A method for removing pyrrolizidine alkaloids from plant extracts

Abstract

The application discloses a method for removing pyrrolizidine alkaloids in plant extracts, which is characterized by comprising the following steps: S1. dissolving the plant extract with water, and then performing solid-liquid separation to obtain a clear liquid; S2. adjusting the pH of the clear liquid to 8.0-9.0, and then taking supernatant after centrifugation; then performing extraction with an organic solvent, and retaining the water phase, namely the water layer liquid; S3. performing vacuum concentration on the water layer liquid, and then performing ion exchange resin elution on the water layer liquid, and collecting the eluate; the elution operation is to first wash with water until the effluent is clear, and then perform elution with acidic ethanol and collection; and S4. performing vacuum concentration and spray drying on the eluate. The removal method significantly improves the total saponin content in some plant extracts to more than 41%, and the removal efficiency of PAs reaches more than 99%. The method can also protect the structure of bioactive substances, and improves environmental protection and economy.

Description

Technical Field

[0001] This invention belongs to the field of plant extract processing technology, specifically relating to a method for removing pyrrolizidine alkaloids from plant extracts. Background Technology

[0002] Pyrrolizidine alkaloids (PAs) are a class of naturally occurring toxins synthesized by flowering plants. They possess hepatotoxic, cytotoxic, carcinogenic, and mutagenic properties, and are among the most serious and widespread endogenous and adulterated toxic components in food and herbal products. They are secondary metabolites produced by plants to defend against adverse effects. Produced in plant roots, they can be transported to various parts of the plant, especially plant organs related to reproduction and growth, and can accumulate in the human body through the food chain.

[0003] Currently, over 350 PAs (excluding their nitrogen oxides) have been identified, of which approximately 50% exhibit hepatotoxicity and can be transmitted through herbs, food supplements, or the food chain, causing poisoning in humans and animals. To remove PAs from plant extracts, most existing technologies employ extraction to remove pyrrolizidine. Patent document CN116212978A also discloses a method for removing pyrrolizidine alkaloids from traditional Chinese medicine. This method collects the column eluent and 2-3 BV of water washing solution; the large volume of elution mixture increases energy consumption during concentration, and prolonged concentration can lead to the degradation of some heat-sensitive components. Furthermore, the purification effect on saponins has not been investigated. Summary of the Invention

[0004] This invention overcomes the shortcomings of existing technologies, such as low efficiency in removing pyrrolizidine alkaloids and the inability to purify saponins. Therefore, this invention provides a method for removing pyrrolizidine alkaloids from plant extracts. This method achieves a high removal rate of pyrrolizidine alkaloids while simultaneously purifying saponins. Furthermore, this method is simple, efficient, and more environmentally friendly.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] This invention provides a method for removing pyrrolizidine alkaloids from plants, comprising the following steps:

[0007] S1. Mix the plant extract with water and collect the clear liquid;

[0008] S2. Adjust the pH of the clarified solution, centrifuge, and collect the supernatant;

[0009] S3. Extract the supernatant with an organic solvent and collect the aqueous phase;

[0010] S4. After concentrating the aqueous phase, elute it sequentially with water and acidic ethanol aqueous solution, and collect the acidic ethanol aqueous solution eluent;

[0011] S5. The acidic ethanol aqueous solution eluent is concentrated and then dried.

[0012] In this invention, the plant extracts include Tribulus terrestris extract, Tribulus terrestris extract, Chrysanthemum extract, Lavender extract, Mimosa extract, Dendrobium extract, Rehmannia glutinosa extract, Polygonatum odoratum extract, and Oleander extract.

[0013] In S1, the content of pyrrolizidine alkaloids in the plant extract is 1715-2500 ppb.

[0014] In S1, the total saponin content of the plant extract is 35-55%.

[0015] In S1, during the mixing process, the mass of the water is 5 to 25 times the mass of the plant extract, for example, 5, 10, 15, 20, or 25 times, preferably 10 to 20 times, for example, 15 or 20 times.

[0016] In S1, during the mixing process, the temperature of the water is 45–75°C, preferably 50–60°C.

[0017] In S1, the stirring speed during the mixing process is 400-600 rpm, and the stirring time is 3-5 h; the preferred stirring speed is 450-530 rpm, and the stirring time is 3.5-4.5 h.

[0018] In S2, the pH value is 8 to 9.

[0019] In S2, the pH adjuster used is at least one of alkali, carbonate, bicarbonate and aqueous solution thereof; preferably at least one of Na2CO3 aqueous solution, NaHCO3 aqueous solution, NaOH aqueous solution and NH4OH aqueous solution, for example Na2CO3 aqueous solution.

[0020] In S2, the concentration of the pH adjuster used for pH adjustment is 1–10 wt%, preferably 3–6 wt%, for example 5 wt%.

[0021] In S3, the extraction operation involves extracting 2 to 3 times with an organic solvent, discarding the organic layer, and retaining the aqueous layer; for example, using ethyl acetate for extraction, discarding the upper ethyl acetate layer, and retaining the lower aqueous layer.

[0022] In step S3, the organic solvent is a conventional solvent in the art, preferably one of n-butanol, ethyl acetate, ethyl formate, and n-hexane, such as ethyl acetate. The amount of the organic solvent used is 0.6 to 1.5 times the volume of the supernatant, preferably 0.8 to 1.2 times, for example, 1 times.

[0023] In S2, the centrifugation speed is 3700-4500 rpm, preferably 4000 rpm; preferably, the centrifugation time is 5-60 min, more preferably 10-30 min, for example 30 min.

[0024] In step S4, after concentration, the concentrated aqueous layer needs to be cooled and then replenished with water to the original volume. Concentration removes the ethyl acetate organic solvent from the aqueous layer, while replenishing with water maintains the original solids concentration, ensuring effective adsorption on the column.

[0025] In S4, the concentration temperature is 65–80°C; the concentration pressure is -0.07–-0.08 MPa; and the concentration is brought to 40–50% Brix.

[0026] In S5, the concentration temperature is 65–80°C; the concentration pressure is -0.07–-0.08 MPa; and the concentration is brought to 40–50% Brix.

[0027] In step S4, the elution process uses a strong acidic cation exchange resin, preferably one of Amberlite IR120, Dowex 50W, 001x4, 001x7, DL08, and DL10; Amberlite IR120 is preferred. The ion exchange resin can remove unadsorbed impurities such as pyrrolizidine alkaloids, proteins, polysaccharides, and tannins.

[0028] In this invention, the pH of the acidic ethanol solution is 3-4; the pH adjuster of the acidic ethanol solution is citric acid, sulfuric acid, or hydrochloric acid, preferably citric acid. The ethanol concentration in the acidic ethanol solution is 80%, with the remainder being water.

[0029] In this invention, the drying method is spray drying; wherein the inlet air temperature of the spray drying is 175-185℃, the outlet air temperature of the spray drying is 95-115℃, and the homogenizing pump frequency of the spray drying is 26-30Hz. The moisture content after spray drying is ≤5%; preferably, the inlet air temperature of the spray drying is 180℃, the outlet air temperature of the spray drying is 100℃, and the homogenizing pump frequency of the spray drying is 30Hz.

[0030] In this invention, the solid-liquid separation operation is a conventional operation in the art, and filtration or centrifugation is preferred.

[0031] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] 1. The method of this invention is simple and rapid, improves removal efficiency, and significantly increases the total saponin content in the product. Ultimately, the PAs removal efficiency is ≥99%, and the PAs content after removal is below 11.2 ppm; it also has the effect of purifying saponins, with the saponin content after purification of several plant extracts exceeding 41%, and reaching over 51.1% in some preferred schemes.

[0034] 2. This invention uses an adsorption method to remove pyrrolizidine alkaloids from plant extracts. This method is gentle, conducive to the structural protection of active substances, and does not introduce new organic impurities, thus ensuring the safety of the final product. In addition, the solvent can be recycled, which also reduces environmental pollution and is more economical and environmentally friendly.

[0035] 3. This method has good continuity and simple process, making it suitable for industrial production. Attached Figure Description

[0036] Figure 1 This is a high-performance liquid chromatography (HPLC) chromatogram comparing the removal of PAs from Tribulus terrestris extract in Example 7.

[0037] Figure 2 The image shows the high-performance liquid chromatography (HPLC) chromatogram of the Polygonatum odoratum extract before PA removal in Example 7.

[0038] Figure 3 The image shows the high-performance liquid chromatography (HPLC) chromatogram of the Polygonatum odoratum extract after PA removal in Example 7. Detailed Implementation

[0039] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0040] Unless otherwise specified, all raw materials and reagents used in the embodiments and comparative examples of this invention are commercially available. The content of each substance before and after removal of the extract can be detected by high-performance liquid chromatography.

[0041] Example 1

[0042] This embodiment investigates the effect of the amount of solvent required to dissolve the extracts on the extraction of total saponins. 500g of Tribulus terrestris extract, chrysanthemum extract, lavender extract, mimosa extract, dendrobium extract, rehmannia extract, polygonatum extract, and oleander extract were weighed and added to water at 50℃ in amounts of 5, 10, 15, and 20 times their respective concentrations (g / g). The mixture was stirred at 500 rpm for 230 min. The extracts were then separated into solid and liquid phases to obtain a clear liquid. The volume of the clear liquid was recorded, and the concentration of total saponins in the clear liquid was measured. The yield of total saponins after dissolution was calculated, as shown in Table 1 below.

[0043] Table 1. Yield of total saponins after dissolution

[0044]

[0045] As shown in Table 1, when the solvent volume was increased by 15 times during dissolution, the total saponin yield in the extract reached over 99%. Further increasing the solvent volume did not significantly increase the total saponin yield. Therefore, 15 times the solvent volume is preferred for dissolution.

[0046] Example 2

[0047] This embodiment investigates the effect of pH on saponin recovery. 500g each of the following extracts were weighed: Tribulus terrestris extract, Chrysanthemum extract, Lavender extract, Mimosa extract, Dendrobium orchid extract, Rehmannia glutinosa extract, Polygonatum odoratum extract, and Oleander extract. 7.5L of water at 50℃ was added, and the mixture was stirred at 500rpm for 230min. The extracts were then separated into solid and liquid phases to obtain a clear liquid. The pH was adjusted to 9.0 with 5wt% sodium hydroxide solution. The mixtures were centrifuged at 4000rpm for 30min, and the supernatant was collected. The volume of the supernatant was recorded, and the concentration of total saponins in the supernatant was measured. The yield of total saponins after dissolution was calculated, as shown in Table 2 below.

[0048] Table 2

[0049]

[0050] Table 2 shows that the recovery rate of total saponins is relatively low when sodium hydroxide aqueous solution is added to the solution to adjust the pH to 6-8, and relatively high when the pH is adjusted to 8-9. Therefore, the preferred pH setting is 9.

[0051] Example 3

[0052] This embodiment investigates the effect of pH adjuster on saponin recovery. 500g each of the following extracts were weighed: Tribulus terrestris extract, Chrysanthemum extract, Lavender extract, Mimosa extract, Dendrobium orchid extract, Rehmannia glutinosa extract, Polygonatum odoratum extract, and Oleander extract. 7.5L of water at 50℃ was added, and the mixture was stirred at 500rpm for 230min. The extracts were then separated into solid and liquid phases to obtain a clear liquid. 5wt% sodium carbonate solution, 5wt% sodium bicarbonate solution, 5wt% sodium hydroxide solution, and 5wt% ammonia solution were added to adjust the pH to 9.0. The mixture was centrifuged at 4000rpm for 30min, and the supernatant was collected. The volume of the supernatant was recorded, and the concentration of total saponins in the supernatant was measured. The yield of total saponins after dissolution was calculated, as shown in Table 3 below.

[0053] Table 3. Recovery rate of total saponins in the supernatant after pH adjustment of the solution.

[0054]

[0055] Table 3 shows that when sodium bicarbonate aqueous solution is added to the solution as a pH adjuster, total saponins do not precipitate or degrade. Replacing them with other adjusters significantly alters the recovery rate of total saponins in the supernatant. Therefore, sodium bicarbonate is the preferred pH adjuster in the solution.

[0056] Example 4

[0057] This embodiment investigates the effect of extractants on the extraction rate of PAs and whether saponins can be extracted. 500g of Tribulus terrestris extract, Chrysanthemum extract, Rehmannia glutinosa extract, Oleander extract, and Polygonatum odoratum extract were weighed and added to 7.5L of water at 50℃. The mixture was stirred at 500rpm for 230min. The extract was then separated into solid and liquid phases to obtain a clear liquid. A 5wt% sodium bicarbonate solution was added to adjust the pH to 8.0–9.0. The mixture was centrifuged at 4000rpm for 30min, and the supernatant was collected. The supernatant was divided into 5 portions and extracted three times (v / v) with equal volumes of n-butanol, ethyl acetate, ethyl formate, and n-hexane, respectively. The aqueous layer was concentrated at 75℃ and -0.08MPa, and the organic solvent was recovered. The organic phase was recorded, and the concentrations of total saponins and PAs in the organic phase were measured. The extraction effects of the five extractants were calculated, as shown in Table 4 below.

[0058] Table 4. Effect of extractant on PAs removal rate

[0059]

[0060]

[0061] As shown in Table 4, among the five extractants, none of them extracted total saponins. Ethyl acetate had the highest extraction rate for PAs. Therefore, ethyl acetate is the preferred extractant.

[0062] Example 5

[0063] This embodiment investigates the adsorption and removal capacity of ion exchange columns for PAs and whether they adsorb saponins. 500g of Tribulus terrestris extract, chrysanthemum extract, Rehmannia glutinosa extract, Nerium oleander extract, and Polygonatum odoratum extract were weighed and added to 7.5L of water at 50℃. The mixture was stirred at 500rpm for 230min. The extract was then separated into solid and liquid phases to obtain a clear liquid. The pH was adjusted to 9.0 with 5wt% sodium bicarbonate solution, and the mixture was centrifuged at 4000rpm for 30min. The supernatant was collected and extracted three times (v / v) with equal volumes of ethyl acetate to recover the organic solvent. The aqueous layer was concentrated at 75℃ and -0.08MPa, and water was added to the original volume. The mixture was then divided into six portions and passed through an Amberlite IR120 and a Dowex ion exchange column, respectively. 50W, 001x4(731), 001x7(732), DL08, and DL10 ion exchange resins were washed with water until the effluent was clear and the pH of the effluent was between 7.0 and 7.5. The column loading and water washing effluents were collected and combined to detect the concentration of PAs and total saponins. Based on the percentage of total PAs content in the column loading effluent and water washing effluent relative to the PAs in the initial extract, the residual amount of PAs and the loss rate of total saponins were calculated as shown in Table 5 below.

[0064] Table 5 Comparison of residual PAs and total saponins in the effluent after cation exchange resin adsorption.

[0065]

[0066]

[0067] Table 5 shows that ion exchange resins such as 001x4 (731), 001x7 (732), DL08, and DL10 adsorbed both total saponins and polysaccharides (PAs) from plant extracts. Amberlite IR120 was more effective than Dowex 50W, and both Amberlite IR120 and Dowex 50W adsorbed total saponins better. Therefore, Amberlite IR120 is preferred as the adsorbent for total saponins.

[0068] Example 6

[0069] This embodiment investigates the effect of pH adjuster in the eluent on the residual amount of PAs / total saponin elution rate. Weigh 500g of Tribulus terrestris extract, Chrysanthemum extract, Rehmannia glutinosa extract, Oleander extract, and Polygonatum odoratum extract, add 7.5L of water at 50℃, stir at 500rpm for 230min, separate the solid and liquid of the extract, obtain the clear liquid, add 5wt% sodium bicarbonate solution to adjust to pH 9.0, centrifuge at 4000rpm for 10min, collect the supernatant, extract the supernatant with equal volume of ethyl acetate (v / v) 3 times, recover the organic solvent, concentrate the aqueous layer at 75℃ and -0.08MPa, add water to the original volume, divide each into 3 equal parts, pass them through 3 ion exchange resins packed with Amberlite IR120, wash with water until the eluent is clear, prepare 3 parts of 80% acidic ethanol eluent, adjust the pH to 4.0 with citric acid, HCl and H2SO4 respectively, elute the cation exchange resin with the eluent, collect the eluent, detect the content and concentration of PAs and total saponins, calculate the elution rate and the residual amount of PAs as shown in Table 6 below.

[0070] Table 6. Effect of pH adjuster in eluent on elution rate

[0071]

[0072] As shown in Table 6, adjusting the pH of the eluent with citric acid, hydrochloric acid, and sulfuric acid has no effect on the elution rate. Considering that hydrochloric acid and sulfuric acid are controlled reagents for easily manufactured drugs, citric acid is preferred as the pH adjuster in the eluent.

[0073] Example 7

[0074] Weigh 500 kg of Tribulus terrestris extract, Chrysanthemum extract, Rehmannia glutinosa extract, Oleander extract, and Polygonatum odoratum extract, add 7500 L of water at 50℃, stir at 500 rpm for 230 min, separate the solid and liquid phases of the extract, obtain a clear liquid, adjust the pH to 9.0 with 5 wt% sodium bicarbonate solution, centrifuge at 4000 rpm for 30 min, collect the supernatant, extract the supernatant three times with an equal volume of ethyl acetate (v / v), concentrate the aqueous layer at 75℃ and -0.08 MPa, recover the organic solvent, add water to the original volume, and then pass the solution through Amberlite. The IR120 ion exchange resin was washed with water until the eluent was clear. The eluent was then eluted through the cation exchange resin with 80% acidic ethanol (pH adjusted to 4.0 with citric acid). The eluent was collected and concentrated under reduced pressure at 75°C and -0.08 MPa to Brix 45%. The solvent was recovered, and the inlet air temperature was adjusted to 180°C, the outlet air temperature to 100°C, and the homogenizer pump frequency to 30 Hz. The mixture was then spray-dried to obtain the extract. The weight was recorded, and the contents of PAs and total saponins were determined.

[0075] Table 7 Comparison of PAs in plant extracts before and after removal

[0076]

[0077] Table 7 shows that after ethyl acetate extraction and column chromatography, the removal rate of PAs in the plant extract reached over 99%, and the purity of total saponins in the extract was also improved. Among these, Figure 1 The image shows a high-performance liquid chromatography (HPLC) chromatogram comparing the removal of pyrrolizidine alkaloids (PAs) from the Tribulus terrestris extract in Example 7. The PAs elution peaks mainly include four peaks with retention times around 29-30 min, 31-33 min, 37-38 min, and 54-57 min. It can be seen that pyrrolizidine alkaloids are essentially removed, with their residues reduced to acceptable levels. The product prepared by this invention is mainly used as a raw material for food and pharmaceuticals, for the comprehensive extraction and separation of effective components from traditional Chinese medicine, removing harmful pyrrolizidine alkaloids, which is beneficial for promoting the high-value utilization of traditional Chinese medicine resources and for human health. Figure 2 The first image shows the high-performance liquid chromatogram (HPLC) of the Polygonatum odoratum extract in Example 7 before alkaloid removal, and the second image shows the HPLC chromatogram of the extract after alkaloid removal in Example 3. The alkaloids included in this invention are: 1) Pyrrolizidine alkaloids: *Seneciocinone*, *Seneciocinone*, *Seneciocinone*, *Seneciocinone*; 2) Ergot alkaloids: dihydroergotinine, ergoconine, ergoconine, ergoconine, ergoconine, ergoconine, ergoconine; 3) Hyoscyamine alkaloids: atropine, scopolamine, hyoscyamine.

[0078] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for removing pyrrolizidine alkaloids from plant extracts, characterized in that, It includes the following steps: S1. After mixing the plant extract with water, collect the clear liquid; the plant extract is selected from one of the following: Tribulus terrestris extract, chrysanthemum extract, lavender extract, mimosa extract, dendrobium extract, rehmannia extract, polygonatum extract, and oleander extract; S2. Adjust the pH of the clarified solution to 8-9, centrifuge, and collect the supernatant; S3. Extract the supernatant with ethyl acetate and collect the aqueous phase; S4. After concentrating the aqueous phase, it is eluted sequentially with water and an acidic ethanol aqueous solution with a pH of 3-4, wherein the ethanol concentration in the acidic ethanol aqueous solution is 70-90%, and the acidic ethanol aqueous solution eluent is collected; the ion exchange resin used for elution is Amberlite IR120. S5. The acidic ethanol aqueous solution eluent is concentrated and then dried.

2. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, The content of pyrrolizidine alkaloids in the plant extract is 1715~2500 ppb; The total saponin content in the plant extract is 35-55%.

3. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, During the mixing process, the mass of the water is 5 to 25 times the mass of the plant extract; And / or, the temperature of the water during the mixing process is 45~75℃; And / or, the stirring speed during the mixing process is 400~600 rpm, and the stirring time is 3~5 hours.

4. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, During the mixing process, the mass of the water is 10 to 20 times the mass of the plant extract; And / or, the temperature of the water during the mixing process is 50~60℃; And / or, the stirring speed during the mixing process is 450~530 rpm, and the stirring time is 3.5~4.5 h.

5. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, The pH adjuster used is at least one of alkali, carbonate, bicarbonate, and their aqueous solutions.

6. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, The extraction process involves extracting with ethyl acetate 2-3 times, discarding the organic layer, and retaining the aqueous layer. And / or, the amount of ethyl acetate used is 0.6 to 1.5 times the volume of the supernatant.

7. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, The centrifugation speed is 3700~4500 rpm; And / or, the centrifugation time is 5~60 min.

8. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, In S4, after concentration, the concentrated aqueous layer needs to be cooled and then replenished with water to the original volume. And / or, in S4, the concentration temperature is 65~80°C; the concentration pressure is... 0.07~ 0.08 MPa; the concentration is brought to Brix 40-50%; And / or, in S5, the concentration temperature is 65~80℃; the concentration pressure is... 0.07~ 0.08 MPa; the concentration is brought to Brix 40~50%.

9. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, The pH adjuster for the acidic ethanol aqueous solution is citric acid, sulfuric acid, or hydrochloric acid.

10. The method for removing pyrrolizidine alkaloids from plant extracts as described in claim 1, characterized in that, The drying method is spray drying; wherein the inlet air temperature of the spray drying is 175~185℃, the outlet air temperature of the spray drying is 95~115℃, and the homogenizing pump frequency of the spray drying is 26~30Hz. And / or, the moisture content after spray drying is ≤5%.

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