Plant Cell Reports

Plant Cell Reports (1992) 11: 6 3 7 - 640

9 Springer-Verlag 1992

Antimalarial activity of A r t e m i s i a annua flavonoids from whole plants and cell cultures K. Chiung-Sheue Chen Liu 1, Shi-Lin Yang 2, M.F. Roberts 2, B.C. Elford 3, and J.D. Phillipson 2 1 School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC z Department of Pharmacognosy, The School of Pharmacy, University of London, London, U K 3 Institute of Molecular Medicine, Oxford, OX3 9DU, U K Received M a r c h 27, 1992/Revised version received September 16, 1992 - Communicated by N. Amrhein

SUMMARY. Cell suspension cultures developed from Artemisia annua exhibited antimalarial activity against Plasmodium falciparum in vitro both in the n-hexane extract of the plant cell culture medium and in the chloroform extract of the cells. Trace amounts of the antimalarial sesquiterpene lactone artemisinin may account for the activity of the n-hexane fraction but only the methoxylated flavonoids artemetin, chrysoplenetin, chrysosplenol-D and cirsilineol can account for the activity of the chloroform extract. These purified flavonoids were found to have ICs0 values at 2.4 - 6.5 x 10SM against P. falciparum in vitro compared with an IC50value of about 3 x 10SM for purified artimisinin. At concentrations of 5 x 10rM these flavonoids were not active against P. falciparura but did have a marked and selective potentiating effect on the antiplasmodial activity of anemisinin.

Keywords. Artemisia annua L., cell culture, methoxylated flavones, potentiation, artemisinin, antimalarial activity. INTRODUCTION. Artemisia annua L., used in traditional Chinese medicine,

has been investigated as part of a search for novel antimalarial agents. The major constituent responsible for its antimalarial activity is the sesquiterpene lactone artemisinin (QHS, qinghaosu) which has been found to be particularly active against chloroquine resistant Plasmodium falciparum in the treatment of cerebral malaria [China Cooperative Researach Group on Qinghaosu 1982;Jiang 1982]. Artemisinin is not readily synthesised and is obtained by isolation from plant material. Alternative sources of artemisinin have been investigated from plant cell cultures [Nair et al. 1986] and in vitro antiplasmodial activity has been demonstrated from extracts ofA. annua cell suspension Correspondence to: M. E Roberts

cultures [Tawfiq et aL 1989]. It has been reported that the antimalarial activity of artemisinin is markedly enhanced by the presence of the methoxylated flavone casticin [Elford et a1,1987].

Investigation of the flavonoids from the CHC13 extract of the aerial parts of A. annua has resulted in the isolation of 17 methoxylated flavonoids. The major constituents were chrysoplenetin 3(.~, chrysosplenol-D (4), cirsilineol (~) and eupatorin (fi) whilst casticin (~) was present only as a minor constituent [Yang et at. 1989]. In the present work the antimalarial activity of plant cell cultures of A. annua and their production of flavonoids and artemisinin is investigated.

MATERIALS AND METHODS Plant Cell Cultures. Seeds ofArteraisia annua L. were surface sterilised for 25 mins in 2% sodium hypochlorite solution containing Triton XI00 (0.1%) (British Drug Houses, London U.K.) followed by thorough washing (3x) with sterile distilled water. The seeds were germinated in sterile conditions and callus initiated by transfer onto agar (London Analytical and Biological Media Lab)containing Murashige and Skoog basal salts (Flow Laboratories U.K.) with sucrose (5%), kinetin (0,1 mg.1-I) and 2,4-dichlorophenoxyacefic acid (1 mg.l-X). The pH was adjusted to 5.8 - 5.9 before sterilisation by autoclaving. The callus cultures were maintained at 25~ under constant illumination and were sub-cultured every four weeks. After three subcultures, the calli (5g) were inoculated into liquid medium (50 ml) of the same ingredients (without agar) in 250 ml conical flasks and m~intained at 25~ on a rotary shaker operated at 120 rpm under constant illumination. The cell suspension cultures were transferred into fresh medium at 4 week intervals and were used for the isolation of constituents after three passages. Extraction of Cell Cultures for Antimalarial Constituents. Cells from suspension cultures were filtered and freeze dried and extracted successively with CHCI3 and MeOH, the conditioned medium from the cultures was extracted with n-hexane. These extracts were used for antimalarial tests and TLC detection of artemisinin. ]hey were also used for the isolation of a series of flavonoids. The chloroform extract was separated on a Sephadex LH20 column (Pharmacia, Sweden) using methanol as eluent. Of the five fractions obtained, fraction 3 contained flavonoids as the major components. This fraction was further separated by chromatography on a silica gel Column (silica gel 5 - 25 lam particle size) using N~ gas at 725 pPa. The elution of the compounds from the column started with chloroform and then continued with an increasing concentration of methanol

638 from 1-50%. The fractions eluted from the column with chloroform contained the flavonoids. These mixtures were separated to give single compounds using HPLC. Thin layer chromatography. Chromatography was carried out using sihca gel GFz~ (Merck Germany) thin layer plates with the following solvent systems and visualisation: 1. CHC13 : isopropanol (9:1) visualisation: 6% H2SO4 for the detection of artemisinin [Tawfiq et al.1989] and 2. CHC13 : MeOH (9:1) visualisation NH s for the detection of flavonoids. These systems were used to investigate extracts by comparison with authentic compounds [Yang et a1,1989]. High pressure liquid chromatography. HPLC analysis was performed using an Altex lsocratic Liquid Chromatograph (Model 330 Beckman, USA) and a p-Bondapak C18 column (7.9 x 300 mm, Waters Assoc. Inc., USA). The mobile phase was 80% MeOH at a flow rate of 1.0 ml.min"1. The column was calibrated with flavonoid reference compounds [Yang et al1989]. The column was also used for separation of the flavonoids which were finally purified using a Sephadex LH20 column. The HPLC Rt values and the yields of the flavonoids were as follows: chrysoplenetin (0.06% dw) 8 min; cirsilineol (0.05% dw) 9.5 min; chrysosplenol-D (0.06% dw) 11.5 min and artemetin (0.07% dw) 14 min. Structures were confirmed by uv, 1H-nuclear magnetic resonance and mass spectrometry data and reference to previously authenticated compounds.

aliquots of which were deposited directly into each well of a 96-well culture plate by means of a calibrated Hamilton syringe with a stepper attachment. Cultures (200 gl, 1 - 3% parasitaemia and 2% haematocrit) were maintained in a gas-tight box and harvested from 96-well micro-culture plates using an LKB harvester, A flat-bed Betaplate scintillation system [Potter et al, 1986] was used to assess incorporated radioactivity. Dose response curves were generated by calculating the mean incorporated activity (:1: I S.E. for 6 wells)/106 parasitised erythroeytes.

RESULTS AND DISCUSSION

Table I. Comparison of levels of major flavonoids in plants and cultures of Artemisia annua

Flavonoid

% DW of Plant

% DW of Suspension Cultures

---

0.07

casticin 2(~

0.01

---

chrysoplenetin 3~

0.04

0.06

chrysosplenol-D ( ~

0.10

0.06

cirsilineol 5(~

0.01

0.05

eupatorin (.~

0.02

---

artemetin ~

R2 0

CH30

C 1-130 ~

~ OI-I

-

~

R

3

0

Artemetin (i) R I = R 2 = R 3 = O C H 3 Cas~icm (2) R I = I/3 = O C H 3. I%2 = O K ChrT~oplenetm (3~ R i = R Z = OCII 3, R 2 = O H Chrysosplsrtol-D (43 R i = O C H q , lq2 = R B = O H

Cirsilmeol (5) R• = H, R 2 = OCH 3, R S = OH Eupatorin (6) R i = H, R 2 = O H , R 3 = O C H 3

Fig. 1. Major flavones of Artemisia annua plants and cell cultures In vitro test for antimalarial activity. A modification [Elford et a11987] of the in vitro assay based on the incorporation of [SH]-hypoxanthine into chloroquine resistant Plasmodium falciparum used by Desjardins et al. [1979] was used. The results shown in Table II were obtained using the modification of the Desjardins et al,assay as developed by O'Neill et al, [1985]. All other assays for antiplasmodial activity used the following modification [Elford et a1,1987] of the original technique with the ITO4 laboratory isolate of human malaria. Details of the culture conditions have been described by Kirk et a1,[1991]. All growth assays were carried out on tightly synchronised ring-stage infected erythrocytes pulse-labelled for 18 to 24 hours within a single cycle of maturation so that any additional effect of the flavones or artemisinin on the merozoite re-invasion process could be examined separately. Purified flavones and artemisinin were separately dissolved and serially diluted in dimethylsulphoxide (Analar BDH), 0.4/al

Detection and isolation of methoxylated flavones and artemisinin. Cell suspension cultures ofA. annua established as given in the Materials and Methods produced a limited number of flavonoids. These were artemetin Q.), chrysoplenetin 3(.3), chrysosplenol-D ~ and cirsilineol (~) (Fig. 1). These constituents were similar to the major methoxylated flavonoids found in the whole plant [Yang et al, 1989] except that casticin Q and eupatorin ~ were obtained only from the whole plant whereas artemetin was obtained only from the cell cultures, The levels of these methoxylated flavonoids obtained from the plant and cell suspension cultures are compared in Table I. The cell cultures did not appear to contain the more polar flavonoids which are found in the whole plant [Yang et a1.1992]. Trace amounts of the sesquiterpene artemisinin, which is mainly responsible for the antimalarial activity of the whole plant were detected by thin layer chromatography [Tawfiq et al 1989] in the n-hexane fraction of the medium from the cell suspension cultures but this was not confirmed by other standard isolation procedures. Antimalarial activity of cell culture extracts. In vitro antimalarial tests carried out on the fractions obtained from initial solvent extracts of both the plant cell material from suspension cultures and the conditioned medium indicated antimalarial activity. P. falciparum growth assays indicated that the n-hexane fraction from the conditioned medium and

639 the CHC13 extract from the filtered and dried cells were active (Table II). This acti ~,:ty could be due to the trace amounts of artemisinin detected in the n-hexane fraction but no artemisinin was detected in the CHC13 extract of the cells. This latter extract contained methoxylated flavonoids, some of which are known to be cytotoxic [Edwards et ai.1979; Arisawa et a/.1991].

Table II. The inhibition of incorporation of (3H)-hypoxanthine into P. falciparum in vitro by extracts of cell suspension of Artemisia annaa

Solvent Extract

In vitro antiplasmodial

activity, ICso ~ag.ml-I n-hexane (medium)

15.6

chloroform

14.5

methanol (cells)

500

Table III. Inhibitory effect of flavonoids alone or with artemisinin, incorporation of [3H]-hypoxanthine into P. falciparum IC~o Flavonoid alone (M x 10-5)

Artemisinin (M x 108) + flavonoid (5 gM)

artemisinin

---

3.3

artemetin 1(~

2.6

2.6

casticin 2(~

2.4

2.6

chrysoplenetin 3~

2.3

2.25

chrysosplenol-D 4~

3.2

1.5

cirsilineol 5~)

3.6

1.6

eupatorin 6(6(6~

6.5

3.0

enhance the antimalarial activity of artemisinin, this suggests a different mechanism of action for these compounds. Antimalarial activity of methoxylated flavones. The effect of artemetin 1(!), casticin (2.), chrysoplenetin 3(3), chrysosplenol-D 4(4) and cirsilineol 5~) on the inhibition of incorporation of [3H]-hypoxanthine into P. falciparum in vitro was therefore investigated since these compounds, except for casticin, were present in the chloroform extract. The results given in Table III and Fig. 2c show that all the flavonoids tested exhibited in vitro antiplasmodial activity. IC50 values were in the range of 2.3 - 6.5 x 10SM. At concentrations of 5 x 10tM these flavonoids had little or no effect on [3H-hypoxanthine incorporation by P. falciparum.

Potentiation of the antimalarial effect of artemisinin, Previous work (Liu et al, 1989) has shown that 5 ~M casticin has a marked and selective potentiating effect on the antiplasmodial activity of artemisinin giving rise to a 3 - 5 fold reduction in the IC50 value. We have now extended our study of other methoxylated flavonoids present in the plant and in cell suspension cultures. Table III and Fig. 2a,b also show the effects artemetin (.1.), casticin 2(~), chrysoplenetin 3(~), chrysosplenol-D 4(4.),cirsilineol 5(~)and eupatorin (.6.)(all at 5 ~M) on the antiplasmodial activity of artemisinin. To some extent, all the methoxylated flavonoids except eupatorin 6(.6)had a comparable potentiating effect on the in vitro antiplasmodial activity of artemisinin. These results are from a series of repeated experiments with further purified flavones. As we have shown previously (Elford et al, 1987), since the flavones have little effect on the dose response curves of chloroquine at concentrations which significantly

Structure related activity. The flavonoids in this study were all methoxylated at C-6 and C-7; however, except for eupatorin where only slight potentiation of artemisinin activity was observed, variations in methoxyl-hydroxyl substitution at C-3, C-3" and C-4" appeared to have limited effect on antimalarial activity. This suggests that for potentiating activity, if there is no substitution at C-3 as in eupatorin or cirsilineol (~) then unsubstituted oxygen at C-4' is required, as in cirsilineol (_~. If C-3 is substituted with a methoxyl, then ring-B may have either C-3', C-4"-dihydroxyl (chrysosplenol-D); C-3'-hydroxyl, C-4'-methoxyl (casticin); C-3"-methoxyl, C-4"-hydroxyl (cluysoplenetin) or C-3', C-4"dimethoxyl (artemetin) for potentiating activity. Why eupatorin has so little effect is not clear from the results. An extension of these results which are for only a limited number of flavonoid substitution patterns would be of interest as a prerequisite to investigations at the biochemical level of the mechanism by which these flavonoids exert their effect on the potentiation of artemisinin antiplasmodial activity. Biological activity. Flavonoids have a wide range of biological activities including the inhibition of selected enzymes such as phosphodiesterases, ATPases and protein kinases [Cody et al. 1988]; inhibition of low-density lipoprotein oxidation [de Whalley et al 1990]; and control of the activation of nodulation genes in Rhizobium [Firmin et aI. 1986].

640

50ooo, (a)

~

40000

20000 ~

o

Control

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k,\\

"E 10000 +eupatodn 0

10-9

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There are a number of questions which have not yet been answered in connection with these studies. It is still not known whether the flavonoids themselves have any significant effects on other events in the cell cycle of the parasite, i.e., reinvasion. Also, the in vivo activity of these flavonoids needs to be tested against P. berghei in mice. Whether the flavonoids which potentiated the in vitro activity of artemisinin against P.falciparum have any clinical significance in potentiating the activity of artemisinin in patients taking traditional medicines containing Artemisia annua is an unproved possibility. The observation that the maximal shift of artemisinin response was produced by chrysolpenol-D, which was found to be the most abundant flavone in plant material, may be pertinent to the traditional use of A. annua in the clinical treatment of malaria. Sufficient flavonoids have not been tested biologically to ascertain the structural requirements of flavonoids which potentiate the activity of artemisinin against P. falciparum. Acknowledgements. Financial support from the National Science Council of Taiwan ROC (K.C.S.C. Lui) and from Glaxo Group Research (S-L Yang) is gratefully acknowledged. B.C.E. is supported by the MRC. Purified artemisinin was a gift of the Minister of Public Health of the Chinese Peoples' Republic, provided via Dr. Peter Trigg, W.H.O., Geneva. Seeds of Artemisia annua L. were kindly supplied by Professor W. Peters, School of Hygiene and Tropical Medicine, University of London.

UQ=

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-~emelJn

REFERENCES 0

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10-4

(M)

Fig 2. Selective effects of a fixed concentration of purified flavone (5x106M) on the incorporation of 3H-hypoxanthine by P. falciparum (ITO4) exposed to increasing concentratiions of artemisinin (10"9 to 10TM). Synchronised ring-stage trophozoites were exposed to artemisinin (+ flavone shown in a and b) or flavone alone (c). Control levels of incorporation in the absence of either the flavone or artemisinin were identical within 5:2% (not shown). Slight antimalarial activity was exhibited by some flavones at 5x106M but this was not sufficient to account for the shift in the dose response curves for artemisinin

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