Biosynthesis of andrographolide in Andrographis paniculata

Phytochemistry 71 (2010) 1298–1304

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Biosynthesis of andrographolide in Andrographis paniculata Nishi Srivastava, Anand Akhila * Central Institute of Medicinal and Aromatic Plants, Lucknow 226 015, India

a r t i c l e

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Article history: Received 7 April 2010 Received in revised form 25 May 2010 Available online 16 June 2010 Keywords: Andrographolide Andrographis paniculata Diterpene lactone Biosynthesis Deoxyxylulose phosphate pathway Mevalonic acid pathway

a b s t r a c t Andrographolide, a diterpene lactone, is isolated from Andrographis paniculata which is well known for its medicinal properties. The biosynthetic route to andrographolide was studied using [1-13C]acetate, [2-13C]acetate and [1,6-13C2]glucose. The peak enrichment of eight carbon atoms in the 13C NMR spectra of andrographolide suggested that deoxyxylulose pathway (DXP) is the major biosynthetic pathway to this diterpene. The contribution of the mevalonic acid pathway (MVA) is indicated by the observed 13C-labeling pattern, and because the labeling patterns indicate a simultaneous contribution of both methyl erythritol phosphate (MEP) and MVA pathways it can be deduced that cross-talk occurs between plastids and cytoplasm. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Andrographis paniculata (Burm. F.) Nees (Acanthaceae) has been used for centuries for the treatment of fever and many infectious diseases. The plant is native to tropical and sub-tropical regions of India and is also used as a pain killer, anti-inflammatory, antibacterial and anti-viral agent (Perry, 1980; Calabrese et al., 2000; Singh et al., 2001). Andrographolide (1) and neoandrographolide (2) have been identified as the main chemical constituents which are responsible for the therapeutics of the plant. Andrographolide, in particular, has shown cytotoxic and cytostatic activity against cancer cells and hepatoprotective activity (Siripong et al., 1992; Clander et al., 1995; Chen et al., 2007; Wang et al., 2009; Akowuah et al., 2006; Muntha et al., 2003; Nanduri et al., 2004; Rao et al., 2004; Srivastava et al., 2004). Though it appears to be a simple diterpene lactone no studies have been carried out on the biogenetic route to andrographolide (1) or neoandrographolide (2) or other related compounds of this plant. Over 30,000 compounds including steroids constitute the family of isoprenoids and reports of new compounds of isoprenoid nature constantly appear in the scientific literature. The biosynthesis of IPP (18) and DMAPP (19) may occur through the mevalonate and DXP pathways (Figs. 1 and 2) in biological systems and IPP (18) is isomerised to DMAPP (19) (Ramos-Valdivia et al., 1997). Since the discovery of the mevalonate pathway an enormous amount of literature has appeared on the biosynthesis of isoprenoid compounds and exhaustive reviews have appeared periodically (Hanson, * Corresponding author. Tel.: +91 522 3056888. E-mail addresses: [email protected], [email protected] (A. Akhila). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.05.022

1984; Banthorpe and Branch, 1985, 1987; Beale and MacMillan, 1988; Beale, 1990a,b, 1991; Dewick, 1995, 1997, 1999, 2002; Hoshino et al., 2004). In most of the literature the biosynthetic pathway to specific terpenoids could only be explained (Akhila and Banthorpe, 1980a,b) after presuming certain facts which might be occurring in the plant cell. These may be the presence of a ‘‘DMAPP Pool” and the movement of IPP and DMAPP across the cell membrane or the plastidial membrane (Hemmerlin et al., 2003). In many cases the incorporation of radio-labeled (14C and 3H) MVA or acetate was very low which could have led to errors while determining the radioactive levels in the compounds. Isotopically labeled MVA and acetate were usually very poorly incorporated into mono-, diand tetraterpenoids such as carotenoids. However, their incorporation into sesqui-, triterpenoids and sterols was much more efficient. Though there was no unequivocal proof to deny the existence of mevalonate pathway but the results always pointed towards an alternative route which could have been taking place in the biosynthesis of isoprenoid compounds. The reassessment of the mevalonate pathway was underway in several laboratories since the early 1990s. Finally the individual studies performed in bacteria and some phototrophic eukaryotes by Rohmer, Eisenreich, Lichtenthaler, Hoeffler and many other workers (Rohmer, 1999; Rohmer et al., 1993, 1996; Lichtenthaler, 1999; Eisenreich et al., 1997, 1998, 2001; Hoeffler et al., 2002) proposed the deoxyxylulose path (DXP pathway) in addition to the MVA pathway which led to IPP and DMAPP (Fig. 2). The biosynthetic relationship between glucose and the two building blocks IPP and DMAPP through the DXP and MVA pathways is shown in Figs. 2 and 3, respectively. It is important to note that glucose may catabolise through different mechanisms in bacteria and

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O

15

16

14 13

HO

16

14

O

13

20

11

11 17

17 1

1

9

2

10

7

8

5

7 6

HO

19

18

10

3

6 HO

9

2

8

5

3

O

12

12 20

O

15

19

18 GlO

HO

1

2 OH

O

O

1

1

*

2

O-

2

*

SCoA

O

O

*

*

O

1 SCoA

*

*

*

*

SCoA

2

O

O

OH

CoA

1,2-13C-Acetate (3) Acetyl Coenzyme A (4) Acetoacetyl Coenzyme A (5)

HMGCoA (6)

6

PPO

* 5

2 4

PPO

3* 6

DMAPP (19)

* 5

2 4

4

-CO2

3*

5 6

IPP (18)

PPO

*

OH

OP

3 *

O

* 2 * 1

MVAPP ( 8)

3ATP * OH

HO

* O

OH

Mevalonic acid (7)

Fig. 1. (*) Denotes tracer from [1-13C]acetate, whereas (d) denotes tracer from [2-13C] acetate.

higher plants (Fig. 3). Various studies have been conducted on the DXP pathway using [1-13C] or [6-13C] glucose as precursors (Eisenreich et al., 1998; Bacher et al., 1999). There are reports that mevalonate has been successfully and efficiently used as a precursor of triterpenes and sterols and has also been incorporated in the monoterpene moiety of the alkaloid loganin, although with a quite lower % of incorporation. Finally Arigoni’s group (Eisenreich et al., 1998) was able to demonstrate that both terpenoid pathways, the mevalonate and the DXP, operate in higher plants. There is evidence that the mevalonate pathway operates in the cytoplasm and the DXP pathway is located in plastids (Arigoni et al., 1997). Thus both the mechanisms are compartmentalised. This compartmental separation is not absolute because there have been examples when mevalonic acid has been found to be incorporated into monoterpenoidal and diterpenoidal moieties which are supposed to be formed in plastids. Schwarz (1994) found that 1–2% of the isoprenoid units of ginkgolides were derived from mevalonic acid. Lichtenthaler reported that cytosolic isoprenoids (sterols) are formed via the DXP/MEP pathway in some green algae (Lichtenthaler et al., 1997). Similarly, the monomers in phytol and carotenoids in Catharanthus roseus were derived from the DXP pathway (Arigoni et al., 1997). There are reports that steroids are preferentially formed from the mevalonate pathway,

whereas monoterpenes, diterpenes, carotenoids and phytol are biosynthesised through the DXP pathway. However, it is very likely that both the pathways exist in higher plants and a ‘crosstalk’ between the two pathways can be explained by the exchange of metabolic intermediates between the cytoplasm and the plastids. The formation of isoprene units through MVA or DXP pathways can be determined by 13C NMR spectroscopy on the basis of different 13 C labeling patterns after incorporation of [1-13C]glucose, [6-13C]glucose or [1,6-13C]glucose into isoprenoids (Figs. 2 and 3) (Rohmer et al., 1993; Schwarz, 1994; Schwender et al., 1997). This methodology helps in elucidating the mixed biosynthetic pathways via MVA and DXP routes (Adam and Zapp, 1998; Adam et al., 1998, 1999). Recently, the DXP and MVA pathways have been studied using 13C-labeled precursors in salvinorin in Salvia divinorum (Kutrzeba et al., 2007), gaudichaudianic acid in Piper gaudichaudianum (Lopes et al., 2007), volatiles from Brassica oleracea (Connor et al., 2008), artemisia ketone and germacrene D in Tanacetum vulgare (Umlauf et al., 2004) and linalool in grape berry (Luan and Wust, 2002). 13 C NMR has been frequently used by the isotope enrichment technique to establish precursor–product relationships (Schneider, 2007). In the present study, the biosynthetic pathway to andrographolide (1) in Andrographis paniculata has been studied using 13C-

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1

H

O

O 2

H

OH

+

Lost as CO2

1C

O-

2

O

C

3

*CH2OP

(a)

1*

Glyceraldehyde-3-phosphate (11) Pyruvate (12)

O

2

3

H

O 4

OH

1*

OP

O

(c)

3

2

*

*

4

2-C-Methyl-D-erythritol 4-phosphate (MEP) (21)

5

1 5

HO

OH

OP

OH

5

*

*

4

1-deoxy-D-xylulose 5-phosphate (20)

OH

*

3

2

Pyruvate moeity

*3 CH

Glyceraldehyde moeity

5

(b)

3

1

OH

4

O

OP

HO

3

2

* OP

OH NH2

NH2

(d) 1 5

2

3

N

N

OH

1*

4

OH

*

N

OPOPO

H

3

OH

P

H

OH

H OH

OH H

(e)

2

OH4

5*

5

3

(g) O

OH

H OH

4

2

* OPP

OH

1-hydroxy-2-methyl-2-(E)-butenyl -4-diphosphate (HMBDP) (25)

OH

2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) (24) (a) DXP Synthase (b) Thiamine diposphate (c) DXP reductoisomerase (d) MEP-Cytidylyltransferase (e) CDP-Me kinase (f) MECDP Synthase (g) HMBDP Synthase

H

1*

O

P

H

2-Phospho-4-(cytidine 5'-diphospho) -2-C-methyl-D-erythritol (CDP-Me2P) (23)

(f) HO

O

OPOPO

O

O

N

5*

4

O OH

H

O

*

2

3

4-(Cytidine 5'-diphospho)-2-C-methyl-Derythritol (CDP-Me) (22)

1

*

O

O

OH

OP

1*

*1

2 4

3

* 5

IPP (18)

2 3

*

5 OPP 4 DMAPP

(19)

Fig. 2. DXP pathway or non-mevalonate pathway believed to exist in plastids and observed in the biosynthesis of mono and diterpenes. *C in glyceraldehyde-3-phosphate and pyruvate is derived from C-1, C-6 of glucose.

labeled precursors. 13C-acetate and 13C-glucose have been the precursors of choice in these studies. According to the findings in the literature (Fig. 1) label derived from C-1 (*), which is the carboxylic carbon of acetate would attain the position at C-3 and C-5 of IPP (18) and DMAPP (19), whereas the label from C-2 (d), which is the methyl carbon of acetate will appear at the C-2, C-4 and C-6 positions of IPP and DMAPP via the MVA pathway. C-1 from mevalonic acid is lost as CO2 during the formation of isoprene units, i.e. IPP and DMAPP. On the other hand (Fig. 3) during the process of glycolysis the glucose (9) molecule is supposed to be bisected at the center producing two molecules possessing C3 carbon chains each. As a matter of convenience we will continue the same numbering in these two molecules which are dihydroxyacetone (10) and glyceraldehyde-3-phosphate (11). C-1, C-2, C-3 as top half of glucose and

C-4, C-5, C-6 as bottom half in dihydroxyacetone phosphate (10) and glyceraldehyde-3-phosphate (11). These two molecules are interconvertible by the action of triose phosphate isomerase. Accordingly, the label from [1-13C]glucose and [6-13C]glucose is likely to occupy the C-1 position of 10 or the C-6 position of 11. Finally, through pyruvate ? acetate ? mevalonate pathway the label (*) from C-1 or C-6 is likely to occupy the C-2, C-4 and C-6 positions of IPP and DMAPP as shown in Fig. 3. As per the other possibility in the DXP pathway the condensation between glyceraldehyde-3-phosphate (11) and the pyruvate (12) produces 1-deoxy-D-xylulose-5-phosphate (20) and the tracer from C-1 (*) or C-6 of glucose sits at C-1 and C-5 of IPP and DMAPP as shown in Fig. 2. Considering all the above facts we designed three separate sets of experiments to study the biosynthetic pathway to andrographo-

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N. Srivastava, A. Akhila / Phytochemistry 71 (2010) 1298–1304 1or 6

*CH OP 2

1* CHO

H

2

HO

3

H

4

OH

H

5

OH

OH

2 or 5

Glycolysis several steps

H

3 or 4

H

O

2 or 5

H HO

3 or 4

H

O OH

1or 6

*CH2OP

H

Glyceraldehyde-3phosphate (11)

Dihydroxyacetone phosphate (10)

6

*CH2OP Glucose-6-phosphate (9) O

O

O

C 2 or 5

1* or 6

1or*6

O

NADH + H +

NAD +

3 or 4

C 2 or 5

C2 or 5

Acetoacetyl-CoA (14)

2 or 5

C

SCoA

*6 1 or

SCoA

O-

C

(a)

CoA-SH

CO2

Acetyl-CoA (13)

O

1 or 6

*CH3

Pyruvate (12)

(b) OH

6* 3

*4 5

O

OH

6*

*

2 1

3

(c) (d)

COOH

*4

SCoA

1

5

3-hydroxy-3-methylglutaryl-CoA (15)

*

2

COOH

HO

OH

6* 3

(e) ATP

*4

(f) ATP

5

*

2 1

COOH

PPO

MVA 5-diphosphate ( 17)

Mevalonate (16)

(g) 6

*

6*

(h) 5

3

5

3

2

4

*

*

OPP

Dimethylallyl diphosphate (19)

2

4

*

*

OPP

Isopentenyl diphosphate (18)

Fig. 3. Mevalonic acid pathway through pyruvate–acetate after the breakdown of glucose. This is believed to occur in cytosol during the formation of C15 and C30 compounds. (*) Denotes carbon from C-1, C-6 of glucose. Carbons in all the skeletons up to structure 14 have been numbered as per numbering in glucose (9). Independent numbering of carbon atoms has been done from HMGCoA (15) to DMAPP (19) for convenience of discussion.

lide (1). The numbering pattern in andrographolide (1) is shown in Fig. 4. The 13C-label from [1-13C]acetate, [2-13C]acetate and [1,6-13C]glucose is shown in Fig. 4 and also summarized in Table 1. 2. Results and discussion A sample of 13C-enriched andrographolide (1) was isolated by CC fractionation of a hot methanol extract of twigs and leaves of A. paniculata that were fed with [1-13C]acetate, [2-13C]acetate, [1,6-13C2]glucose in separate sets of experiments. CC on Si-Gel with increasing % of EtOAc in hexane was performed, fractions yielding andrographolide (1) were concentrated and crystals of 1 were obtained in absolute purity. The 13C-spectra of 1 enriched with 13C and that of a reference sample of 1 (13C at natural abundance) were measured in CDCl3 under identical conditions and the relative enrichment was measured taking into consideration the natural abundance of 1.1% for the carbon with the lowest

13

C enrichment. The results from all the three 13C-labeled precursors are presented in Table 1. As shown in Fig. 4, the feeding experiments using [1-13C]acetate showed an enrichment at eight carbons (C-2, C-4, C-6, C-8, C-10, C-11, C-13 and C-15), whereas the experiment using (2-13C)acetate showed enrichments at twelve carbons (C-1, C-3, C-5, C-7, C-9, C-12, C-14, C-16, C-17, C-18, C-19 and C-20). The signals showing 13C enrichment in the 13C-spectra were clearly distinct from those of the non-enriched positions. All the enriched carbons indicate that the incorporation of acetate up to IPP and DMAPP follow the MVA pathway and thereafter these intermediates cross over the barrier into the plastids where the biosynthesis of diterpene (andrographolide 1) takes place. Interestingly, the andrographolide (1) obtained from the feeding of [1,6-13C2]glucose showed much higher enrichment of 13C peaks at C-2, C-6, C-11, C-15, C-16, C-17, C-18 and C-20. These carbon positions are supposed to be enriched by C-1 and C-6 of glucose through the DXP pathway.

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N. Srivastava, A. Akhila / Phytochemistry 71 (2010) 1298–1304 OPP

IPP

PPO

*Δ

*

*Δ

H

Δ Δ

*

H

IPP

*

*

OPP

DMAPP

*Δ

*

Δ

OPP

GPP

*

Δ

Δ

OPP

*

*

Δ

Δ

Δ

OPP

H

Δ*

Δ

*

Δ

Δ

*Δ H

*

*Δ

H

* H

*

*

Δ

Δ

Δ O

Δ

*

Δ Δ

*

*Δ

* OPP

*

*

Δ

Δ Δ*15

oxidoGGPP

FPP

O

14 13

*

Δ 16

O

*

HO

12

Δ*11

20

Δ Δ* 2

17

1

9

* 10 5

3

7 6

4 HO

18

Δ

*

Δ

*

8

Δ*

19

HO

1

Fig. 4. Biosynthetic routes to andrographolide. (*) Denotes tracer from [1-13C]acetate, whereas (d) denotes tracer from [2-13C] acetate when biogenetic route to andrographolide is achieved from IPP and DMAPP through mevalonate pathway; (4) denotes tracer from 1,6-13C2-glucose if the andrographolide is biosynthesised from DXP (non-mevalonate) pathway.

3. Conclusion

4. Experimental

The biosynthesis of andrographolide (1) in A. paniculata seems to proceed by both MVA and MEP/DXP pathways. The lower enrichment of carbon positions [using (13C)acetate] through the MVA pathway in comparison to very high enrichment of carbon positions [using 1,6-(13C)]glucose] through the MEP/DXP pathway indicates the existence of MVA pathway also. However, the very high enrichment of specific carbons from 1,6-(13C)]glucose confirm that the major biosynthetic pathway to this diterpenoid (1) operates through DXP. This also implies that some biosynthetic steps proceed in different compartments and specific intermediates or precursors cross over through the chloroplast boundary. In case of 1 the diterpenoid skeleton has been biosynthesised from IPP units derived from each pathway. Initially, the first C10 unit is derived from prenylation with DMAPP derived from either MVA or MEP pathways. This is followed by second and third prenylations with IPP. A possible biosynthetic pathway of isoprene units and andrographolide (1) from there onwards in shown in Figs. 2–4.

4.1. General NMR Brucker 300 MHz (CDCl3 1H d 7.24; 13C d 77.0), [1-13C]acetate, [2-13C]acetate, [1,6-13C]glucose (>99% isotopic abundance) were purchased from Sigma–Aldrich, USA. 4.1.1. Plant material and feeding methods Specimens of A. paniculata were cultivated in the agricultural farms of CIMAP, Lucknow, India. Three different methods were tried: (a) Direct-stem injection – 2 months old plants (30 cm high, 6 mm wide) were selected and a needle (0.30 mm) attached to a 500 ll syringe (Hamilton) was inserted up to the middle of the stem (about 3 mm) about 15 cm from the top of the plant. One hundred microlitres of a 1% solution of 13C-labeled precursor (acetate or glucose) was injected over 12 h. The plant was harvested after 15 days and extracted for andrographolide (1). (b) Precursor uptake by plant cuttings under normal environmental conditions –

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N. Srivastava, A. Akhila / Phytochemistry 71 (2010) 1298–1304 Table 1 Incorporation of [1-13C]acetate, [2-13C]acetate, [1,6-13C2]glucose into andrographolide (1) which was obtained from the aerial parts of Andrographis paniculata. Precursor fed Andrographolide C-Chemical shift assignment d (ppm)b 13

C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20

37.8 28.8 79.2 43.4 55.2 24.5 38.4 147.9 56.6 38.0 24.9 146.8 130.2 66.2 75.2 170.3 109.0 23.8 64.2 15.3

a

[1-13C]acetate MVA pathway

[2-13C]acetate MVA pathway

[1,6-13C2]glucose DXP pathway

d (ppm)b

Enrichment factorc (relative enrichment)

d (ppm)b

Enrichment factorc (relative enrichment)

d (ppm)b

Enrichment factorc (relative enrichment)

37.4 28.5 79.0 43.8 55.1 24.5 38.8 148.1 56.3 38.5 24.7 146.9 130.1 66.0 75.0 170.5 109.6 23.3 64.1 15.1

0.2 2.0 0.3 1.2 0.0d 1.6 0.4 1.1 0.2 1.9 1.4 0.2 1.6 0.4 1.3 0.7 0.2 0.3 0.2 0.2

37.3 28.4 79.3 43.5 55.1 24.5 38.5 147.7 56.2 38.3 24.9 146.8 130.1 66.3 75.1 170.3 109.4 23.3 64.4 15.2

1.8 0.3 1.6 0.4 1.7 0.2 1.8 0.4 1.5 0.0d 0.3 1.7 0.5 1.9 0.2 1.7 1.8 1.6 1.5 1.6

37.4 28.4 79.1 43.5 55.1 24.4 38.6 147.8 56.4 38.3 24.8 146.7 130.1 66.2 75.1 170.2 109.1 23.7 64.1 15.2

0.3 4.1 0.4 0.4 0.3 4.4 0.2 0.2 0.5 0.6 4.5 0.0d 0.5 0.7 4.3 4.6 4.2 4.5 0.4 4.3

a

Numbering pattern corresponds to that shown in Fig. 4. Referenced to CDCl3. c Calculated by comparison of relative intensities of signals in the 13C NMR spectra of 13C-labeled 1 and a reference standard with factor was calculated according to formula = (enriched integral  unlabelled integral)/unlabelled integral. d Denotes the carbon used as reference to calculate the integration ratio. b

15–20 cm high plant twigs with intact leaves were grown under non-sterile conditions for about 7 days in 25 ml Hoagland’s medium. The plant cuttings were then transferred to a solution of 1% 13 C-labeled precursors in separate sets of experiments which were supplemented with 2 ml of labeled substrate for 2 weeks and then extracted for the desired compound 1. (c) Precursor uptake by plant cuttings under forced transpiration – 15 cm high plant twigs were maintained on Hoagland’s medium for 7 days and then transferred to a solution of 13C-labeled substrate. This solution was allowed to be taken up by the plant twigs under illumination (a 60 W bulb) and forced transpiration by an air fan. After the uptake of desired precursor the twigs were maintained for 7 days on sterile medium to avoid contamination. The peak enrichment of desired carbons by direct-stem injection method was very poor showing that the fedprecursor could not enter the site of biosynthesis through a proper path. The incorporation of 13C-labeled precursors through stem cuttings under normal conditions and forced transpiration was almost the same.

4.2. Extraction and isolation The plant twigs (500 g) which were fed with 13C-labeled precursors were air-dried, mixed with the dried normal plants and 1 kg of the dried material was extracted with MeOH (5  2 l reflux for 8 h). The combined extracts were concentrated in vacuo to yield 110 g of dark brown residue. The residue was defatted with hexane and then separated by CC on Si-Gel using EtOAc–hexane (as eluents) while increasing the % of EtOAc [0:100; 5:95; 10:90; 15:85; 20:80; 25:75; 30:70; 35:65; 40:60; 45:55; 50:50; 55:45; 60:40; 65:35; 70:30; 75:25; 80:20] and continuously monitoring the eluted fractions for the presence of 1. The fractions obtained from EtOAc–hexane (75:25 and 80:20) afforded white crystals of 1 which were again recrystallised from hot MeOH. The structure of 1 was confirmed by 1H and 13C NMR spectra and its comparison

13

C at natural abundance. Enrichment

with the spectroscopy data available in the literature (Du et al., 2003; Akowuah et al., 2006). Acknowledgments One of us (N.S.) is thankful to CSIR for Research Internship at CIMAP and we are thankful to Director CIMAP for encouragement during the course of this work. References Adam, K.P., Zapp, J., 1998. Biosynthesis of the isoprene units of chamomile sesquiterpenes. Phytochemistry 48, 953–959. Adam, K.P., Thiel, R., Zapp, J., Becker, H., 1998. Involvement of the mevalonic acid pathway and the glyceraldehyde–pyruvate pathway in terpenoid biosynthesis of the liverworts Conocephalum conicum and Ricciocarpos natans. Arch. Biochem. Biophys. 354, 181–187. Adam, K.P., Thiel, R., Zapp, J., 1999. Incorporation of 1-[1-13C]deoxy-D-xylulose in chamomile sesquiterpenes. Arch. Biochem. Biophys. 369, 127–132. Akhila, A., Banthorpe, D.V., 1980a. Biosynthetic origin of gem-methyls of geraniol. Phytochemistry 19, 1429–1430. Akhila, A., Banthorpe, D.V., 1980b. Biosynthesis of the skeleton of pulegone in Mentha pulegium. Z. Pflanzenphysiol. 99, 277–282. Akowuah, G.A., Zhari, I., Norhayati, I., Mariam, A., 2006. HPLC and HPTLC densitometric determination of andrographolides and antioxidant potential of Andrographis paniculata. J. Food Compos. Anal. 19, 118–126. Arigoni, D., Sagner, S., Latzel, C., Eisenreich, W., Bacher, A., Zenk, M., 1997. Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher plants by intramolecular skeletal rearrangement. Proc. Natl. Acad. Sci. USA 94, 10600–10605 (and references cited therein). Bacher, A., Rieder, C., Eichinger, D., Arigoni, D., Fuchs, G., Eisenreich, W., 1999. Elucidation of novel biosynthetic pathways and metabolite flux patterns by retrobiosynthetic NMR analysis. FEMS Microbiol. Rev. 22, 567–598. Banthorpe, D.V., Branch, S.A., 1985. The biosynthesis of C5–C20 terpenoid compounds. Nat. Prod. Rep. 6, 513–524. Banthorpe, D.V., Branch, S.A., 1987. The biosynthesis of C5–C20 terpenoid compounds. Nat. Prod. Rep. 4, 157–173. Beale, M.H., 1990a. The biosynthesis of C5–C20 terpenoid compounds. Nat. Prod. Rep. 1, 25–39. Beale, M.H., 1990b. The biosynthesis of C5–C20 terpenoid compounds. Nat. Prod. Rep. 5, 387–407. Beale, M.H., 1991. Biosynthesis of C5–C20 terpenoid compounds. Nat. Prod. Rep. 5, 441–454.

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