Tolerability and Toxicity of Lipidosterolic Extract of American Dwarf Palm Serenoa Repens in Wistar Rats: Well-Known Extract, New Insight

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European Review for Medical and Pharmacological Sciences 2011; 15: 1311-1317 Tolerability and toxicity of lipidosterolic extract of american dwarf palm Serenoa repens in Wistar rats: well-known extract, new insight

N. DUBORIJA-KOVACEVIC1, V. JAKOVLJEVIC2, A. SABO2, Z. TOMIC2, B. PAJOVIC3, D. PEROVIC3

1Department of Pharmacology and Clinical Pharmacology, Medical School of the University of Montenegro, Podgorica (Montenegro) 2Department of Pharmacology, Toxicology and Clinical Pharmacology, Medical School of the University in Novi Sad Novi Sad (Serbia) 3Clinic for Urology and Nephrology, Clinical Center of Montenegro, Podgorica (Montenegro)

Abstract. – Background and Objectives: cise conclusion about its tolerability and toxici- Serenoa repens extracts (SrE) have been used ty, it should be excluded possible limiting fac- for centuries in the treatment of benign prostatic tors that we identified in this study. hyperplasia (BPH). According to recommendations that each product should be examined Key Words: separately, including its tolerability and toxicity, Serenoa repens, we conducted this study in order to broaden the Tolerability, Toxicity, Wistar rats. current cognition about tolerability and toxicity of SrE, in particular of German brand Prostamol- unoR. Materials and Methods: Twenty-four adult male Wistar rats were randomly distributed into 4 Introduction groups of 6 animals. The first control group (O) received water (1 ml/kgBW) and second control Extracts of Serenoa repens (SrE) derived from group (OO) received olive oil (1 ml/kgb.w.) every day for 30 days. The third and fourth group of partially dried American dwarf palm fruit Saw rats (SR5 and SR10) were treated with SrE (150 palmetto (family Arecaceae), which grows main- and 300 mg/kgb.w. daily) dissolved in olive oil. ly in the southeast United States, from South Tolerability and toxicity of SrE were estimated Carolina to Florida. They are widely available on the basis of daily monitoring of behavior, and their uses in the treatment of patients with body weight gain (BWG), relative weight of liver, benign prostatic hyperplasia (BPH) are rising left kidney, prostate and left testis, and values of 1 general biochemical parameters. Total liver pro- throughout of the world . teins (TLP) and glutathione content in hepato- It is assumed that the major pharmacologically cyte suspension were also determined. active substances are free fatty acids: lauric, oleic, Results: BWG was significantly unchanged in myristic and palmitic2-4. Flavonoids and sterols ex- SR5 and SR10 compared to both controls in all ert anti-inflammatory effects that could also have intervals of measurement and at the end of treat- an impact on the overall improvement of lower ment (p > 0.05). LW/BW ratio was significantly urinary tract symptoms (LUTS)5-6. Although the higher in SR10 compared with O (p < 0.01). Crea- tinine and potassium were significantly higher in precise mechanism of action has not been clarified SR5 compared to O (p < 0.05), but in SR10 were yet, it is found that SrE exerts antiandrogenic, significantly higher compared to both control proapoptotic and anti-inflammatory effects7. groups (p < 0.01). TLP content was significantly Exact data related to the pharmacokinetics of higher in SR5 compared to OO (p < 0.01). The different SrE have not been known till now. Yale content of glutathione in homogeneous suspen- et al8 revealed that SrE acts as strong inhibitor of sion of hepatocytes didn’t alter significantly. Conclusions: Obtained results have expand- CYP2D6, CYP3A4 and CYP2C9 liver enzymes ed the current state of knowledge about the tol- that participate in metabolism of many drugs that erability and toxicity of SrE, in particular of potentially can lead to significant interactions. Prostamol-unoR. For the adoption of a more pre- Markowitz JS et al9 did not find the impact of

Corresponding Author: Natasha Duborija-Kovacevic, MD, PhD; e-mail: [email protected] 1311 N. Duborija-Kovacevic, V. Jakovljevic, A. Sabo, Z. Tomic, B. Pajovic, D. Perovic multiple dose of SrE on the CYP2D6 and 3A4 liv- pharmacotherapy16. Some earlier data also suggest er enzymes. Some other Authors10 came to the that Prostamol-unoR is efficient and well tolerated same conclusion. Singh ZN et al11 evaluated the by most users and is not associated with serious influence of SrE on rat liver function by measur- adverse effects17-18. ing its effects on several enzymes and formation Bearing in mind the above recommendations, of malondialdehyde (MDA), a byproduct of lipid the purpose of this study was to enlarge the cur- peroxidation. Applied doses of 9.14 and 22.86 rent cognition about tolerability and toxicity pro- mg/kgb.w./day didn’t cause a significant differ- file of SrE, in particular of Prostamol-unoR, in ence in animal body weight, enzyme activity, or two dosage regimes in experimental animals. MDA formation at the end of two and four weeks treatment. The majority of adverse effects of SrE is mild, infrequent and reversible, and includes abdomi- Material and Methods nal pain, diarrhea, nausea and fatigue, headache, decreased libido and rhinitis12. Study Design An intensive investigation of SrE has begun Twenty-four sexually mature male Wistar lab- since the eighties. For this purpose, most com- oratory rats, approximately weighing 250-300 g, monly used product has been Permixon, n-hexa- were randomly distributed into 4 groups of 6 ani- ne lipidosterolic extract of French producer mals. The first control group (O) received only Pierre Fabre Medicament. Of all herbal remedies, water (1 ml/kg BW p.o.) and second control Permixon has been subjected to greater scientific group (OO) received olive oil (1 ml/kg BW p.o.) scrutiny and is associated with more clinical tri- every day for 30 days. The third and fourth als and pharmacologic analyses than any other groups of rats (SR5 and SR10) were treated with preparation of SrE7. SrE dissolved in olive oil. Preparations were pre- Today, a large number of preparations contain- pared daily. Animals received the equal volume ing SrE, whether plain or in combination with of solvent (olive oil) per kilogram of BW in other plant extracts and minerals is on the mar- which was dissolved the determined dose of SrE. ket. The composition of these preparations, de- Both control and investigated substances were pending on the manufacturer, is very different in administered every morning by feeding needle. terms of percentage of free fatty acids (from Animals’ bodyweight was measured weekly, so 40.7% to 80.7%) and other ingredients13. the applied dose was adapted to the new mea- Also, the lack of products’ standardization is sured weight. an additional problem. Analyzing six different preparations, Feifer AH et al14 concluded that the Substances and Doses amount of active ingredients ranging from –97% Following substances were used: extract of to +140% compared to the one that was specified Serenoa repens (Prostamol-unoR, soft capsules 30 × on the package. 320 mg, Berlin-Chemie AG, Menarini Group, Foregoing means that the results obtained in Berlin, Germany); olive oil (bottle 50 ml, Pharmacy studies with Permixon could not be applied to the “Biljana”, Novi Sad, Serbia); urethane solution products of other manufacturers. In this regard 25%; sulphosalicylic acid 4%; Ellman’s reagent and recommendations of the 5th International (0.2 mmol/dm3 DTNB* [*5.5’-dithiobis-(2-ni- 3 Consultation on BPH that each product, although trobenzoic acid)] in 0.1 mol/dm K3PO4, pH=8); Bi- derived from the same plant, should be evaluated uret reagent (0.15% CuSO45H2O; 0.6% K, Na-tar- individually and rigorously through both clinical trate, 0.1% KJ dissolved in 0,85 mmol/dm3 NaOH. and pharmacological trials15. In determining the pharmacological dose of German brand Prostamol-unoR (Berlin-Chemie SrE in rats there was used Clark’s formula, by AG, Berlin, Germany) is the most commonly used which dose for rats were calculated in relation to herbal product for BPH in Montenegro. In the form the human dose: 3 of soft capsules containing 320 mg of the active D/kg × √ BW (man) substance, it can be purchased in state and private X = ––––––––––––––––––––––––– 3 pharmacies. Our recent investigation found that its √BW (animal) safety and tolerability during three-month treatment of patients with lower urinary tract symptoms X = dose for animal; BW = bodyweight; D/kg = hu- (LUTS) due to BPH, was comparable to standard man dose

1312 Tolerability and toxicity of Serenoa repens

In order to investigate the tolerability and toxi- Committee regulations of Medical School in cological profile of SrE, obtained value (30 Novi Sad, Serbia, and adhere to the recommen- mg/kg BW/day) was multiplied with five (150 dations from the Declaration of Helsinki. mg/kg BW/day) and ten (300 mg/kg BW/day), that means five and ten times higher dose than Statistical Analysis human recommended daily dose. All the data were expressed as mean ± SD of the mean and were analyzed by analysis of vari- Experimental Procedures ance (ANOVA) for multiple comparisons, fol- After 30 days of treatment, the animals were lowed by Tukey’s test where appropriate. The ac- measured, than anesthetized with 25% urethane cepted level of significance was p < 0.05. solution (2.5 ml/kg intraperitoneally). Blood was collected to prepare serum for biochemical analyses. The liver was measured; a portion of 1 mg was removed for the preparation of ho- Results mogenate, which was used for the determination of total protein and glutathione content in homo- By continual monitoring of experimental ani- geneous suspension of hepatocytes. Left kidney, mals, there wasn’t noticed any change in their prostate and left testis were also separated and behavior that could be attributed to the possible measured. toxicological effect of applied preparations. No Tolerability and toxicity of SrE were estimated animal has died during treatment and food and on the basis of daily monitoring of behavior and water use was similar in all groups. There wasn’t bodyweight gain (BWG) at weekly intervals and observed any change in rats’ behavior during at the end of treatment. Additional parameters weekly measurements and cleaning of the cage. were liver weight/body weight (LW/BW) ratio, However, from the fifth day to the end of one- kidney weight/body weight (KW/BW) ratio, month treatment, almost all rats from SR10 (300 prostate weight/body weight (PW/BW) ratio and mg/kg BW/day) refused application of the ex- testis weight/body weight ratio (TW/BW). The tract by feeding needle and expressed anxiety value of urea, creatinine, electrolytes, transami- and visual aggressiveness during treatment. nases (ALT and AST) and lactate dehydrogenase Bodyweight gain (BWG) in rats that were (LDH) was also measured using standard treated with SrE in two dose regimes compared methodology. to control groups, in intervals of seven days, is For determination of glutathione content in shown on Figure 1. liver homogenate and hepatocyte suspension, we BWG was significantly unchanged in SR5 used a method that was applied by Kapetenovic (150 mg/kg BW/day) and SR10 (300 mg/kg IM et al19 (modified Elman’s method). BW/day) compared to both controls in all inter- Total protein content was measured by a bi- vals of measurement and at the end of thirty-day uretic method, modified by Gornall and Bard- treatment (p > 0.05). Also, there is no statistical- wall.20 ly significant difference in BWG between control groups (p > 0.05). Animals The influence of SrE in two doses on the liver Rats were bred in the vivarium at the Depart- weight/body weight (LW/BW), kidney ment of Pharmacology, Toxicology and Clinical weight/body weight (KW/BW), prostate Pharmacology, Medical Faculty, University of weight/body weight (PW/BW) and testis Novi Sad, Serbia. Animals were kept in standard weight/body weight (TW/BW) ratio in experi- plexiglass cages (six per cage) at standard labora- mental animals is shown in Table I. tory conditions (light period of 12h/day, constant One-month treatment of experimental animals room temperature 21±1°C and humidity with SrE in a higher dose of 300 mg/kgBW daily 55%±1.5%). They were fed by standard laborato- significantly increased the LW/BW ratio compared ry rat feed, produced by the Veterinary Institute with the control group O (p < 0.01), but not with in Zemun, Serbia, with free access to food and OO (p > 0.05). water. Rats were allowed to acclimatize for two The PW/BW ratio was different between con- weeks before the application of preparations. An- trol groups of animals. As shown in Table I, the imal care and all experimental procedures were PW/BW ratio is significanly higher in OO com- carried out in compliance with the Animal Care pared to O (p < 0.05), and values of SR5 and

1313 N. Duborija-Kovacevic, V. Jakovljevic, A. Sabo, Z. Tomic, B. Pajovic, D. Perovic

Figure 1. Bodyweight gain (BWG) in rats chronically treated with SrE (SR5 and SR10) compared to control substances (O and OO), in intervals of seven days, expressed in percentages (%) (Mean ± SD) (p > 0.05). O: first control group (n=6), water (1 ml/kg BW/day p.o.); OO: second control group (n=6), olive oil (1 ml/kg BW/day p.o.); SR5: first experimental group (n=6), extract of Serenoa repens (150 mg/kg BW/day p.o.) dissolved in olive oil; SR10: second experimental group (n=6), extract of Serenoa repens (300 mg/kg BW/day p.o.) dissolved in olive oil.

SR10 are between obtained results, but without The effect of SrE (150 and 300 mg/kg BW/day) statistically significant difference (p > 0.05). on AST, ALT, LDH, total protein content in the liv- One-month treatment with both SrE didn’t cause er (in mg of proteins per g of the liver) and glu- any difference in KW/BW and TW/BW ratio. tathione content in liver homogenates and suspen- SrE in two doses also caused some changes in sions of hepatocytes, is shown in Table III. the values of biochemical parameters that were At first, it must be noted here that one-month analyzed. The results are shown in Table II. treatment with olive oil high significantly re- Five times higher dose of SrE than human sig- duced (p < 0.01) the content of total proteins in nificantly increased (p < 0.05) the values of serum the liver compared to the control O. creatinine and potassium compared to the control Chronic treatment with SrE in a lower dose group O. SrE in tenfold higher daily dose high sig- significantly decreased (p < 0.05) ALT values in nificantly increased (p < 0.01) the creatinine com- relation to the first control group, but AST and pared to the first control group of animals (O), but LDH values remained statistically unchanged significantly (p < 0.05) compared to the OO. The compared to both controls. SrE in dose of 300 same dose high significantly (p < 0.01) increased mg/kg BW/day increased the values of transami- the serum potassium values in relation to both nases and LDH, but the difference didn’t achieve control groups. statistical significance (p > 0.05).

Table I. Body weight (BW) at the end of treatment, liver weight/body weight (LW/BW), kidney weight/body weight (KW/BW), prostate weight/body weight (PW/BW) and testis weight/body weight (TW/BW) ratio in rats who were treated with SrE (150 and 300 mg/kg BW/day p.o.) compared with control groups (O and OO) (Means ± SD, n=6 rats).

BW LW/BW KW/BW PW/BW TW/BW Group (g ± SD) (x10-3) ± SD (x10-3) ± SD (x10-3) ± SD (x10-3) ± SD

O 414.33 ± 6.85 31.87 ± 0.19 3.27 ± 0.05 1.37 ± 0.12 3.60 ± 0.22 OO 415.83 ± 34.17 33.55 ± 2.34 3.27 ± 0.17 1.78 ± 0.27** 3.42 ± 0.53 SR5 417.05 ± 37.89 31.17 ± 2.98 3.55 ± 0.33 1.53 ± 0.17 3.23 ± 0.49 SR10 432.80 ± 33.25 34.38 ± 0.98* 3.13 ± 0.16 1.58 ± 0.24 3.50 ± 0.32

*p < 0.01 vs. first control group (O); **p < 0.05 vs. first control group (O).

1314 Tolerability and toxicity of Serenoa repens

Table II. The values of serum urea, creatinine, potassium, sodium and chloride in Wistar rats who were treated with SrE (150 and 300 mg/kg BW/day) for 30 days and control groups of animals (O and OO) (Means ± SD, n=6 rats).

Urea Creatinine Potassium Sodium Chlorides Group (mmol/l) (µmol/l) (mmol/l) (mmol/l) (mmol/l)

O 9.50 ± 0.45 55.67 ± 4.50 4.10 ± 0.67 153.00 ± 2.83 96.00 ± 0.82 OO 9.18 ± 0.93 55.50 ± 7.61 4.50 ± 0.70 155.50 ± 1.38 101.00 ± 1.15 SR5 9.00 ± 0.91 63.00 ± 5.73* 5.12 ± 0.37* 155.20 ± 0.98 104.00 ± 1.79§ SR10 10.85 ± 0.67 69.50 ±6.54§,# 5.98 ± 0.33§,‡ 153.75 ± 1.64 102.75 ± 1.30§

*p < 0.05 vs. first control group (O); §p < 0.01 vs. first control group (O); #p < 0.05 vs. second control group (OO); ‡p < 0.01 vs. second control group (OO).

Lower dose of 150 mg/kg BW/day increased Prostamol-unoR. In favor of previous assumption, both the content of total proteins and glutathione both doses of SrE didn’t significantly alter BWG in the liver in comparison to control groups, but in SR5 and SR10 compared to both controls in all statistically significant difference was observed intervals of measurement and at the end of treat- only in total protein content compared to second ment (p > 0.05). Although, possible protective ef- control group OO (p < 0.01). fect of olive oil as a solvent, as the relatively small Applying the statistical analysis to the results number of treated animals (n=6), can be limiting in Table III, there hasn’t been found statistically factors in making a final conclusion. significant difference in the values of glutathione Continuously observing the behavior of experi- in liver homogenates between groups of animals mental animals, it was noticed the existence of anx- which were treated with SrE (SR5, SR10) and iety and expressed aggressiveness during per oral control groups (O and OO). application of SrE in group of animals who received the ten times higher daily dose in relation to the human. It could be the consequence of many possible influences of SrE in applied dose (unpleas- Discussion ant taste and/or smell, possible gastrointestinal dis- turbances, some psychotropic effect of high dose of Thirty-day treatment with SrE in five (150 SrE) or influence on some another level, that we mg/kgBW) and ten (300 mg/kgBW) times higher can’t specify on the basis of obtained results. daily doses than human, experimental animals sur- The liver weight/body weight (LW/BW) ratio vived well and none died during treatment. If it is was higher in SR10 compared to both control known that applied dose of 300 mg/kgBW is ap- groups and also with SR5, but achieved statistical- proximately sixty times higher in comparison to ly significant difference (p < 0.01) only in com- human daily dose of SrE and did not exhibit any parison with control that received water (O). This lethal effect in experimental animals during one- could indicate the potential hepatotoxicity of SrE month treatment, it most likely confirms a favor- in applied dose of 300 mg/kg BW/day. However, able toxicological profile of SrE, in particular of it must be noted here that LW/BW ratio was ap-

Table III. AST, ALT, LDH, total protein and glutathione content in liver homogenates and suspension of hepatocytes, measured after one-month treatment with SrE (150 and 300 mg/kg BW/day), compared with control groups (O and OO) (Means ± SD, n = 6 rats).

AST ALT LDH Total liver proteins Glutathione (X ± SD) Group (U/l) (U/l) (U/l) (X ± SD)(mg/g) x10-6 (mol/mg)

O 196.33 ± 9.57 62.00 ± 9.20 2745.00 ± 304.62 371.00 ± 11.43 0.0131 ± 0.0009 OO 207.17 ± 51.65 54.17 ± 16.36 3568.17 ± 1129.25 346.50 ± 9.69* 0.0136 ± 0.0021 SR5 197.20 ± 58.49 50.60 ± 5.78§ 3084.40 ± 772.32 402.50 ± 33.02# 0.0142 ± 0.0016 SR10 279.75 ± 91.00 72.00 ± 13.04 3664.50 ± 1485.01 362.25 ± 10.35 0.0132 ± 0.0013

*p < 0.01 vs. first control group (O); §p < 0.05 vs. first control group (O); #p < 0.01 vs. second control group (OO).

1315 N. Duborija-Kovacevic, V. Jakovljevic, A. Sabo, Z. Tomic, B. Pajovic, D. Perovic proximately the same in the group of animals that Unchanged values of urea, transaminases, lac- received five times higher dose of SrE (150 mg/kg tate dehydrogenase and glutathione content in the BW/day) as in group O, while olive oil in a dose liver (p > 0.05) probably speak in favor of the ab- of 1 ml/kg BW/day also caused a rise of LW/BW sence of oxidative damage and/or reduction of ratio to some degree in relation to group O. Since the functional capacity of the liver under the in- olive oil was used as the solvent for both doses of fluence of applied SrE. Our results are consistent SrE, it can not be ruled out that the increase of with the findings of Singh et al11 who came to the LW/BW ratio in group SR10 has been partially same conclusion by measuring SrE effects on caused by the contribution of olive oil. In these several enzymes and formation of malondialde- terms, a final position on possible hepatotoxicity hyde (MDA), but with too much smaller doses in of SrE in a dose of 300 mg/kg BW/day can not be comparison with ours (9.14 and 22.86 mg/kg made based on the obtained results. BW/day). Five times higher dose of SrE didn’t influence An interesting result is that the lower dose of animals’ behavior, BWG or LW/BW ratio signifi- SrE significantly increased (p < 0.01) the con- cantly (p > 0.05), but its potentially toxic effects tent of total liver protein compared with the can not be excluded because olive oil as a solvent control that received olive oil, while the content could act as hepatoprotective factor, that was of protein does not differ in group SR10 com- shown in some studies.21-23 According to previ- pared to both controls (p > 0.05). Neither here it ously stated, for the adoption of a more precise can not be eliminated the effect of olive conclusion about the possible hepatotoxicity of oil. High significantly reduction of total protein SrE, it would be necessary to eliminate olive oil content in the liver as the result of one-month as a solvent and, if it is possible, give animals the treatment of rats with olive oil in a dose of 1 pure extract. ml/kg BW/day, could be explained as the conse- One-month treatment with olive oil (1 ml/kg quence of the increased hepatic fat accumula- BW/day) significantly increased the prostate tion and fatty infiltration of the liver22-23. Richter weight/body weight (PW/BW) ratio in Wistar et al25 in a similar experimental model have rats compared to animals who received only wa- found the existence of moderate, non-degenera- ter (O) (p < 0.05), but SrE showed the tendency tive fatty infiltration of the liver which started in to decrease those difference. Some studies found periportal spaces. These changes were attributed that increased consumption of both butter and to the effects of monounsaturated oleic acid. In margarine was positively associated with BPH this regard, possible conclusion could be that risk, but no overall association was found with the dose of SrE of 150 mg/kgBW/daily actually respect to consumption of olive oil24. Without acted as a protective factor against fatty infiltra- pathological examination of rats’ prostate and tion of the liver caused by the olive oil. some additional analyses, it can’t be possible to determine precisely at which level olive oil influenced the relative weight of rats’ prostate. Also, additional studies are needed to investigate the Conclusions reduction of effects of olive oil on rats’ prostate by SrE to some extent. If we know that both pro- The findings of this investigation have ex- liferation of prostate tissues and anti-androgenic panded the current state of knowledge about effect of drugs (5-alpha reductase inhibitors and the tolerability and toxicity of SrE, in particular androgen receptor antagonists) need more time of German brand Prostamol-unoR. If we know than one month, it will be interesting to explore that a human dose of SrE is 320 mg daily, that obtained findings with more details. is about 5 mg/kgBW, it can be concluded that Significantly higher values of serum creati- investigated preparation is safe for human use nine, potassium and chloride in experimental ani- most likely. mals which received SrE in both dosage regimes Olive oil as the solvent has been shown as the compared with controls, could be the conse- limiting factor in the precise interpretation of quence of nephrotoxicity and/or skeletal muscle obtained results, because it demonstrated spe- toxicity of SrE in applied doses. Similar results cific effects and probably influenced the tolera- hasn’t been published till know, although the ex- bility and toxicity of SrE to some extent. Also, isting research mainly was done with small doses in order to obtain more exact conclusions, it compared to ours. would be desirable to examine the tolerability

1316 Tolerability and toxicity of Serenoa repens and toxicity of SrE and in another animal model rantium, Echinacea purpurea, milk thistle, and saw over a longer period of time by applying addi- palmetto. Clin Pharmacol Ther 2004; 76: 428-440. tional experimental procedures that were identi- 11) SINGH YN, DEVKOTA AK, SNEEDEN DC, SINGH KK, HA- fied in this study. LAWEISH F. Hepatotoxicity potential of saw palmetto (Serenoa repens) in rats. Phytomedicine 2007; 14: 204-208. –––––––––––––––––––– Acknowledgements 12) AGBABIAKA TB, PITTLER MH, WIDER B, ERNST E. Serenoa repens (saw palmetto): a systematic review of ad- Berlin-Chemie AG (Menarini Group), Berlin, Germany, verse events. Drug Saf 2009; 32: 637-647. was supported doctoral dissertation of Assist. Prof. N. 13) HABIB FK, WYLLIE MG. Not all brands are created Duborija-Kovacevic in 2006 by donation of Prostamol- equal: a comparison of selected components of unoR. 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