samples of stem , root , and leafy branches preserved in 70% ethanol were collected from a population near saipipi , savaii , samoa . a healer preparation from falealupo
prostratin ( 1 ) was detected in each sample , and these materials were used for method development and validation .
another collecting expedition was made to the island of savaii , samoa , in january 2005 .
thirty - six samples , consisting of vouchers and ethanol - preserved pharmaceutical grade collections , were taken from three natural populations near the villages of saipipi , tafua , and falealupo .
six trees were sampled from each population , and gps coordinates , elevation , dbh , height , petiole color , and surrounding vegetation were recorded for each tree sampled . from three plants at each site three different morphological samples were collected : roots ( bark + wood ) , stem ( bark + wood ) , and leafy branches , with fruit and/or flowers if present .
only stem samples were collected from the other three trees at each site for a total of 12 samples per site .
nine stem samples were collected on additional expeditions to the island of tutuila in american samoa in may and november of 2005 for a total of 45 samples including 27 stemwood samples .
samples were shipped in vacuum - sealed aluminum vessels in 70% ethanol ( except for the tutuila island collections , which were shipped in 70% 2-propanol ) .
the alcohol fractions were separated and the plant material air - dried in a fume hood .
vouchers were deposited at the herbarium of the california state university fullerton s department of biological science .
plant material was finely chopped then pulverized with a coffee grinder , and approximately 1 g of dry sample tissue was suspended in 25 ml of acetone in a 55 c bath for 10 min .
4 disk , dried in a savant aes1000 speedvac , suspended in 500 l of 80% ethanol in hplc grade water , and then filtered with a 0.22 m millipore ultrafree mc centrifugal filter device .
nontarget organics were removed from the alcohol fraction with a waters sep - pak c18 cartridge using reversed - phase elution ( conditioning : sequential washes of 5 ml of 52% acetonitrile , 5 ml of 26% acetonitrile , and 2.5 ml of 100% hplc water ; loading : 100 l of sample ; separation : 1 ml of hplc water , 2.5 ml of 26% acetonitrile , and 2.5 ml of 40% acetonitrile ) ; the last fraction was filtered with a 0.22 m ultrafree mc centrifugal filter , dried , and suspended in 40 l of 80% ethanol for injection . the ethanol or 2-propanol fractions and the acetone extract for each of 45 samples were analyzed by hplc in triplicate to measure prostratin ( 1 ) concentration in g per g. prostratin was separated by reversed - phase elution using a waters nova - pak c18 column , 4 m bead , 300  3.9 mm , on a gradient hplc system ( waters 717 automated injector , waters 1525 binary solvent delivery system , and empower data analysis system ) at 30 c .
identification was using a waters 2487 dual - wavelength uv absorbance detector using an authenticated standard ( icn mp biomedicals ) at 254 nm .
aliquots of 10 l of each sample were injected , eluted over 15 min with a linear gradient mixing from 32% to 40% acetonitrile using filtered and degassed hplc grade water ( fisher ) and hplc grade acetonitrile ( sigma chromasolv ) , with a flow of 1.0 ml / min .
method validation was completed in compliance with the specifications in the united states pharmacopoeia ( usp ) , general chapter 1225,(33 ) and meyer.(34 ) ruggedness was evaluated by calculating the precision of biweekly triplicate injections at one concentration during the entire range of the study ( rsd 5.21% ) .
linearity was evaluated by plotting peak area as a function of analyte concentration , and regression analysis was performed : slope = 207.78 ; intercept = 1468.70 ; correlation coefficient = 0.9997 ; residual sum of squares = 716  109  646.17 .
the limit of detection ( lod ) and limit of quantification ( loq ) were determined as 2.5 and 25 pmol , respectively , with a range to 30 nmol .
accuracy ( recovery = 96% ) was calculated by spiking blank matrix with known amounts of 1 in triplicate at five concentrations .
repeatability was assessed with triplicate injections at five concentrations on two consecutive days ( rsd 1.81% ) .
intermediate precision was calculated biweekly over the range of the study with triplicate injections at three concentrations ( rsd 5.87% ) .
the observed concentrations of 1 were ranked , and 95% confidence limits for the distribution of these concentrations around the median were constructed using eq 8.2.2 in snedecor and cochran.(35 ) to determine if the median prostratin ( 1 ) concentrations of all populations were equal , statistical hypotheses were tested using a kruksalwallis h test , the nonparametric analogue of anova .
the resultant h statistic was tested for statistical significance at the p < 0.05 level using standard  tables .
a chi - square test for independence was employed to ascertain if the occurrence of exceptionally high concentration plants was equal between populations and also to determine if the falealupo population had a greater than expected number of exceptionally high - yielding prostratin plants .
we used a similar chi - square procedure to test the tafua and tutuila populations to see if they had a higher than expected number of exceptionally low - yielding 1 plants .
spearman s rank correlation coefficient was calculated to assess the correlation between prostratin concentration and diameter at breast height ( dbh ) .
a kruskalwallis h test was employed to establish if 1 was equally distributed throughout plant parts ( leafy branches , stem , and root ) ; to determine if plants with high stem concentrations also have high concentrations in the leaf or roots , we calculated a spearman s rank correlation coefficient and tested for significant correlation at the p < 0.05 level .
 homalanthus nutans , used by samoan healers to treat hepatitis , produces the antiviral compound 12-deoxyphorbol 13-acetate , prostratin ( 1 ) . 
 prostratin is being developed as an adjuvant therapy to clear latent viral reservoirs , the major obstacle to eradication of hiv - aids within the human body . 
 a validated reversed - phase hplc method was developed to assay concentrations of 1 in h. nutans . 
 a survey of four distinct populations on two different samoan islands revealed significant variability in content . 
 the stem tissue ( range 0.252.6 g / g 1 ) , used by healers in indigenous therapies , gave a higher median concentration of prostratin ( 3.5 g / g ) than root or leaf tissues ( 2.9 and 2.5 g / g , respectively ) . the high variability and skewness of these data indicate that cultivar selection for drug production will be important for this species . 
 the reversed - phase hplc assay will allow plants to be selected for agricultural development and genetic analysis by identifying those individuals above and below a 95% confidence interval for the median concentration . 