Phenylpropanoids from Callus Tissue of Ajuga turkestanica

Article abstract of DOI:10.1007/s10600-019-02608-8

Phenylpropanoids 3,4-dihydroxy-β-phenylethoxy-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-4-O-caffeoyl-β-D-glucopyranoside (1) and 3,4-dihydroxy-β-phenylethoxy-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-4-O-feruloyl-β-D-glucopyronoside (2) were isolated for the first time during studies of the chemical composition of Ajuga turkestanica (Regel) Briq. callus tissue.

Full text of DOI:10.1007/s10600-019-02608-8

DOI 10.1007/s10600-019-02608-8
Chemistry of Natural Compounds, Vol. 55, No. 1, January, 2019
PHENYLPROPANOIDS FROM CALLUS TISSUE
OF Ajuga turkestanica
K. A. Eshbakova,1* R. P. Zakirova, Kh. I. Khasanova,
1
2,4
1
2,3
1
Kh. M. Bobakulov, H. A. Aisa, Sh. Sh. Sagdullaev,
5
and A. M. Nosov
Phenylpropanoids 3,4-dihydroxy-β-phenylethoxy-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranosyl-
(
(
1→3)-4-O-caffeoyl-β-D-glucopyranoside (1) and 3,4-dihydroxy-β-phenylethoxy-O-α-L-arabinopyranosyl-
1→2)-α-L-rhamnopyranosyl-(1→3)-4-O-feruloyl-β-D-glucopyronoside (2) were isolated for the first time
during studies of the chemical composition of Ajuga turkestanica (Regel) Briq. callus tissue.
Keywords: Ajuga turkestanica, callus tissue, phenylpropanoids, glucose, rhamnose, arabinose.
New sources of biologically active compounds must be discovered without damaging natural reserves because of the
rising demand for herbal preparations. Therefore, biotechnological approaches to the production of secondary metabolites
from isolated plant tissues are highly promising [1].
Cell cultures are currently widely used in basic research because biosynthetic processes can be examined without
correlated interactions and monitoring of other tissues and the whole organism. Model systems are developed by characterizing
the physiology of the organ and studying its sensitivity to various hormones.
Ajuga turkestanica (Regel) Briq. is a perennial herbaceous plant of the family Lamiaceae. The species produces
ecdysteroids with broad spectra of pharmacological activity [2] and is endemic to mountains of the Hissar Range.
Previously, A. turkestanica cell culture obtained from plant ovaries was found to biosynthesize in vitro ecdysterone
and turkesterone [3, 4].
The goal of the present work was to study secondary metabolites produced by A. turkestanica callus tissue originating
in leaves, to isolate them pure, to identify them, and to elucidate their structures.
Callus tissue was obtained from leaves of wild A. turkestanica collected in 2014 in Surxondaryo Region near Baysun
village. The culture was grown in medium without added growth regulators and characteristically gave high yields of crude
mass. The growth index in the second year of cultivation was 9.29.
HPLC found that the MeOH extract of biomass from the third year of cultivation contained mainly phenolic compounds
with insignificant contents of ecdysterone and turkesterone. The MeOH extract of A. turkestanica callus tissue afforded several
phenylpropanoid compounds, two of which were phenylpropanoid glycosides lavandulifolioside (1) [5] and leonoside A (2) [6].
1
3
Compound 1 was a dark-yellow resin. Its structure was elucidated by analyzing PMR and C NMR spectra
and HSQC and HMBC experiments. PMR spectra taken in C D N exhibited resonances characteristic of caffeic acid,
5
5
a 3,4-dihydroxyphenylethanoid, and three monosaccharides [5].
1
) S. Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan,
7 M. Ulugbek St., Tashkent, 100170, e-mail: e_komila@yahoo.com; 2) Key Laboratory of Plant Resources and Chemistry of
Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing South Road
0-1, 830011, Urumqi, Xinjiang, P. R. China, e-mail: haji@ms.xjb.ac.cn; 3) Key Laboratory of Xinjiang Indigenous Medicinal
7
4
Plant Resources Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing
South Road 40-1, 830011, Urumqi, P. R. China; 4) University of Chinese Academy of Sciences, 19 Yuquan Road, 100049,
Beijing, P. R. China; 5) K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, 35 Botanicheskaya St.,
Moscow, 127276. Translated from Khimiya Prirodnykh Soedinenii, No. 1, January–February, 2019, pp. 26–28. Original article
submitted July 17, 2018.
2
8
0009-3130/19/5501-0028 ©2019 Springer Science+Business Media, LLC

1
13
TABLE 1. H and C NMR Data of Compounds 1 and 2 (Py-d , δ, ppm, J/Hz)
5
1
2
C atom
δ
Ñ
δ
Í
δ
Ñ
δ
Í
Aglycon
1
2
3
4
5
6
7
8
130.86
117.96
147.65
146.15
117.01
120.94
36.62
–
130.86
117.97
147.66
146.16
117.01
120.94
36.62
–
7.22 (m)
7.22 (m)
–
–
–
–
7.18 (m)
7.18 (m)
6.75 (dd, J = 8.1, 2.0)
2.98 (2Í, t, J = 7.5)
3.91 (dd, J = 16.2, 7.5)
6.76 (dd, J = 8.1, 2.2)
2.99 (2Í, t, J = 7.9)
3.92 (m); 4.31 (m)
71.81
71.83
4
.30 (dd, J = 16.2, 7.5)
Glucose
1
′
′
′
′
′
′
104.76
76.13
80.84
70.62
76.81
62.55
4.81 (d, J = 7.8)
4.02 (dd, J = 8.5, 8.0)
4.47 (m)
104.79
76.15
80.81
70.71
76.83
62.60
4.85 (d, J = 7.8)
4.04 (m)
4.52 (m)
5.72 (t, J = 9.5)
4.03 (m)
4.15 (m); 4.22 (m)
2
3
4
5
6
5.69 (t, J = 9.7)
3.97 (m)
4.11 (m); 4.20 (d, J = 12.1)
Rhamnose
6.41 (s)
4.77 (d, J = 3.2)
1
2
3
4
5
6
′′
′′
′′
′′
′′
′′
102.14
82.47
73.27
74.87
70.49
19.45
102.12
82.48
73.28
74.86
70.48
19.42
6.44 (s)
4.78 (br.s)
4.50 (m)
4.14 (m)
4.43 (m)
4.49 (m)
4.13 (m)
4.40 (m)
1.58 (d, J = 6.1)
1.62 (d, J = 6.1)
Arabinose
5.18 (d, J = 7.5)
4.51 (m)
4.10 (m)
4.23 (m)
3.72 (d, J = 12.0)
.25 (dd, J = 12.0, 2.2)
1
′′′
2′′′
3
′′′
4
′′′
5
′′′
108.01
73.76
75.23
70.17
67.84
108.03
73.75
75.24
70.17
67.87
5.18 (d, J = 5.9)
4.51 (m)
4.10 (m)
4.22 (m)
3.72 (d, J = 12.3)
4.24 (m)
4
Caffeic acid
Ferulic acid
1
2
3
4
5
6
7
8
9
′′′′
′′′′
′′′′
′′′′
′′′′
′′′′
′′′′
′′′′
′′′′
127.44
116.26
148.14
151.06
117.18
122.80
147.22
115.22
167.47
–
–
127.06
112.01
149.42
151.64
117.26
124.29
146.91
115.61
167.40
56.29
–
7.64 (br.s)
7.36 (br.s)
–
–
–
–
7.20 (m)
7.20 (m)
8.03 (d, J = 15.8)
6.73 (d, J = 15.8)
7.18 (m)
7.27 (d, J = 8.1)
8.03 (d, J = 15.8)
6.77 (d, J = 15.8)
–
–
–
3
′′′′-OCH3
3.78 (s)
The aromatic protons of caffeic acid appeared at weak field as a 2H multiplet at 7.20 ppm (H-5″″ and H-6″″); a broad
H singlet at 7.64 ppm (H-2″″); and two olefinic resonances of the acyclic chain as two doublets at 8.03 and 6.73 ppm with
spin–spin coupling constant (SSCC) J = 15.8 Hz. These data were confirmed by resonances in the C NMR spectrum of 1 at
1
1
3
1
47.22 (C-7″″) and 115.22 ppm (C-8″″) (Table 1).
The SSCC (J) of 15.8 Hz indicated that H-7″″ and H-8″″ were positioned trans relative to each other. The PMR
spectrum showed resonances for aromatic protons of the 3,4-dihydroxyphenylethanoid part of the compound as multiplets at
7
.18 (H-5) and 7.22 ppm (H-2) and a doublet of doublets at 6.75 ppm (H-6) with meta- and ortho-constants (J = 2.0, 8.1 Hz).
Furthermore, the spectrum had a 2H triplet at 2.98 ppm (J = 7.5 Hz, H-7) and two doublets of doublets at 3.91 (J = 16.2, 7.5 Hz,
H-8a) and 4.30 ppm (J = 16.2, 7.5 Hz, H-8b) for the ethanoid moiety.
2
9

HO
6'
2
O
O
7
2
''''
O
OH
OH
7
''''
9''''
4'
R
O
1'
8
O
4
6
5''
HO
HO
O
OH
5
''''
CH
3
1''
3''
HO
O
5'''
1: R = OH
O
2: R = OCH3
HO
1'''
3'''
HO
HO
1, 2
Fig. 1. Chemical structures of 1 and 2 and key cross-peaks in an HMBC experiments.
Anomeric protons of the three carbohydrates resonated at 4.81 ppm as a doublet with SSCC J = 7.8 Hz (H-1′), at 6.41
as a singlet (H-1′′), and at 5.18 as a doublet with SSCC J = 7.5 (H-1′′′). The three anomeric protons confirmed that the
compound was a trioside. The other carbohydrate proton resonances were observed at 5.69–3.97 ppm; rhamnose methyl
protons, as a doublet at 1.58 ppm (J = 6.1 Hz).
13C NMR and HSQC spectra of 1 showed resonances for 34 C atoms represented by seven quaternary including one
carbonyl, 22 methine, four methylene, and one methyl C atom.
1
3
The C NMR spectrum at strong field exhibited characteristic resonances for rhamnose methyl at 19.45 ppm and
1
3
a phenylpropanoid methylene. The range 62–82 ppm showed 13 C resonances characteristic of carbohydrates. The three
1
3
anomeric C atoms resonated in the middle of the C NMR spectrum at 102.14 (C-1″), 104.76 (C-1′), and 108.01 ppm (C-1′′′).
1
3
An analysis of the PMR and C NMR spectral data and HSQC and HMBC experiments led to the conclusion that
1
3
compound 1 contained glucose, rhamnose, and arabinose in a 1:1:1 ratio. The aromatic and weak-field parts of the C NMR
spectrum exhibited resonances for phenylethanoid and caffeic acid aromatic C atoms (Table 1).
The positions of the caffeic acid and three carbohydrates in 1 were established using HMBC and chemical shifts of C
atoms. The HMBC spectrum of 1 showed cross peaks H-1′/C-8, H-3′/C-1″, H-4′/C-9″, and H-1′′′/C-2″. Also, H-4′ (5.69 ppm)
1
3
was shifted to weaker field in the PMR spectrum; C-3′ (80.84) and C-2″ (82.47), in the C NMR spectrum. These data
established that glucose was in the phenylpropanoid C-8 position; caffeic acid and rhamnose, on glucose C-4′ and C-3′,
respectively; and arabinose, on rhamnose C-2″.
Acid hydrolysis of 1 produced D-glucose, L-rhamnose, and L-arabinose and mixtures of genin-type products.
The results as a whole gave the structure of 1 as 3,4-dihydroxy-β-phenylethoxy-O-α-L-arabinopyranosyl (1→2)-
α-L-rhamnopyranosyl-(1→3)-4-O-caffeoyl-β-D-glucopyranoside isolated earlier from Stachys lavandulifolia [5] (Fig. 1).
1
13
Compound 2 was identified as leonoside A, the H and C spectral data are given in Table 1 [6].
Phenylpropanoids were previously isolated from seven Ajuga species growing in Japan [6] and from A. turkestanica
for the first time.
EXPERIMENTAL
General Comments. NMR spectra were recorded in C D N on a Varian VNMRS 600 at operating frequency
5
5
1
13
6
00 MHz for H. The internal standard for PMR spectra was TMS (0 ppm); for C NMR spectra, a solvent resonance
(
α-Py-d , 150.35 ppm vs. TMS). UV spectra were obtained on a Shimadzu UV-2550 spectrophotometer. Analytical HPLC
5
used a Dionex Ultimate 3000 instrument with a UV detector and XBridge™ C18 column (4.6 × 250 mm, 5 μm). Preparative
HPLC used a Shimadzu LC-20AT with a UV detector and XBridge™ Prep C18 column (10 × 150 mm, 5 μm). TLC was
performed on Silufol UV 254 chromatographic plates with detection by I vapor, NH vapor, UV light at 254 and 365 nm,
2
3
and vanillin solution (1%) in conc. H SO . Paper chromatography (PC) was carried out on Filtrak No. 11 paper using
2
4
3
0

n-BuOH–AcOH–H O (4:1:5, system 1) and n-BuOH–Py–H O (6:4:3, system 2). Free monosaccharides in PC were detected
2
2
by spraying with aniline hydrogen phthalate.
Callus Tissue Cultivation Conditions. Callus tissue was cultivated at 26°C in the dark in Murashige–Skoog medium
[
7] with added sucrose (30 g/L), meso-inositol (100 mg/L), thiamine HCl (0.4 mg/L), and agar-agar (0.75%) and without
added growth regulator.
Extraction and Isolation of Phenylpropanoid Triglycosides from A. turkestanica Callus Tissue. Air-dried and
milled plant raw material (100 g) was extracted with MeOH (3 × 200 mL) at room temperature. The combined extract was
evaporated in vacuo. The condensed residue (20 g) was chromatographed over a column (1.5 × 150 cm) with ODS (150 g)
using a MeOH–H O gradient (100:1→1:1) to produce fractions A–C that were separated by prep. HPLC using MeOH–H O.
2
2
Compounds 1 and 2 were isolated by prep. HPLC using MeOH (35%)–H O as a dark-yellow amorphous powder,
2
13
C H O (1) and C H O (2). Table 1 lists the PMR and C NMR spectral data (600 MHz).
34
44 19
35 46 19
Acid Hydrolysis. Compound 1 or 2 (10 mg) was hydrolyzed by HCl (5%) in aqueous MeOH (15 mL) for 2 h on
a boiling-water bath. The resulting precipitate of the aglycon was filtered off. The carbohydrate part of the hydrolysate was
neutralized with BaCO and evaporated. PC of the residue using system 2 identified D-glucose, L-rhamnose, and L-arabinose.
3
ACKNOWLEDGMENT
The work was financially supported by the Applied Scientific Research Program of theAS, RUz (Grant FA-A11-T040);
the Foundation for International Science and Technology Collaborative Project Program (Grant No. 2016YFE0120600); and
the Central Asian Center of Drug Discovery and Development, AS, PRC.
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