THE CHEMICAL ANALYSIS OF NOSTREXIN® REVEALS THE PRESENCE OF 5-HYDROXY-L-TRYPTOPHAN, THE TYPICAL GRIFFONIA SIMPLICIFOLIA ALKALOIDS AND THE ABSENCE OF BACTERIAL FERMENTATION COMPOUNDS

Nostrexin® is a Griffonia simplificolia extract characterized by the presence of 5-hydroxy-L-tryptophan along with the typical plant alkaloids. The extract is analyzed by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) for the identification and characterization of the chemical constituents. Table 1 shows the chemical characterization of Nostrexin® performed by HPLC-MS/MS, whereas Figure 1 shows the chemical structure of the most representative Nostrexin® compounds.

Table 1. Chemical composition and mass spectrometric data of the main nitrogen-containing compounds present in Nostrexin®.

Compound MW [M + H]+ m/z
5-hydroxytryptamine (5-HT) 176.0 177.0 158.8 135.8 117.0
5-hydroxy-L-tryptophan (5-HTP) 220.0 221.0 204.0 161.9
hyrtioerectine B 246.0 247.0 229.9 203.9 174.0
griffonine 329.0 329.0 167.9
hyrtiosulawesine 343.0 343.0 228.9 200.9 182.9
Figura-1-EN

Figure 1. Chemical structure of the most representative compounds of Nostrexin®.

The ratio between 5-HTP and the typical G. simplicifolia alkaloids in Nostrexin® is always lower than 10:1, a value that is impossible to reach if the 5-HTP is obtained by bacterial fermentation. In fact, 5-HTP can be produced by heterologous systems; however, the bacterial transformation may lead to tryptophan by-products [e.g., peak E as 1,1′-ethylidenebis(l-tryptophan) and peak UV-5 as 3-(phenylamino)alanine] that have been associated to diseases such as eosinophilia–myalgia syndrome (Belongia et al., N. Engl. J. Med. 1990, 323:357–365; Mayeno et al., Science 1990, 250:1707–1708; Goda et al., Chem. Pharm. Bull. 1992, 40:2236–2238).
Nostrexin® analysis confirms the complete lack of peaks E and UV-5.

THE SEEDS OF GRIFFONIA SIMPLICIFOLIA USED TO PRODUCE NOSTREXIN® ARE IDENTIFIED BY DNA FINGERPRINTING

Existing assay methods for the authentication of G. simplicifolia seeds are laborious, time-consuming, and sensitive to interference from co-occurring materials. In order to ensure reliable supplies of appropriate G. simplicifolia seed material, Biosfered uses an efficient, dependable, and relatively rapid method for assessing the quality of seeds before purchase. Biosfered team, along with mass spectrometry-based analytical methods, has developed a biomolecular characterization of G. simplicifolia seeds by using DNA fingerprinting, as shown below.

Figure 2 shows the nucleotide sequence of the ITS region of G. simplicifolia seeds. In general, the ITS-amplified sequence was about 758 bp long (NCBI GenBank Accession No MH707248) (Figure 2 lane 1). In order to provide a better-defined DNA fingerprint, a PCR–RFLP method was applied. Three different restriction enzymes (MspI, HhaI, and HaeIII) were used to selectively cleave the resulting amplicon. Digestion of the PCR product with MspI generated three fragments: one major fragment of about 471 bp, and two minor fragments of about 57 and 213 bp (Figure 2 lane 2). An HhaI site could also be identified in the ITS region, generating five distinct fragments: the major fragment gave a band of about 319 bp, followed by a band of about 215 bp. Two minor bands were found at about 198 and 240 bp, and there was a faint band at 86 bp (Figure 2 lane 3). Finally, digestion with HaeIII generated three bands: a major band of about 431 bp, followed by two bands of minor concentration of about 198 and 86 bp (Figure 2 lane 4).

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Figure 2. Electrophoresis representation of PCR products generated by primers flanking the internal transcribed spacer (ITS) region of ITS-1 and ITS-4 and PCR–restriction fragment length polymorphism (RFLP) products from G. simplicifolia. Capillary gele electrophoresis (Bioanalyzer) has been used to obtain the data.The figure also shows the nucleotide sequences of the ITS gene spacer region of G. simplicifolia.  For more details, please refer to the paper indicated below (Vigliante et al., Molecules, 2019, 24;1032).

Nostrexin® is always produced from G. simplicifolia seeds identified by DNA fingerprinting using specific PCR–RFLP markers which is used as an unequivocal method for the plant identification. Identified sequences are deposited in GeneBank. DNA fingerprinting offers the advantage of a fast and rather inexpensive method for the unequivocal identification of this plant species long before the chemical analysis is done. Moreover, being restricted to DNA, this analysis it is not affected by environmental factors that might affect the expression of those genes involved in 5-HTP production.

For a complete description of methods and results please refer to the paper:

Vigliante, I., Mannino, G., Maffei, M.E. (2019)
Chemical characterization and DNA fingerprinting of Griffonia simplicifolia Baill.
Molecules, 24:1032(download pdf)

NOSTREXIN® IS A NATURAL SOURCE OF THE SEROTIN PRECURSOR 5-HTP

Nostrexin® has a standardized content (30%) of 5-HTP, a direct precursor and enhancer of the activity of the brain hormone serotonin (5-hydroxytryptamine, 5-HT) (Fellows et al., 1970, Phytochemistry, 9:2389–2396). Tryptophan is first hydroxylated to 5-HTP, then is converted to 5-HT by the enzyme tryptophan decarboxylase (Freedman, 2010, Maturitas, 65:383–385) (Figure 3). The administration of 5-HTP increases 5-HT levels in the central nervous system (CNS) (Kitahama et al., 2002, J. Neural Transm. 109:683–689). A medical food formulation comprised of G. simplicifolia containing 5-HTP is thought to be effective for serotonin-related disorders (Esposito et al., 2015, J. Med. Food 18:916–920; Muszynska et al., 2015, Psychiatr. Polska 49:435–453), including depression (Iovieno et al., 2011, J. Affect. Disord. 130:343–357). Further uses of G. simplicifolia seed extracts include support to insomnia, migraine, headache, and the regulation of appetite leading to weight reduction in obese patients, as well as the regulation of mood, memory, and many other functions (Carnevale et al., 2011, Phytomedicine 18:848–851).

Figura-3-EN

Figure 3. Biosynthetic pathway of serotonin. The amino acid tryptophan is first hydroxylated to 5-hydroxytryptophan and then decarboxylated to obtain serotonin.

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