RSC Advances
Paper
Cambial meristematic cells (CMCs), also known as plant 156.4 (C-5), 111.9 (C-6), 142.8 (C-7), 111.9 (C-8), 154.9 (C-9),
stem cells, are innately undifferentiated cambium cell lines 106.5 (C-10), 23.5 (4-CH3), 21.1 (7-CH3).26
located in the meristems of plants and possess unlimited
proliferation characteristics.18 The performance of CMCs is far
superior to that of dedifferentiated plant cells (DDCs) on the
Identication of transformed products
basis of faster growth rates, higher yields of secondary metab- Aer HPLC analysis, it was seen that four products were formed,
olites and lower variability.19 Moreover, CMC suspension followed by the appearance of four additional peaks at retention
cultures can exhibit stable growth rates and growth properties time of 16.94 (I-1), 16.99 (II-1), 17.80 (III-1) and 22.19 (IV-1) min,
on an industrial scale.19,20 This may likely be attributed to respectively (Fig. 1). Then, four products from the biotransfor-
strikingly greater tolerance to shear stress, which limits the mation of 4-methylcoumarins with C. acuminata CMCs were
growth of cells and reduces cell aggregation in stirred tanks or identied.
air-li bioreactors. The presence of small, abundant vacuoles
Compound I was transformed to I-1 in C. acuminata CMCs. I-
within each CMC facilitates this phenomenon.20,21 In our 1 was obtained as a yellow solid, and its HR-ESI-MS spectrum
previous study, C. acuminata CMCs were induced for the rst showed a pseudo molecular ion peak at m/z 353.1234 [M + H]+
time. Furthermore, the up-regulation of IPI, G10H, ASA1, TSB, (calcd 353.1231 for C17H21O8), indicating a molecular formula
TDC1, TDC2, and STR, which encode the key enzymes in the of C17H20O8. IR (KBr) vmax 3509, 2934, 1722, 1609, 1383, 1285,
biosynthesis pathway of terpenoid indole alkaloids, resulted in 1076 cmꢁ1. The 13C NMR spectrum showed carbon signals of dC
the higher accumulation of camptothecin (CPT) and 10- 160.3, 158.0, 153.6, 152.0, 123.3, 114.1, 113.8, 111.6, 111.1, 18.2
1
hydroxycamptothecin (HCPT) in C. acuminata CMCs than in C. and 8.3. The H NMR spectrum showed proton signals of dH
acuminata DDCs.22 As a new plant culture, Camptotheca acumi- 7.57 (1H, d, J ¼ 8.3 Hz), 7.15 (1H, d, J ¼ 8.3 Hz) and 6.23 (1H, d, J
nata CMCs have not been studied as a biotransformation ¼ 1.1 Hz). The 13C, 1H NMR and IR spectra indicated compound
system to transform exogenous substrates. Therefore, we aimed I-1 was a coumarin similar with compound I. In addition, the
to determine whether plant stem cells with higher enzymatic additional proton signals of dH 4.96 (1H, d, J ¼ 7.2 Hz, H-10),
activity can be used for biotransformation.
3.20–3.36 (4H, m, H-20-50), 3.69 (1H, d, J ¼ 11.4 Hz, H-60a), and
In the present study, the biotransformation of 4-methyl- 3.48 (1H, m, H-60b) and carbon signals of dC 100.6, 77.2, 76.6,
coumarins by C. acuminata CMCs was reported for the rst 73.3, 69.7, and 60.7 were observed in the 1H NMR and 13C NMR
time. Our aim was to explore the potential of C. acuminata spectra of compound I-1 (Table 2), which suggested the presence
CMCs for biotransformation reactions and develop a new and of a glucopyranosyl moiety in I-1. Comparison of the 1H and 13
C
sustainable system on biotransformation of active molecules by NMR data with those of I revealed that compound I-1 is a gluco-
CMCs of plants. Meanwhile, MAO inhibitory activities of all side of I (Table 2).10 In the HMBC spectrum, the correlation of the
biotransformed products were also evaluated. Furthermore, the anomeric proton signal at d 4.96 (H-10) to the carbon signal at
biotransformation time and amount of substrates were opti- d 158.0 (C-7) indicated that the sugar was linked to C-7. The 13
C
1
mized to increase the biotransformation efficiency.
NMR chemical shi of the anomeric carbon, along with the H
NMR chemical shi and spin–spin coupling constant (J ¼ 7.2 Hz)
of the anomeric proton, allowed for the identication of a b-
glucopyranosyl moiety. Based on these data, the structure of
compound I-1 was identied as 4,8-dimethylcoumarin-7-O-b-D-
Results and discussion
Structures of substrates
4-Methylcoumarins were synthesized by a simple and efficient glucopyranoside, which is a new compound.
protocol shown as Table 1.
Compound II was biotransformed by C. acuminata CMCs to
7-Hydroxy-4,8-dimethylcoumarin (I). White solid, mp 263.8– a new compound, namely compound II-1. Compound II-1 was
265.3 ꢀC. HR-ESI-MS m/z: [M + H]+ 191.0703. 13C NMR (125 obtained as a pale solid, with a molecular formula of C17H20O8
MHz, DMSO-d6) dC: 160.5 (C-2), 111.7 (C-3), 153.8 (C-4), 123.1 (C- based on a pseudo-molecular ion peak at m/z 353.1235 [M + H]+
5), 110.7 (C-6), 158.9 (C-7), 109.9 (C-8), 152.8 (C-9), 112.0 (C-10), (calcd 353.1231 for C17H21O8) in HR-ESI-MS. IR (KBr) vmax 3365,
18.2 (4-CH3), 8.0 (8-CH3).23
2830, 1644, 1624, 1385, 1028 cmꢁ1. The 1H NMR and 13C NMR
6-Hydroxy-4,7-dimethylcoumarin (II). Yellow solid, mp data of compound II-1 exhibited an additional glucopyranosyl
207.7–208.3 ꢀC. HR-ESI-MS m/z: [M + H]+ 191.0705. 13C NMR moiety at dH 4.87 (1H, d, J ¼ 7.4 Hz, H-10), 3.14–3.31 (3H, m, H-
(125 MHz, DMSO-d6) dC: 160.2 (C-2), 113.4 (C-3), 152.0 (C-4), 20-40), 3.44 (2H, m, H-50, 60a), and 3.74 (1H, dd, J ¼ 11.4, 4.1 Hz,
117.8 (C-5), 152.7 (C-6), 130.2 (C-7), 117.9 (C-8), 146.1 (C-9), H-60b) and dC 101.5, 77.4, 76.8, 73.3, 70.2, 61.0. Comparison of
108.3 (C-10), 18.0 (4-CH3), 16.2 (7-CH3).24
the 1H and 13C NMR data with those of II revealed that
6,7-Dihydroxy-4-methylcoumarin (III). Yellow solid, mp compound II-1 is a glucoside of II (Table 2).27 In the HMBC
199.5–201.3 ꢀC. HR-ESI-MS m/z: [M + H]+ 193.0495. 13C NMR spectrum, the correlation of the anomeric proton signal at dH
(125 MHz, DMSO-d6) dC: 160.0 (C-2), 109.6 (C-3), 152.8 (C-4), 4.87 (H-10) to the carbon signal at dC 152.1 (C-6) indicated that
119.8 (C-5), 149.7 (C-6), 146.2 (C-7), 115.6 (C-8), 153.8 (C-9), the sugar was linked to C-6. The b-anomer of the glucose was
114.5 (C-10), 18.1 (4-CH3).25
determined by the anomeric carbon at dC 101.5, anomeric
5-Hydroxy-4,7-dimethylcoumarin (IV). White solid, mp proton at dH 4.87, and coupling constant (7.4 Hz). Therefore, the
+
250.9–252.2 C. HR-ESI-MS, m/z: [M + H] 191.0704. 13C NMR structure of compound II-1 was assigned as 4,7-dimethylcou-
ꢀ
(125 MHz, DMSO-d6) dC: 159.8 (C-2), 107.7 (C-3), 154.6 (C-4), marin-6-O-b-D-glucopyranoside.
9450 | RSC Adv., 2019, 9, 9449–9456
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