Acne Research

In the following studies,DHT blockers Gamma Linolenic Acid (GLA), Alpha Linolenic Acid (ALA), and Linoleic Acid were shown to be the most potent known inhibitors of type 1 and 2 forms of 5-alpha reductase and highly effective in decreasing the levels of dihydrotestosterone (DHT), when applied topically. More importantly this is accomplished without affecting any systemic effects.

The acne research study further suggests that these fatty acids can be useful in the treatment of disorders related to dihydrotestosterone including male pattern baldness, acne treatment, and excessive female body hair (hirsuitism).

STUDY A: Growth suppression of hamster flank organs by topical application of gamma-linolenic and other fatty acid inhibitors of 5 alpha-reductase. Liang T; Liao S. Journal of Investigational Dermatology: 1997 Aug; 109 (2): 152-7

ABSTRACT

Certain unsaturated aliphatic fatty acids, such as gamma-linolenic acid, inhibit 5alpha-reductase activity in vitro and in vivo. Hamster flank organ growth, as measured by the increase in the area of pigmented macule, is dependent on androgen. When one of the paired flank organs of a castrated hamster was treated topically with testosterone, the treated organ, but not the contralateral flank organ, became larger and darker. Topical application of gamma-linolenic acid to the testosterone-treated flank organ suppressed this testosterone effect. Other fatty acids that were not inhibitors of 5alpha-reductases were not active. Topical treatment of hamster flank organs with 5alpha-dihydrotestosterone also stimulated the growth of the organ. This 5alpha-dihydrotestosterone-dependent activity, however, was not significantly affected by gamma-linolenic acid, suggesting that flank organ growth was dependent on 5alpha-dihydrotestosterone and that gamma-linolenic acid acted by inhibiting 5alpha-reductase. With intact male hamsters, the endogenous androgen-dependent growth of flank organs is also suppressed by topical treatment with gamma-linolenic acid. The effect of gamma-linolenic acid is localized at the site of its application; topical application of gamma-linolenic acid did not affect the androgen-dependent growth of other organs such as testis, epididymis, seminal vesicle, and prostate. gamma-Linolenic acid, with low toxicity and absence of systemic effect, therefore suggesting a hormonal acne treatment,which may be potentially useful for treatment of androgen-dependent skin disorders.

STUDY B: Androgen action: molecular mechanism and medical application. Liao S . Journal of Formos Medical Association: 1994 Sep; 93 (9): 741-51.

ABSTRACT

Androgen action in many organs, such as prostate and skin, is dependent on the conversion of testosterone by 5 alpha-reductase to 5 alpha-dihydrotestosterone. 5 alpha-Dihydrotestosterone then binds to the androgen receptor to regulate specific gene expression. Inhibitors of 5 alpha-reductase are useful for the selective treatment of prostatic cancer, benign prostate hyperplasia, acne, baldness and female hirsuitism, without affecting spermatogenesis, sexual behavior and smooth muscle growth, that do not require the conversion of testosterone to 5 alpha-dihydrotestosterone. Certain unsaturated fatty acids, such as gamma-linolenic acid, are potent 5 alpha-reductase inhibitors, suggesting a linkage between unsaturated fatty acids and androgen action. Mutations in androgen receptor genes are responsible for many cases of androgen-insensitivity. In some prostate cancer cells, some antiandrogens may act like androgens in stimulating the proliferation of the cancer cells because these antiandrogens can bind to a mutated androgen receptor and transactivate target genes. Prostate cancers are usually androgen-dependent initially but can lose dependency and responsiveness. Tumor cells which are resistant to endocrine therapy ultimately proliferate. Androgen-independent or androgen-repressive cells can arise from androgen-sensitive prostate cancer cells by changes in specific gene expression over time in a clonal isolate. This change in androgen responsiveness was accompanied by a change in androgen receptor expression and transcriptional activity as well as expression of some oncogenes.

STUDY C: Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids. Liang T; Liao S Journal of Biochemistry, 1992 Jul 15, 285 ( Pt 2):, 557-62

ABSTRACT

Human or rat microsomal 5 alpha-reductase activity, as measured by enzymatic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymatic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymatic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells.

In the following studies Saw Palmetto extract was shown to inhibit both type 1 and 2 forms of 5 alpha-reductase (Finasteride or Propecia® only inhibits type 2), was more potent than Finasteride and was able to effectively reduce binding of Testosterone and DHT to their receptors on various tissues. Please note that in the following studies Saw Palmetto Extract is referred to as Permixon or lipido-sterol extract of Serenoa repens (LSESr)

Study A:

Human prostatic steroid 5 alpha-reductase isoforms–a comparative study of selective inhibitors.

This study suggests that Fenistride (active ingredient of Propecia) only inhibited the Type 2 form of 5 alpha reductase where Saw Palmetto extract inhibited both type 1 and 2 forms of 5 alpha-reductase and was more potent than Fenistride. Iehlé C; Délos S; Guirou O; Tate R; Raynaud JP; Martin PM . J Steroid Biochem Mol Biol, 54: 5-6, 1995 Sep, 273-9.

ABSTRACT

The present study describes the independent expression of the type 1 and 2 isoforms of human 5 alpha-reductase in the baculovirus-directed insect cell expression system and the selectivity of their inhibition. The catalytic properties and kinetic parameters of the recombinant isozymes were consistent with published data. The type 1 isoform displayed a neutral (range 6-8) pH optimum and the type 2 isoform an acidic (5-6) pH optimum. The type 2 isoform had higher affinity for testosterone than did the type 1 isoform (Km = 0.5 and 2.9 microM, respectively). Finasteride and turosteride were selective inhibitors of the type 2 isoform (Ki (type 2) = 7.3 and 21.7 nM compared to Ki (type 1) = 108 and 330 nM, respectively). 4-MA and the lipido-sterol extract of Serenoa repens (LSESr) markedly inhibited both isozymes (Ki (type 1) = 8.4 nM and 7.2 micrograms/ml, respectively; Ki (type 2) = 7.4 nM and 4.9 micrograms/ml, respectively). The three azasteroids were competitive inhibitors vs substrate, whereas LSESr displayed non-competitive inhibition of the type 1 isozyme and uncompetitive inhibition of the type 2 isozyme. These observations suggest that the lipid component of LSESr might be responsible for its inhibitory effect by modulating the membrane environment of 5 alpha-reductase. Partially purified recombinant 5 alpha-reductase type 1 activity was preserved by the presence of lipids indicating that lipids can exert either stimulatory or inhibitory effects on human 5 alpha-reductase.

Study B: Testosterone metabolism in primary cultures of human prostate epithelial cells and fibroblasts.

This Study shows that Fenistride and 4-MA inhibited the formation of some testostrone Metabolites (including DHT), where as Saw Palmetto inhibited the formation of all the Testostrone metabolites studied. Délos S; Carsol JL; Ghazarossian E; Raynaud JP; Martin PM. J Steroid Biochem Mol Biol, 55: 3-4, 1995 Dec, 375-83

ABSTRACT

We compare testosterone (T) metabolism in primary cultures of epithelial cells and fibroblasts separated from benign prostate hypertrophy (BPH) and prostate cancer tissues. In all cultures, androstenedione (delta 4) formed by oxidation of T by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) represented 80% of the metabolites recovered. The amounts of 5 alpha-dihydrotestosterone (DHT), formed by reduction of T by 5 alpha-reductase (5 alpha-R), were small: 5 and 2% (BPH) and 8 and 15% (adenocarcinoma) for epithelial cells and fibroblasts, respectively. Northern blot analysis of total RNA from epithelial cells (BPH or adenocarcinoma) attributed the reductive activity to the 5 alpha-reductase type 1 isozyme and oxidative activity to the 17 beta-HSD type 2. In cancer fibroblasts, only little 17 beta-HSD type 2 mRNA was detected. The 5 alpha-reductase inhibitors, 4-MA (17 beta-(N,N-diethyl)carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) and finasteride, inhibited DHT formation with a preferential action of 4-MA on epithelial cells (BPH or adenocarcinoma) and of finasteride on fibroblasts from adenocarcinoma. Neither inhibitor acted on delta 4 formation. On the other hand, the lipido-sterol extract of Serenoa repens (LSESr, Permixon, Saw Palmetto) inhibited the formation of all the T metabolites studied [IC50 S = 40 and 200 micrograms/ml (BPH) and 90 and 70 micrograms/ml (adenocarcinoma) in epithelial cells and fibroblasts, respectively]. These results have important therapeutic implications when selecting appropriate treatment options for BPH.

Study C: The effect of Permixon on androgen receptors.

This study shows that Saw Palmetto extract was able to effectively reduce Binding of Testostrone and DHT to their receptors on various tissues. el-Sheikh MM; Dakkak MR; Saddique A. Acta Obstet Gynecol Scand, 67: 5, 1988, 397-9

ABSTRACT

Permixon, the liposterolic extract of the plant Serenoa Repens is a recently introduced drug for the treatment of benign prostatic hyperplasia. The effect of Permixon on dihydrotestosterone and testosterone binding by eleven different tissue specimens was tested. The drug reduced the mean uptake of both hormones by 40.9% and 41.9% respectively in all tissue specimens. Since hirsutism and virilism are among other gynecological problems caused either by excessive androgen stimulation or excess endorgan response, we suggest that Permixon could be a useful treatment in such conditions and recommend further investigations of the possible therapeutic values of the drug in gynecological practice.

This article refers to the activity of multiple natural ingredients that have the potential for inhibition of 5-alpha-reductase activity. Of the ingredients in the article, dodecyl gallate has the greatest inhibition of 5-alpha-reductase activity in both cell-free and whole cell assays in vitro. The significance of this is that dodecyl gallate inhibits both forms of 5-alpha-reductase (type I and type II) in not only the presence of the enzyme but can penetrate the cell membrane and inhibit the enzyme while the cells are in culture, further supporting the effectiveness of dodecyl gallate. Biochem Pharmacol. 2002 Mar 15;63(6):1165-76.

Structure-activity relationships for inhibuman 5alpha-reductases by polyphenols.

Department of Biochemistry and Molecular Biology, The Ben May Institute for Cancer Research, and The Tang Center for Herbal Medicine Research MC6027, University of Chicago, 5841 S. Maryland, Chicago, IL 60637, USA.

The enzyme steroid 5 alpha-reductase (EC 1.3.99.5) catalyzes the NADPH-dependent reduction of the double bond of a variety of 3-oxo-Delta(4) steroids including the conversion of testosterone to 5 alpha-dihydrotestosterone. In humans, 5 alpha-reductase activity is critical for certain aspects of male sexual differentiation, and may be involved in the development of benign prostatic hyperplasia, alopecia, hirsutism, and prostate cancer. Certain natural products contain components that are inhibitors of 5 alpha-reductase, such as the green tea catechin (-)-epigallocatechin gallate (EGCG). EGCG shows potent inhibition in cell-free but not in whole-cell assays of 5 alpha-reductase. Replacement of the gallate ester in EGCG with long-chain fatty acids produced potent 5 alpha-reductase inhibitors that were active in both cell-free and whole-cell assay systems. Other flavonoids that were potent inhibitors of the type 1 5alpha-reductase include myricetin, quercitin, baicalein, and fisetin. Biochanin A, daidzein, genistein, and kaempferol were much better inhibitors of the type 2 than the type 1 isozyme. Several other natural and synthetic polyphenolic compounds were more effective inhibitors of the type 1 than the type 2 isozyme, including alizarin, anthrarobin, gossypol, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and octyl and dodecyl gallates. The presence of a catechol group was characteristic of almost all inhibitors that showed selectivity for the type 1 isozyme of 5 alpha-reductase. Since some of these compounds are consumed as part of the normal diet or in supplements, they have the potential to inhibit 5 alpha-reductase activity, which may be useful for the prevention or treatment of androgen-dependent disorders. However, these compounds also may adversely affect male sexual differentiation.

The articles below demonstrate the antibacterial effect of Dodecyl Gallate. The high antibacterial effect of Dodecyl Gallate promotes decreased inflammation in acne prone skin since the reduced level of bacteria allows healing process to occur without inflammatory by-products of bacteria.

Bioorg Med Chem. 2003 Sep 15;11(19):4255-62.

Molecular design of multifunctional antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA).

Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA. ikubo@uclink.berkeley.edu

Antibacterial activity of a series of alkyl gallates (3,4,5-trihydroxybenzoates) against Gram-positive bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains was evaluated. Gram-positive bacteria are all susceptible to alkyl gallates. Dodecyl gallate was the most effective against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate was bactericidal against this MRSA strain at any growth stage. This activity was observed even in the chloramphenicol-treated cells, but the rate of decrease of cell number was slower than that in the exponentially growing cells. The bactericidal activity of medium-chain alkyl gallates was noted in combination with their ability to disrupt the native membrane-associated function nonspecifically as surface-active agents (surfactants) and to inhibit the respiratory electron transport. Subsequently, the same series of alkyl protocatechuates (3,4-dihydroxybenzoates) were studied and the results obtained are similar to those found for alkyl gallates. The length of the alkyl chain is not a major contributor but is related to the activity.

Bioorg Med Chem. 2003 Feb 20;11(4):573-80.

Non-antibiotic antibacterial activity of dodecyl gallate.

Department of Environmental Science, Policy and Management, University of California, CA 94720-3112, Berkeley, USA. ikubo@uclink4.berkeley.edu

Dodecyl (C(12)) gallate (3,4,5-trihydroxybenzoate) (1) was found to possess antibacterial activity specifically against Gram-positive bacteria, in addition to its potent antioxidant activity. The time-kill curve study indicates that this amphipathic gallate exhibits bactericidal activity against methicillin resistant Staphylococcus aureus (MRSA) strains. Dodecyl (lauryl) gallate inhibited oxygen consumption in whole cells and oxidation of NADH in membrane preparation. The antibacterial activity of this gallate comes in part from its ability to inhibit the membrane respiratory chain. As far as alkyl gallates are concerned, their antimicrobial spectra and potency depend in part on the hydrophobic portion of the molecule. Bioorg Med Chem Lett. 2002 Jan 21;12(2):113-6.

Anti-MRSA activity of alkyl gallates.

Department of Environmental Science, Policy and Management, University of California, CA 94720-3112, Berkeley, USA. ikubo@uclink4.berkeley.edu

A series of alkyl gallates (3,4,5-trihydroxybenzoates) was found to show antibacterial activity against Gram-positive bacteria including methicillin resistant Staphylococcus aureus (MRSA) strains. For example, dodecyl (C(12)) gallate (1) exhibited bactericidal activity against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate is bactericidal against this MRSA strain. This bactericidal activity comes in part from its ability to inhibit respiratory electron transport systems. The length of the alkyl chain is not a major contributor but plays an important role in eliciting the activity.

This article demonstrates that dodecyl gallate is a powerful antioxidant. The antioxidant effect of dodecyl gallate allows healing of acne-prone skin without inflammation from oxidative radicals of bacteria or the immune system. This reduces the oxidative stress on human skin while making the skin more vibrant and less irritated.

Antioxidant activity of dodecyl gallate

J Agric Food Chem. 2002 Jun 5;50(12):3533-9.

Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112, USA. ikubo@uclinik4.berkeley.edu

Dodecyl (C12) gallate exhibits both potent chain-breaking and preventive antioxidant activity. The pyrogallol moiety is responsible for both activities. Dodecyl (lauryl) gallate prevents generation of superoxide radicals by xanthine oxidase, and this activity comes from its ability to inhibit the enzyme. The inhibition kinetics analyzed by Lineweaver-Burk plots found that dodecylgallate is a noncompetitive inhibitor for the generation of superoxide anion. Dodecyl gallate also inhibits formation of uric acid. The inhibition kinetics analyzed by Lineweaver-Burk plots found that dodecyl gallate is a competitive inhibitor for this oxidation. Mitochondrial lipid peroxidation induced by Fe(III)-adenosine 5′-diphosphate/reduced nicotinamide adenine dinucleotide was inhibited by dodecyl gallate while its parent compound, gallic acid, did not show this inhibitory activity. Dodecyl gallate protected mitochondrial functions and human red blood cells against oxidative stresses, but gallic acid showed little effect. The hydrophobic dodecyl group is largely associated with the preventive antioxidative activity.