Arch Pharm Res Vol 34, No 10, 1657-1661, 2011DOI 10.1007/s12272-011-1010-6

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Effects of Vegetative and Flowering Stages on the Biosynthesis of Artemisinin in Artemisia Species

Abdul Mannan1,2, Ibrar Ahmed1,3, Waheed Arshad1, Izhar Hussain2, and Bushra Mirza1

1Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan, 2Department of Pharmaceutical Sciences,COMSATS Institute of Information Technology, Abbottabad, Pakistan, and 3Institute of Molecular Biosciences, MasseyUniversity, Palmerston North, New Zealand

(Received April 4, 2011/Revised July 11, 2011/Accepted July 20, 2011)

Artemisinin is an endoperoxide sesquiterpene lactone, and has been proven to be very effec-tive in treating drug resistant cases of malaria, cancer, etc. The compound is obtained fromArtemisia species. In the current study, the effects of vegetative and flowering stages onartemisinin production were studied, to determine the proper harvesting time of naturallygrowing Artemisia species with the highest levels of artemisinin. Eight Artemisia speciesalong with two varieties were selected for this analytical work. The results showed that arte-misinin content was high in the leaves of Artemisia indica, A. sieversiana, A. roxburghianavar. roxburghiana, A. roxburghiana var. gratae, and A. parviflora at the flowering stage. Thehighest artemisinin content was measured in the leaves of A. dracunculus var. dracunculus.Upon comparisons of artemisinin content among the individual plant species, the highestamount of artemisinin was again in A. dracunculus var. dracunculus followed by A. sieversi-ana when harvested at the flowering stage. In overall comparisons, the plants at the floweringstage showed high levels of artemisinin, which is deemed the optimum harvesting time ofArtemisia species in Pakistan for maximum artemisinin content.Key words: Artemisia species, Artemisinin, Flowering stage, Vegetative stage

INTRODUCTION

Artemisinin is a multifunctional drug, required inlarge amounts to fight various diseases including mal-aria. The drugs derived from artemisinin have lowtoxicity because they target the protozoan cells thatcontain iron obtained from host hemoglobin. Thus,until the discovery of an effective antimalarial vaccine,artemisinin is the best option against drug resistantmalaria caused by Plasmodium falciparum. The useof artemisinin to treat malaria will continue, even byArtemisia annua extracts, until the disappearance ofresistant strains of Plasmodium (Mannan, 2009).

However, only a very minute concentration of arte-misinin is present in Artemisia annua, which is a

major obstacle in the commercialization of this sec-ondary metabolite (Abdin et al., 2003). It has been re-ported that about 25 Artemisia species are growingnaturally in Pakistan (Qureshi et al., 2002). And re-cently, artemisinin has been reported in many of theseArtemisia species. But again, the highest artemisininwas found in Artemisia annua (Mannan et al., 2010).By determining the proper harvesting time, an effici-ent amount of artemisinin can be obtained from theseArtemisia species.

Banyai et al. (2011) reported that the level of arte-misinin in Artemisia consistently changes. And accord-ing to another report, a dramatic increase in the com-pound occurred in the plant at the flowering stage(Baraldi et al., 2008). The best harvesting time wouldbe when artemisinin reaches its highest level in aplant. It has been reported that biosynthesis of arte-misinin is effected by light intensity and develop-mental stage (Ferreira et al., 1995). Artemisinin levelsin Artemisia plants increase gradually during thephase of vegetative growth and reach a highest level

Correspondence to: Abdul Mannan and Bushra Mirza, Depart-ment of Biochemistry, Quaid-i-Azam University, Islamabad,PakistanTel: 92-51-9064-3108, Fax: 92-51-9064-3136E-mail: abdulmannan_ka@yahoo.com (A. Mannan), bushramirza@qau.edu.pk (B. Mirza)

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in 6 month old plants with a long-day photoperiod(Zhang et al., 2006).

The present study was carried out to investigate arte-misinin content in different naturally growing Artemisiaspecies during their vegetative and flowering stages,in order to determine the effects of the plant develop-mental stages on artemisinin biosynthesis to establisha proper harvesting time.

MATERIALS AND METHODS

A total of eight Artemisia species including two var-ieties of Artemisia roxburghiana were collected duringtheir vegetative and flowering stages from the variousHimalayans, Karakoram, and Hindu Kush ranges ofNorthern Pakistan (Table I). These species were iden-tified at the Herbarium of Islamabad, by a taxonomist,Dr. Rizwana Aleem Qureshi, Department of PlantSciences, Quaid-i-Azam University Islamabad, Pakistan,according to the Flora of Pakistan (Ghafoor, 2002) andby comparisons with already identified herbariumsheets of the same Artemisia species preserved in theherbarium.

Artemisinin was extracted from the leaves, stems,and roots of all Artemisia species by following themethodology of Mannan et al. (2010). One gram ofeach plant tissue was taken and placed in an oven at60oC for three days. The dry weight of each sample wasmeasured, separately. These dried tissues were groundin 5 mL of toluene (Sigma) to make a homogenousmixture and were then placed in a sonicator (ElmaTM)for 30 min. During the sonication process, artemisininwas released into the toluene and separated from thecellular debris by centrifugation at 2000 × g and −8oCfor 20 min (Eppendorf centrifuge, model 5810R). Thesupernatant was decanted, and the whole process wasrepeated again with the pellets. Both supernatantswere pooled; the extracts were air dried before storingat −20oC for the quantification of artemisinin.

For HPLC analysis of artemisinin, dried extracts

and a standard of artemisinin were converted to itsQ260 derivative (Mannan et al., 2010). All the extractswere filtered through a 0.45 µm filter before HPLC in-jection. The mobile phase was prepared by combining45% (v/v) methanol and 55% 0.01 M sodium phosphatebuffer (pH 7.0). All samples were analyzed with aZorbax SB C18 column (150 × 4.6 mm × 5 µm; AgilentTechnologies USA). The flow rate of the mobile phasewas optimized to 1 mL/min. Artemisinin was detectedby using a Diode Array Detector (G1315B-DAD) at anabsorbance of 260 nm and its retention time was 12min.

The Artemisia species were harvested during thevegetative stage for measurement of artemisinin con-tent in the leaves, stems, and roots. The obtained arte-misinin data were compared to artemisinin concentra-tions of the same plant species harvested at the flower-ing stage, which have been already been reported byMannan et al. (2010). The percent dry weight for eachsample was determined, average values were calculat-ed for each part, and the data were analyzed usingANOVA and LSD.

RESULTS

Different authors have claimed different concentra-tions of artemisinin according to the vegetative andflowering periods of plants. The findings of someauthors are contrary to those of others. Woerdenbaget al. (1993) and Acton and Klayman (1985) reportedthe highest artemisinin level in Artemisia at the pre-flowering period while Pras et al. (1991) and Moraleset al. (1993) reported the highest artemisinin level atthe full flowering period. Recently, Arsenault et al.(2010) also reported the highest artemisinin level atthe full flowering stage. They further suggested thatthe differences in artemisinin concentration were dueto the differences in developmental stages and trichomematuration. It was previously confirmed that the com-pound is biosynthesized and then accumulated in the

Table I. Artemisia species collected from Northern PakistanS No Plant scientific name Location name Sampling point

1 A. moorcroftiana Kalam, Swat Saeed Abad2 A. vestita Galyat Donga Gali3 A. vulgaris Kalam, Swat Gatal4 A. indica Shangla, Swat Topseen5 A. sieversiana Soost, Northern Areas Aaeen Abad6 A. roxburghiana var. roxburghiana Rawalakot, Azad Kashmir Mujahid Abad7 A. roxburghiana var. gratae Murree, Rawalpindi Bhurban8 A. parviflora Rawalakot, Azad Kashmir Tararh Khal9 A. dracunculus var. dracunculus Abbass Pur, Azad Kashmir Abbass Pur

Effect of Plant Growth on Artemisinin Biosynthesis 1659

glandular trichome of Artemisia annua plants becauseof its high phytotoxicity to the plants themselves(Olsson et al., 2009).

The current study investigated practical changes inartemisinin levels in various Artemisia species growingin fields with respect to the plants’ vegetative andflowering growth. The obtained results were highlysignificant showing that the developmental stages ofthe plants significantly affected artemisinin levels inthe leaves and stems of all Artemisia species (Fig. 1).However, artemisinin concentrations in the roots werenegligible.

According to the results, artemisinin levels in theleaves of the four Artemisia species along with twovarieties including A. indica, A. sieversiana, A. roxbur-ghiana var. roxburghiana, A. roxburghiana var. gratae,and A. parviflora were increased at the floweringstage. Artemisinin was decreased in the two Artemisiaspecies of A. vestita and A. dracunculus var. dracunculusat this stage. There was no overall significant effect ofartemisinin content in the leaves of A. moorcroftianaand A. vulgaris (Fig. 1). The artemisinin levels in thestems of seven Artemisia species with two varietiesincluding A. moorcroftiana, A. vestita, A. vulgaris, A.indica, A. sieversiana, A. roxburghiana var. roxbur-ghiana, A. roxburghiana var. gratae, and A. parvifloraimproved at the flowering stage. While artemisininconcentration decreased in the stem of A. dracunculusvar. dracunculus at the flowering stage.

In the current study, the highest artemisinin level of0.32 ± 0.03% was measured in the leaves of A. dra-

cunculus var. dracunculus before the flowering period,followed by 0.27 ± 0.01% for the same Artemisia spe-cies but at the flowering period. Although A. indicahad the lowest artemisinin during all periods, theflowering stage showed a slight positive effect onartemisinin content.

By comparisons of artemisinin levels among the in-dividual plant species (leaves, stem, and roots) acrossthe genus, the highest artemisinin levels (0.16% and0.13%) were in A. dracunculus var. dracunculus. Thesecond highest artemisinin level of 0.08% was measuredin A. sieversiana harvested at the flowering stage,followed by 0.06% artemisinin in each sample of A.moorcroftiana, A. vestita, A. roxburghiana var. gratae,and A. parviflora harvested at the flowering stage(Fig. 2).

In an overall comparison, plant growth and flower-ing had positive effects on the biosynthesis of artemi-sinin. The plants had high artemisinin content at theflowering stage, which was deemed the best harvest-ing time.

DISCUSSION

According to the results, the leaves of four Artemisiaspecies including two varieties i.e. A. indica, A. sie-versiana, A. roxburghiana var. roxburghiana, A. rox-burghiana var. gratae, and A. parviflora, showed incre-ases in artemisinin at the flowering stage (Fig. 1).These results are in agreement with a study conduct-ed in Italy, in which Baraldi et al. (2008) claimed that

Fig. 1. Artemisinin concentration in various parts of Artemisia species before flowering and at flowering period (Alphabetson the bars are ranking orders after ANOVA and LSD).

1660 A. Mannan et al.

the artemisinin concentration of leaves at the flower-ing stage was almost double that found before theflowering stage. Another report showed that levels ofartemisinin were considerably effected by differentplant components between strains and these changescontinued during vegetative growth (Laughlin et al.,2002). Ferreira and Janick (1996) analyzed six clonesderived from Chinese material during both vegetativeand flowering stages. Five of the six clones showed thesame or higher levels of artemisinin at the floweringstage. Although the plants from all clones were har-vested on the same date, they were in different stagesof development.

Conversely, the leaves of two Artemisia species, A.vestita and A. dracunculus var. dracunculus, showeddecreases in artemisinin concentration at the flower-ing stage. Much research has been carried out showingthe positive effects of flowering on artemisinin levels.Still, a few scientists have reported the presence ofhigh amounts of artemisinin in leaves just two weeksbefore flowering (Singh et al., 1988; Laughlin, 1994).They further reported that maximum dry leaf massand whole plant yield may not occur until full bloom.

Although the exact mechanism is not known, recent-ly, two separate studies investigated the link betweenflowering and artemisinin. The flowering promoterfactor genes from Arabidopsis, fpf1, and the earlyflowering gene from Arabidopsis, CONSTANS, wereconstitutively expressed in A. annua, and althoughflowering was induced approximately two to threeweeks earlier in the transgenic lines, there was no

corresponding increase in artemisinin biosynthesis.These data suggest that there was no direct regulatorylink between flowering and artemisinin synthesis, andthat some other factor is likely to be contributing tothe observed increase in artemisinin content such asthe time of harvest, light intensity, or developmentalstage, which shift the plant to the reproductive stage(Ferreira et al., 1995; Wang et al., 2004, 2007; Achakzaiet al., 2009; Arsenault et al., 2010).

In this analytical work, the effects of flowering onthe artemisinin content of various Artemisia specieswere observed and five Artemisia species showed in-creases in artemisinin in their leaves at the floweringstage, while two Artemisia species showed decreasesin artemisinin at the flowering stage. For extraction ofartemisinin, Artemisia species should be harvestedwhen they are in full bloom stage, because at thattime, there are maximum leaves as well as flowers onthe plants, which will provide maximum dry mass forextracting artemisinin.

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