METHOD FOR THE EXTRACTION OF PYRROLICIDINAL CALOIDS

Description

[0001] The invention relates to a method for extracting pyrrolizidine alkaloids present in a plant extract of interest.

[0002] Pyrrolizidine alkaloids (PAs) form a class of alkaloids and secondary metabolites characterized by a pyrrolizidine structure composed of two pyrrole rings. These alkaloids are produced by plants and constitute a defense mechanism against herbivorous animals.

[0003] No therapeutic applications have been found for these alkaloids, and it is rather their toxicity that attracts attention.

[0004] It is suspected that plant extracts used in perfumery, cosmetics, pharmaceuticals, and even food may contain pyrrolizidine alkaloids. These natural impurities are particularly undesirable contaminants due to their high toxicity. Consequently, herbal preparations, in general, are subject to monitoring, and a daily ingested dose of 1.5 µg of these impurities appears to be a level that does not present carcinogenic risks.

[0005] Authorities in some countries, particularly in Europe, have published a statement on the analysis of pyrrolizidine alkaloids to ensure the quality and safety of medicines. This statement included a list of plants in which pyrrolizidine alkaloids had been measured. Concentrations are generally low, on the order of a few µg / kg. St. John's wort ( Hypericum herb ) , The Passion Flower ( Passionflower herb) , Chamomile ( Motherwort flower ) , The lady's coat ( Alchemilla herb ) , Licorice ( Licorice root ) , Lemon balm ( Melissa leaf ) , Peppermint ( Peppermint leaf ) , Sage ( Sage leaf ) , Dandelion and root ( Dandelion herb with root ) .

[0006] It is noteworthy, however, that pyrrolizidine alkaloids are likely to be found in all plants and therefore the invention is not limited to the plants and / or extracts of said plants listed above but to all plants and / or extracts likely to contain pyrrolizidine alkaloids.

[0007] The general structure of pyrrolizidine alkaloids includes a pyrrolizidine motif or 1 H-Pyrrolizine, hexahydro- (5 / 5 cyclic system condensed with a nitrogen atom on a bridgehead, Formula 1) called necine (base) and substituted by a hydroxymethyl group at C-1 and a hydroxyl group at C-7 (Formula I).

[0008] When pyrrolizidine is substituted with a hydroxymethyl group at C-1 and a hydroxyl group at C-7 (Formula II), it is called necine, or retronecine when it is unsaturated at position -1,2. This is the basic unit common to all pyrrolizidine alkaloids.

[0009] The two hydroxyl groups are usually esterified with one or two acids called necic acids. These two side chains can also cyclize, forming a macrocyclic structure.

[0010] To date, more than 370 individual pyrrolizidine alkaloid structures from over 560 plant species have been isolated. Based on chemostasis and biogenetic studies, the majority of known pyrrolizidine alkaloids belong to one of four basic skeleton types, whose molecular structures III, IV, V, and VI are illustrated below.

[0011] Furthermore, the breaking of the intracyclic NC bond common to all pyrrolizidine alkaloids can be achieved by synthesis to lead to a fifth pyrrolizidine alkaloid skeleton of the otonecine type (Formula VII) such as, for example, senkirkine.

[0012] The fifth type encompasses the rare and structurally simpler phalaenopsins (pyrrolizidine alkaloids of orchids), which lack the 1,2 double bond and the hydroxyl group in position 7 on the basic necine skeleton (Formula VIII).

[0013] A European study conducted on over a thousand samples identified 35 pyrrolizidine alkaloids in animal samples and 28 pyrrolizidine alkaloids in plant samples, listed in Table 1 below. These 28 pyrrolizidine alkaloids are the ones considered in quality control of plant extracts intended for use in pharmaceuticals, food, cosmetics, or perfumery. Table 1: List of the 28 pyrrolizidine alkaloids monitored in plant-derived samples 1. Echimidine 15. Lycopsamine 2. Echimidine-N-oxide 16. Lycopsamine-N-oxide 3. Erucifolin 17. Monocrotaline 4. Erucifolin-N-oxide 18. Monocrotaline-N-oxide 5. European 19. Backwards 6. Europine-N-oxide 20. Retrorsine-N-oxide 7. Heliotrin 21. Senecionine 8. Heliotrine-N-oxide 22. Senecionine-N-oxide 9. In between 23. Seneciphylline 10. Intermedine-N-oxide 24. Seneciphylline-N-oxide 11. Jacobin 25. Senecivernine 12. Jacobine-N-oxide 26. Senecivernine-N-oxide 13. Lasiocarpine 27. Senkirkin 14. Lasiocarpine-N-oxide 28. Trichodesmin

[0014] All are representatives of the four basic structural types corresponding to formulas III, IV, V and VI.

[0015] Despite the apparent structural diversity of pyrrolizidine alkaloids, their chemical properties are very similar.

[0016] Besides the fact that the 28 pyrrolizidine alkaloids mentioned above belong to four different base types, they all carry hydrocarbon side chains with polar functions, mainly hydroxyl, methoxyl, and ester groups, and their molecular weights are greater than or equal to 300 Da. Thus, in terms of polarity and volatility, which are the key characteristics determining how steam-trainable a substance is, the chemical properties of all pyrrolizidine alkaloids are very closely related and contrast sharply with the properties of the components generally found in plant extracts, particularly essential oils, absolutes, concretes, or resinoids.

[0017] Pyrrolizidine alkaloids are mainly found in two botanical families, Boraginaceae and Asteraceae, and secondarily in Fabaceae, Apocynaceae, Euphorbiaceae, Orchidaceae, and Poaceae.

[0018] In terms of chemical properties, pyrrolizidine alcalcaloids constitute a relatively homogeneous group.

[0019] Most pyrrolizidine alkaloids are mutagenic and induce liver tumors.

[0020] It has been shown in rats that pyrrolizidine alkaloids such as retrorsine, senkirkine, monocrotaline, lasiocarpine and symphytine and several plants ( Tussilago farfara L., Symphytum officinale L., Petasites jalcaloïdes pyrrolizidiniques onicus Maxim. etc.) could cause liver tumors when administered regularly orally. Several alkaloids in the group have also been experimentally proven to be mutagenic and teratogenic.

[0021] Macrocyclic pyrrolizidine alkaloids of types III and V (senecionine, retrorsine, seneciphylline, ridelline) are the most toxic. Next come the diesters, which are more toxic than the monoesters.

[0022] Regular consumption of plants containing pyrrolizidine alkaloids can cause serious liver poisoning. Poisoning by these pyrrolizidine alkaloids can manifest as loss of appetite, pain, abdominal distension, and an enlarged liver (hepatomegaly).

[0023] As an example, we can cite comfrey ( Symphytum officinalis ) which contains pyrrolizidine alkaloids such as intermedine, lycopsamine, 7-acetyl-intermedine and which, due to their toxicity, has its use prohibited in many countries.

[0024] In Guadeloupe, a common plant, the bellflower ( Crotalaria retusaL.) is used to make "bell-ring tea," a folk remedy for many ailments. However, cases of severe poisoning have been reported. Several pyrrolizidine alkaloids have been identified in Crotalaria retusa L. primarily monocrotaline, but also retronecin-N-oxide, retusin, and retusamine. Monocrotaline, found mainly in the seeds, is known to be hepatotoxic, neurotoxic, and genotoxic. This molecule and its metabolites have the ability to cross the blood-brain barrier. Crotalaria retusa L. is notably responsible (with C. crispata ) of Kimberley disease which affects horses in northern Australia (specifically in the Kimberley region).

[0025] Other "medicinal plants" containing these hepatotoxic compounds include coltsfoot, borage, heliotropes, hound's tongue, groundsel, etc.

[0026] Pyrrolizidine alkaloids have been identified in medicinal herbs from China, South America, and Sri Lanka.

[0027] Large-scale poisoning incidents have been reported in Afghanistan, India, and the former USSR due to contamination of wheat crops by Boraginaceae ( Heliotropium lasiocarpum, H. popovii, H. europaeum ) .

[0028] It is understood from the above that there is a need for a quick, effective and safe way to eliminate pyrrolizidine alkaloids potentially present in plant extracts usable in pharmaceuticals, food, cosmetics or perfumery.

[0029] This is one of the aims of the present invention.

[0030] Indeed, the Applicant has shown that a hydrodistillation step of an organic composition containing pyrrolizidine alkaloids makes it possible to eliminate the latter from the composition.

[0031] As shown by the Applicant, it appears that during the hydrodistillation of organic phases including pyrrolizidine alkaloids, such as expressed or hydrodistilled essential oils, the pyrrolizidine alkaloids are not carried away by the water vapor and remain trapped in the aqueous phase.

[0032] Therefore, hydrodistillation is a method of choice for removing pyrrolizidine alkaloids that may be present in an organic composition.

[0033] In particular, the potential presence of pyrrolizidine alkaloids in essential oils, particularly in expressed essential oils, would thus be eliminated by an additional hydrodistillation step.

[0034] Thus, according to a first aspect the invention aims to treat a plant extract, said plant extract of interest being an essential oil, likely to contain pyrrolizidine alkaloids, by hydrodistillation with a view to eliminating said pyrrolizidine alkaloids present in the starting products by dissolution / concentration in an aqueous phase (distillation residues or hydrosol).

[0035] The invention therefore relates to a process for extracting pyrrolizidine alkaloids present in a plant extract of interest, said plant extract of interest being an essential oil, comprising at least one hydrodistillation step.

[0036] By "extraction of pyrrolizidine alkaloids present in a plant extract of interest" is meant the total or partial separation of pyrrolizidine alkaloids present in said plant extract of interest in order to remove them from said plant extract.

[0037] According to the invention, the starting extract, from which it is desired to remove the pyrrolizidine alkaloids, is an essential oil.

[0038] According to one embodiment of the invention, the extract to be treated can be steam-treated to remove the volatile substances contained therein. The steam thus formed can then be directed to a condenser, and the liquid thus collected can separate into two phases: an upper phase, lighter than water which contains the volatile substances, and a lower phase, the aqueous phase, which can be redirected (or not) to the boiler to be recirculated in order to eliminate any residual volatile substances by one or more of the same evaporation and / or condensation and / or separation cycle(s).

[0039] Depending on the variant of the process, it is possible to add a non-polar organic solvent to the condensates obtained during steam treatment (hydrodistillation), which can allow for better decantation.

[0040] According to this variant, the solvent can be chosen from linear, branched, or cyclic hydrocarbons, preferably pentane, hexane, heptane, trimethylpentane, and cyclohexane. These solvents can be used alone or in a mixture with diethyl ether. In the case of a mixture with diethyl ether, the latter can be added in an amount between 25% and 75% of the volume of the nonpolar organic solvent, preferably 50%, expressed as a volume percentage.

[0041] According to this variant, the solvent can be added in an amount between 5% and 20% of the initial volume to be treated, preferably between 10% and 15%, expressed as a volume percentage.

[0042] The extract to be treated, freed from pyrrolizidine alkaloids, can then be recovered and the aqueous phase containing the pyrrolizidine alkaloids can be eliminated.

[0043] Other advantages and features of the invention will become apparent from the non-limiting examples presented below, as well as from the accompanying figures in which: There Figure 1 presents the UPLC-MS profiles of an orange essential oil boosted with APs (a), the same orange essential oil boosted with APs, distilled (b), crude orange essential oil (c), and distilled crude orange essential oil (d). Figure 2 presents the UPLC-MS profiles of an AP-boosted Eucalyptus essential oil (a), the same AP-boosted Eucalyptus essence, distilled (b), crude Eucalyptus essential oil (c) and distilled crude Eucalyptus essential oil (d). Examples Example 1 : Essential oil sample processing of Eucalyptus globulus Labill., of Citrus sinensis (L.) Osbeck and of Citrus x lemon (L.) Burm. f. Operating mode

[0044] In a 500 mL flask, 300 mL of distilled water, a magnetic stir bar, some pumice stones and a weighed quantity of sample between 15 and 30 g are introduced.

[0045] The balloon is then connected to a Clevenger type device and placed in a heated oil bath.

[0046] Add 10 mL of distilled water and 4 to 5 mL of pentane to fill the separator.

[0047] The water supply for the condenser is opened and the mixture is brought to reflux for two hours.

[0048] The heating is then stopped and the mixture is allowed to settle. The organic and aqueous phases separate in the separator and are collected.

[0049] ANALYSIS HR-TOF-MS conditionsThe analyses were performed on a Waters XEVO G2 TOF system. ESI source conditions: capillary voltage: 0.5 kV, cone voltage: 45 V, extraction cone voltage: 4 V, source temperature: 120 °C, desolvation temperature: 400 °C, gas flow rate: 10 L / h, desolvation gas flow rate: 1200 L / h. The analyses were performed in positive mode. The mass spectrometer was calibrated with sodium formate for a mass range of 50 to 1200 Da. Leucine-enkephalin (lockmass) was the standard used for mass correction. The two characteristic leucine-enkephalin masses were verified (278.1141; 556.2771). Acquisitions were performed in MSE mode using argon as the collision gas. Fragmentation : The MSE mode allows the simultaneous acquisition of mass spectra at low and high collision energy, the first allowing the acquisition of a parent ion, the latter producing fragment ions. EXPERIMENTAL

[0050] After dilution of the essential oil extracts in methanol, the pyrrolizidine alkaloids are chromatographed by RP-UPLC system, in elution gradient mode, detected and identified by mass spectrometry and quantified by an external calibration method. Reference substances

[0051] The various pyrrolizidine alkaloids and their oxides are obtained from Sigma-Aldrich. Their purity is given as a minimum of 95%. Reference solutions

[0052] Standard solutions of pyrrolizidine alkaloids are prepared as follows: In a 100 mL volumetric flask (Vf), exactly 10 mg (Ms) of the standard substances are weighed out and the volume is adjusted with methanol. The solutions are homogenized using an ultrasonic bath for 3 min.

[0053] The concentration of the standard solution is given by the following equation: C mg / mL = Ms × Pu / 100 × Vf Ms = Weight of the standard substance; Pu = Purity of the standard substance as a percentage

[0054] Next, this stock solution is diluted until the appropriate concentrations for the detector sensitivity are obtained.

[0055] The standard solution will be prepared for each calibration of the quantitative method. Sample preparation

[0056] In a 20 mL volumetric flask, exactly 1 g (Pe) of the sample is weighed out and the volume is adjusted with methanol. The solution is homogenized for 3 min using an ultrasonic bath, then filtered using a syringe through a 0.25 µm PTFE filter, and then transferred to an injection bottle.

[0057] If necessary, this solution must be successively diluted to fall within an appropriate detector linearity range. Example 2: Study of the presence or absence of pyrrolizidine alkaloids in essential oil samples enriched with pyrrolizidine alkaloids before and after hydrodistillation

[0058] In order to demonstrate the principle of the invention, a quantitative study intended to show the presence or absence of pyrrolizidine alkaloids in samples of essential oil enriched in pyrrolizidine alkaloids was carried out before and after hydrodistillation.

[0059] In this study, commercially available pyrrolizidine alkaloids representing two previously described classes of type II and IV were used, namely: cyclic alkaloids (the most represented class) for senecionine [130-01-8], senecionine-N-oxide [13268-67-2], retrorsine [480-54-6], retrorsine-N-oxide [15503-86-3], jacobine [3870-67-3], jacobine-N-oxide [38710-25-7], seneciphylline [480-81-9], seneciphylline-N-oxide [38710-26-8], and monoester alkaloids for lycopsamine [10285-07-1], lycopsamine-N-oxide [95462-15-0], and intermedine. [10285-06-0], intermedine-N-oxide [95462-14-9]. Preparation of samples enriched with pyrrolizidine alkaloids

[0060] Three essential oils of lemon, orange and eucalyptus, spiked up to 100 ppm of each of the pyrrolizidine alkaloids and their respective oxides, were hydrodistilled separately for two hours according to the protocol described in Example 1. The experiment is carried out in duplicate.

[0061] Before and after hydrodistillation, the organic and aqueous phases are separated and injected in UPLC-HRMS / TOF.

[0062] The results are reported in Table 2 below. Table 2: Fragmentation of APs and their respective oxides obtained by UPLC-HRMS RN CAS compound Molecular formula Exact mass [M+H] +< [MH]- [M+NH4] +< [M+Na] +< Ion son 1 Ion son 2 Intermedine 10285-06-0 C15H25NO5 299.173279 300.18110 298.16545 317.20765 322.16305 156.3 138.3 Intermedine-N-oxide 95462-14-9 C15H25NO6 315.168182 316.17601 314.16036 333.20255 338.15795 Lycopsamine 10285-07-1 C15H25NO5 299.173279 300.18110 298.16545 317.20765 322.16305 156.3 138.3 Lycopsamine-N-oxide 95462-15 C15H25NO6 315.168182 316.17601 314.16036 333.20255 338.15795 Monocrotaline 315-22-0 C16H23NO6 325.152527 326.16035 324.14470 343.18690 348.14230 237.3 120.3 Monocrotaline-N-oxide 35337-98-5 C16H23NO7 341.14745 342.15528 340.13963 359.18182 364.13722 137.4 118.3 Retrosine 480-54-6 C18H25NO6 351.168182 352.17601 350.16036 369.20255 374.15795 138.3 120.3 Retrosine-N-oxide 15503-86-3 C18H25NO7 367.1631 368.17093 366.15528 385.19747 390.15287 136.2 118.2 Senecionine 130-01-8 C18H25NO5 335.173279 336.18110 334.16545 353.20765 358.16305 138.2 120.2 Senecionine-N-oxide 13268-67-2 C18H25NO6 351.16819 352.17602 350.16037 369.20256 374.15796 136.3 118.1 Seneciphylline 480-81-9 C18H23NO5 333.157623 334.16545 332.14980 351.19199 356.14739 138.4 120.3 Seneciphylline-N-oxide 38710-26-8 C18H23NO6 349.15254 350.16037 348.14472 367.18691 372.14231

[0063] The results show the absence of signals corresponding to senecionine, senecionine-N-oxide, retrorsin, retrorsin-N-oxide, jacobin, jacobin-N-oxide, seneciphylline, seneciphylline-N-oxide, lycopsamine, lycopsamine-N-oxide, intermedin, intermedin-N-oxide in the organic phase collected after hydrodistillation. (Table 2).

[0064] These experiments confirm the initial hypotheses. Pyrrolizidine alkaloids are classified as small molecules according to CAS. However, with molecular masses exceeding 300 DA, as well as high boiling points, strong polarity, high solubility, and very low vapor pressure, these parameters hinder their transfer into the oil phase during the steam distillation process.

[0065] Almost all of the pyrrolizidine alkaloids added in the organic phase are found in the aqueous phase, as shown by the results reported in Tables 3 and 4. Table 3: Doped Lemon Essential Oil - Dosage of PAs on the distillate and corresponding aqueous phase φ Organic (distillate) Aqueous (distillation residue) φ Organic (distillate) Aqueous (distillation residue) C [ppm] CT [ppm] C [ppm] C [ppm] CT [ppm] C [ppm] 1 < LoQ 0,806 0,810 < LoQ 0,868 0,839 2 < LoQ 0,662 0,691 < LoQ 0,621 0,605 3 < LoQ 0,656 0,678 < LoQ 0,626 0,698 1: Retrorsin; 2: Retrorsin-N-oxide; 3: Lycopsamine C: Concentration; CT: Theoretical concentration LOQ: 2 ppb Table 4: Doped Lemon Essential Oil - Dosage of PAs on the distillate and corresponding aqueous phase φ Organic (distillate) Aqueous (distillation residue) φ Organic (distillate) Aqueous (distillation residue) C [ppm] CT [ppm] C [ppm] C [ppm] CT [ppm] C [ppm] 1 < LoQ 0.797 0.663 < LoQ 0.828 0.711 2 < LoQ 0.457 0.678 < LoQ 0.593 0.612 3 < LoQ 0.550 0.588 < LoQ 0.597 0.621 4 < LoQ 0.028 0.024 < LoQ 0.030 0.027 1: Retrorsin; 2: Retrorsin-N-oxide; 3: Lycopsamine; 4: Lycopsamine-N-oxide C: Concentration; CT: Theoretical concentration LOQ: 2 ppb

[0066] UPLC-MS profiles targeted at retrorsin and its oxide, and at lycopsamine ( Figure 1, Figure 2 ) show that these substances are absent in crude and distilled lemon essential oil, but also in standardized and distilled essential oil. The same is true for orange and eucalyptus essences.

Claims

1. A method for extracting pyrrolizidine alkaloids present in a plant extract of interest with a view to removing them from said plant extract, said plant extract of interest being an essential oil, comprising at least one hydrodistillation step.

2. The method according to claim 1, comprising the following steps of: - treating the essential oil with steam to entrain the volatile substances, - directing the steam thus formed towards a condenser, the liquid thus collected being separated into two phases: • an upper phase, which contains the volatile substances, and • a lower phase, the aqueous phase, which is, optionally, recirculated in order to remove any residual volatile substances by one or more evaporation and / or condensation and / or separation cycle(s).

3. The method according to claim 2, wherein a non-polar organic solvent is added to the condensates.

4. The method according to claim 3, wherein the non-polar organic solvent is chosen from linear, branched, or cyclic hydrocarbons preferably from pentane, hexane, heptane, trimethylpentane, and cyclohexane.

5. The method according to one of claims 3 or 4, wherein the non-polar organic solvent may be supplemented with ethyl ether in a quantity between 25% and 75%, preferably 50%, of said non-polar organic solvent, by volume percentage.

6. The method according to one of claims 2, 3 or 4, wherein the organic solvent is added in a quantity between 5% and 20% of the initial volume to be treated, preferably between 10% and 15%, by volume percentage.