WO2017099612A1 - Composés marqueurs de miels de leptospermum et leurs procédés d'isolement et de dosage - Google Patents

Composés marqueurs de miels de leptospermum et leurs procédés d'isolement et de dosage Download PDF

Info

Publication number
WO2017099612A1
WO2017099612A1 PCT/NZ2016/050195 NZ2016050195W WO2017099612A1 WO 2017099612 A1 WO2017099612 A1 WO 2017099612A1 NZ 2016050195 W NZ2016050195 W NZ 2016050195W WO 2017099612 A1 WO2017099612 A1 WO 2017099612A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
honey
structural formula
leptospermum
following structural
Prior art date
Application number
PCT/NZ2016/050195
Other languages
English (en)
Inventor
Margaret Anne Brimble
Ralf Christian Schlothauer
Gordana PRIJIC
Jonathan Stephens
Benjamin DANIELS
Original Assignee
Comvita Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from NZ722140A external-priority patent/NZ722140A0/en
Application filed by Comvita Limited filed Critical Comvita Limited
Priority to AU2016365528A priority Critical patent/AU2016365528B2/en
Publication of WO2017099612A1 publication Critical patent/WO2017099612A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D475/00—Heterocyclic compounds containing pteridine ring systems
    • C07D475/02—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution

Definitions

  • marker compounds of Leptospermum honeys novel isotopically labelled marker compounds of Leptospermum honeys and methods of isolation, chemical synthesis and assaying thereof, for use in the verification of the quality and purity of Leptospermum honeys such as Manuka honey.
  • Manuka honey is active against methicillin-resistant Staphylococcus aureus (M RSA) 9 , 10 and increases the susceptibility of MRSA to rifampicin 11 and oxacillin 12 .
  • M RSA methicillin-resistant Staphylococcus aureus
  • Pteridines (1, Figure 1) are derivatives of the pyrazine ⁇ B-dlpyrimidine ring system, the first examples of which were isolated from butterfly wings 18, 19 .
  • 2,4-Diketopteridines are known as lumazines (2), examples of which have been isolated from a range of organisms 20, 21, 22, 23, 24 .
  • lepteridine (3) 25 a known pteridine derivative from
  • a marker compound of Type I isolated from a Leptospermum honey and represented by the following structural formula I:
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 or -C4H9 alkyl group.
  • Leptospermum honey refers to flowers, nectar or honey of the Leptospermum plant species including Leptospermum scoparium, Leptospermum scoparium var. exinium, Leptospermum polygalifolium, Leptospermum submur.
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 or -C4H9 alkyl group where the compound of Type I I contains at least one isotope not having an identical atomic mass to that of the most abundantly occurring isotope of that element in nature.
  • isotope refers to an isotope not having an identical atomic mass to that of the most abundantly occurring isotope of that element in nature, which is understood to have been introduced by means known to those skilled in the art of organic synthesis from commercially available isotopically enriched starting materials.
  • isotopes not having an identical atomic mass to that of the most abundantly occurring isotope of that element in nature include but are not limited to 2 H, 13 C and 15 N.
  • a method of isolation of at least one compound of the Type I as described above comprising the following step: a. a chromatography step in which a fraction is collected by eluting a solution of a honey derived from nectar collected from a Leptospermum flower with at least one elution solvent.
  • a method of assaying and quantifying a Leptospermum honey comprising the following steps:
  • step (c) deriving the concentration of at least one compound of Type I native to the Leptospermum honey via interpolation using the calibration curve generated in step (a) and the mass spectrum generated in step (b).
  • a method of assaying and quantifying a Leptospermum honey comprising the following steps: a. subjecting a Leptospernum honey to a stimulus sufficient to cause fluorescence of at least one compound of Type I as described above present in the Leptospernum honey; and
  • test kit for testing the purity of a Leptospermum honey, the kit comprising at least one compound of Type I or Type I I as described above.
  • Leptospermum honey and represented by the following structural formula I:
  • composition comprising of at least one compound of Type I isolated from a Leptospermum honey and represented by the following structural formula I:
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 or
  • a food supplement to improve physiological oxidative stress comprising at least one compound of Type I isolated from a Leptospermum honey and represented by the following structural formula I :
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 H 9 alkyl group produced via a condensation of an intermediate of the following structural formula II
  • R 1 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H g alkyl group with a compound of the following structural formula III:
  • R 2 and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C4H9 alkyl group produced via an alkylation of the compound represented by the following structural formula IV at position N-3:
  • R 2 and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 H g alkyl group produced via the generation of a transient isocyanate species from a compound of the following structural formula V:
  • R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C4H9 alkyl group, wherein the compound of Type I I contains at least one isotope produced via a condensation of an intermediate of the following structural formula II:
  • R 1 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group with a compound of the following structural formula III:
  • R 2 and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 H 9 alkyl group, produced via an alkylation of the compound represented by the following structural formula IV at position N-3:
  • R 2 and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represent either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 H 9 alkyl group, produced via the generation of a transient isocyanate species from a compound of the following structural formula V:
  • advantages of the marker compounds of Leptospermum honeys and methods of isolation and assaying thereof described herein may comprise:
  • Figure 1 shows the chemical structure of pteridine (1), lumazine (2), lepteridine (3), leptosperin (4);
  • FIG. 2 shows the HM BC (heteronuclear multiple bond correlation) of lepteridine
  • Figure 3 shows a scheme for the synthesis of lepteridine
  • Figure 4 shows the 1H N MR spectra of natural and synthetic lepteridine
  • Figure 5 shows a scheme for the synthesis of 3,6,7-(3- 2 H 3 )trimethyllumazine
  • Figure 6 shows (A) Emission spectra of lepteridine standard ( ), manuka honey (— ), and manuka nectar ( ⁇ ⁇ ) at 330nm excitation wavelength. "X' indicates a non-diagnostic peak. Data shows mean spectra from experiments performed in duplicate. (B) Correlation between lepteridine concentration and fluorescence intensity for 27 manuka honey samples.
  • Figure 7 shows a mass spectrum of a typical manuka honey sample before (A) and after (B)
  • Figure 11 shows independently the HPLC chromatograms for leptosperin (262 nm) and lepteridine
  • Figure 12 shows normalisation of the nectar concentrations with reduced concentrations in both leptosperin and lepteridine in the fully ripened honey compared to the corresponding nectar at start of the dehydration process;
  • C illustrates the distribution of leptosperin and lepteridine loss as a mean percentage of the initial concentration in the nectar;
  • D and E illustrate, respectively, the normalised concentration changes of leptosperin and lepteridine over time at 100% ( ⁇ ), 66% ( ⁇ ), and 33%
  • A L. scoparium nectar content;
  • F illustrates the comparison of the % compound loss between the individual nectar groups for both leptosperin (shaded bars) and lepteridine (unshaded bars).
  • the inventors screened flower nectar and honeys of various floral types found in New Zealand to identify chemicals that were either unique to or in significantly higher concentrations in manuka nectar and mono-floral manuka honey compared to other predominantly mono-floral nectars and honeys.
  • the inventors discovered a marker compounds that could distinguish Leptospermum honey nectar and honey from other floral sources.
  • a marker compound of Type I isolated from a Leptospermum honey and represented by the following structural formula I:
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 -C4H9 alkyl group.
  • R 1 , R 2 , and R 3 are -CH 3 (this compound is referred to as 3,6,7-trimethyllumazine or lepteridine).
  • the Leptospermum honey is selected from the flower group comprising: Leptospermum scoparium, Leptospermum scoparium var. exinium, Leptospermum polygalifolium, Leptospermum sub 1952.
  • a Leptospermum honey certification protocol may include at least one of these Lepteridine analogue as described above either in absolute amounts or in a relative ratio of the marker compounds.
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 or -C4H9 alkyl group where the marker compound of Type I I contains at least one isotope not having an identical atomic mass to that of the most abundantly occurring isotope of that element in nature.
  • a chromatography step in which a fraction is collected by eluting a honey derived from nectar collected from a Leptospermum flower with at least one elution solvent.
  • the compound represented by the formula I can be further purified with a second and subsequent chromatography or other purification steps.
  • the organic elution solvent is a solution of acetic acid or a solution of acetonitrile.
  • organic elution solvents could be used without departing from the scope of the method of manufacture described above such as formic or trifluoroacetic acid or organic alcohols such as Cl-4 linear or branched chain alcohols.
  • the form of chromatography in the first chromatography step is not limited. It is not limited to column chromatography and may take various other forms.
  • a detection means such as an MS detector.
  • a method of assaying and quantifying a Leptospermum honey comprising the following steps: a. deriving a calibration curve for the concentration of a compound of Type I I using mass
  • step (c) deriving the concentration of a compound of Type I native to the Leptospermum honey via interpolation using the calibration curve generated in step (a) and the mass spectrum generated in step (b).
  • a method of assaying and quantifying a Leptospermum honey comprising the following step: a. subjecting a Leptospernum honey to a stimulus sufficient to cause fluorescence of a compound of Type I as described above present in the Leptospernum honey; and
  • a manuka honey certification protocol may include at least two or three marker compounds to increase its robustness and reliability.
  • the method of assying and quantifying would also include the method step c) of determining the authenticity of the Leptospermum honey based on the measured amount of the at least one compound represented by the formula I. Either the absolute amounts could be used in the determination step or the relative ratio of the marker compounds (depending upon the variations of the marker compounds from different regions and/or climatic conditions).
  • test kit for testing the purity of a Leptospermum honey, the kit comprising at least one compound of the Type I or Type II as described above.
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H g alkylgroup, in the manufacture of a medicament for the treatment of disease.
  • composition comprising of at least one compound of Type I isolated from manuka honey and represented by the following structural formula I:
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group for use in the treatment of wounds.
  • a food supplement to improve physiological oxidative stress comprising at least one compound of Type I isolated from a Leptospermum honey and represented by the following structural formula I:
  • R 1 , R 2 , and R 3 independently represents either a -H, -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 H 9 alkyl group produced via a condensation of an intermediate of the following structural formula II: II
  • R 1 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H g alkyl group with a compound of the following structural formula III:
  • R 2 and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • the compound of the structural formula III is in the presence of an acid in a liquid carrier.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 H 9 alkyl group produced via an alkylation of the compound represented by the following structural formula IV at position N-3:
  • R 2 and R 3 independently represents either a - IVCH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 Hg alkyl group produced via the generation of a transient isocyanate species from a compound of the following structural formula V:
  • R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • the transient isocyanate species is that generated by a Curtius, Hofmann, Lossen or Schmidt rearrangement.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 H 9 alkyl group, where the compound of Type I I contains at least one isotope produced via a condensation of an intermediate of the following structural formula II:
  • R 1 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group with a compound of the following structural formula III:
  • R 2 and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H g alkyl group.
  • R 1 , R 2 , and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C 4 H 9 alkyl group, produced via an alkylation of the compound represented by the following structural formula IV at position N-3:
  • R 2 and R 3 independently represents either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • R 1 , R 2 , and R 3 independently represent either a -CH 3 , -C 2 H 5 , -C 3 H 7 or - C4H9 alkyl group, via the generation of a transient isocyanate species from a compound of the following structural formula V:
  • R 2 , and R 3 independently represent either a -CH 3 , -C 2 H 5 , -C 3 H 7 or -C 4 H 9 alkyl group.
  • isotopes not having an identical atomic mass to that of the most abundantly occurring isotope of that element in nature, such as 2 H, 13 C or 15 N, have been introduced by means known to those skilled in the art of organic synthesis from commercially available isotopically enriched starting materials.
  • At least one composition represented by formula I as described above could be used as an internal control to be added as a positive control to determine that the verification protocol is working correctly.
  • the filtrate was divided into two portions of 100 mL and each portion was subjected to SPE using MeOH- H 2 0 + 0.1% HCOOH (1:9, 80 mL) to remove undesired substances.
  • the desired fraction was then eluted using MeOH-H 2 0 + 0.1% HCOOH (4:1, 80 mL).
  • the two fractions were combined and concentrated to give the crude extract (0.23 g) which was further purified by flash chromatography (pet. ether-EtOAc 1:4) to give purified extract (3 mg) as a brown solid.
  • HMBC correlations are from protons stated to the indicated carbon or nitrogen.
  • the molecular formula of the unknown compound was established as CgH 10 N4O 2 by positive ion HRESI MS.
  • the compound was soluble in CD 3 OD and CDCI 3 ; the latter was used for recording N M R spectra due to the presence of a broad resonance at ⁇ 8.55 ppm (H-1) that was not present in spectra recorded in CD 3 OD.
  • This peak was assigned as an amide proton on the basis of its chemical shift and the absence of a distinctive hydroxyl absorption in the IR spectrum.
  • Amino uracil (6) was then treated with sodium nitrite (Na N0 2 ) and acetic acid (AcOH) solution, followed by reduction with sodium dithionite (Na 2 S 2 0 4 ) in the aqueous solvent ammonia (N H 3 ) at 70 °C 41 to give 5,6-diamino-3-methyluracil (7) in 31% yield over two steps.
  • Alternative acids which could be used in the nitrosation first step include hydrochloric acid.
  • An alternative to the first step reduction with sodium nitrite and acetic acid is actalytic hydrogenation using a catalyst such as palladium on carbon or platinum dioxide in an aqueous or organic solvent.
  • lepteridine 3,6,7-trimethyllumazine is a pteridine derivative isolated from Leptospermum honey the isolated compound was named lepteridine.
  • intermediate compound shown above or via transformation of the intermediate compound shown below into a transient isocyanate species including but not limited to those generated by a Curtius, Hofmann, Lossen or Schmidt rearrangement.
  • Amino uracil (6) was then treated with sodium nitrite (Na N0 2 ) and acetic acid (AcOH) solution, followed by reduction with sodium dithionite (Na 2 S 2 0 4 ) in the aqueous solvent ammonia (N H 3 ) at 70 °C 40 to give 5,6-diamino-3-( 2 H 3 )methyluracil (10) in 31% yield over two steps.
  • Alternative acids which could be used in the nitrosation first step include hydrochloric acid.
  • An alternative to the first step reduction with sodium nitrite and acetic acid is actalytic hydrogenation using a catalyst such as palladium on carbon or platinum dioxide in an aqueous or organic solvent.
  • RP-HPLC was performed with an Agilent 1100 using a Jupiter C 18 300 A, 5 ⁇ , 2.0 mm x 250 mm column at a flow rate of 0.2 mLmin 1 with a DAD Detector operating at 262, 280 and 320 nm.
  • a suitably adjusted gradient of 5% B to 100% B was used, where solvent A was 0.1% HCOOH in H 2 0 and B was 20 % A in MeCN. Flash chromatography was carried out using 0.063-0.1 mm silica gel with the desired solvent.
  • TLC Thin layer chromatography
  • Kieselgel F254 Merck silica plates and compounds were visualised using UV irradiation at 254 or 365 nm and/or staining with a solution of potassium permanganate and potassium carbonate in aqueous sodium hydroxide.
  • Preparative TLC was performed using 500 ⁇ , 20 x 20 cm Un iplateTM (Analtech) silica gel TLC plates and compounds were visualised using UV irradiation at 254 or 365 nm. Melting points were determined on a Kofler hot-stage apparatus and are uncorrected.
  • Infrared spectra were obtained using a Perkin-Elmer Spectrum 100 FTIR spectrometer on a film ATR sampling accessory. Absorption maxima are expressed in wavenumbers (cm "1 ).
  • N M R spectra were recorded as indicated on either a Bruker Avance 400 spectrometer operating at 400 MHz for 1 nuclei and 100 MHz for 13 C nuclei, a Bruker DRX-400 spectrometer operating at 400 M Hz for 1 nuclei, 100 M Hz for 13 C nuclei, a Bruker Avance AVI II- HD 500 spectrometer operating at 500 MHz for 1 nuclei, 125 MHz for 13 C nuclei or a Bruker Avance 600 spectrometer operating at 600 M Hz for 1 nuclei, 150 MHz for 13 C nuclei.
  • a Leptospernum honey in the form of Manuka honey was diluted in sterilised distilled water to a concentration of 2 % (w/v).
  • the diluted Manuka honey is placed in microtiter plates and measured in a spectrofluometer (for example a Gemini EM Dual Scanning Microplate Spectrofluorometer manfactured by Molecular Devices Inc coupled to an external computer equiped with SoftMax Pro software) with top down reading for better signal to noise ratio. All samples were incubated and read at room temperature.
  • the diluted honey sample was subjected to a stimulus in the form of an excitation wavelength of 330 nm sufficient to cause fluorescence of lepteridine present in the Leptospernum honey.
  • the presence of lepteridine was detected by measuring an emmission wavelength of 470 nm in a spectrophotmeter.
  • a calibration profile can be derived which can be used in the interpretation of the 470 nm fluorescence value for the purposes determining the authencity of unknown honey samples.
  • Example 5 Lepteridine as the compound responsible for manuka honey fluorescence
  • lepteridine is a fluorescence marker compound of manuka honey
  • the emission spectrum of lepteridine were plotted at 330 excitation wavelength alongside manuka honey and manuka nectar (see Figure 6A).
  • the pattern of all emission spectrum were almost identical, showing elevated fluorescence ranging from 390nm to 590nm and peak fluorescence at 470nm.
  • fluorescence spectrometry is a highly specific analytical technique controlled by two independent wavelengths.
  • this level of similarity provides strong support for lepteridine the responsible compound for M M2 fluorescence (as indicated by the arrow on Figure 6A).
  • another emission band can be observed at 330-650 ex-em wavelength.
  • this peak was non-diagnostic and it is present in all samples including blanks. This is likely to be caused by background interference arising from surface reflection or auto-fluorescence of the wells (Lakowicz, 2006). Therefore, this emission peak should not be interpreted as fluorescence signal from samples.
  • lepteridine is directly relevant to fluorescence displayed at the MM2 wavelength. However, there are chances that multiple compounds could be present in manuka honey which fluoresce at similar wavelengths. If that is the case, spiking lepteridine into manuka honey should not lead to the expected increase in fluorescence (according to Figure 6B).
  • O.C ⁇ g, O.lC ⁇ g, and O.l ⁇ g of lepteridine standard were spiked into 3 manuka honey samples with pre-quantified lepteridine concentration at 0.3C ⁇ g/kg, 0.51 ⁇ g/kg, and 0.7C ⁇ g/kg.
  • Figure 6D plotted fluorescence against lepteridine concentration for spiked manuka honeys samples (— o— ), and compared with earlier results from natural manuka honey samples ( ⁇ — x—-).
  • Our results demonstrate the addition of lepteridine directly led to the expected level of increase in fluorescence. Thus indicating that lepteridine is probably to be the principle compound responsible for M M2 fluorescence.
  • Example 6 Quantification of lepteridine in manuka honey using mass spectrometry
  • lepteridine may be utilised as a fluorescent marker compound for manuka honey.
  • the fluorescence intensity ( ex 330nm - em 470nm) demonstrated strong linear correlation with lepteridine concentration quantified by HPLC (area under curve). However, these results are best validated using a separate quantitative approach.
  • LC-MS/MS tandem mass spectrometry
  • Example 7 Stability of Leperidine as a marker of manuka honey
  • New Zealand manuka (Leptospermum scoparium) honey contains unique nectar-derived compounds useful for its identification. Chemical alterations to these compounds during the honey ripening process are currently unknown. Relative concentration changes of lepteridine and leptosperin (a known marker in the art and used as a reference standard herein) were examined during L. scoparium nectar to honey conversion. Concentration changes of these compounds were often non-linear with respect to increasing sugar concentration. Normalisation relative to an 80 °Brix sugar solution showed a mean percentage loss of 13.66 ⁇ 0.77% for leptosperin and 9.62 ⁇ 1.03% for lepteridine. These two compound losses appeared to be non-enzymatic and independent of floral dilution. The lack of a floral dilution effect on leptosperin and lepteridine losses during nectar to honey conversion strongly reinforces the use of these compounds as chemical markers for authentication of manuka honey.
  • nectar to honey is essentially a two-step process: the hydrolysis of sucrose to glucose and fructose followed by evaporation of excess water.
  • a laboratory simulation of the honey ripening process was carried out to examine the chemical changes that occur during L. scoparium nectar conversion to honey.
  • leptosperin and lepteridine are stable over prolonged storage and heat treatment in honey, the chemical stability of these compounds in L. scoparium nectar during the honey ripening process has not been examined.
  • the inherently higher level of leptosperin a nd lepteridine in nectar suggest chemical changes or physical processes during nectar to honey transformation that result in loss of these compounds in the final honey product. It is possible that these manuka-specific floral markers are modified or broken down following bee enzymatic activity or the physico-chemical changes that take place during the conversion process.
  • nectar dilutions 100%, 66%, and 33% v/v L. scoparium nectar content
  • nectar solution 8 °Brix
  • the principal enzymes from the bee hypopharyngeal glands namely glucose oxidase, a-glucosidase, and ⁇ -glucosidase, were supplemented into nectars in three treatment groups as indicated in Figure 10.
  • the final enzyme concentration of glucose oxidase, ⁇ -glucosidase, and ⁇ -glucosidase were 0.1, 0.0005, and 0.0005 mg/ml respectively.
  • a no-enzyme treatment consisting of nectar only was included as a control.
  • the dehydration process was carried out in a dehydrator at 37 °C with a starting volume of 480 ⁇ .
  • the experiment was carried out in duplicate, and the partially evaporated nectar was subsampled at different time points during the process.
  • the nectar was considered fully ripened when it reached approximately 80 °Brix, and the final honey product was extracted by manual scraping off the wax containers. All nectar and honey samples were stored at -20 °C until analysis.
  • Leptosperin and lepteridine concentrations were quantified on a Dionex UltimateTM 3000 reversed-phase high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific, New Zealand) with diode-array detection (DAD) based on known methods in the art.
  • HPLC high-performance liquid chromatography
  • DAD diode-array detection
  • Honey and nectar samples were diluted in 0.1% v/v formic acid to a final sugar concentration in the range of 1 to 2 "Brix.
  • the injection volume was 3 ⁇ . Separation was carried out on a Hypersil GOLD column (150 ⁇ 2.1 mm; 3 ⁇ particle size) by gradient elution at a constant flow rate of 0.200 ml/min.
  • the binary mobile phase consisted of 0.1% v/v aqueous formic acid (Solvent A) and 80:20 acetonitrile:Solvent A (Solvent B).
  • a 30 min gradient elution programmed was employed: initial (5% B, held 2 min), 14 min (50% B), 16 min (100% B, held 3 min), 20 min (5% B, held 10 min).
  • the col umn was thermostatically controlled at 25 °C.
  • Leptosperin and lepteridine were monitored at 262, and 320 nm, respectively. Identification of these compounds were based on retention time. Under the specified chromatographic conditions, leptosperin has a retention time of 14.1 min, and lepteridine 12.9 min at the respective detection wavelengths.
  • An artificial nectar (20 °Brix) was supplemented with leptosperin and lepteridine chemical standards at a concentration equivalent to 250 mg/kg and 15 mg/kg in honey, respectively.
  • the solution was incubated at 37 °C for two hours in the presence glucose oxidase (0.1 mg/ml), a-glucosidase (0.0005 mg/ml), and ⁇ -glucosidase (0.0005 mg/ml).
  • glucose oxidase 0.1 mg/ml
  • a-glucosidase 0.0005 mg/ml
  • ⁇ -glucosidase 0.0005 mg/ml
  • concentrations of leptosperin and lepteridine are expressed as a weight ratio of the compound of interest/80 °Brix sugar solution in mg/kg.
  • the normalisation by sugar content to 80 °Brix eliminates variance due to differences in sugar content, and therefore allows fair comparison between all samples.
  • Leptosperin and lepteridine concentrations were measured by HPLC-DAD with monitoring at 262 nm and 320 nm, respectively.
  • Figure 11A and B illustrate independently the HPLC chromatograms for leptosperin (262 nm) and lepteridine (320 nm) in the undiluted L scoparium nectar used during this analysis.
  • the major peak with a retention time of 14.1 min corresponds to leptosperin
  • the peak at 12.9 min Figure 11B
  • This nectar carried 9966 ⁇ 13 mg/kg leptosperin and 212 ⁇ 0.4 mg/kg lepteridine.
  • Figure llC shows the relationships between sugar (x-axis) and both leptosperin (— ; left y-axis) and lepteridine (— ; right y-axis) concentrations in L. scoparium nectar during conversion to honey.
  • the data plotted encompassed al l three floral dilution groups at 100%, 66%, and 33% L. scoparium nectar content subsampled at various time points during the dehydration process.
  • the increases in both leptosperin and lepteridine concentrations were non-linear with respect to sugar concentration.
  • the individual correlations were best-fitted to a second-order polynomial function, and were consistent irrespective of the L. scoparium nectar content.
  • nectars ripened in the presence of glucose oxidase (Group 1, 2, and 3) behaved similarly to the nectar control without glucose oxidase (p>0.05). Therefore acidification was unlikely to account for the loss of these compounds.
  • nectar dehydration experiment an artificial nectar (20 "Brix) with supplemented leptosperin and lepteridine at 2500 mg/kg and 150 mg/kg concentration equivalent in honey, respectively, was incubated independently with glucose oxidase, a-glucosidase, and ⁇ -glucosidase at concentrations similar to the nectar dehydration experiment.
  • Analysis by HPLC revealed no significant changes in both compound concentrations following a two-hour incubation at 37 °C (p>0.05), thereby confirming that the observed loss of leptosperin and lepteridine during the nectar dehydration process was not due to hydrolysis by these enzymes.
  • Figure 12C illustrates the distribution of leptosperin and lepteridine loss as a mean percentage of the initial concentration in the nectar. Overall, there was a mean percentage loss of 13.66 ⁇ 0.77% for leptosperin and 9.62 ⁇ 1.03% for lepteridine, which were considerably less compared to the concentration differences in nectar and honey previously reported.
  • Figure 12D and E illustrate, respectively, the normalised concentration changes of leptosperin and lepteridine over time at 100% ( ⁇ ), 66% ( ⁇ ), and 33% ( A ) L scoparium nectar content.
  • the datasets plotted represent combined mean value from all enzyme treatment groups and control. Whilst the concentrations of both compounds at t 3 were significantly reduced (leptosperin, p ⁇ 0.0001; lepteridine, p ⁇ 0.001), one-way ANOVA comparison of the % compound loss between the individual nectar groups revealed no significant differences between the 100%, 66%, and 33% L.
  • the concentration of leptosperin and lepteridine in honey would be expected to correlate with the inherent quantities present in the bulk nectar incorporated into the beehive, thus reinforcing the use of leptosperin and lepteridine as an indicator for florality status of manuka honey.
  • LC-MS/MS quantification HPLC-grade acetonitrile and formic acid were purchased from Merck. Water was purified using the Barnstead Nanopure Diamond laboratory water system. A lOul injection was made of each sample directly onto a 0.3 x 100 mm Zorbax 300SB- C18 column (Agilent, Santa Clara, CA, USA) at 12 ul/min for 6 minutes.
  • the HPLC gradient between Buffer A (0.1% formic acid in water) and Buffer B (0.1% formic acid in acetonitrile) was formed at 6 ul/min as follows: 10% B for the first 3 min, increasing to 25% B by 18 min, increasing to 97% B by 21 min, held at 97% until 24 min, back to 10% B at 25.5min and held there until 30 min.
  • the LC effluent was directed into the lonspray source of QSTAR XL hybrid Quadrupole-Time-of-Flight mass spectrometer (Applied Biosystems, Foster City, CA, USA) scanning from 150-800 m/z.
  • 6-Amino-3-methyluracil (6) 6-Aminouracil (5) (5.18 g, 40.7 mmol) was suspended in HM DS (25 mL) and H 2 S0 4 (0.1 m L) was added. The mixture was heated at reflux for 3 h then concentrated in vacuo. The residue was dissolved in DM F (30 mL), Mel (8.5 mL, 136.5 mmol) was added, and stirring was continued for 72 h at room temperature. The reaction was cooled to 0 °C and NaHC0 3 was carefully added. The mixture was stirred at 0 °C until no more bubbling was observed. The precipitate was filtered, washed with MeOH and H 2 0 and dried to give the title compound 6 (4.49 g, 78%) as a yellow solid which was used without further purification.
  • 6-Amino-3-methyl-5-nitrosouracil 6-Amino-3-methyluracil (6) (1.04 g, 7.40 mmol) was suspended in H 2 0 (10 mL). The suspension was heated at reflux for 2 h then cooled to room temperature. AcOH (4.20 g, 69.9 mmol) was added. A solution of NaN0 2 (1.04 g, 15.1 mmol) in H 2 0 (7 mL) was then added dropwise over 5 min, during which the pale yellow suspension became violet. The mixture was stirred for 30 min before the precipitate was filtered, washed with MeOH and H 2 0 and dried to give the title compound (1.07 g, 85%) as a violet solid which was used without further purification.
  • UV-vis (H 2 0) A max (log ⁇ ) 208 (0.54), 234 (0.45), 329 (0.35);
  • UV-vis (H 2 0) A max (log ⁇ ) 211 (2.77), 231 (2.36), 329 (1.42);
  • 6-Amino-3-( 2 H 3 )methyluracil 6-Aminouracil (1.05 g, 8.29 mmol) was suspended in hexamethyldisilazane (5 mL) and sulfuric acid (0.02 mL) was added. The mixture was heated at reflux for 1.5 h then concentrated in vacuo. The residue was dissolved in dimethylformamide (6 mL) and iodomethane-a ⁇ (0.8 mL, 12.9 mmol) was added, and stirring was continued for 72 h at room temperature. The reaction was cooled to 0 °C and sodium bicarbonate (15 mL) was carefully added.
  • 6-Amino-3-( 2 H 3 )methyl-5-nitrosouracil 6-Amino-3-( 2 H 3 )methyluracil (9) (0.40 g, 2.76 mmol) was suspended in water (5 m L). The suspension was heated at reflux for 2.5 h then cooled to room temperature. Acetic acid (1.68 g, 28.0 mmol) was added. A solution of sodium nitrite (0.46 g, 6.70 mmol) in water (4 mL) was then added dropwise over 5 min, during which the pale yellow suspension became grey. The mixture was stirred for 5 min before the precipitate was filtered, washed with methanol and water and dried to give the title compound (0.39 g, 81%) as a grey solid which was used without further purification;

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Dermatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne de nouveaux composés isolés de miel de Leptospermum et leurs procédés de dosage destinés à être utilisés dans le but de vérifier le lieu d'origine, l'authenticité et la teneur de miels de Leptospermum tels que le miel de mānuka. Les inventeurs ont criblé du nectar et des miels de fleur de divers types floraux rencontrés en Nouvelle-Zélande afin d'identifier des produits chimiques qui ont été soit propres au nectar de mānuka et au miel monofloral de mānuka, soit dans des concentrations significativement plus élevées dans ceux-ci comparativement aux autres nectars et miels essentiellement monofloraux. Grâce à l'exercice de criblage, les inventeurs ont découvert des composés marqueurs susceptibles de faire la distinction entre le nectar de miel et le miel de Leptospermum, et le miel provenant d'autres sources florales.
PCT/NZ2016/050195 2015-12-11 2016-12-12 Composés marqueurs de miels de leptospermum et leurs procédés d'isolement et de dosage WO2017099612A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2016365528A AU2016365528B2 (en) 2015-12-11 2016-12-12 Marker compounds of Leptospermum honeys and methods of isolation and assaying thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
NZ715094 2015-12-11
NZ71509415 2015-12-11
NZ722140 2016-07-13
NZ722140A NZ722140A0 (en) 2016-07-13 Marker Compounds of Leptospermum Honeys and Methods of Isolation and Assaying Thereof

Publications (1)

Publication Number Publication Date
WO2017099612A1 true WO2017099612A1 (fr) 2017-06-15

Family

ID=59013868

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NZ2016/050195 WO2017099612A1 (fr) 2015-12-11 2016-12-12 Composés marqueurs de miels de leptospermum et leurs procédés d'isolement et de dosage

Country Status (2)

Country Link
AU (1) AU2016365528B2 (fr)
WO (1) WO2017099612A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113820435A (zh) * 2021-11-23 2021-12-21 中国农业科学院蜜蜂研究所 水苏碱和左旋水苏碱作为米团花蜂蜜特征性标志物的应用
WO2022005308A1 (fr) * 2020-07-03 2022-01-06 Comvita Limited Compositions anti-inflammatoires, méthodes et utilisations associées
CN114096253A (zh) * 2019-07-04 2022-02-25 康维他有限公司 包含3，6，7-三甲基二氧四氢蝶啶的组合物用于预防、改善或治疗mmp-9相关病症和炎症的用途
WO2023093731A1 (fr) * 2021-11-23 2023-06-01 中国农业科学院蜜蜂研究所 Procédé d'identification de degré de maturité de miel d'acacia

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8759774B2 (en) * 2010-11-29 2014-06-24 Comvita New Zealand Limited Method and apparatus that utilises fluorescence to determine plant or botanical origin characteristics of honey

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8759774B2 (en) * 2010-11-29 2014-06-24 Comvita New Zealand Limited Method and apparatus that utilises fluorescence to determine plant or botanical origin characteristics of honey

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ADAMS C.J. ET AL.: "Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka (Leptospermum scoparium) honey", CARBOHYDRATE RESEARCH, vol. 343, no. 3, 2008, pages 651 - 659, XP022497589 *
ALVAREZ-SUAREZ J.M. ET AL.: "The Composition and Biological Activity of Honey: A Focus on Manuka Honey", FOODS, vol. 3, 2014, pages 420 - 432, XP055389352 *
DANIELS B.J. ET AL.: "Isolation, Structural Elucidation, and Synthesis of Lepteridine From Manuka (Leptospermum scoparium) Honey", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 64, 2016, pages 5079 - 5084, XP055389879 *
EREJUWA O.O. ET AL.: "Honey: A Novel Antioxidant", MOLECULES, vol. 17, 2012, pages 4400 - 4423, XP055389318 *
ETERAF-OSKOUEI T. ET AL.: "Traditional and Modern Uses of Natural Honey in Human Diseases: A Review", IRANIAN JOURNAL OF BASIC MEDICAL SCIENCES, vol. 16, no. 6, 2013, pages 731 - 742, XP055320724 *
RÜCKRIEMEN J. ET AL.: "Identification and Quantitation of 2-Acetyl-1-pyrroline in Manuka Honey (Leptospermum scoparium)", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 63, 2015, pages 8488 - 8492, XP055389868 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114096253A (zh) * 2019-07-04 2022-02-25 康维他有限公司 包含3，6，7-三甲基二氧四氢蝶啶的组合物用于预防、改善或治疗mmp-9相关病症和炎症的用途
EP3993803A4 (fr) * 2019-07-04 2023-08-02 Comvita Limited Utilisation d'une composition comprenant de la 3,6,7-triméthyllumazine pour prévenir, améliorer ou traiter des états associés à une métalloprotéinase matricielle-9 et une inflammation
WO2022005308A1 (fr) * 2020-07-03 2022-01-06 Comvita Limited Compositions anti-inflammatoires, méthodes et utilisations associées
AU2021299167B2 (en) * 2020-07-03 2024-03-21 Comvita Limited Anti-inflammatory compositions, methods and uses thereof
CN113820435A (zh) * 2021-11-23 2021-12-21 中国农业科学院蜜蜂研究所 水苏碱和左旋水苏碱作为米团花蜂蜜特征性标志物的应用
CN113820435B (zh) * 2021-11-23 2022-04-01 中国农业科学院蜜蜂研究所 水苏碱和左旋水苏碱作为米团花蜂蜜特征性标志物的应用
WO2023093731A1 (fr) * 2021-11-23 2023-06-01 中国农业科学院蜜蜂研究所 Procédé d'identification de degré de maturité de miel d'acacia

Also Published As

Publication number Publication date
AU2016365528A2 (en) 2020-12-17
AU2016365528B2 (en) 2022-01-20
AU2016365528A1 (en) 2018-07-05

Similar Documents

Publication Publication Date Title
AU2016365528B2 (en) Marker compounds of Leptospermum honeys and methods of isolation and assaying thereof
García-Estévez et al. First evidences of interaction between pyranoanthocyanins and salivary proline-rich proteins
Jang et al. Flavan-3-ols having a γ-lactam from the roots of Actinidia arguta inhibit the formation of advanced glycation end products in vitro
Monti et al. New insights on the interaction mechanism between tau protein and oleocanthal, an extra-virgin olive-oil bioactive component
Su et al. Identification of three flavan-3-ols from grapes
Quideau et al. DNA topoisomerase inhibitor acutissimin A and other flavano-ellagitannins in red wine
Julianti et al. Quantification of the antiplasmodial alkaloid carpaine in papaya (Carica papaya) leaves
Liang et al. A fluorogenic probe for ultrafast and reversible detection of formaldehyde in neurovascular tissues
Feng et al. Highly selective discrimination of cysteine from glutathione and homo-cysteine with a novel AIE-ESIPT fluorescent probe
Pfau et al. N6-adenyl arylation of DNA by aristolochic acid II and a synthetic model for the putative proximate carcinogen
Beretta et al. A solid-phase extraction procedure coupled to 1H NMR, with chemometric analysis, to seek reliable markers of the botanical origin of honey
Serdiuk et al. Flavonol-based fluorescent indicator for determination of β-glucosidase activity
Filho et al. Inhibitory effect of verbascoside isolated from Buddleja brasiliensis Jacq. ex Spreng on prolyl oligopeptidase activity
CN104771407B (zh) Cdk5抑制剂及其治疗用途
Mayser et al. Pityrialactone-a new fluorochrome from the tryptophan metabolism of M. alassezia furfur
Song et al. A sensitive and selective red fluorescent probe for imaging of cysteine in living cells and animals
Deng et al. Screening of α-glucosidase inhibitors from green tea extracts using immobilized enzymes affinity capture combined with UHPLC-QTOF MS analysis
Krüzselyi et al. Isolation and structural elucidation of a novel brunnein-type antioxidant β-carboline alkaloid from Cyclocybe cylindracea
Ilkei et al. C8-selective biomimetic transformation of 5, 7-dihydroxylated flavonoids by an acid-catalysed phenolic Mannich reaction: Synthesis of flavonoid alkaloids with quercetin and (–)-epicatechin skeletons
Li et al. Structural Characterization of Amadori Rearrangement Product of Glucosylated Nα‐Acetyl‐Lysine by Nuclear Magnetic Resonance Spectroscopy
Brown et al. Analytical procedures for cyclic nucleotides and their associated enzymes in plant tissues
Steklov et al. Facile synthesis of 8-azido-6-benzylaminopurine
Xue et al. Synthesis and evaluation of a novel ‘off-on’chemical sensor based on rhodamine B and the 2, 5-pyrrolidinedione moiety for selective discrimination of glutathione and its bioimaging in living cells
Bouaziz et al. Cinnamtannin B-1 from bay wood exhibits antiapoptotic effects in human platelets
NZ722140A (en) Marker Compounds of Leptospermum Honeys and Methods of Isolation and Assaying Thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16873435

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2016365528

Country of ref document: AU

Date of ref document: 20161212

Kind code of ref document: A

122 Ep: pct application non-entry in european phase

Ref document number: 16873435

Country of ref document: EP

Kind code of ref document: A1