VARIATIONS IN SAW PALMETTO FORMULATIONS IN THE US MARKET
N. M. Enwerem1*, K. Kumar1, F. Ayorinde2, K. R. Scott1 and A.K. Wutoh1
1Department
of Pharmaceutical Sciences and 2Department of Chemistry, Howard University,
College of Pharmacy, 2300 4th Street, NW, Washington DC, 20059-0001
Results: A typical HPLC separation of fatty acids, Lauric acid, Linoleic acid, Myristic acid
and Linoleic acid is shown for acid standards (Fig 2) and in a representative sample
(Fig 4). The on-line UV spectra are shown in Figs 3 and 5 respectively.The retention
times for lauric acid, linoleic acid, myristic acid and oleic acid were 8.4,12.1,13.4,and
16.6 min, respectively. The four marker compounds (Lauric acid, Linoleic acid, Myristic
acid and Oleic acid) showed linearity over the range 62.5 – 1000 µM and the accuracy
and precision data are shown in tables 2 and 3.
242.9
Fig 2
1.00
15.00
AU
241.7
0.30
0.20
0.10
279.4
279.4
279.4
279.4
240.00
260.00
12.00 14.00
Minutes
340.00
360.00
380.00
242.9
Fig 5
Fig 4&5:
Chromatogram of
a represent-ative
sample & it’s online spectra
1.40
1.20
241.7
1.00
0.80
0.60
242.9
0.40
242.9
0.20
220.00
10.00
320.00
1.60
0.00
8.00
300.00
nm
1.80
279.4
279.4
279.4
279.4
0.00
6.00
280.00
AU
Myristic acid - 13.530
220.00
20.00
14.907
11.582
Linoleic acid - 12.175
9.516
0.50
10.602
1.00
Fig 2&3:
Chromatogram of
a mixture of
standards & their
on-line spectra
242.9
0.40
Oleic acid - 16.605
10.00
Minutes
Fig 4
6.993
7.855
Lauric acid - 8.394
AU
242.9
0.00
5.00
1.50
Experimental
Standards and Controls
8 mM of Lauric acid, Myristic acid, Linoleic acid and Oleic acid were prepared in heptane.
1.0 ml of each solution was transferred to a sample tube, vortexed and labeled as mixed working
standard.
1:1 serial dilution of this mixture were prepared in heptane to give calibration standards [1000,
500, 250,125 , 62.5, 31.25, 15.6 and 7.8 M] and quality control standards [62.5 – 1000 M].
The calibration and quality control standards, were derivatized with phenacyl bromide,
triethylamine and acetic acid, using modified method of Adosh et al [4] as shown in Fig. 1.
The derivatized solution was evaporated to dryness under an atmosphere of nitrogen using an
integrated Speed vac system for 30 minutes.
The residue was reconstituted in acetonitrile/water mixture [83/17] and analyzed by HPLC.
Sample: Saw palmetto commercial dietary supplement products were purchased from local
pharmacies, herbal and grocery shops in the District of Columbia and Maryland, USA. To protect
the manufacturers’ identity, the samples are labeled A to J.
Sample Preparation
Five dosage units of each product to be tested were triturated to form a uniform mix.
50 mg of the sample was weighed into a sample tube & extracted with 15 mL of heptane for 1 hr.
The mixture was centrifuged at 800 x g for 10 minutes and filtered.
1 ml of the resulting extract was transferred to a reaction vial, derivatized with phenacyl bromide
(100 L), triethylamine (100  L) and acetic acid (150 L) and analyzed by HPLC.
Chromatography: HPLC measurements were made using a Waters 2695 Alliance HPLC system,
equipped with a 996 photodiode array detector. Separation of the analytes was performed on a 5
m VYDAC, C18 column (250 cm x 4.6 mm id) at a flow rate of 1.0 ml/min using a gradient. The
eluents were monitored at 242 nm at ambient temperature. The column temperature was maintained
at 255º C.The chromatographic separation was accomplished by a gradient solvent system
(Table 1)
0.50
0.00
2.00
Fig 3
0.60
Oleic acid - 16.643
Lauric acid - 8.401
2.00
Aim: To determine the variability in a range of Saw palmetto products in the United States. To do
this, we identified and measured the concentration of four marker compounds [Lauric acid,
Linoleic acid, Myristic acid and Oleic acid] by HPLC with UV detection.
Introduction: The lipidosterolic extract of Saw Palmetto plant or Serenoa repens [Batr] has been
used for many years to treat symptomatic Benign Prostatic Hyperplasia [BPH] throughout Europe
[1,2]. It’s use in the United States is increasing rapidly [1]. Chemical investigation of Serenoa
repens revealed the presence of numerous free fatty acids, fatty acid esters and glycerides,
phytosterols, carbohydrates, and aromatic acids. The n-hexane (lipidosterolic) extracts have been
reported to contain 35,30,11 and 5 % of oleic, lauric, myristic and linoleic acids respectively as
free fatty acids, and less than 2 % of phytosterol, aliphatic alcohols and polypyrenic compounds
[2,3]. The lipidosterolic extract is commonly blended with excipients to form powders with or
without standardization. Saw palmetto preparations are standardized to 20 –25% fatty acids. It is
difficult to quantitate fatty acids directly with UV or fluorescence detection due to lack of
chromophores in the structure. HPLC methods of fatty acids after derivatization, have been
reported in the literature. Various derivatization techniques have also been investigated [4]. The
increasing demand for Saw palmetto has led to the production of various commercial products in
the market. However, it is difficult to find products that are uniform in content. The purpose of this
pilot study was to develop a simple HPLC method for standardization of Saw Palmetto products.
Linoleic acid - 12.136
Myristic acid - 13.454
3.00
AU
Abstract: One of the most popular herbal agents in the US market is Saw Palmetto (Sabal,
Serenoa repens), which is derived from the American dwarf palm tree. Among its important
usages include the treatment for prostrate complaints, bladder irritation, urination problems in
BPH stages I and II. However the significant limiting factor to our understanding of the use
and effectiveness of this phytotherapy is the lack of standardization of the products and
unknown active chemical constituents. It has become essential to standardize these
products,so that the variations between products and within different batches of the same
manufacturer can be assessed. In this study, various finished commercial Saw palmetto
preparations purchased from pharmacies or well reputed health stores in the USA were
analyzed for their content based upon the concentrations of four free fatty acids (oleic,
myristic, linoleic and lauric acid) after solvent extraction with heptane followed by
derivatization with phenacyl bromide/triethylamine in acetone by an HPLC method on a C18
column. The compounds were eluted using Acetonitrile/Water and the eluents were
monitored at a wavelength of 242 nm. The content of these four free fatty acids varied from
0.3 – 31.3 % lauric acid, 0.2- 13 % for linoleic acids; 0.06- 7.78 % for myristic acid and 0.7 –
71.99 % for oleic acid. It was interesting to observe that the concentration of lauric and oleic
acids were more in the soft gel formulations as compared to tablet or capsule. The variability
in concentration of these marker compounds, suggest a need for standardization of saw
palmetto products for maximum benefits to our population.
16.00
240.00
260.00
280.00
300.00
nm
320.00
340.00
360.00
380.00
18.00
Intraday
and interday assay precision and accuracy are shown in Tables 2 and 3.
Variability was below 7 % over the concentration range ( 62.5 – 1000 M) with accuracy
between 99-100% for Lauric acid, 93-103% for Linoleic acid, 89-104%for Myristic acid
and 93-103%for Oleic acid.
Table 3: Interday accuracy & precision (n=4)
Table 2: Intraday accuracy & precision (n=5)
There
was a significant product-to-product variability in the amount of these fatty
acids (Table 4).Total lauric acid varied 92 fold (0.3-31.3 % by weight), Linoleic acid
varied 65 fold ( 0.2 –13 % by weight), Myristic acid varied by 128 fold ( 0.06-7.78 % by
weight) and oleic acid varied by 100 fold (0.7 – 71.99 % by weight). The concentration
of these acids were more in gel preparations than in powder formulations (capsules
and tablet).
Table 4: Concentration of marker compounds in commercial Saw palmetto products
Reaction Scheme
O
O
Br
Time
(mins)
Flow
%A
%B
0.0
1.0
80
20
8.0
1.0
85
15
16.0
1.0
90
10
20.0
1.0
100
0
22.0
1.0
100
0
23.0
1.0
80
10
R
+
+
OH
(CH3CH2-)3N
triethylamine
Free fatty acid:
Phenacyl
bromide
R= CH3(CH2)10
(Lauric acid)
R= CH3(CH2)4CH=CHCH2CH=CH(CH2)
(Linoleic acid)
R= CH3(CH2)12
(Myristic acid)
R = CH3(CH2)7CH=CH(CH2)7
(Oleic acid)
CH3CO2H
Acetic acid
O
R
O
O
Fig. 1
Phenacyl ester
Table 1: Gradient table; (A = acetonitrile, B = water)
Conclusion: Our data show that US Saw palmetto products contain lauric acid,
linoleic acid, myristic acid and oleic acid in various concentrations. The variability in
the concentration of these marker compounds, suggests that standardization may be
necessary for quality assurance .
Acknowledgement: The funding for this project was provided by Center for
Excellence (COE), Howard University, HRSA.
References
[1]http//www.nutritional-supplement-info.com/saw palmetto html. [2] Bombardelli, E. and Morazzoni, P.
(1997) Serenoa repens (Bartram) J.K. Small. Fitoterapia LXVIII:99 - 113. [3] Raynaaud, J.P., Cousse, H.
and Martin, P.M. (2002) Inhibition of type 1 and type 2 5-alpha-reductase activity by free fatty acids,
active ingredients of Permixon. J. Steroid Biochem. Mol. Biol. 82: 233 – 239. [4] Mehta, A., Oeser, A.M.,
Carlson, MG. (1998) Rapid quantitation of free fatty acids in human plasma by high-performance liquid
chromatography. J. Chromatography B 719: 9 - 23.