Chemopreventive Potentials of Ultraviolet Screen Agents in the Epstein-Barr Virus Early Antigen Activation and Nitric Oxide Induced Mouse Skin Carcinogenesis Govind J. Kapadia1*, Harukuni Tokuda2, Magnus A. Azuine1,3, Xang Yang Mou2, Teruo Mukainaka2,

1

• 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

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.
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.
Fig 2
Fig 3
Fig 23 Chromatogram of a mixture of standards
their on-line spectra
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.
Fig 4
Fig 5
Fig 45 Chromatogram of a represent-ative
sample its on-line spectra
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. Its 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.

• 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,
  89-104for Myristic acid and 93-103for Oleic
  acid.

Table 3 Interday accuracy precision (n4)
Table 2 Intraday accuracy precision (n5)

• 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.
• 11 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)

• 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
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
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 1http//www.nutritional-supplement-inf
o.com/saw palmetto html. 2 Bombardelli, E. and
Morazzoni, P. (1997) Serenoa repens (Bartram)
J.K. Small. Fitoterapia LXVIII99 - 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.
Fig. 1
Table 1 Gradient table (A acetonitrile, B
water)