Accepted Manuscript Title: Male enhancement Nutraceuticals in the Middle East market: Claim, pharmaceutical quality and safety assessments Author: Ghada ElHaddad Fatema ElAmrawy Ahmed ElYazbi Ahmed Eshra Mohamed I. Nounou PII: DOI: Reference:

S0378-5173(15)30023-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.07.006 IJP 15006

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

14-5-2015 26-6-2015 1-7-2015

Please cite this article as: ElHaddad, Ghada, ElAmrawy, Fatema, ElYazbi, Ahmed, Eshra, Ahmed, Nounou, Mohamed I., Male enhancement Nutraceuticals in the Middle East market: Claim, pharmaceutical quality and safety assessments.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Male enhancement Nutraceuticals in the Middle East market: Claim, pharmaceutical quality and safety assessments Ghada ElHaddad1, Fatema ElAmrawy1, Ahmed ElYazbi2, Ahmed Eshra1 and Mohamed I. Nounou1,* 1

Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt.

2

Department of Pharmacology, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt.

*

Corresponding author:

Mohamed Ismail Nounou, M.PSc., Ph.D. Assistant Professor Department of Pharmaceutics School of Pharmacy Alexandria University 1 El-Kartum Square Alexandria Egypt. (w) +2 03 4869512 (fax) +2 03 4871668 (cell Egypt) +2 012 21444930 (Cell USA) +1 806 494 5949; (email) [email protected], [email protected]

Gr aphical abstr act

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ABSTRACT The global market is invaded by male enhancement nutraceuticals claimed to be of natural origin sold with a major therapeutic claim. Most of these products have been reported by international systems like the Food and Drug Administration (FDA). We hypothesize that these products could represent a major threat to the health of the consumers. In this paper, pharmaceutical evaluation of some of these nutraceutical products sold in Egypt under the therapeutic claim of treating erectile dysfunction, are discussed along with pharmacological evaluation to investigate their safety and efficacy parameters. Samples were analyzed utterly using conventional methods, i.e.: HPLC, HPTLC, NIR, content uniformity and weight variation and friability. The SeDeM system was used for quality assessment. On the basis of the results of this research, the sampled products are adulterated and totally heterogeneous in their adulterant drug content and pharmaceutical quality. These products represent a major safety threat for the consumers in Egypt and the Middle East, especially; the target audience is mostly affected with heart and blood pressure problems seeking natural and safe alternatives to the well-established Phosphodiesterase 5 Inhibitors (PDE-5Is).

CHEMICALS STUDIED Sildenafil, CID:5212; Vardenafil, CID:110634; Tadalafil, CID:110635 Ammonium formate, CID: 2723923; Acetonitrile, CID: 6342; Methanol, CID: 887

3

KEYWORDS Phosphodiestrase inhibitors; Sildenafil; Clinical Study; SeDeM; Evaluation; Adulteration; Middle East; Nutraceuticals; Tiger King

1 INTRODUCTION

1 2

Nutraceuticals for male enhancement has been used throughout history (Brayn

3

and Smith, 1930; Chauhan et al., 2014). The rising number of people affected by

4

erectile dysfunction (ED) may be a leading factor contributing in the wide use of these

5

nutraceuticals. Remedies for ED and increasing sexual power has been described in

6

ancient cultures like the Pharonic Ebers Papyrus (Brayn and Smith, 1930) and

7

Ayurvedic Texts (Chauhan et al., 2014). Expectedly, by year 2025 approximately 322

8

million will be affected by erectile dysfunction worldwide (Ayta et al., 1999).

9

Although many synthetic drugs are available to treat these problems, some of

10

the drawbacks for these drugs include their high cost and also their ability to provoke

11

serious adverse effects (Chauhan et al., 2014). Effective natural treatments are therefore

12

still in demand (Chauhan et al., 2014). People are interested in seeking a natural

13

substance with the misconception that it could have less or no side effects (Pratap and

14

Rajender, 2012). However, there is little evidence from literature that recommend the

15

use of natural aphrodisiacs for the enhancement of sexual desire and performance

16

(Shamloul, 2010). Unfortunately, several herbal products in the global market for male

17

enhancement were found to be adulterated with undeclared phosphodiestrase 5

18

inhibitors (PDE-5Is): sildenafil, tadalafil, verdenafil and their analogues (Campbell et

19

al., 2013).

2 20

By inspecting the current OTC and tele-advertised nutraceuticals sold in the

21

Egyptian market under the claim of being 100% natural for the treatment of erectile

22

dysfunction, over 50 actual products were found. These products could represent a

23

major threat to the health of the consumers, especially because they are sold with a

24

major therapeutic claim in spite of the fact that they are nutraceuticals.

25

In this research, the illegal products were objectively and carefully analyzed

26

pharmaceutically, pharmacologically and clinically. High performance liquid

27

chromatography (HPLC), high performance thin layer chromatography (HPTLC) and

28

near infra-red (NIR) were used to determine the content of the nutraceuticals under

29

investigation. Physical properties were also assessed using content uniformity and

30

weight variation test together with friability test. Whilst SeDeM analysis was used to

31

investigate the pharmaceutical quality and inter and intra batch variability within

32

products (Aguilar-Diaz et al., 2014). SeDeM analysis was originally designed as a fast

33

preformulation and quality control tool (Aguilar-Diaz et al., 2014). The outcome of the

34

SeDeM analysis is a single diagram per formulation summarizing the overall quality

35

and reproducibility (Aguilar-Diaz et al., 2014).

36

Moreover, in-vivo study on rats was designed to the safety and the possible

37

adverse effects of these products on a biological system along with the possible

38

adulterants bioavailability. Finally, in order to assess the safety and efficacy of the

39

investigated products, a clinical study was designed on health volunteers.

40

Our objective is to assess the 100% natural label claim, pharmaceutical quality

41

and safety of male enhancements nutraceuticals sold in the Egyptian market as a

42

representation of the Middle East region on the time frame from 2013 to 2014.

3

43

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2 MATERIALS AND METHODS 2.1 Materials

45

Two grams of Sildenafil and Vardenafil were a generous gift from Bayer Inc.

46

100 grams of Tadalafil and Vardenafil were purchased from Changland technology co.,

47

Ltd., China. Viagra® 50 mg coated tablets (Sildenafil citrate, Pfizer, USA), Cialis® 20

48

mg tablets (Tadalafil HCL, Eli Lilly, USA) and Levitra® 20 mg tablets (Vardenafil

49

HCL, Bayer, Germany) were purchased from local pharmacies and used as reference

50

materials. Tiger King (Hong Kong wexixin bio-technology, china), Hercules (Hong

51

Kong Tianlong biology, china), Herbal Viagra (Tian bao Tsawo Gang Sanitation,

52

china), Plant Viagra (Tian bao biological engineering, china), and Natural Viagra

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(Qinghai baojiantang pharmaceutical, china) were the test male enhancement products

54

sold in the Egyptian market, purchased from different, strategically located and distant

55

regions. Triplicate packages from three different batches were purchased from each

56

location. All solvents and chemicals used were of analytical grade.

57

2.2 Methodology

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2.2.1 Adulterants’ detection and quantification

59

2.2.1.1 High Performance Liquid Chromatography (HPLC)

60

Agilent HPLC (Agilent-1260, isopump-G1310B, VWD-G1314F, Man inj-

61

G1328C, Germany) was used. Kinetics 5u XB-C18 100A column 250×4.5 mm with

62

security guard ULTRA cartridges HPLC C18 (4.6 mm, Phenomenex, USA) was the

63

stationary phase. The mobile phase used was a 50:50 mixture of acetonitrile: 10 mM

64

aqueous ammonium formate (37C, 1 ml/min flow rate).

4 65

UV-detector was used at wavelength (λmax) of 230 nm. 20 μl injection volume

66

was maintained throughout the analysis. The method was validated for reliable

67

concomitant quantification of Sildenafil, Vardenafil, and Tadalafil in the range of 0.2-

68

0.003125 mg/ml. Tablets were extracted in Methanol as described elsewhere (Zailina

69

et al., 2013).

70

2.2.1.2 High Performance Thin Layer Chromatography (HPTLC)

71

HPTLC was performed on CAMAG HPTLC-P57 Scanner-3 (CAMAG

72

Switzerland). Silica gel 60 F254 aluminum sheets (10*20 cm) were used. A mixture of

73

chloroform: ethanol (90:10) was used as the developing solvent. The chromatogram

74

was recorded at wavelength (λmax) of 254 nm (Al-Tahami, 2014; Naveen Bimal and

75

Sekhon, 2013).

76

2.2.2 Pharmaceutical quality assessment

77

2.2.2.1 Near-Infrared spectroscopy (NIR)

78

NIR spectra were recorded on Bruker™ near-infrared spectroscopy (Bruker-

79

MPA, Germany). Measurements were carried out with an optical resolution 16 cm-1

80

and 32 cm-1 over the spectra range 6200-5600 cm-1 (Vredenbregt et al., 2006; Zhang

81

and Su, 2014).

82

2.2.2.2 Pharmacopeial tests

83

Content uniformity and weight variation (British Pharmacopeia Commission,

84

2014c), dissolution testing (British Pharmacopeia Commission, 2014a) and friability

85

testing (British Pharmacopeia Commission, 2014b) were performed as described by the

86

British Pharmacopeia 2014 (B.P. 2014).

5 87

2.2.2.3 Pharmaceutical assessment technique; SeDeM diagram

88

SeDeM system is based on twelve input parameters and four output parameters.

89

Input parameters are bulk density, tapped density, inter-particle porosity, carr index,

90

cohesion index, Hausner ratio, angle of repose, powder flow, loss on drying,

91

Hygroscopicity, particle size, and homogeneity index (I ). While, output parameters

92

are radius of SeDeM diagram, parameter index (IP), parameters profile index (IPP), and

93

good compressibility index (IGC). SeDeM system was developed in the Service of

94

Development of Medicines Pharmaceutical Technology Unit, Pharmacy and

95

Pharmaceutical Technology Department at University of Barcelona (Spain) by

96

Montserrat Miñarro et al.(Perez et al., 2006; Sune-Negre et al., 2011).

97

The numerical value of the input parameters were obtained experimentally and

98

converted into the radius (r) of a scale from 0 to 10 (Sune-Negre et al., 2011). Detailed

99

calculations of all input and output parameters of SeDeM system are summarized in

100

online appendix 1.

101

2.2.3 Rats’ in-vivo study for safety assessment

102

Thirty male rats were used in this experiment (Wistar rats, animal house in

103

faculty of pharmacy, Alexandria University). Their average weight ranged from 195 to

104

220 g before the experiment. All rats were randomly assigned into three groups,

105

negative control, positive control (Viagra® coated tablets) and test group (Tiger King

106

tablets).

107

The appropriate dose of the formulation administered to each rat was calculated

108

by extrapolating the therapeutic dose for humans on the basis of body surface area ratio

109

using Paget table (Paget and Barnes, 1964). The doses were adjusted each three-day

110

depending on rats’ weight (Triple Beam balance, Spain). Doses were dissolved in

6 111

distillated water and orally administrated via oral needle once daily. The study period

112

was four weeks. The Research Ethics Committee (Institutional Review Board, IRB) at

113

the Faculty of Pharmacy (Alexandria University) approved the study.

114

2.2.3.1 HPLC rats blood analysis (Tripathi et al., 2013)

115

2 ml blood samples were collected 30 and 120 minutes post dosing in

116

Ethylenediaminetetraacetic acid (EDTA) tubes, centrifuged for 10 minutes at 1000 rpm

117

by Tap Top centrifuge-PLC-05, Taiwan. Supernatant was collected in 10 ml falcon

118

centrifuge tube and equivalent volume of acetonitrile was added. Centrifuge tubes were

119

shaken and centrifuged for 10 minutes at 1000 rpm. Supernatant was then filtrated via

120

0.45 μm and 0.22 μm nylon filters. Samples were assayed via HPLC analysis (Section

121

2.2.1.1).

122

Rats were wieghed before and after treatment. Sildenafil citrate concentration

123

(µg/ml) and amount (mg) in each rat plasma sample 30 and 120 miuntes post-product

124

oral administration were quantified.

125

2.2.3.2 Liver and kidney functions assessment (Abbott et al., 2004; Sultana and

126

Najam, 2012)

127

Several biochemical parameters were assessed before and after the study (4

128

weeks). 2 ml of blood samples were collected a week before the initiation of the

129

experiment and a week after the end of the experiment. In order to assess the kidney

130

functions, serum urea and serum creatinine were measured, using fully automated

131

methods on the Chemistry Analyzer Olympus AU400 (USA). Liver functions were

132

assessed by measuring the plasma activities of Alanine Aminotransferase (ALT),

133

Aspartate Aminotransferase (AST) and Gamma-glutamyl transferase (GGT). Those

134

enzymes were assessed on the same fully automated system, Olympus AU400. All

7 135

reagents, standards and controls were purchased from Beckman Coulter Co. (CA,

136

USA). Two levels of quality control material were assayed daily, at the beginning of

137

each run.

138

2.2.3.2.1 Serum urea concentration

139 140

141

Urea was determined kinetically without deproteinization according to the following reaction (Sampson et al., 1980): urease

 2NH4+ + CO2 Urea + H2O  

urease

142 143

+

2NH4 + α–ketoglutarate + NADH + H 2NAD+ 2H2O

   2L-glutamate

+

144

The decrease in sample (T) absorbance (ΔA/min) due to NADH+H+ oxidation

145

was monitored spectrophotometrically at λ 340 nm for 60 seconds, and compared to a

146

standard urea solution (S) of a known concentration (Cs) similarly treated.

147

2.2.3.2.2 Serum creatinine concentration

148

Creatinine was determined, without deproteinization, using Jaffé reaction in a

149

kinetic manner (Ward et al., 1976). The rate of increase in absorbance (ΔA) due to

150

complex formation between creatinine in the sample (T) and alkaline picrate reagent

151

was monitored kinetically over a period of 1 minute at max 500 nm, and compared to a

152

standard creatinine solution (S) of a known concentration (Cs) similarly treated.

153

2.2.3.2.3 Serum Alanine aminotransferase (ALT)

154

155 156

ALT activity was determined as follows (Bergmeyer, 1980): urease  L-glutamate + Pyruvate - oxoglutarate + L-alanine  

Pyruvate + NADH + H+ 

urease



 



Lactate + NAD

8 157

The decrease in absorbance per minute (∆A/min) at 340 nm (due to NADH +

158

H+ oxidation) was monitored kinetically for 3 minutes, with a standard that was treated

159

similarly. The enzyme activity was calculated and expressed in units/L.

160

2.2.3.2.4 Serum aspartate aminotransferase (AST)

161

AST activity was determined as follows (Burtis et al., 2012): urease

 L-glutamate + oxaloacetate -oxoglutarate + L-aspartate 

162

Oxaloacetate + NADH + H+ 

163

urease



 



L-malate + NAD

164

The decrease in absorbance per minute (∆A/min) at 340 nm (due to NADH +

165

H+ oxidation) was monitored kinetically for 3 minutes, with a standard that was treated

166

similarly. The enzyme activity was calculated and expressed in units/L.

167

2.2.3.2.5 Gamma-Glutamyl transferase (GGT)

168

γ-Glutamyl Transferase Reagent was used to measure the γ-glutamyl transferase

169

activity by an enzymatic rate method (Burtis et al., 2012). In the reaction, the γ-glutamyl

170

transferase catalyzes the transfer of a gamma-glutamyl group from the colorless

171

substrate, gamma-glutamyl-p-nitroaniline, to the acceptor, glycyclycine with

172

production of the colored product, p-nitroaniline.

173

The auto-analyzer monitored the change in absorbance at 410 nanometers. This

174

change in absorbance was directly proportional to the activity of γ-glutamyl transferase

175

in the sample and used to calculate and express γ-glutamyl transferase activity in

176

units/L.

9 177

2.2.3.3 Gross toxicity study

178

The gross toxicities of rats were assessed on weekly basis for 36 days and

179

observing righting reflex, skin ulceration, anxiety, hematuria, and loss on hair, loss on

180

activity, tremor, salivation, vomiting, diarrhea, and mortality. Gross toxicity was

181

assessed on a scale system from 0 to 10, indicating the number of rats showing

182

symptoms per group (10 rats) (Sultana and Najam, 2012). Scores were averaged within

183

each group throughout the study period.

184

2.2.4 Statistical analysis

185

One-way ANOVA and Bonferroni post-hoc test were used in the statistical

186

analysis (IBM SAS 9.1). Average and standard deviation (SD) are indicated in all

187

figures.

3 RESULTS

188 189

All tablets and batches of all analyzed products were inspected physically in its

190

intact, fine or course ground form prior to experimentation and analysis. Test products

191

have shown non-uniform and heterogeneous composition in their fine and course

192

ground forms, most significantly in Tiger King batches. Surprisingly, high similarity in

193

morphology was observed between some Plant Viagra and Natural Viagra tablets in

194

their course and fine ground On the other hand, Viagra®, Cialis® and Levitra® have

195

shown homogeneity in their morphological characteristics.

196

3.1 Adulterants’ detection and quantification

197

3.1.1 HPLC

198

The Inter-day precision and accuracy for Sildenafil, Tadalafil and Vardenafil

199

covering concentrations from 50 to 1000 ng/ml ranged from 1.56 to 8.24% and 95.11

10 200

to 103.30% for Sildenafil, from 2.33 to 10.07% and 98.57 to 103.12% for Tadalafil and

201

from 4.79 to 11.71% and 96.21 to 104.65% for Vardenafil respectively. The Intra-day

202

precision and accuracy for Sildenafil, Tadalafil and Vardenafil covering concentrations

203

from 50 to 1000 ng/ml ranged from 2.02 to 4.53% and 98.23 to 110.77% for Sildenafil,

204

from 0.76 to 3.5% and 98.64 to 102.59% for Tadalafil and from 1.54 to 2.86% and 98

205

to 100.36% for Vardenafil respectively.

206 207

Limit of quantification was 50 ng/mL for Sildenafil, Tadalafil and Varenafil,

208

corresponding to the lowest value of the calibration curve. Limit of detection for this

209

system was 20 ng/mL for Sildenafil and 40 ng/ml for both Tadalafil and Varenafil,

210

which was calculated to be the lowest concentration detected with reproducible results.

211

The linear range was found to be 50 – 5000 ng/mL for the three components.

212

Sildenafil citrate was detected in all analyzed products. 111, 42, 70, 54 and 18

213

units (tablet or capsule) of Tiger King, Plant Viagra, Hercules, Herbal Viagra and

214

Natural Viagra were analyzed. The mean values of Sildenafil citrate content in different

215

batches of Tiger King, Hercules, Plant Viagra, Natural Viagra and Herbal Viagra

216

ranged from 48.4 (SD of 21.8) to 76 (SD of 46), 25.9 (SD of 37.4) to 52.5 (SD of 36.9),

217

11.2 (SD of 16.8) to 22.2 (SD of 19.1), 78 (SD of 10.7) to 78.7 (SD of 9.6) and 62.1

218

(SD of 25.9) to 64.3 (SD of 28.2) mg respectively (Fig. 1A).

219

The analysis revealed the existence of two distinct populations within the test

220

products, as shown in fig. 2A. The mean values of Sildenafil citrate content in Tiger

221

King two populations were 35.4 (SD of 6) and 89 (SD of 27.9) mg respectively, and in

222

case of Plant Viagra, 1.4 (SD of 1.9) and 37 (SD of 6) mg respectively. For Hercules

223

two population, the mean values were 1.8 (SD of 1.9) and 73.8 (SD of 15) mg

11 224

respectively. Finally, in case of Herbal Viagra, the mean values were 0 (SD of 0) and

225

71.4 (SD of 9.6) mg respectively.

226

3.1.2 HPTLC

227

Only sildenafil citrate was analyzed via HPTLC, as a confirmatory tool for the

228

HPLC analysis, as all products under experimentation only contained Sildenafil citrate.

229

From the data shown in fig. 1B, the mean content of Sildenafil citrate (mg) in Viagra®

230

(n=22), Tiger King (n=24), Plant Viagra (n=22), Hercules (n=22), Natural Viagra

231

(n=15) and Herbal Viagra (n=28) was 51.27 (SD of 1.7), 11.69 (SD of 19.7), 28.87 (SD

232

of 35.7), 28.02 (SD of 34.2), 72.07 (SD of 9.3), and 63.60 (SD of 14) mg respectively.

233

As shown in fig. 2B, two populations were observed for the products. The mean

234

values of Sildenafil citrate content in Tiger King two populations were 54.86 (SD of

235

1.7) and 3.054 (SD of 0.4) mg respectively. In case of Plant Viagra, the mean values of

236

Sildenafil citrate content in their two populations were 77.62 (SD of 10) and 4.503 (SD

237

of 1.8) mg respectively. For Hercules two population, the mean values of Sildenafil

238

citrate content were 75.04 (SD of 6.3) and 4.515 (SD of 1.1) mg respectively. Finally,

239

in case of Herbal Viagra, the mean values of Sildenafil citrate content in their two

240

populations were 91.27 (SD of 1.6) and 63.60 (SD of 8.7) mg respectively.

241

3.2 Pharmaceutical quality assessment

242

3.2.1 NIR

243

NIR was performed to assess the variability and quality of the examined

244

products. Individual NIR spectra are shown in supporting fig. 1. Comprehensive

245

collective NIR spectra of all products together are illustrated in fig. 3. The Percentage

246

(%) correlation across batches within each brand of each product is shown and

12 247

illustrated in fig. 4A. The percentage correlation cutoff used, which indicates

248

uniformity and consistency within each product, was 96%. The Percentage of identical

249

tablets (96% correlation) across different batches of Viagra®, Tiger King, Hercules,

250

Plant Viagra, Natural Viagra and Herbal Viagra were 100%, 50%, 29%, 67%, 100%

251

and 92% respectively.

252

The percentage of Hercules tablets identical to Tiger King with 90%, 95% and

253

96% correlation were 51%, 9% and 2% respectively. Furthermore, percentage of Herbal

254

Viagra identical to Tiger King with 90%, 95% and 96% correlation were 89%, 47%

255

and 22% respectively. Moreover, percentage of Natural Viagra tablets identical to Tiger

256

King with 90%, 95% and 96% correlation were 93%, 60% and 36% respectively.

257

Finally, percentage of Plant Viagra tablets identical to Tiger King with 90%, 95% and

258

96% correlation were 71%, 18% and 11% respectively. (Fig. 4B).

259

3.2.2 Pharmacopeial tests

260

3.2.2.1 Content uniformity and weight variation

261

Tablets weight across batches ranged from 301 mg to 321 mg (SD of 5.6) for

262

Viagra®, 747 mg to 903 mg (SD of 31.6) for Tiger King, 180 mg to 400 mg (SD of

263

51.6) for Hercules, 713 mg to 769 mg (SD of 13.61) for Herbal Viagra, 449 mg to 785

264

mg (SD of 78.1) for Plant Viagra and 549 mg to 838 mg (SD of 71.7) for Natural Viagra.

265

(Supporting fig. 2A)

266

Tiger King, Hercules, Plant Viagra, Herbal Viagra and Natural Viagra were

267

assessed for content uniformity and weight variation according to the BP 2014. The

268

acceptance and M values pharmacopeial limits are ≤ 15 and within 98.5-101.5

269

respectively.

13 270

The M and acceptance values for weight variations of Viagra® batchs were 98.9

271

and 8.4 (Batch 1), 99.3 and 6.8 (Batch 2) and 98.5 and 5 (Batch 3). The M and

272

acceptance values for weight variations of Tiger King batchs were 101.5 and 324.4

273

(Batch 1), 101.5 and 216.2 (Batch 2), 101.5 and 276.7 (Batch 3) and 101.5 and 365.3

274

(Batch 4). In case of Hercules batches, the M and acceptance values for weight

275

variations were 101.5 and 482.4 (Batch 1), 101.5 and 460.2 (Batch 2) and 101.5 and

276

408.6 (Batch 3). For Herbal Viagra batches, the M and acceptance values for weight

277

variations were 101.5 and 361.6 (Batch 1), 101.5 and 358.4 (Batch 2) and 101.5 and

278

516.3 (Batch 3). The M and acceptance values for weight variations of Plant Viagra

279

were 98.5 and 201.2 (Batch 1), 98.50 and 219.4 (Batch 2) and 98.50 and 189.7 (Batch

280

3). Last, the M and acceptance values for weight variations of Natural Viagra batches

281

were 102.5 and 362.7 (Batch 1), 102.50 and 294.7 (Batch 2), 102.50 and 304.2 (Batch

282

3). Only Viagra® did meet the weight variation pharmacopeial standards. The data are

283

shown in the supporting fig. 2B and 2C.

284

The M and acceptance values for content uniformity of Viagra batchs were

285

99.10 and 6.1 (Batch 1), 99.10 and 6.1 (Batch 2) and 98.60 and 4.6 (Batch 3). The M

286

and the acceptance values for content uniformity of Tiger King batchs were 100.50 and

287

104.7 (Batch 1), 98.50 and 94.4 (Batch 2), 98.50 and 98.7 (Batch 3) and 101.5 and

288

134.14 (Batch 4). For Hercules batches, the M value and the acceptance values for

289

content uniformity were 98.50 and 220.4663 (Batch 1), 101.5 and 173.5 (Batch 2) and

290

98.5 and 215.2017 (Batch 3). The M value and the acceptance values for content

291

uniformity of Herbal Viagra batchs were 101.50 and 131.77 (Batch 1), 101.5 and

292

124.45647 (Batch 2), 101.5 and 211.68 (Batch 3). For Plant Viagra batches, the M and

293

the acceptance values for content uniformity were 98.50 and 147.50 (Batch 1), 98.50

294

and 155.30 (Batch 2) and 98.50 and 140.50 (Batch 3). Last, the M and the acceptance

14 295

values for content uniformity of Natural Viagra batchs were 102.5 and 103.70 (Batch

296

1), 102.50 and 110.30 (Batch 2) and 102.50 and 86.60 (Batch 3). Only Viagra® did meet

297

the content uniformity pharmacopeial standards. The data are shown in the supporting

298

fig. 2B and 2C.

299

3.2.2.2 Dissolution

300

The average amounts of Sildenafil citrate (mg) released from different batches

301

of Viagra were 44.76 (SD of 1.74), 50.79 (SD of 2.39), 51.33 (SD of 1.93), 50.97 (SD

302

of 1.92), 51.05 (SD of 2.25), 51.06 (SD of 2.02) mg after 5, 15, 30, 45, 60 and 120

303

minutes time intervals respectively. For Tiger King batches, the mean amounts of

304

Sildenafil citrate (mg) released were 12.25 (SD of 4.24), 45.78 (SD of 11.34), 57.88

305

(SD of 22.64), 57.85 (SD of 22.8), 57.74 (SD of 26.12), 57.67 (SD of 26.23) mg after

306

5, 15, 30, 45, 60 and 120 minutes time intervals respectively. The mean amounts of

307

Sildenafil citrate (mg) released from different batches of Hercules were 0, 5.74 (SD of

308

9.13), 7.09 (SD of 11.26), 9.18 (SD of 14.59), 9.10 (SD of 14.47), 9.08 (SD of 15.72)

309

mg after 5, 15, 30, 45, 60 and 120 minutes time intervals respectively. For Herbal

310

Viagra batches, the average amounts of Sildenafil citrate (mg) released were 42.37 (SD

311

of 20.34), 55.99 (SD of 10.28), 71.05 (SD of 3.71), 75.08 (SD of 1.48), 87.86 (SD of

312

11.89), and 87.27 (SD of 12.04) mg respectively after 5, 15, 30, 45, 60 and 120 minutes

313

time intervals respectively. The mean amounts of Sildenafil citrate (mg) released from

314

different batches of Plant Viagra were 22.51 (SD of 9.8), 39.21 (SD of 15.7), 51.68 (SD

315

of 27), 57.59 (SD of 26.2), 62.48 (SD of 26.7), 82.53 (SD of 21.2) mg after 5, 15, 30,

316

45, 60 and 120 minutes time intervals respectively. Last, the average amounts of

317

Sildenafil citrate (mg) released from different batches of Natural Viagra were 13.14

318

(SD of 0.5), 14.97 (SD of 1.5), 15.99 (SD of 2.1), 16.89 (SD of 1.9), 17.05 (SD of 1.9),

319

and 17.13 (SD of 2.1) mg after 5, 15, 30, 45, 60 and 120 minutes time intervals

15 320

respectively. Only Viagra® did show consistant release pattern with 50 mgs of

321

Sildenafil citrate released in less than 20 minutes with minimal standard deviation. Data

322

is shown in the supporting fig. 3.

323

3.2.2.3 Friability

324

The average percentages of weight loss for different batches of Viagra, Tiger

325

King, Plant Viagra, Natural Viagra and Herbal Viagra were 0.061 with SD of 0.002,

326

20.78 with SD of 13.28, 12.48 with SD of 95.91, 0.2 with SD of 0.005 and 5.31 with

327

SD of 2.0 respectively. The percent weight loss after friability test pharmacopeial limit

328

is 1%. Only Viagra® and Plant Viagra did pass the BP 2014 friability test. (Supporting

329

fig. 4)

330

3.2.3 Pharmaceutical assessment technique; SeDeM diagram

331

The optimal values for the SeDeM diagram output paramters should be within

332

0-10 for radius average for all parameters, 0.5 for parameter index (IP), 5 for parameter

333

profile index (IPP) and 5 for good compresibility index (ICG). Furthermore, all

334

individual input parameters raduis should be within 0-10. The values of all these

335

paramters, along with the ploted SeDeM

336

variabilities within batches for each product. The results are illustrated in fig. 5 and fig.

337

6.

diagram, can also indicate and detect

338

The radius average for three different Viagra® batches were 5.78, 5.9 and 5.87

339

respectively. The IP value for the three different Viagra® batches were 0.5. The IPP

340

value for the three different Viagra® batches were 5.78, 5.89 and 5.87 respectively.

341

Last, the ICG value for the three different Viagra® batches were 5.54, 5.65 and 5.63

342

respectively.

16 343

In case of Tiger King, the radius average for four different batches were 9.38,

344

8.91, 7.63 and 8.25 respectively. The IP value for the four different batches were 0.58,

345

0.5, 0.59 and 0.59. The IPP value for the four different batches were 9.38, 8.9, 7.63 and

346

8.2 respectively. Last, the ICG value for the four different batches were 9.0, 8.54, 7.32

347

and 7.87 respectively. With respect to the individual input parameters for Tiger King

348

batches used for the construction of the SeDeM diagram, powder flow and homogenity

349

index were far outside of the acceptable range of 0-10 (-10.5 with SD of 0.4 in powder

350

flow and 53.38 with SD of 7.98 for homogenity index). Furthermore, all of the SeDeM

351

diagram output paramters were outside of the recommended values range (0.57 for IP

352

with SD of 0.77, 8.53 for IPP with SD of 0.77 and 8.18 for ICG with SD of 0.74). These

353

results and the heterogenisity of SeDeM diagrams of the four batches of Tiger King

354

show the inconsistancy of Tiger King formulations across batches.

355

In case of Hercules, the radius average for three different batches were 4.93,

356

4.14 and 4.51 respectively. The IP value for the three different batches were 0.67, 0.56

357

and 0.58. The IPP value for the three different batches were 4.9, 4.1 and 4.5

358

respectively. Last, the ICG value for the three different batches were 4.7, 3.94 and 4.32

359

respectively. With respect to the individual input parameters for Hercules batches used

360

for the construction of the SeDeM diagram, powder flow was far outside of the

361

acceptable range of 0-10 (-9.5 with SD of 0.5). Furthermore, all of the SeDeM diagram

362

output paramters were close to the recommended values range (0.6 for IP with SD of

363

0.06, 4.5 for IPP with SD of 0.4 and 4.32 for ICG with SD of 0.38). Compared to Tiger

364

King, Hercules has shown better formulation quality. The SeDeM diagram of the three

365

batches of Hercules are heterogenous, especially in case of batch 1 compared to batch

366

2 and 3.

17 367

In case of Herbal Viagra, the radius average for three different batches were 8.2,

368

8.57 and 8.27 respectively. The IP value was 0.75 for the three different batches. The

369

IPP value for the three different batches were 8.2, 8.6 and 8.3 respectively. Last, the

370

ICG value for the three different batches were 7.87, 8.26 and 7.97 respectively. With

371

respect to the individual input parameters for Herbal Viagra batches used for the

372

construction of the SeDeM diagram, powder flow and homogenity index were far

373

outside of the acceptable range of 0-10 (-6.3 with SD of 1.2 in powder flow and 43.5

374

with SD of 3 for homogenity index). Furthermore, all of the SeDeM diagram output

375

paramters were outside of the to the recommended range (0.75 for IP with SD of 0, 8.37

376

for IPP with SD of 0.21 and 8.03 for ICG with SD of 0.2). These results and the

377

homogencity of SeDeM diagrams of the three batches of Herbal Viagra show its poor

378

formulation and consistancy across batches.

379

In case of Natural Viagra, the radius average for three different batches were

380

10.09, 10.03 and 10.13 respectively. The IP value was 0.25 for the three different

381

batches. The IPP value for the three different batches were 10.09, 10.003 and 10.13

382

respectively. Last, the ICG value for the three different batches were 9.58, 9.53 and

383

9.63 respectively. With respect to the individual input parameters for Natural Viagra

384

batches used for the construction of the SeDeM diagram, homogenity index was far

385

outside of the acceptable range of 0-10 (53.83 with SD of 2.02). Furthermore, all of the

386

SeDeM diagram output paramters were outside of the to the recommended range (0.25

387

for IP with SD of 0, 10.08 for IPP with SD of 0.05 and 9.58 for ICG with SD of 0.05).

388

These results and the homogencity of SeDeM diagrams of the three batches of Natural

389

Viagra show its poor formulation and consistancy across.

390

In case of Plant Viagra, the radius average for three different batches were 9.89,

391

9.77 and 9.85 respectively. The IP value was 0.33 for the three different batches. The

18 392

IPP value for the three different batches were 9.89, 9.77 and 9.85 respectively. Last, the

393

ICG value for the three different batches were 9.49, 9.28 and 9.36 respectively. With

394

respect to the individual input parameters for Plant Viagra batches used for the

395

construction of the SeDeM diagram, homogenity index was far outside of the

396

acceptable range of 0-10 (51.67 with SD of 1.15). Furthermore, all of the SeDeM

397

diagram output paramters were outside of the to the recommended range (0.33 for IP

398

with SD of 0, 9.84 for IPP with SD of 0.06 and 9.38 for ICG with SD of 0.11). These

399

results and the homogencity of SeDeM diagrams of the three batches of Plant Viagra

400

show its poor formulation and consistancy across batches.

401

3.3 Rats in-vivo study

402

3.3.1 HPLC rats blood analysis

403

The mean and SD of Sildenafil citrate amount in mg per rat after 30 min and

404

120 min for placebo (group A) were 0 mg, for Viagra (group B) were 0.11 mg and 0.04

405

(at 30 min), 0.29 mg and 0.21 (at 120 min) respectively, for Tiger King (group C) were

406

0.90 mg and 0.20 (at 30 min), 0.98 mg and 0.20 (at 120 min) respectively. (Supporting

407

fig. 5)

408

The average rats body weight (gm) for placebo, Viagra, and Tiger King groups

409

were 202.4, 214.2, and 219.6 gm respectively on the first day of the experiment before

410

formulation administration. No significant decrease in rats weight was observed post-

411

treatment after the completion of the study in placebo and Tiger King groups. In case

412

of Viagra® group, there was a slight significance increase in rats weight post-treatment

413

after the completion of the study from 214.2 gms on average to 231.2 gms.

19 414

3.3.2 Liver and kidney functions assessment

415

Before the initiation and after the completion of the study, rats’ liver and kidney

416

functions were assessed with respect to creatinine (mg/dl), urea (mg/dl), AST (U/l), and

417

ALT (U/l) plasma levels to evaluate the short-term safety of Tiger King. Placebo,

418

Viagra®, and Tiger King groups showed no significance difference (P > 0.05) in all

419

parameters except in case of creatinine levels (mg/dl) in the Tiger King group, where

420

creatinine levels significantly increased from 0.54 mg/dl (SD of 0.017) to 0.6 (SD of

421

0.03) mg/dl (P ≤ 0.001). One way analysis of variance (ANOVA) and Bonferroni post-

422

hoc test were adopted for the statistical treatment.

423

3.3.3 Gross toxicity study

424

Gross toxicity was assessed on a scale system from 0 to 10, indicating the

425

number of rats showing symptoms per group (10 rats). Scores were averaged within

426

each group throughout the study period. Two rats in Tiger King group suffered form

427

anxiety. Two rats in Placebo group and Viagra® group, and one rat in Tiger King group

428

suffered from diarrhea. One rats in Viagra® group and two rats in Tiger King group

429

suffered from hunched back. One rat in Tiger King and Viagra® groups died during the

430

study.

431

4 DISCUSSION

432

Pharmaceutical, analytical, pharmacological, and clinical studies on the current

433

OTC and teleadvertized nutraceuticals, sold in Egypt market under the claim of being

434

100% natural for male enhancement (treatment erectile dysfunction), were investigated

435

in this thesis. Tiger King, Hercules, Herbal Viagra, Plant Viagra, and Natural Viagra

436

were the selected products in this research project based on their widespread sales in

437

Egypt in General. Three batches of each product were properly sampled from different

20 438

geographical locations in Egypt over a time period from December 2013 to February

439

2014.

440

Similarity in morphology between Plant Viagra and Natural Viagra was

441

obvious. The similarity was in tablet shape and color. In addition, the course and fine

442

ground powders showed clear similarity. This could be explained as these two products

443

are simply originating of the same source sold under different names, prices and brands.

444

In another aspect, the ground tablets color ranged between grey, pink, black and white.

445

The colored powder distribution was obviously different between batches of the same

446

product. For example, the morphology of ground Tiger King batch purchased from

447

Manshiya was different from Tiger King purchased from Sidi Bishr. Moreover,

448

heterogonous morphology was shown inside the tablets in same batch. This confirms

449

the non-uniformity in the tablets production. Hence, the quality of the products was

450

highly questioned.

451

The data acquired from HPLC performed to Tiger King, Hercules, Plant Viagra,

452

Natural Viagra, and Herbal Viagra were confirmed by HPTLC, NIR, content uniformity

453

and weight variation, friability and SeDeM diagram. This definitely proves that these

454

products contained undeclared Sildenafil citrate as active pharmaceutical ingredient for

455

treat erectile dysfunction. However, these products are marketed as 100% natural

456

products without side effects and can be purchased without prescription. This

457

undeclared ingredient have dangerous side effect for consumer.

458

The products showed the same heterogeneous manner within and across

459

batches and even within the same package. Quality control and reproducibility for the

460

products were furthermore assessed by near infrared. Besides, the heterogeneity within

461

the products was assured by in vivo and clinical studies. Hazardous effects on man’s

21 462

health may be caused if individual administered a tablet containing sub dose of

463

sildenafil then decided to administer two tablets, which happen to contain highly toxic

464

dose. A possible scenario, if a user consume one tablet of Tiger King for example,

465

containing zero mgs of Sildenafil and showed no effect, the user may decide later to

466

consume two tables, which by coincidence may contain 50 mgs of Sildenafil combined.

467

The administered dose would be equivalent to one tablet of authentic Viagra. Later, he

468

may use two tablets containing each 200 mgs of Sildenafil (Total of 400 mgs). This

469

amount could be lethal for the user, especially; the target audience are alluded with the

470

fact that these products are natural and safe. The target audiences of these products are

471

individuals with heart and blood pressure problems. Such audiences prefer the natural

472

claim to avoid the use of Viagra, which could be lethal for their case. Such scenario

473

most probably will be lethal and dramatic.

474

Based on the data generated from the NIR correlation analysis of all products,

475

it was suspected that there could be similarities and correlations between some batches

476

of different products. This suspicion was agitated by some of the morphological

477

similarities observed between Plant Viagra and Natural Viagra. Based on this suspicion,

478

all batches of Plant Viagra, Natural Viagra, Hercules and Herbal Viagra were analyzed

479

against Tiger King for possible correlations and similarities. Thus, the NIR results

480

showed the alarming fact that these products could be simply the same, package and

481

sold under different names, prices, shapes and formulations to attract more users and

482

cannibalize the market of male enhancement products.

483

In a similar study by Campbell et al (Campbell et al., 2013), conducted by

484

Pfizer Inc., purported herbal/natural OTC dietary supplements claiming to naturally

485

enhance sexual performance were assessed with respect to availability, cost, origin,

486

categorical content, and adulteration with PDE5 inhibitors. Although no sample

22 487

claimed to include synthetic substances, 74 (81%) contained PDE5-inhibitors,

488

including tadalafil and/or sildenafil (n = 40, of which 18 contained >110% of the highest

489

approved drug product strength) or PDE5-inhibitor analogs (n = 34). In this study,

490

products were only scanned for possible contamination with adulterants. In our study,

491

we, not only quantified the possible PDEI5s presence, but also assessed the

492

pharmaceutical quality of the formulation, inter and intra-batch variability and safety

493

parameters in-vivo. Furthermore, we reported the situation in the Middle-East and

494

highlighted the possible drastic outcomes on the region and globally and the possible

495

deadly scenarios for consumers.

496

4.1 Conclusion

497

The products were proven to contain undeclared Sildenafil citrate with variable

498

heterogeneous amounts ranging from zero to over 180 mgs in a single dose with poor

499

pharmaceutical quality and consistency. These results were confirmed by HPLC,

500

HPTLC, NIR, content uniformity, weight variation, dissolution, friability, and SeDeM

501

diagram analysis as a quality control and assurance tool. Moreover, the heterogeneity

502

of the adulterant API within the products was assured by in vivo study. This may cause

503

health hazards for individuals using these products. Substantially, that these products

504

are marketed with therapeutic claim yet with no physician supervision. Furthermore,

505

the target customers for these products are generally suffering from heart or blood

506

pressure problems, seeking natural, effective and safe alternatives to well-known PDEIs

507

such as Sildenafil, Tadalafil and Verdenafil (Viagra®, Cialis® and Levitra® and their

508

generics) which generally are avoided in their cases. Such products need to be banned

509

from getting to customers.

23 510 511

4.2 Future Directions Based on the findings, we propose some recommendations and future directions

512

to be taken into consideration to correct the current problem:

513

1. The need to aware consumers of the possible lethal consequences of these products

514

free sold, marketed and advertised in the Egyptian market and sold without a

515

prescription. Beside, officially report such results to health authorities in Egypt to show

516

the possible damages.

517

2. The need for strict laws and regulations in place to protect the consumer. These laws

518

has to be active and deals with the registration, marketing, sales and advertisement of

519

nutraceuticals and cosmeceuticals.

520

3. The need to invest in funding and supporting continuing research to investigate the

521

safety and efficacy of other products invading the Egyptian market illegally and sold

522

freely in the Egyptian pharmaceutical market such as female enhancement products and

523

weight loss preparations.

524

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Sune-Negre, J.M., Montoya, E.G., Lozano, P.P., Aguilar Diaz, J.E., Carreras, M.R., Garcia, R.F., Carmona, M.M., Tico, J.R., 2011. SeDeM Diagram: A New Expert System for the formulation of drugs in solid form, in: Vizureanu, P. (Ed.), Expert Systems for Human, Materials and Automation. InTech. Tripathi, A.S., Dewani, A.P., Shelke, P.G., Bakal, R.L., Chandewar, A.V., Mazumder, P.M., 2013. Development and validation of RP-HPLC method for simultaneous estimation of glimepiride and sildenafil citrate in rat plasma-application to pharmacokinetic studies. Drug research 63, 510-514. Vredenbregt, M.J., Blok-Tip, L., Hoogerbrugge, R., Barends, D.M., de Kaste, D., 2006. Screening suspected counterfeit Viagra and imitations of Viagra with near-infrared spectroscopy. Journal of pharmaceutical and biomedical analysis 40, 840-849. Ward, P., Ewen, M., Pomeroy, J.A., Leung, F.Y., 1976. Kinetic urine creatinine determination with the Gemsaec analyzer. Clinical biochemistry 9, 225-227. Zailina, A., Aminah, A., Ambar, Y., 2013. Optimaziation of extraction methods for determination of phosphodiesterase-5 (PDE5) inhibitors in premix coffee. Sains Malaysiana 2, 135-142.

26 630

Figure captions

631 Fig. 1

Average amount of Sildenafil citrate (mg) in indvidual batches of Viagra®, Tiger King, Plant Viagra, Hercules, Natural Viagra and Herbal Viagra analyzed via (A) HPLC. Individual representation of each batch in a bar form is shown. Reversed phase C18 column was used (Column length of 25 cm with C18 guard column) along with actetonitrile:10 mM aqueous ammonium formate as the mobile phase (UV detection at λmax of 230 nm)

(B) HPTLC. A mixture of chloroform: ethanol (90:10) was used as the developing solvent. The chromatogram was recorded at wavelength (λmax) of 254 nm. CAMAG HPTLC-P57 Scanner-3 was used (CAMAG Switzerland)

Fig. 2

Average amount of Sildenafil citrate (mg) in different batches of Viagra®, Tiger King, Hercules, Plant Viagra, Natural Viagra and Herbal Viagra, representing two distinct populations with different means, analyzed via

(A) HPLC. Reversed phase C18 column was used (Column length of 25 cm with C18 guard column) along with actetonitrile:10 mM aqueous ammonium formate as the mobile phase (UV detection at λmax of 230 nm.

(B) HPTLC. A mixture of chloroform: ethanol (90:10) was used as the developing solvent. The chromatogram was recorded at wavelength (λmax) of 254 nm. CAMAG HPTLC-P57 Scanner-3 was used (CAMAG Switzerland)

27 Combined NIR Spectra for different batches of Viagra® (Grey),

Fig. 3

Tiger King (Black), Hercules (Red), Natural Viagra (Blue), Herbal Viagra (Purple) and Plant Viagra (Green). NIR spectra were recorded on Bruker near-infrared spectroscopy (Bruker MPA, Germany). Measurements were carried out with an optical resolution of 16 cm-1 and 32 cm-1 over the spectra range 6200-5600 cm-1

Fig. 4

(A) Percentage Correlation within each brand of Viagra®, Tiger King, Natural Viagra Plant Viagra and Herbal Viagra as reported by quantitative NIR analysis. Bruker near-infrared spectroscopy was used (Bruker MPA, Germany). Measurements were carried out with an optical resolution of 16 cm-1 and 32 cm-1 over the spectra range 62005600 cm-1. Grey shaded area represents correlation within each brand more than 96%. (B) Percentage of Hercules, Herbal Viagra, Natural Viagra and Plant Viagra tablets (Five batches each) identical to a single batch of Tiger King with 90%, 95% or 96% correlation as reported by quantitative NIR analysis. Bruker near-infrared spectroscopy was used (Bruker MPA, Germany). Measurements were carried out with an optical resolution of 16 cm-1 and 32 cm-1 over the spectra range 6200-5600 cm1

Fig. 5 SeDeM diagrams for different batches of Viagra® (a, b and c), Tiger King (d, e and f), Hercules (g, h and i), Herbal Viagra (j, k and l), Plant Viagra (m, n and o) and Natural Viagra (p, q and r)

Fig. 6 Average Parameter values (Radius (R), Parameter index (IP), Parameter profile index (IPP) and Good compressibility index (ICG)) for different batches Viagra®, Tiger King, Hercules, Herbal Viagra, Plant Viagra and Natural Viagra (n = 3-11). One-way ANOVA followed by Bonferroni posthoc test was used in the satisitical analysis (ns for P > 0.05, * for P ≤ 0.05, **for P ≤ 0.01, *** for P ≤ 0.001 and **** for P ≤ 0.0001)

28

Figure 1

29

Figure 2

30

Figure 3

31

Figure 4

32

Figure 5

33

Figure 6

632