In vitro effect of D-004, a lipid extract of the fruit of the cuban royal palm (Roystonea regia), on prostate steroid 5α-reductase activity.

BACKGROUND: D-004, a lipid extract of the fruit of the Cuban royal palm (Roystonea regia), has been found to reduce prostatic hyperplasia (PH) induced with testosterone (T), but not PH induced with dihydrotestosterone (DHT), in rodents, suggesting the inhibition of prostate 5α-reductase activity. OBJECTIVES: The aims of this study were to assess whether D-004 inhibits prostate 5α-reductase activity in vitro and to examine the effects of D-004 on enzyme kinetics. METHODS: This experimental study was conducted at the Pharmacology Department, Center of Natural Products, National Center for Scientific Research, Havana, Cuba. Soluble rat prostate preparations were used as the source of 5α-reductase, and ((3)H)-DHT production was measured to determine prostate 5α-reductase activity. Cell-free rat prostate homogenates were pre-incubated with carboxymethyl cellulose 2% alone (control tubes) or D-004 (0.24-125 µg/mL) suspended in the vehicle (treated tubes) for 10 minutes prior to adding the labeled substrate ((3)H)-T Once the reaction was stopped, sterols were extracted with chloroform and aliquots were applied on silica gel plates developed in benzene-acetone (4:1, v/v). Areas containing DHT were scraped and radioactivity was counted. The median inhibitory concentration (IC50) was determined by measuring the conversion of T to DHT The apparent Michaelis-Menten constant (Km) and Vmax values before and after adding D-004 were determined in kinetic studies using labeled T (0.5-25 µmol/L). RESULTS: Compared with controls, D-004 significantly and dose-dependently inhibited the enzymatic reaction at doses of 1.95 to 125.0 µg/mL) (all, P < 0.05). The IC50 of D-004 required to inhibit 5a-reductase activity was 2.25 µg/mL. Enzyme inhibition was noncompetitive, since D-004 lowered the Vmax from 15.3 to 10.0 nmol DHT/min · mg(-1) protein, while the Km (4.54 µmol/L) was almost unaffected. CONCLUSIONS: D-004 dose-dependently and noncompetitively inhibited in vitro 5α-reductase activity in soluble fractions of rat prostate. Although the extent of the maximal inhibition was high and the value of IC50 was low, the relevance of such inhibition requires further study in vivo.

Plasma levels, tissue distribution, and excretion of radioactivity after single-dose administration of ((3)H)-oleic acid added to D-004, a lipid extract of the fruit of Roystonea regia, in rats.

BACKGROUND: D-004, a lipid extract of the fruit of Roystonea regia, contains a mixture of fatty acids-mainly oleic, lauric, palmitic, and myristic acids, with oleic acid being among the most abundant-that has been found to reduce the risk for prostatic hyperplasia (PH) induced with testosterone (T) in rats. The pharmacokinetic profile of D-004 has not been reported. OBJECTIVE: The objective of this study in rats was to assess plasma levels, tissue distribution, and excretion of total radioactivity (TR) after single-dose administration of oral D-004 radiolabeled with ((3)H)-oleic acid, as a surrogate for the pharmacokinetics of D-004. METHODS: This experimental study was conducted at the Pharmacology Department, Center of Natural Products, National Center for Scientific Research, Havana, Cuba. Single doses of suspensions of ((3)H)-oleic acid 0.16 µCi/mg mixed with D-004 400 mg/kg (radioactive dose/animal 7.2 µCi) were given orally to male Wistar rats weighing 150 to 200 g assigned to the treated or control group. Three rats were euthanized at each of the following times: 0.25, 0.5, 1, 1.5, 2, 4, 8, 24, 48, 72, 96, and 144 hours after study drug administration. After administration, the rats euthanized at the last experimental time point were housed individually in metabolism cages. Urine and feces samples were collected daily. At each time point, blood samples were drawn and plasma samples were obtained using centrifugation. After euthanization, tissue samples (liver, lungs, spleen, brain, kidneys, adipose tissue, muscle, stomach, small and large intestines, adrenal glands, heart, testes, prostate, and seminal vesicles) were quickly removed, washed, blotted, and homogenized. Plasma (100 µL), tissue aliquots (100 mg), feces (10 mg), and urine (100µL) were dissolved and TR was measured. Samples were assayed in duplicate. Results were expressed in µgEq of radio-labeled oleic acid per milliliter of plasma or urine or gram of tissue or feces. Plasma, tissue, feces, and urine samples of rats that did not receive ((3)H)-oleic acid were used as controls. Excretion was expressed as the percentage of the radioactivity excreted via each route with respect to the total radioactive dose administered to each rat. RESULTS: A total of 50 rats were included in the experiment (mean age, 4 weeks; mean weight, 310 g). Absorption was rapid; mean Cmax was 195.56 (31.12) µgEq/mL, and mean Tmax was 2 hours. Thereafter, a biphasic decay of TR was found: a rapid first phase (t1/2α, 1.33 hours), followed by a slower second elimination phase (t1/2ß, 36.07 hours). Radioactivity was rapidly and broadly distributed throughout the tissues, with more accumulating in the prostate than elsewhere. In the first 8 hours, accumulation of TR was greatest in the prostate, followed by the liver, small intestine, and plasma. Subsequently, TR increased in the small intestine, while it decreased in the liver and plasma. In contrast, over the periods of 24 and 144 hours after administration, TR increased in the adipose tissue, while it decreased in the other tissues and plasma. During those intervals, TR was greatest in the prostate, followed by adipose tissue. Mean peak radioactivity in the prostate (562.41 µgEq/g) was reached at 4 hours and decreased slowly thereafter. The prostate had the highest values of t1/2ß and cumulative AUC compared with the other tissues and plasma. Mean (SD) TR was similar in feces (33.48% [4.90%]) and urine (28.96% [5.32%]), with total excretion being 62.40% (5.90%) of the administered dose. CONCLUSIONS: In this experimental study, after single-dose administration of oral D-004 radiolabeled with ((3)H)-oleic acid in rats, TR was rapidly and widely distributed across the tissues, with the prostate having the highest accumulation of radioactivity. Excretion of TR was limited, with similar amounts being excreted in feces and urine. The broad distribution of radiolabeled oleic acid and/or its metabolites suggests (SD) pharmacokinetic rationale for the effectiveness of D-004 in reducing the risk for PH induced with T in rats.