Method for reducing pyrrolizidine alkaloid in alkanet extract through liquid-liquid chromatography

Abstract

The present invention relates to a novel separation process for preparing an extract of Arnebia root having a reduced pyrrolizidine alkaloid content by liquid-liquid chromatography (LLC) using a biphasic binary solvent system obtained by partially mixing a polar organic solvent with water acidified with a strong organic acid, the polar organic solvent is selected from the group consisting of fatty alcohols having 4 to 5 carbon atoms and alkanoates having 3 to 6 carbon atoms.

Description

Technical Field

[0001] The present invention relates to a novel separation process, in particular to a method for significantly reducing pyrrolizidine alkaloids in lithospermum officinale root extract by using liquid-liquid chromatography. Background Art

[0002] Comfrey (Symphytum oficinale L., Boraginaceae) is a well-known medicinal plant that has traditionally been used topically to treat inflammatory joint and muscle conditions (e.g., arthritis, strains, contusions, or sprains). The most widely used topical Comfrey preparations are based on an aqueous ethanol root extract containing a wide range of phytochemical constituents: polysaccharides (25%-30%), allantoin (0.6%-4.7%), phenolic acids (such as caffeic acid (CA, 0.004%), chlorogenic acid (0.012%), rosmarinic acid (0.2%), lithospermic acid, globoidnan A (0.1%), chrysanthemum acid (0.2%), calendula officinalis (0.1 ... A), globulosaccharide B and caffeic acid tetramer isomers (rabdosiin) (hereinafter referred to as CAD oligomers), phytosterols, triterpenes, pyrocatechol tannins (2.4%), minerals, vitamins and pyrrolizidine alkaloids (PA, 0.013-1.2%, or even 3%), such as melanocyte stimulating hormone, acetyl melanocyte stimulating hormone, lycopodiol, acetyllycopodiol, symphytine and their corresponding most abundant N-oxides (PANO). Among these compounds, allantoin with wound healing and immunomodulatory activities and caffeic acid with antioxidant, antibacterial, antiallergic and anti-inflammatory properties are the most abundant. Morphogenic acid derivatives can be considered as active biomarkers of comfrey root preparations. In contrast, PA and PANO produce severe mutagenicity, carcinogenicity and hepatotoxicity, so there are major safety issues in the application of comfrey products. Therefore, the European Medicines Agency has set the daily intake tolerance for herbal products containing or contaminated with PA at a maximum of 0.007 μg / kg per day for skin application (EMA / HMPC / 893108 / 2011 and EMA / HMPC / 572844 / 2009). In practice, this usually results in a total PA tolerance of up to 1 ppm in comfrey extracts used in topical ointments.

[0003] To overcome this safety issue and meet stringent regulatory standards, several strategies have been disclosed to control, reduce or eliminate PA in comfrey extracts and other herbal preparations. First, Mauz et al. used protonated cation exchangers in an alcoholic extract of Petasites hybridus to reduce PA levels, achieving a depletion factor of about 10 times (Mauz et al., Pharm Acta Helv (Swiss Pharmaceutical Journal) 1985; 60: 256-259). When applied to comfrey extracts, this approach also slightly reduced the anti-inflammatory efficacy of PA-depleted products (Andres et al., Planta Med (Medicinal Plants) 1990; 56: 664). Schnecker reported a PA depletion factor of about 50 times in an optimized extraction process characterized by light protection, reduced contact with air, and extremely long contact time (EP0673654A1). In order to cope with the problem of wide polarity range of crude plant extracts, colleagues in Dalian developed a three-liquid extraction method to extract PA from traditional Chinese medicine in the following way: applying a three-phase solvent system consisting of a hydrophobic organic solvent, a hydrophilic alcohol and a salt solution, and adjusting the pH of the aqueous phase to 9-10, they were able to achieve a 90% PA recovery rate (about 10 times depletion factor). It is worth noting that the disclosure (CN105748535A) does not illustrate lithospermum officinale. Regarding the liquid-liquid extraction conditions of PA for analytical purposes, Kopp et al. reported the importance of acidic components: significantly higher PA recoveries can be achieved by using higher concentrations of strong acids (Kopp et al., Planta Med (Medicinal Plants) 2020; 86: 85-90); and Rizzo et al. demonstrated that PA extraction efficiency can be improved by a simple salting-out method (Rizzo et al., J Food Compos Anal (Journal of Food Composition and Analysis) 2022; 108: 104457). In addition, Mroczek et al. reported that the highest PA yield was obtained using a 1% methanol solution of tartaric acid as the extraction solvent in an electric basket at 100°C for 2 hours (Mroczek et al., Chem Anal (Warsaw) 2006;51:567).

[0004] On an industrial scale, Merck uses a complex multi-step process to produce a tailored PA-removed comfrey extract that includes a series of multi-stage extractions, differential precipitation, and strong cation exchange (SCX) column chromatography steps (EP3159002A1). The method was later cleaned up and simplified into a one-pot process by SMC Research (WO2018 / 224518 Al). Both methods can be considered scalable and efficient (typically able to reduce the total PA level of the final extract to less than 1 ppm). However, both technologies rely heavily on the use of large amounts of strong cation exchange resins, which makes the process costly and unsustainable. In addition, colleagues at Frutarom Schweiz AG demonstrated that moderate PA depletion (typical depletion factors of 2-10) can be achieved in different herbal extracts by adsorption on bentonite (WO2020015983A1), while Kopp et al. reported lower success using molecularly imprinted polymers (MIPs) (average PA depletion of 26%-70%) (Kopp et al., Planta Med Int Open 2020; 7: e26-e33). Robertet disclosed a highly selective PA removal method achieved by hydrodistillation of essential oils (EP3922633A1). However, none of these three procedures were applied to comfrey extracts.

[0005] Finally, alternative approaches have emerged aimed at manipulating PA biosynthesis in Comfrey (hairy root cultures) with promising results, however, so far these techniques are still far from reaching industrial scale and applicability (see Zakaria et al., Molecules 2021;26:1498 or Kruse et al., Planta Med 2019;85(14 / 15):1177-1186).

[0006] Liquid-liquid chromatography (LLC) is a unique preparative separation technique that is considered highly suitable for removing unwanted (toxic) contamination from matrices with biological value. For example, Sibal and Luca et al. recently demonstrated the pesticide removal ability of CPC for cannabinoid extracts (see US2019 / 0099697 Al and Luca et al., Ind Crops Prod ("Industrial Crops and Products") 2020; 155: 112726). In addition, the LLC method has been successfully used to separate bioactive comfrey components, such as separating caffeic acid oligomers in a standard hexane / ethyl acetate / methanol / water solvent system operated in descending (dsc) mode (Trifan et al., J Ethnopharmacol ("Journal of Ethnopharmacology") 2020; 262: 113169). Cooper and colleagues separated toxic PAs from the alkaloid fraction of lithospermum root extract in a chloroform / 0.2M phosphate buffer (pH 5.6) solvent system in a CCC machine operated in dsc mode (Cooper et al., J Chromatogr A 1996; 732: 43-50); while Kim et al. reported a dsc CCC method to separate three PAs from alkaloid-rich lithospermum root extract using a hexane / ethyl acetate / methanol / water solvent system acidified with 0.05% trifluoroacetic acid (Kim et al., J Nat Prod 2001; 64: 251-253). It is worth emphasizing that none of these three separation methods seeks to retain the composition or biological activity of the lithospermum crude extract, and the products obtained by these methods are individual lithospermum components. Summary of the invention

[0007] The present invention aims to develop an efficient, scalable and cost-effective separation process by which toxic PA and PANO can be removed from comfrey extracts in such a way that the extract remaining as a product of the process does not lose a significant amount of its original biological activity. We have found that this can be achieved by applying a liquid-liquid chromatography method and a suitable solvent system, i.e., a bioequivalent ethanol-water solution of comfrey extract can be produced in one chromatographic step by appropriate fraction collection.

[0008] The subject of the present invention is therefore a process for preparing a Radix Lithospermi Root extract with a reduced PA content, wherein PA is removed from a crude Radix Lithospermi Root extract by liquid-liquid chromatography (LLC) using a biphasic binary solvent system obtained by partially mixing a polar organic solvent with water acidified to pH 2-3 with a strong organic acid, and the polar organic solvent is selected from either of the following two groups of compounds: a) fatty alcohols having 4-5 carbon atoms; and b) alkanoic acid esters having 3-6 carbon atoms.

[0009] In one embodiment, the polar organic solvent is an aliphatic alcohol having 4-5 carbon atoms, preferably n-butanol, isobutanol, tert-butanol, sec-butanol or n-pentanol.

[0010] In one embodiment, the polar organic solvent is an alkanoate having 3 to 6 carbon atoms, preferably methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate or butyl acetate.

[0011] In one embodiment, the strong organic acid is formic acid, acetic acid or trifluoroacetic acid.

[0012] In one embodiment, the solvent system is n-butanol, isobutanol, tert-butanol or sec-butanol / water acidified with 0.1%-1.0% (v / v) formic acid, acetic acid or trifluoroacetic acid; ethyl acetate / water acidified with 0.1%-1.0% (v / v) formic acid, acetic acid or trifluoroacetic acid; more preferably n-butanol / water acidified with 0.25% (v / v) trifluoroacetic acid, n-butanol / water acidified with 1.0% (v / v) acetic acid, sec-butanol / water acidified with 0.25% (v / v) formic acid, or ethyl acetate / water acidified with 0.25% (v / v) trifluoroacetic acid.

[0013] In one embodiment, the solvent system has the characteristics of fast sedimentation time (<30 seconds), good sample dissolving ability (more than 50 mg of crude lithospermum root extract can be dissolved in 1 mL of the solvent system), and is classified as a Class III or Class IV solvent.

[0014] LLC separations can be achieved in either hydrodynamic (countercurrent chromatography, CCC) or hydrostatic (centrifugal partition chromatography, CPC) devices.

[0015] In one embodiment, the crude extract of Radix Lithospermi is an extract prepared by extracting Radix Lithospermi with an ethanol-water solution, and before performing the LLC, the solvent is evaporated from the crude extract and the obtained solid or oily extract is dissolved in an upper phase rich in a polar organic solvent and / or in an aqueous lower phase.

[0016] In one embodiment, the LLC may include two steps of elution-extrusion, wherein the extract is dissolved in the solvent system, and the extract dissolved in the upper phase is run in an ascending mode, while the extract dissolved in the lower phase is run in a descending mode, and the product fractions of the two runs are combined.

[0017] In one of the embodiments, the LLC is performed in dual mode, wherein the crude extract is dissolved in two phases of the solvent system and co-injected onto the LLC column.

[0018] The process may also comprise an additional step in which the pH of the product fraction obtained with LLC is readjusted to the pH of the crude extract and the salts formed are filtered.

[0019] According to a preferred embodiment, a pharmacopoeia-compliant comfrey root extract can be produced having a depletion factor of at least 300 times, preferably 500 times, more preferably 1000 times, relative to the total PA content of the crude extract.

[0020] The extract produced according to the present invention does not substantially lose its biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are CPC elution profiles (reconstructed fraction maps or heat maps) in ascending order of partition coefficient (Kd) for extruded PA and PANO and selected five comfrey biomarkers eluted in descending mode using n-butanol / water 0.25% (v / v) trifluoroacetic acid solvent system, as described in Example 1. Note that darker colors indicate more concentrated fractions.

[0022] Figure 2 Shown is a chromatogram of an aqueous ethanolic extract of Lithospermum officinale roots obtained by the descending mode bench-scale elution extrusion CPC process described in Example 1. PA: pyrrolizidine alkaloids; PANO: N-oxide derivatives of pyrrolizidine alkaloids.

[0023] Figure 3 Shown are chromatograms of an aqueous ethanol extract of Lithospermum officinale roots obtained by a laboratory-scale dual-mode CPC process: (A) ascending descending direction switching mode; and (B) descending ascending direction switching, as described in Example 2. PA: pyrrolizidine alkaloids; PANO: N-oxide derivatives of pyrrolizidine alkaloids.

[0024] Figure 4 Shown are chromatograms of aqueous ethanol extracts of Radix Lithospermi obtained by pilot elution extrusion CPC method: (A) ascending mode; and (B) descending mode, as described in Example 3. PA: pyrrolizidine alkaloids; PANO: N-oxide derivatives of pyrrolizidine alkaloids.

[0025] Figure 5 Shown are HPLC-MS / MS profiles of the nine most abundant pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides acquired in MRM mode: (A) crude aqueous ethanolic extract of Lithospermum erythrorhizon; and (B) PA-depleted product of the same aqueous ethanolic extract of Lithospermum erythrorhizon after CPC purification, as disclosed in Example 3. DETAILED DESCRIPTION

[0026] The present application is directed to the production of comfrey root extracts having reduced pyrrolizidine alkaloid content.

[0027] The term "pyrrolizidine alkaloids" refers to pyrrolizidine alkaloids (PA) found in the comfrey plant and drugs made therefrom, which are generally retronecine-type pyrrolizidine alkaloids with alkaline characteristics, and generally include melanocyte stimulating hormone, acetyl melanocyte stimulating hormone, lycopodiol, acetyllycopodiol and syringaphrine. As used herein, the term "pyrrolizidine alkaloids" includes its N-oxide derivatives (PANO).

[0028] According to the present invention, "reduced pyrrolizidine alkaloid content" means at least a 2-fold reduction compared to the content found in the original comfrey root extract. This means that the minimum depletion factor of the total PA content is 300-fold, preferably 500-fold, or even 1000-fold. This usually results in a total PA level in the final product extract below the limit of 1 ppm.

[0029] By means of the method disclosed in the present application, it is possible to produce a comfrey root extract that does not substantially lose its biological activity. By this we mean that at the end of the procedure, the level of at least one of the medically useful components and active biomarkers does not drop by more than 50% compared to the original crude extract, and the total phenolic content of the product extract does not drop by more than 70%. The active biomarkers may be caffeic acid oligomers and allantoin. This means that the extract produced according to the present application will be biosimilar to the active ingredients of the pharmaceutical preparation containing the licensed comfrey root extract.

[0030] According to the present application, the aqueous phase of the solvent system is acidified with a strong organic acid to a pH value between 2 and 3, inclusive.

[0031] By "strong organic acid" is meant certain carboxylic acids widely used as acidic modifiers in chromatography practice, the pH of which in 0.1%-1.0% (v / v) aqueous solutions drops below 3. Preferred examples of such strong organic acids are formic acid, acetic acid and trifluoroacetic acid.

[0032] In liquid-liquid chromatography (LLC), both the stationary phase and the mobile phase are liquids, and separation is performed based on the distribution of solutes between these immiscible liquid phases. LLC methods can be implemented in hydrodynamic (also known as countercurrent chromatography, CCC) devices as well as hydrostatic (also known as centrifugal partition chromatography) devices, and a given LLC method can be perfectly transferred between any CCC device and CPC device. A general description of LLC technology can be found in the following books: "Countercurrent Chromatography-The Support-Free Liquid Stationary Phase ("Countercurrent Chromatography-No Support Body Liquid Stationary Phase")" Billardello, B.; Berthod, A; Wilson & Wilson's Comprehensive Analytical Chemistry 38 ("Wilson and Wilson's Comprehensive Analytical Chemistry"); Berthod, A., editor; Elsevier Science Private Limited: Amsterdam (2002).

[0033] In the hydrostatic implementation of LLC (also known as centrifugal partition chromatography, CPC), a series of cells interconnected by tubing are attached to a rotor and filled with a liquid stationary phase, which is held inside the rotor by strong centrifugal forces. The other phase of the biphasic solvent system is used as mobile phase containing the sample to be purified: this phase is fed into the rotor under pressure and is pumped through the stationary phase (i.e., saturated) in the form of tiny droplets. An important feature of CPC is that the flow direction and the role of the phases are interchangeable: when the lower (denser) phase of the biphasic solvent system is used as the stationary phase, CPC is operated in ascending (asc) mode, while when the upper (lighter) phase is immobilized and used as the stationary phase, CPC experiments are performed in descending (dsc) mode.

[0034] Since retention and therefore selectivity in CPC is governed solely by partitioning, once the partition coefficient (K d ), we can fully predict the rate at which each compound (solute) passes through the system. d The determination of the K values ​​is performed routinely during method optimization by the shake flask method and is conventionally expressed as the ratio of the concentration of the compound in the upper phase to its concentration in the lower phase. As a rule of thumb, the K of the target compound in the optimal solvent system is d It should preferably be in the range of 0.5 to 2.0 (the so-called sweet spot), in which the resolving power of CPC is considered to be maximum. Note that in the case of inverted liquid phase action (asc to dsc or dsc to asc), the corresponding K d The reciprocal of the value.

[0035] The solid support-free nature of CPC enables a variety of operating modes that can be flexibly adapted to a given separation task. A typical scenario is that the sample may contain molecules outside the optimal range (K d >4-5) and thus it is not necessary to extend the run time without any benefit. In this case, the so-called elution squeeze-out mode can be applied: after compounds with suitable retention times have eluted, the compounds still retained in the CPC system can be squeezed out by pumping the stationary phase instead of the mobile phase through the rotor. Thereby, fresh stationary phase will replace the stationary phase loaded with the sample and will be eluted out. Another scenario is that compounds can elute at the sweet spot without sufficient resolution: in this case, the role of mobile and stationary phase as well as the flow direction can also be reversed during the separation (descending to ascending and vice versa), resulting in a higher plate number in the system. Typically, this so-called dual mode is used for columns containing a wide range of K d Value of the complex sample of the compound.

[0036] According to the present invention, the CPC process comprises a biphasic solvent system, wherein the combination of the organic solvent component, the pH value of the aqueous component, and the type and amount of the acidic component of the solvent system (i.e., the degree of acidification) is such that the K of most PAs and PANOs is d The values ​​are close to unity (within the optimal point of CPC); while the solvent system also provides K values ​​outside the optimal point range for valuable comfrey biomarkers d The CPC process also includes an operation mode, which can preferably be (i) a classical elution extrusion mode in one embodiment of the present invention; or (ii) a dual mode in another preferred embodiment.

[0037] The rough nature of aqueous ethanol extracts of Lithospermum officinale roots is reflected in the chemical complexity and the wide polarity range of its components. In order to remove minor (trace) components or groups of components from such complex matrices, selective interactions providing high selectivity factors between unwanted and valuable components, as well as efficient separation techniques providing high pedal numbers should be utilized. In CPC, by directly adjusting the pH of the aqueous phase of the solvent system, the partitioning of ionizable compounds can be precisely adjusted and selectivity can be generated.

[0038] The PA in Lithospermum officinale is mainly senecioclase alkaloids, and its pK a Basal values ​​are in the range of 7-8, for example, the pK values ​​of lycopodiol and melanocyte stimulating hormone a The predicted value is 7.82. Acylation of the free hydroxyl groups results in a slight reduction in their basic properties, for example, the pK values ​​of 7-acetyl melanocyte stimulating hormone and 7-acetyllysozolin area The predicted value is 7.22; the corresponding N-oxide derivative actually lost its basicity (pK a In contrast, CAD oligomers are weak carboxylic acids with moderate polarity (pK a 3.6-5), while allantoin can be considered as a very hydrophilic (logP = -3.14) weak acid (pK a =8.48).

[0039] Therefore, in a properly acidified polar solvent system (wherein the pH of the aqueous phase falls between 2 and 3, and wherein the organic phase consists of fatty alcohols or alkanoates that are slightly soluble in water, thereby providing good solubility for the crude comfrey extract), CAD oligomers will exist in a neutral (non-ionized) form and will be enriched in the organic upper phase (K d >3-4), while PA will exist in a fully protonated form and can form ion pairs with acidic components to produce a near unity distribution (K d =0.5-2.0). At the same time, allantoin will definitely be enriched in the aqueous lower phase (K d = 0.1-0.2), however, by increasing the ionic strength of the lower phase it can be forced to partition into the organic phase to a certain extent. Table 1 summarizes the K values ​​of valuable compounds (allantoin, globulosan A and B, rosmarinic acid and caffeic acid tetramer isomers) and toxic compounds (mainly PA and PANO) measured in solvent systems composed according to this method. d ;and Figure 1 A typical partition / elution curve diagram obtained by this method in a preferred embodiment of the present invention is shown. Since retention in LLC is completely governed by partitioning, the partition coefficient unambiguously determines the chromatographic elution curve, and therefore knowing its measured value is fully sufficient to predict the quality of a separation.

[0040] Table 1. Partition coefficients of studied compounds of Lithospermum officinale root extract measured by shake flask method in LLC solvent system suitable for PA removal.

[0041]

[0042] Example

[0043] The following examples are provided by way of illustration of the present invention, but are not intended to limit the scope of the invention as defined in the claims. Table 2 details three preferred embodiments of the present invention, which were performed on two different CPC devices from two different manufacturers (Gilson and RotaChrom) at benchtop and pilot scales (rotor volumes of 250 mL and 2.1 L), respectively.

[0044] Table 2. Summary of parameters for the laboratory and pilot scale CPC processes detailed in the Examples section. UP: upper phase of the solvent system; LP: lower phase of the solvent system.

[0045]

[0046] Example 1

[0047] The input lithospermum root extract to be purified was a crude ethanol-water solution extracted with 60% (v / v) ethanol. Based on HPLC-MS / MS, the PA and PANO content in the lithospermum extract was 300-350ppm. The experimental goal was to reduce the level of PA and PANO in the product as much as possible. Due to the high ethanol content in the input sample, the stationary phase loss in CPC was high, so the sample solvent needed to be evaporated before injection. A laboratory scale CPC (labCPC) run required 50mL of input sample, in which about 0.5g of solid extract was obtained. It was redissolved in 10mL of the upper phase of the solvent system.

[0048] LabCPC experiments were performed on a Gilson PLC 2250 purification system equipped with a Gilson CPC-250 rotor (total volume of 250 ml) controlled by Gilson GliderCPC software. In short, the rotor was filled with the stationary phase (upper phase) at a high flow rate (50 mL / min). The system was balanced with a mobile phase. Subsequently, the sample was injected using a 10 mL annular piece built into the instrument. CPC was operated in the so-called elution extrusion mode. Conventional chromatographic elution was combined with a stationary phase base to recover a high K d Value compound.

[0049] The solvent system used was a n-propanol / water volumetric system acidified with 0.25% (v / v) trifluoroacetic acid. The solvent system was mixed in a separatory funnel before the CPC run. After equilibrium was established, the upper (stationary) phase and the lower (mobile) phase were separated. The main parameters of the method are summarized in Table 3. Due to the wide range of polarity of the valuable components (allantoin to CA oligomers), the CPC method requires relatively long elution times and extrusion times. Figure 2 The CPC chromatogram for this run is presented, and Figure 1 The corresponding elution profiles (heatmaps or fraction plots) for this run are provided, as well as the K values ​​of the comfrey biomarkers determined in the shake flask measurements. d value.

[0050] Table 3. Downward Mode Laboratory Scale Elution Extrusion CPC Process Schedule (Example 1).

[0051]

[0052] After the CPC run, the appropriate fractions (allantoin and caffeic acid oligomers, fractions #7-10 and #40-47) were combined and evaporated to dryness on a rotary evaporator (laboratory scale Heidolph Hei-VAP Value equipped with a Vacuubrand 4c vacuum pump, controlled by a Vacuubrand CVC 3000 vacuum control unit). The final product extract obtained was redissolved in 60% (v / v) ethanol and analyzed by HPLC-MS / MS in a multiple reaction monitoring (MRM) method to determine the levels of PA and PANO: a depletion factor of more than 300 times was achieved in the CPC purified product compared to the input crude extract.

[0053] Example 2

[0054] The input comfrey root extract to be purified was a crude ethanol-water solution extracted with 60% (v / v) ethanol. Based on HPLC-MS / MS, the PA and PANO content in the comfrey extract was 400-500 ppm. The experimental goal was to reduce the levels of PA and PANO in the product as much as possible. Due to the high ethanol content in the input sample, the stationary phase loss in CPC was high, so the sample solvent needed to be evaporated before injection. A labCPC run required 50 mL of input sample, from which about 0.7 g of solid extract was obtained. In order to completely redissolve the input material and avoid disturbing the hydrodynamic equilibrium of the system, equal volumes of upper and lower phases were used to obtain 10 mL (biphasic) sample solutions.

[0055] The LabCPC experiment was carried out on a Gilson PLC 2250 purification system equipped with a Gilson CPC-250 rotor (total volume of 250 mL) controlled by Gilson GliderCPC software. In short, the rotor was filled with 50% upper phase and 50% lower phase at a high flow rate (50 mL / min) without rotation. In the first injection (dual mode, switching from asc to dsc), the system was balanced with the starting mobile phase (upper phase). The sample was injected by using the built-in 10 mL annular piece of the instrument. The so-called dual mode was used: after eluting for 15 minutes, in the last 5 minutes of this run, the asc mode was switched to the dsc mode using a switching valve. In the second injection (dual mode, switching from dsc to asc), exactly the same method settings were applied, except that the run was started in dsc mode and the valve was switched to the asc mode after 15 minutes of elution time.

[0056] The solvent system used was n-propanol in water acidified with 0.25% (v / v) trifluoroacetic acid. The solvent system was mixed in a separatory funnel before the CPC run. After equilibrium was established, the phases were separated. The main parameters of the method are summarized in Tables 4 and 5. Figure 3 The CPC chromatogram for this run is presented.

[0057] Table 4. Laboratory scale dual mode (asc to dsc) CPC process schedule (Example 2, first injection).

[0058]

[0059] Table 5. Laboratory scale dual mode (dsc to asc) CPC process schedule (Example 2, second injection).

[0060]

[0061] After the CPC run, the appropriate fractions (allantoin and caffeic acid oligomers, fractions #13-19 for the first injection and fractions #3-10 for the second injection) were combined and evaporated to dryness on a rotary evaporator (laboratory scale Heidolph Hei-VAPValue equipped with a Vacuubrand 4c vacuum pump, controlled by a Vacuubrand CVC 3000 vacuum control unit). The final product extract obtained was redissolved in 60% (v / v) ethanol and analyzed by HPLC-MS / MS in a multiple reaction monitoring (MRM) method to determine the levels of PA and PANO: a depletion factor of more than 500 times was achieved in the CPC purified product compared to the input crude extract.

[0062] Example 3

[0063] The input comfrey root extract to be purified was a crude ethanol-water solution extracted with 60% (v / v) ethanol. Based on HPLC-MS / MS, the PA and PANO content in the comfrey extract was 400-500ppm. The experimental objective was to reduce the levels of PA and PANO as much as possible. Due to the high ethanol content in the input sample, the stationary phase loss in CPC was high, so the sample solvent needed to be evaporated before injection. A labCPC run required 400mL of input sample, in which about 5.6g of oily extract was obtained. In order to completely redissolve the input material, 100mL (biphasic) sample solution was obtained using the same volume of upper and lower phases. The two phases of the sample solution were separated in a separatory funnel and injected into the CPC rotor in two different modes to reduce the PA content of each run, speed up the method and avoid injection problems and column loss caused by overloading. Therefore, in the ascending mode, the upper phase of the sample solution was injected into the rotor, and in the descending mode, the lower phase of the sample solution was injected into the rotor.

[0064] The purification was performed on a RotaChrom pilot scale CPC (rCPC) system (ECOM ECB2005PC gradient box with computer, ECOM ECP 2300 isocratic pump, ECOM Flash 14DAD 400 UV detector, ECOM cassette with 10-position valve fraction collector, and RotaChrom rCPC rotor (2.1 L total internal volume, 100 extraction units)), which was controlled by ECOMAC software. Briefly, the rotor was filled with the stationary phase and then the system was equilibrated with the mobile phase. The sample solution was injected by using a pump. The rCPC was operated in the so-called elution extrusion mode. Conventional chromatographic elution was combined with stationary phase extrusion to recover the residues with high K d Compounds with high concentrations of 100 μg / mL: After a short elution period of five to six minutes, approximately one rotor volume of stationary phase is pumped through the rotor.

[0065] The solvent system used was a n-propanol / water volumetric system acidified with 0.25% (v / v) trifluoroacetic acid. Before the CPC run, the solvent system was mixed in a separatory funnel. After equilibrium was established, the upper and lower phases were separated. The main parameters of the method are summarized in Tables 6 and 7. Figure 4 Chromatograms from two runs are presented. Both CPC runs used the same method setup, but the operating mode was reversed.

[0066] Table 6. Ascending mode pilot scale elution extrusion CPC process schedule.

[0067]

[0068]

[0069] Table 7. Down mode pilot scale elution extrusion CPC process schedule.

[0070]

[0071] After two CPC runs, appropriate fractions with UV absorbance (alantoin and caffeic acid oligomers, fractions #1-7 when asc is running and fractions #1-10 when dsc is running) are combined. The pH of the combined fractions is measured using an IKA RET Control-vise instrument with an SI Analytics BlueLine pH electrode. After the CPC run, the pH of the combined fractions was 1.79. Although the characteristics of the comfrey root extract are weakly acidic, such acidic pH may eventually lead to the decomposition of CAD oligomers. Therefore, the pH was adjusted to 5.36 using 1M sodium hydroxide solution. The pH-adjusted product fractions were evaporated on a rotary evaporator (laboratory scale Heidolph Hei-VAP Value, with a Vacuubrand 4c vacuum pump controlled by a Vacuubrand CVC 3000 vacuum control unit). After the sample was evaporated to dryness, it was redissolved in ethanol to filter out sodium trifluoroacetic acid salt from the product. The replicate dried final products were analyzed by HPLC-MS / MS in a multiple reaction monitoring (MRM) approach to determine the levels of PA and PANO: depletion factors greater than 500-fold were achieved in the CPC purified product compared to the input crude extract (see Figure 5 and Table 8). In addition, to characterize the chemical equivalence and bioequivalence of the product liquid extract, density, total phenolic content, antioxidant capacity (DPPH and ABTS free radical scavenging activity) and tentative levels of biomarkers have been evaluated using appropriate in vivo experiments and LC-MS (see Table 8). Based on these data, the CPC-purified comfrey extract was shown to be satisfactory (total PA and PANO levels below 1 ppm) and retains its core biological activities relevant to its medicinal use.

[0072] Table 8. Comparative characterization of crude aqueous ethanolic extract of Radix Lithospermi as input sample and PA-depleted product sample of the same aqueous ethanolic extract of Radix Lithospermi after CPC purification (Example 3).

[0073]

[0074] Biomarker levels (LC-MS) and their changes relative to levels before CPC purification (%)

[0075]

Claims

1. A preparation process for reducing the content of pyrrolizidine alkaloids in Lithospermum officinale root extract, characterized in that: Pyrrolizidine alkaloids are removed from a crude extract of Radix Lithospermi by liquid-liquid chromatography (LLC) using a biphasic binary solvent system obtained by partially mixing a polar organic solvent with water acidified to pH 2-3 with a strong organic acid, and the polar organic solvent is selected from any one of the following two groups of compounds: a) fatty alcohols having 4 to 5 carbon atoms; and b) Alkanoic acid esters having 3 to 6 carbon atoms.

2. The method according to claim 1, characterized in that The polar organic solvent is an aliphatic alcohol having 4-5 carbon atoms, preferably n-butanol, isobutanol, tert-butanol, sec-butanol or n-pentanol.

3. The method according to claim 1, characterized in that: The polar organic solvent is an alkanoate having 3 to 6 carbon atoms, preferably methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate or butyl acetate.

4. The method according to claims 1-3, characterized in that: The strong organic acid is formic acid, acetic acid or trifluoroacetic acid.

5. The method according to claim 1, characterized in that The solvent system is n-butanol, isobutanol, tert-butanol or sec-butanol / water acidified with 0.1%-1.0% (v / v) formic acid, acetic acid or trifluoroacetic acid; ethyl acetate / water acidified with 0.1%-1.0% (v / v) formic acid, acetic acid or trifluoroacetic acid, preferably n-butanol / water acidified with 0.25% (v / v) trifluoroacetic acid, n-butanol / water acidified with 1.0% (v / v) acetic acid, sec-butanol / water acidified with 0.25% (v / v) formic acid, or ethyl acetate / water acidified with 0.25% (v / v) trifluoroacetic acid.

6. The method according to claims 1-5, characterized in that: The LLC is performed in a hydrostatic centrifugal partition chromatography (CPC) apparatus or in a hydrodynamic countercurrent chromatography (CCC) apparatus.

7. The method according to claims 1-6, characterized in that: The crude extract of lithospermum erythrorhizon is an extract prepared by extracting lithospermum erythrorhizon medicine with ethanol aqueous solution, and before the liquid-liquid chromatography, the ethanol aqueous solution is evaporated from the crude extract, and the obtained solid or oily extract is dissolved in an upper phase rich in polar organic solvent and / or dissolved in an aqueous lower phase.

8. The method according to claim 7, characterized in that The liquid-liquid chromatography method comprises two steps of elution-extrusion, wherein the extract is dissolved in the solvent system, and the extract dissolved in the upper phase is run in an ascending mode, while the extract dissolved in the lower phase is run in a descending mode, and the product fractions of the two runs are combined.

9. The method according to claims 1-7, characterized in that: The liquid-liquid chromatography was performed in dual mode, wherein the crude extract was dissolved in two phases of the solvent system and co-injected onto a liquid-liquid chromatography column.

10. The method according to claims 1-9, characterized in that The method further comprises re-adjusting the pH of the product fraction obtained by the liquid-liquid chromatography to the pH of the crude extract and filtering the formed salts.

11. The method according to claims 1-10, characterized in that A pharmacopoeia-compliant comfrey root extract is produced having a depletion factor of at least 300-fold, preferably 500-fold, more preferably 1000-fold relative to the total PA content of the crude extract without significant loss of biological activity.

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