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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

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

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

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

r t i c l e i n f o

rticle history:eceived 6 January 2010eceived in revised form 8 February 2011ccepted 9 February 2011

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)

vailable online 9 March 2011

eywords:rtemisiaemperate regionerb

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.

rtemisininil yield

. Introduction

The genus Artemisia, comprised of small herbs and shrubs, isne of the largest and most widely distributed genera of the Aster-ceae family. Members of this genus have a distinctive scent or tastend are of unique botanical and pharmaceutical interest. Amongsteveral species of the genus, Artemisia annua L., commonly knowns ‘qinghao’ or ‘annual wormwood’, is an annual aromatic planthich is luxuriant in growth, erect and with bright green foliage

nd inflorescence of loose panicles. It is a traditional medicinal herb,ative to China and widely cultivated in Asia, America and EuropeOzguven et al., 2008).

A. annua contains many biologically active compounds; theost important is a sesquiterpene lactone with an endoperox-

de bridge called artemisinin. Together with its semi-syntheticerivatives such as arteether, antemether, artesunate and dihy-roartemisinin, this compound has been established as the mostotent antimalarial drug and possesses activity against drug-esistant strains of the malarial parasite (Plasmodium falciparum).urrently, artemether is recommended by the World Health Orga-

ization (WHO) for resistant and cerebral malaria (Wyk and Wink,004). In line with this recommendation, about 56 countries infrica, Asia and South America have actually adopted Artemisinin-ased 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).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.02.004

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

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 77.67

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xperiences temperate climate and is usually warm during sum-er and cold during winter. The monsoon usually breaks in June

nd continues up to September. The soil at the experimental site isandy loam with pH 6.8, 0.42% organic carbon, 145 kg ha−1 avail-ble nitrogen, 10.8 kg ha−1 available P, 130 kg ha−1 exchangeable.

.2. Experimental layout

The experiment was initiated in the first fortnight of March005. The treatments consisted of date of transplanting (DOT) of. annua cv. CIM-Arogya (Khanuja et al., 2005) at monthly inter-als over a period of two cropping seasons (Table 1). The seeds ofhe cultivar were obtained from CIMAP, Lucknow, India. Seedlingsere raised in 1 m × 1 m flat nursery beds by mixing the seedsith vermicompost that were subsequently broadcast over the

oil surface. The beds were thereafter irrigated with water usingprinklers.

Seedlings were transplanted when they were 60 days old with–8 leaves. Transplanting was done in plots of 3 m × 2 m replicatedhrice in a randomized block design at a spacing of 50 cm × 30 cm,iving the total number of plants per plot to be 40. The firstransplanting was done on 15th March 2005 and the last on 15thebruary 2006 in the first cropping year and similarly on the sameates in the second cropping season. The recommended dose ofompound fertilizers, N:P:K at 50:50:50 kg ha−1, respectively, waspplied. Before transplanting full dose of phosphorus, potassium

nd one-third of nitrogen along with vermicompost at the rate of.5 t ha−1 were applied. The remaining N was top dressed in twoqual splits at monthly intervals. The plots were irrigated afterransplanting and further irrigation was provided on requirementuch that moisture was maintained at field capacity.

able 2lant growth attributes, flowering behaviour and seed maturation of Artemisia annua for

Treatments Plant height (cm) Plant canopy (plant type) (

March 267 113

April 258 113

May 239 103

June 159 96

July 128 75

August 112 78

September 121 51

October 114 55

November 109 47

December 73 43

January 158 102

February 266 115

(CD p = 0.05) 11.45 10.55

14.40 29.20 78.009.21 26.10 78.662.40 21.00 73.00

2.63

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 TLC

tank 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),

years 2005–2007.

cm) Days to flower initiation Days to seed maturity

180–190 155160–170 129130–140 100100–110 100

90–100 9060–70 6260–70 26550–60 210

300–310 175280–290 235260–270 200240–250 160

– –

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 37

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ollowed by heating of the plates at 110 ◦C for 15 min on a CAMAGLC plate heater to visualize a pink colour of artemisinin. For quan-ification, TLC spot, corresponding to artemisinin, was measured at40 nm. Calibration curve of artemisinin was constructed by plot-ing concentration versus spot area of the compound (Gupta et al.,996).

.5. Essential oil analysis

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

The oil samples were subjected to GC analysis on a Nucon gashromatograph model 5765 equipped with FID using stationaryhase BP-20 (coated with a carbowax 20M) fused silica column30 m × 0.32 mm × 0.25 �m film thickness). Hydrogen was used as

carrier gas at the rate of 1.0 ml/min. Injector and detector temper-tures were 200 ◦C and 230 ◦C, respectively. The oven temperatureas programmed from 70 ◦C to 230 ◦C at 4 ◦C/min with an ini-

ial hold time of 2 min. Identification of components was done byomparing the retention times and retention indices to standardubstances and by peak enrichment on co-injection with authenticamples. The peak area percentage was computed from the peakreas without applying FID response factor correction.

.6. Statistical analysis

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

. Results

.1. Survival of plants

The data presented in Table 1 reveal the survival percentage ofransplanted seedlings, which was more than 70% throughout theropping year. Maximum survival of the seedlings was recordedhen they were transplanted in the first fortnight of May in both

ropping seasons (87.33%) followed by April transplanted crop83.00%). The least survival percentage was however recorded inhe seedlings that were transplanted in February (73%).

.2. Flowering behaviour and seed maturation

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

0.50 291.61 1732.46 2310.12 29.96

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 listed

in 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

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 4.36

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il were identified. The main components of this oil were camphor34.8%), borneol (11.4%), 1,8 cineole (7.9%), camphene (6.4%) and-caryophyllene (5.8%).

. Discussion

As evident from the result the survival of the plants was max-mum in the May transplanted plants and minimum in Februaryransplanted plants. This could be related to the frost conditionsrevailing in December when the nursery of February transplantedlants was raised which provided unhealthy plants.

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

The data for growth parameters revealed that the tallest andidest plants were observed in the transplants of March and Febru-

ry, respectively, while the shortest and narrowest in Decemberransplants. The low plant height and canopy size may be influ-nced by the chilling winter temperatures in this region whichrevail from mid November to January end. However, these resultsontrasted with similar studies conducted on another A. annuaultivar ‘Jeevanraksha’ in southern India (Bangalore), where a maxi-um plant height of 242 cm was recorded from October transplants

Singh et al., 2009). Additionally, in the North Indian Plains (Luc-now) the plants were recorded tallest (300 cm) in the January

ransplanted crop (Gupta et al., 2002). Whereas, in Turkey Ozguvent al. (2008) recorded the height of A. annua transplanted in theonth of April, with a range of 271.8–290.5 cm in a field experiment

onducted in the Cukurova region.

able 5il 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

: trace (<0.05%).

0.39 4.300.41 15.090.40 22.090.04 1.17

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.

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