Saponins in Calendula officinalis L. – structure, biosynthesis, transport
and biological activity
Anna Szakiel, Dariusz Ruszkowski & Wirginia Janiszowska*
Department of Plant Biochemistry, Warsaw University, ul. Miecznikowa 1, 02-096 Warszawa, Poland;
*Author for correspondence (Tel.: +48-22-5543321; Fax +48-22-5543221; E-mail: firstname.lastname@example.org)
Key words: biosynthesis, oleanolic acid glycosides, structure, transport
Trends in research on Calendula officinalis L. saponins performed in Department of Plant Biochemistry at
Warsaw University are reviewed. Calendula officinalis, a well known medicinal plant, contains significant
amounts of oleanane saponins, which form two distinct series of related compounds, called ‘‘glucosides’’
and ‘‘glucuronides’’ according to the structure of the respective precursor. Both series differ in the pathway
of their biosynthesis and further metabolism, i.e. the rate of formation and stages of possible degradation;
distribution in the single cell and in the whole plant, including accumulation sites; and the possible
physiological role played in the plant according to appropriate biological activities.
Abbreviations: ADP – adenosine 5¢-diphosphate; ATP – adenosine 5¢-triphosphate; Gal – galactose; Glc –
glucose; GlcUA – glucuronic acid; MVA – mevalonic acid; OL – oleanolic acid; Pi – inorganic phosphate;
PPi – inorganic pyrophosphate
One of the most interesting group of secondary
plant isoprenoids are cyclic triterpenoid glycosides
broadly described as saponins, the term refers to
their ability to form a soapy material when con-
centrated. The triterpenes are subdivided into
about 20 groups, depending on their structures
(Mahato et al., 1992). The base structure that is
found in largest number of plants is the pentacyclic
triterpene of oleanane type. The most common
triterpene of this type is oleanolic acid (OL, Figure
1), which occurs both in a free and more often as
glycoside or glycoside ester. Its glycosides differ in
the structure of carbohydrate moieties, containing
sometimes up to 10 monosaccharide units, often in
the form of branched chains. Some of them are
mono- or bidesmosides, i.e. they contain one sugar
chain usually present at C-3 hydroxyl group and
the other attached to C-28 carboxyl group of ole-
anolic acid. Oleanolic acid and its derivatives were
discovered in at least 120 species in the 6 (among
8) subclasses of the Magnoliopsydia class (Liu,
1995) and in only one species of the Liliopsida class
(Crocus sativa) (Bisset and Wichtl, 2001). These
compounds have a wide spectrum of pharmaceu-
tical activities and because of these activities they
are included in some promising drugs of natural
origin (Liu, 1995). Their anti-inflammatory, anti-
allergic, anti-ulcer, immunomodulatory, cytotoxic,
anti-tumor, anti-nociceptive, anti-mutagenic, anti-
antibacterial and trypanocidal activities were de-
scribed in many articles (Finney and Tarknoy,
1960; Miles et al., 1974; Subba Rao et al., 1974;
Dajani et al., 1979; Dargan and Subak-Sharpe,
Phytochemistry Reviews (2005) 4: 151–158
? Springer 2005
1985; Nishino et al., 1988; Inoue et al., 1990; Ta-
naka et al., 1991; Serra et al., 1994; Pinducciu
et al., 1995; Farina et al., 1998).
Marigold, Calendula officinalis L. (Asteraceae)
occurs widely in Europe and North Africa. It is
popular in gardens as a decorative annual species,
but it is also well known for its medicinal prop-
erties, pharmaceutical and cosmetic use. In fact,
this plant has been already applied as a remedy
for many diseases in ancient Egypt and Greece.
Traditionally it has been used topically for many
eruptive skin diseases and abrasions, as well as for
gastric and menstrual discomfort, as a plant with
antiseptic, mild diaphoretic and antispasmodic
properties (Wichtl, 1994; Barnes, 2002). In the
contemporary folk medicine, since 12th century,
marigold flower extracts were commonly used as
external anti-inflammatory agents against suppu-
ration (Kohlmunzer, 1998). During the American
Civil War they were used to speed up the healing
of wounds and to draw out infections. At present,
there are many medical products derived from the
marigold plant applied in the treatment of various
skin tumours, dermatological lesions, ulcers and
swellings (Wichtl, 1994). Extracts from marigold
flowers are still used in ointments, cosmetic
creams and hair-shampoos (Anon., 2001; Paulsen,
The structure of oleanolic acid glycosides
occurring in Calendula officinalis L.
Marigold synthesizes significant amounts of ole-
anane saponins, found not only in flowers but also
in all organs of this plant. These glycosides form
two series of structurally related compounds, i.e.
derivatives of 3-O-monoglucoside of oleanolic acid
(hence named ‘‘glucosides’’) and derivatives of
3-O-monoglucuronide (‘‘glucuronides’’), depend-
ing on the first sugar moiety linked to the C-3
hydroxyl group of oleanolic acid, which is either
glucose or glucuronic acid (Figure 1). Subse-
quently, in various glycosides of both types, either
glucose or galactose moieties form the sugar chain,
which finally contains up to five monosaccharide
units. Moreover, in some representatives of both
series a single glucose molecule is attached also to
the carboxyl group of oleanolic acid, thus creating
bidesmosides. The structure of compounds from
Figure 1. The structure of oleanolic acid glycosides occuring in Calendula officinalis.
both series of oleanolic acid glycosides occurring
in C. officinalis was established at Department of
Plant Biochemistry, Warsaw University (Kasprzyk
and Wojciechowski, 1967; Wojciechowski et al.,
1971). ‘‘Glucuronides’’, known as the series I, were
designated with letters (F, D, D2, C, B, A) and
‘‘glucosides’’, forming the series II – with sub-
sequent Roman numerals from I to VIII.
‘‘Glucuronides’’ occur in relatively big amounts
(up to 2% of the dry mass) in flowers, and in
considerably lower quantity in green organs of the
plant. Lately (Ruszkowski et al., 2003) we have
amounts of ‘‘glucuronides’’ in roots of young
marigold plants. In turn, ‘‘glucosides’’ are accu-
mulated mainly in roots of grown and senescing
plants. They also occur in green organs of the
plant. However, only glycosides I, II, III, VI and
VII can be found in marigold shoots.
Biosynthesis and metabolism of oleanolic acid
Both series of glycosides are synthesized in leaves
(Kasprzyk et al., 1970, 1973). Our previous studies
in marigold leaf cells, the biosynthesis of squalene,
b-amyrin, erythrodiol and oleanolic acid proceeds
in the microsomal fraction via the mevalonate
pathway (Eisenreich et al., 2004). In the micro-
somal fraction the biosynthesis of not only agly-
cone, but also its monoglycosides (I and F) and the
other ‘‘glucosides’’ takes place. Thus, the linkage of
a single molecule of galactose at position C-4¢ of
glucose in monoglucoside I results in formation of
diglycoside II, the addition of subsequent galactose
– forms triglycoside III (Figure 2). However, from
diglycoside II and triglycoside III the pathway
branches, and two pentaglycosides (VI and VII)
appear most probably as a result of adding chains
which contain several carbohydrate residues, i.e.
di-or trisaccharide chains (Kasprzyk et al., 1973).
In turn, the remaining ‘‘glucuronides’’ originate in
the Golgi membranes by sequential additions of
single sugar molecules; additionally, a cytosol en-
zyme is probably involved in glycosylation of car-
boxyl group of oleanolic acid (Wojciechowski,
1975). After the formation the precursor of this
series, i.e. 3-O-monoglucuronide F, either glucose
can be linked in the position C-28 of aglycone or
galactose in the position C-3¢ of glucuronic acid, so
two parallel pathways, forming mono- or bides-
mosides. The rate of the biosynthesis of ‘‘gluco-
sides’’ in leaves is several times greater than that of
‘‘glucuronides’’, although finally the content of
‘‘glucuronides’’ several times exceeds the content of
‘‘Glucuronides’’ arise in leaves slowly but
steadily and then they are transported and accu-
mulated in large amounts in flowers, whereas
‘‘glucosides’’ are synthesized quickly and only two
pentaglycosides (VI and VII) are transported to
roots (Figure 3) (Janiszowska and Kasprzyk,
1974). There they undergo gradual degradation
forming compounds with a decreasing number of
sugar molecules, among them tri- and tetraglyco-
sides (IV and V) not occurring in shoots. Later
deglycosylation leads to the monoglucoside I and
in old plants at the end of vegetation even to free
oleanolic acid. Simultaneously, in old roots the
pentaglycoside VIII appears – the compound not
detected in green organs of the marigold plant.
Our latest results have shown that both series
of glycosides are synthesized in roots of young
marigold plants (Ruszkowski et al., 2003). The
pathway of their biosynthesis is similar, although
not identical, to the pathway occurring in green
organs (Figure 2). Several distinct differences have
been observed between those pathways. Namely,
considering glucuronides – the diglycoside D2,
absent in leaves, appears in roots – pointing to
another possibility of the triglycoside C formation
– as a result of linking a galactose molecule to the
diglycoside D2. Considering glucosides – surpris-
ingly, the pentaglycoside VI is not formed in
roots. The inability of roots to form the penta-
glycoside VI is of great interest, because big
amounts of this compound are actually found in
this organ. In such a case the pentaglycoside VI
found in roots of young marigold plants could be
accumulated there exclusively as a result of its
transport from the leaves and the triglycoside III
might be the end product of this branch of the
pathway. Two hypothesis are the most probable
to explain this phenomenon: (a) the high con-
centration of the accumulated compound inhibits
its own formation by a negative feed-back; (b) the
respective glycosyltransferase is lacking or inac-
tive in root cells.
Distribution in the cell
Our studies on distribution of oleanolic acid gly-
occur mainly in the Golgi membranes, in the
microsomal fraction, cell walls and vacuoles,
whereas glucosides are present mainly in the
less amounts) in cell walls and vacuoles (Janis-
zowska & Kasprzyk, 1977; Szakiel and Kasprzyk,
1989; Szakiel et al., 1995). It is of great interest that
in the cytoplasm is subsequently transported across
the vacuolar membrane – the tonoplast and finally
accumulated in the vacuole. Our detailed studies on
the mechanism of this transport pointed to the
existence of two separate carriers, one common for
all glucosides, the other for glucuronides (Szakiel
across thetonoplastbyanactive, energy-dependent
Figure 2. Biosynthesis of oleanolic acid glycosides (?root,$leaf,leaf and root).
and carrier-mediated mechanism, whereas glucu-
ronides undergo translocation across the tonoplast
of secondary-activated mechanism, whereas the
transport of glucuronides is most probably a facil-
itated diffusion. Since the glycosides belonging to
is not transported into vacuoles, but only binds to
Figure 3. Scheme of translocation of oleanolic acid glycosides in the leaf cell and the plant.
the tonoplast (Janiszowska and Szakiel, 1991), it
seems that the first sugar moiety bound to the C-3
hydroxyl group is decisive of the character of
tonoplast transport of oleanolic acid glycosides in
Calendula officinalis suspension culture
So far, little is known about the existence and pro-
are useful and simple models for studies on the
metabolism of different compounds and its regula-
tion, due to their uniformity and relative ease of
controlling environmental conditions (Pauthe-
Dayde et al., 1990; Sashida et al., 1994). Recently,
for the first time, using various basal media, differ-
ent cytokinin/auxin combinations, explants and
external conditions, we have successfully initiated
oleanolic acid (Grzelak and Janiszowska, 2002). It
to triterpenoids is operative in all established cul-
plant, are synthesized after supplying the cultures
with [3)3H]oleanolic acid (Szakiel et al., 2003a).
The biosynthesis ofoleanolicacidglycosidesaswell
as their release in the extracellular medium are
of the culture. Therefore, it seems to be possible to
use the marigold suspension culture as a model
system to investigate the regulation of oleanolic
glycosides production and excretion. Additionally,
the obtaining of suspension cultures not only pro-
ducing the desired compounds, but also transport-
ing them to the surrounding medium, bears
considerable biotechnological importance.
In a continuation of our systematic studies of
marigold triterpenoids some biological activities of
oleanolic acid and its glycosides were examined.
demonstrate hemolytic action towards red blood
cells (Oda et al., 2000). So no wonder that we have
free aglycone, are hemolytic agents (Janiszowska
et al., 1987). They also exhibit antifungal activity
against Trichoderma viride, however, ‘‘glucurono-
sides’’ are twice more active then ‘‘glucosides’’ while
free oleanolic acid posses only very low activity. The
biological activity of particular glycosides varies
depending on the number and the type of the car-
bohydrate moieties in a glycoside molecule.
these compounds, we have tested their effects on the
sativa L. and Lepidium sativum L., and the mono-
cotyledon Triticum vulgare L. (Ruszkowski et al.,
acid nor 3-O-monoglucuronide F possesses allelo-
pathic activity and the remaining ‘‘glucuronosides’’
demonstrate onlylow activity. Onthe otherhand, 3-
O-monoglucoside I is very active allomon against
dicotyledons. There are no significant activities
exhibited against the monocotyledon species, what
can be understood because C. officinalis has to share
its biotope mainly with dicotyledons.
Recently the antibiotic activity of oleanolic acid
as well as its derivatives against medically impor-
tant Gram negative bacterium Escherichia coli was
observed (Ruszkowski, unpublished data). More-
over, the wormicidal activity of oleanolic acid
glycosides against parasitic nematode Heligmo-
somoides polygyrus was investigated (Ruszkowski
et al., 2004a). Oleanolic acid as well as the mixture
of all ‘‘glucosides’’ exhibits practically the same not
very high wormicidal activity while the mixture of
‘‘glucuronides’’ is very active. More work should
be done to characterize individual ‘‘glucuronides’’
of marigold in order to assign wormicidal effects to
Therefore, several biological activities of the
oleanolic acid glycosides present in Calendula
officinalis are found. Nevertheless, more data are
needed to support and extend present knowledge
and to try satisfactorily answer open questions on
mode of penetration and action of oleanolic acid
glycosides in different organisms.
The present findings additionally support ear-
lier results concerning differences of physiological
functions of the two series of oleanolic acid gly-
cosides in marigold plant. ‘‘Glucuronides’’ are
present in many organs of the plant, but in flowers,
seeds and seedlings in particularly great amounts.
Since these compounds have fungicidal and anti-
bacterial activities, the role they play in germi-
nating seeds and young seedlings can be regarded
as protective against soil pathogens. It seems also
possible that ‘‘glucuronides’’ can be somehow in-
volved in regulation of germination and early
development. In turn, the role played by ‘‘gluco-
sides’’ in roots is connected to their allelopathic
activity. We have demonstrated the transport of
the monoglucoside I from the marigold root to the
Moreover, some amounts of ‘‘glucosides’’, with a
great dominance of the monoglucoside I, were
detected in marigold seeds. These observations are
consistent with our findings about the allelopathic
activities of the oleanolic acid ‘‘glucosides’’.
Anon. (2001) Final report on the safety assessment of Calendula
officinalis extract and Calendula officinalis Int. J. Toxicol. 20:
Barnes J, Anderson L & Philipson J (2002) Herbal Medicines.
Pharmaceutical Press, London, (pp. 103–106).
Bisset NG & Wichtl M (2001) Herbal Drugs and Phytopharma-
ceuticals. CRC Press, Boca Raton 167.
Dajani FZ, Bianchi RG, Casler JJ & Weet JF (1979) Gastric
aldosterone and desoxycorticosterone in rats. Arch. Int.
Pharmacodyn. 242: 128–138.
Dargan DJ & Subak-Sharpe JH (1985) The effect of triterpe-
noid compounds on uninfected and Herpes Simplex virus-
infected cells in culture. I. Effect of cells growth, virus
particles and virus replication. J. Gen. Virol. 66: 1771–1784.
Eisenreich W, Bacher A, Arigoni D & Rohdich F (2004)
Biosynthesis of isoprenoids via the non-mevalonate path-
way. Cell. Mol. Life Sci. 61: 1401–1426.
Farina C, Pinza M & Pifferi G (1998) Synthesis and anti-ulcer
activity of new derivatives of glycyrrhetic, oleanolic and
ursolic acid. Il Farmaco 53: 22–32.
Finney RSH & Tarknoy AI (1960) The pharmacological
J. Pharm. Pharmacol. 12: 49–53.
Grzelak A & Janiszowska W (2002) Initiation and growth
characteristics of suspension cultures of Calendula officinalis
cells. Plant Cell Tiss. Org. 71: 29–40.
Inoue H, Kurosu S, Takeuchi T, Mori T & Shibata S (1990)
Glycyrrhetinic acid derivatives anti-nociceptive activity of
deoxoglycyrrhetol dihemiphthalate and the related com-
pounds. J. Pharm. Pharmacol. 42: 199–200.
Janiszowska W & Kasprzyk Z (1974) Transport of glycosides of
oleanolic acid from shoot to root in Calendula officinalis.
Acta Biochim. Polon. 16: 415–421.
Janiszowska W & Kasprzyk Z (1977) Intracellular distribution
and origin of pentacyclic triterpenes in Calendula officinalis
leaves. Phytochemistry 16: 1919–1923.
Janiszowska W, Jurzysta M & Kasprzyk Z (1987) Biological
activities of oleanolic acid glycosides from Calendula offici-
nalis. 13th Conference on Isoprenoids. Poznan ´ , Poland.
Abstacts p. 100.
Janiszowska W & Szakiel A (1991) The transport of [3)3H]ole-
anolic acid and its monoglycosides to isolated vacuoles of
protoplasts from Calendula officinalis leaves. Phytochemistry
Kasprzyk Z & Wojciechowski Z (1967) The structure of
triterpenic glycosides from the flowers of Calendula officinalis
L.. Phytochemistry 6: 69–75.
Kasprzyk Z, Wojciechowski Z & Janiszowska W (1970)
Incorporation of 1)14C-acetate into glycosides of Calendula
officinalis. Phytochemistry 9: 561–564.
Kasprzyk Z, Janiszowska W & Sobczyk E (1973) Metabolism
of the new series of oleanolic acid glycosides in Calendula
officinalis. Acta Biochim. Polon. 20: 231–235.
Kohlmunzer S (1998) Pharmacognosis Handbook for phar-
macy students. PZWL, Warsaw 319–321.
Liu J (1995) Pharmacology of oleanolic acid and ursolic acid. J.
Ethnopharmacol. 49: 57–68.
Mahato SB, Nandy AK & Roy G (1992) Triterpenoids.
Phytochemistry 35: 2199–2249.
Miles DH, Kokpol U, Zalkow LH, Steindel SJ & Nabors JB
(1974) Tumor inhibitors. I. Preliminary investigation of
antitumors activitity of Saracenia flava. J. Pharmacol. Sci.
Nishino H, Nishino A, Takayasu J, Hasegawa T, Iwashima A,
Hitahahayashi K, Iwata S & Shibata S (1988) Inhibition of
the tumor-promoting action of 12-O-tetradecanoylphorbol-
13-acetate by some oleanane-type triterpenoid compounds.
Cancer Res. 48: 5210–5215.
Oda K, Matsuda H, Murakami T, Katayama S, Ohgitani T &
Yoshikawa M (2000) Adjuvant and haemolytic activities of
43 saponins derived from medicinal and food plants. Biol.
Chem. 381: 67–74.
Paulsen E (2002) Contact sensitization from Compositae-
containing herbal remedies and cosmetics Contact Derm.
Pauthe-Dayde D, Rochd M & Henry M (1990) Triterpenoid
saponin production in callus and multiple shoot cultures of
Gypsophilla sp. Phytochemistry 29: 483–487.
Pinducciu G, Serra C, Cagetti MG, Cotti M, Deida D &
Pinza M (1995) Selective antimicrobial activity of triter-
pene derivates on oral bacteria. Med. Microbiol. Lett. 4:
Ruszkowski D, Szakiel A & Janiszowska W (2003) Metabolism
of [3)3H]oleanolic acid in Calendula officinalis L. roots. Acta
Physiol. Plant. 25: 311–317.
Ruszkowski D, Chouj A, Doligalska M & Janiszowska W
(2004a) The reduction of nematode infective stages viability
under oleanolic acid glycosides of Calendula officinalis L.
roots. International Conference on Saponins, Puawy, Po-
land. Abstracts p. 105.
Ruszkowski D, Uniewicz K, Augus´cin ´ ska E & Janiszowska W
(2004b) The allelopathic properties of oleanolic acid
3-O-monoglucoside secreted by roots of Calendula officinalis
to the soil. Second European Allelopathy Symposium,
Puawy, Poland. Abstracts p. 101.
Sashida Y, Ogawa K, Yamanouchi T, Tanaka H, Shoyama I &
Nishioka I (1994) Triterpenoids from callus tissue of
Actinidia polygama. Phytochemistry 33: 377–380.
Serra C, Lampis G, Pompei R & Pinza M (1994) Antiviral
activity of new triterpene derivaties. Pharmacol Res. 29:
Szakiel A & Kasprzyk Z (1989) Distribution of oleanolic acid
glycosides in vacuoles and cell walls isolated from protop-
lasts and cells of Calendula officinalis leaves. Steroids 53:
Szakiel A, Wasiukiewicz I & Janiszowska W (1995) Metabolism
of [3)3H]oleanolic acid in the isolated Calendula officinalis
leaf cells and transport of the synthesized glycosides to the
cell wall and the extracellular space. Acta Biochim. Polon.
Szakiel A & Janiszowska W (2002) The mechanism of oleanolic
acid monoglycosides transport into vacuoles isolated from
Calendula officinalis leaf protoplasts. Plant Physiol. Bio-
chem. 40: 203–209.
Szakiel A, Grzelak A, Dudek P & Janiszowska W (2003a)
Biosynthesis of oleanolic acid and its glycosides in Calendula
officinalis suspension culture. Plant Physiol. Biochem. 41:
Szakiel A, Ruszkowski D & Janiszowska W (2003b) Excretion
of oleanolic acid glycosides to the medium from the roots of
marigold Calendula officinalis L. Pol. J. Nat. Sci. 1, Suppl.
Subba Rao G, Sinsheimer JF & Cochran KW (1974)
Tanaka S, Uno C, Akimoto M, Tabata M, Honda C &
Kamisako W (1991) Anti-allergic effect of bryonolic acid
from Luffa cylindrical cell suspension culture. Planta Med.
Wichtl M (1994) Herbal Drugs and Phytopharmaceuticals
Medpharm Scientific Publisher, Stuttgart 446.
Wojciechowski Z (1975) Biosynthesis of oleanolic acid glyco-
sides by subcellular fractions of Calendula officinalis seed-
lings Phytochemistry 14: 1749–1753.
Wojciechowski Z, Jelonkiewicz-Konador A, Tomaszewski M,
Jankowski J & Kasprzyk Z (1971) The structure of glyco-
sides of oleanolic acid isolated from the roots of Calendula
officinalis. Phytochemistry 10: 1121–1124.