EP4593978A1

EP4593978A1 - Method for depleting pyrrolizidine alkaloids in comfrey root extracts by means of liquid-liquid chromatography

Info

Publication number: EP4593978A1
Application number: EP23716656.6A
Authority: EP
Inventors: Dóra RUTTERSCHMID; Árpád KÖNCZÖL
Original assignee: Rotachrom Technologies PLC
Current assignee: Rotachrom Technologies PLC
Priority date: 2022-09-29
Filing date: 2023-03-14
Publication date: 2025-08-06
Also published as: WO2024069196A1; HUP2200389A1; CN119947808A

Abstract

Present invention relates to a novel separation process for the preparation of comfrey root extract with reduced pyrrolizidine alkaloid content by means of liquid-liquid chromatography (LLC) using a biphasic binary solvent system obtained by partially mixing a polar organic solvent selected from aliphatic alcohols with 4-5 carbon atoms and alkanoate esters with 3-6 carbon atoms with water acidified with a strong organic acid.

Description

Method for depleting pyrrolizidine alkaloids in comfrey root extracts by means of liquid-liquid chromatography

Technical Field

Present invention relates to a novel separation process, in particular to a process for the significant depletion or remediation of pyrrolizidine alkaloids in comfrey root extracts using liquid-liquid chromatography.

Background Art

Comfrey (Symphytum officinale L., Boraginaceae) is a well-known medicinal plant, traditionally used externally to treat inflammatory joint and muscle disorders (e.g., arthritis, strains, bruises, and sprains). The most widespread topical comfrey preparations are based on hydroethanolic root extracts, containing a wide array 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, globoidnan B, and rabdosiin (referred hereinafter as CA oligomers), phytosterols, triterpenes, pyrocatecholtype tannins (2.4%), minerals, vitamins, as well as pyrrolizidine alkaloids (PAs, 0.013-1.2%, or even 3%), such as intermedine, acetylintermedine, lycopsamine, acetyllycopsamine, symphytine and their corresponding N-oxides (PANOs) as the most abundant ones. Among these compounds, allantoin with wound healing and immunomodulatory activities and caffeic acid derivatives with antioxidant, antimicrobial, anti-allergic, and anti-inflammatory propensities can be considered active biomarkers of comfrey root preparations. In contrast to this, PAs and PANOs exert strong mutagenicity, carcinogenicity, and hepatotoxicity, raising thereby a significant safety concern in the application of comfrey products. Therefore, the European Medicines Agency set the acceptable daily intake of PA-containing or PA- contaminated herbal medicinal products to a maximum of 0.007 pg/kg/day for cutaneous application (EMA/HMPC/893108/2011 and EMA/HMPC/572844/2009). In practice, this typically results in a maximum of 1 ppm allowable total PA level in comfrey extracts utilized in topical creams.

To overcome this safety issue and meet strict regulatory criteria, several strategies have been disclosed to control, reduce, or eliminate PAs in comfrey extracts and other herbal preparations. First, Mauz et al. utilized protonated cation exchanger to reduce the level of PAs in alcoholic extracts of Petasites hybridus thereby achieving a depletion factor ca. 10-fold (Mauz et al., Pharm Acta Helv 1985; 60:256-259). This approach also slightly decreased the antiphlogistic efficacy of the PA-depleted product when applied to comfrey extracts (Andres et al., Planta Med 1990; 56:664). Schnecker reported a ca. 50-fold PA-depletion factor in an optimized extraction process characterized by light protection, reduced contact to air, and extremely long contact time (EP0673654A1). To tackle the wide polarity range of crude plant extracts, co-workers of Dalian have developed a three-liquid phase extraction method to extract PAs from Traditional Chinese Medicines: by applying a three-phase solvent system composed 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 90% recovery rate for PAs (ca. 10-fold depletion factor). Important to note that comfrey was not exemplified in this disclosure (CN105748535A). Regarding the liquid-liquid extraction conditions of PAs for analytical purposes, Kopp et al. reported the importance of the acidic component: significantly higher PA recovery rates can be realized by using stronger acids at higher concentrations (Kopp et al., Planta Med 2020; 86:85-90); while Rizzo et al. demonstrated that PA-extraction efficiency can be increased by a simple salting-out approach (Rizzo et al., J Food Compos Anal 2022; 108:104457). In addition, Mroczek et al. reported the highest PA yield using 1% methanolic solution of tartaric acid as extraction solvent in an electric basket at 100 °C for 2 hours (Mroczek et al., Chem Anal (Warsaw) 2006; 51:567).

At industrial scale, Merck utilizes a complex, multistep process for producing PA-remediated special comfrey extracts, which involves sequences of multistage extraction, differential precipitation, and strong cation exchange (SCX) column chromatography steps (EP3159002A1). This method was later improved and simplified by SMC Research to a one-pot process (WO 2018/224518 Al). Both approaches can be considered scalable and efficient (typically capable to reduce the total PA-level of the final extract to below 1 ppm). However, both techniques are heavily relying on the extensive use of strong cation exchanger resins, making the process costly and unsustainable. In addition, co-workers at Frutarom Schweiz AG demonstrated that a moderate PA-depletion (typical depletion factor of 2-10) can be realized in different herbal extracts by bentonite adsorption (W02020015983A1), while Kopp et al. reported on the usage of molecularly imprinted polymers (MIPs) with inferior success (26-70% PA-depletion in average) (Kopp et al., Planta Med Int Open 2020; 7:e26-e33). Robertet disclosed a highly selective PA-remediation accomplished by hydro-distillation of essential oils (EP3922633A1). However, none of these three procedures were applied to comfrey extracts.

Finally, alternative approaches aiming to manipulate the biosynthesis of PAs in comfrey (hairy root cultures) have also appeared with promising results, however, to date, these techniques are far from industrial scale and applicability (see Zakaria et al., Molecules 2021; 26:1498 or Kruse at al., Planta Med 2019; 85(14/15): 1177-1186).

Liquid-liquid chromatography (LLC) is a unique preparative separation technique, which is considered as highly suitable to remove unwanted (toxic) contaminations from biologically valuable matrices. For instance, Sibal and Luca et al. have recently demonstrated the pesticide remediation capability of CPC on cannabinoid extracts (see US 2019/0099697 Al and Luca et al., Ind Crops Prod 2020; 155:112726). Furthermore, LLC methods were successfully applied for the isolation of bioactive comfrey constituents, such as caffeic acid oligomers in standard hexane/ethyl acetate/methanol/water solvent systems operated in dsc mode (Trifan et al., J Ethnopharmacol 2020; 262:113169). Cooper and co-workers isolated toxic PAs from the alkaloid fraction of a comfrey root extract in chloroform/0.2 M 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 using hexane/ethyl acetate/methanol/water solvent system acidified with 0.05% trifluoroacetic acid to isolate three PAs from an alkaloid- enriched comfrey root extract (Kim et al., J Nat Prod 2001; 64:251-253). It is worth highlighting here that none of these three isolation methods sought to preserve the composition or bioactivity of the crude comfrey extract, the products obtained by these methods were individual comfrey components.

Summary of Invention

The object of the present invention was to develop efficient, scalable, and cost-efficient separation methods by which toxic PAs and PANOs can be removed from comfrey extracts in such a way that the extract remaining as the product of the process does not significantly lose its original bioactivity. We discovered that by applying liquid-liquid chromatographic methods and suitable solvent systems, the above goal can be achieved, i.e., bioequivalent hydroethanolic comfrey extracts can be produced in one chromatographic step with appropriate fraction collection.

The subject of the invention is therefore a process for the preparation of a comfrey root extract with a reduced PA content, in which the PAs are removed from comfrey root extract by liquid-liquid chromatography (LLC) using a biphasic, binary solvent system, which is obtained by partially mixing a polar organic solvent with water acidified with a strong organic acid to pH 2-3, and the polar organic solvent is selected from any of the following two groups of compounds: a) aliphatic alcohols with 4-5 carbon atoms; and b) alkanoate esters with 3-6 carbon atoms.

In one embodiment of the invention, the polar organic solvent is an aliphatic alcohol with 4-5 carbon atoms, preferably n-butanol, isobutanol, tert-butanol, sec-butanol, or n- pentanol.

In another embodiment, the polar organic solvent is an alkanoate ester with 3-6 carbon atoms, preferably methyl acetate, ethyl acetate, n-propyl acetate, i-propyl acetate, or butyl acetate.

Preferably the strong organic acid is formic acid, acetic acid or trifluoroacetic acid.

Preferably 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.

Furthermore, the preferred solvent systems are characterized by fast settling time (<30 sec), good sample solvating ability (more than 50 mg crude comfrey root extract can be dissolved in 1 mL solvent system) and are categorized as Class III and IV solvents.

The LLC separation can be realized in hydrodynamic (counter-current chromatography, CCC) or in hydrostatic (centrifugal partition chromatography, CPC) device.

In one embodiment, the crude comfrey root extract is prepared by hydroethanolic extraction of a comfrey root drug, and prior the LLC is performed, the solvent is evaporated from the crude extract and the resulting solid or oily extract is dissolved in the polar organic solvent-rich upper phase and/or dissolved in the aqueous lower phase.

In this embodiment, the LLC may comprise two elution-extrusion steps, where the extract is dissolved in the solvent system and the extract dissolved in the upper phase is run in ascending mode, while the extract dissolved in the lower phase is run in descending mode, and the product fractions of these two runs are combined.

In another embodiment, LLC is performed in a dual mode where the crude extract is dissolved in both phases of the solvent system and co-injected onto an LLC column. The process may also include an additional step, in which the pH value of the product fraction obtained with LLC is readjusted to the pH value of the crude extract, and the salt formed is filtered.

According to one preferred embodiment, a pharmacopoeia-compliant comfrey root extract with a depletion factor of at least 300-fold, preferably 500-fold, more preferably 1000- fold, with respect to the total PA content of the raw extract can be produced.

The extract produced according to the invention does not significantly lose its bioactivity.

Brief description of Drawings

Figure 1 shows the CPC elution profile (reconstructed fractogram or heatmap) of PAs and PANOs along with five selected comfrey biomarkers in ascending order of partition coefficient (Kd) using the n-butanol/water 0.25% (v/v) trifluoroacetic acid solvent system in elutionextrusion in descending mode as described in Example 1. Note that the darker colour represents the higher concentration in fractions.

Figure 2 shows a chromatogram of a hydroethanolic comfrey root extract obtained by a laboratory-scale elution-extrusion CPC method in descending mode as described in Example 1. PAs: pyrrolizidine alkaloids; PANOs: N-oxide derivatives of pyrrolizidine alkaloids.

Figure 3 shows chromatograms of a hydroethanolic comfrey root extract obtained by a laboratory-scale dual-mode CPC method (A) in ascending-descending direction switch mode; and (B) in descending-ascending direction switch as described in Example 2. PAs: pyrrolizidine alkaloids; PANOs: N-oxide derivatives of pyrrolizidine alkaloids.

Figure 4 shows chromatograms of a hydroethanolic comfrey root extract obtained by a pilotscale elution-extrusion CPC method (A) in ascending mode; and (B) in descending mode as described in Example 3. PAs: pyrrolizidine alkaloids; PANOs: N-oxide derivatives of pyrrolizidine alkaloids.

Figure 5 shows the HPLC-MS/MS profiles acquired in MRM mode of the most abundant nine pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides of (A) a crude hydroethanolic comfrey root extract; and (B) the PA-depleted product of the same hydroethanolic comfrey root extract after CPC purification as disclosed in Example 3. Detailed description of the invention

The invention relates to the production of comfrey root extracts with reduced pyrrolizidine alkaloid content.

The term "pyrrolizidine alkaloids" refers to pyrrolizidine alkaloids (PAs) found in the comfrey plant and in drugs made from it, which are typically retronecine-type pyrrolizidine alkaloids of a basic character, and typically include the compounds intermedine, acetyl- intermedine, lycopsamine, acetyl-lycopsamine and symphytin. As used herein, the term "pyrrolizidine alkaloids" includes their N-oxide derivatives (PANOs).

According to the invention, "reduced pyrrolizidine alkaloid content" means at least a 2- fold reduction compared to the level found in the original comfrey root extract. This preferably means a total PA content with a minimum depletion factor of 300-fold, more preferably 500- fold, even more preferably 1000-fold. This typically results in total PA levels in the final product extract being below the 1 ppm limit.

By mean of the method disclosed in the present invention, comfrey root extracts can be produced that do not significantly lose their bioactivity. By this we mean that the level of at least one of the medically useful components and active biomarkers does not decrease by more than 50% compared to the original crude extract at the end of the procedure, and the total phenolic content of the product extract does not decrease by more than 70%. Active biomarkers are preferably caffeic acid oligomers and allantoin. This means that the extracts produced according to the invention will be biosimilar to the active ingredient of pharmaceutical preparations containing licensed comfrey root extracts.

According to the invention, the aqueous phase of the solvent system is acidified with a strong organic acid to a pH value between 2 and 3, including the extremities.

By "strong organic acid", we mean certain carboxylic acids, widely used as acidic modifiers in chromatographic practice, whose pH value falls below 3 in a 0.1-1.0% (v/v) aqueous solution. Preferred examples of this are formic acid, acetic acid, and trifluoroacetic acid.

In liquid-liquid chromatography (LLC) both the stationary and mobile phases are liquids and separation is based on the partitioning of the solutes between these immiscible liquid phases. LLC methods can be realized in hydrodynamic (called Counter-Current Chromatography, CCC) and in hydrostatic (called Centrifugal Partition Chromatography) devices and a given LLC method is perfectly transferable between any CCC and CPC equipment. General description of the LLC technique can be found in the book „Countercurrent Chromatography - The Support- Free Liquid Stationary Phase" Billardello, B.; Berthod, A; Wilson & Wilson's Comprehensive Analytical Chemistry 38; Berthod, A., ed.; Elsevier Science B.V.: Amsterdam (2002).

In the hydrostatic-type realization of LLC - called Centrifugal Partition Chromatography (CPC) - a series of cells interconnected by ducts are attached to a rotor and filled with the liquid stationary phase, which is immobilized inside the rotor by a strong centrifugal force. The other phase of the two-phase solvent system is used as the mobile phase containing the sample to be purified: it is fed under pressure into the rotor and pumped through the stationary phase in the form of tiny droplets (i.e., percolation). An important feature of CPC is that the direction of the flow and role of the phases are interchangeable: CPC operates in ascending (asc) mode when the lower (denser) phase of the biphasic solvent system is used as stationary phase, while the CPC experiment is performed in descending (dsc) mode when the upper (lighter) phase is immobilized and used as stationary phase.

Because retention and thus selectivity in CPC is governed solely by partitioning, the rate at which each compound (solute) travels through the system is fully predictable once the compound's partition coefficient (Kd) is known. Determination of the Kd value is performed routinely by the shake-flask method during method optimization and by convention 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, Kd of the targeted compounds in an optimal solvent system should preferably be in the range of 0.5-2.0 (the so-called sweet spot), where the resolving power of CPC is considered as maximum. Note that in the case of inverting the role of the liquid phases (asc to dsc or dsc to asc), the reciprocal values of the corresponding Kd values should be considered.

The solid support-free nature of CPC enables numerous operation modes which can be flexibly adapted to the given separation task. One typical scenario is when the sample may contain compounds eluting out of the sweet spot range (Kd>4-5) thus unnecessarily extending the run time without any benefits. In this case, the so-called elution-extrusion mode can be applied: after compounds with suitable retention times are eluted, by pumping the stationary phase instead of the mobile phase through the rotor, compounds still retained in the CPC system can be extruded. Thereby, the fresh stationary phase will displace the stationary phase loaded with the sample which thus will be eluted. Another scenario is when compounds may elute in the sweet spot without sufficient resolution: in this case, the role of the mobile and stationary phase and the flow direction can also be reversed (descending to ascending or vice versa) during the separation, thereby generating higher plate number in the system. Typically, this so-called dual mode is used for complex samples containing compounds with a wide range of Kd values.

According to the present invention, the CPC method comprises a biphasic solvent system, wherein the combination of the organic solvent component, the pH of the aqueous component, and the type and amount of the acidic component (i.e., degree of acidification) of the solvent system result in Kd values around unity (in the sweet spot of CPC) for the majority of PAs and PANOs; while the solvent system simultaneously provides Kd values outside the sweet spot range for valuable comfrey biomarkers thereby generating selectivity (separation factor) between the unwanted and valuable components. This CPC method also comprises the mode of operation, which can be preferably a (i) classical elution-extrusion mode in one embodiment of the present invention; or a (ii) dual mode in another preferred embodiment.

The crude nature of hydroethanolic comfrey root extracts manifests in chemical complexity and its components' wide polarity range. To remove a minor (trace) component or group of components from such a difficult matrix a selective interaction providing a high selectivity factor between the unwanted and valuable components along with an efficient separation technique providing a high plate number should be utilized. In CPC, by directly adjusting the pH of the aqueous phase of the solvent system, the partition of ionizable compounds can be precisely modulated and selectivity can be generated.

PAs in comfrey are predominantly retronecine-type alkaloids with basic p/C_a values in the range of 7-8, e.g., the predicted p/C_a value of lycopsamine and intermedine is 7.82. Acylation of the free hydroxyl group causes slight weakening of their basic character, e.g., the predicted p/C_a value of 7-acetylintermedine and 7-acetyllycopsamine is 7.22; while the corresponding N-oxide derivatives are practically losing their basicity (predicted p/C_a=2.82). In contrast to this, CA oligomers are moderately polar weak carboxylic acids (p/ _a 3.6-5), while allantoin can be considered an extremely hydrophilic (logP=-3.14) weak acid (p/C_a=8.48).

Thus, in a properly acidified polar solvent system, wherein the pH of the aqueous phase falls between 2 and 3 and wherein the organic phase is composed of a slightly water-soluble aliphatic alcohol, or an alkanoate ester, providing good solubility for the crude comfrey extract, CA oligomers will be present in neutral (un-ionized) form, and will enrich in the upper organic phase (Kd>3-4), while PAs will be present in fully protonated form and can form ion-pairs with the acidic component to produce partition around unity (Kd=O.5-2.0). Simultaneously, allantoin will definitely enrich in the lower aqueous phase (Kd=0.1-0.2), however, its partition into the organic phase can be forced to some extent by increasing the ionic strength of the lower phase. Table 1 summarizes Kd values measured in solvent systems composed according to this approach for the valuable (allantoin, globoidnan A and B, rosmarinic acid and rabdosiin) and for the toxic (major PAs and PANOs) compounds; while Fig. 1 shows a typical partition/elution profile obtained by this approach in a preferred embodiment of the present invention. Since retention in LLC is solely governed by partition, the partition coefficients unambiguously determine the chromatographic elution profile, so the knowledge of their measured value is fully sufficient to predict the goodness of the separation.

Table 1. Partition coefficients for the studied compounds of comfrey root extract measured by the shake-flask method in LLC solvent systems suitable for PA-removal.

Partition coefficient (Kd) n-butanol/ , , , , , , ethyl acetate/ n-butano /water sec-butano /water

Compound water 0.25% „ _no7 . . ' _n ' water 0.25%

. , . . 1.0% (v/v) acetic 0.25% (v/v) formic

(v/v) trifluoro- ' ' (v/v) trifluoro- acid acid acetic acid acetic acid

Allantoin 0.19 0.20 0.24 0.04

Intermedine „ „„ „ „„ „ „ „

, . 0.74 0.29 0.48 0.10 lycopsamine

Intermedine-N-oxide „ „ „ „ „„

. , 1.13 0.69 0.65 0.22 lycopsamine-N-oxide

7-acetyl-intermedine

7-acetyl-lycopsamine 1.14 0.48 0.41 0.49 and their N-oxides

Symphytine-N-oxid and isobar 2.81 0.81 1.16 2.10 compounds

Globoidnan B 5.19 4.27 - 8.34

Rabdosiin 6.55 5.27 5.79 23.89

Rosmarinic acid 11.18 11.91 3.91 12.08

Globoidnan A 11.21 13.38 3.59 20.11

Examples

The following examples are provided by way of illustration of the invention and do not intend to restrict the scope of the invention as defined in the claims. Table 2 details three preferred embodiments of the invention on two different CPC devices from two different manufacturers (Gilson and RotaChrom) on benchtop and on pilot-scale (250 mL and 2.1 L rotor volumes), respectively.

Table 2. Summary of laboratory and pilot-scale CPC method parameters detailed in the Examples section. UP: upper phase of the solvent system; LP: lower phase of the solvent system.

Flow

„ CPC device „ , Sample Loading Rot. Run-

^Ex „P and ro *tor Solvent Mode so .lvent . and . per run speed . , rate time

# system . . . . . . . (mL/ . volume inj. solvent (g) (rpm) . (min) min)

„„„ n-BuOH/ elution-

Gilson CPC

1 , water extrusion UP 0.5 2000 10 90

^{250 m L} 0.25% TFA dsc dual

, mode UP&LP 0.7 2000 10 20 n-BuOH/

„ asc-dsc z iison CrC water . "? . ? .

250 L 0.25% TFA _{1Q 2Q} dsc-asc e ^ex^lt^Ur^tuⁱ°siⁿo'n UP/ , L , P „ 22.6

„ , and LP

„ n-BuOH/ dsc

„ RotaChrom „„„„ „„„

³ CPC ? 1 i water 5.6 1000 200 r ' 0.25% TFA elution-

UP/LP extrusion , , 22.6 and UP asc

Example 1

The input comfrey root extract to be purified was a hydroethanolic crude extracted with 60% (v/v) ethanol. The PA and PANO content of this comfrey extract was 300-350 ppm based on HPLC-MS/MS. The aim of the experiment was to reduce the level of PAs and PANOs in the product as much as possible. As the high ethanol content of the input sample caused high stationary phase loss in CPC, evaporation of the sample solvent was necessary before injection. One laboratory-scale CPC (labCPC) run required a 50 mL input sample, of which about 0.5 g of solid extract was obtained. This was redissolved in 10 mL upper phase of the solvent system.

The LabCPC experiment was performed on a Gilson PLC 2250 purification system equipped with a Gilson CPC-250 rotor (total volume of 250 mL) controlled by the Gilson Glider CPC Software. Briefly, the rotor was filled up with the stationary phase (upper phase) at a high flow rate (50 mL/min). The system was equilibrated with the mobile phase. Afterward, the sample was injected by using the built-in 10 mL loop of the instrument. The CPC was operated in the so-called elution-extrusion mode. The regular chromatographic elution was combined with stationary-phase extrusion to recover compounds with high Kd values.

The solvent system utilized was the n-butanol/water solvent system acidified with 0.25% (v/v) trifluoracetic acid. Prior to the CPC run the solvent system was mixed in a separatory funnel. After equilibrium was established, upper (stationary) and lower (mobile) phases were separated. The main parameters of the method are summarized in Table 3. Due to the wide polarity range of the valuable components (allantoin to CA oligomers), the CPC method required a relatively long elution period and a long extrusion period. The CPC chromatogram of the run is presented in Fig. 2, while the corresponding elution profile (heatmap or fractogram) of the run along with the Kd values of comfrey biomarkers determined in shake-flask measurement is provided in Fig. 1.

Table 3. Timetable of a laboratory-scale elution-extrusion CPC method in descending mode (Example 1).

Pump line A Pump line B Rotational Detection

Time Flow rate , . . „ . . . . .. .

. . . . . (upper phase) (lower phase) speed wavelength Mode

(mm) (ml/min) _{% % (rpm) (}„_m)

0.00 10 0 100 2000 220, 254, 280 Dsc

75.0

10 100 0 2000 220, 254, 280 Dsc

900

10 100 0 2000 220, 254, 280 Dsc

After the CPC run appropriate fractions (allantoin and caffeic acid oligomers, fractions #7-10 and #40-47) were pooled and evaporated on a rotary evaporator (laboratory-scale Heidolph Hei-VAP Value with Vacuubrand 4c vacuum pump controlled by Vacuubrand CVC 3000 vacuum control unit) to dryness. The obtained final product extract was redissolved in 60% (v/v) ethanol and analysed by HPLC-MS/MS in a multiple reaction monitoring (MRM) method to determine the level of PAs and PANOs: a depletion factor higher than 300-fold was achieved in the product of the CPC purification compared to the input crude extract.

Example 2

The input comfrey root extract to be purified was a hydroethanolic crude extracted with 60% (v/v) ethanol. The PA and PANO content of this comfrey extract was 400-500 ppm based on HPLC-MS/MS. The aim of the experiment was to reduce the level of PAs and PANOs in the product as much as possible. As the high ethanol content of the input sample caused high stationary phase loss in CPC, evaporation of the sample solvent was necessary before injection. One labCPC run required a 50 mL input sample, of which about 0.7 g of solid extract was obtained. To completely redissolve the input material, and to avoid the disturbance of the hydrodynamic equilibrium of the system, the same volume of the upper and lower phase was used to obtain 10 mL (biphasic) sample solution.

LabCPC experiment was performed on a Gilson PLC 2250 purification system equipped with a Gilson CPC-250 rotor (total volume of 250 mL) controlled by the Gilson Glider CPC Software. Briefly, the rotor was filled up with 50% upper and 50% lower phase at a high flow rate (50 mL/min) without rotation. In the first injection (dual-mode, switching asc to dsc) the system was equilibrated with the starter mobile phase (upper phase). The sample was injected by using the built-in 10 mL loop of the instrument. The so-called dual mode was used: after 15 min elution, asc mode was switched to dsc mode using a switching valve for the last 5 min of the run. In the second injection (dual-mode, switching dsc to asc) the very same method setup was applied except that the run was started in dsc mode and the valve was switched to asc mode after 15 min elution time.

The solvent system utilized was the n-butanol/water solvent system acidified with 0.25% (v/v) trifluoracetic acid. Prior to the CPC run the solvent system was mixed in a separatory funnel. After equilibrium was established, phases were separated. The main parameters of the method are summarized in Tables 4 and 5. The CPC chromatogram of the run is presented in Fig 3.

Table 4. Timetable of a laboratory-scale dual mode (asc to dsc) CPC method (Example 2, 1^st injection).

Flow Pump line A Pump line B Rotational Detection

Time . . . .. . . . .. . rate (upper phase) (lower speed wavelength Mode ^tmin) (mL/min) % phase) ^% (rpm) (nm)

0.00 10 166 6 2000 220, 254, 280 Asc

15.00 10 0 100 2000 220, 254, 280 Dsc

20.00 io 6 166 2000 226, 254, so DSC

Table 5. Timetable of a laboratory-scale dual mode (dsc to asc) CPC method (Example 2, 2^nd injection).

Flow Pump line A Pump line B Rotational Detection

Time . . . .. . . . .. . rate (upper phase) (lower speed wavelength Mode ^tmin) (mL/min) % phase) % (rpm) (nm)

0.00 10 0 100 2000 220, 254, 280 Dsc

15.00 10 100 0 2000 220, 254, 280 Asc

20.00 10 100 0 2000 220, 254, 280 Asc After the CPC run appropriate fractions (allantoin and caffeic acid oligomers, fractions #13-19 in the first injection and fractions #3-10 in the second injection) were pooled and evaporated on a rotary evaporator (laboratory-scale Heidolph Hei-VAP Value with Vacuubrand 4c vacuum pump controlled by Vacuubrand CVC 3000 vacuum control unit) to dryness. The obtained final product extract was redissolved in 60% (v/v) ethanol and analysed by HPLC- MS/MS in a multiple reaction monitoring (MRM) method to determine the level of PAs and PANOs: a depletion factor higher than 500-fold was achieved in the product of the CPC purification compared to the input crude extract.

Example 3

The input comfrey root extract to be purified was a hydroethanolic crude extracted with 60% (v/v) ethanol. The PA and PANO content of this comfrey extract was 400-500 ppm based on HPLC-MS/MS. The aim of the experiment was to reduce the level of PAs and PANO as much as possible. As the high ethanol content of the input sample caused high stationary phase loss in CPC, evaporation of the sample solvent was necessary before injection. One labCPC run required a 400 mL input sample, of which about 5.6 g of oily extract was obtained. To completely redissolve the input material, the same volume of the upper and lower phase was used to obtain ca. 100 mL (biphasic) sample solution. The two phases of this sample solution were separated in a separatory funnel and injected into the CPC rotor in two different modes to reduce the PA content per run, to speed-up the method, and avoid injection problems and column bleeding due to overloading. Thus, the upper phase of the sample solution was injected onto the rotor in ascending mode, and the lower phase of the sample solution was injected onto the rotor in descending mode.

The purification was performed on a RotaChrom pilot-scale CPC (rCPC) system (ECOM ECB2005PC Gradient Box with PC, ECOM ECP 2300 Isocratic Pump, ECOM Flash 14 DAD 400 UV- detector, ECOM Box with 10-position valve fraction collector and a RotaChrom rCPC rotor (2.1 L total internal volume, 100 extraction cells)) controlled by the ECOMAC Software. Briefly, the rotor was filled with the stationary phase, then the system was equilibrated with the mobile phase. The sample solution was injected by using the pump. The rCPC was operated in the so- called elution-extrusion mode. The regular chromatographic elution was combined with stationary-phase extrusion to recover compounds with high Kd values: ca. one rotor volume of the stationary phase was pumped through the rotor after a short, 5.6 min elution period. The solvent system utilized was the n-butanol/water solvent system acidified with 0.25% (v/v) trifluoracetic acid. Prior to the CPC run the solvent system was mixed in a separatory funnel. After equilibrium was established, upper and lower phases were separated. The main parameters of the method are summarized in Tables 6 and 7. The chromatogram of the two runs is presented in Fig. 4. The same method setup was used for both CPC runs, only the operation mode was reversed.

Table 6. Timetable of a pilot-scale elution-extrusion CPC method in ascending mode.

-|-j_me Fl°^w Pump line Pump line Pump line Rotatio- Detection

Output rate A (sample) B (lower D (upper nal speed Wavelength Mode mⁱⁿ (mL/min) % phase) % phase) % (rpm) (nm)

0.00 Waste 200 0 0 100 1000 220, 254, 280 Asc

4.00 Waste 80 100 0 0 1000 220, 254, 280 Asc

5.00 Waste 200 0 0 100 1000 220, 254, 280 Asc

7.00 Fraction 1 200 0 0 100 1000 220, 254, 280 Asc

7.40 Fraction 2 200 0 0 100 1000 220, 254, 280 Asc

7.80 Fraction 3 200 0 0 100 1000 220, 254, 280 Asc

8.20 Fraction 4 200 0 0 100 1000 220, 254, 280 Asc

8.60 Fraction 5 200 0 0 100 1000 220, 254, 280 Asc

9.00 Fraction 6 200 0 0 100 1000 220, 254, 280 Asc

9.40 Fraction 7 200 0 0 100 1000 220, 254, 280 Asc

9.80 Fraction 8 200 0 0 100 1000 220, 254, 280 Asc

10.20 Fraction 9 200 0 0 100 1000 220, 254, 280 Asc

10.60 Waste 200 0 100 0 1000 220, 254, 280 Asc

22.60 Waste 200 0 100 0 1000 220, 254, 280 Asc

Table 7. Timetable of a pilot-scale elution-extrusion CPC method in descending mode. i_me Flow Pump line Pump line Pump line Rotational Detection

Output rate A (samp- B (lower D (upper speed Wavelength Mode

(mL/min) le) % phase) % phase) % (rpm) (nm)

0.00 Waste 200 0 100 0 1000 220, 254, 280 Dsc

4.00 Waste 80 100 0 0 1000 220, 254, 280 Dsc

5.00 Waste 200 0 100 0 1000 220, 254, 280 Dsc

7.00 Fraction 1 200 0 100 0 1000 220, 254, 280 Dsc

7.40 Fraction 2 200 0 100 0 1000 220, 254, 280 Dsc

7.80 Fraction 3 200 0 100 0 1000 220, 254, 280 Dsc

8.20 Fraction 4 200 0 100 0 1000 220, 254, 280 Dsc

8.60 Fraction 5 200 0 100 0 1000 220, 254, 280 Dsc

9.00 Fraction 6 200 0 100 0 1000 220, 254, 280 Dsc

9.40 Fraction 7 200 0 100 0 1000 220, 254, 280 Dsc

9.80 Fraction 8 200 0 100 0 1000 220, 254, 280 Dsc

10.20 Fraction 9 200 0 100 0 1000 220, 254, 280 Dsc

10.60 Waste 200 0 0 100 1000 220, 254, 280 Dsc

22.60 Waste 200 0 0 100 1000 220, 254, 280 Dsc

After the two CPC runs appropriate fractions (allantoin and caffeic acid oligomers, fractions #1-7 in the asc run and fractions #1-10 in the dsc run) with UV-absorbance were pooled. The pH of the pooled fractions was measured using the IKA RET Control-vise apparatus with SI Analytics BlueLine pH-electrode. After CPC run the pH of the pooled fractions was 1.79. Although the character of the comfrey root extract was slightly acidic, such an acidic pH can cause decomposition of the CA oligomers eventually. Thus, the pH was adjusted to 5.36 with 1 M sodium hydroxide solution. The pH-adjusted product fraction was evaporated on a rotary evaporator (laboratory-scale Heidolph Hei-VAP Value with Vacuubrand 4c vacuum pump controlled by Vacuubrand CVC 3000 vacuum control unit). After the sample was evaporated to dryness, it was redissolved in ethanol to filter out the sodium trifluoroacetate salt from the product. The repeatedly dried final product was analysed by HPLC-MS/MS in a multiple reaction monitoring (MRM) method to determine the level of PAs and PANOs: a depletion factor higher than 500-fold was achieved in the product of the CPC purification compared to the input crude extract (see Fig. 5 and Table 8). In addition, to characterize the chemical- and bioequivalence of the product liquid extract, the density, the total phenolic content, the antioxidant capacity (DPPH and ABTS radical scavenging activities), and the tentative level of biomarkers have been assessed using appropriate in vitro tests and LC-MS (see Table 8). Based on these data, the CPC purified comfrey extract proved to be compliant (total PA and PANO level below 1 ppm) and preserved its core bioactivity related to its medicinal usage.

Table 8. Comparative characterization of the crude hydroethanolic root extract of comfrey as input sample and the PA-depleted product sample of the same hydroethanolic comfrey root extract after CPC purification (Example 3). _ Crude comfrey PA-depleted product

Parameter of the Extract root extract of CPC purification

. yellowish .

Appearance solution light yellowish solution

Solvent 60% (v/v) ethanol ethanol

Density (g/cm³) 0.89 0.91 pH 5.99 5.36

Total PA concentration (HPLC-MS/MS, ppm) 299-399 0.34-0.52

Total phenolic content (mg caffeic acid equivalent/g) 151 58

DPPH radical scavenging activity (EC50, pg/mL) 4.2 6.6

ABTS radical scavenging activity (EC50, pg/mL) 2.3 3.3

Level of biomarkers (LC-MS) and their changes relative to the level before CPC purification (%)

• A ..l.lanto.in , h .ig ,h low

(70-95% decrease)

, . moderate

• Rosmannic acid moderate .

(20-30% decrease)

• Globoidnan A high ,

(40-50% decrease)

• G . o.boid.nan B . low ___n/ low , ,

(40-50% decrease)

, , , moderate

• Rabdosiin moderate ___n/ , ,

(50-60% decrease)

Claims

Claims A process for the preparation of comfrey root extract with reduced pyrrolizidine alkaloid content, wherein pyrrolizidine alkaloids are removed from a crude comfrey root extract by liquid-liquid chromatography (LLC) using a biphasic binary solvent system, which is obtained by partially mixing a polar organic solvent with water acidified with a strong organic acid to pH 2-3, and the polar organic solvent is selected from any of the following two groups of compounds: a) aliphatic alcohols with 4-5 carbon atoms; and b) alkanoate esters with 3-6 carbon atoms. The method of claim 1, wherein the polar organic solvent is an aliphatic alcohol with 4-5 carbon atoms, preferably n-butanol, isobutanol, tert-butanol, sec-butanol, or n-pentanol. The method of claim 1, wherein the polar organic solvent is an alkanoate ester with 3-6 carbon atoms, preferably methyl acetate, ethyl acetate, n-propyl acetate, i-propyl acetate, or butyl acetate. The method of claims 1-3, wherein the strong organic acid is formic acid, acetic acid or trifluoroacetic acid. The method of claim 1, wherein 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. The method of claims 1-5, wherein the LLC is performed in a hydrostatic centrifugal partition chromatography (CPC) device or in a hydrodynamic counter-current chromatography (CCC) device. The method of claims 1-6, wherein the crude comfrey root extract is an extract prepared by hydroethanolic extraction of a comfrey root drug, and prior the LLC is performed, the solvent is evaporated from the crude extract and the resulting solid or oily extract is dissolved in the polar organic solvent-rich upper phase and/or dissolved in the aqueous lower phase. The method of claim 7, wherein the LLC method comprises two elution-extrusion steps, where the extract is dissolved in the solvent system and the extract dissolved in the upper phase is run in ascending mode, while the extract dissolved in the lower phase is run in descending mode, and the product fractions of these two runs are combined. The method of claims 1-7, wherein the LLC method is performed in a dual mode where the crude extract is dissolved in both phases of the solvent system and co-injected onto an LLC column. The method of claims 1-9, wherein the method further includes readjusting the pH value of the product fraction obtained with LLC to the pH value of the crude extract, and filtering the salt formed. The method of claims 1-10, wherein a pharmacopoeia-compliant comfrey root extract with a depletion factor of at least 300-fold, preferably 500-fold, more preferably 1000- fold, with respect to the total PA content of the crude extract is produced without significant bioactivity loss.