Investigation of Some Food Additives Containing

Investigation of some food additives containing ... PHARMACIA, vol. 61, No. 3/2014 17

INVESTIGATION OF SOME FOOD ADDITIVES CONTAINING COMPOUNDS WIHT ANDROGENIC ACTIVITY AND THEIR ANALYTI- CAL STUDY St. Ivanova1, D. Obreshkova2, P. Atanasov3, K. Ivanov1, B. Zlatkov1 1Medical University – Plovdiv, Faculty of Pharmacy 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University – Sofia 3Clinic of THKI, UMHATEM „N.I.Pirogov”– Sofia 4Faculty of Pharmacy, Medical University – Plovdiv

Abstract. The market of food additives is growing. There is a big demand of food supplements with androgenic compounds from extracts of Tribulus terrestris Linn., Rhaponticum carthamoides L. or tree Yohimbe. Food additives with androgenic effects very often contain steroidal saponins, ecdysteroids, Yohimbine, De- hydroepiandrosterone (DHEA) or combination of these substances. The steroids are forbidden substances in food supplements. Recent studies show that non – hormonal additives such as vitamins, minerals and aminoacids can contain a not declared on the labels of the products anabolic androgenic steroids: pro- hormones of Testosterone or 19-Nortestosterone. These undeclared substances can cause health risks to consumers and might lead to positive results in sports doping control, especially for Nandrolone metabolite Norandrosterone. This paper reviews investigation of some food additives, containing compounds with androgenic activity like extracts from Tribulus terrestris Linn., ecdysteroids, Yohimbine and DHEA and the most used methods for their analysis.

Key words: additives, androgenic effects, Tribulus, analysis.

Introduction. taining the prohibited growth hormone – releasing The consumption of food supplements and their peptide – 2 are detected [3]. Some authors offer ana- importance in people’s life are constantly increasing lytical strategies for the detection of non – labelled [1]. This review is focused on some food additives anabolic androgenic steroids in nutritional supple- containing compounds with androgenic activity and ments. Recent studies showed that non – hormonal the applied methods for their analysis. In accordance supplements such as vitamins, minerals and amino with the European Community Directive of 2002 acids can contain undeclared anabolic androgenic (Directive 2002/46/EC) and Bulgarian legislation, is- steroids, which can cause health risks to consumers sued by the Ministry of Health 01.08.2005, food ad- [4]. Food additives containing extracts from Tribulus ditives are defined as substances, containing concen- terrestris Linn., ecdysteroids, Yohimbine and DHEA trated nutrients or other elements with a nutritional or possess androgenic effect. physiological effect, alone or in combination, distrib- uted in certain dosage forms designed to enrich the I. Food additives with extract from Tribulus ter- diet. The fact that food supplements are from plant restris Linn. or animal origin, does not make them safe. The food Extracts from Tribulus terrestris Linn. possess additives are tested for efficiency, which is support- antibacterial [5, 6], antifungal [5, 7], antihelmintic ed by scientific evidence, but in some supplements (Tribulosin and β-sitosterol-D-glucoside) [8], analge- the qualitative and quantitative composition of their sic [9], antihypertensive [10], diuretic [11], hypogly- components is not declared [2]. cemic [12], hypolipidemic [13] and cytotoxic (Meth- The steroids are forbidden substances in food ad- ylprotodioscin) [14] activity and are applied for skin ditives. In 2009 and 2010, dietary supplements con- care [15] and human hormones regulation [16, 17]. 18 PHARMACIA, vol. 61, No. 3/2014 St. Ivanova, D. Obreshkova, P. Atanasov, K. Ivanov, B. Zlatkov

Active compounds in extracts from Tribulus ter- Table 1. Food additives with extracts from Tribulus terrestris Linn. are furostanol [18, 19] saponins and oth- restris Linn. er steroidal saponins: Terrestrinins A and B [20], Ter- 1. Bali Mojo caps. Not labelled restrosins A, B, C, D, E [21], Tribulosaponin B [22], Fountain Of Youth HGH lignanamides (Tribulusamides A and B), Tribulosap- 2. Not labelled Complete,700 g powder onin B (Fig. 1), alkaloids, flavonoids rutin, quercetin 3 Irexis caps. Not labelled and kaempferol [23]. In the Bulgarian plant extracts, the main components are Protodioscin, Dioscin (Fig. 4 Libilov (USA), 90 caps. Not labelled 1.), Metylprotodioscin, Prototribestin [24]. The first 5 Natrolex, 60 caps. Not labelled Natural-T (Top Secret Nutrition), standardized preparation is the bulgarian ”Tribestan” 6. Not labelled (Sopharma). More products were developed lately as 90 caps. preparations or food supplements and are used for 7. Virectin, 90 caps. Not labelled impotence disorders. The product UNEX is proposed Test Booster (Blade Nutrition), 8. 100 mg as diuretic [11]. 60 caps. In Table 1. are presented food supplements with 9 Anotesten (Muscletech) 250 mg extracts from Tribulus terrestris Linn. 10 Tribex (Biotest), 60 caps. 250 mg 11 Tribestan (Sopharma), 60 tabl. 250 mg II. Food supplements with ecdysteroids. 12 Tribosten (Thermolife), 60 tabl. 250 mg Food supplements with ecdysteroids are considered to possess androgenic effect. Recent data shows 13. MuscleTech TEST HD, 90 caps. 250 mg that in plants over 250 ecdysteroid analogs have been 14. Everlastin, 30 caps. 300 mg identified. Rich of ecdysteroids is the plant Rhapon- 15. Testogenix, 30 caps. 300 mg ticum carthamoides (Maral root), growing wild in 16. Testosyn, 60 caps. 300 mg Southern Siberia, Kazakhstan, Altay, Eastern Eu- 17. TribuPlex (MRM), 60 caps. 400 mg rope. Animal studies indicate that Maral root may 18. Tribestan (Sopharma), 60 tabl. 500 mg 19 Tribulus (Now), 100 caps. 500 mg 20 Trioxalon 500 (AST), caps. 500 mg Tropinol XP (iForce Nutrition), 21. 563 mg 100 caps. 22. Tribulus (Optimum), 50 caps. 625 mg Tribulus Pro (Universal Nutrition), 23. 625 mg 100 caps. 24. TribX (All Max Nutrition), 90 caps. 750 mg 25. Tribulus (MET-Rx), 90 caps. 750 mg Protodioscin Dioscin Tribulus 2400 (iForce Nutrition), 26. 800 mg 90 caps. 27. Tribulus (Now), 180 tabl. 1000 mg 28. Tribuvar, (S.A.N), 90 tabl. 1000 mg 29. Tribulus Terrestris, 100 caps. 1250 mg have a beneficial effect on memory and learning in rats [25], increases working capacity of skeletal muscles and possesses anabolic and adaptogenic effects in rats [26]. Ecdysteroids include insect moulting hormones, which regulate insect development, and similar chemical structures widely spread through- out plants and affecting various biological functions [27]. 20-Hydroxyecdysone is derivative of Ecdysone (Fig. 2.) and is the most common naturally occurring ecdysteroid, which controls the ecdysis (moult- Fig 1. Chemical structures of Protodioscin and Dioscin. ing) and metamorphosis of arthropods. It is also a Investigation of some food additives containing ... PHARMACIA, vol. 61, No. 3/2014 19 phytoecdysteroid produced by various plants, includ- 3β-reductase and the formation of 3α-ecdysteroids by ing Cyanotis vaga, where its purpose is presumably influence of 3-dehydroecdysteroids 3α-reductase. to disrupt the development and reproduction of in- Food supplements with 20-Hydroxyecdysterone sects. In arthropods the compound acts through the are summarized on Table 2. ecdysone receptor. Although in mammals this receptor is nor included, 20-Hydroxyecdysone may affect III. Food supplements containing Yohimbine. mammalian biological systems. 20-Hydroxyecdysone 1. Effects of Yohimbine. is an ingredient of some bodybuilding nutritional sup- Yohimbine is a mild monoaminooxidase inhibitor plements, which aim to enhance physical performance with stimulant and aphrodisiac effects and blocks the [28], strength or athletic improvement [29]. presynaptic and postsynaptic α2 receptors. Blockade of On Fig. 3 is illustrated the enzymic pathway postsynaptic α2 receptors causes minor corpus caverno- for transformation of 3-dehydroecdysteroids to sum smooth muscle relaxation. The majority of adreno- 3β-ecdysteroids in presence of 3-dehydroecdysteroid- ceptors in the corpus cavernosum are of the α1 type. Blockade of pre – synaptic α2 receptors facilitates the release of several neurotransmitters in the corpus cavernosum – nitric oxide and norepinephrine. Whereas nitric oxide released in the corpus cavernosum is the major vasodilator contributing to the erectile process, norepinephrine is the major vasoconstrictor through stimula- tion of α1 receptors on the corpus cavernosum smooth muscle. Under physiologic conditions, nitric oxide at- tenuates norepinephrine vasoconstriction. The clinical use of Yohimbine in the management of impotence shows a delayed action which is inconsistent with its short apparent half life and thus its activity may reside

Table 2. Food additives with 20-Hydroxyecdysterone. 1. MMUSA T, 30 caps. Not labelled 2. Vemoherb, 90 caps. Not labelled 3. Leuzeia plus, 90 caps. 15 mg 4. Myprotein T Matrix, 180 tabl. 30 mg 5. USN 19 Anabol Testo, 90 caps. 50 mg Supreme Sports Enhancements 6. 250 mg Omnibolic, 120 caps. Fig 2. Chemical structures of Ecdysone and 20-Hydroxy- ecdysone. Ecdysone 20-Hydroxyecdysone 7. Rhaponticum carthamoides solution 20 ml

Fig 3. Enzymic interconversions of 3β-ecdysteroids, 3-dehydroecdysteroids and 3α- ecdysteroids. 20 PHARMACIA, vol. 61, No. 3/2014 St. Ivanova, D. Obreshkova, P. Atanasov, K. Ivanov, B. Zlatkov in possible metabolites that can display a biological half Probable biosynthetic pathway for the formation life longer than that of the parent drug. Continuous ad- of Yohimbe alkaloids is presented on Fig. 4. [32]. ministration of Yohimbine, as opposed to on – demand In studies aimed at measuring the actual content administration, might result in less norepinephrine of Yohimbine versus the one declared on the label, it output due to increased turnover or α1 receptors down was found that Yohimbine content ranged from none regulation via a feedback mechanism. α1 blockers pre- up to 18.8 mg/serving. Quantitative data for the other vent vasoconstriction caused by norepinephrine [30]. Yohimbe alkaloids are not available but would also be Depending on dosage Yohimbine can either increase required in view of their known or potential biological or decrease systemic blood pressure (through vasocon- activity. Yohimbe bark extracts for use in food supple- striction or vasodilation, respectively): small amounts ments should be evaluated based on existing toxico- of Yohimbine can increase blood pressure, while large logical data and data in the chemical specifications. amounts can dangerously lower blood pressure. The No data describing the absorption, distribution, me- therapeutic index of Yohimbine is quite low. The range tabolism and excretion of Yohimbe alkaloids after oral between an effective dose and a dangerous dose is very administration of Yohimbe bark preparations such as narrow [31]. A typical dose for sexual dysfunction is 15 extracts are available. When administered to humans, – 30 mg, whereas 100 mg is considered dangerous. This Yohimbine is rapidly absorbed and its bioavailability may also lead to increasing of panic disorder type reac- is reported to range widely from 7 % to 87 %. In the tions or heart attack. More serious adverse effects may liver Yohimbine undergoes oxidation to its pharmaco- include seizures and renal failure. Yohimbine should not logically active metabolite 11-Hydroxy-yohimbine. be consumed by anyone with liver, kidney, heart disease This metabolic pathway is dependent on the two cy- or a psychological disorder. tochrome P450 polymorphic variants CYP2D6 and 2. Methods for synthesis of Yohimbine. CYP3A4. The great interindividual variability in the Yohimbine and its isomers are monoterpenoid – clinical effects may be attributable to the differences in derived indole alkaloids formed from the initial con- densation of tryptamine with secologanin in the presence of strictosidine synthase to form strictosidine. The tryptamine portion of the alkaloid is derived from the decarboxylation of tryptophan with tryptophan decarboxylase enzyme. Strictosidine, after de- glycosylation with glycosidase, through a series of reactive intermediates forms 4,21-dehydrocorynan- theine aldehyde which undergoes isomerisation and reduction to afford Yohimbine. A laboratory synthesis of Yohimbine is illustrated on Fig. 3.

Fig 4. A Laboratory synthesis of Yohimbine. Fig 5. Biosynthesis of Yohimbine. Investigation of some food additives containing ... PHARMACIA, vol. 61, No. 3/2014 21 the hepatic metabolism. No short – term or subchronic Table 3. Food additives with Yohimbine. toxicity and carcinogenicity studies on yohimbe bark VPX Redline Xtreme Shot, 90 1. Not labelled or its preparations were available [32]. ml solution 3. Yohimbine in food supplements. BPI Sports 1.M.R Vortex, 150 g 2. Not labelled Food additives containing Yohimbine are also con- powder sidered to possess androgenic effects. Yohimbine, the Physique Enhancing Science 3. Not labelled major alkaloid of Yohimbe bark and Raubasine, an- Alphamine, 150 g powder other alkaloid occurring in lower concentrations in USP labs Jack 3 Advanced, 230 4. Not labelled the bark, are used as active ingredients in a number of g powder medicinal products for which adverse effects are de- 5. Cellucor Super HD, 10 caps. Not labelled scribed as headache, nausea, increased urinary urge, Cellucor D4 Thermal Shock, insomnia, anxiety, restlessness, irritability, increase of 6. Not labelled 120 caps. blood pressure and pulse rate, palpitation, dizziness, vomiting, anorexia, gastric complaints, diarrhoea, 7. Evogen Lipocide, 60 caps. Not labelled HIT Supplements Torch, 135 flush, sweating, shivering, allergic reactions, nervous- 8. Not labelled ness, hypotension, tremor, bronchospasm, dysuria, de- caps. Muscle Meds Methyl Burn creased urge, genital pains, exanthema. 9. Not labelled Few states have registered medicinal products con- Extreme, 60 caps. Yohimbine HCl (4 Dimension taining Yohimbine for oral use (tablets). There is a sin- 10. 2.5 mg gle registered medicinal product containing Raubasine Nutrition), 90 caps. Fighter Diet FDXtreme Burn, in Italy (peroral drops or coated pills). In Ireland, the 11. 3 mg use of Yohimbe is not permitted in traditional herbal 60 caps. medicinal products, as it would be controlled as a pre- 12. Lipo-6 (Nutrex), 240 caps. 3 mg scription medicine under the terms of the Medicinal Hydroxycut Hardcore Elite 13. 28 mg Products Regulations. The United Kingdom does not (MuscleTech) powder consider any products containing P. yohimbe or Yo- Hydroxycut Hardcore Elite 14. 28 mg himbine to be classified as foods. In the UK the sale (MuscleTech), 180 caps. and supply of botanicals containing P. yohimbe are 15. Male Fuel (Twinlab), 120 caps. 400 mg controlled under the Medicines (Retail Sale or Sup- 16. Yohimbe Bark (Natrol), 90 caps. 500 mg ply of Herbal Plants Remedies) Order 1977 SI 2130. Kamagra (Borolla), 30 caps. The use of the bark of P. yohimbe is prohibited in the production of foods in the Czech Republic. The use of Yohimbine 50 mg P. yohimbe and its alkaloids is not authorised as food 17. Tribulus terrestris 150 mg or food supplements in Belgium (Royal Decree of 29 Ginseng 150 mg August 1997). Food supplements with P. yohimbe L-Arginine 250 mg herbal preparations are not allowed on the Dutch mar- Kamasutra (Borolla) 30 caps. ket based on the Warenwetbesluit Kruidenpreparaten Yohimbine 5 mg (Letter from EMA, personal communication, April Pyridoxine 6 mg 2013). The Austrian food codex (Codex Alimentarius 18 Zinc aspartate 7 mg Austriacus, Österreichisches Lebensmittelbuch) in- cludes the species P. johimbe in a list of plants not to be Extract of Ginkgo biloba 20 mg used for the production of herbal additives. The Dan- α- Tocopherol 20 mg ish Drogelisten lists Yohimbe (Corynanthe johimbe K. Schum) as a plant that is unacceptable as food regard- less of amount [32]. On Bulgarian market are aviable chloric acid lead to intermediates which are trans- many food additives containing Yohimbine. Food sup- formed to Dehydroepiandrosterone. plements with Yohimbine are presented on Table 3. On Table 4. are summarized additives containing Dehydroepiandrosterone. IV. Food supplements with (Dehydroepiandros- For analysis of different compounds in food sup- terone (DHEA). plements the very often applied are the following 16-Dehydropregnenolone acetate with hydroxy- methods: 1) HPLC with UV – detection for: a) deter- lamine form oxime, which after reaction with hydro- mination of L – Arginine in Tonotyl® solution [33]; 22 PHARMACIA, vol. 61, No. 3/2014 St. Ivanova, D. Obreshkova, P. Atanasov, K. Ivanov, B. Zlatkov

Fig 6. Synthesis of Dehydroepiandrosterone.

Table 4. Additives containing Dehydroepiandrosterone. plied methods are HPLC [40] and GC [41]. DHEA (Blade Nutrition Test Booster), 25 mg Tribulus extracts from roots and fruits after extrac- 1. 180 caps. tion with of acetonitrile are analyzed by reverse phase 2. DHEA (MRM), 90 caps. 25 mg HPLC at the following conditions: gradient elution with mobile phase: phosphoric acid buffer with pH = 3 3. DHEA (Natrol), 300 tabl. 25 mg and acetonitrile, injection volume: 20 μl, flow rate: 1.0 4. DHEA Fast Dissolve (Natrol), 30 tabl. 25 mg ml/min., UV – detection at λ = 203 nm [11]. 5. DHEA MAX (Nutraceutics), 60 tabl. 25 mg For determination of glycosidic furostanolic and 6. DHEA (Ultimate Nutrition), 100 caps. 25 mg spirostanolic saponins from dietary supplements of 7. DHEA (Healthy 'N Fit), 100 caps. 50 mg Tribulus terrestris Linn. is applied an HPLC-ESI- 8. DHEA (MRM), 60 caps. 50 mg MS method using Protodioscin as external standard 9. DHEA (Natrol), 60 tabl. 50 mg [42]. Steroidal saponins in Tribulus terrestris Linn. extracts are quantified by RP HPLC with evaporative 10. DHEA (S.A.N.), 90 caps. 50 mg light scattering detection [43]. 11. DHEA (Ultimate Nutrition), 100 caps. 50 mg Chromatograms of comparative analytical inves- 12. DHEA (PharmaFreak), 28 caps. 60 mg tigation of Tribulus terrestris Linn. preparations from 13. DHEA (Maximum Nutrients), 60 caps. 100 mg different manufacturers [44] are shown on Fig 5. Analysis of methanol extracts of Tribulus ter- b) control of aminoacids in organic foods and food restris Linn. are analyzed by GC – MS, with capil- supplements [34]; c) quality control of content of lary column with dimethyl polysiloxane (30 m x 0.25 Vitamin C and Vitamin B6 in food supplements and mm, 1 μm), oven temperature: 280oC, injection port drugs [3]; 2) gas chromatography with flame ioniza- temperature: 250oC, helium flow rate as 1 ml/min., tion detector for: determination in food supplements ionization voltage: 70 eV. On Table 5 are presented – tablets of total content of aminoacids [36] and of name, retention time and peak areas of the analyzed free aminoacids [37] and determination of total con- by GC – MS components in the methanolic extract of tent and for quantity of free aminoacids in liquid food Tribulus terrestris Linn. additives [38]; 3) TLC – densitometric method for In Tribulus terrestris Linn. extracts spirostanol sap- simultaneous analysis of L – Leucine and L – Valine onin Diosgenin and furostanol saponins are determined in additives [39]. by TLC – densitometric analysis after derivatization with an anisaldehyde sulfuric acid, stationary phase:

V. Methods for analysis of extracts from Tribu- precoated silica gel 60 F254 plates, mobile phase: tolu- lus terrestris Linn. in food supplements. ene : ethyl acetate : methanol = 7: 3 : 1 v/v, detection For the determination of steroidal saponins from at 430 nm [46]. In fruits of Tribulus terrestris Linn. extracts from Tribulus terrestris Linn. the most ap- steroidal saponins Protodioscin and Prototribestin are Investigation of some food additives containing ... PHARMACIA, vol. 61, No. 3/2014 23

VII. Methods for analysis of Yohimbine in supplements. Different methods were adopted for analyzing of Yohimbine hydrochloride, including spectroscopic, voltammetric, polarographic chemiluminometric methods, TLC, HPLC and gas chromatography [52]. For a qualitative and quantitative analysis Yo- himbine in commercial supplements are reported: 1) HPLC-UV-MS technique with atmospheric pressure chemical ionisation and electrospray ionisation [53]; 2) ultra performance liquid chromatography using

Waters Acquity BEH C18 column, gradient elution with mobile phase: 0.1 % v/v aqueous ammonium hydroxide and 0.1 % ammonium hydroxide in methanol and mas detection [54, 55]; 3) non – aqueous Fig 7. Chromatogram of preparation with Tribulus capillary electrophoresis [56] and gas chromatogra- terretris Linn. phy with mass spectrometry [56, 57]. Metabolites Table 5. GC – MS analysis of Tribulus terrestris Linn. [45]. 10- and 11-Hydroxy-yohimbine are analyzed by the normal phase HPLC [58]. Peak area N: t Name of compound According to European Food Safety Authority R [%] (EFSA) there are no official inter-laboratory validat- 3,7,11,15-tetramethyl- 1. 14.52 1.04 ed methods for the determination of Yohimbine and 2-hexadecan-1-ol accompanying alkaloids in food supplements [32]. 2. 16.27 n-Hexadecadienoic acid 8.83 n-Hexadecadienoic acid 3. 16.58 0.74 VIII. Methods for analysis of food supplements ethyl ester with Dehydroepiandrosterone. 4. 18.55 Phytol 0.99 For the determination of Dehydroepiandroster- 5. 18.86 9,12-Octadecadienoic acid 1.86 one are applied HPLC with stationary phase: Zorbax 9,12,15-Octadecadienoic Rx C column, mobile phase: acetonitrile : 0.025 M 6. 18.95 8.58 18 acid phosphate buffer = 60 : 40 v/v, or 75 : 25 v/v, UV 7. 19.26 Octadecanoic acid 2.95 detection at λ = 292 nm [59]. Androstenedione, De- 1.2-benzenedicarboxylic hydroepiandrosterone and Testosterone are analysed 8. 24.82 9.27 acid, diisoocthyl ester by liquid chromatography – mass spectrometry on stationary phase Gemini C column (100 mm x 2.0 9. 31.46 α-Amyrin 65.73 18 mm, 3 μm), injection volume: 50 μl, oven tempera- quantified through HPTLC on precoated silica gel 60 ture: 30 °C [60]. A HPLC with electrospray ionization - mass spectrometric (LC–ESI-MS/MS) method F254 aluminum plates and mobile phase of n – butanol : glacial acetic acid : water = 80 : 6 : 20 v/v [47]. is reported for the simultaneous quantification of Tes- tosterone and its precursor, Dehydroepiandrosterone, VI. Methods for analysis of 20-Hydroxyecdysone after derivatized with 2-hydrazino-1-methylpyridine, in additives. is developed [61]. 20-Hydroxyecdysone is determined by the fol- A gradient RP-HPLC method is developed for the lowing methods: 1) TLC with detection at λ = 254 separation of Dehydroepiandrosterone, its sulfate es- nm or at λ = 366 nm [48]; 2) spraying the TLC plate ter (DHEA-S), its three C7-oxidized metabolites, and with vanillin - sulfuric acid reagent to obtaining tur- its biosynthetic derivates (Androstenedione, Testos- quoise fluorescenting spots at λ = 366 nm [48]; 3) terone, Estradiol, Pregnenolone) [62]. densitometric evaluation at λ = 250 nm in reflectance/ GC-MS analysis of the trimethylsilyl derivatives absorbance mode [49]; 4) HPLC [50, 51]; 5) micellar of the steroids is developed. The limit of detection electrokinetic chromatography [48]. was improved by the addition of a mixture of 1-N,N- diisopropylamino-n-alkanes to the final extract. 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 Corresponding author Danka Obreshkova Faculty of Pharmacy, Medical University - Sofia 2 Dunav Str. 1000 Sofia e-mail: [email protected]