FORM 2
THE PATENTS ACT 1970
j (39 of 1970)
&
Thej Patent Rules 2005
PROVISIONAL SPECIFICATION
(see sections 10 & rule 13)
1. TITLE OF THE INVENTION

"PROCESS FOR ISOLATION AND PURIFICATION OF ARTEMISININ AND OTHER
CONSTITUENTS FROM ARTEMISIA PLANT" 2. APPLICANT (S)
NAME NATIONALITY 'ADDRESS

Almet Corporation Limited INDIAN

PREAMBLE TO THE DESCRIPTION

332. Adhyaru Industrial Estate, Sun Mill Compound, Lower Parel. Mumbai- 400 013.

PROVISIONAL SPECIFICATION
The following specification describes the invention.

FIELD OF INVENTION
The present disclosure relates to a process for isolation and purification of artemisinin from Artemisia plant particularly from Arlemi.sia annua. The present disclosure also relates to isolation of other constituents from the Artemisia plant.
BACKGROUND OF THE INVENTION
Artemisinin is a sesquiterpene lactone endoperoxide which is present in the aerial parts of the plant Artemisia annua (Klayman, DL, et al, J. of Nat. Products, 1984, 47: 715-717). Artemisia annua L (Asleraceae) is a herb of Asiatic origins and has been traditionally grown in China as a medicinal and, more recently in Europe, for its aromatic leaves which are used in flavoring beverages, and that has been also naturalized in India. This species is receiving considerable attention because of the antimalarial activity of artemisinin.
Efforts are being made to make artemisinin derived drugs more cost effective and available worldwide. The chemical synthesis of artemisinin is not commercially viable: therefore the plant Artemisia annua remains the sole source of artemisinin.
Artemisia annua is also valued for its essential oil which has a characteristic sweet aroma and has applications in perfumery, cosmetics, aromatherapy and also as an antimicrobial, dcrmatological and fungicidal agent.
Artemisinin extraction and purification method reported by Klayman DL et al. (J. of Nat. Products, 1984, 47: 715-717) and Rucker G et al. {Planta Medica, 1986, 3:245) involves extraction with petroleum ether followed by chromatographic purification of the relatively crude extract on silica gel. The column is eluted with a mixture of 7.5% ethyl acetate in chloroform to obtain pure artemisinin.
Singh A ct al. {Planta Medica. 1988. 64:475-476) reported extraction of Artemisinin from plant parts with n-hexanc follovvcd by chromatography on
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silica gel column. In this case elution is performed with the mixture of ethyl acetate and hexane.
Both these methods utilized purification of crude plant extract using chromatography on silica gel which is not amenable to scale-up and employs large volumes of solvent which is not economical on production scale.
US Pat. No. 4.952.603 discloses a method for extraction and purification of artemisinin that involves the extraction with hot hexane, followed by partitioning the extract between hexane and acetonitrile-water (20%), removing the water by adding sodium chloride, and evaporating acetonitrile phase to oily residue. Oily residue obtained is subjected to chromatography on silica gel with hexane-ethyl acetate. The eluted oil liquid fraction after rccrystallization gives artemisinin.
Canadian Pat. No. CN 1092073A, discloses the process for isolation of artemisinin which involves leaching of artemisinin from the leaves of Artemisia annua with aqueous ethanol (<70% concentration) followed by extracting the leaching liquid with gasoline containing 30% of benzene or ethyl acetate in a continuous extracting device. Gasoline extract is then treated with activated carbon to decolorize followed by concentrating and crystallizing the artemisinin using ethanol.
US Pat. No. 5.955.084 discloses a process for simultaneous production of artemisinin and essential oil from the plant Artemisia annua. This process involves extraction of artemisinin from the plant material with hexane, followed by partitioning the extract between hexane and acetonitrile-water mixture. Water was removed from acetonitrile followed by extraction in hexane-benzene mixture and evaporating acetonitrile phase. This was followed by chromatographic separation on silica gel to obtain artemisinin. The disclosure further provides the method of extraction of essential oil from the residual 'marc' by hydro-distillation and describes a method for isolation of artemisinic acid and its conversion to artemisinin using photo-oxidation.
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US Pat. No. 6.180,105 discloses a method that involves supercritical extraction of artemisinin from Artemisia annua using liquid carbon dioxide and allowing the carbon dioxide to evaporate from the resultant mixture.
US Pat No. 6.685.972 discloses a method for extraction and purification of artemisinin that involves extraction of herbage of Artemisia annua in ethanol. and extracting the ethanol extract with hexanc. The hexane layer is subjected to activated charcoal treatment to yield dark green oily liquid and finally recrystalli/ation of artemisinin in solvents.
None of the prior art discloses the, direct adsorptive chromatographic purification of artemisinin from non-polar solvent extract of Artemisia plant to recover artemisinin free from oil and chlorophyll. Also none of the prior art discloses the crystallization of artemisinin from acetonitrile-water mixture. Further, no disclosure has been found in prior art related to overall recovery of artemisinin from the actual content in the plant, removal of related products/impurities from artemisinin, and purity of recovered artemisinin. Most of the processes reported in prior art are tedious and involve water removal before chromatographic step on silica gel. None of the processes involve chromatographic step on reusable polymeric adsorbents having high adsorption capacity and resolution efficiency, aimed at increased efficiency of overall process, and giving artemisinin in high yield and purity.
Therefore, development of a new process for industrial production of substantially pure artemisinin in high yield is desirable, further, there is also a need to obtain other useful components of the Artemisia plant extract namely, aretmisnic acid, essential oil, and scopoletin, which if accomplished will increase the overall economy of the process.
Applicants co-pending application No /MUM/2007 discloses the novel
apparatus used for the extraction purpose.
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DESCRIPTION OF THE INVENTION
Accordingly, the prescnl disclosure relates to a process for isolation of artcmisinin. other constituents such as artemisinic acid, scopoletin, and essential oil from Artemisia plant particularly from Artemisia annua. The present disclosure further provides a process for the purification of artcmisinin in high yield and purity, as also the recovery of purified artemisinic acid, essential oil and scopoletin.
The process of isolation of artcmisinin. artemisinic acid, scopoletin and essential oil from Artemisia plant comprises extracting the fresh, semi-dried or dried plant, or any part thereof, of Artemisia annua, preferably the leaves, with a non-polar organic solvent in an extracting device (a Solid-Liquid Lxtractor) wherein the solvent is passed through the column packed with the said plant portion in any suitable form and si/.e. thereby producing the plant extract in a more efficient manner than in any other device such as stirred tank or belt extractors. The plant extract thus produced is used as such, or in a concentrated form, for loading onto a chromatographic column packed with suitable polymeric adsorbent resin, followed by washing the adsorbent resin appropriately to remove unbound and bound impurities in stepwise manner, or all at once, including selective desorption of the essential oil. and then selective desorption of adsorbed artcmisinin, artemisinic acid and scopoletin from adsorbent resin followed finally by regeneration of adsorbent resin for reuse in the next cycle.
1-lutcd artcmisinin is then extracted with solvent such as acetonilrile, ethanol. methanol, isopropanol and mixture thereof or their mixture with water in any suitable proportion, and loaded, as such and/or after concentration to remove solvent and/or appropriate dilution with water, on to a second chromatographic column packed with the same or another polymeric adsorbent resin, followed by washing the adsorbent resin appropriately to remove unbound and/or partially bound impurities, and then selective desorption of adsorbed artemisinin from
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adsorbent resin followed by regeneration of adsorbent resin for reuse in the next cycle.
Alternatively, the plant extract is subjected to liquid - liquid extraction using a polar solvent like acctonitrile ethanol. methanol, isopropanol and mixture thereof with water or without water, whereby the artemisinin is extracted into the polar phase and used with or without distillation for loading onto the second
chromatographic column described above.
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i Artemisinin thus obtained from the eluted or desorbed fraction from the second
chromatographic column was precipitated or crystallized from polar organic
solvent mixture giving more than 98% purity.
In the first part of the disclosed process, the crude extraction step is designed as a novel process to result in maximum \icld of artemisinin in the organic solvent extract from the plant "herbage', and comprises packing the herbage in a column, and passing the extracting solvent through it at an optimum flow rate over a period of time so as to extract all available artemisinin from the herbage. The packed column extraction system is a novel method that provides a far more efficient extraction of herbage at large scale and is equivalent of contacting the herbage many times over if conducted in the traditional batch manner.
The chromatographic purification part of the disclosed process is carried out using adsorbent resins comprising copolymers of divinylbenzene and styrene and/or mcthacrylalc, polymethacrylatc copolymers or polyacrylamide copolymers, natural polymer or modified silica.
The interacting group of the adsorbent matrix may be the part of base matrix, or may be grafted on the matrix by the known activation chemistry, to give hydrophobic, hydrophilic polar and/or ionic characteristics to the adsorbent matrix or resin.
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The adsorption resins used in the two chromatographic steps mentioned above can be identical or different: if different resins arc employed, the difference may reside for example in the particle si/.c. pore si/.e. surface area, and/or the polarity of the surface or solubility index. The optimum rcsin is selected depending on the type of the medium and the characteristics of the impurities present.
The selected operating conditions for chromatographic separations are operating temperature, loading, wash and edition volumes, mobile phase flow rates, composition of mobile phases, and the optimum contact time with the resin. The operating conditions in a step vary depending on the properties of the mobile phase medium and its composition; and therefore, the preferred and suitable conditions are determined experimentally.
The term "related impurities'" means the impurities present in plant leaves and extracted in a non-polar or non-aqueous organic polar solvent, or generated during processing before a said first or second chromatographic step, and may be structurally related to the desired active pharmaceutical ingredient namely here, artcmisinin.
Selective separation of artcmisinin from the first of the two chromatographic columns, results in a partially purified artcmisinin, which is essentially free from the essential oil and chlorophylls. In the experiments the presence of artemisinin and other impurities can be determined and estimated by high performance liquid chromatography (HPLC).
According to one of the embodiments of the present disclosure, the plant component containing artemisinin is extracted in a packed solid extractor wherein the wet. dry or semi-dry herbage is packed and extracted in a upflowing extractant solvent, comprising a mixture of one or more non-polar organic solvents like pentanc. hcxanc. heptane or other hydrocarbon solvents, alone or in mixture with polar solvents like methanol, ethanol or other alcohols, esters or ketones: with extraction being performed at temperatures between 10 to 50°C
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In another embodiment, the adsorption is mostly performed with a packed bed chromatographic column which comprises filling the column with a suitably selected adsorbent, and passing artemisinin extract in an organic solvent through the column pre-equilibrated in the same or different solvent with or without water.
In still another embodiment, the adsorption can be performed in batch contactors, or other variants of column contactors including those from fluidised bed, expanded bed. spouted bed. circulating fluidised bed or other types of moving bed systems, including simulated moving bed systems.
In yet another embodiment, suitable amount of the adsorbent is filled into the column. The feed is pumped into the column at a temperature of 10 to 50°C, preferably 20 to 40°C, and at the rate of 10 to lOOOcm/hr linear flow velocity, preferably 75 to 500 cm/hr linear flow velocity, and the flow through contains essential oil and clution fraction corresponding to the partially purified artemisinin is collected. The adsorbed resin column is washed with mobile phase of same or different composition as that of equilibration mobile phase followed by clution to selectively isolate artemisinin from related impurities, chlorophyll and essential oil.
In one another embodiment, the artemisinin is isolated from impurities in the first chromatographic column not only by a constant mobile phase composition but also by the changing mobile phase composition in an appropriate stepwise or linear gradient of 0.5 to 20 column volumes, preferably 1.0 to 15.0 column volumes, and most preferably 15 to 10 column volumes. The mobile phase preferably contains organic modifiers such as but not limited to methanol, ethanol, isopropanol, acetonitrile, chlorinated organic solvents, toluene, benzene, hexane, petroleum ether, heptane, butyl acetate, ethyl acetate, acetone, and any suitable combination of one or more than one thereof.
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In another embodiment, the extract from the packed bed plant / herbage extractor is directly extracted in a liquid liquid extractor using a polar solvent like acctonitrile in mixture with water or without water, whereby the artemisinin is extracted into the polar phase and used with or without distillation for loading onto the second chromatographic column described below.
In another embodiment, the second chromatographic column mobile phase used may contain water, acctonitrile. acetone, ethyl acetate, methanol, ethanol, isopropanol. letrahydrofuran, 1.4 dioxane, dimethyl formamide and any suitable combination of one or more than one thereof. The concentration of aqueous to organic is 0.001 %v/v to 100 %v/v depending upon the type of polarity of the solvent selected.
In another embodiment, the mobile phase may contain l%v/v to 100%v/v the water. If, water is used in the mobile phases then the mobile phases used in the elution chromatography are miscible to each other.
In the process, the regenerants can be properly selected depending upon the types and amounts of impurities, and the type of the adsorbent resin. For example, alcohols such as methanol, ethanol. isopropanol. acetone and acctonitrile can be used. Other organic solvents are selected from the group of solvents mentioned above in a suitable concentration.
In another embodiment, active ingredient obtained either by the first process and /or alternative second process can be obtained in crystalline form directly from the elute portion i.e. elute containing polar organic solvent by subjecting it to solvent removal whereby product starts crystallizing.
The said invention gave an active ingredient in more than 95% yield and having purity -98% as analyzed by RP-HPLC. The specific characteristics and useful advantages of the present invention are:
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1. Extraction of active ingredient from plant material in compacted upflow solid-liquid extractor so as to make operation less cumbersome compared to batch extraction.
2. Extraction of active ingredient in non-polar organic solvent followed by direct loading of this extract and/or after concentration without any liquid-liquid extraction on regenerablc/rcusable adsorbent resin.
3. Extraction of elule material from first chromatographic column with polar organic solvent along with water and loading of the extract without any water removal on second chromatographic column containing regenerablc/rcusable adsorbent resin.
4. The adsorbent resin used in first and second column are reusable on account of suitable in place regeneration procedure involving passage of suitable rcgencrant media/mobile phase through the chromatographic column/s, the regenerant media/mobile phase can be, but not limited to polar, non-polar organic solvents, and their mixtures with or without water at neutral, alkaline or acidic pH condition.
5. Crystallization of the product from elute fraction of second chromatographic column by solvent removal thereby crystalli/ation/prccipitation of active product from aqueous mixture instead of extracting it in non-polar solvents for crystallization.
6. Overall yield of more than 95% and purity of more than 98% in minimum number of steps.
7. The process of the present invention is also useful for the isolation and purification of other valuable products such as artemisinic acid, scopoletin. and coumarins from Artemisia plant
8. The process of the present invention can be used for isolation and purification of said producl/s from improved verities made through
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genetic manipulation, hybridization, transgenic techniques, and expression into other plant/s by methods known to those skilled in art.
In one embodiment of the present invention, dried herb of Artemisia annua (5 Kg) was packed in compacted upflow solid liquid extractor and extracted by passing 150 liters of hcxanc at 12 lit/hr flow rate. The extract so obtained was concentrated 10 times under vacuum at temperature not exceeding 40 degree Celsius. The concentrated hexane extract was passed through a packed chromatographic column containing 1.5 liter of DIAION HP2MG (Mitsubishi chemical corporation, Japan) adsorbent in down-flow mode at 150 cm/hr flow velocity. I'lowthrough fraction of this column was taken for essential oil recovery, further. 10 bed volume of hcxanc was passed through the column and fractions containing arlcmisinin was collected. The said fractions containing arlcmisinin was concentrated to 1/10Ih volume and extracted three volumes of aqueous acetonitrile (50:50 acetonitrile: water). The combined extract was concentrated and loaded to second chromatographic column containing 0.8 liter of SHPABEADS SP700 (Mitsubishi chemical corporation, Japan) adsorbent matrix. The said column was then washed with 45 %(v/v) aqueous acetonitrile and adsorbed artemisinin was selectively eluted with 50 %(v/v) aqueous acetonitrile. The clucnt so obtained was processed to obtain white 4.2 gm of crystalline artemisinin by evaporative crystallization. Overall yield of process was 97.7% and purity of artemisinin recovered was 99.56% by 11PLC.
In another embodiment, dried leaves of Artemisia annua (lOKg) containing 26.6 gm as artemisinin was packed in upflow solid liquid extractor and extracted by passing 250 liters of hexane at 12 lit/hr flow rate. The extract obtained was concentrated 5 times under vacuum at temperature not exceeding 40 degree Celsius. It was then extracted with 3 volumes of aqueous acetonitrile (1:1 ratio) followed by concentration of aqueous acetonitrile phase to half the volume. This concentrate was then loaded on 1.5 liter SHPABEADS SP700 (Mitsubishi chemical corporation. Japan) adsorbent matrix. Polar and related impurities was
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removed by washing with 45 %(v/v) aqueous acetonitrile and bound artemisinin was selectively eluted with 48 %(v/v) aqueous acetonitrile. The eluted aqueous acetonitrile fractions containing artemisinin was evaporated to yield 25.5 gm white needle shaped crystalline artemisinin accounting about 95.9% recovery with respect to content in dried leaves. Purity of recovered artemisinin was found to be about 99.4% by HPI.C assay.
In yet another embodiment, the raffinate obtained after extraction with acetonitrile (1:1 ratio) was re-extracted with pure acetonitrile and the extract was diluted with water till 1:1 ratio of acetonitrile and water. The said diluted extract was loaded on 1.0 liter of SF.PABKADS SP207 (Mitsubishi chemical corporalion. Japan) adsorbent matrix. The adsorbent column was washed with 50% (v/v) acetonitrile. Bound artemisinic acid and scopoletin was selectively and sequentially eluted with increasing concentration of acetonitrile. The eluted fractions were evaporated at 45 0C under vacuum to obtain substantially pure artemisinic acid (68.2 gm) and scopoletin (32.4 gm).
Tabic 1 shows results obtained on the solid-liquid extractor for the extraction of artemisinin from Artemisia annua leaves.
Table 1: Summary of the parameters and results of artemisinin extraction using
Compacted Upflow Solid Liquid Extractor (CUSLE)
Parameter Result
Artemisinin concentration in solid feed (% w/w) 0.26
Extractant flow rate (ml/min) 150
Quantity of solids (herbage) (Kg) 10
Total solvent volume used (Tit) 200
Artemisinin in extract (gm) 25.2
Artemisinin recovered (%) 96.9

Dated Ml day of April 2007

T.Srinivasan Agent for the Applicant

To
The Controller of Patents.
Patent Office at Mumbai

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