- Email: [email protected]
Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit
Chapter 23
Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit Naïm Stiti1,2, Saïda Triki2 and Marie-Andrée Hartmann1 1
lnstitut de Biologie Moleculaire des Plantes (CNRS UPR 2357), Université de Strasbourg, 28 rue Goethe, 67083 Strasbourg, France Faculté des Sciences de Tunis, Département des Sciences Biologiques, Campus Universitaire, 2092 Tunis, Tunisia
2
23.1 Introduction Olive oil as well as olive leaves have been known for a long time to contain a wide range of sterols and non-steroidal triterpenoids, including erythrodiol, oleanolic acid and maslinic acid (Power and Tutin 1908; Caputo et al., 1974; Itoh et al., 1981), which are oxygenated derivatives of -amyrin (olean-12-en-3-ol), one of the most commonly occurring triterpenes. Sterols and non-steroidal triterpenoids, which belong to the group of terpenoids or isoprenoids, the largest family of natural products, are synthesized via the cytoplasmic acetate/mevalonate pathway and share common precursors up to (3S)-2,3-oxidosqualene (OS) (Seo et al., 1988; Benveniste, 2002). Then, OS serves as a substrate for various OS cyclases, also called triterpene synthases, to form C30 compounds (i.e., comprising six C5-isoprene units). Cycloartenol synthase catalyzes the cyclization of OS folded in the pre-chair-boat-chair conformation, via the protosteryl cation, into cycloartenol, the first cyclic precursor of the sterol pathway (Figure 23.1). About 20 steps are needed to convert cycloartenol in end pathway sterols (Benveniste, 2002). Non-steroidal triterpenoids are assumed to be formed from OS folded in the all-pre-chair conformation, through a series of carbo cationic intermediates (Abe et al., 1993) (Figure 23.1). They are then often metabolized into more oxygenated compounds, which serve as precursors for the synthesis of triterpenic saponins (Mahato et al., 1988). As the cyclization of OS into sterols and non-steroidal triterpenoids represents a branch point between primary and secondary metabolisms, OS cyclases are attractive tools for investigating the physiological roles of non-steroidal triterpenoids. The present study sheds more light on biosynthetic relationships occurring between the sterol and non-steroidal triterpenoid pathways in Olea europaea L. throughout olive fruit ontogeny. Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
23.2 THE OLIVE FRUIT CONTAINs A VAST ARRAY OF STEROLS AND NON-STEROIDAL TRITERPENOIDS Sterols and non-steroidal triterpenoids were isolated from total lipid extracts of olive drupes (i.e., the whole fruit comprising the epicarp, the mesocarp and the endocarp or pit with the seed) as previously described (Hartmann and Benveniste 1987; Stiti et al., 2007). Free sterols, tetracyclic and pentacyclic triterpenes, triterpenic diols as well as the compounds released after hydrolysis of ester conjugates were identified as acetate derivatives by their relative retention time in gas chromatography and their mass spectrometry fragmentation pattern in gas chromatography coupled to mass spectrometry (Rahier et al., 1989; Stiti et al., 2007 and references herein). Mono- and dihydroxy pentacyclic triterpenic acids (HPTAs) were isolated according to Pérez-Camino and Cert (1999) and Stiti et al. (2007) and identified as acetate derivatives of the corresponding methylesters (Stiti et al., 2007).
23.2.1 Sterols Olive drupes were shown to contain a mixture of sterols, with sitosterol as the largely predominant compound (between 70 and 95% of total sterols), and 24-methylcholesterol, stigmasterol and isofucosterol (5-avenasterol) (1–3%). We also identified brassicasterol, 24-methylenecholesterol, 5,24-stigmastadienol, 7-avenasterol and cholesterol. All the usual intermediates of the sterol pathway: squalene, 4-dimethylsterols (cycloartenol and 24-methylenecycloartanol) and 4a-methylsterols (obtusifoliol, cycloeucalenol, 24-methylene and 24-ethylidenelophenol) were found. The occurrence of some less usual sterols such as 24-methyl- and 24-ethyl-lophenol, (24S)24-ethylcholesta-5,25-dien-3-ol (clerosterol) and sterols
211
Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
212
Section | I Lipids, Phenolics and Other Organics and Volatiles
+ H
+ 20
H H
+
17
2,3-oxidosqualene
CCC
dammarenyl cation
C B C
protosteryl cation
H 19
HO
HO
H
+
H H
H H
H
3 2 O
H
+
HO
baccharenyl cation
HO
lupenyl cation
20 + H
HO
oleanyl cation
-12αH
ursanyl cation
-12βH
H HO
HO
9
12
HO
1
7
HO
-9βH
-19H
CH2OH
HO
HO
parkeol
HO
cycloartenol
HO
9
3
14 HO
COOH
15
COOH
COOH
HO
HO
CH2OH
CH2OH
HO
10
4
Sterols COOH HO HO
5
Non-steroidal triterpenoids Figure 23.1 Postulated biosynthetic pathway of non-steroidal triterpenoids from 2,3-oxidosqualene cycIization in the Olea europaea fruit. This figure is adapted from the previously published Scheme 1 (Stiti et al., 2007). CBC and CCC refer respectively to the pre-chair-boat-chair and allpre-chair conformations of oxidosqualene (OS). OS serves as a substrate for the synthesis of either sterols or non-steroidal triterpenes. Cycloartenol synthase catalyzes the formation of cycloartenol, the first cyclic precursor of sterols, via the protosteryl cation. The OS cyclization reaction by non-steroidal triterpene synthases is thought to proceed through generation of several four or five ring-carbocationic intermediates. These carbocationic intermediates are represented in brackets. Oleanane-type triterpenoids, which arise from the oleanyl cation, are by far the predominant compounds, as shown by the widest arrows. The names of the different compounds, which are designated by a number, are given in the legend of Figure 23.2.
with a double bond at C-23 (5,23-stigmastadienol and 24ethyl E-23-dehydrolophenol); at C-11 (5-lanosta-9(11), 24-dien-3-ol) or parkeol and 24-methylene-lanost-9(11)en-3-ol has to be mentioned. All these sterols were also present as esters. However, it is interesting to note that no free parkeol could be detected.
23.2.2 Non-steroidal Triterpenoids Besides sterols, the olive fruit contains a great diversity of triterpenoids. Their structures are shown in Figure 23.2. They include 19 pentacyclic triterpenoids arising from four different carbon skeletons: oleanane-type (1–7) (-amyrin 1, 28-nor--amyrin 2, erythrodiol 3, oleanolic acid 4, maslinic acid 5, -amyrone 6 and -amyrin 7), ursane-type (8–12) (-amyrin 8, 28-nor- amyrin 9, uvaol 10, ursolic acid 11 and -amyrone 12), lupane-type (13–17) (lupeol 13, 3epi-lupeol 14, 3-epi-betulin 15 and 3-epi-betulinic acid 16
and lupenone 17) and taraxane-type (18–19) (taraxerol 18, taraxer-14-ene-3,28-diol 19), as well as two tetracyclic triterpenes with euphane-type (butyrospermol 20) and baccharane-type (bacchar-12,21-dien-3-ol 21) carbon skeletons. Oleanane triterpenoids were largely predominant, with oleanolic and maslinic acids representing by far the major compounds. Pentacyclic triterpenes also occurred as esters, but no acylated triterpenic diols have been found. Thus, more than 40 sterols and non-steroidal triterpenoids have been identified in the olive fruit. Our results are consistent with previous reports about the sterol and triterpenoid composition of olive oil or fruit (Itoh et al., 1981; Chryssafidis et al., 1992; Bianchi et al., 1994; Reina et al., 1997; Ranalli et al., 2002; Stiti et al., 2002; Azadmard-Damirchi et al., 2005; see Chapter 27). However, the occurrence in the olive fruit of taraxer-14-ene-3,28-diol, 3-epi-lupeol and its metabolites, 3-epi-betulin and 3-epi-betulinic acid, had not been reported before.
213
Chapter | 23 Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit
R1
R1 R2
(1) (2) (3) (4) (5) (6) (13) (14) (15) (16) (17)
R
HO
R
(7)
R = α-H, β-OH, R1 = CH3, R2 = H R = α-H, β-OH, R1 = H, R2 = H R = α-H, β-OH, R1 = CH2OH, R2 = H R = α-H, β-OH, R1 = COOH, R2 = H R = α-H, β-OH, R1 = COOH, R2 = OH R = O, R1 = CH3, R2 = H R = α-H, β-OH, R1 = CH3 R = α-OH, β-H, R1 = CH3 R = α-OH, β-H, R1 = CH2OH R = α-OH, β-H, R1 = COOH R = O, R1 = CH3
(8) (9) (10) (11) (12)
R = α-H, β-OH, R1 = CH3 R = α-H, β-OH, R1 = H R = α-H, β-OH, R1 = CH2OH R = α-H, β-OH, R1 = COOH R = O, R1 = CH3
(18) R1 = CH3 (19) R1 = CH2OH R1
R1 H HO
R
H
H HO
HO
(20)
(21)
Figure 23.2 Structures of the non-steroidal triterpenoids identified in the olive fruit. Oleanane-type: (1), b-amyrin; (2), 28-nor-b-amyrin; (3), erythrodiol; (4), oleanolic acid; (5), maslinic acid; (6), b-amyrone; (7), -amyrin; Ursane-type: (8), -amyrin; (9), 28-nor--amyrin; (10), uvaol; (11), ursolic acid; (12), -amyrone; Lupane-type: (13), lupeol; (14), 3-epi-lupeol; (15), 3-epi-betulin; (16), 3-epi-betulinic acid; (17), lupenone; Taraxane-type: (18), taraxerol; (19), taraxer-14-ene-3,28-diol; Euphol-type: (20), butyrospermol; Baccharanetype: (21), bacchar-12,21-dien-3-ol.
23.3 Changes in the Content of Free and Esterified Sterols and Non-Steroidal Triterpenoids Throughout Fruit Development
Drupes from the different batches were analyzed for their content in free and esterified sterols and non-steroidal triterpenoids, but only data corresponding to olives harvested at the stages 12, 18, 21 and 30 WAF are presented here.
Olive fruit were handpicked from all the sides of one olive tree, Olea europaea L. cv Chemlali, at 13 distinct stages of fruit growth and ripening corresponding to 12, 13, 15, 16, 18, 21 23, 25, 27, 29, 30, 32 and 33 weeks after development (WAF). At the time of the first harvest, the lignification of the olive endocarp had ended. Between 12 and 18 WAF, olives were green and progressively increased in size and fresh weight, but in the case of the Chemlali cultivar, these changes were of limited amplitude compared to other olive varieties. At the end of this period, the final fruit size was almost fixed and from the 21st WAF, epidermal color gradually turned from green to purple. Complete maturity was observed after 29 WAF. The 33 WAF stage corresponded to an ‘over maturation’ stage.
23.3.1 Sterols from the 12th to the 18th WAF In the young olive fruit (i.e. between 12 and 18 WAF), free sterols were present as a mixture in which sitosterol was largely predominant (95%), but the usual sterol intermediates, 4,4-dimethyl- and 4-methylsterols, were barely detectable (Table 23.1). A relatively high amount of squalene (700 g g1 dry wt) was detected at 13 WAF, but then rapidly decreased to 40 g g1 at 18 WAF (data not shown). During this period of time, a slight decrease in the total free sterol content of the olive fruit was observed (Table 23.1). The young olive drupes were also found to contain sterols as ester conjugates, with a sterol profile slightly
214
Section | I Lipids, Phenolics and Other Organics and Volatiles
Table 23.1 Changes in free sterols during olive fruit development.
Table 23.2 Changes in esterified sterols during olive fruit development.
Developmental stage Sterol classes
12 WAF
18 WAF
21 WAF
30 WAF
Developmental stage Sterol classes
12 WAF
18 WAF
21 WAF
30 WAF
4,4-dimethylsterols cycloartenol
nd
nd
0.4
3.2
4,4-dimethylsterols cycloartenol
nd
nd
2.7
1.4
24-methylenecycloartanol nd
nd
3.1
26.6
parkeol
nd
nd
16.4
13.4
24-methylenecycloartanol
4.7a
6.6
7.7
10.4
4-methylsterols obtusifoliol
0.5
0.7
1.4
0.7
4-methylsterols obtusifoliol
nd
nd
0.6
0.2
24-methylenelophenol
nd
nd
1.0
0.7
24-methyl-lophenol
nd
nd
0.2
0.1
cycloeucalenol
nd
nd
0.7
0.4
24-methylenelophenol
2.7
2.0
0.8
0.4
0.1
cycloeucalenol
1.6
0.9
0.6
0.6
1.1
1.3
2.7
3.3
24-ethyl-lophenol
nd
nd
0.2
24-ethylidenelophenol
nd
nd
0.9
1.1
24-ethylidenelophenol
4-demethylsterols brassicasterol
0.4a
0.4
0
0
4-demethylsterols 24-methylcholesterol
2.1
2.1
2.7
2.3
1.9
stigmasterol
7.2
7.5
2.4
0.7
0.8
clerosterol
0.8
0.7
0.8
0.7
62.5
62.5
59.2
62.0 4.1
24-methylcholesterol stigmasterol
1.5 1.4
1.4 2.5
2.9 1.3
clerosterol
0.7
1.2
0.8
0.6
sitosterol
sitosterol
95.9
94.4
87.5
61.3
isofucosterol
16.2
15
2.6
5,24-stigmastadienol
0.5
0.6
nd
nd
Total amount (g/g dry wt)
44
100
140
305
isofucosterol Total amount (g/g dry wt)
0 250
0 230
0.2 500
2.7 960
The standard deviation for quantitative determinations was 5%. a
% of total free sterols; nd: not detectable.
different from that of free forms. In particular, sitosterol remained the major compound (63%), but significantly higher relative proportions of 24-methylcholesterol, stigmasterol and isofucosterol were found (Table 23.2). Low amounts of acylated sterol intermediates, especially 24methylenecycloartanol, were present (Table 23.2). Between 12 and 18 WAF, a 2.3-fold increase in the total amount of sterol esters was observed, an increase that equally affected sterol intermediates and end products, indicating that some sterol biosynthesis took place in the very young fruit. However, these newly synthesized sterols were immediately conjugated to a fatty acid and thus removed from the free sterol pathway.
23.3.2 Sterols from the 21st to the 30th WAF From the 21st WAF, dramatic changes were observed in the free sterol pathway. Early biosynthetic intermediates, i.e., squalene, 4,4-dimethyl- (cycloartenol and 24-methylene cycloartanol) and 4-methylsterols (cycloeucalenol, obtusifoliol, 24-methylene- and 24-ethylidenelophenol) as well as late precursors (isofucosterol) began to be detectable
The standard deviation for quantitative determinations was 5%. a
% of total sterols; nd: not detectable.
and a progressive increase in sterol end products was concomitantly observed (Table 23.1 and Figure 23.3A, 21st WAF). Throughout the fruit-ripening process, sterols continued to accumulate, with sitosterol remaining the major compound. At 30 WAF, the free sterol content of the olive fruit amounted to 950 g g1 dry wt, corresponding to a four-fold total increase from the 12th WAF. At this stage, a significant accumulation of some early intermediates, especially squalene (data not shown) and 24-methylenecycloartanol, was observed (Table 23.1 and Figure 23.3A, 30th WAF), indicating a slowing down of the metabolic flux through the sterol pathway. Between 21 and 30 WAF, the content of the olive fruit in sterol esters continued to rise, particularly between 27 and 29 WAF, to give a total amount corresponding to a seven-fold increase during the whole period of fruit development (Table 23.2). At the end of the ripening process (30 WAF), a significant accumulation of 24-methylenecycloartanol and 24-ethylidenelophenol was observed, in agreement with previous data on olive oil (Chryssafidis et al., 1992). It is interesting to note the occurrence of a new compound in the fraction of 4,4-dimethylsterols, which has been identified as parkeol (Table 23.2). This compound was formed concomitantly with cycloartenol and represented 60% of the total esterified 4,4-dimethyl sterols.
Chapter | 23 Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit
215
Figure 23.3 Changes in the content and the composition of free sterols (A) and free non-steroidal triterpenoids (B) throughout olive fruit development. The central part of the figure corresponds to the total content in free sterols and non-steroidal triterpenoids at different developmental stages of the olive fruit (12, 18, 21 and 30th WAF). Sterol pathway: 4,4-dimethylsterols; 4a-methylsterols; 4-demethylsterols. non-steroidal pathway: penta cyclic triterpenes; triterpenic diols; mono- and di-HPTAs.
During the whole period of olive fruit development, it should be pointed out that free sterols remained predominant compared to ester conjugates (Tables 23.1 and 23.2).
23.3.3 Non-steroidal Triterpenoids from the 12th to the 18th WAF At the beginning of fruit ontogeny, besides sterols, the olive fruit were found to contain high levels of the pentacyclic tri terpenes a- and b-amyrins, in a 3:2 ratio, as well as several more oxygenated compounds with additional hydroxymethyl or carboxylic groups (Table 23.3 and Figure 23.3B, 12th WAF). The introduction of a hydroxyl group in C-28 position of - and -amyrins gives rise to uvaol and erythrodiol,
respectively (Figures 23.1 and 23.2). A further oxidation of this hydroxyl group leads to the corresponding ursolic and oleanolic acids. Finally, the introduction of an additional hydroxyl group at the C-2 position of oleanolic acid results in the formation of maslinic acid. In addition to these oleanane- and ursane-type triterpenoids, lupane (3-epi-betulin and 3-epi-betulinic acid) and taraxane (taraxen-14-ene3b,28-diol) derivatives were also formed (Table 23.3). The enzymes involved in all these oxidation reactions have not been characterized yet, but are likely cytochrome P-450 monooxygenases. According to this hypothesis, the pathway should implicate the intermediate formation of the aldehydes 3-hydroxy-5-urs-12-en-28-al and 3-hydroxy5-olean-12-en-28-al. The presence of these compounds has not been checked. However, the occurrence of significant
216
Section | I Lipids, Phenolics and Other Organics and Volatiles
Table 23.3 Changes in free non-steroidal triterpenoids throughout olive fruit development.
Table 23.4 Changes in esterified non-steroidal triterpenes.
Developmental stage Triterpenoid classes
12 WAF
18 WAF
21 WAF
30 WAF
Developmental stage
12 WAF
18 WAF
21 WAF
30 WAF
Pentacyclic triterpenes ß-amyrin
4.3a
-amyrin
5.5
5.5
nd
Nd
2.5
0.1
0.2 ß-amyrin
25.1
13.9
10.9
11.9
28-nor-b-amyrin
2.3
1.5
–
– -amyrin
10.0
21.5
15.9
22.6
a-amyrin
6.0
3.3
–
– taraxerol
33.6a
21.7
11.6
5.6
28-nor-a-amyrin
0.8
0.4
–
– butyrospermol
nd
nd
61.6
59.8
pentacyclic diols taraxerol
–
–
0.1
0.1
lupeol
25.7
37.3
nd
nd
taraxer-14-ene-3ß-28diol
0.2
0.3
–
–
Total amount (g/g dry wt)
7.0
8.8
39
35
erythrodiol
12.5
9.7
0.4
0.5
The standard deviation for quantitative determinations was 5%.
uvaol
7.6
6.2
0.1
–
3-epi-betulin
0.3
0.2
–
–
Mono-HPTAs 3-epi-betulinic acid
0.7
0.6
0.6
0.6
oleanolic acid
39.2
35.8
41.2
37.6
Di-HPTAs ursolic acid
0.2
0.2
0.2
0.2
maslinic acid
25.9
39.3
57.3
60.8
Total amount (g/g dry wt)
3210
2610
3930
2470
a
% of total esterified non-steroidal triterpenes; nd, not detectable.
The standard deviation for quantitative determinations was 10%. a
% of total free non-steroidal triterpenoids.
amounts of 28-nor--amyrin and 28-nor--amyrin, which have no methyl group at C-17 (Figure 23.2), in the pentacyclic triterpene fraction (Table 23.3), might result from the decarbonylation of such aldehydes (Hota and Bapuji, 1994). The somewhat delayed accumulation of maslinic acid (Table 23.3) suggests that the hydroxylation step at C-2 may involve another type of cyt P450-monooxygenase. Between 12 and 18 WAF, the non-steroidal triterpenoid pathway was very efficient as attested by the synthesis of very high levels of these compounds (i.e. 3 mg g1 dry wt) (Table 23.3 and Figure 23.3B18th WAF). During the same period, ester conjugates of pentacyclic triterpenes were barely detectable (0.2% of the corresponding free forms) (Table 23.4). Traces of a- and b-amyrins, taraxerol, -amyrin and lupeol were found, with no change in the total amount of these esters between 12 and 18 WAF (Table 23.4).
23.3.4 Non-steroidal Triterpenoids from the 21st to the 30th WAF Between 21 to 30 WAF, a dramatic decrease in the content of a- and b-amyrins and pentacyclic triterpenic diols was observed (Table 23.3 and Figure 23.3B, 21st WAF). Nonsteroidal triterpenoids were constituted almost exclusively of mono- and di-HPTAs, with oleanolic and maslinic acids as the largely predominant compounds (98% of total triterpenoids) (Table 23.3 and Figure 23.3B, 21st WAF). In the mature olive fruit (30 WAF), a significant decrease in the content of all HPTAs was observed (Table 23.3), indicating that these compounds might be further metabolized, maybe into triterpenic saponins via the involvement of specific glycosyltransferases (Achnine et al., 2005). To our knowledge, saponins from olive tree have not yet been identified. Between 21 and 30 WAF, when amyrins were not any longer formed, esterified conjugates began to progressively accumulate, especially d-amyrin (23% at 30 WAF) (Table 23.4). Concomitantly, a change in the profile of triterpenes could be noticed, consisting in the appearance of a new tetracyclic triterpene, identified as butyrospermol, which represented up to 60% of total esterified triterpenes.
23.4 How is carbon flux regulated between both triterpenic pathways in the olive fruit? Evidence is presented here for the occurrence in the olive fruit of a vast array of sterols and non-steroidal triterpenoids. More than 40 different compounds have been found and the composition of this complex mixture was found to
Chapter | 23 Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit
be strongly dependent on the fruit developmental stage as illustrated in Figure 23.3. Throughout fruit ontogeny, two periods can be clearly distinguished: from 12 to 18 WAF and from 21 to 30 WAF. In the young green olive fruit (between 12 and 18 WAF), most of the available squalene molecules are almost exclusively devoted to the synthesis of a- and bamyrins. These non-steroidal pentacyclic triterpenes were rapidly metabolized into more oxygenated compounds, first into triterpenic alcohols, then into mono- and di-HPTAs. During the same period, no free sterols were formed. However, the sterol pathway remained functional as attested by the formation of sterol esters. Between 21 and 30 WAF, when the epidermal color gradually turned from green to purple, a- and b-amyrins and their hydroxylated derivatives were not present any longer, while the already-formed oxygenated intermediates were converted into mono- and diHPTAs (see the postulated biosynthetic pathway in Figure 23.1). Interestingly, our data also indicate that although a-amyrin was present in excess compared to b-amyrin, oleanane-type compounds as a whole were produced in far higher amounts than ursane-type compounds (Table 23.3). In early stages of fruit development, oleanane-type compounds represented from 84–89% of total non-steroidal triterpenoids and ursane-type compounds, only 10–15%. Between 21 and 30 WAF, ursane-type compounds completely disappeared. Thus, the question of the metabolic fate of a-amyrin in the olive fruit remains to be solved. From the 21st WAF, free and esterified sterols began to be formed and accumulated until the complete maturity of the fruit, but whatever the fruit developmental stage, nonsteroidal triterpenoids remained the major triterpenic compounds, with maslinic acid as the most represented one in the mature fruit (Table 23.3). Taken together, these results clearly indicate that a complex regulation process takes place at the oxidosqualene cyclization step, which represents a branch point between the sterol pathway and the non-steroidal triterpenoid pathway (Figure 23.1). OS serves indeed as a substrate for cyclo artenol synthase, the first enzyme of the sterol pathway, but also for various OS cyclases involved in the synthesis of the different classes of pentacyclic triterpenes. These OS cyclases are designated as mono- or multifunctional enzymes, depending on whether they produce single or several cyclization products (Ebizuka et al., 2003). OS cyclases of the olive fruit have not been characterized yet, but might include a lupeol synthase, a mono-functional enzyme, similar to that identified in the olive leaf (Shibuya et al., 1999) as well as a multifunctional triterpene synthase, such as the OS cyclase recently identified in Olea cell suspension cultures and able to form mainly -amyrin, but also -amyrin and butyrospermol (Saimaru et al., 2007). Mechanisms underlying regulation of the carbon flux through both pathways remain to be elucidated. Such a regulation clearly involves interplay between several partners, including the different OS cyclases (cycloartenol synthase
217
and triterpenes synthases) but also several acyltransferases and maybe glycosyltransferases. Squalene and OS are synthesized in the membranes of the endoplasmic reticulum where cycloartenol synthase (as well as the other enzymes of the sterol pathway) and also probably triterpenes synthases are located. As suggested by the present work, the expression patterns of the different OS cyclases appear to be closely dependent on the stage of the fruit developmental process. Our results clearly indicate that, in early stages, synthesis of pentacyclic triterpenes, by one or several triterpene synthases, occurs concomitantly with acylation of sterols whereas the opposite situation (i.e. free sterol biosynthesis and esterification of pentacyclic triterpenes) is observed in later stages (after the 21st WAF). Thus, these acylation reactions, leading to the removal of the newly synthesized sterol intermediates or pentacyclic triterpenes from each respective pathway appears as a means to direct most of the available OS molecules toward only one pathway. Very little attention has been paid to acyltransferases involved in these reactions. In Arabidopsis, two different enzymes catalyzing the formation of sterol esters, via either a phospholipid (Banas et al., 2005) or a fatty acyl CoA as the acyl donor (Chen et al., 2007), have been recently characterized. The first enzyme seems to be specific to the sterol pathway as it is able to acylate various sterol end products as well as sterol intermediates, but not lupeol or ß-amyrin. The best substrate of the second enzyme was found to be cycloartenol, but whether or not this enzyme is also capable of forming acylated pentacyclic triterpenes has not been determined. It should be pointed out that sterol esters, which are not membrane components, are synthesized concomitantly with triacylglycerols (Stiti et al., 2007) and thus participate with them in the formation of olive fruit oil droplets. In conclusion, further work is needed to investigate more deeply the relationships between both triterpenic pathways in the olive fruit. The elucidation of the roles played in planta by the non-steroidal triterpenoids also appears to be a challenging objective. For example, these compounds are known to be constituents of epicuticular wax crystals and might be involved in plant–insect interactions (Guhling et al., 2006). The rising interest in the valuable biological properties for human health of non-steroidal triterpenoids, including maslinic acid (see Liu et al., 2007 and Chapter 158), constitutes an additional motivation to address these questions.
Summary points Evidence is given here for the occurrence in the olive fruit of a vast array of sterols and non-steroidal triterpenoids, among which oleanane-type compounds are largely predominant. These two classes of compounds are synthesized via the mevalonate pathway and share common precursors.
l
218
Section | I Lipids, Phenolics and Other Organics and Volatiles
The composition of this complex mixture of triterpenoids was found to be closely dependent on the fruit developmental stage. l From the 12th to the 18th WAF, the young green olive fruit contained high amounts of a- and b-amyrins along with hydroxylated pentacyclic alcohols and acids, but no new free sterols were formed. l From the 21st WAF, when the epidermal color progressively turned from green to purple, a- and b-amyrins were not present any longer whereas free sterols began to be synthesized, indicating a re-direction of the carbon flux from the non-steroidal pathway toward the sterol pathway. Between 21 and 30 WAF, a two-fold increase in the content of free and esterified sterols was observed. Concomitantly, non-steroidal triterpenoids were represented almost exclusively by oleanolic and maslinic acids. l These data clearly indicate that a complex regulation process takes place at the oxidosqualene cyclization step. l
References Abe, L., Rohmer, M., Prestwich, G.D., 1993. Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chem. Rev. 93, 2189–2206. Achnine, L., Huhman, D.V., Farag, M.A., Sumner, L.W., Blount, J.W., Dixon, R.A., 2005. Genomics-based selection and functional characterization of triterpene glycosyltransferascs from the model legume Medicago truncatula. Plant J. 41, 875–887. Azadmard-Damirchi, S., Savage, G.P., Dutta, P.C., 2005. Sterol fractions in hazelnut and virgin olive oils and 4,4-dimethylsterols as possible markers for detection of adulteration of virgin olive oil. J. Am. Oil Chem. Soc. 82, 717–725. Banas, A., Carlsson, A.S., Huang, B., Lenman, M., Banas, W., Lee, M., Noiriel, A., Benveniste, P., Schaller, H., Bouvier-Nave, P., Stymne, S., 2005. Cellular sterol ester synthesis in plants is performed by an enzyme (phospholipid:sterol acyltransferase) different from the yeast and mammalian acyl-CoA:sterol acyltransferases. J. Biol. Chem. 280, 34626–34634. Benveniste, P., 2002. Sterol metabolism. American Society of Plant Biologists, Rockville. http://www.bioone.org/archive/i1543-8120-38-1.pdf/ Bianchi, G., Pozzi, N., Vlahov, G., 1994. Pentacyclic triterpene acids in olives. Phytochemistry 37, 205–207. Caputo, R., Mangoni, L., Monaco, P., Previtera, L., 1974. New triterpenes from the leaves of Olea europaea. Phytochemistry 13, 2825–2827. Chen, Q., Steinhauer, L., Hammerlindl, J., Keller, W., Zou, J., 2007. Biosynthesis of phytosterol esters: identification of a sterol-Oacyltransferase in Arabidopsis. Plant Physiol. 145, 974–984. Chryssafidis, D., Maggos, P., Kiosseoglou, V., Boskou, D., 1992. Composition of total and esterified 4a-monomethylsterols and triterpene alcohols in virgin olive oil. J. Sci. Food Agric. 58, 581–583.
Ebizuka, Y., Katsube, Y., Tsutsurni, T., Kushiro, T., Shibuya, M., 2003. Functional genornics approach to the study of triterpene biosynthesis. Pure Appl. Chem. 75, 369–374. Guhling, O., Hobl, B., Yeats, T., Jetter, R., 2006. Cloning and characterization of a lupeol synthase involved in the synthesis of epicuticular wax crystals on stem and hypocotyl surfaces of Ricinus communis. Arch. Biochem. Biophys. 448, 60–72. Hartmann, M.A., Benveniste, P., 1987. Plant membrane sterols: isolation, identification and biosynthesis. Methods Enzymol. 148, 632–650. Hota, R.K., Bapuji, M., 1994. Triterpenoids from the resin of Shorea Robusta. Phytochemistry 35, 1073–1074. Itoh, T., Yoshida, K., Yatsu, T., Tamura, T., Matsumoto, T., 1981. Triterpene alcohols and sterols of spanish olive oil. J. Am. Oil Chem. Soc. 58, 545–550. Liu, J., Sun, H., Wang, X., Mu, D., Liao, H., Zhang, L., 2007. Effects of oleanolic acid and maslinic acid on hyperlipidemia. Drug Dev. Res. 68, 261–266. Mahato, S.B., Sarkar, S.K., Poddar, G., 1988. Triterpenoid saponins. Phytochemistry 27, 3037–3067. Perez-Camino, M.C., Cert, A., 1999. Quantitative determination of hydroxy pentacyclic triterpene acids in vegetable oils. J. Agric. Food Chem. 47, 1558–1562. Power, F.B., Tutin, F., 1908. The constituents of olive leaves. J. Chern. Soc. Trans. 93, 891–904. Rahier, A., Benveniste, P., 1989. Mass spectral identification of phytosterols. In: Nes, W.D., Parish, E. (eds) Analysis of Sterols and Other Biologically Significant Steroids. Academic Press, New York, pp. 223–250. Ranalli, A., Pollastri, L., Contento, S., Di Loreto, G., Iannucci, E., Lucera, L., Russi, F., 2002. Sterol and alcohol components of seed, pulp and whole olive fruit oils. Their use to characterise olive fruit variety by multivariates. J. Sci. Food Agric. 82, 854–859. Reina, R.J., White, K.D., Jahngen, E.G., 1997. Validated method for quantitation and identification of 4,4-desmethylsterols and triterpene diols in plant oils by thin-layer chromatography-high resolution gas chromatography-mass spectrometry. J. AOAC Int. 80, 1272–1280. Saimaru, H., Orihara, Y., Tansakul, P., Kang, Y.-H., Shibuya, M., Ebizuka, Y., 2007. Production of triterpene acids by cell suspension cultures of Olea europaea. Chem. Pharm. Bull. (Tokyo) 55, 784–788. Seo, S., Yoshimura, Y., Uomori, A., Takeda, K., Seto, H., Ebizuka, Y., Sankara, U., 1988. Biosynthesis of triterpenes, ursolic acid, and oleanolic acid in tissue cultures of Rabdosia japonica Hara fed [5-13C2H2 J mevalonolactone and [2-13C2H3] acetate. J. Am. Chem. Soc. 110, 1740–1745. Shibuya, M., Zhang, H., Endo, A., Shishikura, K., Kushiro, T., Ebizuka, Y., 1999. Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases. Eur. J. Biochem. 266, 302–307. Stiti, N., M’Sallem, M., Triki, S., Cherif, A., 2002. Etude de la fraction insaponifiable de l’huile d’olive de differcntcs varietes tunisiennes. Riv.ltal. Sostanze Grasse 79, 357–363. Stiti, N., Triki, S., Hartmann, M.A., 2007. Formation of triterpenoids throughout Olea europaea fruit ontogeny. Lipids 42, 55–67.
Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit Naïm Stiti1,2, Saïda Triki2 and Marie-Andrée Hartmann1 1
lnstitut de Biologie Moleculaire des Plantes (CNRS UPR 2357), Université de Strasbourg, 28 rue Goethe, 67083 Strasbourg, France Faculté des Sciences de Tunis, Département des Sciences Biologiques, Campus Universitaire, 2092 Tunis, Tunisia
2
23.1 Introduction Olive oil as well as olive leaves have been known for a long time to contain a wide range of sterols and non-steroidal triterpenoids, including erythrodiol, oleanolic acid and maslinic acid (Power and Tutin 1908; Caputo et al., 1974; Itoh et al., 1981), which are oxygenated derivatives of -amyrin (olean-12-en-3-ol), one of the most commonly occurring triterpenes. Sterols and non-steroidal triterpenoids, which belong to the group of terpenoids or isoprenoids, the largest family of natural products, are synthesized via the cytoplasmic acetate/mevalonate pathway and share common precursors up to (3S)-2,3-oxidosqualene (OS) (Seo et al., 1988; Benveniste, 2002). Then, OS serves as a substrate for various OS cyclases, also called triterpene synthases, to form C30 compounds (i.e., comprising six C5-isoprene units). Cycloartenol synthase catalyzes the cyclization of OS folded in the pre-chair-boat-chair conformation, via the protosteryl cation, into cycloartenol, the first cyclic precursor of the sterol pathway (Figure 23.1). About 20 steps are needed to convert cycloartenol in end pathway sterols (Benveniste, 2002). Non-steroidal triterpenoids are assumed to be formed from OS folded in the all-pre-chair conformation, through a series of carbo cationic intermediates (Abe et al., 1993) (Figure 23.1). They are then often metabolized into more oxygenated compounds, which serve as precursors for the synthesis of triterpenic saponins (Mahato et al., 1988). As the cyclization of OS into sterols and non-steroidal triterpenoids represents a branch point between primary and secondary metabolisms, OS cyclases are attractive tools for investigating the physiological roles of non-steroidal triterpenoids. The present study sheds more light on biosynthetic relationships occurring between the sterol and non-steroidal triterpenoid pathways in Olea europaea L. throughout olive fruit ontogeny. Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
23.2 THE OLIVE FRUIT CONTAINs A VAST ARRAY OF STEROLS AND NON-STEROIDAL TRITERPENOIDS Sterols and non-steroidal triterpenoids were isolated from total lipid extracts of olive drupes (i.e., the whole fruit comprising the epicarp, the mesocarp and the endocarp or pit with the seed) as previously described (Hartmann and Benveniste 1987; Stiti et al., 2007). Free sterols, tetracyclic and pentacyclic triterpenes, triterpenic diols as well as the compounds released after hydrolysis of ester conjugates were identified as acetate derivatives by their relative retention time in gas chromatography and their mass spectrometry fragmentation pattern in gas chromatography coupled to mass spectrometry (Rahier et al., 1989; Stiti et al., 2007 and references herein). Mono- and dihydroxy pentacyclic triterpenic acids (HPTAs) were isolated according to Pérez-Camino and Cert (1999) and Stiti et al. (2007) and identified as acetate derivatives of the corresponding methylesters (Stiti et al., 2007).
23.2.1 Sterols Olive drupes were shown to contain a mixture of sterols, with sitosterol as the largely predominant compound (between 70 and 95% of total sterols), and 24-methylcholesterol, stigmasterol and isofucosterol (5-avenasterol) (1–3%). We also identified brassicasterol, 24-methylenecholesterol, 5,24-stigmastadienol, 7-avenasterol and cholesterol. All the usual intermediates of the sterol pathway: squalene, 4-dimethylsterols (cycloartenol and 24-methylenecycloartanol) and 4a-methylsterols (obtusifoliol, cycloeucalenol, 24-methylene and 24-ethylidenelophenol) were found. The occurrence of some less usual sterols such as 24-methyl- and 24-ethyl-lophenol, (24S)24-ethylcholesta-5,25-dien-3-ol (clerosterol) and sterols
211
Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
212
Section | I Lipids, Phenolics and Other Organics and Volatiles
+ H
+ 20
H H
+
17
2,3-oxidosqualene
CCC
dammarenyl cation
C B C
protosteryl cation
H 19
HO
HO
H
+
H H
H H
H
3 2 O
H
+
HO
baccharenyl cation
HO
lupenyl cation
20 + H
HO
oleanyl cation
-12αH
ursanyl cation
-12βH
H HO
HO
9
12
HO
1
7
HO
-9βH
-19H
CH2OH
HO
HO
parkeol
HO
cycloartenol
HO
9
3
14 HO
COOH
15
COOH
COOH
HO
HO
CH2OH
CH2OH
HO
10
4
Sterols COOH HO HO
5
Non-steroidal triterpenoids Figure 23.1 Postulated biosynthetic pathway of non-steroidal triterpenoids from 2,3-oxidosqualene cycIization in the Olea europaea fruit. This figure is adapted from the previously published Scheme 1 (Stiti et al., 2007). CBC and CCC refer respectively to the pre-chair-boat-chair and allpre-chair conformations of oxidosqualene (OS). OS serves as a substrate for the synthesis of either sterols or non-steroidal triterpenes. Cycloartenol synthase catalyzes the formation of cycloartenol, the first cyclic precursor of sterols, via the protosteryl cation. The OS cyclization reaction by non-steroidal triterpene synthases is thought to proceed through generation of several four or five ring-carbocationic intermediates. These carbocationic intermediates are represented in brackets. Oleanane-type triterpenoids, which arise from the oleanyl cation, are by far the predominant compounds, as shown by the widest arrows. The names of the different compounds, which are designated by a number, are given in the legend of Figure 23.2.
with a double bond at C-23 (5,23-stigmastadienol and 24ethyl E-23-dehydrolophenol); at C-11 (5-lanosta-9(11), 24-dien-3-ol) or parkeol and 24-methylene-lanost-9(11)en-3-ol has to be mentioned. All these sterols were also present as esters. However, it is interesting to note that no free parkeol could be detected.
23.2.2 Non-steroidal Triterpenoids Besides sterols, the olive fruit contains a great diversity of triterpenoids. Their structures are shown in Figure 23.2. They include 19 pentacyclic triterpenoids arising from four different carbon skeletons: oleanane-type (1–7) (-amyrin 1, 28-nor--amyrin 2, erythrodiol 3, oleanolic acid 4, maslinic acid 5, -amyrone 6 and -amyrin 7), ursane-type (8–12) (-amyrin 8, 28-nor- amyrin 9, uvaol 10, ursolic acid 11 and -amyrone 12), lupane-type (13–17) (lupeol 13, 3epi-lupeol 14, 3-epi-betulin 15 and 3-epi-betulinic acid 16
and lupenone 17) and taraxane-type (18–19) (taraxerol 18, taraxer-14-ene-3,28-diol 19), as well as two tetracyclic triterpenes with euphane-type (butyrospermol 20) and baccharane-type (bacchar-12,21-dien-3-ol 21) carbon skeletons. Oleanane triterpenoids were largely predominant, with oleanolic and maslinic acids representing by far the major compounds. Pentacyclic triterpenes also occurred as esters, but no acylated triterpenic diols have been found. Thus, more than 40 sterols and non-steroidal triterpenoids have been identified in the olive fruit. Our results are consistent with previous reports about the sterol and triterpenoid composition of olive oil or fruit (Itoh et al., 1981; Chryssafidis et al., 1992; Bianchi et al., 1994; Reina et al., 1997; Ranalli et al., 2002; Stiti et al., 2002; Azadmard-Damirchi et al., 2005; see Chapter 27). However, the occurrence in the olive fruit of taraxer-14-ene-3,28-diol, 3-epi-lupeol and its metabolites, 3-epi-betulin and 3-epi-betulinic acid, had not been reported before.
213
Chapter | 23 Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit
R1
R1 R2
(1) (2) (3) (4) (5) (6) (13) (14) (15) (16) (17)
R
HO
R
(7)
R = α-H, β-OH, R1 = CH3, R2 = H R = α-H, β-OH, R1 = H, R2 = H R = α-H, β-OH, R1 = CH2OH, R2 = H R = α-H, β-OH, R1 = COOH, R2 = H R = α-H, β-OH, R1 = COOH, R2 = OH R = O, R1 = CH3, R2 = H R = α-H, β-OH, R1 = CH3 R = α-OH, β-H, R1 = CH3 R = α-OH, β-H, R1 = CH2OH R = α-OH, β-H, R1 = COOH R = O, R1 = CH3
(8) (9) (10) (11) (12)
R = α-H, β-OH, R1 = CH3 R = α-H, β-OH, R1 = H R = α-H, β-OH, R1 = CH2OH R = α-H, β-OH, R1 = COOH R = O, R1 = CH3
(18) R1 = CH3 (19) R1 = CH2OH R1
R1 H HO
R
H
H HO
HO
(20)
(21)
Figure 23.2 Structures of the non-steroidal triterpenoids identified in the olive fruit. Oleanane-type: (1), b-amyrin; (2), 28-nor-b-amyrin; (3), erythrodiol; (4), oleanolic acid; (5), maslinic acid; (6), b-amyrone; (7), -amyrin; Ursane-type: (8), -amyrin; (9), 28-nor--amyrin; (10), uvaol; (11), ursolic acid; (12), -amyrone; Lupane-type: (13), lupeol; (14), 3-epi-lupeol; (15), 3-epi-betulin; (16), 3-epi-betulinic acid; (17), lupenone; Taraxane-type: (18), taraxerol; (19), taraxer-14-ene-3,28-diol; Euphol-type: (20), butyrospermol; Baccharanetype: (21), bacchar-12,21-dien-3-ol.
23.3 Changes in the Content of Free and Esterified Sterols and Non-Steroidal Triterpenoids Throughout Fruit Development
Drupes from the different batches were analyzed for their content in free and esterified sterols and non-steroidal triterpenoids, but only data corresponding to olives harvested at the stages 12, 18, 21 and 30 WAF are presented here.
Olive fruit were handpicked from all the sides of one olive tree, Olea europaea L. cv Chemlali, at 13 distinct stages of fruit growth and ripening corresponding to 12, 13, 15, 16, 18, 21 23, 25, 27, 29, 30, 32 and 33 weeks after development (WAF). At the time of the first harvest, the lignification of the olive endocarp had ended. Between 12 and 18 WAF, olives were green and progressively increased in size and fresh weight, but in the case of the Chemlali cultivar, these changes were of limited amplitude compared to other olive varieties. At the end of this period, the final fruit size was almost fixed and from the 21st WAF, epidermal color gradually turned from green to purple. Complete maturity was observed after 29 WAF. The 33 WAF stage corresponded to an ‘over maturation’ stage.
23.3.1 Sterols from the 12th to the 18th WAF In the young olive fruit (i.e. between 12 and 18 WAF), free sterols were present as a mixture in which sitosterol was largely predominant (95%), but the usual sterol intermediates, 4,4-dimethyl- and 4-methylsterols, were barely detectable (Table 23.1). A relatively high amount of squalene (700 g g1 dry wt) was detected at 13 WAF, but then rapidly decreased to 40 g g1 at 18 WAF (data not shown). During this period of time, a slight decrease in the total free sterol content of the olive fruit was observed (Table 23.1). The young olive drupes were also found to contain sterols as ester conjugates, with a sterol profile slightly
214
Section | I Lipids, Phenolics and Other Organics and Volatiles
Table 23.1 Changes in free sterols during olive fruit development.
Table 23.2 Changes in esterified sterols during olive fruit development.
Developmental stage Sterol classes
12 WAF
18 WAF
21 WAF
30 WAF
Developmental stage Sterol classes
12 WAF
18 WAF
21 WAF
30 WAF
4,4-dimethylsterols cycloartenol
nd
nd
0.4
3.2
4,4-dimethylsterols cycloartenol
nd
nd
2.7
1.4
24-methylenecycloartanol nd
nd
3.1
26.6
parkeol
nd
nd
16.4
13.4
24-methylenecycloartanol
4.7a
6.6
7.7
10.4
4-methylsterols obtusifoliol
0.5
0.7
1.4
0.7
4-methylsterols obtusifoliol
nd
nd
0.6
0.2
24-methylenelophenol
nd
nd
1.0
0.7
24-methyl-lophenol
nd
nd
0.2
0.1
cycloeucalenol
nd
nd
0.7
0.4
24-methylenelophenol
2.7
2.0
0.8
0.4
0.1
cycloeucalenol
1.6
0.9
0.6
0.6
1.1
1.3
2.7
3.3
24-ethyl-lophenol
nd
nd
0.2
24-ethylidenelophenol
nd
nd
0.9
1.1
24-ethylidenelophenol
4-demethylsterols brassicasterol
0.4a
0.4
0
0
4-demethylsterols 24-methylcholesterol
2.1
2.1
2.7
2.3
1.9
stigmasterol
7.2
7.5
2.4
0.7
0.8
clerosterol
0.8
0.7
0.8
0.7
62.5
62.5
59.2
62.0 4.1
24-methylcholesterol stigmasterol
1.5 1.4
1.4 2.5
2.9 1.3
clerosterol
0.7
1.2
0.8
0.6
sitosterol
sitosterol
95.9
94.4
87.5
61.3
isofucosterol
16.2
15
2.6
5,24-stigmastadienol
0.5
0.6
nd
nd
Total amount (g/g dry wt)
44
100
140
305
isofucosterol Total amount (g/g dry wt)
0 250
0 230
0.2 500
2.7 960
The standard deviation for quantitative determinations was 5%. a
% of total free sterols; nd: not detectable.
different from that of free forms. In particular, sitosterol remained the major compound (63%), but significantly higher relative proportions of 24-methylcholesterol, stigmasterol and isofucosterol were found (Table 23.2). Low amounts of acylated sterol intermediates, especially 24methylenecycloartanol, were present (Table 23.2). Between 12 and 18 WAF, a 2.3-fold increase in the total amount of sterol esters was observed, an increase that equally affected sterol intermediates and end products, indicating that some sterol biosynthesis took place in the very young fruit. However, these newly synthesized sterols were immediately conjugated to a fatty acid and thus removed from the free sterol pathway.
23.3.2 Sterols from the 21st to the 30th WAF From the 21st WAF, dramatic changes were observed in the free sterol pathway. Early biosynthetic intermediates, i.e., squalene, 4,4-dimethyl- (cycloartenol and 24-methylene cycloartanol) and 4-methylsterols (cycloeucalenol, obtusifoliol, 24-methylene- and 24-ethylidenelophenol) as well as late precursors (isofucosterol) began to be detectable
The standard deviation for quantitative determinations was 5%. a
% of total sterols; nd: not detectable.
and a progressive increase in sterol end products was concomitantly observed (Table 23.1 and Figure 23.3A, 21st WAF). Throughout the fruit-ripening process, sterols continued to accumulate, with sitosterol remaining the major compound. At 30 WAF, the free sterol content of the olive fruit amounted to 950 g g1 dry wt, corresponding to a four-fold total increase from the 12th WAF. At this stage, a significant accumulation of some early intermediates, especially squalene (data not shown) and 24-methylenecycloartanol, was observed (Table 23.1 and Figure 23.3A, 30th WAF), indicating a slowing down of the metabolic flux through the sterol pathway. Between 21 and 30 WAF, the content of the olive fruit in sterol esters continued to rise, particularly between 27 and 29 WAF, to give a total amount corresponding to a seven-fold increase during the whole period of fruit development (Table 23.2). At the end of the ripening process (30 WAF), a significant accumulation of 24-methylenecycloartanol and 24-ethylidenelophenol was observed, in agreement with previous data on olive oil (Chryssafidis et al., 1992). It is interesting to note the occurrence of a new compound in the fraction of 4,4-dimethylsterols, which has been identified as parkeol (Table 23.2). This compound was formed concomitantly with cycloartenol and represented 60% of the total esterified 4,4-dimethyl sterols.
Chapter | 23 Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit
215
Figure 23.3 Changes in the content and the composition of free sterols (A) and free non-steroidal triterpenoids (B) throughout olive fruit development. The central part of the figure corresponds to the total content in free sterols and non-steroidal triterpenoids at different developmental stages of the olive fruit (12, 18, 21 and 30th WAF). Sterol pathway: 4,4-dimethylsterols; 4a-methylsterols; 4-demethylsterols. non-steroidal pathway: penta cyclic triterpenes; triterpenic diols; mono- and di-HPTAs.
During the whole period of olive fruit development, it should be pointed out that free sterols remained predominant compared to ester conjugates (Tables 23.1 and 23.2).
23.3.3 Non-steroidal Triterpenoids from the 12th to the 18th WAF At the beginning of fruit ontogeny, besides sterols, the olive fruit were found to contain high levels of the pentacyclic tri terpenes a- and b-amyrins, in a 3:2 ratio, as well as several more oxygenated compounds with additional hydroxymethyl or carboxylic groups (Table 23.3 and Figure 23.3B, 12th WAF). The introduction of a hydroxyl group in C-28 position of - and -amyrins gives rise to uvaol and erythrodiol,
respectively (Figures 23.1 and 23.2). A further oxidation of this hydroxyl group leads to the corresponding ursolic and oleanolic acids. Finally, the introduction of an additional hydroxyl group at the C-2 position of oleanolic acid results in the formation of maslinic acid. In addition to these oleanane- and ursane-type triterpenoids, lupane (3-epi-betulin and 3-epi-betulinic acid) and taraxane (taraxen-14-ene3b,28-diol) derivatives were also formed (Table 23.3). The enzymes involved in all these oxidation reactions have not been characterized yet, but are likely cytochrome P-450 monooxygenases. According to this hypothesis, the pathway should implicate the intermediate formation of the aldehydes 3-hydroxy-5-urs-12-en-28-al and 3-hydroxy5-olean-12-en-28-al. The presence of these compounds has not been checked. However, the occurrence of significant
216
Section | I Lipids, Phenolics and Other Organics and Volatiles
Table 23.3 Changes in free non-steroidal triterpenoids throughout olive fruit development.
Table 23.4 Changes in esterified non-steroidal triterpenes.
Developmental stage Triterpenoid classes
12 WAF
18 WAF
21 WAF
30 WAF
Developmental stage
12 WAF
18 WAF
21 WAF
30 WAF
Pentacyclic triterpenes ß-amyrin
4.3a
-amyrin
5.5
5.5
nd
Nd
2.5
0.1
0.2 ß-amyrin
25.1
13.9
10.9
11.9
28-nor-b-amyrin
2.3
1.5
–
– -amyrin
10.0
21.5
15.9
22.6
a-amyrin
6.0
3.3
–
– taraxerol
33.6a
21.7
11.6
5.6
28-nor-a-amyrin
0.8
0.4
–
– butyrospermol
nd
nd
61.6
59.8
pentacyclic diols taraxerol
–
–
0.1
0.1
lupeol
25.7
37.3
nd
nd
taraxer-14-ene-3ß-28diol
0.2
0.3
–
–
Total amount (g/g dry wt)
7.0
8.8
39
35
erythrodiol
12.5
9.7
0.4
0.5
The standard deviation for quantitative determinations was 5%.
uvaol
7.6
6.2
0.1
–
3-epi-betulin
0.3
0.2
–
–
Mono-HPTAs 3-epi-betulinic acid
0.7
0.6
0.6
0.6
oleanolic acid
39.2
35.8
41.2
37.6
Di-HPTAs ursolic acid
0.2
0.2
0.2
0.2
maslinic acid
25.9
39.3
57.3
60.8
Total amount (g/g dry wt)
3210
2610
3930
2470
a
% of total esterified non-steroidal triterpenes; nd, not detectable.
The standard deviation for quantitative determinations was 10%. a
% of total free non-steroidal triterpenoids.
amounts of 28-nor--amyrin and 28-nor--amyrin, which have no methyl group at C-17 (Figure 23.2), in the pentacyclic triterpene fraction (Table 23.3), might result from the decarbonylation of such aldehydes (Hota and Bapuji, 1994). The somewhat delayed accumulation of maslinic acid (Table 23.3) suggests that the hydroxylation step at C-2 may involve another type of cyt P450-monooxygenase. Between 12 and 18 WAF, the non-steroidal triterpenoid pathway was very efficient as attested by the synthesis of very high levels of these compounds (i.e. 3 mg g1 dry wt) (Table 23.3 and Figure 23.3B18th WAF). During the same period, ester conjugates of pentacyclic triterpenes were barely detectable (0.2% of the corresponding free forms) (Table 23.4). Traces of a- and b-amyrins, taraxerol, -amyrin and lupeol were found, with no change in the total amount of these esters between 12 and 18 WAF (Table 23.4).
23.3.4 Non-steroidal Triterpenoids from the 21st to the 30th WAF Between 21 to 30 WAF, a dramatic decrease in the content of a- and b-amyrins and pentacyclic triterpenic diols was observed (Table 23.3 and Figure 23.3B, 21st WAF). Nonsteroidal triterpenoids were constituted almost exclusively of mono- and di-HPTAs, with oleanolic and maslinic acids as the largely predominant compounds (98% of total triterpenoids) (Table 23.3 and Figure 23.3B, 21st WAF). In the mature olive fruit (30 WAF), a significant decrease in the content of all HPTAs was observed (Table 23.3), indicating that these compounds might be further metabolized, maybe into triterpenic saponins via the involvement of specific glycosyltransferases (Achnine et al., 2005). To our knowledge, saponins from olive tree have not yet been identified. Between 21 and 30 WAF, when amyrins were not any longer formed, esterified conjugates began to progressively accumulate, especially d-amyrin (23% at 30 WAF) (Table 23.4). Concomitantly, a change in the profile of triterpenes could be noticed, consisting in the appearance of a new tetracyclic triterpene, identified as butyrospermol, which represented up to 60% of total esterified triterpenes.
23.4 How is carbon flux regulated between both triterpenic pathways in the olive fruit? Evidence is presented here for the occurrence in the olive fruit of a vast array of sterols and non-steroidal triterpenoids. More than 40 different compounds have been found and the composition of this complex mixture was found to
Chapter | 23 Sterols and Non-steroidal Triterpenoids of the Developing Olive Fruit
be strongly dependent on the fruit developmental stage as illustrated in Figure 23.3. Throughout fruit ontogeny, two periods can be clearly distinguished: from 12 to 18 WAF and from 21 to 30 WAF. In the young green olive fruit (between 12 and 18 WAF), most of the available squalene molecules are almost exclusively devoted to the synthesis of a- and bamyrins. These non-steroidal pentacyclic triterpenes were rapidly metabolized into more oxygenated compounds, first into triterpenic alcohols, then into mono- and di-HPTAs. During the same period, no free sterols were formed. However, the sterol pathway remained functional as attested by the formation of sterol esters. Between 21 and 30 WAF, when the epidermal color gradually turned from green to purple, a- and b-amyrins and their hydroxylated derivatives were not present any longer, while the already-formed oxygenated intermediates were converted into mono- and diHPTAs (see the postulated biosynthetic pathway in Figure 23.1). Interestingly, our data also indicate that although a-amyrin was present in excess compared to b-amyrin, oleanane-type compounds as a whole were produced in far higher amounts than ursane-type compounds (Table 23.3). In early stages of fruit development, oleanane-type compounds represented from 84–89% of total non-steroidal triterpenoids and ursane-type compounds, only 10–15%. Between 21 and 30 WAF, ursane-type compounds completely disappeared. Thus, the question of the metabolic fate of a-amyrin in the olive fruit remains to be solved. From the 21st WAF, free and esterified sterols began to be formed and accumulated until the complete maturity of the fruit, but whatever the fruit developmental stage, nonsteroidal triterpenoids remained the major triterpenic compounds, with maslinic acid as the most represented one in the mature fruit (Table 23.3). Taken together, these results clearly indicate that a complex regulation process takes place at the oxidosqualene cyclization step, which represents a branch point between the sterol pathway and the non-steroidal triterpenoid pathway (Figure 23.1). OS serves indeed as a substrate for cyclo artenol synthase, the first enzyme of the sterol pathway, but also for various OS cyclases involved in the synthesis of the different classes of pentacyclic triterpenes. These OS cyclases are designated as mono- or multifunctional enzymes, depending on whether they produce single or several cyclization products (Ebizuka et al., 2003). OS cyclases of the olive fruit have not been characterized yet, but might include a lupeol synthase, a mono-functional enzyme, similar to that identified in the olive leaf (Shibuya et al., 1999) as well as a multifunctional triterpene synthase, such as the OS cyclase recently identified in Olea cell suspension cultures and able to form mainly -amyrin, but also -amyrin and butyrospermol (Saimaru et al., 2007). Mechanisms underlying regulation of the carbon flux through both pathways remain to be elucidated. Such a regulation clearly involves interplay between several partners, including the different OS cyclases (cycloartenol synthase
217
and triterpenes synthases) but also several acyltransferases and maybe glycosyltransferases. Squalene and OS are synthesized in the membranes of the endoplasmic reticulum where cycloartenol synthase (as well as the other enzymes of the sterol pathway) and also probably triterpenes synthases are located. As suggested by the present work, the expression patterns of the different OS cyclases appear to be closely dependent on the stage of the fruit developmental process. Our results clearly indicate that, in early stages, synthesis of pentacyclic triterpenes, by one or several triterpene synthases, occurs concomitantly with acylation of sterols whereas the opposite situation (i.e. free sterol biosynthesis and esterification of pentacyclic triterpenes) is observed in later stages (after the 21st WAF). Thus, these acylation reactions, leading to the removal of the newly synthesized sterol intermediates or pentacyclic triterpenes from each respective pathway appears as a means to direct most of the available OS molecules toward only one pathway. Very little attention has been paid to acyltransferases involved in these reactions. In Arabidopsis, two different enzymes catalyzing the formation of sterol esters, via either a phospholipid (Banas et al., 2005) or a fatty acyl CoA as the acyl donor (Chen et al., 2007), have been recently characterized. The first enzyme seems to be specific to the sterol pathway as it is able to acylate various sterol end products as well as sterol intermediates, but not lupeol or ß-amyrin. The best substrate of the second enzyme was found to be cycloartenol, but whether or not this enzyme is also capable of forming acylated pentacyclic triterpenes has not been determined. It should be pointed out that sterol esters, which are not membrane components, are synthesized concomitantly with triacylglycerols (Stiti et al., 2007) and thus participate with them in the formation of olive fruit oil droplets. In conclusion, further work is needed to investigate more deeply the relationships between both triterpenic pathways in the olive fruit. The elucidation of the roles played in planta by the non-steroidal triterpenoids also appears to be a challenging objective. For example, these compounds are known to be constituents of epicuticular wax crystals and might be involved in plant–insect interactions (Guhling et al., 2006). The rising interest in the valuable biological properties for human health of non-steroidal triterpenoids, including maslinic acid (see Liu et al., 2007 and Chapter 158), constitutes an additional motivation to address these questions.
Summary points Evidence is given here for the occurrence in the olive fruit of a vast array of sterols and non-steroidal triterpenoids, among which oleanane-type compounds are largely predominant. These two classes of compounds are synthesized via the mevalonate pathway and share common precursors.
l
218
Section | I Lipids, Phenolics and Other Organics and Volatiles
The composition of this complex mixture of triterpenoids was found to be closely dependent on the fruit developmental stage. l From the 12th to the 18th WAF, the young green olive fruit contained high amounts of a- and b-amyrins along with hydroxylated pentacyclic alcohols and acids, but no new free sterols were formed. l From the 21st WAF, when the epidermal color progressively turned from green to purple, a- and b-amyrins were not present any longer whereas free sterols began to be synthesized, indicating a re-direction of the carbon flux from the non-steroidal pathway toward the sterol pathway. Between 21 and 30 WAF, a two-fold increase in the content of free and esterified sterols was observed. Concomitantly, non-steroidal triterpenoids were represented almost exclusively by oleanolic and maslinic acids. l These data clearly indicate that a complex regulation process takes place at the oxidosqualene cyclization step. l
References Abe, L., Rohmer, M., Prestwich, G.D., 1993. Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chem. Rev. 93, 2189–2206. Achnine, L., Huhman, D.V., Farag, M.A., Sumner, L.W., Blount, J.W., Dixon, R.A., 2005. Genomics-based selection and functional characterization of triterpene glycosyltransferascs from the model legume Medicago truncatula. Plant J. 41, 875–887. Azadmard-Damirchi, S., Savage, G.P., Dutta, P.C., 2005. Sterol fractions in hazelnut and virgin olive oils and 4,4-dimethylsterols as possible markers for detection of adulteration of virgin olive oil. J. Am. Oil Chem. Soc. 82, 717–725. Banas, A., Carlsson, A.S., Huang, B., Lenman, M., Banas, W., Lee, M., Noiriel, A., Benveniste, P., Schaller, H., Bouvier-Nave, P., Stymne, S., 2005. Cellular sterol ester synthesis in plants is performed by an enzyme (phospholipid:sterol acyltransferase) different from the yeast and mammalian acyl-CoA:sterol acyltransferases. J. Biol. Chem. 280, 34626–34634. Benveniste, P., 2002. Sterol metabolism. American Society of Plant Biologists, Rockville. http://www.bioone.org/archive/i1543-8120-38-1.pdf/ Bianchi, G., Pozzi, N., Vlahov, G., 1994. Pentacyclic triterpene acids in olives. Phytochemistry 37, 205–207. Caputo, R., Mangoni, L., Monaco, P., Previtera, L., 1974. New triterpenes from the leaves of Olea europaea. Phytochemistry 13, 2825–2827. Chen, Q., Steinhauer, L., Hammerlindl, J., Keller, W., Zou, J., 2007. Biosynthesis of phytosterol esters: identification of a sterol-Oacyltransferase in Arabidopsis. Plant Physiol. 145, 974–984. Chryssafidis, D., Maggos, P., Kiosseoglou, V., Boskou, D., 1992. Composition of total and esterified 4a-monomethylsterols and triterpene alcohols in virgin olive oil. J. Sci. Food Agric. 58, 581–583.
Ebizuka, Y., Katsube, Y., Tsutsurni, T., Kushiro, T., Shibuya, M., 2003. Functional genornics approach to the study of triterpene biosynthesis. Pure Appl. Chem. 75, 369–374. Guhling, O., Hobl, B., Yeats, T., Jetter, R., 2006. Cloning and characterization of a lupeol synthase involved in the synthesis of epicuticular wax crystals on stem and hypocotyl surfaces of Ricinus communis. Arch. Biochem. Biophys. 448, 60–72. Hartmann, M.A., Benveniste, P., 1987. Plant membrane sterols: isolation, identification and biosynthesis. Methods Enzymol. 148, 632–650. Hota, R.K., Bapuji, M., 1994. Triterpenoids from the resin of Shorea Robusta. Phytochemistry 35, 1073–1074. Itoh, T., Yoshida, K., Yatsu, T., Tamura, T., Matsumoto, T., 1981. Triterpene alcohols and sterols of spanish olive oil. J. Am. Oil Chem. Soc. 58, 545–550. Liu, J., Sun, H., Wang, X., Mu, D., Liao, H., Zhang, L., 2007. Effects of oleanolic acid and maslinic acid on hyperlipidemia. Drug Dev. Res. 68, 261–266. Mahato, S.B., Sarkar, S.K., Poddar, G., 1988. Triterpenoid saponins. Phytochemistry 27, 3037–3067. Perez-Camino, M.C., Cert, A., 1999. Quantitative determination of hydroxy pentacyclic triterpene acids in vegetable oils. J. Agric. Food Chem. 47, 1558–1562. Power, F.B., Tutin, F., 1908. The constituents of olive leaves. J. Chern. Soc. Trans. 93, 891–904. Rahier, A., Benveniste, P., 1989. Mass spectral identification of phytosterols. In: Nes, W.D., Parish, E. (eds) Analysis of Sterols and Other Biologically Significant Steroids. Academic Press, New York, pp. 223–250. Ranalli, A., Pollastri, L., Contento, S., Di Loreto, G., Iannucci, E., Lucera, L., Russi, F., 2002. Sterol and alcohol components of seed, pulp and whole olive fruit oils. Their use to characterise olive fruit variety by multivariates. J. Sci. Food Agric. 82, 854–859. Reina, R.J., White, K.D., Jahngen, E.G., 1997. Validated method for quantitation and identification of 4,4-desmethylsterols and triterpene diols in plant oils by thin-layer chromatography-high resolution gas chromatography-mass spectrometry. J. AOAC Int. 80, 1272–1280. Saimaru, H., Orihara, Y., Tansakul, P., Kang, Y.-H., Shibuya, M., Ebizuka, Y., 2007. Production of triterpene acids by cell suspension cultures of Olea europaea. Chem. Pharm. Bull. (Tokyo) 55, 784–788. Seo, S., Yoshimura, Y., Uomori, A., Takeda, K., Seto, H., Ebizuka, Y., Sankara, U., 1988. Biosynthesis of triterpenes, ursolic acid, and oleanolic acid in tissue cultures of Rabdosia japonica Hara fed [5-13C2H2 J mevalonolactone and [2-13C2H3] acetate. J. Am. Chem. Soc. 110, 1740–1745. Shibuya, M., Zhang, H., Endo, A., Shishikura, K., Kushiro, T., Ebizuka, Y., 1999. Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases. Eur. J. Biochem. 266, 302–307. Stiti, N., M’Sallem, M., Triki, S., Cherif, A., 2002. Etude de la fraction insaponifiable de l’huile d’olive de differcntcs varietes tunisiennes. Riv.ltal. Sostanze Grasse 79, 357–363. Stiti, N., Triki, S., Hartmann, M.A., 2007. Formation of triterpenoids throughout Olea europaea fruit ontogeny. Lipids 42, 55–67.