Quality Management of Nutraceuticals - American Chemical Society

Chapter 14

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 12, 2014 | http://pubs.acs.org Publication Date: December 17, 2001 | doi: 10.1021/bk-2002-0803.ch014

A Quantitative HPLC Method for the Quality Assurance of Goldenseal Products in the U.S. Market 1

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Mingfu Wang , Nanqun Zhu , Yi Jin , Nathan Belkowitz , and Chi-Tang Ho 2

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Research and Development Department, Quality Botanical Ingredients Inc., 500 Metuchen Road, South Plainfield, NJ 07080 Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901-8520

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A rapid and quantitative method of quality assurance for the marker phytochemicals and bioactive components in products containing materials derived from Goldenseal has been developed. B y using a Luna phenyl-hexyl column with a mixture of sodium lauryl sulfate-phosphoric acid-water— isopropanol-acetonitrile as mobile phase, the two marker compounds, hydrastine and berberine were well separated at ambient temperature in 20 minutes. The alkaloid contents of six commercial available Goldenseal root and Goldenseal/ Echinacea supplements were also analyzed.

Goldenseal (Hydrastis canadensis L.) is an herbaceous perennial with a short horizontal rhizome bearing multiple roots, two or three palmatilobate leaves and a unique terminal, greenish-white flower. Over the years, it has also been referred to by a large number of other names, including yellow root, ground raspberry, eye-balm, yellow paint, wild turmeric and yellow eye. The © 2002 American Chemical Society

In Quality Management of Nutraceuticals; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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200 plant grows wild in the eastern part of North America, but it is mostly cultivated now. Goldenseal was used extensively by Native Americans (the Cherokee people) as an herbal medication and clothing dye. Its root has been listed in the U S Pharmacopoeia from 1803 to 1926. It is also listed in the 10 edition of the French pharmacopoeia which specify that the part to be used "consists of the dried rhizome and roots and that they must contain not less than 2.5% hydrastine calculated relative to the dried drug (1). However the aerial part is also sold in the U S A nutraceutical market in different formula. Goldenseal is now a top selling herb in North America. Its medicinal uses centered on its ability to soothe the mucous membranes of the respiratory, digestive, and genitourinary tracts in inflammatory conditions induced by allergy or infection. It can be found in many formulations, including treatment of nasal congestion, mouth sores, eye and ear infections, and as a topical antiseptic. It is commonly believed to help boost the immune system, increase the efficacy of other medicinal herbs, and for this purpose, it is usually formulated together with Echinacea. The isoquinoline alkaloids are the major components in goldenseal root, including hydrastine (1.5-4%) berberine (0.5-6%), canadine, candaline and other minor alkaloids (2-4). Goldenseal also contains fatty acids, resin, polyphenols acids, chlorogenic acid, meconin and a small amount of volatile oil. Modern pharmacological research has found that the alcoholic extract of Hydrastis canadensis L . root is well known for its vasoconstrictive effects ( i , J). And a number of immunomodulatory effects have been attributed to goldenseal (6) and the alcoholic extract of goldenseal root also showed reversible smooth muscle relaxant activity (7,8). One of the major alkaloids, berberine has a long history of medicinal use in both Ayuvedic and Chinese medicine. It has demonstrated significant anti-microbial activity against various organisms including bacteria, viruses, fungi, protozoans, helminths and chlamydia (1,9). Currently the clinical uses of berberine include bacterial diarrhea, intestinal parasite, and ocular trachoma infection. In addition to similar action, hydrastine is vasoconstrictive, stimulates bile secretion and involuntary muscles in human and display sedative and hypotensive effects (5). Due to the activity of berberine and hydrastine, the quantities of berberine and hydrastine are therefore used as a quality control measure in standardized herbal and nutraceutical preparation. Although the analysis of berberine in other botanicals have been studies extensively (10-14). Surprisingly, very few groups have ever reported on the quantity of berberine and hydrastine found in herbal raw materials or commercially available dietary supplements containing goldenseal. Leone et al. (75) recently reported an H P L C determination of the major alkaloids extracted from Hydrastis canadensis L , but this method is not validated, the peak


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separation and shape are not good. Caslavska and Thormann reported in 1998 the analysis of isoquinoline alkaloids in goldenseal by capillary electrophoresis coupled with mass spectrometry (16). In this research a new, simple and validated ion-pair high-performance chromatography method for simultaneous determination of berberine and hydrastine (structures shown in Scheme 1) in goldenseal herbs, goldenseal roots and dietary supplements containing goldenseal root extract was developed.

Hydrastine

Berberine

Scheme 1. Structures of hydrastine and berberine

Materials and Methods

Materials Hyrastine hydrochloride and berberine hydrochloride and sodium lauryl sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). Phosphoric acid 85%, H P L C grade acetonitrile, water and isopropanol were obtained from Aldrich Chemical Co. (Milwaukee, WI). Goldenseal roots and goldenseal herbs (aerial part) were obtained from commercial suppliers in the North American market. Several goldenseal roots, goldenseal/Echinacea dietary supplements were purchased from local retailers in New Jersey.


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Instrumentation and Chromatographic Conditions Analytical H P L C analyses were performed on a Hewlett-Packard 1100 modular system equipped with an autosampler, a quaternary pump system, a photodiode array detector and a HP Chemstation data system. A prepacked 150x4.6 mm (3 μ particle size, 00F-4256-E0) Luna phenyl-hexyl analytical column (Phenomenex, Torrance, C A ) were operated with a mobile phase consisting of acetonitrile, isopropanol, water and phosphoric acid (34:10:56:0.2, v/v/v/v). Sodium lauryl sulfate, an ion-pairing agent, was added to the mobile phase to achieve a final concentration of 5 m M . The mobile phase was delivered at a flow rate of 1.2 mL/minute at room temperature. The absorption spectra were recorded from 200 to 400 nm for all peaks; quantitation was carried out at a single wavelength of235 nm.

Standard Preparation M i x 250 m L methanol, 250 m L water and 10 m L concentrated HC1 to make a 50% methanol acidic extraction solvent. Accurately weigh about 15 mg hydrastine hydrochloride and 15 mg berberine hydrochloride into 25 m L volumetric flask, record the weight (correcting for water and HC1). A d d approximately 15 m L 50% methanol acidic solution and sonicate for 15 minutes. Allow the flask to cool to room temperature and fill to full volume with 50% methanol acidic solution. Measure 5 m L above solution and transfer to a 25 m L volumetric flask and diluted to the full volume using 50% methanol acidic solution (standard solution).

Preparation of Samples For herbs, accurately weigh about 400 mg goldenseal (aerial part), for goldenseal root, accurately weigh 100 mg, for herbal extract (10% total alkaloids), accurately weigh about 50 mg into 50 m L volumetric flask. For goldenseal dietary supplements, weigh 20 capsules or tablets and record the average value for each capsule or tablet, then those capsules and tablets were pulverized and 100 mg of these samples were added into a 50 m L volumetric flask. Add appropriately 35 m L of 50% methanol acidic solution and sonicate for 25 min and shake for 15 min. Allow the flask to cool to room temperature and fill to volume with 50% methanol acidic solution. Using a disposable syringe and 0.45 μπι filter, filter the sample into an H P L C vial.


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Results and Discussion A Luna phenyl-hexyl column (150x4.6mm, 3 μ particlei size) was selected for the ion-pairing H P L C . Various mobile phase systems 'were evaluated to achieve a satifactory seperation of berberine and hydratine. A mixture of sodium lauryl sulfate, acetonitrile, isopropanol, water and phosphoric acid (85%) (0.5:34:10:56:0.2, M/v/v/v/v) was found to be the best. Chromatograms for the goldenseal herb, goldenseal root and goldenseal with Echinacea were showed in Figures 1, 2 and 3, respectively. N o interfering peaks were noted for Hydrastis canadensis L and good resolution was achieved among all the alkaloids. The retention time for hydrastine and berberine were approximately 8.7 and 16.7 minutes, respectively.

System Suitability System suitability was checked by performing 10 replicate analyses of one goldenseal sample within the same working day and then check the percent relative standard deviation (%RSD) of the retention time and peak area for both peaks. The % R S D of the retention time for hydrastine and berberine were 0.67% and 0.88%, respectively. The % RSD for the peak area of hydrastine and berberine were 0.32% and 0.30%. It indicated this method is suitable for the analysis of goldenseal.

Precision The precision of the extraction procedure was validated using one goldenseal herb sample. Six samples, weighing about 400 mg, were extracted as described above. A n aliquot of each sample was then injected and quantitated. The average amount of hydrastine was 0.286% with an R S D of 2.10%. The average amount of berberine was 0.366% with an R S D of 1.48%. Suggesting the method has excellent precision.

Percent Recovery The recovery was determined by removing 25 m L of extraction solvent from all 6 extractions done in precision test and replacing it with 25 m L of fresh extraction solvent. These samples were then re-extracted and reanalyzed for hydrastine and berberine. A value of exactly one half (1/2) the value of the original extraction validates 100% recovery. The average amount of hydrastine recovered from the 6 extracts was 0.286% with an RSD of 2.10%. The average amount of hydrastine recovered from the re-extraction of these samples χ 2,



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Figure 1. Representative HPLC chromatogram of goldenseal herb (aerial part). Peak at 0.656 min: hydrastine; at 16.680 min: berberine.

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Figure 2. Representative HPLC chromatogram of goldenseal root. Peak at 0.688 min: hydrastine; at 16.673 min: berberine

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Figure 3. Representative HPLC chromatogram of Goldenseal/Echinacea. Peak at 0.665 min: hydrastine; at 16.687 min: berberine



207 was 0.278% with an R S D of 0.79%. Assuming the average amount extracted in the re-extraction is 100%, the average amount recovered in the original extraction is then validated to be 102.9%. The average amount of berberine recovered from the 6 extracts was 0.366% with an R S D of 1.48%. The average amount of berberine recovered from the re-extraction of the samples χ 2, was 0.372% with an R S D of 1.02%. Assuming the average amount extracted in the re-extraction is 100%, the average amount recovered in the original extraction is then validated to be 98.4%. This proves the excellence of recovery for this method.

Linear Dynamic Range The linear dynamic range for hydrastine was determined by preparing a standard curve consisting of 9 standard solutions covering a range from 0.0011.0 mg/mL. The linear dynamic range for berberine was determined by preparing a standard curve consisting of 9 standard solutions covering a range from 0.0006-0.8 mg/mL. The linearity range for hydrastine was determined as 0.002-1 mg/mL with correlation coefficient 0.9996. The linearity range for berberine was determined as 0.002-0.8 mg/mL with correlation coefficient 0.9992 (the calibration curves for hydrastine and berberine are showed in Figures 4 and 5, respectively.). Therefore for hydrastine a result greater than 1.0 mg/mL is considered to be outside the linear range of the assay. The sample must be diluted and reassayed. A result less than 0.002 mg/mL must be reported as negative. For berberine, a result greater than 0.8 mg/mL is considered to be outside the linear range of the assay. The sample must be diluted and reassayed. A result less than 0.002 mg/mL must be reported as negative.

Ruggedness The ruggedness of the assay was demonstrated by comparing the results obtained from two different chemists. A l l extractions and standard preparations were done according to the method described above. Two chemists got similar results for one goldenseal sample (one is 5.15% total alkaloids, another is 5.10% total alkaloids).

The Content of Alkaloids in Goldenseal Herbs and Goldenseal Roots Over twenty goldenseal root and herb samples were tested. Hydrastine and berberine were found to exist in both herbs and roots. But the content were quite different, in herb (aerial part) samples, hydrastine ranged in content from 0.27-0.29%, berberine ranged in content from 0.36-0.39%. While in goldenseal



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Figure 5. Calibration curve for berberine. root, hydrastine ranged in the content from 2.25-3.32% and berberine ranged from 2.61% to 3.75%. In the market, goldenseal is also sold as 5% or 10% alkaloid extracts. Most of the extracts we tested met the claim, but half of the extract contained only berberine, hydrastine were either not observed or the content was very low. They are suspected not to be the right species, because goldenseal usually contains almost equal amounts of berberine and hydrastine. On the other hand, berberine exists in quite a number of herbs such as Coptis Chinese, Berberis aquifolium, Berberis vulgaris, Berberiis aristata, etc. and these herbs usually contain either no or very low content of hydrastine. Representative chromatograms of the wrong species were showed in Figures 6, 7 and 8.



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Figure 7. Representative HPLC chromatogram of a wrong species sample B.



Figure 8. Representative HPLC chromatogram of a wrong species sample C.

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Evaluation of Commercial Sample (Finished Products) Although goldenseal has been considered as safe alternatives to conventional drugs for many years, its dosage has not been set. So it is not surprised to notice that the contents of alkaloids are different from products to prodcuts covering a range of 1.25 mg to 57 mg total alkaloid per serve. In the US market, goldenseal is usually formulated together with Echinacea for immune system boosting. We tested the alkaloids contents of three commerical Goldenseal/Echinacea products, no chromatographic interference was noted for Echinacea (Echinacea angustifolia and Echinacea purperea) or other ingredients. Two of them matched the label claim for total alkaloids (but one products only contained berberine and hydrastine was not detected), one failed with low alkaloid content. Two goldenseal root products were also tested for the total alkaloids, one was found to contain 55.5 mg alkaloids (calculated as hydrastine and berberine, label claim is 57 mg per serve), the other was found to contain 27.1 mg alkaloids (calculated as hydrastine and berberine, while the label only claimed 10 mg hydrastine/per serve).

Conclusion A rapid and quantitative method of quality assurance of hydrastine and berberine contents in products containing materials derived from Goldenseal has been developed. By using a Luna phenyl-hexyl column with a mixture of sodium lauryl sulfate-phosphoric acid-water-isopropanol-acetonitrile as mobile phase, this method showed excellent linearity, accuracy and precision. The alkaloid contents of goldenseal roots, goldenseal root extracts, commercial available goldenseal roots and goldenseal/echinacea supplements were also analyzed. For the goldenseal root extracts (5% and 10% alkaloids), quite a lot of samples only contained berberine or the concentrations of hydrastine were very low. These samples may not be the goldenseal.

References 1. 2.

3. 4.

Bruneton, J. Technique & Documentation-Lavoisier 1995, 740-741. Gentry, E . ; Jampani, H.B.; Keshavarz-Shokri, A ; Morton, M . D . ; Velde, D.; Telilepalli, H . ; Mitscher, L . A . ; Shawar, R.; Humble, D.; Baker, W. Journal of Natural Products 1998, 61, 1187-1193. Gleye, J.; Stanishlas, E . Plantes Medicinaleset Phytotherapie 1972, 6, 306-310. Gleye, J.; Ahond, Α.; Stanislas, E . Phytochemistry 1974, 13, 675-676.


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5. 6.

7.


8. 9. 10. 11. 12. 13. 14.

Shideman, F.E. Bull. National Formulary Comm. 1950, 18, 3-9. Rehman, J.; Dillow, J . M . ; Carter S . M . ; Chou, J.; Le, Β.; Maisel A . S . Echinacea angustifolia and Hydrastis canadensis, Immunology Letters, 1999, 68, 391-395. Cometa, M . F . ; Galeffi, C.; Palmery, M. Phytotherapy Research, 1996, 10: Supplement1,S56-S58. Cometa, M . F . ; Abdel-Haq, H.; Palmery, M. Phytotherapy Research 1998, 12: Supplment 1, S83-S85. Anonymous, Alternative Medicine Review 2000, 5, 175-177. Misaki, T.; Sagara, K.; Ojima, M.; Kakizawa, S.; Oshima, T.; Yoshizawa, H . Chem. Pharm. Bull. 1982, 30, 354-357. L i n , S.-J.; Tseng, H.-H.; Wen, K . - C . ; Suen, T.-T. J. Chromatography 1990, 730, 17-23. Liu, Y.-M.; Sheu, S.-J. J. Chromatography 1992, 623, 196-199. Luo, G . ; Wang, Y.; Zhou, G . ; Y u , Y. Journal of Liquid Chromatography 1991, 13, 3825-3832. Lee, H.S.; Eom, Y . E . ; Eom, D.O. Journal of Pharmaceutical & Biomedical Analysis 1999, 21, 59-63.

15. Leone, M . G . ; Cometa, M . F . ; Palmery, M.; Saso, L . Phytotherapy Research 1996, 10, S45-S46. 16. Sturm, S.; Stuppner, H . Electrophoresis 1998, 19, 3026-3032.


Quality Management of Nutraceuticals - American Chemical Society

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