STUDIES TO IMPROVE PRODUCTION, CONTENT AND EXTRACTION EFFICIENCY IN THE PRODUCTION OF ARTEMISININ AND ITS ANALOGS I INTRODUCTION 1.1 BACKGROUND INFORMATION POST-HARVEST DRYING TREATMENT EFFECTS ON AMTIMALARIAL CONSTITUENTS OF ARTEMIASIA ANNUA L. Author: J.C. Laughlin Keyword s: qinghao, annual wormwood, malaria, artemisinin (qinghaosu), artemisinic (qinghao) acid, essential oil, sun drying, shade drying, dark drying. Abstract: Two field experiments were carried out in cool temperate maritime latitudes in NW Tasmania (41ºS) to assess whether wilting and drying Artemisia annua plants in the field after harvest had any detrimental effects on artemisinin (the source of important antimalarial drugs) or its precursor artemisinic acid. A third field experiment studied the effect of steam distillation of A. annua for its essential oil, prior to oven drying, on artemisinin and artemisinic acid. In the first two experiments whole plants were cut off at the base and left in situ for 1, 3 and 7 days (Experiment 1) and for 7, 14 and 21 days (Experiment 2). Experiment 2 included two additional treatments: (i) shade drying whole plants under ambient conditions in the field for 21 days and (ii) drying leaves, detached at harvest, for 21 days under ambient conditions inside in the dark. The effects of all of these treatments were compared with oven drying (35ºC) leaves which had been detached immediately after harvest.
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STUDIES TO IMPROVE PRODUCTION, CONTENT AND EXTRACTION EFFICIENCY IN THE PRODUCTION OF ARTEMISININ AND ITS ANALOGS
I INTRODUCTION 1.1 BACKGROUND INFORMATION
POST-HARVEST DRYING TREATMENT EFFECTS ON AMTIMALARIAL CONSTITUENTS OF ARTEMIASIA ANNUA L.Author: J.C. Laughlin
Keywords:
qinghao, annual wormwood, malaria, artemisinin (qinghaosu), artemisinic (qinghao) acid, essential oil, sun drying, shade drying, dark drying.
Abstract: Two field experiments were carried out in cool temperate maritime latitudes in NW Tasmania (41ºS) to assess whether wilting and drying Artemisia annua plants in the field after harvest had any detrimental effects on artemisinin (the source of important antimalarial drugs) or its precursor artemisinic acid. A third field experiment studied the effect of steam distillation of A. annua for its essential oil, prior to oven drying, on artemisinin and artemisinic acid. In the first two experiments whole plants were cut off at the base and left in situ for 1, 3 and 7 days (Experiment 1) and for 7, 14 and 21 days (Experiment 2). Experiment 2 included two additional treatments: (i) shade drying whole plants under ambient conditions in the field for 21 days and (ii) drying leaves, detached at harvest, for 21 days under ambient conditions inside in the dark. The effects of all of these treatments were compared with oven drying (35ºC) leaves which had been detached immediately after harvest.Field drying for 1, 3 or 7 days had no adverse effect on either artemisinin or artemisinic acid in Experiment 1 and all leaf concentrations were similar to oven drying. Field drying for 7 days in Experiment 2 also gave artemisinin and artemisinic acid levels similar to oven drying. However there was a trend for sun-, shade- and dark drying for 21 days to give higher artemisinin than oven drying although artemisinic acid was unaffected. Distillation of A. annua plants for oil extraction, prior to oven drying at 35ºC, resulted in nil to negligible leaf concentration of artemisinin but artemisinic acid was unaffected. Field drying may be a way of reducing the cost of antimalarial drugs and the dual production of oil and artemisinic acid is a possibility.
Artemisinin Oil
Bella Mira Essential Oil : Artemisinin Essential Oil
Classification: Artemisia annua Essential Oil
Other Names: Wormwood Essential Oil, Sweet Annie Essential Oil, Capillaris Essential Oil, Chinese Wormwood Essential Oil, Wormwood Oil, Wormwood Extract, Artemisinin Extraction, Artemisia
Interesting Facts: In times past Artemisinin was thought to counteract poison. Artemisinin was also strewn about chambers to repel moths, fleas and other insects. When rumors of plague breaking out in London hit the streets in 1760, merchants reported running out of Artemisinin due to the huge public demand.
The use of Artemisinin in beverages dates back many centuries, perhaps as far back as the Saracens. Various methods of consumption have been used throughout history, including mixing Artemisinin essential oil with beer or adding wormwood seeds to the distillation of whisky.
Most famous however is the mixing of the wormwood drug absinthe with anise to produce the intoxicating beverage known as absinthe. Overuse of this drink had devastating effects in Europe in the 18th century, with overindulgence known to have brought about paralysis.
Wormwood is employed today in the making of vermouth, and accounts for this drink’s characteristic bitter flavor.
Medicinal Use: Artemisinin essential oil is beneficial for respiratory congestion and infection, catarrh, anti-cancer, anti-malarial properties. Artemisinin essential oil effective against intestinal parasites. Artemisinin essential oil is also effective as a uterine and womb tonic. Artemisinin Essential oil treats urinary tract infections caused by the Klebsiella bacteria. Artemisinin essential oil is also very effective against River Blindness (Onchocerciasis). Artemisinin Essential oil aids in the secretion of bile thus soothing intestinal tissues and helping with fat digestion. May aid in the discomfort of gall stones and assist in the cleansing of the gall bladder and liver.
Constituents: alpha-pinene (0.032%), camphene (0.047%), ß-pinene (0.882%), myrcene (3.8%), 1,8-cineole (5.5%), artemisia ketone (66.7%), linalool (3.4%), camphor (0.6%), borneol (0.2%), and ß-caryophyllene (1.2%).
Simon, J.E., D. Charles, E. Cebert, L. Grant, J. Janick, and A. Whipkey. 1990. Artemisia annua L.: A promising aromatic and medicinal. p. 522-526. In: J. Janick and J.E. Simon (eds.), Advances in new crops. Timber Press, Portland, OR.
Artemisia annua L.: A Promising Aromatic and Medicinal*
James E. Simon, Denys Charles, Ernst Cebert, Lois Grant, Jules Janick, and Anna Whipkey
INTRODUCTIONArtemisia annua L. (annual wormwood, sweet wormwood, sweet annie), a highly aromatic annual herb (Fig. 1) of Asiatic and eastern European origin, is widely dispersed throughout the temperate region (Bailey and Bailey 1976, Simon et al. 1984). The species has naturalized in the United States and is sold on a limited scale as a dried herb for the floral and craft trade where it is used as an aromatic wreath. The plant has traditionally been grown in China as a medicinal and, more recently in Europe for its aromatic leaves which are used in flavoring beverages.
Recent research in the Peoples Republic of China with traditional herbal medicine has brought attention to A. annua, the source of qinghaosu (artemisinin), a compound that shows promise as an anti-malarial agent (Klayman 1985). Artemisinin has also been reported to be a potent plant inhibitor with potential as a natural herbicide (Duke et al. 1987, Chen et al. 1987). Artemisinin and its derivatives, artemether and artesunate, have been studied for their efficacy as antimalarial agents. In in vitro trials conducted in China (WHO 1981), all three compounds have been effective against the erythrocytic stages of two chloroquine-resistant Hainan strains of Plasmodium falciparum, the malarial parasite, at lower minimum effective concentrations than chloroquine, the most commonly used drug. Artemisinin and its derivatives have effectively treated malaria and cerebral malaria in human subjects with no apparent adverse reactions nor side effects (Klayman 1985). With P. falciparum developing resistance to chloroquine and pyrimethamine/sulfonamide (WHO 1981), alternative treatments based on new compounds such as artemisinin and its derivatives are actively being sought. While artemisinin and its derivatives may be synthesized (Zhou 1986, Xu et al. 1986), the synthetic compounds are unlikely to be economically competitive with the naturally derived plant products (Schmid and Hofheinz 1983, Xu et al. 1986).
The relatively low content of artemisinin in cultivated European and New World types of A. annua has been a limiting factor for the isolation and evaluation of artemisinin on a technical scale. Artemisinin yields of 0.06% have been extracted from samples of A. annua collected in the United States (Klayman et al. 1984) which are low for commercial exploitation. Yields of extracted artemisinin from the above-ground portions of the plant have ranged from 0.01% to 0.5% (w/w) in the People's Republic of China (WHO 1981). Although artemisinin yield varies with environmental and management conditions (WHO 1981), specific effects are unknown. The extent of genetic variation on artemisinin content was also poorly understood.
Since 1984, we have been evaluating the plant s production potential determining its horticultural characteristics, and developed a rapid assay to determine artemisinin content from crude plant materials in anticipation of future improvement programs. Essential
(volatile) oil composition was characterized in order to evaluate A. annua as a source of aroma chemicals for the fragrance industry
CHEMISTRY
ArtemisininArtemisinin is a secondary or natural plant metabolite identified as a sesquiterpene lactone endoperoxide (Klayman et al. 1984). Artemisinin has been synthesized and its structure and absolute stereochemistry established by Zhou (1986) and Xu et al. (1986), but biosynthetic pathways and the mechanisms and regulation processes are unknown. Analysis of artemisinin is difficult because the compound is unstable, concentrations in the plant low, the intact molecule stains poorly, and other compounds in the crude plant extracts interfere in its detection. A method to analyze artemisinin in crude plant extracts based upon high pressure liquid chromatography (HPLC) with reductive mode electrochemical detection was first developed by Acton et al. (1985) and later modified by Charles et al. (1990). This latter method is highly sensitive, rapid, and should be of value in analyzing large numbers of samples as needed in crop improvement programs.
Using this later method, we evaluated our germplasm collection of A. annua for artemisinin content to determine whether there was genetic variability that could be exploited and to identify plant lines and individual plants with high concentrations (Charles et al. 1990). Wide variation in artemisinin content was observed with accessions in our collection ranging from 0.003 to 0.21%, and with individual plants ranging from 0.00 to 0.39% (dry weight basis). This data suggests that artemisinin productivity could be enhanced by further selection and breeding.
Essential Oils Essential oils of A. annua can be extracted via steam distillation in units similar to that used in the commercial distillation of peppermint and spearmint. Essential oils of A. annua were extracted in our studies via hydrodistillation with a modified clevenger trap (Simon and Quinn 1988) and chemically characterized by gas chromatography (GC) analysis using a fused silica capillary column (12 m x 0.2 mm id) with a OV 101 (Varian, polydimethylsiloxane) bonded phase. Direct injection of 0.5 ml of essential oil samples with He as a carrier gas (100:1 split vent ratio) and oven temperatures held isothermal at 80°C for 2 min and then programmed to increase at 3°C/min to 210°C gave complete elution of all peaks (sensitivity 10-10). The injector and detector temperatures were 210°C and 300°C, respectively. Confirmation of essential oil constituents was based upon comparison of retention time with standards and via GC/Mass Spectroscopy analysis.
Essential oils of A. annua are comprised of many constituents with the major compounds including (in relative % of total essential oil) alpha-pinene (0.032%), camphene (0.047%), ß-pinene (0.882%), myrcene (3.8%), 1,8-cineole (5.5%), artemisia ketone (66.7%), linalool (3.4%), camphor (0.6%), borneol (0.2%), and ß-caryophyllene (1.2%).
Location of Natural Products Essential oils and artemisinin were assumed to be associated with secretory cells based on the association of mono- and sesquiterpenes with well-defined secretory structures (Croteau 1986, Henderson et al. 1970). The relative distribution of artemisinin is shown in Table 1. Leaves had 89% of the total artemisinin in the plant with the uppermost foliar portion of the plant (top 1/3 of growth at maturity) containing almost double that of the lower leaves (Charles et al. 1990). Kelsey and Shafizadeh (1980) had reported that 35% of the mature leaf surface is covered with capitate glands which contain most of the monoterpenes and virtually all of the sesquiterpene lactones. Essential oils from A. annua are similarly distributed, with 36% of the total from the upper third of the foliage, 47% from the middle third, and 17% from the lower third, with only trace amounts in the main stem side shoots, and roots.
HORTICULTURE
Response to Plant Spacing and Nitrogen Application The response of A. annua to plant spacing and nitrogen fertilization was evaluated in 1985 and 1986 with three populations established from transplants: high density, 30 cm x 30 cm (111,111 plants/ha); intermediate density, 30 cm x 60 cm (55,555 plants/ha); and low density, 60 cm x 60 cm (27,778 plants/ha). Plants in each spacing received three levels of nitrogen fertilization (0, 67, and 134 kg N/ha) applied as a preplant broadcast application.
Plants from the densely populated treatment (111,111 plants/ha) produced an average fresh weight of 275 g/plant, as opposed to 430 g/plant from the intermediate and 750 g/plant from the lowest populations. However, total biomass (fresh weight kg/ha), was greatest from the higher density population (Table 2). Plants of the most densely populated treatments were slightly taller, produced less side shoots, and had longer internodes with little lateral growth than the lower densities. Number of side shoots per main stems decreased as density increased. While yield increased with added nitrogen, the greatest growth (herbage and essential oil content) was obtained with 67 kg N/ha (Table 2).
Increasing density tended to increase essential oil production on an area basis, but highest essential oil yields (85 kg oil/ha) was achieved by the intermediate density at 55,555 plants/ha receiving 67 kg N/ha (Table 2). Artemisinin content was not analyzed in this study because the instrumentation and method of analysis had not yet been developed in our laboratory
Influence of Planting Date and Harvest Time Seedlings of A. annua were transplanted into a central Indiana field on April 27, May 17, June 10, and July 13, 1987, and plant samples (for growth and essential oil) were obtained every two weeks until the first frost (Fig. 2, 3). The May transplanting date produced the highest fresh yields and tallest plants, while the May and June plantings had the highest percentage of essential oil. Regardless of planting date, all plants began to flower by mid-August, with maximum concentration of essential oil produced in mid-
September (peak flowering stage). Results from the 1988 growing season (data not presented) were similar to those obtained in 1987.
Tissue Culture Plantlets of A. annua were regenerated using shoot tips of mature field grown plants. Shoot tips were surface sterilized and cultured in Murashige and Skoog (MS) media with factorial combinations of 2,4-D (0, 0.1, 1.0 mg/liter) and benzyladenine (BA) (0, 0.1, 1.0 and 10.0 mg/liter). Both axillary and adventitious shoots were produced in all the treatments. BA alone at 10 mg/liter produced the greatest number of shoots, but the most normal shoots were obtained at 1.0 mg/liter. Shoots were easily multiplied on MS medium with 1 mg/liter BA and subculturing at 4 week intervals. Artemisinin was detected from in-vitro shoots of A. annua, in concentrations from 0.03 to 0.05% (dry weight basis).
All shoots rooted when dipped in commercial preparations of 0.3% indolebutyric acid and placed in potting soil under mist conditions. Acclimatization to in vivo conditions was easily achieved. This technique will permit rapid increases of individual plant selections.
PROSPECTS
We have found that A. annua is relatively easy to grow and that very high biomass yields (35t/ha) can be obtained in the Midwest. Plant spacing had a highly significant affect on biomass yield and plant architecture. Wide variation in artemisinin content has been found in our germplasm collection with accessions reaching 0.21%, and individual plants as high as 0.39% (dry weight basis). This suggests that high artemisinin yields lines could be developed by further selection and breeding. The commercialization of A. annua in this country is dependent upon whether artemisinin or its derivatives will be approved for use in the treatment of malaria. Domestic production of A. annua for the extraction and processing of artemisinin for export should be considered as overseas markets develop and if they will purchase imported materials.
Artemisia has potential as a source of essential oils and we have obtained oil yields of 85 kg/ha. Uses of the essential oil from A. annua in the fragrance industry would provide an additional market and a new use for this species.
REFERENCES
Acton, N. and D.L. Klayman. 1985. Artemisinin, a new sesquiterpene lactone endoperoxide from Artemisia annua. Planta Medica 47:442-445
Acton, N., D.L. Klayman and I.J. Rollman. 1985. Reductive electrochemical HPLC assay for artemisinin (Qinghaosu). Planta Medica 47:445-446.
Charles, D.J., J.E. Simon, K.V. Wood and P. Heinstein. 1990. Germplasm variation in artemisinin content of Artemisia annua L. using an alternative method of artemisinin analysis from crude plant extracts. J. Nat. Prod. 53:157-160.
Chen, P.K, G.R Leather and D.L. Klayman. 1987. Allelopathic effect of artemisinin and its related compounds from Artemisia annua. Plant Physiol. 83S. Abstr. 406.
Croteau, R. 1986. Biochemistry of monoterpenes and sesquiterpenes of essential oils. In: Craker, L.E. and J.E. Simon (eds.). Herbs, spices, and medicinal plants: Recent advances in botany horticulture and pharmacology. Oryx Press, Phoenix, AZ. Vol. 1:81-133.
Duke, S.O., K.C. Vaughn, E.M. Croom Jr. and H.N. Elsohly 1987. Artemisinin, a constituent of annual wormwood (Artemisia annua), is a selective phytotoxin. Weed Sci. 35:499-505.
Henderson, W, J.W. Hart, P. How and J. Judge. 1970. Chemical and morphological studies on sites of sesquiterpene accumulation in Pogostemon cablin (Patchouli). Phytochemistry 9:1219-1228.
Kelsey, R.G. and F. Shafizadeh. 1980. Glandular trichomes and sesquiterpene lactones of Artemisia nova (Asteraceae). Biochem. Syst Ecol. 8:371-377.
Klayman, D.L. 1985. Qinghaosu (Artemisinin): an antimalarial drug from China. Science 228:1049-1055.
Klayman, D.L., A.J. Lin, N. Acton, J.P. Scovill, J.M. Hock W.K. Milhous and A.D. Theoharides. 1984. Isolation of artemisinin (qinghaosu) from Artemisia annua growing in the United States. J. Nat. Prod. 47:715-717.
Schmid, G., and W. Hofheinz. 1983. Total synthesis of qinghaosu. J. Amer. Chem. Soc. 105:624-625.
Simon, J.E. and J. Quinn. 1988. Characterization of essential oil of parsley J Agric. Food Chem. 36:467-472.
Simon, J.E., A.F. Chadwick and L.E. Craker. 1984. Herbs: an indexed bibliography, 1971-1980. The scientific literature on selected herbs, and aromatic and medicinal plants of the temperate zone. Archon Books, Hamden CT.
Staff of L.H. Bailey Hortorium. 1976. Hortus third. MacMillan Publ., New York. World Health Organization. 1981. Report of the Fourth Meeting of the Scientific
Working Group on the Chemotherapy of Malaria. Beijing, People's Republic of China, October 6-10, 1981.
Xu, Xing-Xiang, J. Zhu, Da-Zhong Huang and Wei-Shan Zhou. 1986. Total synthesis of arteannuin and deoxyarteannuin. 1986. Tetrahedron 42:819-828.
Zhou, Wei-Shan. 1986. Total synthesis of arteannuin (quinghaosu) and related compounds. Pure Appl. Chem. 58:817-824.
*Journal Paper No. 12,032, Purdue Univ. Agr. Exp. Sta., West Lafayette, IN 47907. This research was supported in part by a grant from the Purdue University Agricultural Experiment Station (Specialty Crops Grant No. 014-1165-0000-65178).
Table 1. Relative distribution of artemisinin in Artemisia annua L. (Data from Charles et al. 1990)
Plant part sampledz Artemisinin % of total artemisinin
(% dry wt) /plant
Leaves
Upper third 0.15 41.7
Middle third 0.09 25.0
Lower third 0.08 22.2
Side shoots 0.04 11.1
Main stem trace —
Roots absent —
Seedsy 0.04 —
zEight weeks after transplanting prior to flowering.ySeeds collected from 13 week old plants after transplanting.
Table 2. Fresh weight and oil yield of Artemisia annua in response to plant spacing and nitrogen application (average yields from 1985 and 1986).
N (kg/ha)
Density (no plants/ha) 0 67 134
Biomass yield (t/ha)
27,778 21 23 24
55,555 23 30 28
111,111 30 35 33
Essential oil yield (kg oil/ha)
27,778 56 61 56
55,555 67 85 69
111,111 78 78 83
Fig. 2. Influence of planting date and harvest time on fresh weight of Artemisia annua, 1987.
Fig. 3. Influence of planting date and harvest time on oil yield of Artemisia annua, 1987.
1.2 MALARIA PARASITE
1.3 MALARIAL DRUGS
1.3.1 ARTEMISININ COMBINATION THERAPY
2 LITERATURE REVIEW2.1 PRODUCTION PER HA
Harvesting and treatment
POST-HARVEST DRYING TREATMENT EFFECTS ON AMTIMALARIAL CONSTITUENTS OF ARTEMIASIA ANNUA L.Author: J.C. Laughlin
Keywords:
qinghao, annual wormwood, malaria, artemisinin (qinghaosu), artemisinic (qinghao) acid, essential oil, sun drying, shade drying, dark drying.
Abstract: Two field experiments were carried out in cool temperate maritime latitudes in NW Tasmania (41ºS) to assess whether wilting and drying Artemisia annua plants in the field after harvest had any detrimental effects on artemisinin (the source of important antimalarial drugs) or its precursor artemisinic acid. A third field experiment studied the effect of steam distillation of A. annua for its essential oil, prior to oven drying, on artemisinin and artemisinic acid. In the first two experiments whole plants were cut off at the base and left in situ for 1, 3 and 7 days (Experiment 1) and for 7, 14 and 21 days (Experiment 2). Experiment 2 included two additional treatments: (i) shade drying whole plants under ambient conditions in the field for 21 days and (ii) drying leaves, detached at harvest, for 21 days under ambient conditions inside in the dark. The effects of all of these treatments were compared with oven drying (35ºC) leaves which had been detached immediately after harvest.Field drying for 1, 3 or 7 days had no adverse effect on either artemisinin or artemisinic acid in Experiment 1 and all leaf concentrations were similar to oven drying. Field drying for 7 days in Experiment 2 also gave artemisinin and artemisinic acid levels similar to oven drying. However there was a trend for sun-, shade- and dark drying for 21 days to give higher artemisinin than oven drying although artemisinic acid was unaffected. Distillation of A. annua plants for oil extraction, prior to oven drying at 35ºC, resulted in nil to negligible leaf concentration of artemisinin but artemisinic acid was unaffected. Field drying may be a way of reducing the cost of antimalarial drugs and the dual production of oil and artemisinic acid is a possibility.
2.2 METHODS TO IMPROVE CONTENT OF ARTEMISININ
2.3 TECHNOLOGIES OF PRODUCTION
AbstractArtemisinin, an endoperoxide containingsesquiterpene lactone from Artemisia annua, hasproven very effective in treating drug resistantcases of malaria and cancer. To counter thepresent low content in leaves and uneconomicalchemical synthesis, alternate ways to produceartemisinin have been sought. Inspite ofextensive work in this area, artemisinin remainselusive in dedifferentiated and differentiatedcultures of A. annua. This work reports the firstsuccessful approach for production of artemisininby cell cultures of A. annua cultures of Indianvariety of A. annua. Various precursors ofterpenoid biosynthesis by isoprenoid pathwaywere incorporated to study their influence onartemisinin biosynthesis. Artemisinin content
was maximally increased by 2.0 times, incomparison to control, when mevalonic acid (50mg/L) was added as precursor. Various biotic andabiotic elicitors were also tested at differentconcentration. A maximum increase of 3.47 timesin artemisinin accumulation was attained whenmethyl jasmonate (5 mg/L) was added. Basedon these results, an integrated bioprocess forproductivity enhancement of artemisinin wasdeveloped. A maximum artemisininaccumulation of 96.8 mg/L artemisinin wasproduced on supplementation of mevalonic acidand methyl jasmonate as selected precursor andelicitor respectively, which was 4.79 times higherin productivity than control callus cultures (20.2mg/L).
Enhanced artemisinin production by cell culturesof Artemisia annuaAshish Baldi* 1 and V.K. Dixit 2
1 Department of Biochemical Engineering and Biotechnology,Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India2 Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar (M.P.) 470003, India* For correspondence - [email protected], Artemisia annua, Hairy roots,Agrobacterium rhizogenesIntroductionMalaria is a serious endemic disease inmany parts of the world, affecting 5% of theWorld’s population. About 40% of the world’spopulation is at stake of malaria. In spite ofavailability of extensive chemotherapeuticarmamentarium to combat this disease,widespread emergence of resistant strains of theparasite to drugs used is a global concern (1). AChinese medicinal herb, Artemisia annua L., hasevoked wide interest for its artemisinin (AN)content, a serquiterpene lactone, which iseffective against both chloroquine-resistant andsensitive strains of P. falciparum as well ascerebral malaria with high safety profile. It isalso reported as chemotherapeutic agent againstvarious types of cancers such as leukemia andcolon cancer etc. Significant biological activityand novel chemical structure have promptedefforts in developing a series of derivatives whichare more potent than the parent compound, suchas artesunate, artemether, arteether (2) andsodium salt of artelinic acid (3). Highest ANcontent has been reported in leaves (0.01-0.5%)in Chinese varieties of plant (4). It was later
introduced in India, cultivated and naturalizedin Kashmir and Lucknow (5) but showed lowAN content. Uneconomical synthesis of AN (6)Current Trends in Biotechnology and Pharmacy, Vol. 2 (2) 341-348 (2008)ISSN 0973-8916and low yield from natural source (7) hadtriggered search for alternative means to produceAN in cell cultures. Results from experimentswith undifferentiated and differentiated culturesof A. annua till now are rather disappointing withrespect to the AN production, as only traces ofthis compound have been found (8-12) Till thedate, highest AN content of 0.42% DW in hairyroots was reported by Weathers et al. (13). Fewresearchers reported a very low level (14,15) orabsence of AN in hairy root cultures (16).Feeding of precursors and elicitation hasproved to be an effective way to enhancesecondary metabolites in plant cell cultures. Anumber of precursors such as sodium acetate (17-19), mevalonic acid lactone, casein acidhydrolysate (20) and elicitors like jasmonates(21), salicylates (22), chitosan and gibberlic acid(20) have been investigated to enhance yield ofplant based secondary metabolites. Effect ofsingle precursor/elicitor on a particular cell linewas investigated in most of these studies. Butconsidering the complexicity of AN biosynthesis,it is reasonable to examine the combined effectof these yield enhancement strategies. For thisreason, the present study was undertaken on cellsuspension cultures of A. annua. The aim of thiswork was to develop a high yielding starterculture for AN production in cell cultures. Thisappears to be the very first successful study onproduction of AN in cell cultures developed fromIndian variety of A. annua.Material and MethodsGermination of seedsSeeds of Indian variety of Artemisia annuawere sterilized by treating first with 90% alcoholfor 3 s, and followed by 0.1% (w/v) mercuricchloride treatment for 5 m. Seeds were thenrinsed thoroughly 3 times in sterile distilled waterand 10 seeds per Petri plate were allowed togerminate on padding of sterile filter paper andabsorbent cotton, moistened with 15 ml of liquidMurashige and Skoog’s (MS) medium (23)supplemented with 3% (w/v) sucrose. Seeds werethen incubated at 25±2° under 2200 lux intensityof light and 16/8 h light/dark photoperiod in a
growth chamber. Germination started within 2-3 d.Callus culturesHigh yielding callus culture of A. annuawas developed from aseptically germinatedseedlings and maintained on MS mediumsupplemented with NAA and Kn (0.5 mg/L ofeach) as growth regulators.Establishment of growth and productionkineticsGrowth and production kinetics of A. annuacell culture were established. Callus cultureswere allowed to grow on MS media with 0.5 mg/L each of NAA and Kn, and solidified with 1%agar. Experiments were carried out in 250 mLErlenemeyer flasks containing 50 mL of MSsolid medium with 2.0 g/L inoculum on a dryweight basis. Cultures were incubated at 252Cunder 16/8h light/dark cycle. All cultures wereanalyzed, in triplicate, for biomass and ANcontent at an interval of 5 d.Enhancement of artemisinin productionPrecursor additionTo improve AN production in calluscultures, some known precursors of ANbiosynthesis [sodium acetate (SA), mevalonicacid (MA), casein acid hydrolysate (CAH)] andcholesterol (CH) were added. Stocks of sodiumacetate and casein acid hydrolysate wereprepared in sterile double distilled water and thepH was adjusted to 5.7. Cholesterol andmevalonic acid were dissolved in acetone and95% ethanol respectively. Filter sterilizedprecursors were added to the culture medium on10th d at following concentrations: sodiumacetate, 20, 50 and 100 mg/L; mevalonic acid,342 Baldi and Dixit20, 50 and 100 mg/L; cholesterol, 20, 50and 100 mg/L; casein acid hydrolysate, 0.1, 0.5and 1.0 g/L. Cultures were harvested on 25th d,in duplicate, and analyzed for biomass and ANcontent. In control cultures, equal volume ofsolvent for precursor was added aseptically toculture medium.Elicitor treatmentSome known abiotic elicitors for plantbased secondary metabolites such as signalingmolecules [(methyl jasmonate (MJ), acetylsalicylic acid (ASA)], metal ion [calcium ascalcium chloride (CC)] and blooming agent[gibberlic acid (GA)] were also tested for their
effect on AN content. Stock solutions of acetylsalicylic acid, and calcium chloride wereprepared by dissolving them in sterile doubledistilled water and adjusting the pH to 5.7.Methyl jasmonate was dissolved in 95% ethanol.Abiotic elicitors were aseptically added tocultivation medium at the followingconcentrations: methyl jasmonate: 1,2,5 and 10mg/L; acetyl salicylic acid: 10, 20, 30, and 50mg/L; calcium chloride: 50,100 and 150 mg/L;and gibberlic acid: 5, 10 and 20 mg/L.Biotic elicitors [yeast extract (YE), chitosan(Ch)] were tested at 10, 20, and 50 mg/Lconcentrations by addition of 50 L, 100 Land 250 L of respective stock solutions of yeastextract and chitosan individually.. Yeast extractwas added as aqueous stock (10 mg/ml) havingpH 5.7. For preparation of chitosan, 1 g of crabshell chitosan was dissolved in 2 ml of glacialacetic acid (by dropwise addition) at 60C for aperiod of 15 minutes.; the final volume was madeup to 100 ml with water and pH of the solutionwas adjusted to 5.7 with 1 M NaOH to get a stocksolution of 10 mg/ml.Following the elicitor treatment on 20th day,cultures were harvested on 25th day, in triplicate,and analyzed for biomass and AN content.Integrated bioprocess for productivityenhancementResults from both yield enhancementstrategies viz. addition of precursor and elicitationwere integrated in order to develop a bioprocessfor synergistic enhancement of artemisininproductivity in callus cultures of A. annua.Cultures were cultivated on solid MS mediumwith NAA and Kn (0.5 mg/L of each) at 272Cwith a photoperiod of 16/8h light/dark cycle).Selected precursor (mevalonic acid @ 50 mg/L)and elicitor (methyl jasmonate @ 5 mg/L) wereadded on 10th and 20th day of cultivationrespectively. Cultures were harvested in triplicateon 25th day and analyzed for biomass and ANproduction.AnalysisFor dry cell weight estimation, cells wereharvested and allowed to dry at 602C untilconstant weight was achieved. Sodium derivativeof AN was analyzed by HPLC with somemodification in the method of Zhao and Zheng(24). For analysis of AN, 100 mg of dried andpowdered cells were allowed to macerate with
2.0 mL diethyl ether for 48 h. The filtrate wasthen dried under vacuum and the residue wasdissolved in 1.0 mL of methanol. This was thentreated with 4.0 ml NaOH (0.2% w/v) andincubated at 502C for one h with occasionalshaking to get a sodium derivative of AN.Results and DiscussionCell cultures of A. annua of Indian varietywith significant AN accumulation wereestablished for very first time. Complete growthand production profiles of A. annua cells wereestablished and given as Fig. 1. Relatively longerlog phase of 10 day was observed in batchkinetics. Maximum biomass (16.8 g/L) andartemisinin (20.2 mg/L) were obtained on 25th
day of cultivation. Artemisinin was produced incallus cultures.Current Trends in Biotechnology and Pharmacy, Vol. 2 (2) 341-348 (2008) 343ISSN 0973-8916intracellularly and most productionoccurred during the active growth phase of A.annua cells.Few known precursors for terpenoidbiosynthesis were added to the media on 10th dof cultivation. Their effect on AN production isgiven in Fig. 2. Sodium acetate, mevalonic acidand higher concentration of casein acidhydrolysate had positive effect on AN productionFigure 2 Effect of precursor addition on artemisininproduction by A. annua callus cultures (Averagevalues are given, error bars are represented as verticallines)Figure 1 Growth and artemisinin production profileof A. annua cells (Average values are given, errorbars are represented as vertical lines)A maximum of 32.2 mg/L artemisinin wasproduced on addition of 50 mg/L sodium acetate.It is known to be a potential precursor for varioussecondary metabolites. It has been found toincrease secondary metabolites like azadirachtin(25); astaxanthin (26); taxol (17,18,27); crocin(19) and phenylethnoid glycosides (28). Theincreased production of AN is assumed to beattributed to the increased Acetyl-Co A level asa result of sodium acetate feeding, which maylead to the improved secondary metaboliteproduction by enhanced life activities (29).Mevalonic acid is an intermediate in thebiosynthesis of isoprene units. AN content wasincreased maximally by 2.0 times (40.4 mg/L),in comparison to control, when mevalonic acid(50 mg/L) was added. However, this is not in
agreement with earlier findings (8). Thisenhancement might be due to direct supply ofmevalonic acid to synthesize AN at enhancedlevel.Casein acid hydrolysate serves as anadditional source of amino acids andoligopeptides. With 0.5 g/L, a slight enhancementof the AN content was found, but it remainsalmost constant after this. Indirect precursor,cholesterol, had a negative effect on biomass aswell as AN production. This might be due todiversion of metabolic pathway to othercompounds.Various biotic (chitosan and yeast extract)and abiotic elicitors (gibberlic acid, calciumchloride, acetyl salicylic acid and methyljasmonate) were also tested at differentconcentration to enhance AN yield. Effect ofthese elicitors on AN accumulation is given inFig. 3. Acetyl salicylic acid, methyl jasmonate,and gibberlic acid had resulted in significantimprovement of AN production.Gibberlic acid is a plant growth regulator,capable of inducing blooming (30). AN content344 Cell Cultures of Artemisia annuaFigure 3 Effect of elicitor addition on artemisininproduction by A. annua callus cultures (Average valuesare given, error bars are represented as verticallines)in intact plant are maximum just before or at thetime of flowering (31), so this compound wasadded to callus cultures. Earlier, addition ofgibberlic acid has been reported not to affect theAN content of A. annua shoot cultures (8) but apositive effect was also observed (20).Acetyl salicylic acid also significantlyimproved AN production as a maximum increaseof 2.81 times (56.8 mg/L) was attained whenacetyl salicylic acid (30 mg/L) was added. Thiscould be attributed to the fact that salicylic acidand related compounds act as signalingmolecules and play essential role in many plantdefence reactions (32). It is known to regulatethe expression of various defense related genes(33,34) and has an important role in SystemAcquired Resistance (SAR) and inhibitor ofethylene biosynthesis (35). AN being the defensechemical of plants could be regulated with thepresence of these signal molecules.Addition of methyl jasmonate resulted inan increased AN production of 70.2 mg/L at 5.0
mg/L concentration. Exogenous application ofJA signaling compounds, including jasmonicacid, methyl jasmonate, as well as theirconjugated compounds to the plant cell cultureor intact plant stimulates biosynthesis ofsecondary metabolites (36-38). Elicitor-inducedindole alkaloids in C. roseus (39), phytoalexinbiosynthesis in rice (40), indole glucosinolatesbiosynthesis in Arabidopsis (41), and hthujaplicinin Mexican cypress cell culture (42)support the idea that JA signaling is a mediatorof secondary product accumulation. Chemicalstructure of jasmonic acid related molecule alsoknown to play an important role in elicitationand is very specific in nature (43).Addition of yeast extract also slightlyenhanced AN levels. The possible reason couldbe attributed to presence of some cations like ZNand Co (44), which could act as abiotic elicitors.Although addition of calcium ions did notimprove AN content in callus cultures. Areduction in the AN content was observed byaddition of chitosan. This might be due to changein cell membrane permeability as evident frombrowning of cells.Based on these results, an integratedbioprocess for synergistic enhancement ofartemisinin was developed and experimentallyimplemented. A maximum of 96.8 mg/Lartemisinin was produced on supplementation ofmevalonic acid lactone (50 mg/L on 10th day)and methyl jasmonate (5 mg/L on 20th day) asselected precursor and elicitor respectively,which was 4.79 times higher in productivity thancontrol cultures. A comparative effect of yieldenhancement strategies tried out in present studyis given in Table-1.Yield enhancement Artemisininstrategy production (mg/L)High yielding cell 20.2line selectionPrecursor addition 40.4Elicitation 70.2Integrated bioprocess 96.8Table 1:Comparison of yield enhancement strategiesCurrent Trends in Biotechnology and Pharmacy, Vol. 2 (2) 341-348 (2008) 345ISSN 0973-8916ConclusionThis study represents the first successfulcell culture based approach for the productionof artemisinin from Indian variety of seeds of A.annua. Artemisinin content was found to be
superior to that reported earlier (11,15). Theproblem of low content in plant cell cultures wasaddressed through addition of precursor andelicitation, which resulted in significantimprovement in artemisinin production.Integrated yield enhancement strategycomprising of addition of mevalonic acid asprecursor and methyl jasmonate as elicitor atoptimum concentrations resulted in synergisticenhancement of artemisinin accumulation. Thepresent study indicates the potential of thesebiotechnology-based methodologies for massproduction of artemisinin. Further work relatedto large scale production of artemisinin atbioreactor level under different configurationsand various modes of cultivation are underprogress.AcknowledgementsThe authors are thankful to Prof. PamelaWeathers, Department of Biology andBiotechnology, Worcester Polytechnic Institute,Worcester, USA for her valuable guidance andgenerous gift of standard artemisinin.References1. White, N. and Pukrittayakamee, J. (1993)Clinical malaria in tropics, S. Med. J. Aust.,159: 197-203.2. Titulaer, H.A.C., Zuidema, J. and Lugt , C.B.(1991) Formulation and pharmacokinetics ofartemisinin and its derivatives, Inter. J. Pharm.,69, 83–92.3. Lin, A., Klayman, D.L. and Milhous, W.K.(1987). Antimalarial activity of new watersoluble dihydroartemisinine derivatives. J.Med. Chin., 30: 2147–2150.4. Nair, M.S.R., Acton, N., Klayman, D.L.,Kendrick, K., Basile, D.V. and Mante, S.(1986). Production of artemisinin in tissueculture of Artemisia annua. J. Nat. Prod., 49:504-507.5. Singh, A., Vishwakarma, R.A. and Husain, A.(1988). Evaluation of Artemisia annua strainsfor higher artemisinin production. Planta Med.,54: 475-476.6. Avery, M.A., Chong, W.K.M. and Jennings-White, C. (1992). Stereoselective totalsynthesis of ( + )-artemisinin, the antimalarialconstituent of Artemisia annua L. J. Am. Chem.Soc., 114: 974-979.7. E1Sohly, H.N., Croom Jr, E.M., El-Feraly, F.S.and El-Sherei, M.M. (1990). A large-scaleextraction technique of artemisinin fromArtemisia annua, J. Nat. Prod., 53: 1560-1564.
8. Martinez, B.C. and Staba, E.J. (1988). Theproduction of artemisinin in Artemisia annuaL. tissue cultures. Adv. Cell Cult., 6, 69-87.9. He, X.C., Zeng, M.Y., Li, G.F. and Liang, Z.(1983). Callus induction and regeneration ofplantlets from Artemisia annua and changes inqinhaosu contents. Acta Bot. Sin., 25: 87-90.10. Kudakasseril, G.J., Lam, L. and Staba, E.J.(1987). Effect of sterol inhibitors on theincorporation of ~4C-isopentenyl pyrophosphateinto artemisinin by a cell-free systemfrom Artemisia annua tissue cultures andplants. Planta Med., 53: 501-502.11. Jha, J., Jha, T.B. and Mahato, S.B. (1988).Tissue culture of Artemisia annua L.: Apotential source of an antimalarial drug. Curr.Sci., 57: 344-346.12. Tawfiq, N.K., Anderson, L.A., Roberts, M.F.,Phillipson, J.D., Bray, D.H. and Warhurst, D.C.(1989). Antiplasmodial activity of Artemisiaannua plant cell cultures. Plant Cell Rep., 8:425-428.13. Weathers, P., Cheetham, R.D., Follansbee, E.and Tesh, K (1994). Artemisinin production bytransformed roots of Artemisia annua. Biotech.Lett., 16: 1281-1286.14. Jaziri, M., Shimomur, A.K. and Yoshimatsu, K.(1995). Establishment of normal andtransformed root cultures of Artemisia annuaL. for artemisinin production. J Plant Physiol.,145: 175-177.346 Baldi and Dixit15. Paniego, N.B. and Giulietti, A.M. (1996).Artemisinin production by Artemisia annua L.transformed organ cultures. Enz. Microb.Tech., 18: 526-530.16. Kim, N.C., Kim, J.G., Lim, H.J., Hahn, T.R.and Kim, S.U. (1992). Production of secondarymetabolites by tissue culture of Artemisiaannua L.J. Korean Agri Chem Soc., 35, 99-105.17. Wu, Z.L., Yuan, Y.J., Liu, J.X., Xuan, H.Y., Hu,Z.D., Sun, A.C. and Hu, C.X. (1999). Study onenhanced production of taxol from Taxuschinensis var. mairei in biphasic-liquid culture.Acta Bot. Sin., 41: 1108-1113.18. Yuan, Y.J., Wei, Z.J., Wu, Z.L. and Wu, J.C.(2001). Improved taxol production insuspension cultures of Taxus chinensis var.mairei by in situ extraction combined withprecursor feeding and additional carbon sourceintroduction in an airlift loop reactor.Biotechnol. Lett., 23: 1659-1662.19. Zeng, Y., Yan, F., Tang, L. and Chen, F. (2003).Increased crocin production and inductionfrequency of stigma-like structure from floral
organs of Crocus sativus by precursor feeding.Plant Cell Tiss. Org. Cult., 72: 185-191.20. Woerdenbag, H.J., Lfiers, Jos F.J., van Uden,W., Pras, N., Malingr, Th..M. and Alfermann,A.W. (1993). Production of the newantimalarial drug artemisinin in shoot culturesof Artemisia annua L. Plant Cell Tiss. Org.Cult., 32: 247-257.21. Walker, T.S., Bais, H.P. and Vivanco, J.M.(2002). Jasmonic Acid induced hypercinproduction in Hypericum perforatum L. (St.John wort). Phytochem., 60: 289-293.22. O’Donnell, P.J., Calvert, C., Atzorn, R.,Wasternack, C., Leyser, H.M.O. and Bowles,DJ. (1996). Ethylene as a signal mediating thewound response of tomato plants. Science, 274:1914-1917.23. Murashige, T. and Skoog, F. (1962). A revisedmedium for rapid growth and bioassays withtobacco tissue cultures, Physiol. Plant., 51: 473-497.24. Zhao, S.S. and Zeng, M.Y. (1985). Studies onthe analysis of qinghaosu by high-pressureliquid chromatograph and spectrometry(HPLC). Planta Med., 51: 233-237.25. Balaji, K., Veeresham, C., Srisilam, K. andKokate, C. (2003). Azadirachtin, a Novelbiopesticide from cell cultures of Azadirachtaindica. J Plant Biotechnol., 5: 121-129.26. Orosa, M., Franqueira, D., Cid, A. and Abalde,J. (2001). Carotenoid accumulation inHaematococcus pluvialis in mixotrophicgrowth, Biotechnol. Lett., 23: 373-378.27. Fett-Neto, A.G., Zhang, W.Y. and Dicosmo, F.(1994). Kinetics of taxol production, growthand nutrient uptake in cell suspensions of Taxuscuspidata. Biotechnol. Bioeng., 44: 205-210.28. Ouyang, J., Wang, X.D., Zhao, B. and Wang,Y.C. (2005). Enhanced production ofphenylethanoid glycosides by precursor feedingto cell culture of Cistanche deserticola. Proc.Biochem., 40: 3480-3484.29. Lu, D.P., Zhao, D.X., Huang, Y. and Zhao, Q.(2001). The effect of precursor feeding onflavonoids biosynthesis in cell suspensioncultures of Saussurea medusa. Acta BotYunnanica., 23: 497-503.30. Evans, F.J. (1989). Trease and Evans’Pharmacognosy, Balliire Tindall, London.31. Woerdenbag, H. J., Pras, N., Bos, R., Visser,J.F., Hendriks, H. and Malingr, Th.M. (1991).Analysis of artemisinin and relatedsesquiterpenoids from Artemisia annua L. bycombined gas chromatography/massspectrometry. Phytochem. Anal., 2: 215-219.32. Durner, J., Shah, J. and Kleesing, G.F. (1997).
Salicylic acid and disease resistance in plants,Trends Plant. Sci., 6: 266-274.33. Malamy, J., Carr, J.P., Klessig, D.F. and Raskin,I. (1990). Salicylic acid: A likely endogenoussignal in the resistance response of tobacco toviral infection. Science. 250: 1001-1004.34. Shualev, V., Leon, J. and Raskin, I. (1995). Issalicylic acid a translocated signal of systemicacquired resistance in tobacco?, Plant Cell, 7:1691-1701.35. Dong, X. (1998). SA, JA, ethylene and diseaseresistance in plants. Curr. Opin. Plant Biol., 1:316-323.Current Trends in Biotechnology and Pharmacy, Vol. 2 (2) 341-348 (2008) 347ISSN 0973-891636. Gundlach, H., Muller, M.J., Kutchan, T.M. andZenk, M.H. (1992). Jasmonic acid is a signaltransducer in elicitor-induced plant cellcultures. Proc. Natl. Acad. Sci. U. S. A. 89:2389– 2393.37. Mueller, M.J., Brodschelm, W., Spannagl, E.and Zenk, M.H. (1993). Signaling in theelicitation process is mediated through theoctadecanoid pathway leading to jasmonicacid. Proc. Natl. Acad. Sci. U.S.A. 90: 7490–7494.38. Tamogami, S., Rakwal, R. and Kodama, O.(1997). Phytoalexin production elicited byexogenously applied jasmonic acid in riceleaves (Oryza sativa L.) is under the control ofcytokinins and ascorbic acid. FEBS Lett., 412:61–64.39. Menke, F.L.H., Parchmann, S., Mueller, M.J.,Kijne, J.W. and Memelink, J. (1999).Involvement of the octadecanoid pathway andprotein phosphorylation in fungal elicitorinducedexpression of terpenoid indole alkaloidbiosynthetic genes in Catharanthus roseus.Plant Physiol., 119: 1289–1296.40. Nojiri, H., Sugimori, M., Yamane, H.,Nishimura, Y., Yamada, A. and Shibuya, N.(1996). Involvement of jasmonic acid inelicitor-induced phytoalexin production insuspension-cultured rice cells. Plant Physiol.,110: 387–392.41. Brader, G., Tas, E. and Palva, E.T. (2001).Jasmonate-dependent induction of indoleglucosinolates in Arabidopsis by culturefiltrates of the nonspecific pathogen Erwiniacarotovora. Plant Physiol., 126: 849–860.42. Zhao, J. and Sakai, K. (2001). Multiplesignaling pathways mediate fungal elicitorinduced h-thujaplicin accumulation inCupressus lusitanica cell cultures. J. Exp. Bot.,54: 647–656.43. Wang, W. and Zhong, J.J. (2002). Manipulation
of ginsenoside heterogeneity in cell cultures ofPanax notoginseng by addition of jasmonates.J. Biosci. Bioeng., 93: 48-53.44. Suzuki, T., Mori, H., Yamame, T. and Shimuzu,S. (1985). Automatic supplementation ofminerals in fed-batch culture to high cell massconcentration. Biotech. Bioeng., 27: 192-201.348 Cell Cultures of Artemisia annua
Comparative Assessment of Technologiesfor Extraction of ArtemisininA summary of report commissioned throughMalaria Medicines Ventures (MMV)Malcolm CutlerFSC Development Services LtdAlexei Lapkin and Pawel K. PlucinskiDepartment of Chemical EngineeringUniversity of BathAugust 20062IntroductionTraditionally the majority of Artemisia annua grown worldwide is processed through solvent extraction,using hexane and petroleum ether. The only other considered alternative has been super critical CO2.Whilst petroleum ether and hexane are cheap to buy, both solvents represent a considerable safetyhazard and could be harmful to the environment.Following the world wide exposure Artemisia a. has received as a treatment for malaria, a number ofother technologies which claim to have greater efficiency, are safer or more environmentally friendly,are now being promoted. These processes were initially developed for the extraction of essential oils,fragrances and other pharmaceutical products, but initial trials on Artemisia a. have been successfuland indicate that they could be used as an alternative to the existing technologies.The lack of accurate data on hexane extraction, together with the emergence of these newtechnologies, makes it difficult for new and existing Artemisia producers to assess the efficiency,financial viability, safety and environmental impacts of the individual processes, and thereby select theprocess which is best for their application.The main objectives of this study have therefore been to highlight and examine the new technologieswhich could be used for the extraction of artemisinin and to develop a benchmarking procedurethrough which these new technologies can be compared to existing extraction methods e.g. hexane.The following review is based on the full paper which has been submitted for peer review and
publication in the Journal of Natural Products. A link to the full paper will be included on this websiteas soon as it has been published.Executive SummaryExtraction of artemisinin from Artemisia annua is currently mainly performed using hydrocarbonextraction processes. Due to the risk to human health, poor environmental performance, and thedangers of processes involving large volumes of volatile combustible fluids, there is a need to developalternative processes that would be able to compete in terms of efficiency and cost, and have little ornone of the drawbacks associated with hydrocarbon solvents. Extraction with supercritical carbondioxide (scCO2), ethanol, ionic liquids, and hydrofluorocarbon HFC-134a are compared against thebaseline case of extraction with hexane-ethyl acetate mixed solvent. For a variety of reasons a limitednumber of other solvents and/or technology providers have not been included, as insufficient data wasforthcoming,The study identified a number of key parameters relevant to the majority of stakeholders. Theseparameters were used in the multi objective assessment of the extraction technologies. Mainparameters included efficiency of primary extraction, potential for developing a compact ‘back-of-atruck’plant, capital cost of plants with specific biomass throughput, risk to human health and theenvironment, and potential for multi-crop operation. Amongst the compared alternative technologies,HFC-134 based extraction has been commercialised on the pilot (0.5 m3 vessels) and low-throughputindustrial (1 m3 vessels) scales, scCO2 extraction has been demonstrated on 1 L scale and pilot testsare underway. Considerable discrepancy in the available data on ethanol extraction was identified.Extraction of natural products with ionic liquids is an emerging technology and only initial lab tests datawas available. Therefore, the study compares the performance of a developed optimised process(hexane extraction) with performance of developing processes (scCO2 and HFC-134a) with that ofemerging new technology (ionic liquids).Extraction with HFC-134a and ionic liquids were shown to be the most promising replacement forhexane extraction. Ionic liquids have the potential to outperform hexane extraction in all main criteria.However, further research is urgently needed to develop methods of efficient regeneration of thesolvent, its recovery from the spent biomass and optimisation of the ratio of solvent to biomass.Extraction with HFC-134a provides much cleaner extracts with higher concentration of artemisinin andtherefore should lead to simpler recovery of artemisinin from the primary extract. The process iseconomic, low risk and has low environmental impact, with the main concern being the need for tightcontrol of the solvent inventory and of its recovery from the spent biomass. Extraction with ethanolwas shown to be consistently worse than that with hexane.The main area of uncertainty remains the process of recovery of artemisinin from the primary extractsproduced by the different extraction processes.3General aspects of artemisinin extraction
Artemisinin compounds have been predominately found in the upper parts of the Artemisia annuaplant, with the concentration of artemisinin said to peak just before or during full flowering, thedifference being attributed to climatic conditions, plant variety, or other, yet undetermined, factors.1More specifically, artemisinin and its precursor artemisinic acid have been shown to be localised in theglandular trichomes on the leaf surface.2,3 The main consequences of this are that (i) it may not benecessary to mechanically crush the plants prior to extraction for reasons other than to increase thepacking density, and (ii) the artemisinin content depends on the age of the leaf, since in older leavesthe glands were often found to be ruptured. Repeated harvesting of young leaves from the same plantwas shown to considerably increase the amount of artemisinin produced per area.4 Due to thephysico-chemical properties of artemisinin (low thermal and chemical stability of the endoperoxidefunction, low polarity and, hence, poor solubility in water and good solubility in organic solvents – seeAnnex III, Table 1), its extraction with non-polar solvents is necessarily complicated by simultaneousextraction of essential oils, chlorophylls and waxes. Therefore, the extraction step must be followed byseparation of artemisinin from the initial liquor. This is generally achieved by sequential crystallisationfrom an alcohol solution. Figure 1 shows the processing steps for artemisinin extraction by hexaneethylacetate mixed solvent with an identified system boundary for comparative assessment. The stepof separation of artemisinin from the raw extract is common to all extraction processes, however, thereare significant variations in the times and purification steps. See Annex III, Table 2, for artemisininproperties according to the monograph and Table 3, typical buyer specification..Figure 1. Scheme of artemisinin extraction with hexane-ethyl acetate mixed solvent.
Baseline process: extraction with hexane/petroleum etherIn the simplest hexane batch extraction, dried crushed leaf is soaked three-four times in fresh portionsof warm (30 – 45 ºC) hexane or petroleum ether, each extraction cycle taking between 10-48 hours.5,6
Under flow conditions (solvent percolation through packed biomass bed) at the same temperature theduration of each cycle can be reduced to 90-120 min.7 Different extraction regimes i.e., batch,percolation and continuous are shown in Figure 2. In order to improve the efficiency of extraction, asmall amount of co-solvent ethyl acetate can be added to the main non-polar hydrocarbon solvent.This increases the solubility of artemisinin in the solvent mixture by about two orders of magnitude.81 Laughlin, J. C.; Heazlewood, G. N.; Beattie, B. M. In Artemisia, Wright, C. W., Ed. Taylor & Francis: London and New York,2002.2 Duke, M. V.; Paul, R. N.; Elsohly, H. N.; Sturtz, G.; Duke, S. O. Int. J. Plant Sci., 1994, 155, 365-372.3 Duke, S. O.; Paul, R. N. Int. J. Plant Sci., 1993, 154, 107-118.4 Kumar, S. Natl. Acad. Sci. Lett., 2005, 28, 325-338.5 El-Sohly, H. N.; Jr, E. M. C.; El-Feraly, F. S.; El-Sherei, M. M. J. Nat. Products, 1990, 53, 1560-1564.6 Haynes, R. K. Current Topics In Medicinal Chemistry 2006, 6, 509-537.7 Khanuja, S. P. S., Personal Communication, 2006.8 Reitz, H.; Hill, C. Potential for the extraction and sale of artemisinin: Tanzania and/or Kenya; TechnoServ Tanzania: Arusha,
Tanzania, 5.10.2004, 2004.4Following extraction, the solvent is drained and spent biomass must be stripped of the residualsolvent. Biomass is said to absorb solvent in the ratio of 1 L·kg-1.9 Stripping of the solvent can beachieved by simple evaporation in air under natural convection, which is potentially hazardous andleads to the release of significant quantities of environmentally harmful volatile hydrocarbon, or moreefficiently by steam stripping followed by condensation and recovery of solvent. The recovery and reuseof the solvent reduces the environmental impact and improves the cost-effectiveness of theprocess. Vacuum stripping may also be used to avoid potential biomass decomposition under steamand to avoid downstream water-solvent separation.The obtained crude extract is flash-evaporated to 10 % of its initial volume and the remaining liquor isleft to stand at ambient temperature over ca. 48 h to crystallise crude artemisinin, allowing decantingof the liquor. Crude artemisinin is washed with warm hexane to remove the waxes and otherprecipitated impurities. In order to remove the waxes artemisinin is re-crystallised several times fromethanol-water azeotrope (95 wt% ethanol) in the presence of activated carbon adsorbent, followed byvacuum evaporation.9 Further purification is achieved by chromatography. An alternative method ofseparating artemisinin from the initial hexane extraction involves liquid-liquid extraction of artemisininrelated compounds from hexane into acetonitrile.5,10 This method is not considered due to thehazardous nature of acetonitrile to the environment and human health, rendering its large scale useunacceptable.Figure 2. Schematic diagrams of extraction plant options.
Batch and percolation extraction with alternative solvents: ethanol and ionicliquidsOrganic ionic liquids (organic equivalent of molten salts) is a new class of solvents, characterised bynegligible vapour pressure, nonflammability and possibility to tune solvation properties over a verybroad range. Since ILs lack two major drawbacks of the hydrocarbon solvents: vapour pressure andflammability, these solvents are often cited as a ‘green alternative’. Ionic liquids have been reported asa very good reaction medium for many organic reactions catalysed by chemical catalysts, as well asbio-catalysts. Despite a fairly recent development of this field of research, several chemical processesbased on ionic liquids have already been commercialized. However, there are very few publications onthe application of ionic liquids in extraction of bio-molecules, see review.11 The assessment is basedon the preliminary study undertaken by Bioniqs Ltd (UK) and funded by MMV.Two out of five screened ionic liquids showed promising performance: N,N-dimethylethanolammoniumoctanoate (DMEA oct, 1) and bis(2-methoxyethyl)ammonium bis(trifluoromethylsulfonyl)imide (BMOEA
bst, 2). The extraction process is similar to a standard liquid-solid extraction and was performed in a9 Vries, D. P. J. d.; Chan, P. N. G., Development and application of anti-malaria drugs, based on artemisinin, in Vietnam. 1998.10 El-Feraly, F. S.; El-Sohly, H. N. 4,952,603, 28.08.1990, 1990.11 Zhao, H.; Xia, S.; Ma, P. J. Chem. Techhl. Biotechnl., 2005, 80, 1089-1096.5batch regime at 25 ºC. Extraction with the DMEA oct solvent reached maximum solute concentrationafter 30 minutes of extraction. The observed concentration of artemisinin in solution was similar to thatobtained in the benchmark experiment with hexane at the same temperature.H3C N+
CH3 OHH O (CH2)6O-
CH3
H3CONH2
+ OCH3
NS - SOOOOF3C CF3
1 N,N-dimethylethanolammonium octanoate 2 bis(2-methoxyethyl)ammoniumbis(trifluoromethylsulfonyl)imideIn the case of the solvent 2 the rate of extraction was considerably slower than that with 1. However,the maximum concentration of artemisinin in solution was higher by 23 %.The obtained rate ofextraction with 2 was similar to the rate of extraction with n-hexane at the same temperature. Thus, incomparison with hexane, ionic liquid 1 gave a similar efficiency of extraction at a considerably fasterrate, whereas ionic liquid 2 gave a higher extraction efficiency at the same rate.The process of separation of artemisinin from the raw extract involved partitioning with water atambient temperature. This causes simultaneous separation of the oil fraction and crystallisation ofartemisinin. Crystallisation allows a separation of 82 % of the total extracted amount of artemisinin; theremainder is assumed to be lost with the oil phase. The crystals are 95 % artemisinin (by NMR) andare essentially free of solvent (not detectable by NMR). Separation was achieved in about 10 minutes.Work is now being undertaken to further refine the process since there are alternative options forrecovery of artemisinin from the primary extract and regeneration of ionic liquid solvent and solventrecovery from spent biomass had still to be optimised.A recent study of extraction by ethanol aqueous azeotrope at room temperature claimed highefficiency of extraction.12, Ethanol is potentially an attractive solvent due to its wide spread availabilityfrom renewable feedstocks. This is especially important for processes that are predominantly focusingon locally sourced materials to improve the overall process sustainability. A potential constraint on the
use of ethanol as a process solvent is its use as a spirit. This can be resolved by using a spikedsolvent, which is the customary practice in the EU. However, there are similar concerns with the use ofethanol as in the case of hexane: it is a flammable solvent, with high toxicity and high risk in use.The process based on ethanol extraction involves three sequential extractions with fresh solventportions followed by flash evaporation of solvent to reduce the volume of the primary extract. Someprocess optimisation is possible to reduce the ratio of solvent to biomass, as described in the originalpublication. It should be stressed that a very recent study of ethanol extraction of artemisinin in apressurised percolator extractor was unable to replicate high extraction efficiencies.13
Continuous extraction with supercritical CO2 and hydrofluorocarbon HFC-134aExtraction of artemisinin by scCO2 or sub-critical liquid CO2 has been described in the literature14
and large-scale trials are currently being undertaken. The efficiency of extraction of artemisinin frombiomass is reported to be quantitative, rapid and with higher selectivity compared with thehydrocarbon solvents extraction, based on the gram scale laboratory tests. However, there is widevariability in the efficiencies of extraction with scCO2 cited in the open literature, dependent on thescale of extraction, use of co-solvents, temperature and pressure of extraction, and superficial velocityof the solvent in the extractor. Thus, a lower efficiency than that obtained with a hydrocarbon solventwas reported in the absence of a hydrophilic co-solvent,14c whereas an earlier patent14b gives a widerange of extraction efficiencies, between 25-100 %, depending on the operating conditions. Such awide variation can be attributed to the accuracy of the analytical methods of determining artemisininconcentrations, variability in the operating conditions (pressure and temperature, duration of12 Rodrigues, R. A. F.; Foglio, M. A.; Júnior, S. B.; Santos, A. d. S.; Rehder, V. L. G. Quim. Nova 2006, 29, 368-372.13 Freyhold, M. van, Personal Communication, 2006.14 (a) Kohler, M.; Haerdi, W.; Christen, P.; Veuthey, J.-L. J. Chromatography A., 1997, 785, 353-360; (b) Wheatley, G. W.;Chapman, T. B. US 6,180,105B1, 2001; (c) Quispe-Condori, S.; Sánchez, D.; Foglio, M. A.; Rosa, P. T. V.; Zetzl, C.; Brunner,G.; Meireles, M. A. A. J. Supercritical Fluids, 2005, 36, 40-48; (d) Pulz, O. DE 10336056A1, 2005; Mengal, P.; Zwegers, J.;Monpon, B. French Patent 2,706,166, 1993.6extraction, concentration of co-solvent), variability in the water content of dry biomass, and variabilitybetween and within biomass samples. A typical value of overall extraction efficiency, including thesecondary purification by crystallisation, is about 80 %. The duration of extraction cycle dependsgreatly on the scale of extraction, use of co-solvents as well as more specific aspects of extractordesign that influence optimal solvent mass flow rate. Thus, 20 min extraction cycle was quoted for ca.1 L scale in the case of scCO2-ethanol system, whereas detailed kinetic study of artemisinin extractionwith scCO2 without co-solvents showed extraction times up to 2 h on a 0.2 L scale. A typical scCO2
process scheme is shown in Fig 2 – continuous extraction, pressure driven.Hydrofluorocarbon HFC-134a (1,1,1,2-tetrafluoroethane) isclassed as non-flammable and has zero ozone depleting potential. Itis amongst the most studied and utilised materials for which there isa life-cycle impact study.15 In Europe, Japan and USA, HFC-134a is
accepted by regulatory bodies for use as a solvent in the extractionof food flavourings. One drawback of the solvent is its high globalwarming potency factor – 1300 times larger than that of carbondioxide. Therefore, complete recycle and capture of the solventwithin a process is of significant importance.Hydrofluorocarbons are gases under normal conditions and areliquefied at relatively low pressures. Therefore, these solvents areideally suited for continuous extraction processes, when depressurisationof the solvent results in a rapid separation of theextracted material. Because of the modest pressures and lowoperating temperatures, the energy required for continuous depressurisation/pressurisation cycle is not high, resulting in lowenergy costs, low operating costs, and low greenhouse gasemission due to energy duty. Furthermore, re-circulation of solventcan be achieved without pumps, by establishing an isobariccondensation-evaporation cycle, thus avoiding the need forexpensive capital investment in thepump and the compressor. In thiscase the flow-rate of solvent dependson the efficiencies of condenser andevaporator, as well as percolationproperties of the packed biomass, see process scheme in Figure 2. Acommercial extraction plant (Phurua Natural Oils Limited) based on thisprinciple has been operating in Thailand since 2004.16
Extraction of natural compounds by (3) 1,1,1,2-tetrafluoroethane (HFC-134a) and (4)iodotrifluoromethane (ITFM) have been reported in theliterature,17 although not specifically of artemisinin.Physical properties of ITFM and HFC-134a are quitesimilar. However, because of the presence of weaker Chalogenbond with iodine, there are potential toxicityissues with the use of ITFM. More specifically, acutetoxicity of ITFM itself was found only in exposure to veryhigh concentrations (>25 %vol), but there is a significantrisk of cardiac sensitization at levels of exposure of 0.2%vol (2000 ppm) and there are suspicions of potentialcarcinogenic effect.18 ITFM has poor stability on sunlight,in the presence of artificial UV light and at temperaturesabove 100 ºC; its decomposition is facilitated in thepresence of copper and moisture.18b The products of15 McCulloch, A.; Lindley, A. A. Int. J. Refrigeration 2003, 26, 865-872.16 Wilde, P. F., Personal Communication, 2006.17 (a) Wilde, P. F. US Patent 5,512,285, 1996; (b) Wilde, P. F.; Skinner, R. E.; Ablett, R. F. WO 03/090520 A2, 2002; (c) Corr, S.J. Fluorine Chem., 2002, 118, 55-67.18 (a) Iodotrifluoromethane: toxicity review. http://www.nap.edu/catalog/11090.html (09.07.2006); (b) McCain, W. C.; Macko, J.Toxicity review for iodotrifluoromethane (CF3I). http://www.bfrl.nist.gov/866/HOTWC/HOTWC2003/pubs/R9902725.pdf(09.07.2006).Pilot plant for continuous extraction using HFCCC 134a at Ineos Fluor Ltd.
FFFFHHC
IFFF3 4A compact continuous extraction facility using HFC-134a.(Peter Wilde Associates Ltd.)7ITFM decomposition are HF, HI and COF2
which are highly toxic themselves and canreact further with organic matter leading toacute toxicity. Relatively poor stability ofITFM requires specific safety measuresduring storage and use. HFC-134a is aconsiderably more stable compound and hasbeen subject to long-term (5 yrs) stabilitytrials for its pharmaceutical applications.There are also significant differences in theextraction efficiency and prices. Forcomparison, 100 g of HFC-134a and ITFMwere quoted at £71.8 and £236 respectivelyby Aldrich catalogue in July 2006 (note thatAldrich prices cannot be used for scaling andonly given for comparison purposes). Basedon the data reported in the patent literature,ITFM is a much stronger solvent than HFC-134a - HFC-134a extracts little waxes andheavier oils, which are effectively extracted with the ITFM solvent. Due to this difference in solvationproperties, HFC-134a is expected to be more selective towards artemisinin than ITFM. By combiningITFM with co-solvents, including HFC-134a, it is possible to regulate the extraction efficiency.The data reported in this study are based on the information kindly provided by Ineos Fluor Ltd. Thesedata are supported by similar results obtained by Peter Wilde Associates Ltd.Comparison of extraction efficiencyBased on the available data for each extraction process, the efficiency of extraction relative tobiomass artemisinin content was estimated. The other important criteria are the duration of thecomplete extraction cycle, running and capital cost. These data are shown in Table 1. Among theseprocesses HFC-134a and ionic liquids based extractions may offer the cheapest running cost andcompetitive capital cost with the hexane extraction. Note, that capital cost includes the price of solventinventory.Table 1. Efficiency of extraction (mass artemisinin in crude crystalline extract relative to mass of artemisinin inbiomass), duration of extraction cycle (extraction to biomass exhaustion + loading of fresh biomass), running cost(includes only the cost of electricity and natural gas for heating for the main extraction process), capital cost(includes only equipment for the main extraction and solvent inventory).Extractionefficiency / %Duration ofextraction cycle / hRunning cost / €·kg-1
artemisininCapital cost for 2.5·106 kg
(biomass)·annum-1 / m€Hexane 60 8-10 28 0.7Ethanol 73 7 47 1.0Ionic liquids 79 2.5-6 22 0.3-1.0scCO2 82 3-6 42 4.1HFC-134a >62 6 19 1.0In order to compare the different extraction technologies, it is also necessary to compare the risk andsafety, environmental performance (green house gas emissions), and potential risk to human health.These parameters were normalised against the hexane baseline case and shown in Figure 3.Extraction with HFC-134a and ionic liquids compare best against hexane extraction. Green house gasemissions in the case of HFC-134a is due to the residual solvent in spent biomass, which representsannual loss of < 5 % of solvent inventory.Based on the extraction cycle time, the feasibility of a mobile ‘back-of-a-truck’ extraction facility wasassessed. Extraction with hexane and ethanol were deemed unfeasible due to too long period of timerequired to process the amount of biomass produced by several small holder farmers. In other cases,the small scale plants are potentially feasible. In the case of HFC-134a a 0.4 m3 compact plant hasalready been demonstrated.Commercial HFC-134a based extraction facility, Phurua Natural Oils Ltd,Thailand. (Courtesy of Peter Wilde Associates Ltd).80255075100125HFC-134aHexanescCO2Ionic liquidEthanolOperating costsCapital costsRisk & Safety GHG emissionsToxicity280Figure 3. Multi objective comparison of extraction technologies. Base line case of hexane extraction is 50 oneach axis. All parameters are scaled relative to hexane. An increase in performance, e.g. lower cost or risk, ortoxicity, is given by lower values.
Conclusions and RecommendationsThe main objectives of this study were to highlight new technologies which could be used for theextraction of artemisinin and to develop a bench marking procedure through which these newtechnologies could be compared to existing extraction methods e.g. hexane. In the case of ionicliquids extraction, which is an emerging technology, additional research had to be undertaken toprovide sufficient data for this comparison to be made.Whilst it is recognised that this study is not exhaustive its objectives have been achieved and mostimportantly, specific areas have been identified where additional support to technology providers,
together with other research, will bring speedy and practical results for end users.The results show that the new technologies, specifically HFC-134a and ionic liquids, have particularpotential, being equal or better in extraction efficiency, extraction time, running and capital costs thanhexane. Both technologies are also safer and are potentially more environmentally friendly (acceptingthat care has to be taken to ensure complete recycling and capture of the solvents).HFC-134a is a proven technology for the extraction of a wide range of other natural products and it isrecommended that support is provided to enable Artemisia field trails to be undertaken as soon as ispossible.Ionic Liquids show considerable promise and with additional research it is expected that a specificionic liquid can be identified which combines both high efficiency and speed of extraction. Theimmediate problem of regeneration of the ionic liquid solvent and solvent recovery from the biomass isnow being investigated. The use of ionic liquids in small scale, mobile, plants has been consideredand their potential replacement for hexane in existing extraction facilities should be furtherinvestigated.Mention must also be made of scCO2 which exhibits high efficiency and speed of extraction. Itslimitation is the higher capital and running costs, together with the need for experienced managementdue to the higher operating pressures, which will largely restrict it to larger, possibly multi-extraction,facilities.9A high level of interest has been expressed in the possibility of designing a viable, smaller, portableextraction facilities e.g. 20 tonne biomass throughput. The study shows that this is feasible and that aunit using HFC-134a has been operating for some time extracting other natural products. Potentiallyionic liquids could also be suitable for a mobile unit. The limitation for all technologies, to date, is thelong extraction time which would make such a unit non-viable. If the extraction time can be reduced,together with the possibility of undertaking an initial ‘rough’ primary extraction which can be ‘refined’ ina central process, a mobile unit could be viable. Recommended ongoing research includes work onreducing extraction and solvent recycling times and discussions with end users as to the futurepotential for small scale extraction.In the course of the study a number of other significant problems/opportunities have been identified.These are listed below and action is being advised/implemented to resolve these issues:1) Flexibility of extraction. Annex 1 details some of the pressures and limitations whichextractors face when implementing an artemisinin only i.e. a mono-extraction facility, andthe potential opportunities available through a multi-extraction facility approach. Inparticular is the long term potential for bio-refineries which use the whole plant biomass(including multi cropping).Action: Growers and extractors should investigate local projects involving bio-fuel andbio-refineries. Contact can also be made to the technology providers.2) Post harvest treatment, drying and storage of leaves – very little work has beenundertaken on this subject and variations in moisture content could have significant
effects on the extraction process (which has still to be fully quantified for the newtechnologies). There is also evidence that artemisinin levels can increase during field‘wilting’ prior to leaf stripping.Action: Trials with end users are now being planned.3) There is a requirement for independent artemisinin testing facilities to undertake purityand specification tests. The facility will also help extractors refine their process to meetthe process needs of their buyers.Action: Discussions are now being held with a number of institutions4) The confidence level of new and existing growers and extractors is low due to the recentlarge variations in artemisinin prices and uncertainties over the future demand/supply ofACTs. Whilst there is an urgent ‘market’ need for more ACTs, the organisation andfunding for their supply through the predominant public sector must be improved, clarifiedand speeded up. Until the access situation for ACTs is resolved growers, extractors andACT manufacturers are unlikely to make the investments necessary to meet long termsupply demands. This could also potentially result in the increase in supply of lowerquality and counterfeit drugs.Action: Organisations including WHO/RBM, MMV, Gates Foundation, are now very awareof this situation and actions are being planned/implemented.Comments and suggestions from readers of this report are requested and encouraged. Pleasecontact Malcolm Cutler on [email protected] or [email protected] who will either answer them directly orpass them to the relevant source. Questions relating directly to a specific technology should bedirected to the company(s) involved – see below for contacts.10TECHNOLOGY PROVIDERS - CONTACT DETAILSExtraction with HFC-134aIneos FluorRuncorn Technical CentrePO Box 13The HeathRuncornCheshire, WA7 4QFUKwww.ineosfluor.comContact: Simon GardnerE-mail:[email protected] ResearchRothamstedHarpendenHerts AL5 2JQUKContact: Bhupinder KhambayE-mail:[email protected] Associates Ltd“The Ol Factory”91 Front StreetSowerbyThirsk, YO7 1JPUKwww.wildeandcompany.co.ukContact: Peter WildeE-mail:[email protected]
Extraction with scCO2
Essential Nutrition LtdBank HouseSaltgrounds RoadBroughEast Yorkshire, HU15 1EFUKwww.essentialnuitritian.co.ukTom Chapman - E-mail: [email protected] Newbould – E-mail: [email protected] with Ionic LiquidsBioniqs LtdBiocentreYork Science ParkYork, YO10 5DGUKwww.bioniqs.comAdam Walker - E-mail: [email protected] Sullivan - E-mail: [email protected] 1
Flexibility of Extraction TechnologiesThe Need for Flexibility:Major producers/extractors may be able to make a financial and technical case for a one productextraction process i.e. 100% Artemisia, even with limited annual use. Smaller extractors will, however,almost certainly have to identify other crops which they can extract, during compatible seasons, withthe same equipment. This ability to process multi crops could be particularly essential with smallmobile extraction units.All extraction technologies represent a major investment in both fixed and working capital. The projectpromoter or investor, be they a local company or an international pharmaceutical, need to ensure areturn on this capital, which can only be achieved if there is an acceptable supply of raw material,matched by market demand. Outside of China and Vietnam Artemisia production is relatively new andwhilst the market has considerable potential to grow, there are a number of factors (over and abovelocal conditions, infrastructure etc) which a promoter/investor should investigate/ take into account,before committing to the project i.e. undertaking due diligence:1) Artemisia is only just beginning to emerge as a viable crop and much has still to be learntconcerning seed varieties, crop husbandry and post harvest technology. In addition the regions whereit is being grown vary greatly, with different climates, growing conditions and levels of infrastructure.Production is also widely spread over commercial farms and small holders.With any new crop this situation is not uncommon and with time there will be greater understanding ofproduction methods which, through “lessons learnt”, trials and development will result in consistencyand quality of production. In the meantime growers will most likely experience a steep learning curve.
2) At the present time the market is experiencing considerable uncertainty due, in part, to it beinglargely dependant on donor and aid funding (for the public sector). Predictions of present demand aretherefore effected more by the availability of funds, and their being utilised efficiently, rather than theultimate market need. This has already lead to under/over supply of artemisinin and consequenthighs/lows in prices, a situation which does not provide farmers with the confidence that they can relyon a long term, viable and sustainable market for their Artemisia crop. Therefore farmers, especiallycommercial farmers, will be very cautious of investing in Artemisia, particularly if they can achievegreater stability and returns from other crops. Small holders may have a more short term view but ifthey are “persuaded” to grow a new crop, such as Artemisia and in a few years are told that there isno market or the margins are below other cash crops, they are unlikely to return when the marketimproves. They have been let down too many times before!3) Much has also been recently published about the work surrounding synthetic or bioengineeredendoperoxides, or other promising antimalarial drugs and vaccines. Whilst the wording often used bythe press appears to promise that these advances will result in a total answer to the treatment ofmalaria, reality in likely to be very different. It is generally agreed that in the medium to long term theywill play an important role, but the majority of treatments will still rely on naturally produced artemisinin.This, often confused, situation does little to help give confidence to both Artemisia growers andinvestors in artemisinin extraction facilities.4) Buyers of artemisinin are presently limited to a small number of pharmaceutical companies.Sales through the public sector, financed through the Global Fund are presently limited to twoartemisinin based products with only one, Coartem, being a widely prescribed ACT. Delays inapproving other suppliers (and funds) are seriously restricting the expansion of Artemisia and theintroduction of new extraction facilities. Local ACT production with GMP certified facilities (following1st stage extraction which does not have to be GMP approved)), especially in Africa, could alsopotentially increase the number of ACTs dispersed.125) Whilst, to date, there have been no reported cases of resistance to Artemisia basedtreatments it has to be considered in the long term, given the history of malaria parasite resistance toother compounds. There are however rumours of reduction in efficacy in regions where Artemisiabased mono-therapies have been used for some years. It is for this reason that the WHO is nowstrongly resisting the continued supply of Artemisia based mono therapy treatments.6) The increase in yields per hectare of dry leaf and artemisinin, though new crop husbandry andseed developments, could result in greater levels of financial returns per hectare, which would allowfor mechanical harvesting/drying. This could then make Artemisia production viable for developingcountries (economies of scale), to the detriment of production in Africa. Although this is at the momentonly a hypothetical possibility, it could well become a reality in the future.An example of a crop which ‘bloomed’ and then ‘bust’ is pyrethrum. In the 1960’s East Africa was amajor producer, but with the advent of cheaper synthetic products the market for the natural productcrashed. Growers had to find other crops and extraction facilities shut, or in a few cases started toprocess other crops. Ironically one of these old facilities in Uganda is now reportedly being
refurbished to extract artemisinin, whilst the market for natural pyrethrum has started to increaseagain.It is for the above reasons that promoters/investors would well be advised to explore the possibility ofmulti use at an early stage in their planning.It should also be stressed that the extraction facility is only one part of the production chain andtherefore if multi use is planned, the growing of the necessary additional crops must be planned ingood time to meet the demand of the factory and market.What Other Products Can Be Considered?The range of other potential products will largely be dictated by where the extraction facility is locatedi.e. the climatic, growing conditions and the market. Most recent Artemisia production has been intropical climates, albeit at altitudes which allow temperate crops to be grown. The harvest times willalso have to complement those of Artemisia. Many essential oil producers also work closely with themarket on a contract basis, much in the same way as with artemisinin.Suitable crops can probably be best divided into three categories:1) Lower volume, high value herbs, spices, trees etc producing essential type oils e.g. BlackPepper, Paprika, Cinnamon, Fenugreek, Buchu, Pyrethrum, prunus Africana, roses2) Higher volume crops such as tea, coffee, tobacco again producing essential oils3) Commodity crops such as oil seed crops producing larger volumes of marketable oils for thefood, consumer and energy industry.Of particular interest is the development of the ‘bio-refinery’ principle where as large a proportion ofthe plant as possible is utilised to produce a range of compounds used as final products and asfeedstock to energy and chemicals industries. With Artemisia this could include separation of theessential oils to produce camphor, artemisia acid and other high-value compounds. The remainingbiomass i.e. stems and leaves after processing, could be used to produce bio-ethanol.The concept of the Biorefinery has been proposed for a number of years. The basic idea is verysimple; like oil, biofeedstocks contain a number of valuable building blocks. At present we cannotselectively separate these components in the way that oil is separated in the refining process intogasoline, kerosene, diesel, solvent fractions, naphta, light olefins, paraffins, etc. The reason why it ismore difficult in the case of biofeedstocks, is due to the complexity of the molecules and large numberof oxygen chemical functions, which present difficulty to conventional chemical approaches. At thesame time there is a growing demand for biodiesel, bioethanol, natural drugs and fragrances. Toincrease sustainability of the biofeedstock processes it is necessary to attempt to utilise the plantsmore completely. For example, seed oil can be used to produce biodiesel, lubricants or solvents,whereas remaining cellulosic biomass can be used to produce bioethanol or energy in combined heatand power plants (CHP) and can also potentially be converted into chemical intermediaries.13In Africa there are presently a number of interesting developments concerning the production ofJatropha (Jatropha integerrima – also known as Peregrina), which is an evergreen shrub now beingdeveloped (in plantations) for the extraction of its oils, primarily for bio-fuels. However, the plantextracts are also used for medicinal purposes, as a dye and the oil seed cake as a fertiliser. A reviewof the Jatropha websites will provide considerable information on the growing of and uses for, theplant. Also detailed are companies such as D1 Oils (www.d1plc.com) who have developments inAfrica.
Promoters and investors in new artemisinin extraction facilities therefore have a range of optionswhich could directly effect the viability of their facility. In the case of a dedicated artemisinin supplychain it may be that the extraction of other products does not make sense in the short/medium term,but in the long term may minimise some of the risks of being dependent on a sole product/crop.Other, smaller, facilities may only be viable if they can be used for different extractions over the year.The availability of new artemisinin extraction technologies could help increase the range ofequipment/solvents which will enable promoters/investors to make the decision if, or when, to makethis decision i.e. plan projects which will involve multi product extraction.Technology Choices:As this study has concentrated on artemisinin extraction the following is a brief indication as to theranges of products extracted and their suitability for a multi extraction plant, based on artemisinin.Contact should be made with the manufacturers for more information or a review of existing literaturefor the established solvents.Hexane and other solid-liquid, low pressure solvents:Other than through steam distillation, this group of solvents are the most widely used in a very widerange of extractions. Considerable information on their use can be identified through existingliterature, much of which can be gathered through the web.The study has identified the limitations of the use of hexane (and similar solvents), particularly due toits poor safety and environmental properties. However, hexane has the ability to extract a wide rangeof compounds, but with limited selectivity, and has relatively low capital investment.Supercritical CO2:scCO2 is an established technology, used to extract a wide range of products/compounds. As withhexane a literature review will identify the products successfully extracted by this technology e.g.essential oils, caffeine etc.The viable application of scCO2 technology to a multi-product plant will be very much dependent onthe scales and costs of equipment. Due to the higher operating pressures and therefore the highergrade/quality of components required, resulting in higher costs, it will normally only be viable for alarge scale plant. However, with improvements in technology and reduced manufacturing costs (forinstance in the Far East or South Africa) scCO2 technology could eventually be a viable option for amedium scale multi product extraction facility.Ionic Liquids:Ionic liquid is an emerging technology which to date has been trialled on only a limited number ofproducts. However, with a large and growing number of ionic liquids being identified, the potential for‘tuning’ the solvent to the specific extraction need is very attractive (although each new biomass mayrequire a different solvent for optimal process). For more information and advice contact should bemade with Bioniqs (www.bioniqs.com).Hydroflourocarbons (HFCs):HFC-134a has been used for a number of years to extract essential oils and other components from a
wide range of herbs, spices and flowers. Little or no adjustment is required to switch between differentbiomasses although extraction/cycle times will need optimising for each product (as with hexane andscCO2). Cleaning of the plant between different extractions can also be undertaken quickly and14efficiently. Annex II identifies products which have been successfully extracted using HFC-134asolvents. Further information can be obtained from:• Ineos Fluor (www.ineosfluor.com)• Phurua natural oils, Thailand (www.phurunaturaloils.com)• Wilde Associates Ltd (www.wildeandcompany.co.uk)• Bhubinder Khambay ([email protected])Ethanol:Ethanol is a very polar solvent and therefore has limited applicability. However, there are biofeedstockextraction processes based on ethanol. Its application is potentially effected by taxation regulations.Because of the natural limitation of the solvent, it is perhaps less suited towards multi-crop plants.15Annex IIThe following is a list of products/materials which have reportedly been extracted successfullyusing HFC134a. Contact should be made with the companies listed for more information on theseextractions and potential new product extraction.AgarAjowainAmbretteAniseedAngelica seedArtemisia Annua- Extraction and purification of artemisininAstaxanthin from shell fish & algiBaies roses (Schinus terebinthifolius L.)Basil – sweet (Ocimum basilicum spp)Black Pepper (Piper nigrum)Borage seedBrandy flavourBear flavourBuchu (Agathosma betulina)CaffeineCalendula- extraction and enhancement of isoharmentin 3-O glycoside content -Cannabis- extraction and purification tetrahydrocannabinol and cannabidiolCarawayCardamum seed (Elettaria cardomomum)Celery seedChampee oilChilliChocolateCinnamon- Extraction and fractionation (Cinnamomum zeylanicum)CloveCoffee oilCoriander seed (Coriandrum sativum)Cumin (Cuminum cyminum)Echium seedEvening Primrose seedFennel seed
FenugreekFrangipanierGalanga(l) – (Alpinia galangal, Languas galangal (Linn) Stuntz))GalbanumGarlicGinger oilGinko Biloba (Leaf)Ginseng RootGreen tea (Camellia sinensis)HopsJasmine concrete/grandiflorum/sambacLabdanumLemon peelLovage root/seedLycopene (from tomato skin)MaceMeadow foamMelissaMograMustard seedMyrrhNeem seedNoot katone from citrus oils16NutmegOats (rolled)Oolong tea (Camellia sp.)Orange peelOrris root (Iris pallida)PaprikaParsleyPatchouli (Pogostemon cablin (Blanco) benth)Phormium TenaxPilea MicrophyllaPink pepper berry oilPiperinePyrethrum flowersPyrethrum (olio resin)RaisonsRose oilRosemary- Extraction & enhancing rosmarinic & carnustic acidSage- S.LavandulaefoliaS. prpureaS.fruticosaS.officinalis LSandal wood (Santalum album)Saw PalmettoSea Onion (Siciliana Marittima)Star Anise (Illicium verum)St John’s WartTobacco Leaves- Extraction, purification of nicotine and adsorption onto ion-exchange polymer(chewing gum manufacture)Tonka beansTorreya NuciferaTuberose oilTurmeric
Vanilla (Vanilla planifolia)VetiverWheat germ oilWhiskey flavourWormwoodZanthoxylum Americanum - Genola17Annex IIITable 1. Physico-Chemical Properties of ArtemisininParameter ValueMolecular weight / g·mol-1 282.3Melting point / ºC 156-157Thermal stability in non-polar solvents / ºC 150Solubility in water @ pH 7 / g·L-1 0.063Solubility in water @ pH 7, 37 ºC / g·L-1 0.048*
Solubility in ethanol @ 21 ºC / g·L-1, 12Solubility in ethyl acetate @ 20 ºC / g·L-1 100Solubility in hexane @ 40 ºC / g·L-1 0.46Solubility in hexane – ethyl acetate (5 %vol) / g·L-1 33Solubility in N,N-dimethylethanolammonium octanoate / g·L-1 82Solubility in bis(2-methoxyethyl)ammonium bis(trifluoromethylsulfonyl)imide / g·L-1 110Octanol/water partitioning coefficient / log P 2.94* Value for triclinic crystals obtained by recrystallisation from cyclohexane; recrystallisationfrom EtOH (50 %vol) solution yielded orthorhombic crystals with the lower and slowersolubility in water.Table 2. Artemisinin properties according to monographArtemisinin content 97.0-102.0 (by IR) 98.0-102.0 (by TLC)TM / °C 151 - 154
[ ]200 C
D α +75 - +78° 10 mg⋅mL-1 solution in dehydrated ethanolLoss on drying < 5 mg⋅g-1 At 80 °CSulfated ash < 1 mg⋅g-1
Table 3. Typical artemisinin buyer specificationAppearance Colourless to almost white crystalline powderPurity by HPLC NLT 99% (or greater)Melting point / ºC 150 – 153Loss on drying at 80 ºC NMT 0.5% w/wResidue on ignition NMT 0.5% w/wOptical rotation +75 to +78There are no named impurities.2.31
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