Chromones and coumarins from Saposhnikovia divaricata (Turcz.) Schischk. Growing in Buryatia and Mongolia and their cytotoxicity

Article abstract of DOI:10.1016/j.jep.2019.112517

Ethnopharmacological relevance: Saposhnikovia divaricata (family Apiaceae) a traditional medicinal plant distributed in many provinces of China, is well known for the pharmaceutical value and has been used for rheumatic arthritis, and anxiety in children. Antiviral, antioxidant and antiproliferative activities were also mentioned. The application of this plant are recorded in the Chinese Medicine (CM) classical text the Shen Nong's Materia Medica (Shen Nong Ben Cao Jing). In this monograph S. divaricata (syn Radix Ledebouriella divaricata) is graded as a premium-grade herb, with their broad-spectrum of therapeutic applications for the treatment of cough, common cold, arthralgia, as well as in rheumatic disorders. Aim of the study: To isolate and identify chemical constituents (chromones and coumarins) from S. divaricata, collected in Buryatia and Mongolia and to study their in vitro anticancer activity against MEL-8, U-937, DU-145, MDA-MB-231 and ВТ-474 cell lines. Materials and methods: An 40% aqueous ethanol extract of the roots of S. divaricata was prepared and further successively fractionated by extraction with petroleum ether, diethyl ether, tert-butyl methyl ether and ethyl acetate. The obtained extracts were subjected to a series of chromatographic separations on silica gel for isolation of individual compounds. Isolated compounds were tested for their cytotoxicity with respect to model cancer cell lines using the conventional MTT assays. Results: Total of 15 individual compounds: coumarins scopoletin 2, bergapten 3, isoimperatorin 4, marmesin 5, (+)-decursinol 9, (?)-praeruptorin B 10, oxypeucedanin hydrate 11, chromones: hamaudol 6, cimifugin 7, 5-О-methylvisamminol 8, chromone glycosides: prim-O-glucosylcimifugin 12, sec-O-glucosylhamaudol 13, 4′-O-β-D-glucopyranosyl-5-О-methylvisamminol 14, 4′-O-β-D-glucopyranosylvisamminol (15) and also polyyne compound panaxinol 1 were isolated and characterized. The structure of dihydropyranocoumarin 10 was confirmed by X-ray diffraction analyses. HPLC-UV method was used for determination of the content of most abundant chromones 7, 12 and 14 in the roots of S. divaricata, collected in Mongolia. Compounds 3–11 and 13, 14 were evaluated for their cytotoxicity with respect to model cancer cell lines. All the compounds were non-toxic in the hemolysis test. Conclusion: This report about the phytochemical profiles of S. divaricata growing in Mongolia and Buryatia led to the identification of 14 compounds including coumarins and chromones. The available coumarins and chromones may serve as new leads for the discovery of anticancer drugs.

Full text of DOI:10.1016/j.jep.2019.112517

Journal Pre-proof
Chromones and coumarins from Saposhnikovia divaricata (Turcz.) Schischk. Growing
in Buryatia and Mongolia and their cytotoxicity
Bayarma M. Urbagarova, Elvira E. Shults, Vasilii V. Taraskin, Larisa D. Radnaeva,
Tatyana N. Petrova, Tatyana V. Rybalova, Tatyana S. Frolova, Andrey G. Pokrovskii,
Jamsranjav Ganbaatar
PII:
S0378-8741(19)33777-8
https://doi.org/10.1016/j.jep.2019.112517
JEP 112517
DOI:
Reference:
To appear in: Journal of Ethnopharmacology
Received Date: 22 September 2019
Revised Date: 15 December 2019
Accepted Date: 24 December 2019
Please cite this article as: Urbagarova, B.M., Shults, E.E., Taraskin, V.V., Radnaeva, L.D., Petrova,
T.N., Rybalova, T.V., Frolova, T.S., Pokrovskii, A.G., Ganbaatar, J., Chromones and coumarins from
Saposhnikovia divaricata (Turcz.) Schischk. Growing in Buryatia and Mongolia and their cytotoxicity,
Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2019.112517.
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O
O
O
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CH3
anticancer
compounds
O
O
H3C
HO
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CH3
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Saposhnikovia divaricata (Turcz.) Schischk.

Chromones and coumarins from Saposhnikovia divaricata (Turcz.) Schischk.
growing in Buryatia and Mongolia and their cytotoxicity
Bayarma M. Urbagarova a,b, Elvira E. Shults c*, Vasilii V. Taraskin b, Larisa D. Radnaeva a,b
,
Tatyana N. Petrova c, Tatyana V. Rybalova c,d, Tatyana S. Frolova d,e, Andrey G. Pokrovskiid,
Jamsranjav Ganbaatar f
a Banzarov Buryat State University, 24a Smolina St., Ulan-Ude, 670000, (3012)448255;
b Baikal Institute of Nature Management, Siberian Branch, Russian Academy of Sciences, 6 Sakhyanovoi St., Ulan-
Ude, 670047, (3012)434997;
c
Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Acad.
Lavrentyev Ave. 9, 630090 Novosibirsk, Russia; e-mail: schultz@nioch.nsc.ru;
Novosibirsk State University, Pirogova Str. 1, 630090 Novosibirsk, Russia;
d
e
FRC Institute of Cytology and Genetics, Acad. Lavrentyev Ave. 10, 630090 Novosibirsk, Russia;
f Institute of Chemistry and Chemical Technology, Mongolian Academy of Sciences, Ulan-Bator
Ethnopharmacological relevance: Saposhnikovia divaricata (family Apiaceae) a traditional
medicinal plant distributed in many provinces of China, is well known for the
pharmaceutical value and has been used for rheumatic arthritis, and anxiety in children.
Antiviral, antioxidant and antiproliferative activities were also mentioned. The application
of this plant are recorded in the Chinese Medicine (CM) classical text the Shen Nong’s
Materia Medica (Shen Nong Ben Cao Jing). In this monograph S. divaricata (syn Radix
Ledebouriella divaricata) is graded as a premium-grade herb, with their broad-spectrum of
therapeutic applications for the treatment of cough, common cold, arthralgia, as well as in
rheumatic disorders.
Aim of the study: To isolate and identify chemical constituents (chromones and coumarins)
from S. divaricata, collected in Buryatia and Mongolia and to study their in vitro anticancer
activity against MEL-8, U-937, DU-145, MDA-MB-231 and ВТ-474 cell lines.
Materials and methods: An 40% aqueous ethanol extract of the roots of S. divaricata was
prepared and further successively fractionated by extraction with petroleum ether, diethyl
ether, tert-butyl methyl ether and ethyl acetate. The obtained extracts were subjected to a
series of chromatographic separations on silica gel for isolation of individual compounds.
Isolated compounds were tested for their cytotoxicity with respect to model cancer cell
lines using the conventional MTT assays. Results: Total of 15 individual compounds:
coumarins scopoletin 2, bergapten 3, isoimperatorin 4, marmesin 5, (+)-decursinol 9, (-)-
praeruptorin B 10, oxypeucedanin hydrate 11, chromones: hamaudol 6, cimifugin 7, 5-О-
methylvisamminol 8, chromone glycosides: prim-O-glucosylcimifugin 12, sec-O-
glucosylhamaudol 13, 4'-O-β-D-glucopyranosyl-5-О-methylvisamminol 14, 4'-O-β-D-
glucopyranosylvisamminol (15) and also polyyne compound panaxinol 1 were isolated and
characterized. The structure of dihydropyranocoumarin 10 was confirmed by X-ray
diffraction analyses. HPLC-UV method was used for determination of the content of most
abundant chromones 7, 12 and 14 in the roots of S. divaricata, collected in Mongolia.
Compounds 3-11 and 13, 14 were evaluated for their cytotoxicity with respect to model
cancer cell lines. All the compounds were non-toxic in the hemolysis test. Conclusion: This
report about the phytochemical profiles of S. divaricata growing in Mongolia and Buryatia
led to the identification of 14 compounds including coumarins and chromones. The
available coumarins and chromones may serve as new leads for the discovery of anticancer
drugs.
Keywords: Saposhnikovia divaricata, chromones, coumarins, pyranocoumarins, liquid
chromatography, cytotoxicity

1. Introduction
Saposhnikovia divaricata (Turcz) Schischk. (Apiaceae) is widely found in the northern and
northeastern territories of China and cultivated in many other provinces. As a traditional Chinese
medicines the dried root of S. divaricata (named “Fang-feng”) and ethanol extract are widely
used in the clinic, and about 8% of prescriptions in the Chinese Pharmacopoeia contain RS (the
dried root of S. divaricata). Saposhnikovia divaricata has been ranked as a top-grade herb by
Pharmacopoeia of People’s Republic of China (2015) (Li et al, 2002). The dried root and
rhizoma of S. divaricata has been used as a crude drug in several formulae in Japanese
traditional medicine (Kampo medicine) (Sasaki et al., 1982). In recent years, the
pharmacological activity and chemical constituents of RS have been extensively studied by
researchers worldwide (Kreiner et al., 2017). Anti-inflammatory, analgesic, antipyretic,
antiallergic, antioxidant, antitumoral and anti-viral activities have been shown on different
Saposhnikovia extracts in previous studies (Wang et al., 1991; Wang et al., 1999; Okuyama et
al., 2001; Kuo et al., 2002; Tal and Cheung, 2007; Khan et al., 2013; Kong et al., 2013; Salman
and Shik, 2013; Yua et al., 2015; Chun et al., 2016; Yang et al., 2015). The study of the
chemical composition of Saposhnikovia divaricata led to the release of chromones, coumarins,
unsaturated fatty acids, volatile oils, and polyacetylene compounds (Sasaki et al., 1982; Baba et
al., 1987; Okuyama et al., 2001; Kang et al., 2008; Yu et al., 2009; Gui et al., 2011; Kim et al.,
2011; Yang et al., 2015; Chen et al., 2018). Recently, polysaccharides have been recognized as
immune-regulatory and antineoplastic components in RS (the dried root of S. divaricata) (Yang
et al., 2016).
In Russian Federation S. divaricata is distributed in Eastern Siberia (Republic of Buryatia,
Trans-Baikal Territory) and in the Far East (Pimenov and Ostroumova, 2012). Chemical
investigation of this plants growing in Mongolia and Eastern Siberia was very limited and
considered only with analyzes of the lipid fraction (Urbagarova et al., 2017).
For isolation of the active compounds several pretreatment strategies have been investigated
Currently, pretreatment methods such as stem, ammonia, CO2, hot water extraction, etc. ( Zhao
et al., 2016, 2017; X. Qiao et al., 2018) are applied. For isolation of the active compounds from
the roots of S. divaricata we applied the organosolvent pretreatment using with solvents of
increasing polarity: the the typical advantages of this process was the fact that the the organic
solvents could be easy to be recycled by distillation.
The aim of this study was to examine the constituents of extractive compounds, especially
coumarins and chromones of the root of S. divaricata collected in Buryatia (Mukhorshibirsky
District, Gashey village, 2015) and Mongolia (Khentii Province, Bayan Uul village, 2015).
Additionally, we also analyzed the content of the most abundant chromones in populations of S.
divaricata growing in Mongolia (three Samples, Khentii Province, Bayan Uul village, 2015;
Khentii Province, Berkh village, 2014 and Khentii Province, Berkh village, 2013). One of the
objectives of the present paper was to investigate the cytotoxic activity of the accessible
coumarins and chromones from S. divaricata.
2. Materials and methods
2.1. Plant material
The roots of Saposhnikovia divaricata were collected from Republic of Buryatia,
Mukhorshibirsky District, Gashey village, Russia (51004′05.5"N 107037′04.9"E), on the 24th
July 2015, and from Khentii Province, Bayan Uul village, Mongolia (47021′94.0"N
109009′37.4"E), on the 30th July 2015. The plants were identified by Dr. Oleg Anenkhonov.
Voucher specimens No. 018404 and 018405 are deposited at the UUH – Herbarium of the

Institute of General and Experimental Biology SB RAS, Ulan-Ude, Buryatia, Russian
Federation).
2.2. Chemicals and reagents
Extractive compounds were isolated by column chromatography on silica gel (0.063-0.200
mm, Acros or 60-0.120 mm, Merck KGaA, Darmstadt, Germany) and eluated with chloroform,
chloroform-ethanol (v/v=100:0; to 0:l); chloroform-methanol (v/v=80:0; to 1:10); benzene-EA
(v/v=10:1; to 1:10), petroleum ether-acetone (v/v=5:1; to 0:1). Preparative TLC was carried out
on glass plates (30×30 cm with 2 mm thick sorbent layer; silica gel F254 35- 70µm, Across-
Organics) or on Silica gel 60 F254 plates (Qingdao Marine Chemical, Inc.). The purity of
separation was monitored by TLC on Silufol UV254 plates using various solvent systems, and
spots were visualized by detection under UV light or by treatment with iodine vapor. The purity
of the desired compounds was determined by HPLC using an Agilent 1100 series HPLC
instrument (Agilent, Waldbronn, Germany). The solvents benzene (C6H6), petroleum ether (PE),
ether, t-butylmethyl ether (МТBE), chloroform (CHCl3), methanol (MeOH), ethyl acetate
(EtOAc, EA) and dioxane were purified according to standard methods and distilled immediately
before use. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], were obtained
from Sigma-Aldrich Chem. Co. (St. Louis, MO, USA).
2.3. General methods
13
NMR spectra were acquired on Bruker AV-400 (1H: 400.13 MHz, C: 100.78 MHz), Bruker DRX
500 (1H: 500.13 MHz, 13C: 125.76 MHz) or Bruker AV-600 (1H: 600.30 MHz, 13C: 150.95 MHz) in
CDCl3 or a mixture of CDCl3-CD3OD using tetramethylsilane (TMS) as an internal standard. The 1H and
13C NMR spectra were assigned using two dimensional spectroscopy techniques; COSY
(correlated spectroscopy), HSQC (heteronuclear single-quantum correlation spectroscopy) and
HMBC (heteronuclear multiple-bond correlation spectroscopy). IR spectra were recorded by
means of the KBr pellet technique on a Bruker Vector-22 spectrometer. UV spectra were
obtained on an HP 8453 UV-Vis spectrometer in EtOH solutions (c 10-4 mol/l). Elemental
analysis was carried out on a Carlo-Erba 1106 CHN-analyzer. Mass spectra were recorded on a
Thermo Scientific DFS high-resolution mass spectrometer (evaporator temperature 150-220oC,
EI ionization at 70 eV). Melting points were determined on the Mettler Toledo FP900 thermo
system (USA). The specific rotation values [α]D were determined on a PolAAr 3005 polarimeter
(Germany), and expressed in (deg·ml)/(g·dm), while concentration was expressed in g per 100
ml of solution. X-ray structural study of compound 17 was performed on a Bruker KAPPA
APEX II diffractometer with a two-dimensional CCD detector (MoKα radiation with graphite
monochromator, ω-φ-scanning).
2.4. Producing areas, preparation of extracts and isolation of compounds
Two Samples (I) – collected in Buryatia (Mukhorshibirsky District, Gashey village, 2015),
and II – collected in Mongolia (Khentii Province, Bayan Uul village, 2015) were studied. The
plant materials (250 g) were air-dried, powdered and successively extracted with 95% ethanol
(4×500 mL, with heating on a water bath at 45oC for 6 h), respectively. The combined extract
was concentrated under reduced pressure to the crude extracts. Those extracts I and II were
suspended in distilled H2O (150 mL) and then partitioned sequentially with equal volume of PE
(3×50 mL), ether (3×50 mL), МТB (3×50 mL) and EA (3×50 mL). Evaporation of the obtained
organic extracts I and II in vacuo gave 8 soluble fractions, the yield of which is presented in
Table 1.
Table 1. Yield of extracted Substances from Saposhnikovae divaricata (%)

Solvent for extraction or
distribution
Sample
I
II
Ethanol
14.4
18.0
Distribution of ethanol extract in organic solvent-water
PE
Ether
(MTBE)
0.52 (1.3 г)
0.85 (2.12 г)
0.48 (1.2 г)
0.64 (1.6 г)
1.25 (3.12 г)
1.18 (2.95 г)
(EA)
0.89 (2.22 г)
1.46 (3.65 г)
Separation of ether extract. The ether extract of the Sample I (2.12 g) was subjected to silica
gel column chromatography (0.063-0.200 mm, 4×330 cm, 72 g) eluted with CHCl3-EtOH
(v/v=100:0, 50:1, 20:1, 10:1, 5:1) to afford fractions A–E. Chromatography of Fraction A (340
mg) afforded 63 mg (3% w/w) of panaxinol 1. Fraction B (660 mg) was subjected to silica gel
column chromatography (200–300 mm, eluent CHCl3-EtOH) to afforded 4 subfractions.
Scopoletin (2) (5% w/w, 108 mg) and compound 1 (7% w/w, 127 mg) were isolated from
subfractions 1,2. Chromatography of residue of combined fraction 1 and subfractions 3-4 (560
mg) (benzene-EA, 10:0.5→1:10), produced five subfractions, gave compound 1 (7.8% w/w, 164
mg), bergapten (3) (0.95% w/w, 20 mg), and isoimperatorin (4) (0.8%, 17 mg). Repeat
chromatography of fractions C (400 mg), D (415 mg) and E (180 mg) (CHCl3-EtOH,
100:0.5→1:1), produced five subfractions, which after NMR analysis were crystallized from a
mixture of ether-EA, 1:1. Additionally, compounds 2 (2.6%, 40 мг), 3 (0.95%, 20 mg), and also
marmesin (5) (1.2%, 25 mg), hamaudol (6) (3%, 64 мг), cimifugin (7) (1%, 21 мг) and and 5-О-
methylvissaminol (8) (0.8%, 17 mg) were isolated.
By the chromatography of ether extract of Sample II (3.12 g) compounds 1 (13%, 400 mg),
2 (2.8%, 92 mg), 3 (1%, 32 mg), 4 (1.5%, 48 mg), 5 (1.6%, 53 mg), 6 (1.9%, 60 mg), 7 (4.5%,
140 mg) and 8 (1.8%, 56 mg) were isolated. So, a characteristic future of ether extract was the
polyacetylene compound 1.
Separation of ТBМE extract. The ТBМE extract of the Sample 1 (1.2 g) was subjected to
silica gel column chromatography (0.063-0.200 mm, 2×330 cm, 40 g) eluted with CHCl3-EtOH
(v/v=100:1→50:1, 20:1, 10:1, and 2:1) to afford fractions A–D. Repeat chromatography of
fraction A (320 mg) (benzene: EA, 10:0.5→10:1) gave 4 subfractions. Compounds 5 (2.6%, 31
mg), 6 (2%, 25 mg) and (+)-decursinol (9) (1.8%, 22 mg) were isolated by TLC plate
chromatography of the first subfraction. After repeated chromatography of fraction B (450 mg)
(benzene:EA, 10:0.5→10:1) compounds 4 (2%, 27 mg), 6 (6%, 73 mg), 7 (1,2%, 15 мг) and (-)-
preruptorin B (10) (3.4%, 41 mg) were obtained. Fraction C (140 mg) was purified repeatedly by
silica gel column chromatography (eluted with a gradient of CHCl3-EtOH (100:1, 80:1, 50:1,
10:1, v/v) produced four subfractions, which finally, after trituration with ether gave compounds
6 (2%, 26 mg), 8 (2.4%, 30 mg), oxypeucedanin hydrate (11) (1%, 15 mg), and prim-O-
glucosylcimifugin (12) (1%, 12 mg) and sec-O-glucopyranosylhamaudol (13) (2%, 24 mg),.
Column chromatography of fraction D (230 mg) (eluted with a gradient of CHCl3-EtOH (100:1,
80:1, 50:1, 10:1, 5:1) afforded 5 subfractions; after trituration with ether compounds 8 (4.8%, 60
mg), 10 (3.3%, 40 mg), 11 (7 mg), 12 (2%, 24 mg) and 4'-O-β-D-glucopyranosyl-5-О-
methylvisamminol (14) (0.9%, 11 mg) were isolated. Repeatedly chromatography from the
residues of all MTBE fractions (~0.4 g) on silica gel column (eluted with a gradient of CHCl3–
EtOH (80:1, 50:1, 10:1, 5:1, 0:1), afforded 5 subfractions, from which compounds 8 (3.5%, 42
mg), 12 (3%, 36 mg), 13 (2%, 24 mg), 14 (3.6%, 44 mg) and 4'-O-β-D-
glucopyranosylvisamminol (15) (3%, 36 мг) were obtained.
By the chromatography of TMBE extract of Sample II (2.95 g) compounds 4 (4%, 118 mg), 5
(3.5%, 103 mg), 6 (5%, 147 mg), 7 (6%, 178 mg), 8 (4.5%, 131,5 mg), 9 (4.2%, 124 mg), 10

(2.8%, 86 mg), 11 (2.1%, 62 mg), 12 (6%, 176 mg), 13 (3%, 88 mg), 14 (8%, 236 mg) and 15
(5%, 147 mg) were isolated.
Separation of EA extract. The EA soluble fraction of the Sample 1 (2.22 g) was subjected to
silica gel column chromatography, eluted with CHCl3-EtOH (v/v=100:0, 50:1, 10:1, 5:1, and
1:10) to give five fractions (A: 260 mg, B: 180 mg, C: 750 mg, D: 640 mg, E: 310 mg). Column
chromatography on silica gel of combined fraction A and B (440 mg) (eluent CHCl3-EtOH) gave
additionally compounds 4 (1.3%, 31 mg), 6 (1.0%, 22 mg), 9 (1.2%, 28 mg), 10 (1.8%, 40 mg),
and 11 (1.4%, 34 mg). By column chromatography on silica gel of fraction C (750 mg) (eluted
with a gradient of CHCl3–EtOH (80:1, 50:1, 10:1, 5:1, 1:5) followed by TLC plates purification
of all 5 subfractions compounds 6 (1.5%, 33 mg), 7 (0.8%, 18 mg), 12 (4%, 88 mg), 13 (1.0%,
22 mg), and 14 (2%, 44 mg) were isolated. Column chromatography on silica gel of fraction D
(640 mg) (eluted with a gradient of CHCl3–EtOH (80:1, 60:1, 40:1, 30:1, 20:1, 10:1, 7:1, 5:1,
1:5) gave 9 subfractions, triturated of which with ether afforded compounds 5 (1%, 23 mg), 8
(0.5%, 11 mg), 9 (0.9%, 18 mg), 10 (2%, 44 mg), 11 (1%, 22 mg), 12 (1.5%, 33 mg), 13 (3%, 66
mg), 14 (2%, 45 mg), 15 (2.5%, 55 mg). Further separation of the combined residues from
fraction D and fraction E (610 mg) (eluted with a gradient of CHCl3–MeOH (100:1, 80:1, 50:1,
10:1, 5:1, 1:5) and triturated of the obtained 6 subfractions with ether gave additionally 5 (1.1%,
25 mg), 6 (1%, 22 mg), 8 (0.5%, 123 mg), 12 (2%, 44 mg), 13 (1%, 21 mg), 14 (1%, 22 mg), and
15 (2%, 45 mg).
By a series of column and TLC chromatography of EA extract of Sample II (3.65 g)
compounds 4 (0.5%, 18 mg), 5 (0.8%, 29 mg), 6 (6%, 219 mg), 7 (2.5%, 91 mg), 8 (1.9%, 69
mg), 9 (2.8%, 102 mg), 10 (2.2%, 80 mg), 11 (1.7%, 62 mg), 12 (8%, 292 mg), 13 (1.8%, 66
mg), 14 (4.5%, 164 mg) and 15 (3.3%, 133 mg) were isolated. Collected fractions were checked
by TLC (and 1H NMR) and similar secondary metabolites containing ones were combined.
2.5. Structure determination of isolated compounds
Compounds 1 (panaxynol), 3 (bergapten), 4 (isoimperatorin) and 5 (marmesin) were
identical with the authentic samples (Ganbaatar et al., 2013).
Scopoletin (2), colorless needles; m.p. 201-203°С (EtOH); [m.p. 202-204°С (aq. MeOH)];
1H NMR (CDCl3, 400 MHz), δ: 7.68 (1H, d, J=9.6 Hz, Н-4), 7.11 (1Н, s, Н-8), 7.05 (1Н, s, Н-
5), 6.30 (1H, d, J=9.6 Hz, Н-3), 3.78 (3Н, s, СН3О); 13C NMR (CDCl3, 100 MHz), δ: 161.48 (C-
2, C), 154.12 (C-7, C), 143.87 (C-6, C), 144.03 (C-4, CH), 116.21 (C-4а, C), 112.14 (C-3, CH),
110.82 (C-8a, C), 109.34 (C-5, CH), 104.3 (С-8, CH), 56.48 (СН3О); HRMS m/z 193.0499, [M+]
(calcd for С10Н9O4, 193.0501).
Hamaudol (6). Yellowish needles; m.p. 200-203 °С (ЕtOH); [α]D -18.6 (c 0.32; CHCl3)
[m.p. 202-202.5 °С (ЕtOH); [α]D -22 (c 0.46; CHCl3) Sasaki et al., 1982]; 1H NMR (CDCl3, 600
MHz), δ: 6.31 (1H, s, H-8), 5.98 (1H, br s, H-3), 3.87 (1H, t, J=5.6 Hz, H-3′), 2.98 (1H, dd,
J=16.8 and 5.6 Hz, H-4′), 2.73 (1H, dd, J =16.8 and 5.6 Hz, H-4′), 2.32 (3H, br s, 2-CH3), 1.39
13
(3H, s, 2′-CH3), 1.35 (3H, s, 2′-CH3); C NMR (CDCl3, 150 MHz), δ: 182.76 (C-4, C), 167.03
(C-2, C), 160.08 (C-5, C), 159.98 (C-7, C), 156.29 (C-8a, C), 108.62 (C-3, CH), 105.37 (C-4a,
C), 103.55 (C-6, C), 94.51 (C-8, CH), 78.45 (C-2′, C), 69.02 (C-3′, CH), 27.12 (C-4′, CH2),
24.95 (C-2′-CH3), 22.08 (C-2′-CH3), 20.59 (2-CH3); HRMS m/z 277.1069, [M+] (calcd for
С15Н17O5, 277.1076).
Cimifugin (7). Colorless needles; m.p. 98-100°С (ЕtOH); [α]D +81.5 (c 0.85; EtOH) [m.p.
102-102.5 °С (ЕtOH); [α]D +79.2 (c 0.8; CHCl3) Kondo et al., 1972]; UV λmax (EtOH) nm: 230
(3.98), 245 (sh, 3.27), 250 (4.15), 292 (4.08); IR υmax(KBr) cm-1: 3360 (OH), 1656 (C=O), 1620,
1580, 1500 (C=C); 1H NMR (CDCl3+CD3OD, 600 MHz), δ: 6.44 (1H, s, H-8), 6.14 (1H, s, H-3),
4.61 (1H, dd, J=9.2 and 8.0 Hz, H-2′), 4.32 (2H, s, CH2OH), 3.78 (3H, s, OCH3), 3.22 (1H, dd,
J=16.1 and 9.2 Hz, H-3′), 3.35 (1H, dd, J=16.1 and 8.0 Hz, H-3′), 1.13 (3H, s, 4′-CH3), 1.10 (3H,
s, 4′-CH3); 13C NMR (CDCl3+CD3OD, 150 MHz), δ: 177.24 (C-4), 167.55 (C-2, C), 162.21 (C-
5, С), 160.45 (C-7, C), 156.37 (C-8a, C), 117.75 (C-6, C), 112.07 (C-4a, C), 109.01 (C-3, CH),

94.28 (C-8, CH), 91.91 (C-2′, CH), 71.10 (C-4′, C), 60.93 (OCH3), 60.81 (CH2OH), 28.38 (C-3′,
CH2), 25.01 (CH3), 25.08 (CH3); HRMS m/z 306.1118, [M+] (calcd for С16Н18O6, 306.1102).
5-О-Methylvissamminol (8). Colorless needles; m.p. 138-140°C (PE-ЭА, 5:1, v/v); [α]D
+79.2 (c 0.8; EtOH) [m.p. 141-142 °С; [α]D +91.8 (CHCl3) Sasaki et al., 1982]; UV λmax (EtOH)
nm: 233 (3.36), 248 (4.09), 256 (sh, 4.17), 289 (3.76); IR υmax(KBr) cm-1: 3365 (OH), 1658
1
(C=O), 1626, 1605, 1595 (C=C). H NMR (CDCl3+CD3OD, 600 MHz), δ: 6.51 (1H, s, H-8),
5.95 (1H, br s, H-3), 4.70 (1H, dd, J=9.0 and 8.8 Hz, H-2′), 3.95 (3H, s, OCH3), 3.28-3.42 (2H,
m, H-3′), 1.33 (3H, s, 4′-CH3), 2.25 (3H, br s, 2-CH3), 1.22 (3H, s, 4′-CH3); 13C NMR
(CDCl3+CD3OD, 150 MHz), δ: 177.65 (C-4, C), 163.61 (C-2, C), 165.75 (C-5, C), 160.11 (C-7,
C), 156.48 (C-8a, C), 119.08 (C-6, C), 112.52 (C-4a, C), 111.38 (C-3, CH), 93.81 (C-8, CH),
92.58 (C-2′, CH), 70.56 (C-4′, C), 60.75 (OCH3),29.02 (C-3′, CH2), 26.16 (CH3), 25.54 (CH3),
22.11 (CH3); HRMS m/z 290.1142, [M+] (calcd for С16Н18O5, 290.1154).
(+)-Decursinol (9). Colorless needles; m.p. 96-99°C (ether); [α]D +16.9 (c 1.4; CHCl3) {[α]D
+10.8 (c 1.0, CHCl3) Lemmich et al., 1969}; 1H NMR (CDCl3, 500 MHz), δ: 7.51 (1H, d, J=9.6
Hz, Н-4), 7.11 (1H, br s, Н-5), 6.72 (1H, s, Н-10), 6.16 (1H, d, J=9.6 Hz, Н-3), 3.80 (1H, dd,
J=5.7 and 4.8 Hz, Н-7), 3.09 (1H, ddd, J =16.8, 4.8 and 1.6 Hz, Н-6), 2.82 (1H, ddd, J=16.8, 5.7
13
and 1.2 Hz, Н-6), 2.05 (1H, br s, OH), 1.35 (3H, s, CH3), 1.31 (3H, s, CH3); C NMR (CDCl3,
125 MHz), δ: 161.89 (C-2, C), 156.96 (C-9a, C), 154.49 (C-10a, C), 143.56 (C-4, CH), 129.19
(C-5), 116.81 (C-5a, C), 113.52 (C-10, CH), 113.36 (C-4a, C), 105.16 (C-3, CH), 79.23 (C-8, C),
69.32 (C-7, CH), 31.08 (C-6, CH2), 25.62 (CH3), 22.41 (CH3); HRMS m/z 246.2249, [M+] (calcd
for С14Н14O4, 246.2253).
(-)-Praeruptorin B (10), Colorless orthorhombic crystals; m.p. 138-140°C (EtOH); [α]D
-
23.1 (c 0.4; CHCl3); 1H NMR (CDCl3, 600 MHz), δ: 7.58 (1H, d, J=9.6 Hz, H-4), 7.36 (1H, d, J =
8.4 Hz, H-5), 6.80 (1H, d, J=8.4 Hz, H-6), 6.69 (1H, d, J=4.8 Hz, H-10), 6.21 (1H, д, J = 9.6 Hz,
H-3), 6.11 (1H, br d, J = 7.0 Hz, H-6''), 6.02 (1H, br d, J = 6.8 Hz, H-6'), 5.44 (1H, d, J=4.8 Hz,
H-9), 1.98 (3H, d, J=7.0 Гц, H-7''), 1.95 (3H, d, J=6.8 Hz, H-7'), 1.82 (3H, br s, H-5''), 1.84 (3H,
br s, H-5'), 1.48 (3H, s, CH3-C-8), 1.44 (3H, s, CH3-C-8); 13C NMR (CDCl3, 150 MHz), δ:
166.45 (C-2', C), 166.22 (C-2'', C), 159.75 (C-2, C), 156.65 (C-6a, C), 154.3 (C-10b, C), 143.15
(C-4, CH), 138.45 (C-6'', CH), 139.88 (C-6', CH), 129.14 (C-5, CH), 127.29 (C-4', C), 127.00
(C-4'', C), 114.32 (C-6, CH), 113.23 (C-3, CH), 112.42 (C-4a, C), 107.49 (C-10a, C), 77.41 (C-8,
C), 70.09 (C-9, CH), 60.95 (C-10, CH), 25.34 (C8-CH3), 22.47 (C8-CH3), 20.39 (C-3', CH3),
20.31 (C-3'', CH3), 15.75 (C-7', CH3), 15.56 (C7'', CH3); HRMS m/z 426.3241, [M+] (calcd for
С24Н26O7, 426.3256).
Oxypeucedanin hydrate (11). Yellowish needles; m.p. 130-133°С (ЕtOH); [α]D +21.5 (c
0.81; CHCl3); [m.p. 130.5-131oC; [α]D +13 (c 0.9, acetone) Nielsen, Lemmich, 1964]; 1H NMR
(CDCl3, 600 MHz), δ: 8.18 (1H, d, J = 9.8 Hz, H-5), 7.61 (1H, br d, J = 2.5 Hz, H-2), 7.12 (1H,
br s, H-9), 6.99 (1H, dd, J=1.2 and 2.5 Hz, H-3), 6.28 (1H, d, J = 9.8 Hz, H-6), 4.56 (1H, dd, J =
3.2, 10.2 Hz, H-2'), 4.42 (1H, dd, J = 7.8, 10.2 Hz, H-2'), 3.89 (1H, dd, J=7.8, 3.2 Hz, H-3'), 1.37
(3H, s, CH3), 1.31 (3H, s, CH3); 13C NMR (CDCl3, 150 MHz), δ:161.28 (C-7, C), 158.43 (C-9а,
C), 152.66 (C-8а, C), 148.71 (C-4, C), 145.26 (C-2, CH), 139.16 (C-5, CH), 114.54 (C-3а, C),
113.18 (C-6, CH), 107.16 (C-4а, C), 104.85 (C-3, CH), 95.11 (C-9, CH), 79.08 (C-4', C), 76.17
(C-3', CH), 74.31 (C-2', CH2), 26.62 (CH3), 25.12 (CH3); EI-MS m/z: 304.1038 (M+) (calcd for
С16Н16O6, 304.1025).
prim-O-Glucopyranosylcimifugin (12). Colorless amorphous solid; [α]D +10.2° (c 0.86;
EtOH); [m.p. 141-142 °С; {[α]D +12.6 (c 1.03; MeOH) Sasaki et al., 1982}; UV λmax (EtOH)
nm: 231 (4.06), 246 (3.91), 254 (4.11), 291 (3.81); IR υmax(KBr) cm-1: 3520, 3456, 3358, 3340
(OH), 1658 (C=O), 1620, 1605, 1585 (C=C); 1H NMR (CDCl3+CD3OD, 600 MHz), δ: 6.51 (lH,
s, H-8), 6.15 (lH, br s, H-3), 4.92 (1H, d, J = 8.0 Hz, H-1-Glc), 4.71 (1H, dd, J=9.5 and 8.0 Hz,
H-2′), 3.08-3.29 (2H, m, H-3′), 3.30-3.87 (6H, m, Glc), 3.98 (3H, с, OСН3), 4.62 (2H, уш.с, 2-

CH2), 1.43 (3H, s, CH3), 1.36 (3H, s, CH3); 13C NMR (CDCl3+CD3OD, 150 MHz), δ: 180.05 (C-
4), 165.25 (C-5, C), 162.68 (C-2, С), 161.77 (C-7, C), 157.34 (C-8a), 119.34 (C-6), 112.67 (C-
4a), 111.57 (C-3), 104.31 (Glc-1′′), 95.08 (C-8), 93.15 (C-2′), 78.51 (Glc-3′′), 78.16 (Glc-5′′),
75.45 (C-4′), 72.61 (Glc-2′′), 71.91 (Glc-4′′), 63.82 (Glc-6′′), 60.96 (5-OCH3), 30.62 (C-3′),
25.61 (CH3-C-4′), 25.28 (CH3-С-4′). Found, %: C, 56.25; H, 6.18 (calcd for C22Н28O11, С, 56.41;
H, 6.02; %).
sec-O-Glucopyranosylhamaudol (13). Yellow needles; m.p. 225-228°C (ether); [α]D -32.8 (c
1
0.82, EtOH); [m.p. 229-230 °С; [α]D -48.5 (c 0.68; MeOH) Sasaki et al., 1982]; H NMR
(CDCl3+CD3OD, 500 MHz), δ: 6.38 (1H, s, H-8), 6.02 (1H, уш.с, H-3), 5.02 (1Н, d, J = 7.4 Hz,
H-1′′), 3.95 (1H, dd, J = 7.2 and 6.2 Hz, H-6′′), 3.88 (1H, m, H-2′′), 3.75 (1Н, dd, J=6.2 and 5.0
Hz, H-3′), 3.68 (1H, m, H-5′′), 3.58 (1H, dd, J=7.2 and 5.2 Hz, H-6′′), 3.28-3.44 (2H, m, H-
3′′,4′′), 3.02 (1H, dd, J =16.9 and 5.0 Hz, H-4′), 2.68 (1H, dd, J =16.9 and 6.2 Hz, H-4′), 2.28
13
(3H, br s, 2-CH3), 1.48 (3H, s, 2′-CH3), 1.41 (3H, s, 2′-CH3). C NMR (CDCl3+CD3OD, 150
MHz), δ: 184.43 (C-4, C), 165.54 (C-2, C), 161.41 (C-5, C), 160.23 (C-7, C), 158.11 (C-8a, C),
116.56 (C-6, C), 110.82 (C-4a, C), 109.58 (C-3, CH), 101.12 (Glc-1′′, CH), 96.16 (C-8, CH),
78.51 (C-2′, C), 78.26 (Glc-3′′, CH), 77.98 (Glc-5′′, CH), 75.29 (Glc-2′′, CH), 73.11 (C-4′, CH2),
71.86 (Glc-4′′, CH), 62.86 (Glc-6′′, CH2), 27.54 (C-3′, CH), 22.76 (CH3), 22.32 (CH3), 20.72
(CH3 при С-2); Found, %: C, 57.88; H, 6.11 (calcd for C21Н26O10, С, 57.93; H, 5.98; %).
4'-O-β-D-Glucopyranosyl-5-О-methylvisamminol (14), Colorless needles; m.p. 140-143°C
(ether); [α]D +78.5 (c 0.92; EtOH) [m.p. 149-151°С (water); [α]D +88.2 (c 1.66; MeOH) Sasaki
1
et al., 1982]; H NMR (CDCl3+CD3OD, 500 MHz), δ: 6.62 (1H, s, H-8), 6.07 (1H, br s, H-3),
5.04 (1Н, d, J=7.8 Hz, H-1′′), 4.70 (1H, dd, J=9.2 and 8.4 Hz, H-2′), 3.95 (3H, c, OCH3), 3.81-
3.95 (2H, m, H-2′′, 6′′), 3.58-3.72 (2H, m, H-5′′,6′′), 3.20-3.50 (4H, m, 2-H-3′, H-3′′, H-4′′), 2.16
13
(3H, br s, 2-CH3), 1.50 (3H, s, CH3), 1.41 (3H, s, CH3); C NMR (CDCl3+CD3OD, 125 MHz),
δ: 176.38 (C-4, C), 164.48 (C-5, C), 163.96 (C-2, C), 160.09 (C-7, C), 157.67 (C-8a, C), 117.21
(C-6, CH), 111.35 (C-4a, C), 109.84 (C-3, CH), 100.08 (Glc-1′′, CH), 95.24 (C-8, CH), 88.95
(C-2′, C), 78.59 (Glc-3′′, CH), 78.16 (Glc-5′′, CH), 74.56 (Glc-2′′, CH), 74.28 (C-4′, CH2), 71.42
(Glc-4′′, CH), 63.09 (Glc-6′′, CH2), 28.26 (C-3′, CH), 24.14 (CH3), 23.46 (CH3), 20.66 (CH3 при
С-2); Found, %: C, 58.25; H, 6.18 (calcd for C22Н28O10, С, 58.40; H, 6.24; %).
4'-O-β-D-Glucopyranosylvisamminol (15). Colorless needles, m.p. 136-139°C (ether); [α]D
+42.4 (c 0.38; EtOH); 1H NMR (CDCl3+CD3OD, 500 MHz), δ: 6.41 (1H, s, H-8), 6.01 (1H, br s,
H-3), 5.09 (1Н, d, J=7.8 Hz, H-1′′), 4.85 (1H, dd, J=9.4 and 8.2 Hz, H-2′), 3.82-4.01 (2H, m, H-
2′′, 6′′), 3.56-3.76 (2H, m, H-5′′,6′′), 3.18-3.50 (4H, m, 2-H-3′, H-3′′, H-4′′), 2.18 (3H, br s, 2-
CH2), 1.48 (3H, s, CH3), 1.40 (3H, s, CH3); 13C NMR (CDCl3+CD3OD, 125 MHz), δ: 176.87 (C-
4, C), 165.45 (C-5, C), 163.29 (C-2, C), 159.87 (C-7, C), 156.43 (C-8a, C), 118.68 (C-6, CH),
111.89 (C-3, CH), 112.29 (C-4a, C), 98.92 (Glc-1′′, CH), 94.84 (C-8, CH), 89.92 (C-2′, C), 78.77
(Glc-3′′, CH), 77.09 (Glc-5′′, CH), 77.25 (C-4′, CH2), 75.11 (Glc-2′′, CH), 71.32 (Glc-4′′, CH),
62.38 (Glc-6′′, CH2), 24.33 (C-3′, CH), 23.42 (CH3), 22.34 (CH3), 21.38 (CH3 при С-2); Found,
%: C, 57.38; H, 6.25 (calcd for C21Н26O10, С, 57.53; H, 5.98; %).
2.6. X-Ray analysis and identification of the absolute configuration of coumarin 10.
Suitable crystals of compound 10 were grown from EtOH. Crystallographic data for 10:
C24H26O7, orthorhombic, P212121, a 10.6252(9), b 14.287(1), c 15.187(2) Å, V 2305.3(3) Å, Z 4,
dcalcd 1.229 g·cm3, µ(Mo-Kα) 0.09 mm–1, (1.00 × 0.25 × 0.17) mm3, transmission 0.7681 –
0.8620. Refinement parameters wR2 = 0.1206, S = 1.025 for all 4093 reflections (R = 0.0423 for
3119 observed reflections F > 4σ). Reflection intensities were corrected for absorption by
SADABS program (Sheldrick, 1997). The positions of the hydrogen were calculated
geometrically and refined in riding model.
Crystallographic data for the structure have been deposited at the Cambridge
Crystallographic Data Centre as supplementary publication no. CCDC 1889707. Copy of the
data can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge

CB21EZ, UK (fax: +44 122 3336033 or e-mail: deposit@ccdc.cam.ac.uk; internet:
www.ccdc.cam.ac.uk).
Hydrolysis of (-)-praeruptorin B 10 in basic medium. A stirred solution of 10 (240 mg,
0.684 mmol) in dioxane (20 mL) was treated with KOH solution (40 mL, 0.5 N, 20.0 mmol),
heated at 60°C for 2 h, cooled, treated with H2SO4 (13 mL, 10%) until the pH was 4, and
extracted with CHCl3 (4 × 10 mL). The extract was dried over MgSO4, the solvent was filtered
and removed in vacuo to produce an oily residue that, according to analytical TLC (eluent
hexane:AcOEt, 1:1 v/v), contained a mixture of angelic acid and cis- and trans-khellactones (Rf
0.8, 0.6, and 0.5, respectively). This residue was dissolved in CHCl3 (3 mL) and separated by
TLC plates (eluent hexane:AcOEt (1:2 v/v). Angelic acid 16 (40 mg) and a mixture of cis- 17
and trans-khellactones 18 (140 mg, 81%) (the cis/trans ratio according of NMR spectra was
1.5:1) were obtained. A solution (80 mg) of lactones 17, 18 in CHCl3 (0.5 mL) was subjected to
preparative TLC on Sorbfil UV 254 plates (10 × 20 cm) with elution by hexane:AcOEt (1:1 v/v).
Trans-18 and cis- 17 khellactones, respectively, were collected by elution from the sorbent with
EtOAc. Solvent was removed in vacuo to produce the crystalline compounds
.
1
Angelic acid (16): mp 42-44°C [m.p. 42-44oC (Fraser, 1960)]; H NMR (CDCl3+CD3OD,
500 MHz), δ: 6.20 (кв.кв, 1H, q q, J = 7.4 и J = 1.4, C3H), 2.01 (3H, d q, J =7.4 and 1.4, C4H3),
1.88 (3Н, quintet, J = 1.4, C2-CH3).
о
о
(-)-cis-Khellactone (17): m.p. 171-173 С (ether), [α]D -78.6 (с 0.9, СНСl3) [m.p. 165 С
(benzene), [α]D = -70.6 (с 0.98, СНСl3), Reisch and Voerste, 1994]. 1H NMR (CDCl3, 600 MHz),
δ: 7.62 (1H, d, J=9.4 Hz, H-4), 7.36 (1H, d, J = 8.0 Hz, H-5), 6.80 (1H, d, J=8.0 Hz, H-6), 6.24
(1H, д, J = 9.4 Hz, H-3), 5.25 (1H, dd, J=5.0 и 4.8 Hz, H-10), 4.44 (1H, d, J=4.8 Hz, 10-ОH),
3.91 (1H, dd, J=5.0 and 5.8 Hz, H-9), 3.30 (1H, br d, J=5.8 Hz, 9-OH), 1.46 (3H, s, CH3-C-8),
1.42 (3H, s, CH3-C-8); 13C NMR (CDCl3, 150 MHz), δ: 160.68 (C-2, C), 156.45 (C-6a, C),
154.76 (C-10b, C), 143.98 (C-4, CH), 129.14 (C-5, CH), 114.32 (C-6, CH), 113.23 (C-3, CH),
112.42 (C-4a, C), 107.49 (C-10a, C), 79.11 (C-8, C), 70.87 (C-9, CH), 61.22 (C-10, CH), 24.82
(C8-CH3), 21.87 (C8-CH3).
(+)-trans-Khellactone (18): m.p. 180-182 оС (ether), [α]D + 17.2 (с 0.4, СНСl3) {[α]D + 18.1
(с 0.6, СНСl3), Duh et al., 1991}.
2.7. HLPC method for the determination of cimifugin (7), prim-O-glucopyranosylcimifugin
(12) and 4'-O-β-D-glucopyranosyl-5-О-methylvisamminol (14)
The standard of chromones 12 and 14 were purchased from ANPEL Laboratory
Technologies Inc. (Shanghai, China). The content of the basic substance is ≥ 99.9%. The standard
of cimifugin 7 (95.00%) (C) was obtained in the laboratory of medical chemistry at the N.N. Vorozhtsov
Novosibirsk Institute of Organic Chemistry SB RAS. Standard solutions with a concentration of 0.2
mg.mL-1 were prepared by dissolving an exact weighted portion in methanol. Three Samples (II),
(III) and (IV) collected in Mongolian Khentii Province, (Bayan Uul village, 2015, II and Berkh
village, 2013, III and 2014, IV) were analyzed for the determination of compounds 12, 7 and 14.
Each Sample was extracted with 50% aq. MeOH solution by ultrasonication (USDNA Ultrasonic
Cleaner) at room temperature for 30 min, after cooling. The extraction step was repeated and the
extracted solution was combined and filtrated through analytical filter paper. The filtrate was
evaporated to dryness using a rotary evaporator at 40 °C and the dried extract was dissolved in
5.0 mL of acetonitrile. The sample was finally filtrated through a 0.45 µm membrane filter and
then injected into the HPLC system.
HPLC was performed using a Milichrom A-02 high-performance liquid chromatograph
(Econova, Novosibirsk), consisting of a vacuum degasser, binary pump, an autosampler and a
UV spectrometric detector at wavelengths of 210-300 nm. A ProntoSIL-120-5-C-18AQ column
(75×2 mm, 5 µm). The isocratic mobile phase consisted of 4M solution of LiClO4–acetonitrile
(60:40, v/v) was used to elute for 40 min., the flow rate was kept at 1.0 mL/min. Column
temperature was kept constant at 40 °C. UV detection: 210-300 nm.

The identification of compounds was carried out by analysis of the absorption spectra of
standard solution and test Samples, by the retention volume (Vr), and by spectral ration at seven
wavelengths (Sx/S210). The homogeneity of the chromatographic peak was estimated by the
spectral angle between the multidimensional vectors the coordinates of which respond to the
spectral rations of chromones 7, 12 or 14 to the Sample (in degrees). The quantitative content of
chromones was calculated by peak areas using the Alpha Spectrum program.
The data acquired from the representative chromatographic profiles of Samples (II), (III),
(IV) were given in Supp. part (Fig. S1, S2, S3). Chromones 7, 12 and 14 were unambiguously
verified with the chemical reference Standards (SI, Fig. S4-S12).
2.8. Cell culture and determination of cytotoxicity
The human cancer cells of the MEL-8 (melanoma cell lines), U-937 (human monocytes),
prostate cancer cell line (DU-145) and human breast cancer cell lines (BT-474, MDA-MB-231)
were used in this study. The cells were cultured in the RPMI-1640 medium that contained 10%
embryonic calf serum, L-glutamine (2 mmol/L), gentamicin (80 lg/ml), and lincomycin (30
mg/ml) in a CO2 incubator at 37oC. The tested compounds were dissolved in DMSO and added
to the cellular culture at the required concentrations. Three wells were used for each
concentration. The cells which were incubated without the compounds were used as a control.
Cells were placed on 96-well microliter plates and cultivated at 37 oС C in 5% CO2/95% air for
72 h. The cell viability was assessed through an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-phenyl-
2H-tetrazolium bromide] conversion assay. 1% MTT was added to each well. Four hours later
DMSO was added and mixed for 15 min. The 50% growth inhibition dose (GI50) was determined
using the conventional MTT assay, which allows to estimate the number of survived cells
spectrophotometrically (Wilson et al., 1990). Optical density (D) of the samples was measured
on a BioRad 680 multi-well spectrophotometer (USA) at the wavelength of 450 nm. The 50%
cytotoxic dose GI50) of each compound (i.e., the compound concentration that causes the death
of 50% of cells in a culture, or decreases the optical density twice as compared to the control
wells) was calculated from the data obtained. The experiments were repeated in triplicate.
2.9. Hemolysis Test
To evaluate the toxicity of test compound the hemolysis test was performed. Human
erythrocytes were obtained from fresh citrated blood by centrifugation at 2500 g for 10 min.;
washed three times with PBS and resuspended in the same solution. Aliquots of the cell
suspension with the respective red blood cell number of 107 were added to the PBS containing
different concentrations of the compounds in the range of 10-100 uM. After mixing them gently,
each probe was incubated at 37 ºC for 10 min and then centrifuged at 5000 g for 10 min. Finally,
the absorbance of the hemoglobin released into the supernatant was measured at 540 nm with a
Varian Cary-300 (Agilent). The percentage of hemolysis was expressed as the ratio of
hemoglobin in the supernatant of the sample solutions related to the hemoglobin concentration
after the complete hemolysis of erythrocytes. The dose-response curve was determined using
different concentration of SDS as detergent (positive control). Two negative controls were used:
PBS for SDS dose-response curve and 1% of DMSO in PBS (DMSO as a solvent). As a result
the concentration inducing hemolysis in 50% of the erythrocyte population (HC50) was
subsequently calculated. Experiment was conducted independently three times in triplicate.
Statistical analysis
Statistical processing of the results was performed using the Microsoft Excel-2007,
STATISTICA 6.0, and GraphPad Prism 5.0 software. The results are given as an average value ±
a deviation from the average (mean±standard error of the mean (SEM)). Reliability of
differences (p) was estimated using the Student t test. The differences with p < 0.05 were
considered as reliable.

3. Results and discussion
The plant material was extracted using the solvents listed in Table 1. The components
extracted by ethanol were distributed in PE-H2O, ether-H2O, MTBE-H2O and EA-H2O, which
allow to separate the reserve sugars and also isolate the glycoside containing compounds (Shults
et al., 2017). Table 1 shows the yield of the total extracted substances from Samples (I) and (II).
The PE extract (1.3 and 1.6 g for Samples I and II respectively) was separated by column
chromatography. The main component of those extracts was the polyacetylenic compound
panaxinol 1 (Fig. 1). In addition, free sterols, their esters and fatty acids described previously
were identified in those extracts (Urbagarova et al., 2017). The overall isolated yield of
compound 1 of PE and ether fractions from Samples I and II was 0.21 and 0.29% respectivelly.
The ether, TBME and EA extracts were subjected to a series of chromatographic separations
on silica gel. The known compounds are coumarins: scopoletin (2) (isolated yield was 0.06 and
0.04% from Samples I and II respectively), bergapten (3) (isolated yield was 0.016 and 0.012%
from Samples I and II respectively), isoimperatorin (4) (isolated yield was 0.03 and 0.07% from
Samples I and II), marmesin (nodakenitin) (5) (isolated yield was 0.04 and 0.07% from Samples
I and II), (+)-decursinol (9) (isolated yield was 0.03 and 0.1% from Samples I and II), (-)-
praeruptorin B (10) (isolated yield was 0.07% from Samples I and II), oxypeucedanin hydrate
(11) (isolated yield was 0.03 and 0.05% from Samples I and II), and chromones hamaudol (6)
(isolated yield was 0.11 and 0.17% from Samples I and II), cimifugin (7) (isolated yield was 0.02
and 0.16% from Samples I and II), 5-О-methylvisamminol (8) (isolated yield was 0.08 and
0.09% from Samples I and II), chromone glucosides: prim-O-glucopyransylcimifugin (12)
(isolated yield was 0.09 and 0.19 % from Samples I and II), sec-O-glucopuranosylhamaudol (13)
(isolated yield was 0.05 and 0.06 % from Samples I and II), 4'-O-β-D-glucopyranosyl-5-О-
methylvisamminol (14) (isolated yield was 0.07 and 0.16 % from Samples I and II), 4'-O-β-D-
glucopyranosylvisamminol (15) (isolated yield was 0.05 and 0.11 % from Samples I and II) were
isolated (Fig. 1). It should be noted that gradient extraction made it possible to concentrate
coumarin 11 and chromones 7, 12, 13, 14 and 15 in MTBE and EA extracts from the plant roots
and chromone 8 in ether and MTBE extract. Samples from Buryatia and Mongolia are differ in
content of chromones hamaudol 6 and cimifugin 7. The isolated yield of coumarins 9 and
chromones 6, 7, 12, 14 and 15 of Mongolian sample II was significantly higher (0.09-0.19%)
than of plants from the Buryatian Sample I (0.02-0.09%). The Buryatian population was
characterized by low contents of cimifugin 7.
It is well known, that chromone glycosides 12 and 14 are the two most abundant chromones in S.
divaricata, and the total amounts of these two compounds are used as the chemical index for
evaluating the quality of Radix Saposhnikovia in the Chinese Pharmacopoeia. The content of
compounds 12 and 14 in raw materials should be at least 0.24% (Chinese Pharmacopoeia, 2015).
Liquid chromatography (LC) analysis is an important technique for the identification and
analysis of herbal components (Kim at al., 2011; Ou-Yang et al., 2014; Chen at al., 2018; Yi et
al., 2018; Vestena et al., 2019). The HPLC-UV method was used for the determination of
mentioned components 12 and 14 and also cimifugin 7 in three populations of S. divaricata,
collected in Mongolia (Samples II, III and IV). The chromatographic profile of Samples II, III
and IV was shown in Fig. S1, S2 and S3). All results of injection precision, repeatability and
stability test indicated that this method was adequate, valid and applicable. It could be seen that
some differences were found in the quantitative results. The content of chromone 12 in Samples
II and III ranged from 0.55 to 0.74% (1.23±0.12 – 1.89±0.19 mg.g-1), and the content of
compound 14 was 0.55% (1.98±0.15 mg.g-1) in both Samples. Control Sample purchased in
China was characterized by lower contents of glycosides 12 and 14 (0.27 and 0.19%
respectively). Previously, results indicated that the specimen’s origin played an important role in
the content diversity of S. divaricata. The content of both chromone glycosides was in the range
from 0.31 ± 0.011 to 0.56 ± 0.021 % determined by HPLC (Scherübl at al., 2013). The Samples
collected in the Yunnan province have the lowest total content of chromones (0.23%); the
northeast and north regions were found to be the best sourcing locations (total content 0.98%)

(Liao et al., 2014). Additionally, we isolated furocoumarins and pyranocoumarins of linear 3, 4,
5, 9, 11 and angular 10 structure. Characteristically, the isolated yield of (+)-decursinol 9 was
about 0.1% from the Mongolian population. The availability of this compound is important in
terms of its neuroprotective activity (Skalicka-Woźniak et al., 2016). The
dihydropyranocoumarin (-)-praeruptorin 10 are of interest for obtaining anticancer drugs (Chen
et al., 2017).
From MBTE and EA extracts we isolated the angular dihydropyranocoumarin (-)-
praeruptorin B (10). Structure of this compound was elucidated on the basis of detailed
spectroscopic analyses. The small difference of the chemical shifts for the geminal dimethyl
groups in the 1H NMR spectrum (0.02 ppm) and the small vicinal coupling constant between H-9
– H-10 (J = 4.8 Hz) (Song et al., 2012) argued in favor of the cis-configuration of the angeloyl
groups. The compound (+)-praeruptorin B [anomalin, (9R,10R)-enantiomer] has been reported to
be a crystalline compound isolated from Ligusticum elatum (Padha et al., 1995). We were able to
produce a crystalline sample of compound 10 (Fig. 2). The analysis of molecular geometry was
performed using PLATON program (Spek, 2003). The bicyclic coumarin nucleus is highly
planar (accuracy of ±0.030(3)Å). The fused dihydropyran ring has a distorted half-chair
conformation: while the C10 atom lies in the plane of the coumarin fragment, and the O7 atom
deviates slightly from it (0.040(3) Å), the C8 and C9 atoms go to 0.412(3) and -0.295(3) Å. The
substituent in positions С9 and С10 are in cis-orientation to each other, torsion angle
O1'C9C10O1" is 43.3(2)°. The geometry of the two angeloyloxy substitutes is different, as
indicated by their torsion angles. So, at position C9 interplanar angle between the fragments
С9О1'C2'O3' and С2'C4'(C5')C6'C7' is 27.7°, while for the substituent in C10 the same angle is
44.8°. These variations in the substituent geometry seems to be due to intramolecular hydrogen
bonds (Suppl., Table S1). The crystal structure of compound 10 is characterized by week
intramolecular hydrogen bonds С-Н…О, the main bifurcate С4-Н…O3'(О3'') with distances of
2.61(2.62) Å (the sum of van der Waals radii of 2.68 Å) forming molecular chains along the b
axis can be distinguished.
The absolute configurations of this type of coumarin compounds was usually determined by
acidic or alkaline hydrolysis in combination of gel column chromatographic separation By
alkaline hydrolysis of compound 10 three compounds: angelic acid (16), (+)-cis- (17) and (+)-
trans- (18) khellactones were isolated (Scheme 1). Obviously, the formation of (+)-trans-
khellactone (18) appeared as the result of epimerization of the chiral atom C-10 (Kong et al.,
1996).

OCH3
H3CO
HO
4
9
5
3
6
7
2
O
O
O
O
O
2
3
1
OH
5'
OH
O
RO
4'
4
HO
O
O
O
5
3
O
2'
2
CH3
O
O
5
6
R = H
O
O
O
13 R = Glc
4
H3CO
O
HO
4
6
4
5
5
3'
H3CO
7
3
2
4'
3
O
6
2'
RO
8
7
2
10
O
O
O
O
5
6
4
CH3
O
3'
2'
4'
3
2
HO
9
O
O
8
R=H;
14 R = Glc
OR
HO 6"
7
R = H
5''
12 R = Glc
Glc =
O
4"
OH
1''
2"
3''
HO
4
5
6
3
OH
2
OH
6a
O
O
O
10a
HO
7
8
4'
3'
10
3''
9
O
2'
2''
O
7''
CH3
O
O
OH
O
4''
O
2'
6''
H35C''
3'
4
4
3'
2'
5
6
5
8
3
5'
3
4'
4'
GlcO
CH3
2
2
H
7
O
CH3
H37C'
9
O
O
O
O
6'
H
15
10
11
Fig. 1. Structures of isolated compounds 1–15.
Fig. 2. The molecular structure of (9S,10S)-dihydro-8,8-dimethyl)-2-oxo-2H,8H-benzo
[1,2-b:3,4-b ]dipyran-9,10-diyl) 2-methyl-2butanoate (10). The thermal ellipsoids are drawn
at the 30% probability level.
′

Fig. 3. Compounds isolated by alkaline hydrolysis of dihydropyranocoumarin 10.
So, we describe the isolation of the angular dihydropyranocoumarin (-)-praeruptorin B (10)
from extracts of S. divaricata and obtaining of (-)-cis-khellactone (17) - a perspective compound
for constructing anticancer and anti-HIV agents (Chen et al., 2017).
The coumarins 4, 5 (Yim et al., 2005; Ganbaatar et al., 2013; Lee et al., 2017), 10 (Liang et
al., 2010) and several chromones (L. Zhang et al., 2017; Ma S.-Y. et al., 2018; Duan Y.-D. et al.,
2019) have shown promising anticancer properties against a series of cancer cells (e.g. leukemia,
prostate, breast, or lung, among others), by inhibiting the growth and proliferation of such cells
as well as inducing apoptosis. The cytotoxic activity of the isolated compounds 3-11 and 13, 14
was evaluated against a panel of five different human cancer cell lines using conventional MTT
assay. The cytotoxicity was determined by measuring the concentration inhibiting human tumor
cell viability by 50% (GI50). Doxorubicin and Cisplatin were used as the reference drugs in this
study. The hemolytic activity for all tested compounds has been also investigated. The results are
presented in Table 2.
Table 2. Cytotoxicity of isolated coumarins 3-5, 9-11, and chromones 6, 7, 8, 13, 14.
Compo-
und
HC50,
uM
Cytotoxicity (GI50, µM) against cell lines[a,b]
MEL-8
U-937
DU-145
ВТ-474
MDA-MB-231
>100
>100
>100
>100
>100
>100
26.45±2.08
28.32±2.31
21.32±1.23
36.68±3.12
40.46±2.36
35.68±2.38
38.46±1.38
13.12±1.06
15.20±1.08
12.35±2.08
89.06±8.22
34.58±1.62
37.55±4.06
20.56±2.43
27.82±1.86
29.34±1.68
28.24±4.72
9.18±0.49
6.46±0.87
17.43±1.08
19.16±1.83
28.17±3.06
68.72±3.97
10.41±0.86
8.46±0.65
3
4
5
6
7
8
20.12±2.06
16.48±0.86
35.64±8.52
29.40±2.35
54.21±4.38
>100
10.54±1.08
36.17±1.82
9.42±0.37
51.32±3.45
42.24±1.38
9
>100
>100
>100
>100
NT
39.26±3.12
25.16±2.38
52.56±2.43
60.14±4.64
5.11±0.56
38.81±3.57
22.45±2.78
87.17±0.86
41.18±2.66
0.20±0.06
10.02±0.48
11.56±0.65
56.82±1.96
55.64±3.16
15.28±0.78
16.56±1.34
59.58±0.82
28.91±4.08
2.63±0.78
8.76±0.58
24.62±1.65
56.31±1.64
39.81±4.18
7.91±0.54
10
11
13
14
]
19.82±[c
Doxorubicin
[a] GI50: concentration at which 50% growth inhibition of tumor cells is observed after 72 h incubation.
[b] The experiments were repeated in triplicate.
[c] The cytotoxicity of anticancer drug Cisplatin (Mozad et al., 2017);
NT – not tested
From the results the following points can still be concluded. All the tested compounds were
non toxic for human erythrocytes (HC50 >100 uM). It is noteworthy to mention that linear
furocoumarins 3, 4, 5, 10 and 11 exhibited cytotoxic activities in respect to model cancer cell
lines and compounds 3, 5, 11 were characterized by selective cytotoxicity towards prostate

cancer cells DU-145 and were more potent than Cisplatin. Linear pyranocoumarin 9 possessed
selective inhibition for melanoma cancer cell lines, and this compound also inhibits the grown of
DU-145 cell lines in micromolar concentration and was two time more potent than Cisplatin.
Angular dihydropyranocoumarin 10 exhibits cytotoxicity against DU-145 and MDA-MB-231
cells. The cytotoxicity of coumarin 10 on this cancer breast cell lines was comparable to those of
standard drug Doxorubicin. Among the isolated chromanes cimifugin 7 and 5-О-
methylvisamminol 8 shown selectivity on the human breast cancer cells ВТ-474 and MDA-MB-
231; the activity of chromone 8 was comparable to those of Doxorubicin. The chromone
glycosides 13 and 14 had weak cytotoxicity in compared with compound 6, 8. The latter effect is
unsurprising and is in consent with the literature data (Ma S.-Y. et al., 2018) for various
chromone glycosides. The obtained data revealed that structural modification of natural
chromones on the hydroxyl function can be highly perceptible for improve the cytotoxic profile
of the compounds.
4. Conclusion
In the present study, we have isolated and structurally characterized seven coumarins and seven
chromones from Saposhnikovia divaricata growing in Mongolia and Buryatia. To the best of our
knowledge, chemical constituents from the Mongolian and Buryatian populations of S.
divaricata is reported for the first time. The structure of angular dihydropyranocoumarin (-)-
praeruptopin B (10) was confirmed by the X-ray data. The roots of S. divaricata growing in
Mongolia could be characterized by the significant contents of conventional marker compounds
of this species prim-O-glucopyranosylcimifugin (12) and 4'-O-β-D-glucopyranosyl-5-О-
methylvisamminol (14) (0.50-0.74% by HPLC-UV method). Of interest is also the high contents
and the easiness of isolation of linear pyranocoumarin (+)-decursinol (9), angular
dihydropyranocoumarin (-)-praeruptopin B (10) and chromenes hamaudol (6) and cimifugin (7)
in Mongolian populations of S. divaricata. The in vitro cytotoxicity assay observed that
coumarins 3, 4, 5, 9 and 11 exhibited cytotoxic activity in respect to prostate cancer cell lines
DU-154 and were more potent than the standart drug Cisplatin. The linear
dihydropyranocoumarin 9 shown good activity against melanoma cells. The most active
chromone 5-О-methylvisamminol 8 demonstrated selectivity towards breast cancer cells and its
activity towards . Results showed that the chromone 8 could be readily isolated from the ether
and MTBE extracts. Further studies are needed to understand the mechanism of anticancer
activity of several natural compounds. In addition, the study of cytotoxicity towards the cancer
cells provided cytotoxic lead compounds of chromone and coumarin nature for structural
modification and biological study.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Authors’ contribution
Elvira E. Shults, participated in research design, conducting the experiments, data analyses and
drafting the manuscript. Bayarma M. Urbagarova, Vasilii V. Taraskin, Larisa D. Radnava
and Jamsranjav Ganbaatar participated in research design, interpretation of data and revising
drafts of the manuscript. Vasilii V. Taraskin and Larisa D. Radnava supervised the whole study.
Andrey G. Pokrovsky and Tatyana S. Frolova obtained the cytotoxicity data and also contributed
through design and preparation of the manuscript. Elvira E. Shults and Tatyana N. Petrova
participated in the separation of extractive compounds and its identifications. Tatyana V.
Rybalova obtained the X-ray data. HPLC analysis part of the study were applied by Bayarma M.
Urbagarova and Vasilii V. Taraskin. All authors read and approved the final manuscript.

Acknowledgment
The authors are grateful to Dr. Oleg A. Anenkhonov, Institute of General and Experimental
Biology SB RAS, Ulan-Ude, Republic of Buryatia, Russia, for the help in plant identification and and
geographical consultations; also our thank goes to Dr. Victor V. Chepinoga, V.B. Sochava Institute of
Geography SB RAS, Irkutsk, Russia, for the photo of Saposhnikovia divaricata.
Authors would like to acknowledge the Multi-Access Chemical Service Center SB RAS for spectral
and analytical measurements.
This work was performed under partial financial support from the Russian Foundation for Basic
Research (Projects No 17-33-50176 and 19-53-44003) and Project № МК-501.2019.4 of the RF
President Grant for government support of young Russian scientists
.
Appendix A. Supplementary data
Supplementary material to this article can be found online at https://
doi.org/10.1016/j.jep.2019…
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Coumarins and chromones from Saposhnikovia divaricata growing in Mongolia and
Buryatia.
Structure and absolute configuration of dihydropyranocoumarin (-)-praeruptopin B.
Cytotoxic activities of isolated coumarins and chromones against human cancer cell lines.