Immunodetection of artemisinin in Artemisia annua cultivated in hydroponic conditions

Phytochemistry, Vol. 33, No. 4, pp. 821-826. 1993 Printedin Great Britain.

IMMUNODETECTION CULTIVATED MONDHER JAZIRI,* BILLO DIALLO,?

003 1 -9422/93 $6.00 + 0.00 0 199j PergamonPress Ltd

OF ARTEMISININ IN HYDROPONIC MAURICE VANwwLEN,t

IN ARTEMISIA CONDITIONS

JACQUES HOMI?S,$

ANNUA

KAYO YOSHIMATSU

and KOICHIRO SHIMOMURA~ Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan; TLaboratory of Pharmacognosy and Bromatology, Universite Libre de Bruxelles, CP 205-4, Campus Plaine, B-1050 Bruxelles, Belgium; SLaboratory of Plant Morphology, Universite Libre de Bruxelles, Chaussee de Wavre 18.50,B-l 160 Bruxelles, Belgium (Receioed 9 September 1992) Key Word Index--Artemisia

oxyartemisinin

annua; Asteraceae; ELISA, HPLC; hydroponics; synthesis; artemisinin distribution; artemisinin.

sesquiterpenoid;

de-

Abstract-A highly specific and sensitive ELISA method was developed for the detection and semi-quantitative determination of artemisinin and its structurally related compounds in crude extracts of Artemisia annua. The antibodies were raised in rabbits using a lo-succinyldihydroartemisinin-BSA conjugate as immunogen. The peroxide linkage in the artemisinin molecule was critical in determining the antibody specificity. The working range of the assay was from 0.02 to 10 ng per assay. The cross-reacting material in crude plant extracts was evaluated by chromatographic methods combined with the immunoassay method. The distribution of artemisinin equivalents in five-weekold A. annua plants cultivated in hydroponic conditions was investigated. The highest artemisinin equivalent content (1.12% drywt) was found in the leaves of the upper parts of the plant.

INTRODUCTION

Malaria is still a major health problem in many developing countries. It is recognized that new drugs are urgently needed because of the resistance of Plasmodium to the current chemotherapy (chloroquine and melloquine) [ 11. Artemisinin (I), a sesquiterpene lactone containing a peroxide bridge, was isolated from the aerial parts of Artemisia annua L. [2]. It is effective against both chloroquine-resistant and -sensitive strains of P. falsiparum, and cerebral malaria [3]. The relatively low yield of artemisinin in A. annua has been one of the limiting factors for its clinical use. In addition, synthesis of this compound is not yet an economic feasibility [4]. Many laboratories are engaged in developing tissue cultures of A. annua to produce this active constituent [S-7]. However, in order to understand the biosynthetic pathway, knowledge on the distribution of precursors and closely related compounds to artemisinin in A. annua plant and tissue cultures is of interest. Regarding the analytical methods, artemisinin lacks a UV or fluorescent chromophore as well as functional groups for direct derivatization. Therefore, the development of sensitive and specific analytical methods for detection and determination of artemisinin and structurally related com*Present address: Laboratory of Plant Morphology, Universite Libre de Bruxelles, Chausste de Wavre 1850, B-1160 Bruxelles, Belgium. 4Author to whom correspondence should be addressed.

pounds is desirable. Several analytical methods have been reported for the determination of artemisinin such as (i) derivatization with acid or basic solutions to give UVabsorbing compounds followed by HPLC analysis of the derivatives [8-lo], (ii) HPLC with reductive electrochemical detection using either thin-layer gold mercury amalgam or dropping mercury electrodes [11-133, (iii) HPLC with reductive amperometric detection [14], and (iv) GC with an indirect detection of artemisinin based on the linear relationship obtained between the concentration of artemisinin and the respective peak areas for each of the two thermolabile degradation products [lS]. We now report that an indirect ELISA method has been established to detect artemisinin and closely related compounds in crude extracts of A. annua. The method was applied to investigate the distribution of artemisinin equivalents in five-week-old plants cultivated in hydroponic conditions. RESULTS AND DISCUSSION

Synthesis of conjugates

The presence of the peroxide function in the A-ring of artemisinin is a structural characteristic of this compound. The conjugation of artemisinin to the protein carrier was therefore achieved in three steps (Fig. 1) namely, reduction of artemisinin (1) to dihydroartemisinin (2) (step 1), succinylation of (2) (step 2) and conjugation of lo-succinyldihydroartemisinin (3) to the protein

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Fig. 1. Synthesis

of artemisinin

conjugates.

carriers (step 3) by the mixed anhydride method (for BSAconjugate as an immunogen) and by the carbodiimide method (for OVA-conjugate as a coating conjugate). ELISA for artemisinin

Four rabbits were used for immunization with the BSA-conjugate. All the antisera tested by ELISA showed a positive immuno-response against artemisinin. A typical standard curve for artemisinin is shown in Fig, 2. The working range of the assay extended from 0.02 to 10 ng per assay (=0.07 to 35 pmol per 50 ~1) with a final dilution of the antiserum = l/31250. Since the assay was designed for the analysis of crude extracts from plants and tissue cultures, it is particularly important to assess the extent to which the antiserum used cross-reacted with other artemisinin-related compounds. Inhibition studies showed that the antiserum

4 R= -cw&% 5 R= -cc&

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6 deoxyartemisinin

bound artemisinin (I), dihydroartemisinin (2), arteether (4) and artemether (5) equally well. Indeed, all of these compounds have the same A- and B-ring structure as artemisinin; the difference between these compounds is at the C-10 position. It was assumed that the protein used for conjugation at this position masked part(s) of the Cring of artemisinin. Therefore, it is not surprising that the antibodies failed to distinguish artemisinin from compounds 2,4 and 5. On the other hand, deoxyartemisinin (6) (lacking the peroxide linkage) exhibited no significant cross-reaction; thus the peroxide bridge in the molecule was critical in determining the antibody specificity. In addition, in order to demonstrate the specificity of the anti-artemisinin antiserum, crude extracts of 15 Asteraceae species were assayed by ELISA. Among these species tested, only A. annua showed a positive immunosignal. Evaluation of cross-reacting material in crude plant extracts

A hexane extract prepared from dried leaves of A. annua was subjected to silica gel CC and 25 fractions were Fig. 2. Standard

ELISA

curve

for artemisinin. B/Be is the Percentage of binding in the presence of artemisiain. Bo is 100% binding in the absence of artemisinin. The inserted figure gives a linearized Logit -log plot. Logit (B/Be) indicates ln[(B/Bo)/(lOO -B/Be)]. The standard deviation values at each pomt were below 0.9.

obtained and assayed by ELISA (Fig. 3). Fraction 9 eluted with hexane-chloroform (3: 17) showed the highest value. This fraction was subjected to HPLC for further investigation. Among the 20 fractions isolated by preparative HPLC, only three had a significant immunopositive signal (Fig. 4). The fraction corresponding to the

Immunodetection

of artemisinin

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[Fraction number] Fig. 3. Distribution of immuno-reactive constituents from Artemisia nnnua leaf extract separated by silica gel column chromatography.

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4

5

6

7

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10 11 12 13 14 15 16 17 18 19 20 Fraction number

Fig. 4. Distribution of immuno-reactive constituents of fraction 9 eluted from the silica gel column (see Fig 3) and separated by HPLC.

peak at R, 16.2 min which showed the highest response (91% of the total signal) was identified as artemisinin. It was confirmed by co-chromatography (HPLC) with an authentic sample using a photodiode array detector. Therefore, the immunoassay was found to be suitable for the specific and sensitive detection of artemisinin in crude extracts. In comparison with HPLC methods, pre-purification steps (solvent partition, CC fractionation) and derivatization were not required and the lower limit detection was 12.5 times lower (5 ng ml- ’ for HPLC with

reductive electrochemical detection [ 141 and 0.4 ng ml- i by immunoassay). Distribution old plants

of artemisinin equivalent in jive-week-

It has been reported that yields of artemisinin from the aerial parts of A. annua varied from a poor yield of 0.01% dry weight (in the European, Indian and American type) to a high yield of 0.5% dry weight (in the Chinese type)

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III five-week-old conditions.

[16]. Recently, up to 1.2% dry weight of artemisinin was reported in A. annua growing in Vietnam [17]. Singh et al. [18] reported the evaluation of A. annua strains for higher artemisinin production. It was concluded that the artemisinin yield was positively correlated with plant age. Due to the extreme sensitivity and specificity of the immunoassay, it was also possible to investigate the distribution of artemisinin in a single plant of A. annua. The artemisinin equivalent was evaluated in five-weekold plants cultivated in hydroponic conditions. The highest artemisinin equivalent was found in the leaves of the upper part of the plant (1.12% dry wt), almost 2.5 times higher than those of the other two portions (middle and lower) of the plant (Fig. 5). The artemisinin equivalent content in the main stem was 0.005% dry weight. These results agreed with those of Acton et al. [ 131 and Charles et al. [19] who showed that the highest artemisinin content determined by HPLC was found in the leaves compared to the stem. In addition, using polyclonal antibodies which could recognize the compounds closely related to artemisinin, 0.001% dry weight of artemisinin equivalent was detected in the root of five-week-old plant. TLC analysis showed that artemisinin was not detectable in the root extract. It has been reported that a low amount of artemisinin was accumulated in the roots of plants and of plantlets regenerated from callus cultures which were established from leaf explants [20]. The immunodetection of the unknown artemisinin-related compound(s) in fractions from root extract separated by preparative HPLC is now in progress. EXPERIMENTAL Synthesis of conjugates. (i) Reduction of 1 to 2. Compound 1(26 mg) was treated with NaBH, in EtOH (1 ml)

Lower parts of the plant analysed by ELJSA

plants of Artemisia unnua cultivated

in hydroponic

at 4” for 40 min. The reaction was stopped by addition of H,O (10 ml) and the mixt. extracted with CHCl, (3 x 10 ml). After evapn under red. pres. and purification by prep. TLC with CHCl,-MeOH (19: l), 23 mg of 2 (R, 0.66) was obtained. The structure of the synthesized compound was confirmed by ‘H NMR and EI-MS data. (ii) Synthesis of 3. Succinic anhydride (200 mg) was added to a soln of 2 (23 mg in 1 ml of dry pyridine). After one week incubation at room temp., the soln was evapd, the residue dissolved in H,O and then extracted with CHCl,. After evapn, the residue was purified by prep. TLC using CHCl,-MeOH (9: 1) and 20 mg of 3 (R, 0.86) was obtained. (iii) Synthesis of 3-BSA. The conjugate was synthesized by the mixed anhydride method [21]. Compound 3 (15 mg) was dissolved in DMSO (1 ml) and tri-nbutylamine (100 111)and cooled to 4’. After 30 min, isobutylchloroformate (25 ~1) was added and the mixt. kept at 4” for a further 15 min. The mixt. was then added dropwise to a soln of BSA (30 mg) in H,O (5 ml) which had been adjusted to pH 9.5 and cooled to 4’. The pH of the mixt. was adjusted immediately to 7-7.5 with NaHCO, (1 M) and kept overnight at 4”. After centrifugation, the supernatant was dialysed against dist. H,O (4 x 5 1;24 hr at 4”). (iv) Synthesis of 3-OVA. The conjugate was synthesized by the carbodiimide method t-221.A soln of 3 (5 mg) dissolved in DMSO (0.5 ml) was added to a soln of I-ethyl-3-(dimethylaminopropyl) carbodiimide (20 mg) in H,O (1 ml) which had been adjusted to pH 4.7 with HCl. After incubation for 1 hr at room temp., OVA (20 mg) in acid H,O soln (2 ml) was added. The mixt. was stirred for 16 hr at 4” and finally dialysed against H,O (4 x 5 1; 24 hr at 4”). Immunization schedule and antiserum production. Four rabbits were immunized with the BSA-conjugate emulsified with Freund’s complete adjuvant to give a final cone of 1 mgml- ‘. Freshly prepd mixt. (1 ml) was given

825

Immunodetection of artemisinin in 5-6 dorsal intradermic injections. Boosting injections were made at 2-week intervals in Freund’s incomplete adjuvant. Blood was collected 1 week after the third boosting injection. The blood was allowed to coagulate overnight at 4” and antiserum was obtained by centrifugation for 20 min at 20000 g. The antiserum was stored at - 20”. Bufferfor ELISA. Coating buffer: carbonate/bicarbonate pH 9.5 (0.05 M). Washing buffer: Pi-buffered saline (phosphate 0.01 M, NaC10.15 M, pH 7.2-7.4) containing 0.5 ml Tween 20 (PBS-T). ELISA procedure. Microtitration plates were coated overnight with 50 ~1 OVA-conjugate (0.3 pg ml- ‘) dissolved in coating buffer. The plates were then washed x 5 with PBS-T. The wells were satd by 100 ~1 of PBS-T containing 0.1% (w/v) of casein (C-PBS-T) and incubated for 2 hr at room temp. The plates were washed again and 50~1 of anti-artemisinin antiserum (diln l/31250 in CPBS-T) was added to each well. After incubation for 2 hr at room temp., the plates were washed x 5 with PBS-T and 50 ~1 of goat anti-rabbit peroxidase labelled (diln l/3000 in T-PBS) was added to each well. After incubation for 2 hr at room temp., the plates were again washed prior to adding 50plwell-’ of TMB peroxidase substrate soln (Kirkegaard & Perry Laboratories). After incubation for 30 min in the dark at room temp., the reaction was stopped by adding 50 ~1 of phosphoric acid (1 M) and the activity of the enzyme bound to the solid phase was measured by the A at 450 nm. For inhibition studies, serial diln of the standard, or samples to be assayed, was made in C-PBS-T, and serum in appropriate diln was added. After incubation overnight at 4”, 50 ~1 of the mixt. was added to each well on pre-coated plates and incubated for 2 hr at room temp. A standard of artemisinin was included on each plate and the artemisinin equivalent content in samples was calculated from each standard curve. Synthesis of6. Artemisinin (1) (20 mg) was treated for 24 hr with activated Raney Nickel (W4) (100 mg) in EtOH (2 ml) under H,. After filtration and evapn, the hexane-sol. material was purified by silica gel CC. The structure of the major product eluted by n-hexane was confirmed by ‘HNMR and EI-MS data (direct comparison with previously published data [23]). Plant material and cultivation in hydroponic conditions. Artemisia annua seeds obtained from the Walter Reed

Army Institute of Research, Washington, U.S.A., were surface sterilized with 2% (w/v) NaOCl for 10 min and washed x 2 with sterile dist. H,O. The seeds were sown in hydroponic facilities under the following conditions: 22”, 14 hr light/day (300 ~Em-2sec-‘). After 5 weeks of cultivation, plants were harvested (ca 45 cm height) and divided into 3 parts: upper, middle and lower part (15 cm each). The parts were sepd into shoots (cal cm), leaves, main stems and roots and lyophilized. ELISA for plant material. Dried material (lo-20 mg) was used for extraction with n-hexane. An aliquot of the n-hexane extract was evapd, the residue dissolved in DMSO, then diluted with an appropriate vol. of C-PBS and subjected to ELISA.

Fractionation of leaf extract. Dried leaf powder (10 g) was macerated in n-hexane (20 ml). After filtration and evapn, the residue was dissolved in n-hexane (10 ml). An aliquot (1 ml equivalent to 100 mg dry wt leaf material) was fractionated by silica gel CC. Elution was started with n-hexane and then continued with n-hexane containing increasing amounts of CHCI,. Fractionation was monitored by TLC, using CHCls-EtOAc (93:7). Visualization was carried out by spraying the plate with a 10% H,SO, soln and heating. Authentic artemisinin appeared as a blue fluorescent spot when examined under UV light at 355 nm. Aliquots from each fr. thus obtained were evapd, the residue dissolved in DMSO and a suitable diln made in C-PBS-T for ELISA. The fr. eluted with n-hexane-CHCl, (3: 17) which showed the highest response in ELISA was subjected to prep. HPLC using a TSK gel ODS 120T column (4.6 x 250 mm), particle size 5 pm, MeCN-H,O gradient (40-90% MeCN), flow rate 0.8 mlmin-‘, column temp. 35”, detection at 215 nm (artemisinin R,= 16.2 min). Each of the 20 frs sepd by HPLC were assayed by ELISA. Acknowledgements-The

authors thank Mr T. Kitazawa and MS A. Laurant for technical assistance. This work was supported in part by the ‘Fonds de la Recherche Fondamentale Collective’, Belgium and by Special Cooperation Funds for Promoting Science and Technology from Science and Technology Agency, Japan. REFERENCES

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