Science dossier

Manuka honey anti-microbial spray

There is over 28 years of scientific knowledge condensed in this research. It identifies an alternative origin of methylglyoxal and the potential anti-inflammatory activity of Manuka honey and it's potential anti-cancer properties based on its ability to modulate HDAC activity due to the CEL motif on lysine residues due to MGO reacting with lysine residues in royal jelly proteins in Manuka honey.

The development of a process to extract royal jelly colloidal particles from Manuka honey (royal jelly proteins) was required to produce sufficient biomaterials for clinical evaluation. The number of particles isolated per ml is shown in Figure 1.

Royal jelly protein 1 has some remarkable properties that have been outlined in the scientific literature. I have not tested my product for any of these activities clinically in randomized controlled clinical trials or gold standard RCT's and I am not claiming that my product will cure you of any of these things. #antihypertension #hypocholesterolemic #antibiotic # antitumor #cell groth promoting #prolonging longevity #protecting the immune system 

The unique pharmacological effects of MRJP1 in humans, and they provide insights into the mechanisms underlying its physiological functions in bees, including caste differentiation, learning ability, colony organisation, and development. Importantly, MRJPs have great potential as drug candidates for promoting human health, due to their hypocholesterolemic, antihypertension, antibiotic, anti-tumour, and cell growth-promoting activities, and their ability to prolong longevity and protect the immune system. For example, the MRJP1 glycoprotein harbours three antimicrobial jelleine peptides.

Analysis of colloidal nanoparticles using QNano (Izon)

Figure 1: ΩH ଓଁEE ħΛVE analysis of royal jelly colloidal nanoparticles using qNano instrument (Izon). External analysis was performed to validate the numbers of bioavailable royal jelly particles isolated. Analysis demonstrated 10 billion royal jelly colloidal particles per millilitre.

Zeta sizer analysis of colloidal nanoparticles in OH BEE HAVE

Figure 2: Zeta analysis of royal jelly colloidal nanoparticles in ΩH ଓଁEE ħΛVE.

zeta analysis sizes of the colloidal nanoparticleszeta analysis sizes of the colloidal nanoparticles2

Figure 3: Zeta sizer analysis of royal jelly colloidal nanoparticles in ΩH ଓଁEE ħΛVE

Zeta potential of ΩH ଓଁEE ħΛVE Manuka honey derived royal jelly colloidal particles at pH 5.9 was determined to be -0.34. Previously, it had been determined that Manuka honey royal jelly colloidal particles had a pI around 4.3. These findings suggest that the royal jelly colloidal particle composition changes to produce a new pI around a pH of 5.9. This is most likely due to MGO modification of arginine, lysine and cysteine residues in the proteins.

Zeta potential of OH BEE HAVE colloidal nanoparticles Figure 4: Zeta potential for royal jelly proteins in ΩH ଓଁEE ħΛVE

ΩH ଓଁEE ħΛVE contains small protein particles that contain royal jelly proteins and apisimin that are around 100 nm in size. The structure of the particles have been determined (Figure 5).

royallactin

Figure 5: Royal jelly protein 1, apisimin and 24 methylene cholesterol

The royal jelly colloidal nanoparticles isolated from Manuka honey contain a number of proteins, phenolics, minerals, lipids and sterols (potentially 24 methylene cholesterol, not confirmed by scientific testing) and carbohydrates (N-acetylglucosamine (NAG) bound to ASN-144 on MRJP1 providing anti-hypertensive activity) This is implied by scientific evidence and has not been proven to be the case with OH BEE HAVE. One of the proteins apisimin which hydrophobic lipid-like protein, which coats the major royal jelly proteins making them compatible with skin. It absorbs quickly.  The royal jelly particles are stable up to 55 degrees Celsius as outlined in the scientific literature. They have a negatively charged surface enabling their suspension in solution but they also aggregate below pH 5.0 as the histidine residues on the surface of the protein become positively charged (ionized) below this pH value. 

 colloidal nanoparticles from Manuka honey

Figure 5: Nanosight close-up image of royal jelly colloidal nanoparticle isolated from Manuka honey and bottled at 1 billion royal jelly particles per mL in ΩH ଓଁEE ħΛVE. 

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Composition of honey protein colloidal nanoparticles

colloidal nanoparticles in honey

Figure 6: Colloidal nanoparticles in honey were identified back in the 1930s.

honey colloids analysis

Figure 7: Honey colloidal analysis.

The royal jelly particles were rediscovered again in 2017.

Published: 09 August 2017 Active macromolecules of honey form colloidal particles essential for honey antibacterial activity and hydrogen peroxide production Katrina Brudzynski, Danielle Miotto, Linda Kim, Calvin Sjaarda, Liset Maldonado-Alvarez & Henryk Fukś Scientific Reports volume 7, Article number: 7637 (2017).

They were also purified and their structure determined recently using x-ray crystallography. The H like structure determined and the ratios of MRJP1 : apisimin : 24 methylene cholesterol (4:4:8).

royal jelly protein structure

When the isolated royal jelly particles were analysed using the Nanosight instrument the following observations were made. 

Figure 8: The royal jelly protein colloidal nanoparticles isolated from Manuka honey are put into a bottle of ΩH ଓଁEE ħΛVE. Video produced using the NanoSight instrument (University of Auckland).

Photo-reduction / photo-oxidation cycle

Radical chemistry occurs in honey once dissolved in water. This is usually triggered by glucose oxidase producing hydrogen peroxide. However, this is not the case in Manuka honey as MGO inhibits glucose oxidase (J Med Food. 2014 Feb;17(2):290-3. doi: 10.1089/jmf.2012.0201. Epub 2013 Nov 5. Methylglyoxal may affect hydrogen peroxide accumulation in manuka honey through the inhibition of glucose oxidase. Majtan J1, Bohova J, Prochazka E, Klaudiny J). Hydrogen peroxide reacts with the iron present in honey producing hydroxyl radicals (anti-microbial) present in glucose oxidase active honeys. 

Brudzynski K, Lannigan R. (2012). Mechanism of Honey Bacteriostatic Action Against MRSA and VRE Involves Hydroxyl Radicals Generated from Honey's Hydrogen Peroxide. Front Microbiol. 2012 Feb 7;3:36.

As MGO inhibits glucose oxidase this is not the anti-microbial mechanism of action present in Manuka honey. MGO concentration correlates with the anti-microbial properties, however, MGO alone is not the active. 

Manuka honey activities 

Figure 9: A wide range of functional compounds are present that have a range of biological activities.

 Manuka honey analysis

Figure 10: Manuka honey analysis

 The presence of Fe and phenolics in Manuka honey provides an opportunity for the following chemistry.

 Radicals

Figure 11: Potential for radicals is modulated through hydrogen peroxide and reduced Fe (iron). The reduction of iron from Fe3+ to Fe2+ is mediated via coordination chemistry and pi electrons in the aromatic ring of the phenolic compounds bound to the royal jelly colloidal nanoparticles.

The photo-reduction by light is part of the photo-Fenton cycle that generates hydroxyl radicals. The presence of dimer dihydroxyacetone (DHA), polymeric glyoxal and various methylglyoxal structures means that these compounds also perform coordination chemistry with iron and undergo photo-reduction / photo-oxidation cycles to generate hydroxyl radicals. The breaking of bonds in molecules by the hydroxyl radicals releases photons of light as shown in the following video.

Figure 12: Methylglyoxal + FeSO4 + H2O2 + Phenyllactate. On a microscope slide at 10x magnification with DAPI light filter cube (Excitation 357 nm, UVA). The production of bio-photons via coordination chemistry produces interesting light based energy system. 

Confirmation of radical chemistry in operating in Manuka honey during dissolving in water and exposure of UV light via the photo-reduction using UVA. Only on dilution does changes in radical chemistry occur within Manuka honey. The material appears stable to UV light prior to dilution.

 NBT reaction in diluted honey

Figure 13: NBT reaction with diluted Manuka honey detection of superoxides

Radical chemistry cascade

Figure 14: Radical chemistry cascade

It is proposed that radical chemistry is responsible for the generation of methyl syringate from benzoic acid as we have seen increased concentrations of methyl syringate after UV exposure.

Conversion of benzoic acid into methyl syringate 

Figure 15: Proposed UV induced production of methyl syringate.

MGO reactions with proteins also generate a range of radicals. 

Hyung-Soon Yim, Sa-Ouk Kang, Yung-Chil Hah, P. Boon Chock, and Moon B. Yim. (1995).¶ Free Radicals Generated during the Glycation Reaction of Amino Acids by Methylglyoxal A MODEL STUDY OF PROTEIN-CROSS-LINKED FREE RADICALS. The Journal of Biological Chemistry. Vol 270 No. 47, issue 24 pages 28228-28233.

The presence of polymeric glyoxal in pollen isolated from Manuka honey and its cleavage by either the hydroxyl radical or methyl radical provides an identity of the origin of DHA and MGO.

glyoxal polymer

glyoxal polymer 2 MALDI TOF MS

Figure 16: Polymeric glyoxal present in Manuka honey pollen. A) glyoxal structure and MALDI TOF MS polymer, B) bright field analysis of pollen grain on LED Evos FL microscope, C) Qdot longpass fluorescence analysis of pollen grain isolated from manuka honey on LED Evos FL microscope, D) DAPI filter set fluorescence analysis of pollen grain isolated from manuka honey on LED Evos FL microscope and E) spectral profile of fraction containing the pollen grain that gave the polymeric material.

Pollen grains changes in fluorescence over time during maturation of Manuka honey 

Figure 17: A) Manuka pollen isolated from the flower under bright field, B) Manuka pollen isolated from the flower under DAPI LED light source fluorescence analysis, C) A young Manuka honey pollen isolated and fluorescence analysis, D) Single pollen grain isolated from an aged Manuka honey fluorescence analysis, E) Single pollen grain isolated from an aged Manuka honey bright field analysis, F) Aged Manuka honey isolated pollen, G) Aged Manuka honey isolated pollen increased magnification fluorescence analysis and H) Aged Manuka honey isolated pollen increased magnification bright field analysis

There are changes in fluorescence in pollen grains over time during Manuka honey maturation. This provides further evidence for the origin of MGO in Manuka honey.

Fluorescence of pollen grains

Figure 18: Fluorescence of pollen grains in Mature Manuka honey

Radical chemistry is touted as bad in the health and wellness industry. However, I think we need a rethink. Honey is the food of the Gods.

After the light induced generation of radicals a solution remained on the microscope slide. When light of 357 nm was used to irradiate the solution a structure was generated. Changing the wavelength of light back to white light full spectrum the structure was released and photons of light were released. 

 

My current thinking is that radicals are critical for many biological processes including quantum entanglement where radicals cascade from one molecule to another producing complex structures based on energies in an environment. Further supporting evidence will be provided to the beneficial functional roles for various radicals.

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