STUDY OF THE EFFECT OF AN EXTRACT OF Serenoa repens on the production of the 5-α reductasa enzyme 1) ROLE OF 5-α-ALFA-REDUCTASA 5-α reductasas (5-α-R) are a family of enzymes involved in steroid metabolism. Inhibiting the synthesis of these enzymes has been used in the treatment of benign prostate hyperplasia, prostate cancer and male pattern baldness. In the latter application, the most commonly used drugs are steroids such as Dutasteride and Finasteride. Serenoa repens extract has been described in numerous publications as an inhibitor of the synthesis of the 5-α-R enzyme that prevents the conversion of testosterone into dihydrotesoterone, although until now, it has not been possible to demonstrate this inhibition in vivo. This extract is the basis of the VR6 DEFINITIVE HAIR product. 2) STUDY OF THE EXTRACT OF Serenoa repens present in VR6 DEFINITIVE HAIR product CNCE INNOVACIÓN and BIOPOLIS have collaborated on a project that analyzes the inhibiting effect of the synthesis of the 5-α-R enzyme of Caenorhabditis elegans from the extract of Serenoa repens used in the VR6 DEFINITIVE HAIR product. This work was divided into three stages: i) to design an in vivo detection system in C. elegans of the 5-α-R enzyme; ii) to carry out a proof of concept of the in vivo inhibiting effect of the synthesis of this enzyme using the common drug Dutasteride; and iii) to study the inhibiting effect of the extract of Serenoa repens included in VR6 DEFINITIVE HAIR in this evaluation system. The results are discussed below. 2.1. Development of a protocol for the detection and quantification of the inhibition of the synthesis of the 5-α-R enzyme in C. elegans The first objective of this stage was to develop a methodology that would allow for detecting the presence in C. elegans of the enzyme being studied. To this end, a search was carried out in databases and it was determined that C. elegans possesses at least three possible gene candidates to codify 5-α-R (F19H6.4, CELE-ZK1251.3 and CELEF42F12.3). The sequence of each one of the proteins was aligned with the human protein SRD5A2 that codifies the 5-α-R enzyme, determining an approximate sequence homology of 41% that should permit detecting the presence of the C. elegans enzyme using the antibody against the human enzyme. Following this reasoning, in the second step, the presence of the protein equivalent to human SRD5A2 in C. elegans cultures grown under standard conditions (NG medium) was determined using a western blot. For this, worms in different stages of development were chosen: i) young adult worms (1-day-old adults), ii) 2-day-old adults and iii) 4-day-old adults. As shown in Figure 1, it was possible to detect the protein in all the developmental stages analyzed. The molecular size of the protein was close to 75KDa. The largest band size observed corresponded to a greater load capacity in the gel. Pag. 1
Figure 1. Detection of 5-α-R protein of C. elegans in different stages of development. In short, the development of this stage enabled having a valid methodology for the detection and quantification of the 5-α-R protein of C. elegans. 2.2. Evaluation of the 5-α-R inhibiting effect of the drug Dutasteride in C. elegans In this step, it was determined if the drug Dutasteride, used to treat benign prostate hyperplasia and baldness, produces an inhibition of the synthesis of the 5-α-R protein of C. elegans, evaluating doses of 1, 10, 50 and 100 μg/ml in 2-day-old and 4-day-old C. elegans adult worms. As can be seen in Figure 2, Dutasteride produced a partial inhibition of the synthesis of the 5-α-R protein of C. elegans at all the doses tested on 2-day-old worms. Figure 2. Inhibiting effect of Dutasteride on the synthesis of 5-α-R of C. elegans at two-day-old adult stage. Pag. 2
The inhibition percentage, calculated on the basis of the net intensity of the electrophoretic band, was 42.3% for a Dutasteride dose of 1 and 10 μg/ml and 65.2% for a dose of 50 and 100 μg/ml (Figure 3). This indicates a dose-response inhibiting effect of the drug. An inhibiting effect was not determined in 4-day-old adults. Figure 3. Relative quantification of luminescence intensity starting at 10 μl of C. elegans (2-day-old adults) protein preparations treated with Dutasteride. In short, to determine the inhibiting effect of a treatment, it is necessary to carry out an analysis of a concrete developmental stage of the nematode (the adult at two days). In addition, the drug Dutasteride (1-100 μg/ml dose) is valid as positive trial control. 2.3. Evaluation of the inhibiting effect of the extract of Serenoa repens used in VR6 DEFINITIVE HAIR on the synthesis of the 5-α-R enzyme of C. elegans A variety of evidence exists in the literature that demonstrates the inhibiting effect of this extract on the synthesis of 5-α-R in human prostatic cellular lines, as well as its effectiveness in treating baldness. The trials were carried out under distinct experimental conditions. 2- and 4-day-old nematodes were analyzed, applying doses of 5 and 10 mg/ml. The results (Figure 4) indicated a clear inhibiting effect of the expression of the synthesis of the 5-α-R enzyme in two-day-old adult nematodes fed the Serenoa repens extract. This effect was not observed in 4-day-old nematodes. Pag. 3
Figure 4. Inhibiting effect of Serenoa repens extract on the synthesis of the 5-α-R enzyme of C. elegans in the adult stage at 2 days. The estimated inhibition percentage was 73.2% with a dose of 5 mg/ml and 86.5% with a dose of 10 mg/ml (Figure 5), thus demonstrating a dose-dependent effect. Figure 5. Left panel. Relative quantification of the luminescence intensity starting from 10 μl C. elegans (two-day-old adults) protein preparations treated with Serenoa repens extract. Right panel. Confirmation of load control through detection of the Tubulin protein constituent expression. In short, in this stage, it was determined that the extract of Serenoa repens present in the VR6 DEFINI- TIVE HAIR product has an inhibiting effect on the synthesis of the 5-α-R enzyme of C. elegans at doses of 5 and 10 mg/ml. This effect is detected specifically in 2-day-old adults, the same as occurs with the drug Dutasteride. Pag. 4
3) CONCLUSIONS A protocol has been developed that permits detecting and quantifying the expression of the 5-α-R protein of C. elegans in 2-day-old adults. The system is valid for scrutinizing compounds or extracts with the possible inhibiting effect of the synthesis of this protein. It was determined that the drug Dutasteride reduces the expression of the synthesis of the 5-α-R enzyme of C. elegans at doses of 1, 10, 50 and 100 μg/ml. The inhibition percentages vary between 42 and 65%. The Serenoa repens extract reduces the expression of the synthesis of the 5-α-R enzyme of C. elegans at doses of 5 and 10 mg/ml in 73 and 86%, respectively. Given the presence of this extract in the commercial product VR6 DEFINITIVE HAIR, it is very likely that the action of this product on reducing the production of 5-α-R is the cause of the positive effect of this product on baldness. Pag. 5