroxitoluene) and NaHCO3 were added as antioxidants. The crude extract was filtered, evaporated to dryness in a Rotavapor at 35ºC and re-dissolved in ethyl-ether.
Saponification was carried out in ethyl-ether overnight and under permanently stirring by adding 30% w/v KOH in methanol to a final concentration of 15% w/v KOH. The carotenoids were extracted with ethyl-ether from a saline solution to neutral pH. The total carotenoid extract was evaporated, brought to a fixed volume and used to quantitative evaluation of carotenoids and for separation by chromatographic methods [2, 3].
b. Quantitative evaluation of total carotenoids
The concentration of total carotenoids was calculated using the following equation: X= (A x Y x 1000) (2500 x 100), A= absorbance (λ max= 450 nm), 2500= A1%1cm (specific absorbance of coloured carotenoids), X= weight of carotenoids in the sample (mg), Y= volume of the sample (μl)[2, 3].
The total carotenoids extract was fractionated on open alumina column – Al2O3 grade III with 6% water. Three fractions were collected: F I – 100% petroleum ether; F II – 100% petroleum ether – 50% ethyl-ether; F III – 100% ethyl ether. Each fraction was collected, evaporated and submitted to thin layer chromatography (TLC) and HPLC analysis.
The separation was performed in a HPLC system including a Kontron System 322 pump, a reversed phase Nucleosid 120-5 C 18 250x4,6 column and a Waters 990 Photodiode Array Detector.
The mixture of solvents was programmed (flow 1 ml/min) as follows:
0------------10 min. (90% A--- 50% A) (10% B---50% B)
10-----------30 min. (50% B---10% A) (50% A---90%A)
In order to identify the carotenoids, we compared the retention times (tR) of the samples with those of the standards separated in the same conditions and we also compared the maximum absorption wavelengths (fig. 1, table I).
The maximum absorption wavelengths of identified carotenoids
Maximum absorption wavelengths of standards using the described HPLC method
Maximum absorption wavelengths of samples using the described HPLC method
422, 444, 473
428, 450, 474
428, 450, 474
422, 444, 473
425, 452, 479
418, 438, 467
The fraction collected from alumina open column were separated on silica plates (Kieselgel Merck) using a mixture with 3%, 7% and 15% acetone in petroleum ether. Individual carotenoid standards were used for comparison.
RESULTS AND DISCUSSION
a. Quantitative analysis
The total carotenoids content is 62.56 μg/g in dried flowers (ED), 372.20 μg/g in fresh ligulated flowers (EL) related to the dry vegetal product and 69.49 μg/g in fresh tubular flowers (ET) related to the dry vegetal product.
Content of total carotenoid
Ligulate flowers (EL)
Tubular flowers (ET)
Fraction I (carotenes)
Fraction II (monoxanthophills)
Fraction III (dixantophylls)
b. Qualitative analysis
Our determinations showed that the ligulate and tubular flowers have a very similar carotenoid profile. The concentration of total carotenoids in fresh ligulated flowers is much higher than in dried flowers so the qualitative analysis (HPLC) is outlined just for the fresh vegetal product. Fraction I in EL and ET is poor. The most important compound is trans-β-carotene (peak 1), with tR = 20.53 min. Cis- β-carotene (peak 2) and α-carotene (peak 3) are present in very small amounts (fig. 2). Fraction II in EL and ET contains mostly α-cryptoxanthin (tR= 15.9 min.) and traces of β-cryptoxanthin. Fraction III contains epoxides: neoxanthin, violaxanthin (tR=7.6), unidentified compound (peak 3) and lutein (tR=11.12 min.) (peak 4) and some other unidentified compounds (probably epoxides) (fig.3).
In all the extracts, lutein is the most important compound (in average 46.8% from total carotenoids).
The percentage of carotenoids in tubular flowers (HPLC assay)
Retention time (min.)
* Absorbance units/sec
The extracts were poor in carotenes (7.5 % of the total extract) and we could identify only β-carotene and α-carotene. In the second fraction there were identified α-cryptoxanthin and β-cryptoxanthin. The most important carotenoid identified in the third fraction was lutein – around 47%. The same fraction contains: zeaxanthin, neoxanthin, violaxanthin and other unidentified carotenoidic epoxides.
Anthemis tinctoria is a rich source of carotenoids, mainly lutein (in average 46.8%). The ligulate and tubular flowers have a very similar carotenoid profile. The content is lower in dried flowers than in fresh flowers, probably due to degradation of carotenoids during the storage. The fresh flowers contain 46.86 mg carotenoids/100 g related to the dry vegetal product and the dried flowers contain 6.25 mg carotenoids/100 g. There were identified carotenoids from carotene series (β-carotene), monoxantophyll series (α- and β-criptoxantine), dixantophyll series (luteine) and epoxidic series (neoxanthine and violaxanthine).
Bohlmann F., Rleine K.M., Uber einige neue Acetylen verbindungen aus der Sattung Anthemis L., Chem. Ber., 1999, 2096-2103