In the Laboratory
Detection and Quantification of Valerenic Acid in Commercially Available Valerian Products
W
Ruth H. Douglas, Ciaran A. Muldowney, Rabab Mohamed, Fiona Keohane, Catherine Shanahan, and John J. Walsh* Pharmacognosy, School of Pharmacy, University of Dublin, Trinity College, Dublin 2, Ireland; *
[email protected] Pierce V. Kavanagh Department of Pharmacology and Therapeutics, University of Dublin, Trinity College, Dublin 2, Ireland
Valerian has been used for hundreds of years for its sedative and antispasmodic properties. It also has carminative, hypnotic, and anxiolytic effects (1, 2). Traditionally, it had the synonym of “all-heal” (1), indicating its wide range of therapeutic effects. It is generally supplied as the aqueous extract, alcoholic extract, or in the dried-root form. Modern interest is in its use as a natural sedative and hypnotic. The European Medicines Evaluation Agency, as well as several national pharmacopoeias, now states the use of valerian for relief of temporary, mild nervous tension and for temporary difficulty in falling asleep (1). Several clinical trials have been carried out that confirm valerian’s sedative effects (3–7). There is some evidence that the pharmacological effects of valerian are due to synergistic activity of its many phytochemical constituents (2). The sesquiterpenoid compounds, including valerenic acid (Figure 1), its acetoxy- and hydroxy-derivatives, and valerenone, are significant contributors to the actions of valerian (2, 8, 9). Valerenic acid has been shown to be active physiologically (8). The practical part of the project focuses on the valerenic acid content of commercial valerian products. The Project This experiment has formed part of our practical curriculum for the past three years as a three-hour practical at fourth-year undergraduate level. It emphasizes the need for a selective extraction step on certain herbal medicinal products before analysis on the constituent(s) of interest can commence. The experiment has also proven to be an invaluable exercise in gaining knowledge about herbal medicines and how they can be used as learning aids for carrying out spectroscopic and chromatographic techniques.
Figure 1. Structure of valerenic acid.
www.JCE.DivCHED.org
•
This experiment provides an opportunity for undergraduate students of pharmacy, medicine, herbal medicine, pharmaceutical chemistry, medicinal chemistry, and organic chemistry to improve their knowledge and skills pertaining to the analysis of conventional and herbal medicines. It also addresses the scarcity of published lab experiments that describe the use of GC–MS as an analytical tool for the analysis of herbal medicinal products (10). In this experiment, several valerian-containing products sold in pharmacies are evaluated to verify that they contain Valeriana officinalis by identifying the presence of valerenic acid, which is only found in this species of Valeriana (9). Valerenic acid is a key constituent partly responsible for the herb’s observed activity. Since this sesquiterpene acid is stable and robust, it is amenable to identification and quantitation by GC–MS. In this project, the student will use a validated method for the selective extraction of acidic constituents from the neutral and basic compounds. The second part of the experiment focuses on the method of analysis of the extracts. Chromatographic as well as spectroscopic techniques are utilized. The use of retention time to tentatively identify the presence of valerenic acid in the assigned product is also demonstrated. In addition, the student gains experience of interpreting mass spectra. Results and Discussion This laboratory was divided into two sections: (i) preparation of samples and standards for analysis and (ii) identification and quantitation of valerenic acid in valeriancontaining herbal medicines by GC–MS analysis. The standard solution was prepared by dissolving valerenic acid1 (1 mg) and the internal standard, 4-phenylbutyric acid (1 mg), in 1 mL of methanol. The assay samples were prepared by accurately weighing approximately 0.5 g of the powdered formulation and transferring it to a methanol solution (1 mL), containing 1 mg of the internal standard. The standard and assay preparations were subjected to the same extraction procedure, which involved the addition of aqueous NaOH (6 mL, 0.1 M) to each preparation, vigorously shaking the resulting mixtures for 10 minutes, followed by a centrifugation step to deposit the insoluble material onto the bottom of the tubes. The supernatant (4 mL from each preparation) was transferred to fresh tubes, and water (3 mL) was added to each. The solutions were acidified using concentrated HCl (0.4 mL), and the acidic compounds of interest were extracted from each preparation using ethyl acetate
Vol. 84 No. 5 May 2007
•
Journal of Chemical Education
829
In the Laboratory Table 1. Quantity of Valerenic Acid in Commercial Products Product
Valerenic Acid Mass/mg
Percent in Product
Peaceful Night
0.061
0.012
Good 'n' Natural
0.184
0.037
Nature’s Way
0.185
0.037
Solgar
0.234
0.047
Valerina Day-time
0.429
0.086
(6 mL). After evaporation of the solvent, the resulting residue from each preparation was prepared for GC–MS analysis by dissolving it in methanol (0.5 mL) and transferring 50 µL of each solution to separate microcentrifuge tubes that contained 50 µL of the methylating reagent, MethElute (0.2 M trimethylanilinium hydroxide in methanol solution, TMAH).2,3 The Saturn GC–MS instrument was equipped with an RTX 35 column (30 m × 0.25 mm i.d.) Helium was used as the carrier gas at a flow rate of 1 mL兾min. The injector temperature and the GC–MS transfer line were maintained at 260 ⬚C. The column temperature was initially held at 100 ⬚C for 6 minutes, and then increased at a rate of 15 ⬚C per minute for the next 13.33 minutes, before maintaining a temperature of 300 ⬚C for a further 10 minutes. The standard and assay solution, 2 µL of each, were separately injected into the instrument. In the standard preparation, the respective retention times for the internal standard and valerenic acid were 11.03 and 15.21 minutes. The peak corresponding to valerenic acid was identified on the basis of its molecular mass. The m兾z values and relative intensities (RI) of all peaks in the mass spectrum of methylated valerenic acid in the standard preparation are EI–GC–MS m兾z (RI): 248 (100), 233 (10), 216 (45), 201 (22), 189 (30), 173 (18), 161 (33), 145 (24), 133 (30), 119 (22), 105 (35), 91 (15), 77 (9), 67 (9), 53 (10), 41 (8).
The important features of the mass spectrum of methyl valerenate include the molecular ion peak, (M)+ at m兾z 248, as well as fragment ions corresponding to the loss of CH3 (m兾z 233), CH3OH (m兾z 216), and COOCH3 (m兾z 189). The mass spectrum of the corresponding peak in the gas chromatogram of the assay preparation showed identical ions to those of the methylated valerenic acid in the standard preparation, indicating that this peak was solely due to valerenic acid. GC–MS analysis is a common means of verifying the presence of a compound of interest. Within a complex mixture, if there is any doubt that the chromatographic peak is attributable to more than one component, then it is necessary to investigate the purity of that sample, by using GC–MS.
830
Journal of Chemical Education
•
The respective peak areas of valerenic acid and internal standard were recorded and the peak area ratios of valerenic acid to internal standard were calculated for both the standard and assay preparations. The content of valerenic acid (mg) in each commercial valerian product, ma, was calculated in accordance with the following formula ma = ms
Pa Ps
where ms is the mass of valerenic acid in the prepared standard, Pa is the peak area ratio for the assayed product, and Ps is the peak area ratio for the standard preparation. The percentage of valerenic acid in each product was then calculated. In Table 1, the mass and percentage content of valerenic acid in five readily available valerian-containing products sold in pharmacies is detailed. Hazards The experiment involves the use of some potentially hazardous reagents. Methanol, ethyl acetate, and TMAH are toxic if inhaled, swallowed, or absorbed through skin. Prevent contact with skin and clothes; avoid inhaling vapor; use in a fume hood. 4-Phenylbutyric acid (internal standard) is harmful by inhalation, contact with skin, and if swallowed. Use in a fume hood. Aqueous NaOH and hydrochloric acid are corrosive and irritant to the respiratory system. Avoid contact with the skin and eyes and use in a fume hood. Conclusion The use of GC–MS allowed for the unambiguous identification and quantification of valerenic acid in the valerian products analyzed. An average content of 0.044% valerenic acid was calculated. The content of valerenic acid was found to vary considerably in the products analyzed, emphasizing to the student the importance of standardizing herbal medicinal products. W
Supplemental Material
Instructions for the students, notes for the instructor, ion chromatograms and a mass spectrum are available in this issue of JCE Online. Notes 1. Valerenic acid is obtainable from Extrasynthese, Z. I. Lyon Nord, B.P. 62, 69726 Genay Cedex, France. 2. TMAH is obtainable from Sigma-Aldrich Ireland Ltd. Airton Road, Tallaght Dublin 24. 3. The methyl ester derivatives of these compounds are more suitable to analysis by GC–MS as they produce sharp, discrete peaks in the chromatogram.
Vol. 84 No. 5 May 2007
•
www.JCE.DivCHED.org
In the Laboratory
Literature Cited 1. Barnes, J.; Anderson, L. A.; Phillipson, J. D. Herbal Medicines: A guide for Health-Care Professionals; 2nd ed.; Pharmaceutical Press: London, 1996. 2. Ernst, E. Herbal Medicine: A Concise Overview for Professionals; Butterworth-Heinemann: Oxford, 2000; pp 12–50. Ziegler, G.; Ploch, M.; Miettinen-Baumann, A.; Collet, W. Eur. J. Med. Res. 2002, 11, 480–486. Schulz, H.; Stolz, C.; Muller, J. Pharmacopsychiatry 1994, 27, 147–151. 3. Ziegler, G.; Ploch, M.; Miettinen-Baumann, A.; Collet, W. Eur. J. Med. Res. 2002, 11, 480-486. 4. Schulz, H.; Stolz, C.; Muller, J. Pharmacopsychiatry 1994, 27, 147–151.
www.JCE.DivCHED.org
•
5. Kamm-Kohl, A. V.; Jansen, W.; Brockmann, P. Medwelt 1984, 35, 1450–1454. Vorbach, E. U.; Gortelmeyer, R.; Bruning, J. Psychopharmakotherapie 1996, 3, 109–115. 6. Vorbach, E. U.; Gortelmeyer, R.; Bruning, J. Psychopharmakotherapie 1996, 3, 109–115. 7. Balderer, G.; Borbély, A. A. Psychopharmacology 1985, 87, 406–409. 8. Dewick, P. M. Medicinal Natural Products A Biosynthetic Approach, 1st ed.; John Wiley & Sons, Ltd.: West Sussex, United Kingdom, 1997; pp 152–175. 9. Houghton, P. J. J. Pharm. Pharmacol. 1999, 51, 505–512. 10. Sobel, R. M.; Ballantine, D. S.; Ryzhov, V. J. Chem. Educ. 2005, 82, 601–603.
Vol. 84 No. 5 May 2007
•
Journal of Chemical Education
831
