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Authenticating Saw Palmetto extract: a new approach: this communication will demonstrate a new approach for the effortless and efficient authentication and quality control of commercial Saw Palmetto extracts.

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Saw Palmetto berry (Serenoa repens [W. Bartram] Small [Arecaceae]; Syn: Sabal serrulata, [Michx.] Nutt. ex Schult. & Schult. f.; Serenoa serrulata [Michx.] G. Nichols) is native to the Southeastern US and was used by local Native Americans as a diuretic and sexual tonic, as well as for stomach ache and dysentery. (1) Today, Saw Palmetto and, especially, Saw Palmetto Extract (SPE) are the most popular and widely used phytotherapeutic agents for the treatment of symptoms related to benign prostatic hyperplasia (BPH). (2) Consequently, in 2002, Saw Palmetto-based supplements were the fifth bestselling herbal dietary products in US mainstream outlets.1 From 2003 to 2007, the aggregated (wild and cultivated) annual harvest of dried Saw Palmetto berry grew from 715 to 2625 tons. (3)

There is a large amount of evidence and clinical trials that confirm its efficacy. (4-8) It is suggested that SPE functions as a mild inhibitor of 5[alpha]-reductase (5[alpha]-reductase converts testosterone into dihydrotestosterone, which is in turn linked to the development of BPH). (9) And, it has been shown that the anti-BPH activity of SPE is comparable with the commonly used 5[alpha]-reductase inhibitor, Finasteride (Proscar), although it is still uncertain whether the inhibition of 5[alpha]-reductase is the sole mechanism of action of SPE. (10) The main components of lipophilic SPE are fatty acids (70-95%), phytosterols (0.2-0.5%) and long-chain alcohols (0.15-0.35%). (11) However, it is still undecided which components or group(s) of components are responsible for the anti-BPH activity.

Although SPE is sold as a medicine in Europe, it is considered to be a dietary supplement in the United States and, for this reason, is less strictly regulated. Regrettably, both its wide popularity and weak regulation are encouraging the rising market presence of low quality, adulterated SPE products. It has been reported, for instance, that in two out of six tested commercially available samples of SPE, the level of total fatty acids was found to be less than 20%, which clearly indicates product adulteration. (12) Based on these data and our past experience, we believe that this is just the tip of the iceberg; therefore, it is important for the nutraceutical industry (especially for the manufacturers of finished supplement forms) to be armed with a routine test method to confirm the authenticity of SPE. In general, the most common method for plant oil adulteration is its dilution with less expensive plant oils. If carefully formulated, it is possible to keep the fatty acid profile within specifications; thus, the test for total fatty acids (TFA) may not show adulteration at all. (Here, TFA means the sum of all free fatty aids [FFA] and all bound [esterified] fatty acids [BFA]; TFA=FFA+BFA.)

Fortunately, unlike the majority of plant oils, the extract--derived from ripe Saw Palmetto berries--has a very high content of free fatty acids (FFA) and, correspondingly, a rather low content of bound fatty acids (BFA) in the form of glycerides, along with an insignificant amount of ethyl esters. As the usual acidity of SPE is very high compared with common plant oils, we had recommended using the Acid Value (AV) as a simple criterion for the preliminary quality evaluation of ripe Saw Palmetto Extract. (13) By contrast, the FFA concentration in the ripe Saw Palmetto berry depends on a specific lipase, the activity of which changes during the ripening period, so the acidity of the extract is not a constant value. (14) This means that the AV test alone cannot fully support the TFA test in the SPE authentication task. Interestingly, no regulatory documents worldwide specify the acidity or FFA content in SPE, whereas TFA content was traditionally an important quality control parameter for both Saw Palmetto berry and its extract. This is reflected in some compendial monographs. (11,15)

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Alternatively, it has been suggested that in addition to TFA, FFA content could be of practical use for SPE quality evaluation. (16,17) The authors suggested analysing FFA in SPE by silylation followed by GC separation. Several other methods have also been applied for the analysis of FFA in Saw Palmetto Extract. They are as follows:

* A process that includes the preliminary separation of FFA from SPE by preparative LC followed by direct GC analysis (18)

* High Performance TLC (19)

* Supercritical Fluid Chromatography (20)

* Direct esterification of FFA using the Alltech methylation reagent (unfortunately, reported for the FFA profile only) (21)

* HPLC of fatty acids' UV-absorbing derivatives, such as phenacyl bromide esters. (22)

The common disadvantage of all the above mentioned methods is that they are not as convenient as the existing compendial TFA tests; they require prolonged analysis times and/or relatively expensive equipment (GC and LC columns, TLC plates, etc.) and reagents, as well as instrumentation other than GC. In this paper, we suggest a new approach to FFA determination in SPE. The basic concept is derived from the well-known fact that the derivatization of glycerides (or other esters) to Fatty Acid Methyl Esters (FAME) can be catalysed by either an acid or an alkali. By contrast, free acids can also be esterified and quantitatively converted into their corresponding FAME, but this reaction is catalysed by acids only. Thus, an acid-catalysed treatment of SPE converts both BFA and FFA into FAME, allowing the measurement of all individual fatty acids regardless of their initial form (free or bound); following alkali-catalysed transmethylation, only the bound fatty acids are converted into FAME. Therefore, running both reactions for a split sample of SPE allows for the calculation of the percentages of both individual ([FFA.sub.i(%)]) and total ([FFA.sub.t(%)]) free fatty acids (Formulas 1 and 2).

[FFA.sub.i(%)] = [FA.sub.i(%)] - [BFA.sub.i(%)] (1)

[FFA.sub.t(%)] = [summation] [FFA.sub.i(%)] (2)

Where [FA.sub.i(%)] is the percentage of the individual fatty acid in either form and [BFA.sub.i(%)] is the percentage of the same fatty acid in bound form only.

In other words, instead of direct FFA derivatization, we suggest using a much simpler selective BFA derivatization, in addition to the mandatory TFA test. As both tests are well developed, and can be done consecutively using the same GC column, the obvious advantages of the recommended approach compared with the methods discussed above are shorter testing times, no need for extra analytical equipment and/ or expensive chemicals and, accordingly, lower costs per analysis.

Experimental

All tests were done on three regular production lots of USPlus[R] Saw Palmetto Extract manufactured at Valensa International from the 2006 and 2007 Saw Palmetto berry crop. Dried ripe berries were extracted by Supercritical Fluid Extraction technology using pure carbon dioxide. The fatty acid methyl esters used as standards were purchased from Sigma-Aldrich Corporation (St Louis, MO, USA) and Fluka Chemical Corporation (Milwaukee, WI, USA). Chemicals and solvents were purchased from Fisher Scientific (Pittsburgh, PA, USA) and VWR International (West Chester, PA, USA), and the gas chromatographic column was purchased from Phenomenex (Torrance, CA, USA). Gas chromatography was done using an AutoSystem XL gas chromatograph manufactured by Perkin-Elmer (Shelton, CT, USA). Titrations were done on a Metrohm 798 MPT Titrino Autotitrator (Herisau, Switzerland).

Typical chromatographic conditions were as follows: capillary column with the following parameters; length = 30 m; ID = 0.32 mm; film thickness = 0.25 m; liquid phase = USP phase G16 (100% polyethylene glycol). Instrument setup: carrier gas = helium; carrier flow = 2 mL/ min; detector = FID; detector temperature = 300[degrees]C; injector temperature = 350[degrees]C; split rate = 1:60; injection volume = 0.5-1.0 .L. Temperature program: initial = 80[degrees]C; hold = 1 min; ramp 1 = 80-220[degrees]C at 20[degrees]/min; hold = 4 min; ramp 2 = 220-240[degrees]C at 20[degrees]/min; hold = 1 min. Total runtime = 14 min.

TFA Test (modified USP procedure (11)). Standard Solution for TFA ([SS.sub.TFA]) and Internal Standard Solution for TFA ([ISS.sub.TFA]) were prepared as directed in USP Saw Palmetto Extract monograph (United States Pharmacopeia, 2007). Test Solution for TFA ([TS.sub.TFA]): 100 mg of SPE was accurately weighed in a glass tube and mixed with 4 mL of a 5% solution of [H.sub.2]S[O.sub.4] in methanol, closed firmly and heated for 2 h at 100[degrees]C. After cooling to room temperature, 10 mL of saturated sodium chloride solution was added to the mixture followed by the addition of 5.0 mL of hexanes and 1.0 mL of an ISS, and vigorously shaken for 2 min. After separation of the layers, a portion of the upper layer was taken for injection. Equal volumes of [SS.sub.TFA] and [TS.sub.TFA] were separately injected. The percentage of each individual fatty acid in the SPE ([FA.sub.i(%)]) was calculated using Formula 3.

[FA.sub.i(%)] = 500 x C x [R.sub.U] x [M.sub.A] / [R.sub.S] x [M.sub.E] x W (3)

Where C is the concentration (mg/mL) of the respective methyl ester in the [SS.sub.TFA], W is the sample weight (mg), [R.sub.U] and [R.sub.S] are the ratios of the responses of the relevant methyl ester peak and the internal standard peak obtained from the chromatogram of the [TS.sub.TFA] and [SS.sub.TFA], respectively, and [M.sub.A] and [M.sub.E] are the molecular weights of the relevant fatty acid and its methyl ester, respectively.

Test for BFA (based on EU method (23)). Internal Standard Solution for BFA ([ISS.sub.BFA]): 5.0 mL of [ISS.sub.TFA] was diluted to 25.0 mL with hexanes. Standard Solution for BFA ([SS.sub.BFA]): 5.0 mL of [SS.sub.TFA] was diluted to 25.0 mL with hexanes. Test Solution for BFA ([TS.sub.BFA]): accurately weighed SPE (200 mg) was put in a 50 mL centrifuge tube and mixed with 5.0 mL of hexanes and 1.0 mL of [ISS.sub.BFA];1 mL of 2N potassium hydroxide solution in methanol was added, the solution was vortexed for 2 minutes and 10 mL of saturated sodium chloride solution was added, shaken and then centrifuged for 1 h at 12000 RCF. The upper layer was transferred into a glass tube, washed with 20 mL of water and allowed to separate. A portion of the upper layer was taken for injection. Equal volumes of [SS.sub.TFA] and [TS.sub.TFA] were separately injected. The percentage of each individual bound fatty acid in SPE ([BFA.sub.i(%)]) was calculated using Formula 4.

Where C is the concentration (mg/ mL) of the respective methyl ester in the [SS.sub.BFA], W is the sample weight (mg), [R.sub.U] and [R.sub.S] are the ratios of the responses of the relevant methyl ester peak and the internal standard peak obtained from the chromatogram of the [TS.sub.BFA] and [SS.sub.BFA], respectively, and [M.sub.A] and [M.sub.E] are the molecular weights of the relevant fatty acid and its methyl ester, respectively.

[BFA.sub.i(%)] = 500 x C x [R.sub.U] x [M.sub.A] / [R.sub.S] x [M.sub.E] x W (4)

Results and Discussion

Three tested lots were marked A to C and analysed. All tests were run in triplicate, and the relative standard deviation was less than 2%. Each individual free fatty acid ([FFA.sub.i(%)]) was calculated using Formula 1.

Measurement and calculation results are summarized in Table I. In addition, to confirm the accuracy of our measurements, we also measured the Acid Value (AV) of the same SPE samples. The AV of fats and fixed oils is defined as the number of milligrams of potassium hydroxide required to neutralize the free fatty acids in 1.0 g of the substance (United States Pharmacopeia, 2000). (24) Thus, the expected [AV.sub.calc] can be calculated based on the percentage of individual free fatty acids obtained by GC ([FFA.sub.i(%)]) and their molecular weights (MW) (Formulas 5-7).

[FFA.sub.i(mmol/g)] = 10[FFA.sub.i(%)] / MW (5)

[FFA.sub.t(mmol/g)] = [summation][FFA.sub.i(mmol/g)] (6)

[AV.sub.calc] = [FFA.sub.t.mmol/g)] x 56.11 (7)

Where [FFA.sub.i(mmol/g)] is the number of mmol of the individual free fatty acid in 1 g of SPE and [FFA.sub.i(mmol/g)] is the total number of mmol of all free fatty acids in 1 g of SPE.

A comparison of the calculated AV ([AV.sub.calc]) with the AV numbers obtained by titration ([AV.sub.titr]) is also shown in Table I. The calculated and measured AV results are rather close; the relative average deviation (RAD) is between 0.9% and 1.2%. The fact that the measured AV numbers were always slightly higher than the calculated AV numbers can be explained by the presence of traces of other acids that were not detected or identified by GC. As a consequence, we can state that any substantial mismatch of calculated and measured acid values is a sign of SPE adulteration. It was also interesting to compare the fatty acid profiles for free and total fatty acids. It emerged that the profiles for FFA and TFA in most cases are nearly identical, although negligible variations were sometimes detected (Table II). Although quite predictable, this fact indicates that lipase in Saw Palmetto berry acts non-selectively. This observation allows for the simple identification of adulterated saw palmetto extract by comparing the FFA and TFA profiles and paying attention to any profile discrepancies.

Conclusion

We have shown that the amount of free fatty acids in Saw Palmetto extract can be calculated as the difference between total fatty acid content and bound fatty acid content. The profiles for free fatty acids and total fatty acids are almost identical; therefore, non-authentic Saw Palmetto Extracts can be detected if these profiles are discrepant. It is suggested that any substantial mismatch of calculated and measured acid values can be a sign of SPE adulteration. The recommended approach is simple and inexpensive enough to be adopted by the majority of quality control laboratories in the nutraceutical industry.

Acknowledgments

We thank Valensa International for its support of the present work, Dr John Minatelli for his valuable comments and advice, as well as Vagan and Levon Mikaelian for editing the manuscript.

For more information

Gary Mikaelian and Magdalena Sojka

Valensa International

2751 Nutra Lane

Eustis, Florida 32726-6961, USA.

Tel. +1 352 357 2004

g.mikaelian@valensa.com

www.valensa.com.

References

(1.) M. Blumenthal, et al., "The ABC Clinical Guide to Herbs," American Botanical Council, Austin, Texas, USA (2003) p 409.

(2.) A.E. Gordon and A.F. Shaughnessy, "Saw Palmetto for Prostate Disorders," Am. Fam. Physician 67, 1281-1283 (2003).

(3.) American Herbal Products Association, "Tonnage Survey of Select North American Wild-Harvested Plants, 2004-2005," American Herbal Products Association, Silver Spring, Maryland, USA, (2007) p 7.

(4.) P. Boyle, et al., "Meta-Analysis of Clinical Trials of Permixon in the Treatment of Symptomatic Benign Prostatic Hyperplasia," Urology 55, 533-539 (2000).

(5.) F.C. Lowe and J.C. Ku, "Phytotherapy in Treatment of Benign Prostatic Hyperplasia: A Critical Review," Urology 48, 12-20 (1996).

(6.) M. O'Hara, et al., "A Review of 12 Commonly Used Medicinal Herbs," Arch. Fam. Med. 7, 523-536 (1998).

(7.) Y.A. Pytel, et al., "Long-term Clinical and Biologic Effects of the Lipidosterolic Extract of Serenoa repens in Patients with Symptomatic Benign Prostatic Hyperplasia," Adv. Ther. 19, 297-306 (2002).

(8.) T.J. Wilt, et al., "Saw Palmetto Extracts for Treatment of Benign Prostatic Hyperplasia: A Systematic Review," JAMA 280, 1604-1609 (1998).

(9.) L.S. Marks, et al., "Tissue Effects of Saw Palmetto and Finasteride: Use of Biopsy Cores for in situ Quantification of Prostatic Androgens," Urology 57, 999-1005 (2001).

(10.) J-C. Carraro, et al., "Comparison of Phytotherapy (Permixon) with Finasteride in the Treatment of Benign Prostate Hyperplasia: A Randomized International Study of 1098 Patients," Prostate 29, 231-240 (1996).

(11.) United States Pharmacopoeia 30, 982-983 (2007).

(12.) S.J. Currier, P.D. Johnston and K.J. Gorelick, "Herbal Medicines," Sci. Med. 7, 40-43 (2000).

(13.) G. Mikaelian, et al., "Preliminary Quality Examination of Saw Palmetto Extract," Nutra. Bus. Technol. 2, 64-65 (2006).

(14.) E. Neuzil and H. Cousse, "Le Palmier Scie Serenoa repens. Aspects Botaniques et Chimiques," Bull. Soc. Pharm. Bordeaux 132, 121-141 (1993).

(15.) European Pharmacopoeia 5, 2398-2400 (2004).

(16.) S.L. De Swaef and A.J. Vlietinck, "Simultaneous Quantitation of Lauric Acid and Ethyl Laureate in Sabal serrulata by Capillary Gas Chromatography and Derivatization with Trimethyl Sulphoniumhydroxide," J. Chromatography A 719, 479-482 (1996).

(17.) M. Ganzera, E.N. Croom, Jr and I.A. Khan, "Determination of the Fatty Acid Content of Pumpkin Seed, Pygeum and Saw Palmetto," J. Medicinal Food 2, 21-27 (1999).

(18.) F.K. Habib and M.G. Wyllie, "Not All Brands are Created Equal: A Comparison of Selected Components of Different Brands of Serenoa repens Extract," Prostate Cancer Prostatic Dis. 7, 195-200 (2004).

(19.) T. Halkina and J. Sherma, "Determination of Sterols and Fatty Acids in Prostate Health Dietary Supplements by Silica Gel High Performance Thin Layer Chromatography with Visible Mode Densitometry," J. Liq. Chromatogr. Rel. Technol. 30, 2329-2335 (2007).

(20.) S.I. De Swaef, W. Kleibohmer and A.J. Vlietinck, "Supercritical Fluid Chromatography of Free Fatty Acids and Ethyl Esters in Ethanolic Extracts of Sabal serrulata," Phytochem. Anal. 7, 223-227 (1996).

(21.) S.E. Adams and R. Bartram, "Gas Chromatographic Analysis of Free Fatty Acids in Saw Palmetto," in The Application Notebook (Alltech Associates, Inc., Deerfield, Illinois USA, 2003). p 49.

(22.) A. Mehta, A.M. Oeser and M.G. Carlson, "Rapid Quantitation of Free Fatty Acids in Human Plasma by High-Performance Liquid Chromatography," J. Chromatography B 719, 9-23 (1998).

(23.) European Commission Regulation (EEC) 2568/91, Annex XB, Method A, 64-65 (1991).

(24.) United States Pharmacopoeia 24, 1869 (2000). Ingredients for Women's Health
Table I: Measurement and calculation results for FA, BFA and FFA,
and comparison of the calculated and measured acid values of SPE

        Lot#                         A                     B

     Fatty Acid              FA     BFA    FFA     FA     BFA    FFA

Caproic (%)                  2.0    0.3    1.7     1.9    0.2    1.7
Caprylic (%)                 2.4    0.4    2-0     2.3    0.3    2.0
Capric (%)                   2.9    0.5    2.4     2.8    0.4    2.5
Lauric (%)                  28.0    5.0   23.0    27.9    3.7   24.2
Myristic (%)                10.6    1.9    8.7    10.5    1.4    9.0
Palmitic (%0                 8.1    1.5    6.6     8.1    1.1    7.0
Palm itoleic (%)             0.1    0.0    0.1     0.1    0.0    0.1
Stearic (%)                  1.8    0.4    1.4     1.9    0.3    1.6
Oleic (%)                   30.9    6.0   24.9    31.2    4.4   26.8
Linoleic (%)                 4.2    1.0    3.2     4.5    0.7    3.8
Linolenic (%)                0.6    0.1    0.5     0.6    0.1    0.5
Total (%)                   91.6   17.1   74.5    91.7   12.6   79.1
[FFA.sub.t] (mmol/g) *                    3,278                 3,468
[AV.sub.calc] (mgKOH/g) **                1,839                 194.6
[AV.sub.titr] (mgKOH/g)                  187.8                 198.0
RAD (%)                                   1.01                   0.9

Lot#                                   C

Fatty Acid                    FA      BFA     FFA

Caproic (%)                   2.0     0.3     1.7
Caprylic (%)                  2.4     0.3     2-1
Capric (%)                    2.7     0.4     2.3
Lauric (%)                   26.8     4.0    22.8
Myristic (%)                 10.2     1.5     8.7
Palmitic (%0                  8.1     1.3     6.8
Palm itoleic (%)              0.1     0.0     0.1
Stearic (%)                   1.8     0.3     1.5
Oleic (%)                    31.3     5.1    26.2
Linoleic (%)                  4.2             3.3
Linolenic (%)                 0.6     0.1     0.5
Total (%)                    90.2    14.2    76.0
[FFA.sub.t] (mmol/g) *                       3.330
[AV.sub.calc] (mgKOH/g) **                   186.8
[AV.sub.[titr] (mgKOH/g)                     191.2
RAD (%)                                       1.2

Table II: Comparison of total (free + bound) fatty acid (FA)
and free fatty acid (FFA) profiles of Saw Palmetto Extracts.

     Lot#                   A               B               C

  Fatty Acid       FA      FFA     FA      FFA     FA      FFA

Caproic (%)        2.2     2.3     2.1     2.1     2.2     2.2
Caprylic (%)       2.6     2.7     2.5     2.5     2.7     2.8
Capric (%)         3.2     3.2     3.1     3.2     3.0     3.0
Lauric (%)        30.6    30.9    30.4    30.6    29.7    30.0
Myristic (%)      11.6    11.7    11.3    11.4    11.3    11.4
Palmilic (%)       8.8     8.9     8.8     8.8     9.0     8.9
Palmitoleic (%)    0.1     0.1     0.1     0.1     0.1     0.1
Stearic (%)        2.0     1.9     2.1     2.0     2.0     2.0
Oleic (%)         33.7    33.4    34.0    33.9    34.7    34.5
Linoleic (%)       4.6     4.3     4.9     4.8     4.7     4.3
Linolenic (%)      0.7     0.7     0.7     0.6     0.7     0.7
Total (%)         100.0   100.0   100.0   100.0   100.0   100.0
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Title Annotation:male health
Author:Mikaelian, Gary; Sojka, Magdalena
Publication:Nutraceutical Business & Technology
Geographic Code:1USA
Date:Mar 1, 2009
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