JP4339699B2 - Method for isolating artemisinin from Artemisia annua - Google Patents

Description

  The present invention relates to a method for isolating artemisinin, an antimalarial agent, from a medicinal herb of Artemisia annua plant. This method consists of extracting the herb with ethanol, partitioning the extract into water and hexane, followed by evaporating and crystallizing artemisinin from the hexane phase to produce substantially pure artemisinin.

  Artemisinin (Kinhaos) is a sesquiterpene lactone endoperoxide having strong antimalarial activity (see Non-Patent Document 1). Clinical studies on artemisinin and its semi-synthetic derivatives, artemether, artesunate and arteether, have been conducted in malaria, including malaria with severe complications, multidrug resistant malaria and brain malaria. It has been used for treatment. Because of structural differences from commonly used antimalarial drugs, the problems of resistance and cross-resistance (which severely limit the effectiveness of such drugs today) are not observed for artemisinin and its derivatives. Recently, methods for chemical synthesis of artemisinin have been reported, but economical large-scale synthesis is not yet possible. Thus, Artemisia annua is still the only source of industrial artemisinin. The present invention provides a quick, simple and economical method for isolating artemisinin from Artemisia annua plants.

  Older methods (see Non-Patent Document 2 and Non-Patent Document 3) included a step of subjecting a relatively crude extract to silica gel chromatography after extraction of plant material with petroleum ether. The column was eluted with a chloroform mixture containing 7.5% ethyl acetate to obtain pure artemisinin.

  Thereafter, Shin, A, Vishwakarma, ARA, Hussein, A (see Non-Patent Document 4) extract a plant part with n-hexane, and then subject the extract to chromatography on a silica gel column. reported. In this case, elution was performed with a mixture of ethyl acetate and hexane. In these methods, the main disadvantage was that these techniques depended on the chromatography of the relatively crude extract on silica gel. This requires a large solute to adsorbent ratio of 1:44 and is expensive.

  Another artemisinin extraction method includes the steps of extracting plant material with hexane, and subsequently partitioning the extract between hexane and acetonitrile, followed by chromatography of the acetonitrile phase on silica gel (see US Pat. ). While slow extraction of artemisinin in hexane requires a longer period of thermal leaching, for the distribution process, acetonitrile is a relatively expensive solvent, increasing the cost of artemisinin production. Another disadvantage of this method is that column chromatography is unavoidable to obtain artemisinin. Yet another disadvantage is that artemisinic acid predominates, and artemisinic acid tends to elute with artemisinin, thus affecting the purity of artemisinin.

  Jane, Dc, Tandon, S, Bacni, Rs, Sidiquee, MS, Kahal, Api, Sharma, Rp, Kumar, S, Batacharya, Akei (see Patent Document 2) Proposed simultaneous production method. In this method, the basic methodology of artemisinin extraction involves extracting the plant material with hexane followed by partitioning the extract between hexane and acetonitrile and then chromatographing the acetonitrile phase on silica gel. In addition, this method suggests the extraction of essential oils from residual mar by hydrodistillation and also describes a method for the isolation of artemisinic acid and its conversion to artemisinin. The disclosed method as a whole is a complex fusion of several chemical reactions. However, because hexane is used as an extraction solvent and the extract is distributed between hexane and acetonitrile and then chromatographed, the method remains untouched, such as time consuming and expensive.

  A method for extracting aretannuin from Artemisia annua was reported by Zang, Jay, Fan, Di and Ma, X (see Patent Document 3). This involves leaching Artemisia annua leaves with aqueous ethanol (<70% concentration) and then extracting the leachate with gasoline containing 30% benzene or ethyl acetate using a continuous extractor. Next, the gasoline extract was decolorized by treatment with activated carbon, and then artemisinin was concentrated and crystallized using ethanol. In this method, in order to clean the used ethanol for reuse, it is necessary to fractionate a large volume of aqueous ethanol with gasoline containing 30% benzene or ethyl acetate. This transfers most of the compound extracted with the ethanol: water mixture to the gasoline and benzene mixture. Crystallization occurs by concentrating the gasoline mixture and then crystallizing artemisinin with ethanol. The use of commercial grade benzene during the process is not appropriate due to their possible toxic effects. The process cannot be reused further because it used a mixture of solvents during the transition.

  Wheatley G.D. and Chapman T.B. (see Patent Document 4) include a step of extracting dried Artemisia annual herbs using liquid carbon dioxide and evaporating the carbon dioxide from the resulting mixture. Disclosed a method for producing artemisinin. This method relies on liquid carbon dioxide and can only be obtained from factories that prepare liquid carbon dioxide (very expensive equipment).

  Reference may also be made to the Applicant's US patent (Patent Document 5) describing a method for co-production of artemisinin and essential oil from Artemisia annua plants. In the foregoing invention, artemisinic acid is separated from artemisinin before being treated with a base.

  Reference may also be made to Applicant's co-pending application Ser. No. 09 / 538,892, which discloses an agricultural method for maximizing artemisinin yield of Artemisia annua plants. However, this application focuses on agricultural methods for planning plant growth, not on artemisinin extraction methods.

Applicant's extraction with n-hexane petroleum ether is a commonly used solvent, which is time consuming and requires thermal leaching. These solvents are inherently harmful. Since then, most methods have used chromatography to obtain artemisinin, which has been a major cause of higher production costs.
El Ferrari, Fs, Elsori, HN, 1990, US Pat. No. 4,952,603. Jane, Dishy, Tandon, S, Bacuni, Rs, Sidikue, MS, Kahal, Api, Sharma, Arpii, Kumar, S, Batacharya, Akei, 1999, US Pat. No. 5,955,084. Zang, Jay, Fan, D and Ma, X (1994) Patent No. CN1092073A. Wheatley G.W. and Chapman T.B., 2001, US Pat. No. 6,180,105. Council of Scientific and Industrial Research, US Pat. No. 5,995,084. Clayman Diel, 1985, Science 228, 1049. Clayman, Diel, Lynn, AJ, Acton, N, Scoville, Jaypee, Hoch, JM, Millhouse, W. and Theohalides, Aid, 1984, Journal of Natural Products 47: 715-717. Lacquer, Gee, Mayer, Earl and Mans, Dee, 1986, Planta Medica, 3: 245. Shin, A, Vishwakarma, Aray, Hussein, A, 1988, Planta Medica 64, 475-476.

  The main objective of the present invention is to provide a simple, quick, cost effective and practical method for the isolation of artemisinin from Artemisia annua plants without the use of chromatography. Such a method produces substantial amounts and purity of artemisinin and obviates the disadvantages of the prior art.

DETAILED DESCRIPTION OF THE INVENTION Accordingly, the present invention provides a method for preparing artemisinin from Artemisia annual. The method
(i) extracting a plant part of Artemisia annua using a non-aqueous polar solvent;
(ii) collecting the polar solvent by concentrating the extract of step (i),
(iii) adding water to the concentrated extract as described above;
(iv) adding a hydrocarbon solvent to partition the aqueous extract of step (iii) into an aqueous layer and an organic layer;
(v) separating the organic layer from the solution of step (iv);
(vi) a step of concentrating the organic layer of step (v) to obtain an oily liquid;
(vii) adding ethyl acetate to the oily liquid of step (vi),
(viii) treating the solution of step (vii) with a conventional dye absorbing material to remove green pigmentation;
(ix) filtering the solution to remove the dye absorbing material; and
(x) evaporating the solution followed by crystallization to obtain pure artemisinin.

  In one embodiment of the invention, dried leaves are collected for extraction in step (i).

  In another embodiment of the invention, the leaves are ground prior to extraction.

  In yet another embodiment of the invention, the non-aqueous polar solvent is selected from the group consisting of ethanol, methanol, acetone, methyl isobutyl and hexane.

  In yet another embodiment of the invention, the non-aqueous solvent is ethanol.

  In a further embodiment of the invention the ratio of plant parts to non-aqueous solvent is in the range of 1: 5 to 1:10.

  In another embodiment of the invention, the extraction of the plant part is achieved by leaching the herb with ethanol for 4-6 hours.

  In another embodiment of the invention, the herb leaching is achieved at a temperature of 20 ° C to 25 ° C.

  In one embodiment of the invention, in step (ii) the volume of ethanol extract is reduced from 1/20 to 1/100 of its original volume.

  In another embodiment of the invention, the solvent recovered after concentration in step (ii) is reused in step (i) of the process.

  In yet another embodiment of the invention, in step (iii) water is added 4 times the amount of concentrated ethanol extract.

  In yet another embodiment of the present invention, the hydrocarbon solvent used in step (iv) is selected from the group consisting of pentane, hexane and heptane.

  In a further embodiment of the invention, the hydrocarbon solvent used in step (iv) is hexane.

  In another embodiment of the invention, in step (iv) the aqueous ethanol extract is partitioned into hexane in a ratio of 1: 1 or 2: 1.

  In another embodiment of the invention, in step (v) the organic layer is concentrated from 1/20 to 1/100 of its original volume.

  In an embodiment of the invention, 10-20% (v / v) ethyl acetate is added to the concentrated hexane solution to induce crystallization of artemisinin from the liquid in step (vii).

  In another embodiment of the invention, the dye absorbing material is selected from the group consisting of celite, activated carbon and charcoal.

  In yet another embodiment of the invention, in step (x), artemisinin is obtained by crystallization after slow evaporation.

  The method developed in the present invention provides an improved method of isolating artemisinin from Artemisia annua plant populations. Our method involves cold extraction with ethanol / methanol, on the one hand it can rapidly extract artemisinin quickly, while the energy is compared to the usual extraction methods using hexane or petroleum ether. save. Extraction with hexane or ether was very slow and required hot leaching over several hours. Partitioning of the extract between water and hexane is economical because 60% to 70% of the extract's impurities remain in the aqueous layer and the artemisinin is completely transferred to the hexane layer, and the extract is concentrated for artemisinin. It is a sufficiently inventive method to do this. Therefore, the hexane fraction obtained can be used directly for crystallization after the decolorization step in order to obtain artemisinin without performing chromatography. Therefore, the invention provides a novel approach to the isolation of artemisinin from its natural source Artemisia annual plant population without using chromatography. The mild conditions used for extraction provide a clear plant extract and avoid possible degradation of artemisinin. As a result of the present invention, the method can reduce the recovery of artemisinin per unit biomass to approximately half the cost of the reported method and is also suitable for small scale industries.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS According to the method of the present invention, Artemisia annual ground leaves are used for 4 to 6 hours using a 5 to 10 times volume of non-aqueous solvent selected from ethanol or methanol. Extracted by continuous leaching. The extraction method may be repeated 3 to 5 times using the same solvent ratio to ensure maximum extraction of artemisinin from the herb. The resulting extract can be concentrated from 1% to 5% of the original volume by distillation under vacuum. The recovered solvent may be used again in the extraction method. Excess water (4 times the reduced volume of ethanol extract) is added to the concentrate to make it 80% aqueous, and then the contents are partitioned between water and hexane. In the partitioning, the aqueous content and hexane can be used in a ratio of 1: 1 or 2: 1 v / v. Partitioning the aqueous content with a non-polar solvent can be repeated 3 to 5 times using the same solvent ratio to ensure maximum transfer of artemisinin to the hexane fraction. The mixed hexane fractions are pooled together and then distilled under vacuum (to recover the solvent for reuse) to 1-5% of its original volume. This concentrated liquid can be a light green to dark green oily liquid. Ethyl acetate (10-20% v / v) is added to it. In order to remove green pigmentation, the liquid is treated with activated carbon, which is a 1-3% v / v dye-absorbing material. The yellow liquid obtained after removing the activated carbon (by filtration) can be subjected to evaporating crystals to obtain substantially pure artemisinin without the use of chromatography.

  As an embodiment of the present invention, extraction of dried crushed leaves with non-aqueous ethanol may have several advantages over previously used hexane or diethyl ether solvents. In addition to rapid extraction, ethanol has a low extraction amount of fatty substances that are considered an obstacle in the purification and crystallization process.

  In one embodiment of the invention, the extract is distributed between water and hexane to concentrate the extract with respect to the artemisinin and as many as possible from the concentrate to facilitate crystallization of artemisinin. Remove impurities.

  According to another embodiment of the invention, the partitioning of the ethanol extract is carried out with a proportion of water and hexane in the range (1: 1 or 2: 1), the partitioning process being carried out into the hexane layer. It can be repeated 3-5 times to ensure maximum transfer of artemisinin. The dispensing step substantially transfers the artemisinin to the hexane layer with a concomitant reduction in the amount of material. That is, only 30% to 35% by weight of the original extract is transferred to the hexane layer.

  According to yet another embodiment of the invention, the hexane fraction is subjected to crystallization by slow evaporation after the hexane fraction is concentrated and mixed with 10-20% (v / v) ethyl acetate. Can be used directly.

  In the prior art, artemisinin is obtained by hot extraction with hexane, petroleum ether, which takes time to extract. Heat extraction of plant leaves using the above solvents is dangerous, and extracts more colored substances and fatty substances, which impedes purification. On the other hand, extraction with ethanol at room temperature is relatively quick and prevents simultaneous extraction of colored substances and fatty substances. By partitioning the hexane extract between hexane and 20% aqueous acetonitrile, artemisinin is transferred to the aqueous acetonitrile layer. Water is separated from the aqueous acetonitrile layer by adding sodium chloride to saturation prior to chromatography. In the dispensing process, relatively expensive acetonitrile increases the production cost of artemisinin. Liquid: The combination of water and hexane that has been used for liquid distribution easily produces two distinct layers with larger density differences. This is also the preferred solvent system for the distribution of artemisinin for economic reasons compared to the prior art hexane and acetonitrile. As a result of the partition, only 30% to 35% of the extract remains in hexane so that the extract is concentrated for artemisinin. While artemisinin can be directly crystallized using evaporative crystallization without proceeding to chromatography, it is not possible for the acetonitrile layer consisting of post-partition artemisinin in the previously described method.

An improved method for producing artemisinin, the subject of this patent, is
1. Extraction with ethanol is quick and does not take much time.
2. Extraction saves energy because no heating is required during the process,
3. Partitioning the extract between water and hexane to obtain artemisinin is a better option compared to acetonitrile and hexane because it is economical,
4). The hexane layer obtained after partitioning can be used for artemisinin crystallization without chromatography.
5. As a whole process, reduce artemisinin production costs by 30% to 40% over the prior art.
6). By using commercial grade hexane during extraction, it is possible to avoid contamination of artemisinin with toxic chemicals,
7). There is no need to install expensive equipment to liquefy carbon dioxide,
8). Since the method does not require very large infrastructure, the extraction of artemisinin can be easily planned in rural areas,
9. As the whole process, the solvent used in the process is recovered and reused, which is efficient and economical.
Some advantages were given.

  The invention will be further described with reference to the following examples. These examples are given by way of illustration and should not be construed to limit the scope of the invention in any way.

Solvent selection for artemisinin extraction In order to select an efficient solvent for the extraction of artemisinin, different solvent systems were tested. To that end, 10 g of Artemisia Annua dry powder leaves were extracted in separate tubes using methanol, 20% aqueous methanol, ethanol, 20% aqueous ethanol or hexane as solvent. Ten ml of solvent was taken from the tubes at 2, 4 and 6 hours of incubation. The fraction was evaporated to dryness on a water bath, the extract was dissolved in 2.0 ml of solvent and spotted (5 μl) on a TLC plate with pure artemisinin dissolved in 1 mg / ml individual solvent taken as a control. Was attached. The TLC was performed in a glass TLC tank saturated with mobile phase hexane: diethyl ether (1: 1) and the plate was developed to a height of 15 cm. The plate is then dried and the spots soak the plate in a developing reagent (glacial acetic acid: sulfuric acid: anisaldehyde, 50: 1: 0.5) followed by the appearance of a pink spot of artemisinin at 110 ° C. for 15 minutes. The plate was visualized by heating. For quantification, TLC spots corresponding to artemisinin were scanned with a 540 nm and 610 nm dual wavelength TLC scanner. The results obtained are presented in the form of% artemisinin extracted into the solvent and are summarized in Table 1. It is clear from the table that pure ethanol and pure methanol extract the artemisinin more quickly than hexane, but it is likely that the artemisinin is degraded or converted to other unknown compounds after a period of time, so The concentration begins to decrease with the methanol and ethanol extracts compared to the hexane extract. Mixing water with methanol caused severe decay of the artemisinin extract. Ethanol was more efficient at extracting more artemisinin in less time than both methanol and hexane. Based on low price and rapid extraction efficiency, ethanol is a suitable solvent.

Distribution of artemisinin Pure artemisinin (0.2 g) was dissolved in different test tubes containing a 1: 1 (v / v) mixture of two immiscible solvents, hexane and aqueous ethanol (ie ethanol mixed with water). . Here, the ethanol concentration was changed from 20% to 80%. After artemisinin was completely dissolved in the mixture, the two layers of hexane and ethanol were separated using a separatory funnel. Individual layers from different tubes were tested for the presence of artemisinin content by TLC as in Example 1. The results showing the% of artemisinin fraction separated between each test tube and partitioned between the hexane layer and the aqueous ethanol layer are given in Table-2.

  This example suggests that artemisinin can be selectively transferred from Artemisia annual ethanol extract to hexane by maintaining the above ratio. From the hexane fraction, artemisinin can be crystallized directly using known evaporative crystallization methods.

  Artemisia Annua dry ground leaves (100 g) were extracted by continuous leaching for 4 hours with 600 ml ethanol at a temperature of 30 ° C. The extraction process was repeated 4 times using the same solvent ratio. The combined extracts were concentrated under vacuum to reduce its volume to 50 ml and the volume was made up to 250 ml by adding water to it. The resulting mixture was partitioned four times with 250 ml of hexane. The combined hexane layer was reduced under vacuum to 5% of its original volume (to recover hexane) to give a dark green oily liquid. This liquid was mixed with 20% v / v ethyl acetate and then treated with 1% w / v activated carbon. The liquid was filtered through Whatman 3 mm filter paper to remove the activated carbon. After filtration, the deep yellow liquid obtained was evaporated and crystallized. The obtained white needle-like crystals were separated by a vacuum filtration device and weighed. The total crystal obtained was 603 mg.

  Artemisia Annua dry ground leaves (100 g) were extracted by continuous leaching for 4 hours with 1.0 liter of ethanol at a temperature of 30 ° C. The extraction process was repeated 4 times using the same solvent ratio. The combined extracts were concentrated under vacuum to reduce its volume to 80 ml and the volume was made up to 400 ml by adding water to it. The resulting mixture was partitioned 4 times with 400 ml of hexane. The mixed hexane layer was reduced under vacuum to a volume of 50 ml to obtain a dark green oily liquid. This liquid was mixed with 20% v / v ethyl acetate and then treated with 1% w / v activated carbon. This liquid was filtered as in Example 3 and evaporated to crystallize. The obtained artemisinin crystals weighed 605 mg.

  Artemisia annual dry ground leaves (100 g) were extracted as in Example 3. The concentrated 50 ml extract was made up to 250 ml by adding water to it. The resulting mixture was partitioned five times with 125 ml hexane. The mixed hexane layer was reduced under vacuum to 50 ml of dark green oily liquid. This liquid was mixed with 20% v / v ethyl acetate and then treated with 1% w / v activated carbon. This liquid was filtered through Whatman filter paper to remove the activated carbon. After filtration, the resulting deep yellow liquid was evaporated and crystallized. The obtained white needle crystal was separated and weighed. Total artemisinin obtained was 601 mg.

  Extraction of Artemisia annual leaves was performed as described in Example 3, but in separate vials at various temperatures of 20 ° C., 30 ° C., 40 ° C. and 50 ° C. conditions. Each 50 ml concentrated extract obtained after concentration under vacuum was made up to 250 ml by adding water individually to them. The resulting mixture was partitioned three times with 250 ml of hexane. The hexane layers were reduced under vacuum to 5% of their original volume to give a dark green oily liquid. Each liquid was mixed with 20% v / v ethyl acetate and then treated with 1% w / v activated carbon. The liquid was filtered and the resulting deep yellow liquid was individually evaporated and crystallized in separate vials. The obtained artemisinin crystals were separated and weighed. Crystal yields were 410 mg, 603 mg, 600 mg and 550 mg at temperatures of 20 ° C., 30 ° C., 40 ° C. and 50 ° C., respectively.

  Artemisia Annua dry ground leaves (1.0 kg) were extracted by continuous leaching for 4 hours with 6 liters of ethanol at a temperature of 30 ° C. The extraction process was repeated 4 times using the same solvent ratio. The combined extracts were concentrated under vacuum to reduce the volume to 400 ml and the volume was made up to 2.0 liters by adding water to it. The resulting mixture was partitioned three times with 2.0 liters of hexane. The combined hexane layer was reduced under vacuum to 300 ml of dark green oily liquid (to recover hexane for reuse). This liquid was mixed with 60 ml of ethyl acetate and then treated with 1% w / v activated carbon (to remove green pigment deposits). The deep yellow liquid obtained after filtration was evaporated and crystallized. Artemisinin crystals were separated and weighed. A total of 5.95 g of artemisinin crystals were obtained.

  Artemisia annual dry ground leaves (100 g) were extracted by continuous leaching for 4 hours with 600 ml of methanol at a temperature of 30 ° C. The extraction process was repeated 4 times using the same solvent ratio. The combined extracts were concentrated under vacuum, the volume was reduced to 50 ml and the volume was made up to 250 ml by adding water to it. The resulting mixture was partitioned three times with 250 ml of hexane. The mixed hexane layer was reduced to 5% of its original volume to give a dark green oily liquid. This liquid was mixed with 20% v / v ethyl acetate and then treated with 1% w / v activated carbon. The liquid was filtered using Whatman filter paper to remove the activated carbon. After filtration, the resulting deep yellow liquid was evaporated and crystallized. As a result, white needle-like crystals were separated by a vacuum filter and weighed. The obtained artemisinin crystal was 320 mg.

Claims (16)

A method for preparing artemisinin from Artemisia annua, comprising:
(I) a step of cold-extracting Artemisia annua plant parts at a temperature of 20 ° C to 50 ° C using a non-aqueous polar solvent selected from methanol and ethanol
(Ii) concentrating the extract of step (i) to recover the polar solvent;
(Iii) adding water to the concentrated extract to obtain an 80% aqueous extract;
(Iv) adding a hydrocarbon solvent to partition the 80% aqueous extract of step (iii) into an aqueous layer and an organic layer;
(V) separating the organic layer from the solution of step (iv);
(Vi) concentrating the organic layer of step (v) to obtain an oily liquid;
(Vii) adding ethyl acetate to the oily liquid of step (vi),
(Viii) treating the solution of step (vii) with a conventional dye-absorbing substance to remove green pigmentation;
(Ix) filtering the solution to remove the dye absorbing material, and (x) evaporating the solution followed by crystallization to obtain pure artemisinin;
Including methods.   The method according to claim 1, wherein the dried leaves are used for extraction in step (i).   The method of claim 2, wherein the leaves are powdered prior to extraction. The method of claim 1, wherein the non-aqueous polar solvent is ethanol. The method of claim 1, wherein the ratio of plant parts to non-aqueous polar solvent is in the range of 0.1 to 0.2 (g / ml) .   The method according to claim 1, wherein the extraction of the plant part is performed by leaching the herb with ethanol for 4 to 6 hours.   The method of claim 1, wherein in step (ii) the volume of the polar extract is reduced from 1/20 to 1/100 of its original volume.   The method according to claim 1, wherein in step (ii), the solvent recovered after concentration is reused in step (i) of the method. The process according to claim 5 , wherein in step (iii) water is added four times the amount of concentrated ethanol extract.   The process according to claim 1, wherein in step (iv) the hydrocarbon solvent used is selected from the group consisting of pentane, hexane and heptane.   The process according to claim 1, wherein in step (iv) the hydrocarbon solvent used is hexane. 6. The method of claim 5 , wherein in step (iv), hexane is added to the aqueous ethanol extract , thereby partitioning the aqueous and hexane layers in a 1: 1 or 2: 1 ratio.   The process according to claim 1, wherein in step (v) the organic layer is concentrated to 1/20 to 1/100 of its original volume. In step (vii), to induce crystallization of artemisinin from the liquid, 10~20% (v / v) ethyl acetate is intended to be added to the hexane solution is the concentrate of claim 5, wherein Method.   The method of claim 1, wherein the dye-absorbing material is selected from the group consisting of celite, activated carbon and charcoal.   The process according to claim 1, wherein in step (x) the artemisinin is obtained by crystallization following slow evaporation.