Chemical structures, production and enzymatic transformations of sapogenins and saponins from Centella asiatica (L.) Urban

Chemical structures, production and enzymatic transformations of sapogenins and saponins from Centella asiatica (L.) Urban

    Chemical structures, production and enzymatic transformations of sapogenins and saponins from Centella asiatica (L.) Urban Robert Aze...
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    Chemical structures, production and enzymatic transformations of sapogenins and saponins from Centella asiatica (L.) Urban Robert Azerad PII: DOI: Reference:

S0367-326X(16)30168-X doi: 10.1016/j.fitote.2016.07.011 FITOTE 3450

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

9 June 2016 22 July 2016 27 July 2016

Please cite this article as: Robert Azerad, Chemical structures, production and enzymatic transformations of sapogenins and saponins from Centella asiatica (L.) Urban, Fitoterapia (2016), doi: 10.1016/j.fitote.2016.07.011

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ACCEPTED MANUSCRIPT Chemical structures, production and enzymatic transformations of sapogenins and saponins from Centella asiatica (L) Urban.

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Robert Azerad Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris Descartes, 75006 Paris, France

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Email adress : [email protected]; [email protected]

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Conflicts of interest: The author has no conflict of interest to declare.

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ACCEPTED MANUSCRIPT

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Chemical structures, production and enzymatic transformations of sapogenins and saponins from Centella asiatica (L) Urban.

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Abstract

Centella asiatica (L.) Urban is a medicinal herb traditionally used in Asiatic countries for its

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multiple therapeutic properties, essentially due to its accumulation of specific pentacylic triterpenoid saponins, mainly asiaticoside and madecassoside and the corresponding sapogenins. This review summarizes the updated knowledge about the chemical structures

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of about forty centelloids, found as minor metabolites in Centella, and all derived from ursane and oleane ring patterns. Similarly, the most recent genetic and enzymatic features

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involved in their biosynthesis is reviewed, in relation with their biotechnological production developed, either from in vitro plant cultures or undifferentiated cells, in order to be independent of natural sources and to provide a continuous and reliable source of

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centelloids. Finally, a short survey of the biotransformations of some centelloids, either in

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animal, human or microorganisms is reviewed.

Keywords: centelloids, asiaticoside, madecassoside, pentacyclic triterpenes, biotechnological production, biotransformations.

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ACCEPTED MANUSCRIPT

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Chemical structures, production and enzymatic transformations of sapogenins and saponins from Centella asiatica (L) Urban.

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1.Introduction

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Centella asiatica (L.) Urban, a perennial herbaceous and stoloniferous plant of the Apiaceae family (Umbelliferae) (Fig.1), originates from moist areas of tropical or subtropical regions such as southern east Asia. It was then introduced in India, Sri Lanka, South Africa and

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Madagascar, and is also present in southeast United States and eastern south America (Fig.2). This plant accumulates in its aerial part large quantities of ursane- or oleane-type ©asiatica is used as a medicinal plant in ayurvedic medicine and traditional chinese

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medicine under the names “Gotu kola”, “Antanan”, “Pegagan”, “Brahmi”, “Indian

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Pennywort”, …etc.

Fig.1: Centella asiatica (©Bùi Thụy Đào Nguyên,Wikimedia commons)

Triterpene pentacyclic saponins are common secondary plant metabolites, also frequently found in endophytes, and are synthesized via the isoprenoid pathway to produce a hydrophobic cyclic triterpenoid structure (aglycone or sapogenin) further conjugated with one or several hydrophilic sugar chains (saponin). The remarkable biological activities of saponins have been attributed to these double structural characteristics.

3

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ACCEPTED MANUSCRIPT

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Fig. 2: Geographical distribution of native populations of Centella asiatica (adapted from www.cabi.org). The Centella saponins include mainly asiaticoside and madecassoside and their

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corresponding sapogenins asiatic and madecassic acids, plus a number of minor centelloids and their deglycosylated derivatives[1]. However several chemotype variations in the production of those metabolites due to origin and growth conditions have been described.

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Preparations of C. asiatica or their purified saponins are used in traditional and alternative

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medicine due to the wide spectrum of pharmacological activities associated with these secondary metabolites. In plants, the Centella pentacyclic triterpenoids can be regarded as

infections.

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protective compounds due to their antifungal and antimicrobial activities against pathogen

Based on the plant properties in traditional medicine, the Centella saponins are used as crude

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extracts or pure compounds for a number of medicinal activities, e.g. wound healing and treatment of venous insufficiency. They are also used by the cosmetic industry as a basis of anti-ageing and antioxidant creams. Exhaustive reviews about the multiple biological activities of centelloid compounds have been published [1- 9]. Extraction of these plant-derived pharmacologically active compounds is not always the first choice option for production because of their seasonal formation and weak concentrations. Furthermore, their complex structures make chemical synthesis an economically uncompetitive option. A biotechnological production constitutes an alternative and possibly efficient method for obtaining a sustainable and reliable source of centelloside compounds[10]. The depletion of the wild stock of C.asiatica by unrestricted exploitation and high industrial demand has prompted the development of centelloside production by in vitro cultures. Moreover, the production of such secondary metabolites by cultured cells may provide another advantage, especially when it improves the production of desired 4

ACCEPTED MANUSCRIPT compounds, thus the most recent biotechnological approaches to increase the concentrations of the centelloside metabolites are discussed. In addition, the few studies about the enzymic transformations of centelloids in human,

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animal, and microorganisms are evocated, including hydrolysis of the sugar moiety,

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hydroxylation and acetylation reactions. The biological activity of these metabolites

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compared with that of the parent centelloids, when available, will be also reported. 2-The structural range of pentacyclic triterpenoid compounds found in C.asiatica The basic structure of the sapogenins from C. asiatica is exclusively derived from two

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pentacyclic triterpenoid subtypes, the ursane and the oleane series according to the methyl substitution pattern on C-19 and C-20 (Fig. 3). Further diversity results from the presence of

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double bonds mainly occurring at C12-C13, C13-C18 or C20-C21, hydroxylation in various positions and glycosylation which may consist in the introduction of mono- or oligosaccharides in different positions of the ring at functional substituents, including esters of the carboxylic acid group.

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Some naturally acetylated compounds have been also described.

20

20

CE P

19

18

11

R1

H

AC

5

4

HO R2

6

H

23

17 16

11

CO2R4

28

R1

R3

7

A

H

5 4

HO

H

14

17 16

CO2R4 28

15 10

3

R2

13

1 2

8

22

12

9

15

10

2 3

H

14

9

1

18

22

12

13

21

19

21

8 7

6

B

H 23

R3

Fig. 3: Current substitution patterns of the centelloids of the ursane family (A) and the oleane family (B): R1, R2, R3= H or OH, R4=H or oligosaccharide.

About 40 triterpenoid compounds of C. asiatica derived from the ursane or oleane structural subtypes have been reported until to-day in the literature, although there exist duplicate names and contradictory findings probably arising from analytical mistakes. Table I proposes a dichotomic classification of these compounds, based on their ring substitution, the hydroxylation positions, the double bond(s) position(s), and the structure of the glycosyl moiety, with their usual and canonical denominations.

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ACCEPTED MANUSCRIPT Quantitatively, the main saponins include asiaticoside and madecassoside, but there are also a number of minor centellosides, such as thankuniside, scheffoleoside, centellasaponins, etc. Similarly, the most abundant sapogenins are asiatic and madecassic acids. However, the

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content and relative proportion of triterpene components in C. asiatica may considerably

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change according to the geographical location and environmental conditions of the plant (see section 5). The oligosaccharide moiety of the sapogenins may be diverse but mainly consists

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in a Rha-(1-4)-Glc-(1-6)-Glc- oligosaccharide linear chain esterifying the carboxylic acid group at C-28. C. asiatica also contains a number of other bioactive secondary compounds, including other terpenic compounds[11], volatile oils, flavonoids, tannins, phytosterols,

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mucilages, resins, free amino acids, fatty acids, and sugars[2,12,13].

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-------------------------------------------------------------------------------------------------------Table I: Structures of the pentacyclic triterpenoid compounds and their glycosides reported in Centella asiatica -------------------------------------------------------------------------------------------------------Ursane subtype with 

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I.1 

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31

29

30

R1

AC

25 1

2 3

4

10

24

6

23

14

9

5

R4

18

R2 R3

8 7

15

HO HO

21 22 17 16

28

S1

OH

20

19

12 11 26 13

HOH2C O HO

HOH2C HO HO

CO2R5 S2

O O OH HO HO

O OH

O O OH HO HO

O OH

27 HO HO HO S3

HO O OH

O HO

OH

S4

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ACCEPTED MANUSCRIPT

R2 (C-23) -OH

R3 (C-6) -H

R4 (C-3) -OH

R5 (C-28) -H

Current denomination Asiatic acid

-OH

-OH

-H

-OH

-S1

Asiaticoside

-OH

-OH

-H

-OH

-S2

Asiaticoside E

-OH

-OH

-H

-OH

-S3

Quadranoside IV

-OH

-OAc

-H

-OH

-S1

Asiaticoside C 23-O-Acetylasiaticoside

-OH

-H

-H

-OH

-S1

Asiaticoside D

-OH

-H

Madecassic acid, Brahmic acid, 6hydroxy asiatic acid Madecassoside Brahmoside Brahminoside Asiaticoside A 23-O-Acetyl Madecassoside

Canonical denomination 2α,3β,23-trihydroxyurs-12-en-28-oic acid 2α,3β,23-trihydroxyurs-12-en-28-oic acid-O--L-rhamnopyranosyl -(1-4)O--D-glucopyranosyl-(1-6)-O-Dglucopyranosyl ester 2α,3β,23-trihydroxyurs-12-en-28-oic acid-O--D-glucopyranosyl-(1-6)O-D-glucopyranosyl ester 2α,3β,23-trihydroxyurs-12-en-28-oic acid-O--D-glucopyranosyl ester 23-O-acetyl-2α,3β-23-trihydroxy urs -12-en-28-oic acid-O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester 2α,3β-dihydroxyurs-12-en-28-oic acid-O--L-rhamnopyranosyl-(1-4)O--D-glucopyranosyl-(1-6)-O-Dglucopyranosyl ester 2α,3β,6β,23-tetrahydroxyurs-12-en28-oic acid

Ref [14]

2α,3β,6β,23-tetrahydroxyurs-12-en28-oic acid-O--L-rhamnopyranosyl(1-4)-O--D-glucopyranosyl-(1-6)O-D-glucopyranosyl ester 23-O-acetyl-2α,3β,6β,23tetrahydroxyurs-12-en-28-oic acid-O-L-rhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester 2α,3β,6β,23-tetrahydroxyurs-12-en28-oic acid-O--D-glucopyranosyl(1-6)-O-D-glucopyranosyl ester 2α,3β,6β,23-tetrahydroxyurs-12-en28-oic acid-O--D-glucopyranosyl ester 2α,3β,23-tetrahydroxyurs-12-en-28oic acid-O--L-rhamnopyranosyl-(14)-O--D-glucopyranosyl-(1-6)-OD-glucopyranosyl ester, 23-O--Dglucopyranosyl ether 2α,3β,6β-trihydroxyurs-12-en-28-oic acid 2α,3β,6β-trihydroxyurs-12-en-28-oic acid-O--L-rhamnopyranosyl-(1-4)O--D-glucopyranosyl-(1-6)-O-Dglucopyranosyl ester 3β,23-dihydroxyurs-12-en-28-oic acid-O--L-rhamnopyranosyl-(1-4)O--D-glucopyranosyl-(1-6)-O-Dglucopyranosyl ester

[14-24]

[14-16]

-OH

-OH

-OH

-S1

-OH

-OAc

-OH

-OH

-S1

-OH

-OH

-OH

-OH

-S2

Centellasaponin B

-OH

-OH

-OH

-OH

-S3

Centelloside C

-OH

-OS3

-OH

-OH

-S1

Centellasaponin G

-OH

-H

-OH

-OH

-H

Madasiatic acid

-OH

-H

-OH

-OH

-S1

Centellasaponin C

-H

-OH

-H

-OH

-S1

Asiaticoside F Zemoside A

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-OH

CE P

SC R

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-OH

D

-OH

AC

-OH

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R1 (C-2) -OH

[17-19]

[20-21] [17,18,22]

[18]

[14-23]

[22]

[16,19]

[25]

[26]

[16] [16,17,21]

[18,19] [21,22]

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ACCEPTED MANUSCRIPT

R2 (C-23) -OH

R3 (C-6) -OH

R4 (C-3) -OH

R5 (C-28) -H

Current denomination Isothankunic acid

-H

-OH

-OH

-OH

-S1

Isothankuniside

-OH

-OH

-OH

-OS4

-H

30

20 22

CO2R5

8

5 4

R2 R3 (C-23) (C-6)

-OH

-OH

-OH

-H

-OH

=O

-H

-OH

-OH

-OH

-OH

R6 (C24) -OH

R7 (C30) -H

Current denomination

-H

R4 (C20) -H

R5 (C28) -S1

-H

-H

-S1

-OH

-H

Scheffursoside F

-H

-S1

-H

-H

Scheffuroside B

-H

-H

-S1

-H

-OH

Asiaticoside G

-H

-OH

-H

-H

-H

Centellasaponin F

AC

R1 (C-2)

R2 R3

D

23

27

TE

24

6

16

7

CE P

HO

15

[29]

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10

3

17 28

9

2

R6

18 14

1

[27]

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21

19

R1

3,6,23-trihydroxy-12-en-28-oic acid O--L-rhamnopyranosyl-(1-4)O--D-glucopyranosyl-(1-6)-O-Dglucopyranosyl ester 2,3,6,23-tetrahydroxyurs-12-en28-oic acid 3-O-L-arabinosyl ether

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R7

29

25

Ref [27,28]

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-

R4

12 11 26 13

Canonical denomination 3,6,23-trihydroxy-12-en-28-oic acid

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R1 (C-2) -H

-

Canonical denomination

Ref

2α,3β,2324-tetrahydroxyurs12-en-28-oic acid-O--Lrhamnopyranosyl-(1-4)-O-D-glucopyranosyl-(1-6)-OD-glucopyranosyl ester 2α,3β,24-trihydroxyurs-12-en28-oic acid O--Lrhamnopyranosyl(1-4)-Dglucopyranosyl(1-6)--Dglucopyranoside ester 2α,3β-dihydroxy-23-oxo-urs12-en-28-oic acid O--Lrhamnopyranosyl(1-4)-Dglucopyranosyl(1-6)--Dglucopyranoside ester 2α,3β,23,30-tetrahydroxyurs12-en-28-oic acid-O--Lrhamnopyranosyl-(1-4)-O-D-glucopyranosyl-(1-6)-OD-glucopyranosyl ester 2α,3β,20,23-tetrahydroxyurs12-en-28-oic acid

[26]

[19,30]

[19,30]

[21]

[31]

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ACCEPTED MANUSCRIPT Ursane subtype with 

I.2

30 29 20

19

21

28

1 10

2

8

5 4

27

7

6

R2 R3

23

R1 (C-2) -OH

R2 R3 (C-23) (C-6) -OH -H

-OH

-OH

Current denomination Isoasiatic acid

R5 (C-28) -H -S1

Isoasiaticoside

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D

-H

Ref Canonical denomination 2α,3β, 23-trihydroxyurs-20-en-28-oic [32] acid 2α,3β, 23-trihydroxyurs-20-en-28-oic [19,32] acid O--L-rhamnopyranosyl-(1-4)-O-D-glucopyranosyl-(1-6)-O-Dglucopyranosyl ester

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24

16

MA

3

HO

15

9

IP

CO2R5

14

R1

17

SC R

25

T

22 18

12 11 26 13

Ursane subtype with and 

CE P

I.3

30

29

AC 25

R1

1

2

24

R1 (C-2) -OH

8

15

21 17

22 28

16

CO2R5

27

7

5 6

4

HO

14

9

10

3

18

12 11 26 13

20

19

23

R2 R3

R2 (C-23) -OH

R3 (C-6) -H

R5 (C-28) -S1

Current denomination Centelloside E

Canonical denomination (23,23)-trihydroxyursa-6,12diene 28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)--Dglucopyranosyl ester

Ref [19]

9

ACCEPTED MANUSCRIPT Ursane subtype with 

I.4

30 29 20 21

T

19

12 26 13

1

8

10

27 5

3

R3 (C-6) -H

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R2 (C-23) -OH

Current denomination Centellasaponin J

R5 (C-28) -S1

Canonical denomination (23,23)-trihydroxyursa-5-ene 28oic acid O--L-rhamnopyranosyl(1-4)-O--D-glucopyranosyl-(1-6)-D-glucopyranosyl]

Ref [33]

TE

D

R1 (C-2) -OH

R2 R3

23

MA

24

7 6

4

HO

15

9

2

28

CO2R5

16

14

R1

17

18

SC R

11

25

IP

22

I.5

Ursane subtype without double bond 30

CE P

R4

29

19

R1

AC

25 1

2 3

4

HO 24

R1 (C-2) -OH

10

5

6

23

14

9

8

15

21 22

18

12 11 26 13

20

17 16

28

CO2R5

27

7

R2 R3

R2 (C-23) -OH

R3 (C-6) -H

R4 (C-20) -OH

R5 (C-28) -H

Current denomination -

Canonical denomination 23,2023-tetrahydroxyurs28-oic acid

Ref [31]

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ACCEPTED MANUSCRIPT Oleane subtype with 

II.1 

30

29 20

19

14 10 5

4

HO 24

23

8 6

15

T

27

7

R2 R3

R2 (C-23) -H

R3 (C-6) -OH

R4 (C-28) -S1

-OH

-OH

-OH

-H

-OH

-OH

-OH

-S3

-OH

-OH

-OH

-S2

-OH

-OH

-OH

-OAc

-OH

Current denomination -

Terminolic acid Chebuloside II

Centelloside D

-OH

-S1

Asiaticoside B

AC

CE P

TE

R1 (C-2) -OH

NU

3

9

MA

1 2

CO2R4

16

D

R1

IP

25

22 17 28

SC R

18

12 11 26 13

21

-OH

-S1

23-O-acetyl asiaticoside B

-OS3

-OH

-S1

Centellasaponin H

-OH

-OH

-H

-H

Arjunolic acid

-OH

-OH

-H

-S1

Scheffoleoside A

Canonical denomination Ref [19,21] 2,3,6-trihydroxyolean-12-en28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)--Dglucopyranosyl ester 2,3β,6β,23-tetrahydroxyolean -12- [24] en-28-oic acid 2,3β,6β,23-tetrahydroxyolean -12- [19] en-28-oic acid O-Dglucopyranosyl ester [19] 2,3β,6β,23-tetrahydroxolean-12en-28-oic acid O --Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester 2,3β,6β,23-tetrahydroxyolean -12- [16,24] en-28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester (2α,3β,6β)-23-acetyloxy 2,3,6[22] trihydroxyolean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1-4)-O-βD-glucopyranosyl-(1-6)-β-Dglucopyranosyl ester. 2,3β,6β,23-tetrahydroxyolean -12- [26] en-28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester, 3-O--D-glucopyranosyl ether [30,34] 2,3β,23-trihydroxyolean -12-en28-oic acid 2,3β,23-trihydroxyolean -12-en- [16,26,30] 28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester

11

ACCEPTED MANUSCRIPT R1 (C-2) -H

R2 (C-23) -OH

R3 (C-6) -OH

R4 (C-28) -H

-H

-OH

-OH

-S1

Current denomination -

Ref [16] [16]

30

SC R

IP

T

Centellasaponin D

Canonical denomination 3β,6β,23-trihydroxyolean -12en-28-oic acid 3β,6β,23-trihydroxyolean -12en-28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester

29 20

19

22

14 1

9 10

3 4

R1 (C-2) -OH

23

R2 (C-23) -OH

27

7

R2 R3

R3 (C-6) -OH

R4 (C-28) -S1

CE P

24

6

Current denomination Centellasaponin E

Canonical denomination 3-oxo-2,6,23-trihydroxyolean-12-en-28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester

Ref [33]

AC

O

8

5

16

D

2

15

TE

R1

28

CO2R4

MA

25

17

18

12 11 26 13

NU

21

12

ACCEPTED MANUSCRIPT II.2 Oleane subtype with   30

29

T

20 21

IP

19

22

14

R1

1 10

2 3

24

8

5 4

HO

15

9

6

23

28

CO2R4 16

27

7

NU

25

17

SC R

18

12 11 26 13

R2 R3

Current denomination

R2 (C-23) -OH

R3 (C-6) -H

R4 (C-28) -H

Centella sapogenol A

-OH

-OH

-H

-S1

Centellasaponin A

-H

-OH

-OH

-H

-H

-OH

CE P

TE

D

MA

R1 (C-2) -OH

-S1

Centellasaponin D

Ref [35] [26,35,36]

[16] [16]

AC

-OH

-

Canonical denomination 2,3β,23-trihydroxyolean 13(18)-en-28-oic acid 2,3β,23-trihydroxyolean 13(18)-en-28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester 3β,6,23-trihydroxyolean 13(18)-en-28-oic acid 3β,6,23-trihydroxyolean 13(18)-en-28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester

13

ACCEPTED MANUSCRIPT II.3 Oleane subype with and  30

29 20 21

19

28

1

9 10

3

5 4

HO 24

6

23

27

7

R2 R3

R2 (C-23) -OH

R3 (C-6) -H

Current denomination Centellasaponin I

R4 (C-28) -S1

Canonical denomination 2,3β,23-trihydroxyurs-5,12dien-28-oic acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)-O-Dglucopyranosyl ester

Ref [33]

TE

D

MA

R1 (C-2) -OH

8

NU

2

16

15

CO2R4

IP

14

R1

17

SC R

25

T

22 18

12 11 26 13

CE P

II.4 Oleane subype with and  30

29

20 21

19

R1

1

2 3

24

14

9

10

5

4

HO

R1 (C-2) -OH

AC

25

18

12 11 26 13

6

23

8

15

22 17 28

16

CO2R4

27

7

R2 R3

R2 (C-23) -OH

R3 (C-6) -H

R4 (C-28) S1

Current denomination Centelloside E

Canonical denomination 2,3,23-Trihydroxyursa-6,12dien-28-oic Acid O--Lrhamnopyranosyl-(1-4)-O--Dglucopyranosyl-(1-6)--Dglucopyranosyl ester

Ref [19]

14

ACCEPTED MANUSCRIPT 3- Analytical Methods Centelloids were initially detected, characterized and sometimes quantified by thin layer chromatography[37-39]. More sophisticated methods have been further developed for the

T

characterization and identification of the Centella saponins and sapogenins. However, their

IP

weak UV absorption in the 200-205 nm region did not facilitated their detection, separation

SC R

and quantification [40]. Adsorption and separation on macroporous resins [41] followed by reverse phase HPLC/MS methods, eventually coupled with evaporative light scattering detection[42], are now currently employed, allowing the quantitative measurement of the different compounds.

NU

Detailed HPLC separation conditions can be found in numerous dedicated papers [19,32,37,43-50] including improved separation methods using -cyclodextrin as a mobile

MA

phase additive[51,53]. High speed countercurrent chromatographic techniques have also been elaborated for the scaled-up separation and production of pure compounds[54,55]. These chromatographic methods, in combination with high field systematic 1H and 13C

D

NMR studies[22,24,31,32,49, 56,57] have lead to the isolation and identification of a

TE

number of several minor saponin and saponoside components. Chemometry by 1H-NMR has been recently suggested to discriminate Centella varieties and close species and to

CE P

investigate the effect of growth conditions on metabolite contents[58]. 4-Biosynthesis of centelloid triterpenes

AC

In plants, triterpenoids are synthesized via the isoprenoid pathway resulting from the condensation of five-carbon building blocks designated as IPP (3-isopentenyl pyrophosphate) and DMAPP (dimethylallyl pyrophosphate) derived from the mevalonate pathway (fig. 4). IPP and DMAPP undergo head-to-tail condensation to GPP (geranyl pyrophosphate), and addition of another IPP unit leads to FPP (farnesyl pyrophosphate). Head-to-head condensation of two FPP units leads to squalene (C30), which is subsequently oxygenated to 2,3-oxidosqualene (OSQ). OSQ is the common precursor of triterpenoid saponins and phytosterols. The next step is the enzymic cyclization of OSQ to give the cyclic triterpenoids. In this process internal bonds are introduced into the oxidosqualene backbone through double bonds migrations, resulting in the formation of polycyclic molecules containing several 5and 6- membered rings[59]. The formation of different internal linkages into a chair–chair–

15

ACCEPTED MANUSCRIPT chair conformation during backbone cyclization by oxidosqualene cyclases (OSCs) gives rise to the vast array of triterpene saponin skeletons[60-62] which is found in nature.

OPP

DMPP

GPP

OPP

SC R

IPP

IP

CoA S

T

O

OPP

+

OPP

NU

DMPP

OPP

MA

FPP

FPP

OPP

Squalene

O

AC

CE P

TE

D

+

2,3-oxidosqualene

HO

-amyrin

Oleanes

-amyrin

Ursanes

Dammarenyl cation

Fig. 4: Initial steps in the biosynthesis of saponin aglycones In the case of C. asiatica, it has been suggested that specific OSCs imperfectly succeed in preventing alternative ways of cyclization into predetermined end products. This concept is justified by the observation of numerous minor centelloids accompanying specific major products of OSCs, which results from increasingly sensitive detection methods. Moreover there is no clear evidence about the nature of final cyclic intermediates in the formation of ursane or oleane derived sapogenins Some data have been recently obtained about the genes and the enzymes required for different steps of centelloid biosynthesis. A detailed sequencing and mapping of the transcriptome associated with the secondary metabolism of C. asiatica has been 16

ACCEPTED MANUSCRIPT published[63]. By in silico approach, an attempt to modelize the protein structures of cycloartenol synthetase (the first enzyme on the way to sterols in plants) and -amyrin synthetase, to identify a specific modulator of cycloartenol synthetase, has been recently

T

published, with the project to control the sterol metabolism and overproduce the terpenoid

IP

metabolites by channeling the OSQ substrate in C. asiatica cell cultures[64]. The results of this study suggest that ketoconazole can possibly function as a negative modulator of sterol

SC R

biosynthesis, allowing the over production of triterpenoid secondary metabolites in C. asiatica.

Several genes, including the jasmonate-responsive genes involved in the biosynthetic

NU

pathway in C.asiatica have been cloned, such as hydroxymethyl glutaryl-CoA reductase (CaHMGr) [65], farnesyl diphosphate synthase (CaFs)[66], squalene synthase

MA

(CaSQs)[67,68], oxidosqualene synthase CaOSQs[69], oxidosqualene cyclase (CaOSQc) and a putative -amyrin synthase (CabAs)[70] later identified as a dammarenediol synthase (CaDDs) after cloning and expressing in yeast[71].

D

The functionalization of the different rings of sapogenins conferring further diversity mainly

TE

by oxidation and hydroxylation(s) is certainly mediated by unknown cytochrome P450dependent monooxygenases. Moreover, little is known about the specific glycosyl

CE P

transferases which are probably responsible for triterpenoid glycosylation[72]. Glycosylation is particularly important as the introduction of a sugar chain is critical for the biological activity of saponins. The oligosaccharide chains are probably synthesized by

AC

sequential addition of single sugar residues to the aglycone. Among 24 Cytochromes P450 and 13 Uridine diphosphoglycosyl transferases (UGT) candidates identified from a Centella database, 3 of the P450s and 1 UGT were selected as the most likely candidates to be involved in the methyl jasmonate-induced centelloside synthesis[73].

5-Production of centelloid triterpenes Recent and comprehensive reviews on the biotechnological production of centellosides have been published[1,10]. Whole plant extracts are currently prepared and used for their biological activities. They have been the subject of numerous studies of centelloside composition and identification in local chemotypes and environmental different conditions (see section 5.1). Propagation of whole plants leading to artificial in vitro cultures have been successfully carried on[74-76], making the production process independent from natural sources (see section 5.2). More specifically, micropropagation in defined media (see section 17

ACCEPTED MANUSCRIPT 5.3) can be used for the preparation of calli and plant cells suspension cultures from which secondary metabolites are extracted[76,77]. The advantage of this method is that it can provide a continuous and reliable source of natural products.

T

Owing to the commercial impact of their production, a number of studies about the

IP

parameters influencing the yield of Centella sapogenins in whole plant or cell cultures have been carried out: geographical origin, environmental factors, temperature, illumination,

SC R

mineral nutrition, etc…have been examined. In addition, the effect of a number of elicitors has been intensively and successfully investigated to increase the production of some of the more useful centellosides, such as asiaticoside and madecassoside (see section 5.4).

NU

Ultimately, studies on the biosynthetic pathway of triterpenoid synthesis in Centella and metabolomic studies have been recently carried out to understand the formation of these

MA

specific terpenoids, as a possible approach to control biosynthetic pathways leading to centellosides. The flux and its regulation through these pathways has to be elucidated to obtain a better understanding of the biochemical conversions that will allow the

TE

D

manipulation and exploitation of secondary products synthesized by C. asiatica.

5.1. Chemotypes and cultural yields

CE P

Chemical differences between varieties in plants of the same species (chemotypes) are common and variation in secondary metabolites has been also observed with identical phenotypes and growth conditions, depending on plant origin. Not surprisingly, significant

AC

differences in active constituents (mainly asiatic, madecassic acids and their saponins) have been observed between samples of C. asiatica originating from different geographical regions, possibly as a result of genomic diversity[78]. Moreover, the centelloside biosynthesis seems to be essentially localised in the leaves. In all samples, the level of centelloside content found in the roots of whole plants remains very low[46]. Depending on the origin of the plant material, the total saponins can account for 1- 8% of the constituents [2]. In a study of whole plant samples of C. asiatica of Indian origin, most of them had an average asiaticoside content around 3 mg/g DW, a madecassoside content of 15-20 mg/g and a total triterpene content of about 30 mg/g DW[43]. Variable asiaticoside contents in five lines of C. asiatica from India have been also reported [37]. In whole plant cultures on liquid synthetic media for 5 weeks, the best yields obtained from C.asiatica of Korean origin were 8.9 and 9.1 mg/g DW of madecassoside and asiaticoside, respectively [79]. Similarly, in the fringed and smooth leaf phenotypes of C.asiatica of Malaysian origin 18

ACCEPTED MANUSCRIPT the triterpenoid content was highest in leaves, with asiaticoside (7.9 and 11.5 mg/g DW) and madecassoside (9.7 and 16.5 mg/g DW), respectively[46]. In Thailand, the content of pentacyclic triterpenes in whole plants of C. asiatica varied

T

according to the place of cultivation and the harvesting period: the highest content of total

IP

pentacyclic triterpenes was 37.2 mg/g dry powder[80].

In Madagascar, two foliar morphotypes were identified: morphotype A with small reniform

SC R

leaves, found in the east, and morphotype B with large round leaves found in the west (Fig.5). Morphotype A produces a higher biomass, and is twice as rich in centellosides as morphotype B. Significant variations in biomass yield and centelloside content are observed

NU

depending on the month and the year of collection. Populations located at around 1000 m altitude on the eastern side of Madagascar (Mangoro region), in a sub-humid climate,

MA

appeared to be more productive (127 mg/g DW in the leaves of morphotype A) corresponding to the highest total triterpenoid content reported so far in the literature

AC

CE P

TE

D

[74,81].

Fig. 5: Foliar morphotypes of Centella asiatica from Madagascar Island. Left: Morphotype A with small reniform leaves (eastern island). Right: Morphotype B with larger round leaves (western island). Similar phenotypes of C. asiatica from Southern Africa were compared in relation to the levels of triterpenoid saponins. Analysis of callus, cell suspensions and leaves by means of TLC showed only slight but significant differences in the asiatic acid, madecassic acid and their glycoside derivatives for both phenotypes[82]. The four main metabolites occur with a free acids/glycosides ratio of approximately 1:2.5 in leaf cell cultures to 1:5 for leaf tissue of C. asiatica from Madagascar[74]. A study aimed to identify chemotypes of C. asiatica from 19

ACCEPTED MANUSCRIPT sixty accessions from a wide geographical region in south India and the Adaman Islands grown under identical environment showed madecassoside and asiaticoside contents of 1 to 56 mg/g DW and 1 to 17 mg/g DW respectively. Three accessions showed non-detectable

T

levels of one or both centellosides[83]. Similarly, a remarkable qualitative and quantitative

IP

variability was observed in the terpenoid profiling of several discrete chemotypes of Indian varieties of C. asiatica[84]. Recently, three genetic groups, distinct from Indian and South

SC R

African genotypes, and polyploid populations containing diploid and tetraploid plants with active sexual reproduction have been identified in Madagascar[85]. Tetraploid plants of C. asiatica from Thailand obtained by colchicine treatment exhibited positive trends in both

NU

biomass and triterpenoid production[86,87].

However, there is no report related to the variability in the content of the minor centelloside

MA

compounds. 5.2. In vitro plant cultures

D

Conventional extraction from wild whole plants is industrially limited due to environmental and geographical issues. In addition, owing to the growth of wild populations of C. asiatica

TE

in swampy regions which may be contaminated with heavy metal pollutants and other harmful chemical residues, about 80% of the collected herb is normally rejected by pharma

CE P

companies due to unacceptable heavy metal and microbial loads[88,89]. Moreover vegetative propagation of C. asiatica is not sufficient to meet the needs of pharmaceutical industries. Thus the production of the desired secondary metabolites by in vitro cultures

AC

could be considered as an alternative technology and allow a year-long cultivation in controlled conditions with constant quality and quantity[90,91]. In vitro plant culture by micropropagation offers the possibility of obtaining desirable compounds as well as ensuring a sustainable conservation and rational utilisation of natural biodiversity. Micropropagation is a clonal propagation because the plants are reproduced asexually and genetically uniform. The principle lies on the capacity of cells from calli, obtained from leaves, internode shoot segments or axillary buds, to grow, multiply and differentiate to generate plantlets which can produce roots and be transferred to soil culture. The micropropagation procedure is undoubtedly useful to disseminate the best clones of C.asiatica and raise a stock of stable, genetically homogenous and healthy plant material for field cultivation. Early studies on the establishment of in vitro liquid and semisolid whole plant cultures and optimisation of media, macroelements and sucrose concentrations allowed a production of 20

ACCEPTED MANUSCRIPT madecassoside + asiaticoside close to 15 mg/g DW[79]. An efficient protocol for in vitro propagation of C. asiatica through axillary shoot proliferation from nodal explants has been recently described and optimized [92]. Multiple shoots were induced from nodal segments

T

on semi-solid media containing various concentrations of 6-benzylaminopurine (BAP) and

IP

naphthalene acetic acid (NAA). This study was proposed as an efficient tool for mass production of C.asiatica and its conservation. In vitro grown shoots produced 1.02 mg/g FW

SC R

and 0.47 mg/g FW asiatic acid, respectively, whereas leaf calli produced a maximum amount of 1.46 mg/g FW asiatic acid[93]. A number of other successfull methods of plant regeneration from plants or callus cultures of Centella, employing various inducers

NU

(IBA,BAP, NAA or kinetin) and their effect on triterpenoid saponin content have been described [74,76,88,90,94-104,]

MA

In a recent study, conditions to cultivate C. asiatica plants in hydroponic systems have been reported[75]. HPLC analysis of the crude triterpenoid extract of the harvested leaves showed the presence of 11, 1.7, 36.6 and 6.3 mg/g DW of madecassoside, asiaticoside, madecassic

D

acid and asiatic acid respectively. The mean asiaticoside content of shoots grown in vitro for

TE

35-days (3.80 mg/g DW) was only 20% lower than that of shoots of 6-month-old plants grown in vivo (4.40 mg/g DW). Moreover, asiaticoside accumulation in multiple shoot

CE P

cultures of C. asiatica was studied as a function of nutrient manipulations and shown to reach 7.5 mg/g DW in optimised culture medium[88]. Hairy roots is a plant disease caused by infection with Agrobacterium rhizogenes or Agrobacterium tumefaciens and characterized by high growth rate, unlimited proliferation

AC

and genetic stability. Hairy roots cultures, which may grow in fermenters, have been frequently employed for the metabolic engineering and are considered to be an useful way to produce secondary metabolites[105-107]. However, hairy root cultures of C. asiatica were initially said to produce unsignificant amounts of centellosides[46,108]. On the other hand, a more recent report indicated that Agrobacterium-transformed hairy root culture enhanced the production of asiaticoside up to 172% compared to untransformed calli[109]. Interestingly, it was shown that untreated roots from plants of C. asiatica of Indian origin may contain up to 10.7 mg/g of asiaticoside, whereas adventitious roots obtained from leaf-derived callus may contain a slightly higher level of asiaticoside (11.4 mg/g)[110]. A large-scale production by adventitious roots of C. asiatica should be considered as a reliable and continuous in vitro source of asiaticoside.

5.3. Calli and cell suspensions

21

ACCEPTED MANUSCRIPT Calli and derived cell suspensions of C. asiatica, established by several researchers[82,111,112] have proved to be potential systems for obtaining centellosides[90], albeit not highly productive. The inability to detect asiaticoside and madecassoside in calli

T

was initially reported by several groups who failed to detect asiaticoside in undifferentiated

IP

cells of C. asiatica. Interestingly, in contrast to these observations, Nath and Buragohain[111] reported that calli and cell suspension cultures of C. asiatica of Indian

SC R

origin did, in fact, synthesise asiaticoside. The production of the main centellosides in calli and cell cultures of two C. asiatica phenotypes from South Africa was studied, obtaining values of 1.5–2.5 mg/g DW of asiatic acid, 1.5–3 mg/g DW of madecassic acid, 14–29 mg/g

NU

DW asiaticoside, and 13–28.4 mg/g DW madecassoside[82].

Cell suspension cultures are initiated by shaking grown callus in the callus production liquid

MA

until a suspension of free cells was formed. Cell growth and proliferation in flasks depended on sucrose addition and agitation for mixing to prevent biomass sedimentation without producing cell wall breakdown[113]. Asiaticoside production reached a maximum value of

D

45.35 mg/g DW (more than 4 times the leaves content) after 24 days. The same Centella cell

TE

suspension culture was recently conducted in a 5-L adapted bioreactor to investigate the growth and asiaticoside accumulation under various conditions[114]. The optimized

CE P

conditions for asiaticoside production have been explored, with particular attention to the agitation speed, aeration rate, and inoculum size. Cell biomass and asiaticoside production peaked at day 24. Too high agitation speeds destroy the cell walls and subsequently reduce asiaticoside biosynthesis. The optimum speed was 150 rpm, with an optimum aeration rate

AC

of 2.5 L/min, affording an asiaticoside yield of 62.14 mg/g DW. 5.5. Manipulation of centelloside production in whole plants, cell and tissue cultures One approach used to favour the production of specific secondary metabolites is to add biosynthetic precursors to the culture of calli or cell suspensions. C. asiatica cell suspension cultures are able to convert -amyrin into centellosides: 7 days after supplementing the cell culture with this potential precursor, 42% had been transformed into centellosides (asiatic acid, madecassic acid and the corresponding saponins) promoting a four-fold increase, with a very usual centelloside pattern[115]. Another strategy, the increase of the centellosides production in whole plants or cell cultures by using added auxins, growth modulators or the stress induced by elicitors, has received more attention. Asiaticoside production decreased by application of a range of auxins and cytokinins on growing plants, and only thidiazuron (TDZ), a plant growth regulator, had an

22

ACCEPTED MANUSCRIPT enhancing effect may be through an increase in biomass[116]. A number of root morphotypes was produced by rhizogenesis from C.asiatica leaf explants by using a combination of auxins (IBA/IAA/NAA) in different concentrations as well as different carbon sources. The enhanced accumulation of centellosides was correlated with the expression of key genes in their biosynthetic

T

pathway[84].

IP

A significant increase in centelloside content has been observed in in vitro C. asiatica plant

SC R

cultures[117,118] and hairy root cultures[108] elicited with 100 μM methyl jasmonate (MeJA). The highest asiaticoside production was obtained after elicitation in cultures treated with 0.1 mM MJ and 0.025 mg/l TDZ [118,119]. In MeJA-elicited cell suspension cultures,

NU

the centelloside production observed in the stationary growth phase reached 1.11 mg/g DW, compared to 0.16 mg/g DW in the control cultures. A usual centelloside pattern was observed, with madecassoside as the main compound, followed by asiaticoside[69,120]. In

MA

another elicitation study, the greatest enhancement of asiaticoside production in C. asiatica cell cultures was achieved with 100 μM salicylic acid, followed by yeast extract, supplied to the medium at day 10 of culture[121].

D

Another elicitation strategy is to simulate pathogenic attacks by challenging plant in vitro

TE

cultures with constituents of fungi or bacteria[122,123]. As a fungal elicitor, a Trichoderma harzianum culture filtrate added to the growth medium of Centella shoots resulted in 9.63

CE P

mg/g-DW asiaticoside, that was about 2.4 folds the content of the unelicited control[124]. Pectin, yeast extract and heavy metals salts, particularly Cu++ ion, have been also successfully used as elicitors[109,117]. Whereas highly productive Centella cell cultures in

AC

fermenters have been described[114], no attempt to overproduce asiaticoside or other centelloids in fermentors by using elicited cell or tissue cultures has been published until today.

Metabolomic studies of C. asiatica cell cultures have been recently carried out after the addition of MeJA to investigate the effect of the elicitor on the biosynthesis of asiaticoside and madecassoside aglycones[125,126]. The metabolic changes observed suggest that MeJA treatment reprogrammed the terpenoid pathway by modulating the corresponding gene expression and increasing the flux into the triterpene saponins while decreasing the flux into phytosterols[68,112].

6- Biotransformations of centelloid triterpenes 6-1 Biotransformations in animal and human

23

ACCEPTED MANUSCRIPT There are very few data in the literature about the fate of centelloid compounds ingested by animals or humans. A relatively early investigation of the metabolism of the main centelloids by oral administration in rat was conducted by Chazeaud et al.[127]. Only asiatic

T

acid and madecassic acid, presumably released by hydrolysis of the corresponding

IP

centellosides by the microflora of the gut were absorbed. Sulfate and glucuronide derivatives were detected as biliary or fecal metabolites. In more recent studies the transformation of

SC R

asiaticoside by intestinal flora was investigated and three metabolic products were found and identified both in vitro and in vivo in the urine indicating the conversion to asiatic acid by successive hydrolyses of the trisaccharide side-chain[128,129].

NU

Pharmacokinetic studies on asiatic acid or asiaticoside have been conducted in humans and animals[130] and all concluded that the bioavailability of the saponins was very low due to

MA

their weak solubility and low absorption. Similar results were reported with madecassoside[131]. However, after hydrolysis of asiaticoside for example, in the intestinal track, asiatic acid may permeate through intestinal membranes and may be effectively

D

metabolized in.liver. Asiatic acid is also rapidly metabolized in rat liver microsomes[132].

TE

Recently, the in vivo metabolism of asiatic acid and madecassic acid in zebrafish was investigated, as a model for human metabolism[133], using a sensitive LC/MSn method.

CE P

Nineteen phase I metabolites of asiatic and madecassic acid formed by hydroxylation, dehydrogenation, dihydroxylation, or dehydroxylation reactions and various combinations of such reactions (Fig. 6) were characterized by mass spectrometry after zebrafish was

AC

exposed to the drug.

CO2H

HO HO

CO2H

HO HO

HO

HO asiatic acid

madecassic acid

O

HO HO

OH

O

O

HO

O

HO HO

HO

OH

24

ACCEPTED MANUSCRIPT

Fig.6: Some of the identified biotransformations of asiatic acid or madecassic acid observed in zebra fish

T

6.2. Enzymatic and microbial transformations

IP

It has been shown that microbial transformation can be an efficient approach to expand the chemical diversity of natural products, and particularly terpenoid compounds[134,135]

SC R

Asiatic acid was converted by Alternaria longipes AS 3.2875 into three compounds[136]: 2,3,24,30-tetrahydroxyurs-12-en-28-oic acid, 2,3,22,24-tetrahydroxyurs-12-ene-28oic acid, and -pentahydroxyurs-12-ene-28-oic acid (Fig. 7). When

NU

incubated with Penicillium lilacinum ACCC31890 or Fusarium equiseti CGMCC 3.3658 it gave a single hydroxylated product, 2,3,15,24-tetrahydroxyurs-12-en-28-oic acid[137].

MA

Streptomyces griseus CGMCC 4.18 afforded three derivatives: 2,3,21 ,24tetrahydroxyurs-12-en-28-oic acid, 2,3,24,30-tetrahydroxyurs-12-en-28-oic acid and 2,3,24-trihydroxyurs-12-en-28,30-dioic acid[137]. These modifications at C-21, C-15 and

D

C-30 dramatically decreased the cytotoxicity of asiatic acid on several human cancer cell

CE P

TE

lines.

AC CO2H

HO HO HO

CH2OH

CO2H

HO

A. longipes AS. 3.2875

CH2OH

HO

P. lilacinum ACC 31890 F.equiseti CGMCC 3.3658

CO2H

HO

+

CO2H

HO

+ HO

HO HO

HO

HO

CO2H

HO OH

HO HO

Asiatic acid

CO2H

CH2OH OH

S. griseus CGMCC 4.18

CO2H

HO HO

CO2H

HO

HO

HO HO

CO2H

HO

HO

HO

Fig. 7: Microbial transformation of asiatic acid byAlternaria longipes[136] , Penicillium lilacinum, Fusarium equiseti or Streptomyces griseus[137] New oxidation products, 2-keto,3,24-dihydroxy-urs-12-en-28-oic acid, 2-oxo,3,15-24trihydroxy-urs-12-en-28-oic acid, and 3-oxo,2,15,24-trihydroxy-urs-1,12-dien-28-oic acid

25

ACCEPTED MANUSCRIPT were obtained from the biotransformation of asiatic acid by the fungus Fusarium avenaceum

SC R

IP

T

AS 3.4594 (Fig. 8) selected among twenty five fungal strains[138].

NU

Fig. 8: Microbial transformation of asiatic acid by Fusarium avenaceum.

The transformation of asiatic acid by an endophytic fungus Umbelopsis isabellina afforded

MA

two products: a 7β-hydroxylated asiatic acid (2α,3β,7β,24-tetrahydroxyurs-12-ene-28-oic

D

acid) and an unusual derivative, the 2α,3β,7β,24-tetrahydroxyurs-11-ene-28,13-lactone[139].

CO2H

CO2H

HO

TE

HO

CE P

HO

O

+ OH

HO

HO

O

HO

HO

HO

OH HO

Fig. 9: Microbial transformations of asiatic acid by Umbellopsis isabellina.

AC

The transformation of asiaticoside by Nocardia sp. NRRL-5646 (a strain already used for the biotransformation of several other pentacyclic triterpenes[134,140,141]) showed the hydrolysis of the carbohydrate chain of asiaticoside, and asiatic acid was converted to a sulfate ester[142]. A similar hydrolysis was also described using Aspergillus niger strains: chebuloside II, centelloside C and asiatic acid-28-O-β-D-glucopyranoside were detected in the biotransformation products of total glucosides of Centella asiatica[143]. Another transformation of asiaticoside into three metabolites, in addition to asiatic acid, by Aspergillus niger has been also reported[144]. Various enzymic modifications of the carbohydrate chain of asiaticoside have been performed[145]. The enzymes produced by Fusarium oxysporum, an -L-rhamnosidase and a -D-glucosidase were used sequentially for the synthesis of derhamno-asiaticoside and derhamno-degluco-asiaticoside, then a -1,4-galactosyltransferase was subsequently used for their galactosylation. A new selectively acetylated compound, esterified on the terminal 26

ACCEPTED MANUSCRIPT rhamnose 4-OH was exclusively obtained using the CalB lipase (Novozym 435) in the presence of vinylacetate as an acetyl donor. Moreover a laccase from Trametes pubescens coupled with TEMPO as a chemical mediator catalyzed the selective oxidation of the free

T

primary hydroxy group of the internal glucopyranosyl moiety, resulting in a new

IP

glucuronide derivative.

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7. Conclusion and prospects

Due to its medicinal properties, interest in C. asiatica has increased over the years and there have been significant results on the enhanced production of the centellosides, as well as the

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cloning of genes in their biosynthetic pathway. Beside the asiatic acid and the madecassic acid glycosides, a number of minor derivatives have been isolated and fully chemically

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characterized. The production of centelloid compounds and expression of biosynthetic genes in leaves and roots and non-differentiated cells (calli or cell suspensions) have been investigated. A significant positive action of elicitors, and particularly MeJA, has been

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demonstrated. Although generally centellosides accumulation in plants is higher than in cell

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suspensions or hairy root cultures, a number of approaches have been developed to produce centellosides in in vitro systems, with the inherent difficulty to scale up the process. Their

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biosynthetic pathway has been fully characterized up to the formation of 2,3-oxidosqualene, but its subsequent cyclization by different OSCs and the final steps remain unclear. The complete metabolic pathways for these triterpenoids should be now completely elucidated to obtain a better understanding of the biochemical steps and regulations, and particularly the

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final steps of ring functionalization and glycosylation that will allow the manipulation of centellosides synthesis in C. asiatica. Recent transcriptomic and metabolomic studies of C. asiatica have identified several genes that are overexpressed under elicitation. Also, different C. asiatica genes have been sequenced and cloned, but their application in in vitro systems using metabolic engineering techniques remains limited. New transcriptomic, proteomic, and metabolomic studies will allow researchers to further explore the centelloside biosynthesis pathway, identify the genes that encode the key enzymes, and modify and regulate their expression. Such studies can also ascertain how the secondary metabolite pattern in a culture is changed by elicitation and how the carbon flux is regulated and oriented to the compounds of interest in order to establish highly productive biotechnological systems. The resources of biosynthetic genes overexpression and genetic and metabolic engineering by means of recombinant DNA technology have not yet been fully explored and might 27

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This research did not receive any specific grant from funding agencies in the public,

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commercial, or not-for-profit sectors.

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145. D. Monti, A. Candido, M. M. Cruz Silva, V. Kren, S. Riva, B. Danieli, Biocatalyzed generation of molecular diversity: selective modification of the saponin asiaticoside. Adv. Synth. Catal., 2005. 347: 1168–1174.

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Conflicts of interest: The author has no conflict of interest to declare.

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Graphical abstract

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