This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Industrial Crops and Products 34 (2011) 860– 864

Contents lists available at ScienceDirect

Industrial Crops and Products

jo ur nal homep age: www.elsev ier .com/ locate / indcrop

Influence of planting date on growth, artemisinin yield, seed and oil yield ofArtemisia annua L. under temperate climatic conditions

R.K. Vermaa,∗, Amit Chauhana, R.S. Vermaa, A.K. Guptab

a Central Institute of Medicinal and Aromatic Plants, Resource Centre Purara, PO, Gagarigole 263 688 Bageshwar, Uttrakhand, Indiab Central Institute of Medicinal and Aromatic Plants, PO CIMAP, 226 015 Lucknow, Uttar Pradesh, India

a r t i c l e i n f o

Article history:Received 6 January 2010Received in revised form 8 February 2011Accepted 9 February 2011Available online 9 March 2011

Keywords:ArtemisiaTemperate regionHerbArtemisininOil yield

a b s t r a c t

Artemisia annua L. is an annual aromatic antibacterial herb, with effective antimalarial properties due tothe presence of artemisinin. The intention of the present study was to establish plant survival, growthattributes, yield attributes and artemisinin yield of A. annua cv CIM – Arogya with different transplant-ing months in two cropping seasons (March 2005–February 2006 and March 2006–February 2007)under temperate climatic conditions of Himalaya, India. Artemisinin yield in the dried leaves was foundmaximum amongst the plants that were transplanted in March (24.39 kg ha−1) and minimum in thosetransplanted in November (3.39 kg ha−1).

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The genus Artemisia, comprised of small herbs and shrubs, isone of the largest and most widely distributed genera of the Aster-aceae family. Members of this genus have a distinctive scent or tasteand are of unique botanical and pharmaceutical interest. Amongstseveral species of the genus, Artemisia annua L., commonly knownas ‘qinghao’ or ‘annual wormwood’, is an annual aromatic plantwhich is luxuriant in growth, erect and with bright green foliageand inflorescence of loose panicles. It is a traditional medicinal herb,native to China and widely cultivated in Asia, America and Europe(Ozguven et al., 2008).

A. annua contains many biologically active compounds; themost important is a sesquiterpene lactone with an endoperox-ide bridge called artemisinin. Together with its semi-syntheticderivatives such as arteether, antemether, artesunate and dihy-droartemisinin, this compound has been established as the mostpotent antimalarial drug and possesses activity against drug-resistant strains of the malarial parasite (Plasmodium falciparum).Currently, artemether is recommended by the World Health Orga-nization (WHO) for resistant and cerebral malaria (Wyk and Wink,2004). In line with this recommendation, about 56 countries inAfrica, Asia and South America have actually adopted Artemisinin-based combination therapies (ACTs) as either their first or second

∗ Corresponding author. Tel.: +91 5222358723; fax: +91 5222357136.E-mail address: rajeshcimap@rediffmail.com (R.K. Verma).

line antimalarial treatment wherever the common quinoline andsulphadoxinepyrimethamine based drugs are no longer effective(Olliaro and Taylor, 2004). This has fuelled an increased demandfor ACTs several fold within the past years. Not surprisingly, thisdemand will continue to increase to several hundred million treat-ments within the next few years, which in itself has increased theinternational demand for artemisinin derivatives, leading to supplyshortages that are not likely to be met soon (Brisibe et al., 2008).The foliage and inflorescence of A. annua plants also yield an essen-tial oil, which has potential to be used in perfumery, cosmetics andaromatherapy and has also been reported to possess antifungal andantimicrobial activities (Woerdenbag et al., 1993; Wright, 2002).

As the synthetic production of artemisinin is not feasible theonly viable source of artemisinin is from the plant, A. annua. Inthe present study the plant was introduced to sub-temperate hillsof the western Himalayan region of India with the aim to deter-mine the best date of planting for optimal growth, biomass yield,artemisinin and essential oil contents, and essential oil and seedyields, which have not been reported up until now.

2. Materials and methods

2.1. Experimental site

The field experiment was conducted at the experimental farmof the Central Institute of Medicinal & Aromatic Plants, ResourceCentre, Purara, Bageshwar, Uttrakhand, India. The experimentallocation is situated at an altitude of 1250 m above sea level and

0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.indcrop.2011.02.004

Author's personal copy

R.K. Verma et al. / Industrial Crops and Products 34 (2011) 860– 864 861

Table 1Date of sowing, transplanting and percent survival of Artemisia annua.

Treatments Date of transplanting (DOPT) Mean temperature (◦C) Mean percent survival

I Year (2005–2006) II Year (2005–2006) Mini. Maxi.

March 15.03.05 15.03.06 1.00 19.10 77.66April 15.04.05 15.04.06 3.32 23.50 83.00May 15.05.05 15.05.06 8.00 24.50 87.33June 15.06.05 15.06.06 10.00 30.20 81.00July 15.07.05 15.07.06 12.40 32.00 78.33August 15.08.05 15.08.06 17.80 33.80 78.33September 15.09.05 15.09.06 16.60 34.20 82.00October 15.10.05 15.10.06 15.00 30.50 81.00November 15.11.05 15.11.06 15.00 23.40 77.67December 15.12.05 15.12.06 14.40 29.20 78.00January 15.01.06 15.01.07 9.21 26.10 78.66February 15.02.06 15.02.07 2.40 21.00 73.00(CD p = 0.05) – – 2.63

experiences temperate climate and is usually warm during sum-mer and cold during winter. The monsoon usually breaks in Juneand continues up to September. The soil at the experimental site issandy loam with pH 6.8, 0.42% organic carbon, 145 kg ha−1 avail-able nitrogen, 10.8 kg ha−1 available P, 130 kg ha−1 exchangeableK.

2.2. Experimental layout

The experiment was initiated in the first fortnight of March2005. The treatments consisted of date of transplanting (DOT) ofA. annua cv. CIM-Arogya (Khanuja et al., 2005) at monthly inter-vals over a period of two cropping seasons (Table 1). The seeds ofthe cultivar were obtained from CIMAP, Lucknow, India. Seedlingswere raised in 1 m × 1 m flat nursery beds by mixing the seedswith vermicompost that were subsequently broadcast over thesoil surface. The beds were thereafter irrigated with water usingsprinklers.

Seedlings were transplanted when they were 60 days old with6–8 leaves. Transplanting was done in plots of 3 m × 2 m replicatedthrice in a randomized block design at a spacing of 50 cm × 30 cm,giving the total number of plants per plot to be 40. The firsttransplanting was done on 15th March 2005 and the last on 15thFebruary 2006 in the first cropping year and similarly on the samedates in the second cropping season. The recommended dose ofcompound fertilizers, N:P:K at 50:50:50 kg ha−1, respectively, wasapplied. Before transplanting full dose of phosphorus, potassiumand one-third of nitrogen along with vermicompost at the rate of2.5 t ha−1 were applied. The remaining N was top dressed in twoequal splits at monthly intervals. The plots were irrigated aftertransplanting and further irrigation was provided on requirementsuch that moisture was maintained at field capacity.

2.3. Observations

In each plot 15 plants were selected randomly for observationson growth attributes such as plant height, canopy, days to flow-ering, and days to seed maturation (Table 2). Half the number ofplants in each plot was harvested when 10–15% of the plants flow-ered by cutting at a height of 60 cm above the ground and the otherhalf was left until seed maturation for seed yield. Biomass yield wasrecorded by manual separation of leaves and was calculated on thebasis of leaf/stem ratio after which the leaves were kept in the shadefor drying. The dried samples of leaves were stored in air tight poly-thene bags at room temperature for analysis of artemisinin content,which was undertaken following standard procedures (Gupta et al.,1996). On the basis of the value derived from artemisinin contentand from leaf yield, the theoretical yield of artemisinin in kg perhectare was calculated. The data presented in this paper are basedon two cropping seasons (that is, pooled data for 2005–2007).

2.4. Artemisinin analysis

Plant material (0.1 g) was sonicated with 5 ml n-hexane for15 min, filtered, evaporated and redissolved in 1.0 ml n-hexane andwere subjected to HPTLC (CAMAG, Switzerland and win CATS soft-ware) with CAMAG TLC Scanner 3 and recoated silica gel Plates 60F254 (Merck, Germany) with a layer thickness of 0.25 mm were used.A stock solution of pure artemisinin (1.0 mg ml−1) was prepared inn-hexane and different amounts of it were applied on TLC plates(20 cm × 20 cm). Chromatography was carried out in a glass TLCtank saturated with the mobile phase n-hexane:diethyl ether (1:1)and the plates were developed to a height of about 15 cm. Plateswere taken off, dried and spots were visualized by immersing theplates (CAMAG) immersion device in a freshly prepared mixtureof glacial acetic acid:concentrated H2SO4:anisaldehyde (50:1:0.5),

Table 2Plant growth attributes, flowering behaviour and seed maturation of Artemisia annua for years 2005–2007.

Treatments Plant height (cm) Plant canopy (plant type) (cm) Days to flower initiation Days to seed maturity

March 267 113 180–190 155April 258 113 160–170 129May 239 103 130–140 100June 159 96 100–110 100July 128 75 90–100 90August 112 78 60–70 62September 121 51 60–70 265October 114 55 50–60 210November 109 47 300–310 175December 73 43 280–290 235January 158 102 260–270 200February 266 115 240–250 160(CD p = 0.05) 11.45 10.55 – –

Author's personal copy

862 R.K. Verma et al. / Industrial Crops and Products 34 (2011) 860– 864

Table 3Yield attributes of Artemisia annua for years 2005–2007.

Treatments Fresh leaf yield (t/ha) Dry leaf yield (t/ha) Seed yield (kg/ha)

March 6.51 2.92 264April 4.43 1.99 222May 3.54 1.78 189June 2.43 1.09 174July 1.59 0.75 134August 1.45 0.66 83September 1.16 0.52 57October 1.11 0.50 31November 1.10 0.49 37December 1.10 0.50 29January 3.60 1.61 173February 5.4 2.46 231(CD p = 0.05) 0.17 0.12 29.96

followed by heating of the plates at 110 ◦C for 15 min on a CAMAGTLC plate heater to visualize a pink colour of artemisinin. For quan-tification, TLC spot, corresponding to artemisinin, was measured at540 nm. Calibration curve of artemisinin was constructed by plot-ting concentration versus spot area of the compound (Gupta et al.,1996).

2.5. Essential oil analysis

Essential oil was extracted by hydrodistillation for 3 h using aClavenger apparatus. The oil content (w/v, %) was estimated on afresh weight basis. The oil samples obtained were dehydrated overanhydrous sodium sulphate and kept in a cool and dark place beforeanalysis.

The oil samples were subjected to GC analysis on a Nucon gaschromatograph model 5765 equipped with FID using stationaryphase BP-20 (coated with a carbowax 20M) fused silica column(30 m × 0.32 mm × 0.25 �m film thickness). Hydrogen was used asa carrier gas at the rate of 1.0 ml/min. Injector and detector temper-atures were 200 ◦C and 230 ◦C, respectively. The oven temperaturewas programmed from 70 ◦C to 230 ◦C at 4 ◦C/min with an ini-tial hold time of 2 min. Identification of components was done bycomparing the retention times and retention indices to standardsubstances and by peak enrichment on co-injection with authenticsamples. The peak area percentage was computed from the peakareas without applying FID response factor correction.

2.6. Statistical analysis

The experimental data were analysed using the ‘analysis ofvariance’ technique. Estimation of the significance of differencesbetween means was based on a probability of p < 0.05 (Snedecorand Cochran, 1989).

3. Results

3.1. Survival of plants

The data presented in Table 1 reveal the survival percentage oftransplanted seedlings, which was more than 70% throughout thecropping year. Maximum survival of the seedlings was recordedwhen they were transplanted in the first fortnight of May in bothcropping seasons (87.33%) followed by April transplanted crop(83.00%). The least survival percentage was however recorded inthe seedlings that were transplanted in February (73%).

3.2. Flowering behaviour and seed maturation

The data in Table 2 showed that both flowering and seedmaturity varied with the date of transplanting. There was flower

initiation as early as 50–60 days in plants that were transplantedin October. On the other hand, it took 300–310 days in those trans-planted in November. In case of seed maturation the plants whichwere transplanted in August showed minimum number of days forseed maturation (62 days) while those transplanted in Septembershowed maximum number of days (265 days).

3.3. Growth parameters

The data on influence of transplanting date on growth param-eters namely, plant height and canopy is provided in Table 2. Theplant height ranged from 73 to 267 cm. The maximum plant height(267 cm) was recorded in plants transplanted on 15th March ascompared to the shortest in plants transplanted on 15th December.While the canopy was widest in February plant (115 cm) followedby March and April transplanted plant (113 cm each). The lowestwidth of the canopy was recorded in December transplanted plants(43 cm).

3.4. Yield parameters

Yield attributes of A. annua cv. CIM-Arogya from temperateregion of western Himalaya are presented in Table 3. In thepresent study fresh and dry leaf yields were maximum in theMarch-transplanted plants (6.51 t ha−1 and 2.92 t ha−1, respec-tively) followed by those transplanted in February (5.4 t ha−1 and2.46 t ha−1, respectively). The lowest fresh and dry leaf yieldswere obtained amongst the November and December transplantedplants. Similar trends were also recorded in the seed yield in thepresent study. The highest seed yields were recorded in Febru-ary and March transplanted plants (231 and 264 kg ha−1) whilethe lowest seed yield was obtained in the December transplantedplants (29 kg ha−1).

The artemisinin content in A. annua dried leaves is presentedin Table 4. The artemisinin content showed a small and consis-tent variation throughout the two cropping years. The artemisinincontent ranged from 0.83% in the March transplanted plants to0.63% in June transplanted plants. Because of the small variationin artemisinin content the highest yield of artemisinin (Table 4)occurred at the same time as maximum leaf yield (Table 3).

The yield of artemisinin was also influenced by the time oftransplanting and showed maximum yield from spring (March)transplants (24.39 kg ha−1) and least in winter (November) trans-plants (3.39 kg ha−1).

The quality characteristics and quantity [the quantity is listedin Table 4 and is not discussed directly below] of the essential oilobtained from A. annua cv. CIM Arogya is presented in Table 5. Theessential oil obtained from A. annua grown in temperate condi-tions of Himalaya was analysed by GC-FID. The essential oil contentamounted to 0.38%. Sixteen components accounting for 73.1% of the

Author's personal copy

R.K. Verma et al. / Industrial Crops and Products 34 (2011) 860– 864 863

Table 4Artemisinin and oil yield of Artemisia annua for years 2005–2007.

Treatments Artemisinin (%) Artemisinin (kg/ha) Oil (%) Oil yield (kg/ha)

March 0.83 24.39 0.40 26.88April 0.79 15.76 0.40 17.88May 0.72 12.97 0.40 14.27June 0.63 6.87 0.40 9.95July 0.64 6.22 0.41 6.68August 0.66 4.38 0.44 6.38September 0.73 3.82 0.40 4.67October 0.74 3.70 0.43 4.82November 0.72 3.39 0.39 4.36December 0.71 3.56 0.39 4.30January 0.69 11.22 0.41 15.09February 0.74 18.22 0.40 22.09(CD p = 0.05) 0.07 1.49 0.04 1.17

oil were identified. The main components of this oil were camphor(34.8%), borneol (11.4%), 1,8 cineole (7.9%), camphene (6.4%) and�-caryophyllene (5.8%).

4. Discussion

As evident from the result the survival of the plants was max-imum in the May transplanted plants and minimum in Februarytransplanted plants. This could be related to the frost conditionsprevailing in December when the nursery of February transplantedplants was raised which provided unhealthy plants.

The flowering and seed setting was more or less comparablewith the climates of subtropical regions that occur in the Indo-Gangetic plains area (Kumar et al., 2004).

The data for growth parameters revealed that the tallest andwidest plants were observed in the transplants of March and Febru-ary, respectively, while the shortest and narrowest in Decembertransplants. The low plant height and canopy size may be influ-enced by the chilling winter temperatures in this region whichprevail from mid November to January end. However, these resultscontrasted with similar studies conducted on another A. annuacultivar ‘Jeevanraksha’ in southern India (Bangalore), where a maxi-mum plant height of 242 cm was recorded from October transplants(Singh et al., 2009). Additionally, in the North Indian Plains (Luc-know) the plants were recorded tallest (300 cm) in the Januarytransplanted crop (Gupta et al., 2002). Whereas, in Turkey Ozguvenet al. (2008) recorded the height of A. annua transplanted in themonth of April, with a range of 271.8–290.5 cm in a field experimentconducted in the Cukurova region.

Table 5Oil composition of Artemisia annua.

S.N. Compounds Area (%)

1. �-Pinene 0.92. Camphene 6.43. 1,8 cineole 7.94. Cis-�-ocimene 0.25. �-Terpinene t6. 3-octanol 1.77. Trans-sabinene hydrate 0.98. Artemisia alcohol 0.29. Camphor 34.810. Linalool 0.411. �-Caryophyllene 5.812. Terpinen-4-ol 1.013 �-Terpineol t14. Borneol 11.415. Germacrene-D 1.416. Caryophyllene oxide 0.1

Total identified 73.1

t: trace (<0.05%).

The yield attributes of A. annua cv. CIM-Arogya were alsoaffected by the temperature. The maximum fresh and dry leafyields were recorded in spring (March) transplants while the low-est in November and December transplants this may possibly bedue to the temperature falling in winter (cold stress). Comparableresults of high growth yield were earlier reported from Switzerland(Delabays et al., 1993), Germany (Liersch et al., 1986) and USA(Charles et al., 1990).

The consistent variation within two cropping season as recordedin the results was contrasted with an artemisinin content whichranged from 0.42% to 1.12% in Lucknow and 0.43% to 0.94% in Banga-lore, respectively, in the cultivar ‘Jeevanrakshak’ of A. annua (Kumaret al., 2004; Singh et al., 2009). Although the artemisinin content ofthe cultivar ‘CIM-Arogya’ was found to be lower (0.83%) when cul-tivated in a north temperate region it was more even in variation,ranging from 0.63 to 0.83% establishing it as a more stable cultivarwhich may be due to its genetic makeup.

Moreover the yield of artemisinin also showed a similar trendand was highest in spring transplants and least in winter trans-plants and least in winter transplants which was opposite to thetrend of transplanting in Bangalore (Singh et al., 2009).

Further, earlier studies on essential oil composition of A. annuafrom subtropical region of India also showed similar qualitativecomposition as the oil of A. annua investigated in present study,but the relative percentages of components were different (Bagchiet al., 2003). The essential oil in the present investigation was akinto Vietnamese Artemisia oil which was found to be rich in Camphor(Teixeira da Silva, 2004).

To conclude, A. annua is a very important source of artemisininworldwide. Research on cultivation, breeding and chemical extrac-tion processes for obtaining higher yield and artemisinin contentare currently under way in many countries. In warm climates ofthe world, this plant is easily cultivated; however the biologicallyactive compounds in the plant are affected by temperature, ecolog-ical factors, cultivation methods and plant ontogeny. This study hasshown that it is possible to grow A. annua L. and that it has poten-tial to be a profitable crop in the temperate western Himalayanregion. The spring transplanted crops performed better than theautumn/winter transplanted crops in terms of both growth andyield parameters.

Acknowledgements

The authors are grateful to the Director, CIMAP for providingencouragement and financial help during the study.

References

Bagchi, G.D., Haider, F., Dwivedi, P.D., Singh, A., Naqvi, A.A., 2003. Essential oilconstituents of Artemisia annua during different growth periods at monsoonconditions of subtropical north Indian plains. J. Essent. Oil Res. 15, 248–250.

Author's personal copy

864 R.K. Verma et al. / Industrial Crops and Products 34 (2011) 860– 864

Brisibe, E.A., Uyoh, E.A., Brisibe, F., Magalhaes, P.M., Ferreira, J.F.S., 2008. Buildinga golden triangle for the production and use of artemisinin derivatives againstmalaria in Africa. Afr. J. Biotechnol. 7 (25), 4884–4896.

Charles, D.J., Simon, J.E., Wood, K.V., Heinstein, P., 1990. Germaplasm variationin artemisinin content of Artemisia annua using an alternative method ofartemisinin analysis from crude plant extracts. J. Nat. Prod. 53, 157–160.

Delabays, N., Benakis, A., Collet, G., 1993. Selection and breeding for high artemisinin(quinghaosu) yielding strains of Artemisia annua. Acta Horticult. 330, 203–207.

Gupta, M.M., Jain, D.C., Verma, R.K., Gupta, A.P., 1996. A rapid analytical method forthe estimation of artemisinin in Artemisia annua. J. Med. Arom. Plant Sci. 18, 5–6.

Gupta, S.K., Singh, P., Bajpai, P., Ram, G., Singh, D., Gupta, M.M., Jain, D.C., Khanuja,S.P.S., Kumar, S., 2002. Morphogenetic variation for artemisinin and volatile oilin Artemisia annua. Ind. Crops Prod. 16, 217–224.

Khanuja, S.P.S., Paul, S., Shasany, A.K., Gupta, A.K., Darokar, M.P., Gupta, M.M., Verma,R.K., Govind, R., Kumar, A., Lal, R.K., Bansal, R.P., Singh, A.K., Bhakuni, R.S., Tandon,S., 2005. Genetically tagged improved variety ‘Cim-Arogya’ of Artemisia annuafor high artemisinin yield. J. Med. Arom. Plant Sci. 27, 520–524.

Kumar, S., Gupta, S.K., Singh, P., Bajpai, P., Gupta, M.M., Singh, D., Gupta, A.K., Ram,G., Shasnay, A.K., Sharma, S., 2004. High yields of artemisinin by multi-harvestof Artemisia annua crops. Ind. Crops Prod. 19, 77–90.

Liersch, R., Soicke, C., Stehr, C., Tullner, H.V., 1986. Formation of artemisinin inArtemisia annua during one vegetative period. Planta Med. 52, 387–390.

Olliaro, P.L., Taylor, W.R., 2004. Developing artemisinin based drug combinations forthe treatment of drug resistant falciparum malaria: a review. J. Postgrad. Med.50, 40–44.

Ozguven, M., Sener, B., Orhan, I., Sekeroglu, N., Kirpik, M., Kartal, M., Pesin,I., Kaya, Z., 2008. Effects of varying nitrogen doses on yield, yield compo-nents and artemisinin content of Artemisia annua L. Ind. Crops Prod. 27 (1),60–64.

Singh, R., Puttanna, K., Rao, P., Gupta, A.K., Gupta, M.M., 2009. Evaluation of influ-ence of planting date on growth, artemisinin yield and seed yield of Artemisiaannua under Bangalore agro-climatic conditions. Arch. Agron. Soil Sci. 55 (5),569–577.

Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods, 8th ed. Iowa State Univer-sity Press, Ames, IA.

Teixeira da Silva, J.A., 2004. Mining the essential oils of the Anthemideae. Afr. J.Biotechnol. 3 (12), 706–720.

Woerdenbag, H.J., Bos, R., Salomons, M.C., Hendriks, H., Pras, N., Malingre, T.M.,1993. Volatile constituents of Artemisia annua L (Asteraceae). Flavour Frag. J.8, 131–137.

Wright, C.W., 2002. Artemisia. Taylor and Francis Inc., New York.Wyk, B.E., Wink, M., 2004. Medicinal Plants of the World. Timber Press, Portland,

OR, p. 188.