Production Technologies of Pharmacologically Active Sesquiterpene Lactones

Sesquiterpene lactones form a large group belonging to natural terpenoids series, generally found in plants of Asteraceae family and exhibiting anti-tumor, antiviral, immunostimulant, antifungal, antimicrobial, anti-inflammatory, antimutagenic, growth stimulating, antifeedant effects. Therefore, search for new compounds with a broad spectrum of pharmacological activity in this series provides the opportunities for effective and conceptually new drug design. The basis of the technology for the isolation of sesquiterpene lactones is the extraction of raw materials with various organic solvents, followed by chromatographic purification. Sesquiterpene lactones have no common properties that can be used in their isolation. Some of them are well soluble in non – polar solvents, others-only in polar, in this regard, the methods of isolation of sesquiterpene lactones are diverse. The greatest number of sesquiterpene lactones is isolated from leaves and flowers, slightly less – from roots and bark. Therefore, the development of methods for their isolation is associated with the selection of solvents and optimization of the extraction mode. Unfortunately, very few medicines based on sesquiterpene lactones are produced by the pharmaceutical companies today. Complexity of introduction of pharmacologically active sesquiterpene lactones technology into pharmaceutical production is in imperfection of their isolation methods from plant raw material, their purification and separation from obtained extracts. Production technologies of the patented medicines "Santonin", "Alanton" on the basis of sesquiterpene lactones are multiphase, labor-intensive, implying the use of many toxic organic solvents which is against the international GMP standards.


Introduction
The first sesquiterpene γ-lactone used in medical practice as original antihelminthic drug was α-santonin (1). The organization of pharmaceutical plant in the Southern Kazakhstan was caused firstly by source of santonin raw materials (1), namely the availability of industrial stocks of Artemisia cina Berg.
During the first stage anthodia of Artemisia cina Berg. are soaked in water and mixed with the lime containing at least 60% of calcium oxide. α-Santonin is dissolved in alkalis with the lactone ring opening and forms a salt of santonic acid.
During the second stage a sevenfold alkaline washing of santonic acid calcium salt by water occurs. The content of α-santonin in the extract varies from 0.7 to 1.4%.
During the third stage after completion of extraction and discharge of the extract into an extractor, sharp steam is applied and essential oil is distilled with water vapor. The received oil is settled and dried up over sodium sulfate. Once the distillation of essential oil is over, the extractor is The crushed and air dry raw materials of Artemisia taurica Willd. are extracted with hot water at a temperature of 75-80 °С within 30 min. Then water extraction is treated with chloroform, then poured together and evaporated. Sulfuric ether is added to the received thick dark brown crystallizing substance till the setting of settlement stopped. The settlement was dissolved in chloroform, twice washed with 5% Na 2 CO 3 *10H 2 O solution to separate resinous substances of an acid nature, and twice with water. Chloroform is distilled, the res- Production of "Alanton" substance from the dry crushed roots and rhizomes of Inula helenium L. proceeds the following way [3]: • extraction is carried out by percolation with 85% ethyl alcohol (with the ratio 1:10 of raw material to extracting agent); • the received extract is evaporated under vacuum, then pour together the water residues. Terpenoid fraction is extracted from the water residue by methylene chloride. The methylene chloride extracts are discharged, poured together, dehydratedby the calcined sodium sulfate within 5-6 h, filtered and evaporated to receive 1/4 from the initial volume; • the obtained solution is purified by column chromatography on aluminum oxide. Elution is done by methylene chloride, then the received eluate is evaporated till the solvent is fully removed. The residue is a thick dark yellow substance; • as the final stage of Alanton production,10-fold of ethyl alcohol (96%) is added to the residue, mixed, cooled up to 0-5 °C, and allowed to remain for 24 h till the full sedimentation. Then it is unloaded. The yield of α-santonin during the extraction stage is 95%.
During the fourth stage the concentrated extract, which contains santonic acid calcium salt, pitch, and other extractives, is acidified by nitric acid. Thus, calcium nitrate and santonic acid are formed, latter of which gradually transforms into α-santonin. Raw α-santoninis tenfold washed out with water to receive a neutral reaction, then centrifuged, transferred to a drying cabinet, and dried at a temperature of 66-68 °C. The yield at this stage is about 80%.
The fifth production stage implies purification of technical α-santonin with the repeated crystallization from ethyl alcohol and further treatment on a custom designed filtering apparatus. After crystallization of α-santonin is centrifuged and washed out with the distilled water. The yield of pure α-santonin at this stage is 80-84%. Total yield of α-santonin depends on the content of target compound in the source raw materials and on the number of performed recrystallizations.
Thus, production technology of sesquiterpene lactone α-santonin is labor-intensive, using a multiple process for isolation. Due to the creation of comparative effective antihelminthic drugs α-santonin was withdrawn from production.
idue is dissolved in alcohol, activated charcoal is added, and boiled within 5 min, then filtered. After cooling the crystals are sucked away and repeatedly recrystallized from alcohol. Tauremisin yield is 0.2% in calculation for air-dry raw materials.
However, use of toxic solvents such as chloroform, benzene is against GMP standards. filtered and washed out by the quadruple amount of gasoline cooled up to 0-5 °C on the filter. The settlement is being dried during 10-12 h. Dried alanton is crushed in a ball mill, sifted and packed up. Alanton yield is 1.3% in terms of air dry raw materials. The content of sesquiterpene lactones in the drug should be not less than 95%. The disadvantages of this production method are its time consumption and low yield of a target product.
Babayev N.F. and Serkerov S.V. [6] offered an alternative method of Alanton substance production. The production technology to receive the sum of sesquiterpene lactones alantolactone (3) and isoalantolactone (4) from plant raw materials according to this method is carried out by 2-fold extraction from roots and rhizomes of Inula helenium L. by mixture of ethanol and hexane (1:4) when boiling with the backflow condenser, the weight ratio of roots and rhizomes with extracting agent is1:5 for 3 h. The obtained extracts are poured together, filtered from mechanical impurities, in doing so they precipitate out. The maximum amount of sesquiterpene lactones crystals is formed within 24 h at room temperature. They are yellowish crystals of the purified sum of sesquiterpene lactones alantolactone (3) and isoalantolactone (4) with a quantitative ratio 2:1 and amelting point of 86-88 °C. When using this method, isolation time of lactones is reduced from 192 to 58 h, whereas the yield increases up to 1.7-1.9%. Plekhanova N.V. et al. [4] suggested a production method of alantolactone from the roots of Inula macrophylla Kar. et Kir. ( = I. grandis Schrenk) which includes the following stages: • extraction by acetone within 4 h at room temperature (at the ratio 1:5 of raw material to extractant); • evaporation of extract to receive a thick mass; • treatment of the received thick extract with a mixture of petroleum ether: benzene (7:2.8) at a ratio of the sum of extractives and mixture (9:1). Solvents mixture is distilled up to 40% of the volume, and crystals are separated. Alantolactone yield from Inula macrophylla Kar. et Kir. is 1.12%.
Thus, the proposed production method of alantolactone (3) from Inula macrophylla Kar. et Kir. has a number of advantages, for example: considerable simplification of the process and short cutting some operations (water vapor distillation, using hot acid and alkali).
The disadvantage of the proposed method is the use of flammable solvents of acetone and petroleum ether, as well as toxic benzene.
The production technology of alantolactone reference sample (3) has been developed which includes the following stages: extraction of essential oil from rhizomes and roots of Inula helenium L. by distillation with water vapor with simultaneous extraction in chloroform; isolation of the sum of sesquiterpene lactones by column chromatography on silica gel (eluent-mixture petroleum ether-ethyl acetate 9:1) [8].
Trenafilova A. et al. [7] conducted chemical studies of the roots of Inula helenium L. using the ultrasonic extraction. Based on the results of these experiments, the optimal parameters for a quantitative yield of alantolactone (3) and isoalantolactone were determined (4). 70% Ethyl alcohol was used as an extractant, ultrasonic treatment was done within 30 min at extraction temperature 25 °С. As a result, alantolactone (3) and isoalantolactone (4) were isolated from Inula helenium L. extract with the yield 18.04 and 12.77 mg/g, respectively.
In Kharkiv experimental plant GNCLS (Ukraine) technology of production of "Alanton" includes extraction of raw roots and rhizomes of Inula helenium L. by gasoline and infused with occasional stirring for 24 h. Then the gasoline extract (the first sink) is pumped into the container, and the raw material is re-filled with fresh solvent and insisted again, then the extract (the second sink) is combined with the previous one. This extraction operation is carried out three times, as a rule. Then, the solvent is distilled off, and the remaining evaporated extract (cube concentrate) is dissolved in a 4.5-fold volume of 95% alcohol, treated with activated carbon, and filtered. After filtering, the socalled alcoholic mother liquor is evaporated to half volume and the Alanton crystallizes at a temperature of 8-10 °C. The product is air dried and then washed with cold gasoline to remove yellow impurities, and a white or white crystalline substance with a yellowish hue [9] is obtained.
Summarizing the literature data on the methods for the isolation of alantolactone and isoalantholactone from the roots of Inula helenium L., it can be noted that the preferential method is an alcohol extraction using ultrasonic treatment, which makes it possible to avoid using costly organic solvents.
One of the medicines widely studied in a number of world scientific centers is an antimalarial "Qinghaosu" drug developed by the Chinese scientists based on sesquiterpene lactone artemisinin (5) which had been isolated from Artemisia annua L. [10]. Martinez-Correa H.A. et al. [11] carried out extraction from the aerial parts of Artemisia annua L. using two methods. In the first case, various solvents were used: carbon dioxide CO 2 (40 MPa/60 °C), ethanol (25 °C) and water (60 °C). In the second case, two-phase extraction was carried out as is shown: at first carbon dioxide CO 2 extraction (40 MPa, 60 °C), and then extracted with the use of ethanol at 25 °C or water at 60 °C (second step) under atmospheric pressure (Scheme). Ethanol extracts were received the following way: 3 g of raw materials (thick extract after SFE) were dissolved in 10 ml ethanol at a temperature of 25 °C within 42 h and mixed in a shaker. Then the mixture was filtered and repeatedly extracted in 10 ml ethanol on the centrifuge at gravitational acceleration 2543, 25 °C within 5 min. Water extracts were received the following way: 3 g of raw materials (thick extract after SFE) dissolved in 60 ml of water. Mixture was stirred within 10 min at 60 °C, then on the centrifuge within 10 min at centrifuge at gravitational acceleration 10174. After vacuum filtration they received the extract. For supercritical process, 7 g of raw materials were used; extraction was carried out with the following parameters at 40 MPa, 60 °C and 4 × 10 -5 kg/s of CO 2 . Two fractions of supercritical extract were received: extract fraction and a non-polar fraction. The content of artemisinin in extracts and raw materials of Artemisia annua L. is given in Table 1.
It is clear from the data provided in Table 1 that the yield of artemisinin is comparable in the received CO 2-, and ethanol extracts.
Research of Martinez-Correa et al. [11] provides comparative data on extraction of artemisinin from Artemisia annua L. using the following methods: maceration, ultrasonic, supercritical, and microwave extraction by carbon dioxide (   * -The content of artemisinin was defined according to HPLC analysis. As it`s clear from Table 2, comparative quantitative yield of artemisinin is observed at extraction by hexane (in Soxhlet apparatus) and liquid carbon dioxide (CO 2 ).
Authors [18] proved that ultrasonic extraction from Artemisia annua L. raw materials by hexane at the following parameters: 40 kHz, 25 °C, 60 min, allowed them to receive a higher yield (by 58% higher, according to HPLC analysis) of artemisinin (5) in contrast with the usual soaking under the same conditions. It should be noted that if we increase time up to 120 min, there will be a decrease of artemisinin content.
Authors of the work [20] developed a method for the production of artemisinin (5) without column chromatography, for this purpose ultrasonic extraction of raw Artemisia annua L. with ethyl alcohol was performed. Extraction was carried out on an ultrasonic extractor from China Ningbo Zhenguo Pharmaceutical Equipment Manufactur-ing Co. model TCLX200, by the following technology: dry, crushed to the size of 60-80 mesh, the raw material is placed in an ultrasonic extractor, filled with 80% ethyl alcohol (hydromodule 1:17) and extracted at a frequency of 35 kHz, power at 1000 W, temperature at 40 °C for 30 min. The obtained extract was filtered, petroleum ether was added to the filtrate in a 5:1 ratio and treated at a radiation frequency of 30 kHz, a power of 1000 W, a temperature of 25 °C for 15 min. The ether layer was separated, passed through a column filled with activated carbon and concentrated under reduced pressure, the concentrate was poured into a crystallizer. The fallen artemisinin crystals are recrystallized from 80% ethyl alcohol. The degree of extraction of artemisinin from the raw material by this method is 97.25%.
Various methods for isolating artemisinin from Artemisia annua L. are given in the available patent and scientific and technical literature.
Number of companies [21][22][23][24] developed several methods for extracting artemisinin from raw materials using organic solvents, such as: hexane, mixtures of ethyl acetate: No. 6 Extraction Solvent Oil and petroleum ether: gasoline under vacuum, and other companies of China, Italy, and researchers from Germany [25][26][27][28][29][30][31] used as an extragent: butane, СО 2 -gas, supercritical carbon dioxide and water, supercritical carbon dioxide in combination with microwave extraction, and also supercritical carbon dioxide with a modifier (ethyl alcohol). Scientists from Yunnan Normal University proposed a method for obtaining artemisinin without extraction [32]. Sanofi (France) has developed a longer-term method for producing artemisinin in a semisynthetic way from dihydroartemisinic acid [33].
Thus, different effective methods are used for production of artemisinin from Artemisia annua L., in particular, the use of extraction by supercritical carbon dioxide combined with microwave treatment of 70% ethanol, and without the use of column chromatography of the target substance with supercritical carbon dioxide, followed by recrystallization from hot 75% ethanol.
Based on a sesquiterpene lactone arglabin (6), the original drug Arglabin was developed in the International Research and Production Holding "Phytochemistry" and is currently produced at Karaganda Pharmaceutical Plant. The production method of Arglabin was patented in 11 countries of the world, namely Japan, China, the USA, Great Britain, Germany, Switzerland, France, Austria, Italy, the Netherlands and Sweden [34].
In IRPH "Phytochemistry" an effective and environmentally-friendly technology of isolation and purification of the sesquiterpene lactone arglabin from Artemisia glabella Kar. et Kir was developed according to GMP requirements. It was experimentally determined that isolation technique of arglabin from carbon dioxide extract of Artemisia glabella Kar. et Kir. using a centrifugal partitioning chromatography is optimal for the preparative production and deployment in the manufacture of substance on its basis.
At the same time, the optimal parameters of the extraction of Artemisia glabella Kar. et Kir. raw material were determined using СО 2 -gas in the supercritical state, providing a quantitative yield of arglabin (6).
The use of supercritical carbon dioxide extraction from Artemisia glabella Kar. et Kir. herb for extraction of arglabin has significant advantages in comparison with chloroform extraction (Table 3).
To increase the productivity, automatize, reduce the process duration, and exclude toxic solvents, a production technology of arglabin native substance has been developed (6) which involves the centrifugal partitioning chromatography [35].
Initially, arglabin was isolated from CO 2 -extract of Artemisia glabella Kar. et Kir. with a centrifugal partitioning chromatography in two stages: 1. Purification using accelerated centrifugal chromatography of FCPC-5000 distribution.
2. Recrystallization of technical arglabin. It has been experimentally determined that the method of arglabin extraction from СО 2 extract of Artemisia glabella Kar. et Kir. with the use of centrifugal chromatography of the distribution, which characterized by better productivity, full automation, shorter duration of the process in comparison with column chromatography. This method does not require the use of sorbents and high-purity solvents. Thus, the developed technology made it possible to ensure the yield of arglabin from the CO 2 extract to 30% and more than 2% of the plant material with a purity of the target substance of at least 99.0%.
The developed technology of isolation and purification of sesquiterpene lactone arglabin is introduced by the Karaganda pharmaceutical plant for the production of the original drug "Arglabin".
Thapsigargin (7) is sesquiterpene lactone isolated from Thapsia garganica L. [36,37], the increasing interest in it arose with the discovery of its ability to inhibit the sarco-endoplasmic reticulum calcium ATPase (SERCA) pump. The inhibition of this pump produces a high concentration of calcium in the cytosol, which leads to apoptosis. Several analogues of thapsigargin have been obtained, and a prodrug, thapsigargin peptide conjugate (Mipsagargin), has been designed. At the present, it undergoes clinical trials as antitumor agent.
Isolation and purification of thapsigargin are held using both classical and modern methods of extraction and chromatographic purification.
Appendino G. et al. [38] isolated sesquiterpene lactone thapsigargin from roots of Thapsia garganica L. by the following method: the powdered roots were extracted three times with acetone, the extracts were combined and evaporated. The thick extract was chromatographed on a column of silica gel with petroleum ether-ethyl acetate (7:3) and thapsigargin with a yield of 1.3% per the air-dry raw material was obtained.
Authorized system of the thapsigargin production from acetone extract of the aerial part of Thapsia garganica L. using the Speed-Extractor E-914 and centrifugal chromatography of the distribution was developed by the authors [39]. The development of thapsigargin is carried out in two ways, differing in the extraction stage, according to the following scheme: a) Extraction by maceration: the air-dry raw material is extracted twice with acetone at room temperature for 12 h. The obtained extracts are combined and evaporated. The extract yield is 2.46% per air-dry raw materials.
b) The air-dry raw material is being extracted twice with acetone in the accelerated extractor E-914 for 20 min at a pressure of 10 MPa. The obtained extract is evaporated. The yield of the extract is 2.64%.
Separation of extracts is carried out by liquid-liquid chromatography method, for this purpose the sample of extract is dissolved in mixture of mobile and stationary phases (1:1, concentration is 0.25 g/ml). Separation is carried out in solvents system composed of cyclohexan-ethylacetate-methanol-water (19:1:15:5), downwards, with the following parameters: elution rate from 5 to 13 ml/min, centrifuge at gravitational acceleration 140 to 358. When separating the extract obtained by the maceration method, thapsigargin is isolated with a yield of 1.67% per air-dried raw materials. Separation of the extract obtained using an accelerated extractor produces thapsigargin with a yield of 1.46% per air-dry raw materials.
Thus, methods using classical extraction and chromatographic separation methods that do not yet provide a quantitative yield of the target compound predominate in the isolation and purification of the sesquiterpene lactone thapsigargin.
Similarly, "brother" of thapsigargin, trilobolide (8) sesquiterpene lactone with similar biological properties, was prepared by classical extraction methods [40,41] of the plant material (roots and seeds) by ethyl acetate, after prepurification of the material by petroleum ether. Alternatively, the patent [40] claims the extraction with supercritical carbon dioxide in fluid state doped with ethanol, performed in a specialized commercially available device. The SFE preparation with carbon dioxide doped with ethanol was later further developed using laboratory and semi-pilot plant professional equipment [42]. After both methods of isolation the crude product is effectively purified by crystallisation from several solvent mixtures.

Conclusions
Most methods of isolation sesquiterpene lactones from plant extracts use traditional column chromatography on silica gel with the subsequent re-chromatography of the obtained fractions is used together with the preparative high performance liquid chromatography, what makes the cost of received substances more expensive.
Based on the conducted literature review, it is apparent that besides the efficiency and advantages of supercritical fluid, microwave and ultrasonic extraction techniques, their deployment definitely reduces the cost-effectiveness of sesquiterpene lactones production process.
Thus, the application of innovative methods to isolate and purify sesquiterpene lactones, such as supercritical fluid extraction, ultrasonic extraction, liquid-liquid chromatography, helps to simplify technological processes, reduce production costs, thereby to increase labor productivity and reduce the cost of the original drug.