FARMACIA, 2010, Vol.58, 3

308

CONTRIBUTION TO THE STUDY OF THE PHARMACEUTICAL QUALITY OF SOME CHAMOMILE COMERCIAL SAMPLES. NOTE I. THE ANALYSIS OF THE VOLATILE OIL OANA CIOANCĂ*, ANA CLARA APROTOSOAIE, ADRIAN ŞPAC, MONICA HĂNCIANU, URSULA HELENA STĂNESCU

University of Medicine and Pharmacy “Gr. T. Popa” Iasi, Faculty of Pharmacy, 16 University Str., Iasi, 700117 *corresponding author: oana.cioanca@gmail.com

Abstract Chamomile is well known and widely used for its therapeutic purposes. It is

generally approved that natural products not necessarily mean safe or pure products, thus the aim of our research was to assess the degree to which chamomile tea manufacturers comply with the regulations imposed by the European pharmaceutical distribution market (Romania being part of the European Union) of such a tea. In the present study we present the results obtained in the phytochemical analysis of the volatile oils extracted from 10 commercial samples of chamomile tea. Important qualitative and quantitative differences were noted for the separated volatile fractions.

Rezumat Muşeţelul este o plantă medicinală foarte cunoscută şi extrem de utilizată în scop

terapeutic. Se ştie că produse naturale nu înseamnă produse sigure sau neimpurificate, de aceea scopul cercetării noastre a fost acela de a urmări gradul în care producătorii de ceaiuri medicinale respectă regulile impuse de comercializarea prin reţeaua farmaceutică europeană (România fiind membră a Uniunii Europene) a acestor tipuri de produse. În cadrul studiului de faţă vom prezenta rezultatele obţinute în urma studiului fitochimic a uleiurilor volatile extrase din 10 mostre de ceai de muşeţel comercializat. Au fost observate diferenţe calitative şi cantitative importante între fracţiunile volatile separate.

Keywords: chamomile, volatile oil, differences, quality Introduction Chamomillae flos represents one of the most common medicinal

teas used both for it’s pleasant taste as well as for therapeutic purposes, but most of the vegetal material commercialized by pharmacies has poor quality. Thus, the aim of our study was to investigate this aspect, pursuing the requirements the chamomile flowers should comply with the European Pharmacopoeia (PhEu) [10].

We used nine commercial samples of chamomile bought from pharmacies before Romania became an European Union (EU) member, and

FARMACIA, 2010, Vol.58, 3

309

a German producer sample was added. Our research was centered on: the pharmacognostic macroscopic study of the vegetal material (identification of the vegetal and mineral impurities, the congruence of the product to what the chamomile inflorescences should be); the phytochemical analysis (the composition of the volatile oil, flavones and polyphenolic acids); and the microbiological contamination. In this paper, we are going to present the results obtained for the study of the volatile fraction extracted from the 10 chamomile samples.

Materials and methods The material was represented by 9 commercial chamomile samples

(bought from pharmacies in April-May 2005) from different inland producers. The samples were codified C1-C9 bearing in mind the packaging, tea-bags and 50-100g sacks (for the last type of samples using the term “bulk”). The sample C0 (tea-bags) made by Sidroga, found only in pharmacies within EU countries, was included in the study. For this last sample, the flowers are of cultivated chamomile and comply with the PhEu requirements [2,3,12].

The volatile oils were obtained by hydro-distillation in a Neo-Clevenger type apparatus from the 10 samples of Chamomillae flos, according to the method described in the Romanian Pharmacopoeia Xth Edition [11].

Methods: We established the extraction yield, the colour and aspect

characteristics for each oil sample. The chemical analysis pursued the identification of the terpenoidic

compounds from the volatile fractions, using thin layer chromatography (TLC) followed by gas chromatography with mass spectrometry detection (GC-MS) [1].

GC analysis of the samples was performed on a Gas Chromatograph type Agilent 6890 with 5975 Mass Selectiv Detector, HP 5 MS capillary column (30 m x 0.25 mm x 0.25 µm) with helium as mobile phase at a constant flow of 1 mL/min (average velocity of 36 cm/sec). Sample solutions were injected (0.2 µL) in split mode (1:10) at 260°C. The column temperature was lineary programmed from 35 - 260°C at 10°C/min. Transfer line was heated at 260°C. Evaluation of the results was performed according to the software (ChemStation) including a mass spectral library (Wiley), which was used for identifying organic compounds extracted from

FARMACIA, 2010, Vol.58, 3

310

the oils. The identification of the components was based on the comparison of their mass spectra in the apex of each peak with those of the analytical standards from Willey Mass Spectral Library. For quantification purposes the percent area was reported.

Reagents: All chemical and reagents were of analytical grade or of

chromatographic quality and were purchased from Merck (Darmstadt, Germany), Sigma Aldrich (Seelze, Germany) or Fluka (Buchs, Switzerland).

Results and discussion

Calculating the extraction yield for the volatile oils separated by hydro-distillation from the vegetal material we obtained the results presented in table I and figure 1.

Table I The volatile oil content and aspect for the chamomile samples

Code Producer Type Volatile oil content (%) Aspect Colour

C0 Sidroga s 1.15 slightly viscous liquid indigo-blue

C1 Belin s 0.58 slightly viscous liquid blue-indigo C2 Celmar s 0.43 viscous liquid blue-indigo C3 Fares s 0.43 viscous liquid blue-indigo C4 Ciprod s 0.29 slightly viscous liquid green-blue C5 Hofigal s 0.36 slightly viscous liquid blue-green C6 Digitalis v 0.72 extremely viscous liquid dark blue-indigo C7 Cyani v 2.15 slightly viscous liquid blue-green C8 StefMar v 0.43 slightly viscous liquid blue-green C9 Plafar

Botosani v 0.72 slightly viscous liquid light blue

where: s - tea-bag; v - „bulk” package

Figure 1

Graphic representation of the extraction yield for the volatile oil from the chamomile samples

FARMACIA, 2010, Vol.58, 3

311

Bearing in mind the obtained values we can state that two of the samples, C4 and C5, did not comply with the pharmacopoeial (both Ph.Eu and FR X) standards [10,11] that require a minimum amount of 0.4 mL of volatile oil for 100g of dried vegetal material. Overall, the volatile oil content of the chamomile samples varies between 0.29 – 2.15%. Also, comparing the volatile oil content of the tea-bagged samples we notice that this is lower than the amount registered for the “bulk” samples. Regardless of the Sidroga sample (from EU) the first category had a content of 0.29% to 0.58%, whereas the “bulk” samples provided quantities varying between 0.72% and 2.15% of volatile oil. This fact is in accordance with what we already know about the volatile fractions: for a vegetal material that is grounded, the higher the powdering degree, the higher loss of volatile compounds, especially when the tea-bag material is permeable and there is no other protection (tea bags wrapped in individual waxed envelopes, included in a box with a plastic wrapper, as for the C0 sample).

Assessing the physical aspects of the 10 volatile oils, the one obtained from the Sidroga sample had the organoleptic characteristics that best comply with the standards for the chamomile volatile oil (table I). The color of the oil samples varies from blue-indigo (C1-C3) to light-blue (C9) and blue-green (C5- macroscopic analysis revealed the presence of the leaves and stalk), with important differences regarding the mobility – from slightly viscous (C1, C4, C5) to extremely viscous for the C6 sample. The TLC for the volatile oils on Kieselgel G60 (mobile phase toluene: ethyl acetate 95:5) revealed the images presented below, in figure 2.

Figure 2

TLC for chamomile volatile oil samples; C0-C9 = samples; standards: standard chamomile volatile oil, bisabolol and bornyl acetate

We noticed the lack of uniformity regarding the chromatographic aspects, even if the samples C1-C3 might have a similar compound spectra. The presence of azulene (Rf=0.94) in all samples (excepting sample C5) is important not only for the colour of the oil, but it has a certain importance in the antiinflammatory action of the volatile fraction. C0 sample lacks

FARMACIA, 2010, Vol.58, 3

312

bisabolol (Rf=0.41), but for most of the samples is present. At Rf=0.5 there is a brown spot which, probably belongs to the spiroethers, that for C4-C6 and C8 samples fades out. The GC-MS allowed us to identify a large number of compounds; the main components are presented in table II.

Table II Main compounds identified in the chamomile volatile oils–selective table

Area (%) RT* (min.)

Compound C0 C1 C2 C3 C4 C5 C6 C7 C8 C9

5.692 α-pinene 0.03 - 0.01 - - - 0.01 - 0.04 0.01 6.480 6-methyl-5-hepten-2-one 0.05 - 0.01 - - - 0.01 - - -

7.112 p-cymene 0.02 - 0.05 0.30 - 0.05 0.04 - 0.05 0.04 7.173 limonene 0.04 0.07 0.03 0.12 0.04 - 0.04 - 0.04 0.03 7.233 1,8-cineol 0.02 - - - - - 0.02 - 0.12 - 7.649 artemisia ketone - 0.15 0.09 - - 0.08 - - 0.31 - 7.995 artemisia-alcohol 0.03 0.07 0.04 0.07 - - 0.03 - 0.15 0.01

8.393 α-thujone - 0.10 - - - - 0.07 - - 0.13

9.129 L-menthone 0.10 0.41 0.29 - - - 0.14 - - -

9.285 isomenthone 0.12 0.51 0.30 - - - - - - -

9.319 l-borneol - - - - 0.05 0.12 - - 0.33 0.23

11.016 trans-anethole 1.29 - - 0.32 0.10 0.25 0.07 - - 0.04

12.496 berkheyaradulene 0.28 0.26 0.21 - 0.24 - 0.16 - - 0.20

13.137 pirethryn I - - 0.07 - - - - - -

13.232 trans-β-farnesene 14.07 1.13 1.12 3.93 2.49 2.71 4.32 11.34 3.24 11.13

13.448 alloaromadendrene - 0.45 0.76 - - - 0.09 - - 0.17

13.786 β-selinene 0.59 - 0.47 - - - 0.18 - - 0.62

13.630 α-curcumene 0.36 - 0.18 - - - 0.13 - - 0.20

13.682 germacrene D 1.11 0.41 0.21 0.66 0.17 0.17 0.28 - 0.33 0.89

13.561 (E,Z)-α-farnesene 0.40 0.24 - - - - - - - 0.51

13.760 eremophyllene - - - - - - - - 0.47 0.42

13.881 biciclogermacrene - - 0.16 0.48 - 0.20 0.30 - 0.54 0.87

14.340 cis-α-bisabolene 0.12 - - - - - - - - -

14.348 α-muurolene - - - - - - - - - 0.06

14.868 (-)-spathulenol 2.50 5.01 5.27 10.75 6.53 2.56 2.90 3.61 3.00 2.80

15.733 α-bisabololoxide B 8.34 21.38 14.42 20.49 3.43 4.29 13.58 3.47 3.63 2.69 16.002 α-bisabolol - 0.95 - 1.57 0.85 0.99 - 1.87 1.00 -

16.062 bisabolonoxide - 10.64 8.75 11.14 10.28 13.46 - 31.53 13.25 26.20

16.106 β-bisabolene 21.92 - 0.11 - - - 0.07 - - 0.09

16.616 chamazulene 8.25 2.92 4.97 5.71 0.26 1.84 11.48 3.64 1.76 0.38

16.772 bisabololoxide A 10.76 7.77 9.08 9.60 7.72 10.66 10.29 19.76 10.24 11.59

18.131 en-yne-dicycloether 3.13 1.19 0.71 3.29 1.61 1.23 0.84 1.61 1.65 1.53

20.719 ambretolide - - - 1.36 - - - - - -

24.286 octadecane - - - - - - 0.18 - 0.87 0.02

24.987 tetracosane 0.14 0.31 0.28 - 1.36 12.51 0.65 - 2.01 0.46

25.316 eicosane 0.07 - 0.02 - 0.54 6.13 0.08 1.73 2.18 0.04

* RT – Retention time (minutes).

FARMACIA, 2010, Vol.58, 3

313

As shown in the table, the volatile oils had a complex chemical composition, being mainly constituted of sesquiterpenes, aliphatic compounds, spiroethers and, excepting C7, monoterpenes. The compound spectra as well as the quantity of each component varies a lot from one sample to another. As a matter of fact, the only compounds found in all samples were: chamazulene, en-in-dicycloether, α-bisabololoxide A, α-bisabololoxide B, t-β-farnesene and spathulenol - a compound important for its antifungal properties, the first 5 being considered the major specific components for the chamomile volatile oil.

Excepting the samples C4 and C5, sesquiterpenes were the major fraction (38.45% - 77.16%) of the volatile oil (the literature mentions values of 75-90%). The noted differences are not only quantitative, but qualitative too. For example, alpha-bisabolol and its oxides A and B represent the most important oxygenated sesquiterpenes with pharmacologic activity; but as the internal standard Sidroga (C0) the samples C2, C6 and C9 lack α-bisabolol. Another major sesquiterpene-oxide identified in samples C1-C5, C7-C9 is bisabolonoxide. Regarding the oxygenated sesquiterpenes distribution, a recent study showed that: α-bisabolol, bisabololoxide B and bisabolonoxide are specific for the ligulate and tubular florets, whereas spathulenol and bisabololoxide A are found in the receptacle and tubular florets [7]. All volatile oils contained t-β-farnesene as a major hydrocarbonated sesquiterpene, distinguished in high concentrations in the leaves and roots of Chamomilla recutita [5]. For C0, C1, C2, C4, C6 and C9 samples we identified another hydrocarbonated sesquiterpene – berkheyaradulene, that is specific for the roots of Berkheya radula (Harv.) de Willd. [4] and the Silphium perfoliatum rhizome [9], a common Asteraceae species for our country.

En-yne-dicycloether, with important spasmodic and antimicrobial activity, was present in all samples, the tea-bagged samples containing similar amounts (1.61%-C7, 1.65%-C8, 1.53%-C9) excepting C6 (0.86%), whereas the “bulk” samples the differences were significant: 0.71%-C2 comparing to 3.29%-C3 or 3.13%-C0.

The main identified monoterpenes, even if poorly represented, were: artemisia-alcohol, artemisia-ketone, l-borneol and limonene. Limonene found in all samples excepting C5 and C7 is characteristic rather to the volatile oil from the stems, wheres linalool (C0, C2) belongs to root oil [5]. The thujone from C1, C6, C9 is a compound found in the ligulate florets, where is highly concentrated [7].

FARMACIA, 2010, Vol.58, 3

314

Conclusions The qualitative and quantitative chemical study of the chamomile

samples revealed that the extracted volatile oils differ a lot from one sample to the other and the composition is not well preserved for the samples packed as tea-bags unless included in waxed envelopes. In the same time, the chemical analysis showed the existence of compositional differences more or less important, the only compounds found in all samples were: chamazulene, en-yne-dicycloether, α-bisabololoxide A, α-bisabololoxideTB, t-β-farnesene and spathulenol. Also, the existence of nonspecific chamomile compounds: menthone (C0, C1, C2, C6), isomenthone (C0, C1, C2), trans-anethole (C0,C3-C6), pirethryn I (C2) and ambretolide (C3), suggest that the samples have been contaminated or inadequately processed.

References 1. Benedec D., Oniga I., Oprean R., Tamas M., Chemical composition of the essential oils

of Ocimum basilicum L. cultivated in Romania, Farmacia, 2009, 57(5), 625-629. 2. Bauer R., Czygan FC., Franz G., Ihrig M., Nahrstedt A., Sprecher E.:

Qualitatsanspruche an rational anwendbare Phytopharmaka. Deutsche Apotheker Zeitung, 1993, 133, 4105-4108

3. Pavel M., Rădulescu V., Carolina Ilieş D., GC-MS analysis of essential oil obtained from the species Thymus comosus Heuff. Ex Griseb. (Lamiaceae), Farmacia, 2009, 57(4), 479-484.

4. Bohlmann F., Le Van N., Pickardt J. Naturally occuring derivatives, 108. On an anormal sesquiterpene from Berkheya radula (Harv.) De Willd, Chemische Berichte. 2006, 110 (12), 3777-3781.

5. Das M., Ram G., Singh A., Mallavarapu R.G., Ramesh S., Ram M., Kumar S. Volatile constituents of different plant parts of Chamomilla recutita L. Rausch grown in the indo-Gangetic plains. Flavour and Fragrance Journal. 2002, 17 (1), 9-12

6. Gacea O., Hancianu M., Aprotosoaie C., Spac A., Dorneanu V., Stanescu U.: Acta Horticulturae. 2007, 749, 175-179.

7. Tirillini B., Pagiotti R., Menghini L., Pintore G. Essential oil composition of ligulate and tubular flowers and receptacle from wild Chamomilla recutita (L.) Rausch. grown in Italy, Journal of Essential Oil Research. 2006, 18 (1), 42-46.

8. Wagner H., Bladt S., Zgainski E.M.,1983. Drogenanalyses, Berlin: 32-33, 184-185. 9. Wolski T., Kowalski R., Mardarowicz M. Chromatographic analysis of essential oil

occuring in inflorescences, leaves and rhizomes of Silphium perfoliatum L. Herba Polonica., 2000, 46 (4), 235-242.

10. *** European Pharmacopoeia, VIth Edition, EDQM, Strasbourg, 2007, 2340-2344. 11. *** Romanian Pharmacopoeia, Xth Edition, Ed. Medicala, Bucuresti, 1993, 334-335. 12. ***Chamomile Industrial Profiles. Edits. Franke R., Schilcher H. CRC Press Taylor &

Francis Group. 2005, 21-24, 70-71, 239-240. Manuscript received: July 23rd 2009