TSINGHUA SCIENCE AND TECHNOLOGYISSN 1007-0214 08/20 pp567-572Volume 10, Number 5, October 2005

Crocin Synthesis Mechanism in Crocus sativus

YANG Bo ( ), GUO Zhigang ( )**, LIU Ruizhi ( )

Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

Abstract: Saffron (Crocus sativus) cells can synthesize crocin, crocetin digentiobiosyl ester, in suspension

cultures. The crocin family biosynthesis mechanism was studied using high pressure liquid chromatography

(HPLC) to determinate the glucosyltransferase activity and to develop a method for synthesizing medicine

from saffron cells. Previous studies indicated that two glucosyltransferases might be involved in the forma-

tion of crocetin glucosyl- and gentiobiosyl-esters. GTase1 formed an ester bond between crocetin carboxyl

groups and glucose moieties while GTase2 catalyzed the formation of glucosidic bonds with glucosyl ester

groups at both ends of the molecule. These enzymes can catalyze the formation of crocetin glucosides in vi-

tro. GTase1 activity is higher during the first four days of crocin glucosides biosynthesis, but decreases after

four days. The formation and accumulation of crocin increase during the first six days and stabilized on the

eighth day.

Key words: saffron; crocetin; crocetin di-neapolitanosyl ester; glucosyltransferase; glycosylation

Introduction

The crocin family is a group of carotenoid esters that

occur naturally in saffron stigmata and gardenia

fruits[1]

. These yellow pigments are very soluble in

water and have a strong dyeing capacity. They have a

powerful ability to quench free radicals, so they may

present anticancer activity. Products of the crocin

family are expected to be useful in foods and

pharmaceuticals[2]

, but commercial utilization of

saffron pigments is restricted by its prohibitive price

and limited availability. Therefore, plant cell cultures

have been suggested as an attractive alternative to

produce crocin derivatives.

The crocin family consists of glucosyl and

gentiobiosyl esters of crocetin, a dicarboxylic 20-

carbon carotenoid (Fig. 1). Crocetin is converted into

crocetin glucosides in saffron stigmata and gardenia

fruits. Exogenously-added crocetin encapsulated in

maltosyl- -cyclodextrin is taken up by the cells,

glucosylated and then stored in vacuoles[3]

.

A pathway for glucosylation of encapsulated cro-

cetin has been reported along with some properties of

the glucosyltransferase involved in crocin synthesis

(Fig. 2). Cote et al.[4]

indicated that the glucosylation

of crocetin into crocin involves two glucosyltrans-

ferases: GTase1 which catalyzes the glucose transfer

on both carboxylic ends of crocetin and GTase2 which

helps glucosidic bonds to form gentiobiosyl esters.

GTase1, which is responsible for the formation of cro-

cetin monoglucosyl and diglucosyl esters, is the main

focus of this paper.

The most common methods used to assay enzyme

activity include the use of radioactive sugars, identifi-

cation of the products by thin-layer chromatography

(TLC) or paper chromatography and high pressure liq-

uid chromatography (HPLC) with a linear gradient[5]

.

Recently, the development of HPLC technology has

resulted in increasing use of HPLC to analyze enzyme

activity.Received: 2003-11-07

To whom correspondence should be addressed.

E-mail: guozhig@tsinghua.edu.cn; Tel: 86-10-62785603

This paper reports on the glucosyltransferase activi-

ties in saffron cells during biosynthesis. Saffron cells

Tsinghua Science and Technology, October 2005, 10(5): 567 572568

were cultured in a suspension synthesis medium, with

the enzyme activities analyzed using HPLC. The

crocin biosynthesis mechanism was discussed based on

the enzyme activity data.

Fig. 1 Structure of crocetin and its glucosides[3]

Fig. 2 Multi-step pathway proposed for crocetin glucosides biosynthesis by adding glucose moieties to crocetin

1 Materials and Methods

1.1 Chemicals and reagents

Crocetin, uridine 5’-diphosphoglucose and 6-O- -

maltosyl- -cyclodextrin were purchased from Sigma

Chemical Co. (St. Louis, USA). The PD-10 column

was from Phamacia (Wikstroms, Sweden). The PREP-

ODS (H) kit (4.6 mm 250 mm) column was pur-

chased from Shimadzu Co. (Tokyo, Japan). Albumin

fraction was from Dingguo Biotechnology Devel-

opment Center (Beijing, China). The water filter and

the PT filter (0.45 m) were purchased from Xingya

Purification Material Factory (Shanghai, China). The

methanol was HPLC grade and other chemicals were

analytical reagent grade.

1.2 Cell culture

Saffron (Crocus sativus L.) cells were produced from

well-established cell lines. The cells were subcultured

in solid growth medium for 20 days, and then 10 g

fresh wt. of cells were transferred into 50 mL fresh liq-

uid synthesis medium in 200-mL flasks. The culture

medium was Murashige and Skoog plant salt mixture

with elicitors. The cell suspension culture was main-

tained at 25 in the dark on an orbital shaker operat-

ing at 120 r/min. The cells grown on solid medium

(control) were harvested before transferring and the

liquid synthesis cells were harvested by filtration on

the 2nd, 4th, 6th, and 8th days, and then lyophilized

and stored at 35 .

YANG Bo ( ) et al Crocin Synthesis Mechanism in Crocus sativus 569

1.3 Crocin analysis

Lyophilized cells were pooled, ground, and weighed to

400 mg per sample. 400 mg of ground cells were ex-

tracted with 20 mL 60% (v/v) methanol at 25 for

24 h on an orbital shaker operating at 120 r/min. After

collecting the supernatant, the cells were extracted

with another 10 mL 60% (v/v) methanol with the same

conditions for 24 h. The two obtained fractions of ex-

tracts were pooled, filtered, and analyzed by HPLC

(Shimadzu-Class-10Avp, Japan).

1.4 Glucosyltransferase extraction and assay

A 1-mg/mL stock solution of crocetin was prepared by

encapsulation of the substrate with maltosyl- -

cyclodextrin in a 1:3 molar ratio as described by

Dufresne et al.[6]

All extraction procedures were carried out at 4 or

on ice. For enzymatic extraction, 1 g of ground lyophi-

lized cells were mixed with 10 mmol/L polyethylene

glycol-4000 and 20 mmol/L ethylenediaminetetraace-

tic acid in 15 mL 200 mmol/L Tris-HCl buffer at pH

7.4. The cells with extractant were ground on ice for

30 min. The extract was centrifuged at 10 000g for

30 min at 4 . The supernatant was desalted with a

PD-10 column and eluted in 100 mmol/L Tris-HCl

buffer at pH 7.4.

The standard reaction mixture consisted of 160 L

reaction buffer (100 mmol/L Tris-HCl pH 7.4),

200 mol/L encapsulated crocetin, 2.5 mmol/L UDP-

glucose, and 60 L desalted extract, in a final volume

of 400 L. The reaction was initiated by adding

enzyme extract. The mixtures were incubated for 30

min at 40 . The reaction was stopped by adding

400 L ethanol. The reaction products were separated

and quantified by reverse phase HPLC (Shimadzu-

Class-10Avp, Japan) on an ODS (Shimadzu, Japan)

column.

Protein content was determined using the Bradford

method[7]

with a spectrophotometer (UV/VIS spectro-

photometer specord 2000, Germany) using albumin

fraction as a standard.

1.5 HPLC analysis

1.5.1 Enzyme analysisThe gradient program was 0-10 min, a linear gradient

from 1% (v/v) acetic acid in water to 100% methanol;

10-20 min, a linear gradient from 100% methanol to

1% (v/v) acetic acid in water; 20-25 min, 1% (v/v) ace-

tic acid in water until the end. The flow rate was kept

constant at 1.0 mL/min. The eluate absorbance was

monitored at 440 nm.

1.5.2 Crocin analysis1) The flow rate was kept constant at 1.0 mL/min. The

eluate absorbance was monitored at 250 nm. The gra-

dient program was 0-25 min, the methanol concentra-

tion increased linearly from 0% to 50% (v/v), and then

50% methanol maintained for 5 min. After that, the

methanol concentration increased linearly from 50% to

80% in 10 min, and finally decreased to 0% in 5 min.

2) The flow rate was kept constant at 1.0 mL/min.

The eluate absorbance was monitored at 440 nm. The

gradient program was 0-10 min, a linear gradient from

1% (v/v) acetic acid in water to 100% methanol; 10-20

min, a linear gradient from 100% methanol to 1% (v/v)

acetic acid in water; 20-25 min, 1% (v/v) acetic acid in

water until the end.

2 Results and Discussion

The glucosyltransferases can transfer glucose moieties

to the free carboxyl or glucosyl groups. During crocin

synthesis, the glucosyltransferases transform crocetin

into its related glucosyl- and gentiobiosyl- esters in the

presence of UDP-Glc. Cote et al.[3]

showed that the

formation of crocetin glucosides was a step-wise addi-

tion of glucose moieties in the following sequence:

monoglucosyl ester, monogentiobiosyl and diglucosyl

esters, gentiobiosyl glucosyl ester, and finally crocin

(Fig. 2). Crocetin was transformed into crocetin gluco-

sides upon addition of enzyme extract into the mixture

of UDP-Glc and encapsulated crocetin. This work ana-

lyzed the change of enzymatic activity during crocin

synthesis using HPLC to study the crocin synthesis

mechanism.

Two esters, A and B, were identified in the experi-

mental results from three peaks in the HPLC elution

profile of the enzyme reaction at 440 nm. The retention

time of the middle peak was 15.2 min. According to

the crocetin standard profile at 440 nm (Fig. 3), this

middle peak was related to crocetin. The other two

peaks’ retention times were 14.1 min and 16.2 min,

corresponding to peak 6 and peak 8 in Fig. 4.

Tsinghua Science and Technology, October 2005, 10(5): 567 572570

Comparison of Figs. 3 and 4 suggests that these two

peaks are crocetin monoglucosyl ester and crocetin di-

glucosyl ester[8-11]

. In this paper, these two peaks were

identified as ester A and ester B because of the absence

of standards for these two substances.

Fig. 3 HPLC elution profile of enzyme reaction (a) and of crocetin standard (b) at 440 nm

Fig. 4 HPLC elution profile of saffron cell extract at440 nm

2.1 Consumption of crocetin and formation ofester A and ester B at different reaction times

Figure 5 shows that the consumption of crocetin and

the accumulation of ester A and ester B increased

gradually with time during the enzymatic reaction. The

amount of ester B was lower than that of ester A

during the whole reaction time. This result indicates

that ester A was more easily formed than ester B. In

addition, previous studies have shown that crocetin

monoglucosyl ester formed most easily with most

products in this enzymatic reaction. Therefore, ester A

is suggested to be crocetin monoglucosyl ester. Ester B

was probably formed after the formation of ester A, so

ester B is suggested to be crocetin diglucosyl ester

according to the proposed pathway (Fig. 2).

Fig. 5 Consumption of crocetin and formation of crocetin glucosides at different reaction times

2.2 Comparison of enzyme activities, consumptionof crocetin, and formation of glucosides

In this paper the percentage of the consumption of cro-

cetin per mg protein is defined as the enzyme activity.

The change of enzyme activity during the culture

shown in Fig. 6 shows that the enzyme activity was the

highest just after the cells were transferred from the

subculture medium into the synthesis medium. The

activity then decreased for two days, increased until

the fourth day, and then decreased gradually again

after the fourth day. This result indicates that there

might have been some glucosyltransferases in the

original cells before the cells were transferred into the

synthesis medium, but crocin was not formed in the

cells due to absence of crocetin (HPLC analysis). The

enzyme activity may have decreased during the first

two days due to the abrupt change of culture conditions

which occurred when the cells were transferred from

the solid subculture medium to the liquid synthesis

medium. The synthesis medium may then contain

some inducers promoting the formation of carotenoids

and their precursors, causing the glucosyltransferase

activity to increase again after two days and reach a

maximum on the fourth day, after which the enzyme

activity began to decrease.

YANG Bo ( ) et al Crocin Synthesis Mechanism in Crocus sativus 571

Fig. 6 Conversion ratio of crocetin and formation of crocetin glucosides in biosynthesis

The results in Fig. 6 also show that the formation of

ester A was highest before the cells were transferred.

This result further indicates that ester A may be the

most easily formed crocetin monoglucosyl ester. The

formation of ester A approximately accorded with the

changes in enzyme activities (crocetin consumption).

Ester B formed more slowly before the cells were

transferred, but the formation rate then gradually in-

creased after the transfer. The amount of ester B was

less than that of ester A before the fifth day, but then

exceeded that of ester A after the fifth day and re-

mained greater for most of the rests. The fourth day

was a turning point for formation of both crocetin ester

A and ester B as the formation of ester A began to de-

crease while the formation of ester B still to slowly in-

crease after the fifth day. This suggests that ester A

may be a precursor of ester B, thus ester A would de-

crease as ester B increased. The amounts of both ester

A and ester B increased from the second to the fourth

days, perhaps indicating that the synthesis of ester A

was the first step of the synthesis of crocin. The syn-

thesis of ester A continued with a part of ester A con-

verted to ester B.

Ester A and ester B are the simplest crocetin gluco-

sides and, unfortunately, crocin was not synthesized in

the enzyme reaction. There were probably two reasons

for the lack of crocin: GTase2 perhaps did not exist in

the enzyme extract or the GTase2 might have been de-

activated during the extraction and reaction processes.

Cote et al.[4]

reported that the involvement of two glu-

cosyltransferases in crocin biosynthesis was supported

by the different activities of the enzymes in saffron

plant organs. Though both enzymes were constitutive,

GTase1 activity was higher in the corms while GTase2

was more active in the sprouts. The catalysis of

GTase2 was not identified in the enzyme extracts from

the cells synthesizing crocin, so the conditions for

GTase2 catalysis must differ from those for GTase1,

i.e., GTase2 catalysis occurs only when crocetin gluco-

sides (including crocetin monoglucosyl ester and cro-

cetin diglucosyl ester) reach some required amount.

However, since the amount of transformed crocetin

glucosides was very limited in our experiment, observ-

able GTase2 catalysis did not occur.

2.3 Changes in crocin in saffron cells duringsynthesis

The crocin, cis-crocetin digentiobiosyl ester biosynthe-

sis is shown in Fig. 7. The results show that the crocin

content was very low initially but increased rapidly

with time. Crocetin first appeared after the cells were

transferred into the culture medium (Fig. 7), reached a

maximum level on the second day, and then rapidly

decreased after the second day as the quantity of crocin

increased. These results can be compared with the glu-

cosyltransferases activity measurements where the en-

zyme activity decreased a little during the first two

days, but then increased to a maximum on the fourth

day. After four days the formation of ester A slowed,

but the total enzyme activity still remained high be-

cause of the conversion to ester B. The accumulation

of crocin in the cell culture showed that the crocin

level increased actually with time until the eighth day.

The configuration and color of the cells changed

gradually from a buff color to red from the second to

the sixth days with the deepest red on the eighth day.

After the eighth day, the cells began to age and their

color began to darken, so the synthesis time was lim-

ited to 6-8 days.

Fig. 7 Accumulation of crocin (250 nm) and crocetin(440 nm) in saffron cells in culture (The initial amount of crocetin is 32 pmol)

Tsinghua Science and Technology, October 2005, 10(5): 567 572572

3 Conclusions

The experimental results show that saffron cells

contain glucosyltransferases, which catalyze the

reaction of crocetin and UDP-Glc and transform to

crocetin glucosides or crocin in vitro. The crocetin was

first transformed to crocetin monoglucosyl ester, then

to other glucosides, until crocin was finally

synthesized. Two glucosyltransferases might be

involved in the formation of crocetin glucosyl- and

gentiobiosyl-esters. One formed ester bonds between

the crocetin carboxyl groups and glucose moieties

while the other catalyzed the formation of glucosidic

bonds with the glucosyl ester groups at both ends of

the molecule. During the culture, the GTase1 activity

was higher during the first four days and then

decreased, but the biosynthesis of crocin was faster

during the first six days, particularly from the second

to the sixth days, which indicates that the GTase1 had

its greatest effect in the first four days. The GTase2

activity was not examined because the crocetin

monoglucosyl ester content was too low. However,

GTase2 may also have its greatest effect from the

second to the sixth days when the crocin formation rate

was the greatest.

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