Tech Bull Fac Agr Kagawa Univ , Vol 46, No 2, 135-140, 1994
REGULATION OF THE PRODUCTIVITY OF ANTHOCYANINS
AND ROSMARINIC ACID FROM CULTURED CELLS OF
PERILLA FRUTESCENCE
VAR CRISPA (LABIATAE)
Hirotoshi TAMURA, Masahiro FUJIWARA, Kyoko
TAKAHASHI
a n d HiroshiSUGISAWA
Production of secondary metabolites from Perzlla frutescence var crtspa was conducted by cultured cells in a suspension culture and on an agar medium of Gamborg B5 medium Anthocyanin and rosrnarinic acid contents in the cutured cells were approximately 6mg gFW-' and 15mg gFW-', respectively Productivity of both metabolites was affected by adding a synthetic cytokinin, N-(2-chloro-4-pyridy1)-N'-phe- nylurea The effect of N-(2-chloro-4-pyridy1)-N7-phenylurea suppressed the production of anthocyanin (35%, productivity) and stimulated the production of rosmarinic acid (200%) Potassium nitrate increased the production of both metabolites, especially anthocyanins (180%) The lack of phosphate salt in Gamborg B5 medium led to the 200% rosmarinic acid production and the 100% anthocyanin production
Key Words: Pertlla frutescence var crtspa, Labiatae, malonylshisonin, shisonin, rosmarinic acid, N-(2-chloro-4-pyridy1)-N'-phenylurea, inhibitor of anthocyanin synthesis, regulation of the production of metabolites
Introduction
Anthocyanins in t h e akachirimen-shiso (Perzlla frutescence var crzspa) leaves have been used a s t h e coloring materials in pickles with U m e Natural pigments produced by cultured cells a r e potential substances for food additives Recently Koda e t a1 reported t h e properties of pigment from cultured cells of Perzlla frutescens a s food colors(') Rosma~inic acid has anti-inflammatory activity and a c t s a s a n antioxidant for spices T h e s e chemicals a r e useful metabolites for our life
Chemical structure of anthocyanins and rosmarinic acid in t h e intact plant have been elucidated in past d e ~ a d e ( ~ , ~ ) Secondary metabolites from cultured cells of akachirimen-shi- so (Perzlla frutescence var crzspa) w a s extracted Malonylshisonin, shisonin and rosmarinic acid a s s a m e a s those in intact plant were isolated a s t h e main m e t a b o ~ i t e s ( ~ ) Ohta e t a1 reported t h e anthocyanin formation in a callus induced under t h e blue light i l l ~ m i n a t i o n ( ~ ) Rosmarinic acid production w a s reported by
B E
Ellis et a1 ') andM H
Zenk e t a1 (*) in suspension cell cultures of Coleus blumez (Labiatae)T h i s paper describes t h e regulation of t h e anthocyanin formation and rosmarinic acid formation by using a cytokinin, N-(2-chloro-4-pyridy1)-N'-phenylurea (CPPU) and changing t h e mineral composition in Gamborg
B5
medium136 Tech Bull Fac Agr Kagawa Univ, Vol 46, No2, 1994
Materials and Method
Induction of callus from the leaves of Pertlla frutescence var crzspa T h e induction of undifferentiated cells from the leaves of Pertlla frutescence var crzspa was conducted on the Gamborg B5 medium a s reported in the previous paper(4) T h e leaves were inoculated on the Gamborg B5 agar medium containing 20% coconut milk, naphthaleneacetic acid (NAA, 5mg L-') and kinetin ( l m g
L-')
under 2000 lux at 25"C, and then 0 15 grams of callus tissue were subcultured under the same conditions about every 30 days The metabolites were analysed by HPLC at 40 days after subcultureSuspension culture Suspension culture of Perzlla frutescence var crtspa was conducted in the 2 times diluted Gamborg B5 (1/2 B5) medium solution T h e cultured cells (0 5g) were incubated with 30mL of 1/2 B5 with 1% sucrose, 2 5mg
L-'
NAA and 0 5mgL-'
kinetin T h e suspension cells were cultured at 25°C under 2000 lux for 20 days Incubation flasks were rotated at the rate of 80 cycles min-'Extraction of anthocyanins and rosmarinic acid: typical example Cultured cells (4 45 grams) were homogenized with a minimum amount of water for a few minute at 0°C and then anthocyanins and rosmarinic acid were extracted with an equal volume of cooled 50% aqueous methanol containing 1% trifluoroacetic acid or 50% acetonitrile containing 1% trifluoroaectic acid Extracts were filtrated and then the residue was again immersed in the same volume of the solvent T h e combined extracts (11 5mL) were stored in refrigerator until used
One gram of the fresh callus weight (gFW) corresponded to 0 045 gram of the dry weight (gDW) Maximum content of the anthocyanins reached to lOmg
~ F W - '
at 39th generation cells This amount should correspond to be 222mg g ~ ~ - ' (22 2%) On the other hand, rosmarinic acid contents were decreased from 3mg g ~ ~ to 1 5 m g - ' g ~ ~ - ' along with subcultureAnalytical instruments and analytical conditions for metabolites Quantitative analysis of anthocyanin was conducted by a HITACHI Model 200-15A Spectrophotometer at 520 nm using malvin chloride (Aldrich Chemical Co L t d ) as a standard sample Rosmarinic acid was analyzed by a JASCO 880 gradient HPLC equipped with a JASCO 875
UV
detector under the following conditions: column CI8, 5pm Develosil (250mmx
4 6mm i d ); temperature maintained at 40C; detector JASCO 875UV
set at A=290 nm; mobile phase, A: AcOH: CH3 CN: Hz 0 : H3 PO4 (8: 10: 80 5: 1 5); mobile phase,B:
AcOH: CH, CN: Hz 0 : H3 PO4 (20: 25: 53 5: 1 5) T h e flow rate of the eluent was 1mL min-' T h e analysis was accomplished by a linear gradient elution from solvent A to solvent B (30min)Result and Discussion
The effect of the constituents in basal medium of Gamborg B5 on the regulation of the anthocyanin and rosmarinic acid formation
T h e effect of the concentration of phosphate (NaH2 PO4 ) and nitrogen containing salts ((NH4 ), SO4 , KNOB ) on the regulation of the anthocyanins and rosmarinic acid production was examined as shown in Table 1 and Table 2 It has been reported that inorganic phosphate (Pi) is one of the most effective factors in the control of the growth and metabolism of plant
H TAMURA et a1 : Regulation of the productivity of the metabolites of Pertlla sp 137
Table 1 Effect of phosphate salts on the anthocyanin and rosmarinic acid formation"
phosphate agar mediumb liquid mediumc
salt relative relative concentration
(mM) relative relative concentration
N ~ H ~ P O , fresh weight anthocyanin rosmarinic acid fresh weight anthocyanin rosmarinic acid
0 60 59 119 67 100 216 0
1
117 92 111 129 91 150 0 5 114 91 104 136 96 147 1 2 100 100 100 100 100 100 2 0 109 69 86 132 71 ' 127 3 . 0 116 20 37 128 76 128a
:
cultured on agar medium and in liquid medium of Gamborg B5 with NAA 5 mg L-I, kinetin Img L-I and 20 % coconut milkb
:
control: fresh weight, 1 8 2 g/tube; anthocyanin content, 4 23 mg gFW-'; rosmarinic acid, 0 91 mg gFW-'c
:
control: fresh weight, 2 05 g/cup; anthocyanin content, 10 15 mg gFW-'; rosmarinic acid, 1 14 mg gFW-'Table 2 Effect of ammonium and nitrate salts on the anthocyanin and rosmarinic acid formation"
nitrogen agar mediumc liquid mediumd
saltb
relative relative concentration relative relative concentration NO,-:NH,+ fresh weight anthocyanin rosmarinic acid weight anthocyanin rosmarinic acid
1
:
0 101 121 116 84 184 131 20 1 94 124 102 84 194 114 12 1 100 100 100 100 100 100 5 : l 101 92 70 82 134 96 1 : l 58 9 37 65 54 46 1:
5 38 11 28 40 39 40 0 : l 36 9 22 20 22 7a cultured on agar medium and in liquid medium of Gamborg B5 with NAA 5 mg L-I, kinetin Img L-' and 20 % coconut milk
b ' total concentration of nitrogen atom was 27 mM for agar medium and 1 3 5 mM for liquid
medium, respectively
c
:
control. fresh weight, 2 81 g/tube; anthocyanin content, 2 37 mg gFW-'; rosmarinic acid, 2 22 mg gFW-'d control: fresh weight, 4 19 g/cup; anthocyanin content, 3 06 mg gFW-'; rosmarinic acid, 2 05 mg gFW-'
cells(g) Accumulation of secondary metabolites under the phosphate-reduced conditions has been reported for alkaloids, anthocyanin and p h e n o ~ i c s ( ' ~ - ' ~ ) T h e reduced concentration of phosphate salts intensely increased cyanidin 3-glucoside and peonidin 3-glucoside production in t h e cultured cells of Vztts s p ( " ) However, cultured cells from Perzlla frutescence w e r e not affected against t h e anthocyanin production On t h e other hand, rosmarinic acid production w a s intensely increased by t h e lack of phosphate salts This tendency w a s remarkable in t h e suspension culture Growth w a s slightly decreased on lacking phosphate salt Phosphate salt w a s found to be a effective reagent acting a different way between anthocyanin and rosmarinic acid formations
138 Tech Bull Fac Agr Kagawa Univ, Vol 46, No2, 1994
formation rather than rosmarinic acid formation This tendency was remarkable in the suspension culture High concentration of ammonium salt ( > I 3 5mM) strongly inhibited the growth and the production of both metabolites Changing the total concentration of potassium nitrate added as the only nitrogen constituent of the modified Gamborg B5 medium did not increase the anthocyanin and rosmarinic acid formation On the other hand, lower than 20mM of potassium nitrate inhibited the growth, and the anthocyanin and rosmarinic acid formation
The effect of N-(2-chloro-4-pyridyI)-N'-phenylurea on the anthocyanins and rosmarinic acid production
As shown in Table 3, the production of anthocyanins was inhibited at a higher concentration of N-(2-chloro-4-pyridy1)-N'-phenylurea (CPPU) At the same time, rosmarinic acid formation increased two times more than that of the control callus Callus induced on Gamborg B5 medium with lppm CPPU and 5ppm NAA could not produce
a
detectable amount of anthocyanins at the callus induction stage and after several times of subculture T h e callus color was greenish However, the amount of rosmarinic acid at the 11th genelation was 1 2mgL-'
Biogenesis of rosmarinic acid was found by Ellis et a1 ( I 3 ) from the tn vtvo experiment in Mentha arvense and Mentha ptpertta This experiment indicated that inhibition and suppression of anthocyanin formation might switch the biogenetic pathway into the accumulation of rosmarinic acid since anthocyanins and rosmarinic acid have a common metabolic pathway a s shown in Fig 1 This inhibitive stimulation by CPPU might affect the stage after chalcone synthesis of anthocyanin formation because phenylalanine ammonia lyase activity was still high with 2 5mgL-'
CPPU (data not shown) Inhibition of the anthocyanin formation, and promotion of the rosmarinic acid formation by CPPU have not been reported elswhereIntegration effect of the lack of phosphate salt in Gamborg B5 medium and the addition of 2 5mg
L-'
CPPU could not observed at all Growth was suppressed 30% and the production of anthocyanins and rosmarinic acid was also suppressed 10% and 30%, respectively This medium conditions might give a heavy stress to the cellsFurther experiments about the regulation of the productivity of secondary metabolites will have to be done to get the natural unlimited resources by using cultured cells
Table 3 Effect of N-(2-chloro-4-pyridy1)-N'-phenylurea on the production of rosmarinic acid and anthocyanins"
growth regulator relative relative concentrationc
( P P ~ fresh weightc anthocyanin rosmarinic acid
N A A
:
CPPUb
= 5 : 0 102 158 102 = 5 : O1
100 49 146 = 5 : 1 83 37 204 = 5 : 5 81 34 204 controlN A A
:
kinetin = 5 : 1 100 100 100a
:
cultured on the agar medium of Gamborg B5 b:
N-(2-chloro-4-pyridy1)-N'-phenylureac
:
control: fresh weight, 1 76 g/tube; anthocyanin content, 2 46 mg gFW-I; rosmarinic acid, 1 03 mg gFW-'H TAMURA et a1 : Regulation of the productivity of the metabolites of Pertlla sp HO*".
-
OHF*'
shikimic acid HOOC-CO-SCOA C02H malonyl CoA . a ' tyrosine phenylalanineF.'
HO \ C02H HO pcoumaric acidCHS
DOPA '*, caffeic acid
HO I
rosmarinic acid OH
anthocyanidin Fig 1 Biogenetic pathway of anthocyanin and rosmarinic acid
,---
1----
-2-Route 1 for rosmarinic acid formation w a s proposed by Ellis and Towers in 197013' Route 2 for rosmarinic acid formation w a s proposed by Petersen e t a1 in 198714'
Acknowledgment
T h e a u t h o r s wish t o t h a n k Kyowa Hakkou Kogyo L t d Tokyo, J a p a n for g e n e r o u s gift of
CPPU
T h i s w o r k w a s s u p p o r t e d in part by g r a n t from t h e Yamazaki S p i c e R e s e a r c h & Pro- motion Foundation, Tokyo, J a p a nReferences
(1) KODA, T , ICHI, T , SEKIYA, J : Properties ofpigment from cultured plant cells of Pertlla frutescens as food colors Ntppon Shokuhtn Kogyo Gakkat sht 39, 845 -849 (1992)
(2) KONDO, T , TAMURA H , YOSHIDA, K and GOTO, T : Structure of malonylshisonin, a genuine pigment in purple leaves of Pertlla octmozdes var crtspa Benth Agrtc Btol Chem 53, 797-800 (1989)
(3) OKUDA, T , HATANO, T , AGATA, I and NISHIBE,
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(4) TAMURA, H , FUJIWARA, H and SUGISAWA, H :
Production of phenyl-propanoids from cultured callus tissue of the leaves of akachirimen-shiso (Pertlla sp ) Agrtc Btol Chem 53, 1971 -1973 (1989)
140 Tech Bull Fac Agr Kagawa Univ, Vol 46, No 2, 1994 induction, growth of Pertlla callus tissues and
selection of Perilla pigment producing strain Bull Faculty o f Home Economtc s , K t n ~ o Gakutn Untverstt y (Japan) 24, 67-74 (1984) (6) RAZZAQUE, A and ELLIS, B E : Rosmarinic acid
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(8) ZENK, M H , El-SHAGI, H and ULBRICH, B :
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(9) LI X N and ASHIHARA, H: Effects of inorganic phosphate on sugar catabolism by suspension- cultured Catharanthus roseus Phytoc hemt str y 29, 497-500 (1990)
(10) KNOBLOCH, K
H ,
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(lo
TAMURA, H and SUGISAWA, H : Production of anthocyanins from cultured cells of Muscat Bailey A Fragrance Journal (Japan) 19, 48-53 (4) (1991)(12) HIROSE, M , YAMAKAWA, T , KODAMA, T and KOMAMINE, A : Accumulation of betacyanin in Phytolacca americana cells and of anthocyanin in Vitis sp cells in relation to cell division in suspension cultures Plant Cell Phystol 31, 267-271 (1990)
(13) ELLIS, B E and TOWERS, G H N : Biogenesis of rosmarinic acid in Mentha Btoc hem J 11 8, 291 -297 (1970)
(141 PETERSEN, M and ALFERMANN, A W : T w o new enzymes of rosmarinic acid biosynthesis from cell cultures of Coleus blumet hydroxyphenylpy- ruvate reductase and rosmarinic acid synthase Z Naturforsch, C Btosct 43, 501-504 (1988)
(received May 31, 1994)
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