Agmapure®

AGMAPURE® by GENABOLIX

- 1 -

AGMAPURE®

Trademark: AGMAPURE®

Product Name: Agmatine Sulfate

CAS NO: 2482-00-0

Other Names:

C5 H14 N4 •H2 O4 S Guanidine, N-(4-aminobutyl)-, sulfate (1:1) Guanidine, (4-aminobutyl)-, sulfate (1:1) (8CI,9CI);

MF:

C5H16N4O4

Molecular Weight: 228.27g/mol

Solubility in Water: 50g/L (25 °C)

Density: 1.328±0.06 g/cm3 MP: 234-238°C

I. Introduction of AGMAPURE® Recently Agmatine has gained wide interest as a breakdown metabolite

of Arginine with a multitude of physiological functions. Agmatine Sulfate, the commonly used stable salt of Agmatine, is a promising supplement rapidly drawing the attention of the athletic performance market.

Agmatine Sulfate is thought to improve nutrient partitioning thus leading to more glycogen storage and increased water retention within the muscle. It also increases the level of nitric oxide (NO), which can increase the "PUMP" that athletes look for when weight training. Although Arginine also has this function, the cost of it is quite expensive and, if you want obvious effects, you need at least 6 times more dose than Agmatine, no matter in what form of salt or ester it is.

Attracted by such an application prospect, we’ve developed the new generation product of Agmatine Sulfate: AGMAPURE®.

Currently, AGMAPURE® is the unique Green Agmatine Sulfate in sports nutrition market. It’s in high purity, produced by natural biological process and away from the neurohazard impurity Arcaine, which exists in almost all the other brands of Agmatine Sulfate.


- 2 -

II. AGMAPURE® VS. Putrescine-related Agmatine Sulfate

Why Genabolix® introduced AGMAPURE® into the currently crowded market of Agmatine Sulfate?

Because till now, almost all the competitor products of Agmatine Sulfate use the same synthesis process, starting from notorious putrescine!

Production Process of

Putrescine-related Agmatine Sulfate

Putrescine is a foul-smelling organic compound related to cadaverine. Both of putrescine and

cadaverine are produced by the breakdown of amino acids in living and dead organisms, and both are toxic in large doses. The two compounds are not only responsible for the foul odor of putrefying flesh, but also contribute to the odor of such processes as bad breath and bacterial vaginosis.

The putrescine will finally become hardly removable impurity in final Agmatine Sulfate. It may be in the form of putrescine monosulfate, or even base form, which will be quite unidentified impurity in final Agmatine Sulphate and can’t be purified with current technology, because its chemical structure is highly similar to that of Agmatine. Although some companies can turn out a max purity of 98%, there is still detectable impurity, Arcaine, which is harmful to human brain and will increase the risk of amnesia.

We adopt natural compound as raw material, and turn it into AGMAPURE® by

completely natural biological process. Therefore, AGMAPURE® is clean and safe, without introducing in any unwanted and unknown impurity. It has been demonstrated by our purity test.

Production Process of AGMAPURE®


- 3 -

III. Purity Test of AGMAPURE® & Putrescine-related Agmatine Sulfate

Purpose and Scope The purity test describes the research of impurity profile of AGMAPURE® and the Putrescine-related Agmatine Sulfate to show the difference between them.

Method Description

Method: HPLC-UV(for purity determination) Instrument Type: Aglient 1260 Column: C18 25cm Wavelength: 195nm Method: HPLC-DAD/MS(for identification determination) Instrument Type: Aglient 1260 Column: C18 25cm

Test Results

Putrescine-related Agmatine Sulfate has one impurity at RRT about 1.14 with percent about 10% by normalization calculation method. Furthermore, within MS identified results, the major peak shows the Agmatine characteristic ion segment (114.1 and 131.1). Whatsoever, the impurity peak show 173.1 segment, which complies with the molecular of Arcaine (1,4-diguanidinobutane, CAS# 544-05-8). Arcaine is a standard tool for setting up amnesia animal models.

The purity test of AGMAPURE® doesn’t show any characteristic peak at all. It is Pure!!!


- 4 -

No matter how accurately the process parameters are set and controlled, the byproduct Arcaine (1, 4-diguanidinobutane, CAS#:

544-05-8), a harmful chemical, always remains in the final Agmatine product unavoidably.

Let’s take a look at the reaction mechanism. Agmatine Sulphate is usually synthesized with Putrescine and S-Methylisothiourea by

chemical method, which gives Agmatine Sulphate as main product by controlling the equivalent of S-Methylisothiourea. The mechanism is

an addition-elimination reaction between the amino group of Putrescine and S-Methylisothiourea. But during the process of preparation of

Agmatine Sulphate, the amino group of the product Agmatine Sulphate can also react with S-Methylisothiourea to produce a small quantity

of Arcaine Sulphate as byproduct because of the material adding method and competitive reaction speed, which is very difficult to control in

organic process.

But for the biotransformation process of AGMAPURE®, the specificity of the utilized enzyme is in high efficiency, so there is no byproduct

produced during the process.

The table above is the purity analysis of two types of Agmatine Products with HPLC-UV method. The results are also distinct.

Arcaine is a harmful chemical to human brain. It’s a competitive antagonist of the polyamine binding site at the N-methyl-D-aspartic acid

receptor (NMDAR). As all know, memories are initially fragile, but become more stable with time. Such an observation has led to the

concept of memory consolidation, which refers to the stabilization of a new memory over time by neural processes activated by recently

learned information. The process of consolidation depends on glutamatergic transmission (da Rosa MM et al. 2012). The activation of

glutamatergic NMDAR in the hippocampus, amygdala and other brain areas play an important role in early consolidation of the memory. In

some researches (Raquele Kipper Mariani, et al. 2011), scientists prove the administration of Arcaine, by opioid mechanisms, blocks the

activation site of NMDAR, and impairs the memory performance!

Due to the technological limitations, Arcaine is introduced in almost all the Putrescine-related Agmatine Sulfate products. We have


- 5 -

validated quite a few Putrescine-related Agmatine Sulfate products from different competitors with HPLC method. As expected, they all

contain the stubborn impurity: Arcaine, even the Agmatine Sulfate from Sigma (Cat#: A7127). According to many neurobiology researches,

Arcaine is the standard tool for setting up amnesia animal models. Therefore, we consider the introduction of Arcaine impurity in Agmatine

Sulfate will increase the risk of memory impairment.


- 6 -

IV. Functions of Agmatine Sulfate and the Mechanism

Agmatine is a ubiquitous naturally occurring

molecule (Tabor and Tabor 1984). It is

biosynthesized by decarboxylation of the amino

acid Arginine. Agmatine is found in low amounts

in many food stuff derived from plants, fish, and

animal products. Animal studies demonstrate

that the salt of Agmatine, Agmatine Sulfate, is

absorbed by the gastrointestinal tract and

distributed in the body (Molderings, Heinen et al.

2003). Agmatine initially attracted attention as an

endogenous ligand at imidazoline receptors and

α2-adrenoceptors (Li, Regunathan et al. 1994).

Independent of binding to those receptors,

Agmatine induces a variety of physiological and

pharmacological effects exhibiting a great

therapeutic potential.

Relations of the Arginine, Agmatine and the Polyamine pathways


- 7 -

-Agmatine Decreases blood pressure and heart rate Agmatine as endogenous ligand of imidazoline and α2-adrenoceptors participates in the regulation of blood pressure and heart rate. The stimulation of imidazoline

and/or α2-adrenoceptors will suppresses the norepinephrine and epinephrine that mediate a number of physiological responses to endogenous catecholamines such as hypotension, sedation, inhibition of insulin release, anti-lipolysis and platelet aggregation (Gonzalez et al., 1996, Zhao & Ren,2005). Also imidazoline and/or α2-adrenoceptors are reported to related to reduction in calcium influx and potassium efflux, reducing the rate of depolarization and the amplitude of action potential in the heart, through the inhibition the increase in the intracellular [Ca2+] by blocking L-type voltage dependent Ca2+ channel (Li et al., 1999) (Li et al.2000a, 2002a,b)

Finally, the decrease in systemic blood pressure in rats and rabbits after intravenous administration of Agmatine was sensitive to the nitric oxide synthase (NOS) inhibitor L-NAME (Haulica et al.,1999), suggesting an involvement of nitric oxide (NO).

-Agmatine has antinociceptive effects Agmatine binds to NMDA receptors and acts as an antagonist at NMDA receptor channels (Gadotti et al., 2006), and NMDA antagonists are known to block opioid

withdrawal symptoms (Trujillo & Akil, 1994). Agmatine reversed the up-regulation of the NR1 subunit of NMDA receptors in the nucleus accumbens inmorphine dependence without changing NR1 subunit expression per se (Wang et al., 2011).

-Agmatine Represses the tumor cell growth There is evidence that an impairment of intracellular agmatine homeostasis is crucially involved inmalignant cell proliferation in solid tumors and leukemia (Molderings et

al., 2004; Molderings, 2006; Wolf et al., 2007; Haenisch et al., 2008). In vivo, in human colon cancer tissue agmatine content and in parallel the expression of arginine decarboxylase (ADC) were distinctly reduced compared with that in the normal tissue adjacent to the malignant tumor (Molderings et al., 2004). Decrease of intracellular agmatine content by RNA interference targeting ADC in human intestinal tumor cells resulted in a significant increase of proliferation (Molderings, 2006).

-Potential diuretic Agmatine can be formed and metabolized in rat kidney (Lortie et al., 1996). Administration of exogenous agmatine caused an increase in urinary flow rate by increasing osmotic clearance and a cAMP dependent inhibition of urea absorption in the terminal rat inner medullary collecting duct, effects attributed to binding to imidazoline recognition sites (Smyth & Penner, 1995; Penner & Smyth, 1996; Rouch & Kudo, 2002). Agmatine also produced renal vasodilatation and increases in nephron glomerular filtration rate (Lortie et al., 1996; Schwartz et al., 1997). This effect was not altered by denervation (Lortie et al., 1996) and may be mediated via a constitutive NO synthase- dependent mechanism, since it was abolished by the NO synthase inhibitors NG-monomethyl-L-arginine and nitro-L-arginine methyl ester (Schwartz et al., 1997).

-Agmatine boosts muscle pumps Agmatine sulfate appears to boost muscle pumps in a couple different ways. First, Agmatine sulfate may improve nutrient partitioning, leading to more muscle glycogen

(carbohydrates stored in muscle tissue) stored and consequently more water retained in the muscle. This can lead to a full look to the muscle and increase muscle pumps. Second, Agmatine sulfate also appears to increase nitric oxide production by acting as a competitive inhibitor of the enzyme nitric oxide synthase (Demady DR & Jianmongkol S, et al.). The nutrient partitioning effects of agmatine may be due to its possible ability to increase the insulin response to carbohydrates, and it may also have something to do with the increased blood flow to the muscle experienced with increased nitric oxide production. Agmatine sulfate may play a role in the hypothalamic control of luteinizing hormone and growth hormone release, leading to increased levels of these hormones (Zarandi M & Serfozo P, et al.).


- 8 -

V. Differences among Agmatine, L-Arginine and L-Citrulline

Agmatine, L-Arginine and L-Citrulline are all ingredients for bodybuilding products, but Agmatine is different from the other two

ingredients in many ways:

1) To achieve ideal functions, the oral dosage of Agmatine is much less than Arginine and Citrulline.

2) Besides the bodybuilding function, Agmatine also possesses potent ability as neuroprotectant and insulin modulator.

3) Although L-Arginine has multiple beneficial functions, its cost is actually expensive. Because after oral taken, 50%-75% of L-Arginine is

converted to Ornithine and urea instead of nitric oxide. If you want obvious effects, you need much more dose no matter in what form of salt

or ester it is.

4) L-Citrulline has some bodybuilding and pre-workout effects by converted to L-Arginine.

Ingredient Recommended Daily Dosage Functions Mechanism

Agmatine 750mg~1000mg

√ Building muscle and body tissue √ An intermediate product in the synthesis of polyamines related to nitric oxide synthesis √ Inducing the release of peptide hormones √ Acting as a neuroprotectant √ Stimulates the release of insulin and uptake of calcium in pancreatic cells leading to improved insulin sensitivity

√ Agmatine is an intermediate in polyamine biosynthesis. √ Agmatine is α2-adrenergic receptor agonist.√ Agmatine is imidazoline receptor agonist. √ Agmatine blocks some cation ligand-gated channels to protect brain.

L-Arginine 6~9g √ Building muscle and body tissue √ Producing nitric oxide √ Stimulating the secretion of growth hormone (at high dosage)

√ Precursor for the synthesis of nitric oxide.

L-Citrulline 3~4g √ Building muscle and body tissue √ Producing nitric oxide √Precursor for the synthesis of L-Arginine.

VI. Implications of AGMAPURE®

In preclinical studies, low toxicity of Agmatine has been observed in cultured neurons (Gilad et al., 1996), as well as after intraperitoneal


- 9 -

injection to rats (Satriano et al., 1998). In addition, Agmatine was taken in gram-doses for years in the US bodybuilding community

unrestrained as a putative effective dietary supplement to increase muscle mass and it is yet used in high doses in combination medicine as

an aphrodisiac without reported complications.

AGMAPURE® is produced by natural biological process and it contains no putrescine-related impurity. Therefore, the application of

AGMAPURE® as potential sports nutrition is much safer than any other putrescine-related Agmatine Sulfate product in the market. Besides

this advantage, supplementation with AGMAPURE® may improve athletic performance, increase muscle size, fullness, and vascularity, and

may also increase fat loss. It may also have a couple of additional benefits like protection against free radical damage and an

antidepressant effect. All these implications show a really promising application prospect.

VII. Reference Gadotti, V. M., Tibola, D., Paszcuk, A. F., Rodriques, A. L. S., Calixto, J. B., & Santos, A. R. S. (2006). Contribution of spinal glutamatergic receptors to the antinociception caused by agmatine in mice. Brain Res 1093n, 116–122. Gilad, G. M., Gilad, V. H., & Rabey, J. M. (1996). Arginine and ornithine decarboxylation in rodent brain: coincidental changes during development and after ischemia. Neurosci Lett 216, 33–36. Gonzalez, C., Regunathan, S., Reis, D. J., & Estrada, C. (1996). Agmatine, an endogenous modulator of noradrenergic neurotransmission in the rat tail artery. Br J Pharmacol 119, 677–684. Haenisch, B., von Kügelgen, I., Bönisch, H., Göthert, M., Sauerbruch, T., Schepke, M., et al. (2008). Regulatory mechanisms underlying agmatine homeostasis in man. Am J Physiol Gastrointest Liver Physiol 295, G1104–G1110. Haulica, I., Bild, W., Iliescu, R., Georgescu, R., & Frunza, F. (1999). Preliminary research on possible relationship of NO with agmatine at the vascular level. Rom J Physiol 36, 67–79. Li, G., Regunathan, S., Barrow, C. J., Eshraghi, J., Cooper, R., & Reis, D. J. (1994). Agmatine: an endogenous clonidine-displacing substance in the brain. Science 263, 966–969. Li, Q., Shang, Z. L., Yin, J. X.,Wang, Y. H., & He, R. R. (2002). Effect of agmatine on intracellular free calcium concentration in isolated rat ventricular myocytes. Acta Physiol Sin 54, 467–472. Li, X. T., He, R. R., Liu, S., Liu, L. L., Zhang,W. L., Zhao, H., et al. (2000). Electrophysiological effects of agmatine on human atrial fibers. Life Sci 66, 2351–2356. Li, X. T., Duan, H. R., & He, R. R. (2000). Inhibition by agmatine on spontaneous activity of rabbit atrioventricular node cells. Acta Pharmacol Sin 21, 931–935. Lortie, M. J., Novotny, W. F., Peterson, O. W., Vallon, V., Malvey, K., & Mendonca, M. (1996). Agmatine, a bioactive metabolite of arginine. Production, degradation, and functional


- 10 -

effects in the kidney of the rat. J Clin Invest 97, 413–420. Molderings, G. J., A. Heinen, et al. (2003). "Gastrointestinal uptake of agmatine: distribution in tissues and organs and pathophysiologic relevance." Ann N Y Acad Sci 1009: 44-51. Molderings, G. J., Kribben, B., Heinen, A., Schröder, D., Brüss, M., & Göthert, M. (2004). Intestinal tumor and agmatine (decarboxylated arginine). Low content in colon carcinoma tissue specimens and inhibitory effect on tumor cell proliferation in vitro. Cancer 101, 858–868. Penner, S. B., & Smyth, D. D. (1996). Natriuresis following central and peripheral administration of agmatine in the rat. Pharmacology 53, 160–169. Rouch, A. J., & Kudo, L. H. (2002). Agmatine inhibits arginine vasopressin-stimulated urea transport in the rat inner medullary collecting duct. Kidney Int 62, 2101–2108. Sastre, M., Galea, E., Feinstein, D., Reis, D. J., & Regunathan, S. (1998). Metabolism of agmatine in macrophages: modulation by lipopolysaccharide and inhibitory cytokines. Biochem J 330, 1405–1409. Schwartz, D., Peterson, O. W., Mendonca, M., Satriano, J., Lortie, M., & Blantz, R. C. (1997). Agmatine affects glomerular filtration via a nitric oxide synthase dependent mechanism. Am J Physiol 272, F597–F601. Smyth, D. D., & Penner, S. B. (1995). Renal I1-imidazoline receptor-selective compounds mediate natriuresis in the rat. J Cardiovasc Pharmacol 26(Suppl 2), S63–S67. Trujillo, K. A., & Akil, H. (1994). Inhibition of opiate tolerance by non-competitive Nmethyl- D-aspartate receptor antagonists. Brain Res 633, 178–188. Tabor, C. W. and H. Tabor (1984). "Polyamines." Annu Rev Biochem 53: 749-90. Wang, X. F., Wu, N., Su, R. B., Lu, X. Q., Liu, Y., & Li, J. (2011). Agmatine modulates neuroadaptations of glutamate transmission in the nucleus accumbens of repeated morphine-treated rats. Eur J Pharmacol 650, 200–205. Wolf, C., Brüss, M., Hänisch, B., Göthert, M., von Kügelgen, I., & Molderings, G. J. (2007). Molecular basis for the antiproliferative effect of agmatine in tumor cells of colonic, hepatic, and neuronal origin. Mol Pharmacol 71, 276–283. Zarandi M, Serfozo P, Zsigo J, Bokser L, Janaky T, Olsen DB, Bajusz S, & Schally AV. (1992). Potent agonists of growth hormone-releasing hormone. Part I. International Journal of Peptide and Protein Research. 39(3), 211-7. Kalra SP, Pearson E, Sahu A, & Kalra PS. (1995). Agmatine, a novel hypothalamic amine, stimulates pituitary luteinizing hormone release in vivo and hypothalamic luteinizing hormone-releasing hormone release in vitro. Neuroscience Letters. 194(3), 165-8. Demady DR, Jianmongkol S, Vuletich JL, Bender AT, & Osawa Y. (2001). Agmatine enhances the NADPH oxidase activity of neuronal NO synthase and leads to oxidative inactivation of the enzyme. Molecular Pharmacology. 59(1), 24-9. da Rosa MM, Mello CF, et al. Opioid mechanisms are involved in the disruption of arcaine-induced amnesia by context pre-exposure. Neurobiol Learn Mem. 2012 Mar;97(3):294-300. Raquele Kipper Mariani, Carlos Fernando Mello, et al. Effect of naloxone and morphine on arcaine-induced state-dependent memory in rats. Psychopharmacology (2011) 215:483-491.

Agmapure®

Healthcare

Transcript of Agmapure®