Introduction
Chronic osteomyelitis causes damage to bone tissue and bone marrow and results in
significant morbidity and mortality. It was revealed in vitro that quercetin derivatives and other flavonoids effectively inhibit osteoclast proliferation
and alkaline phosphatase activity and stimulate osteoblast proliferation [1]
[2].
Saussurea controversa DC. (Asteraceae) is a plant popularly used among the people of Siberia for the treatment
of purulent wounds and diseases of the musculoskeletal system. Extracts of S. controversa exhibit anti-inflammatory and immunomodulatory activity in experimental osteomyelitis
[3]
[4]. The anti-inflammatory [5]
[6] and immunomodulatory [7] activity of the extracts may be due to phenolic compounds, particularly flavonoids,
which are described in several studies.
It is of interest to investigate flavonol glycosides from S. controversa as potential sources of its activity in cases of osteomyelitis. This study describes
the isolation of five glycosides of quercetin (1-5) from S. controversa leaves, the determination of their structures, and their effect on bone marrow and
bone tissue in experimental osteomyelitis.
Results and Discussion
The aqueous ethanol extract from S. controversa dry leaves (0.6 kg) was evaporated in a vacuum to remove ethanol completely, and
the residue was extracted successively with chloroform, ethyl acetate, and butanol.
When the butanol was removed, a yellow amorphous powder easily precipitated and was
separated by decantation and dried (butanol precipitate). After separating the butanol
precipitate on silica gel, we sequentially isolated quercetin-7-О-α-L-rhamnoside-3-О-β-D-glucoside (1), quercetin-3-О-β-D-diglucoside-О-α-L-rhamnoside (2), and quercetin-3-rutinoside (3). The following rechromatography on microcrystalline cellulose resulted in the isolation
of quercetin-7-О-α-L-rhamnoside-3-О-β-D-xyloside (4), and quercetin-7-О-β-D-glucoside-3-О-α-L-rhamnoside-О-β-D-glucoside (5) ([Fig. 1]). The proportional relationship of flavonoids in the butanol precipitate by weight
was 2 (1): 8 (2): 10 (3): 1 (4): 4 (5).
Fig. 1 The structure of flavonoids 1-5 from S. controversa.
The structures of aglycones and sugar residues in the compounds were determined by
comparing their spectroscopic data with published values [8]
[9]
[10]. The data for 13C NMR spectra of aglycones in all obtained substances were consistent with data previously
described for quercetin. Proton and carbon spectra of compounds 1-3 and 5 contain signals characteristic for glucose and rhamnose. In compounds 3 and 5, signals in the 13C NMR spectrum at 104.74, 104.67, and 102.05 (3), and at 105.09, 105.03, and 102.41 (5), as well as the number of protons in the 1H NMR spectrum and data for 1H-1H COSY 2D NMR, indicate the presence of two residues of glucose and one of rhamnose.
In compounds 1 and 5, signals in the 13C NMR spectrum at δ 68.20 (1) and δ 68.16, 68.19 (5) as well as a doublet of anomeric protons from glucose in the 1H NMR spectrum at δ 5.00 (J=7.5 Hz; 1 and 5) and a singlet from rhamnose at δ 4.55 (1) indicate the terminal position of these sugars. At the same time, the signal of
H-1 of a rhamnosyl moiety at δ 4.51 (5) and the interaction of the signal with H-2 of the pyranosyl moiety indicates the
position between C-3 of the aglycone and a molecule of glucose. In addition, the 1H-1H COSY of 5 shows the interaction of the proton of rhamnose at C-6 and protons of the phenol
nucleus at C-2, 5, and 6. The key COSY 1H-1H correlations in the spectra of new compound 5 are given in Fig. 1S, Supporting Information.
In the 1H NMR spectra of both substances there is a signal at δ 12.30 (1) and δ 12.28 (5), characteristic of a free 5-OH group, and there is not a signal characteristic of
a free 7-OH group. In the electronic spectrum, 1 and 5 were absent a bathochromic shift with sodium acetate, but it was present when adding
zirconium chloride solution and it disappeared with the subsequent addition of citric
acid, which confirms the free 5-OH position and that it was occupied by 3-OH and 7-OH.
In the 1H-1H COSY of 5, interaction protons at C-1 of glucose and C-2 of rhamnose were observed, which determines
the nature of the glycosidic bond between sugar residues at 3-OH (С-1→С-2) in compound
5. In the same spectrum, the interaction of the protons of rhamnose with only one residue
of glucose was observed. Thus, the position at 7-OH occupied a glucose residue.
In compounds 2 and 3, by contrast, signals in 13C NMR spectra at δ 68.55, 68.51 (2) and δ 68.55 (3) as well as doublets of anomeric protons in the 1H NMR spectrum at δ 5.15 (J=8.0 Hz) and δ 5.14 (2 and 3, respectively) indicate the position of the glucose between C-3 of the aglycone and
a molecule of rhamnose, while a singlet of rhamnose of H-1 at δ 4.55 (2 and 3) indicates the terminal position of this sugar. Signals in the 1H NMR spectra for 5-OH δ 12.35 (2 and 3) and 7-OH δ 7.87 (2), δ 7.95 (3) indicate the free position of these groups. Data for the 1Н-1Н COSY, 1Н-13С COHX, and COLOC 2D NMR spectra of compounds 2 and 3 enabled the nature of the glycosidic bond between molecules of glucose and rhamnose
at 3-OH to be established: С-6→С-1 (2 and 3). The β-configuration of glucose residues and the α-configuration of rhamnose were proven based on the respective magnitudes of their
constant J.
From this data, the structure of compound 1 was identified as 5,3/,4/-trihydroxyflavone-7-О-α-L-rhamnopyranoside-3-О-β-D-glucopyranosidе [11], 2 as 5,7,3/,4/-tetrahydroxyflavone-3-О-β-D-glucopyranoside-(1→6)-β-D-glucopyranoside-(1→6)-О-α-L-rhamnopyranoside [12], 3 as 5,7,3/,4/-tetrahydroxyflavone-3-О-β-D-glucopyranoside-(1→6)-О-α-L-rhamnopyranoside [9], and 5 as 5,3/,4/-trihydroxyflavone-7-О-β-D-glucopyranoside-3-O-α-L-rhamnopyranoside-(1→2)-О-β-D-glucoyranoside. The flavonoid structure 5 is similar to quercetin 3-O-rutinoside-7-O-glucoside [13], but the position of the sugar at C-3 is different. We did not find data on flavonoid
5 in the literature.
The sugar components of 4 (xylose and rhamnose) were determined after acid hydrolysis by identification with
reliable samples in the butanol-pyridine-water system (6:4:3, detector: anilin phthalate
solution). Electronic spectra with ionizing reagents showed the absence of an expressed
bathochromic shift with a sodium acetate and zirconium chloride solution in the presence
of citric acid, which indicates the bonding of sugar residues at positions 3 and 7
of the flavone ring. This was confirmed by the absence of a signal that is characteristic
for a free 7-OH group in the 1H NMR spectrum. The signals for five carbon atoms from one of the sugars in the 13C NMR spectrum are consistent with those for xylose [14]
[15]. The presence of a singlet at δ 4.53 in the 1Н NMR spectrum and the signal at δ 17.61 in the 13C NMR spectrum confirm that the second sugar is rhamnose. From this data, the structure
of compound 4 was identified as 5,3/,4/-trihydroxyflavone-7-О-α-L-rhamnopyranoside-3-О-β-D-xylopyranoside (PubChem CID:44259241). Flavonoids 1, 2, 4, and 5 were described for the first time for the genus Saussurea. Flavonoid 5 was described for the first time ever.
The effect of flavonoids from S. controversa on bone marrow and bone tissue was researched in experimental osteomyelitis. The
development of experimental osteomyelitis led to a decrease in the TNM in the bone
marrow of rats by 38% compared to the intact group ([Table 1]). The decrease in the TNM was driven primarily by a 58% decrease in the number of
lymphocytes and by an average decrease of 34% in the number of young and mature cell
forms of the granulocytes and erythrocytes. As a result of experimental osteomyelitis
in rats, the number of megakaryocytes increased by a factor of four; furthermore,
a large number of destroyed lymphocytes was observed. Greater inhibition of myelopoiesis
was observed after antibiotic treatment in the bone marrow of rats with experimental
osteomyelitis.
Table 1 Bone marrow cells of rats with experimental osteomyelitis before (group 2) and after
the introduction of Cefazolin (group 3) and SCFG (group 4).
|
Indicator
|
The number of cells×106 on the femur (М±m, n=6)
|
|
Group 1
|
Group 2
|
Group 3
|
Group 4
|
|
Erythroblasts
|
0.64±0.15
|
0.45±0.11
|
0.21±0.031
|
0.37±0.07
|
|
Pronormoblasts
|
3.66±0.62
|
1.33±0.061
|
0.61±0.031
|
1.46±0.331, 3
|
|
Basophilic normoblasts
|
4.68±0.23
|
3.08±0.16
|
1.56±0.181
|
3.93±0.513
|
|
Polychromatophilic normoblasts
|
13.91±1.26
|
9.52±0.941
|
7.72±0.341
|
9.66±0.771
|
|
Oxyphilic normoblasts
|
11.20±1.48
|
9.10±0.59
|
5.60±0.371
|
8.32±0.90
|
|
The mitoses of erythrocytes
|
1.92±0.09
|
1.37±0.08
|
0.80±0.071
|
1.44±0.14
|
|
The total number of erythrokaryocytes
|
36.01±3.84
|
24.85±1.951
|
16.50±1.001
|
25.19±2.761, 3
|
|
Myeloblasts
|
1.65±0.22
|
0.58±0.041
|
0.19±0.031
|
0.70±0.11
|
|
Promyelocytes
|
3.88±0.50
|
0.98±0.281
|
2.04±0.262
|
1.75±0.302
|
|
Myelocytes
|
3.66±0.85
|
1.62±0.411
|
1.46±0.171
|
1.90±0.651
|
|
Metamyelocytes
|
4.66±0.67
|
3.00±0.341
|
1.66±0.241
|
4.83±0.252, 3
|
|
Rod-nuclear neutrophils
|
14.18±1.37
|
6.50±0.951
|
4.05±0.381
|
12.88±1.102, 3
|
|
Segmentonuclear neutrophils
|
10.98±1.29
|
12.10±1.14
|
5.49±0.641
|
12.29±1.61
|
|
Eosinophils
|
5.04±0.60
|
5.07±0.40
|
3.15±0.301
|
4.79±0.33
|
|
Basophils
|
0.46±0.01
|
0.28±0.01
|
0.20±0.01
|
0.36±0.01
|
|
The mitosis of myelocytes
|
1.51±0.18
|
0.64±0.05
|
0.26±0.031
|
0.62±0.13
|
|
The total number of granulocytes
|
49.55±5.41
|
30.77±3.621
|
18.20±2.081
|
40.15±4.902, 3
|
|
Monocytes
|
4.26±0.52
|
5.46±0.73
|
2.84±0.332
|
2.85±0.492
|
|
Megakaryocytes
|
0.46±0.01
|
1.96±0.331
|
0.27±0.012
|
0.95±0.322
|
|
Lymphocytes
|
16.70±1.81
|
6.94±1.001
|
4.07±0.351
|
16.34±1.832, 3
|
|
The plasma cells
|
1.02±0.43
|
1.96±0.40
|
0.56±0.112
|
1.83±0.33
|
|
Maturation index of neutrophils
|
0.48±0.01
|
0.30±0.01
|
0.43±0.05
|
0.34±0.01
|
|
Maturation index of erythrokaryocytes
|
0.70±0.01
|
0.75±0.01
|
0.80±0.05
|
0.70±0.01
|
|
Leucoerythroblastic ratio
|
1.96±0.05
|
1.73±0.05
|
1.46±0.051
|
2.45±0.052
|
|
TNM
|
91.50±1.75
|
56.00±2.901
|
38.12±2.6012
|
73.20±2.652, 3
|
Note: The superscripts 1-3 indicate p ≤ 0.05 in comparison with groups 1-3, respectively
After the course of using the SCFG in the proportional relationship of 2 (1): 8 (2): 10 (3): 1 (4): 4 (5), in a dose of 10 mg/kg, the number of bone marrow granulocytes and lymphocytes increased
significantly, indicating stimulation of myelopoiesis under these conditions. Using
the SCFG led to an average decrease of 51% in the number of megakaryocytes.
During a morphological study in the femoral diaphysis of rats on the 36th day after the modeling of osteomyelitis (group 2), signs of pronounced inflammation
were noted. Leukocytic infiltration and hyperemia of blood vessels were observed in
bone marrow spaces. Necrosis and autolysis of bone plates in the cortical and trabecular
layers with the formation of sequesters were discovered. The normal structure of osteons
was disrupted ([Fig. 2]).
Fig. 2 The section of the femoral diaphysis of rats on the 36th day after modeling of osteomyelitis without treatment showed signs of inflammation
without apparent activation of osteogenesis a; after treatment with antibiotic, signs of inflammation remained and there was a
slight activation of osteogenesis b; after the SCFG treatment, activation of osteoblastic processes was observed at the
location of dead osteons c. Staining was done with hematoxylin and eosin, ×200.
In the group of rats treated with Cefazolin (group 3), diffuse inflammation was observed,
but its intensity was lower. Discomplexation of bone plates remained. The process
of bone resorption prevailed over the process of osteogenesis.
In the group of rats treated with SCFG (group 4), a decrease in the intensity of inflammatory
processes was noted. Signs of regenerative processes (activation of endoosteum and
periosteum, the formation of granulation tissue) were visible throughout the femur,
but more pronounced in the epiphysis. Most of the bone plates had a normal structure
and uniform mineralization.
Thus, flavonoids are a potential remedy for integrated treatment of osteomyelitis
by stimulating myelopoiesis and the regeneration of bone tissue.
Despite the wide potential for the application of species of Saussurea in medicine, the chemical composition and biological activity of many of them are
poorly understood. The people of Siberia successfully apply S. controversa as anti-inflammatory and immunomodulatory agents, as well as in bone pathology, but
the structure of the active molecules, including flavonoids, which have a number of
biological effects, still has not been established. A complex of five quercetin glycosides
isolated from S. controversa improves the condition of bone marrow and enhances reparative processes in bone tissue
during standard antibiotic treatment of osteomyelitis. The nature of aglycones and
sugar residues of flavonoids was established using NMR 1H-1H, and 1H-13C with careful comparison of the data. COSY, COLOC, and ROESY experiments allowed
for establishing the point of attachment of sugar residues and the nature of the glycosidic
linkages. Compounds 1, 2, 4, and 5 had not been described previously for the genus Saussurea. Flavonoid 5 had not been described previously in the literature.
Materials and Methods
General experimental procedures
The NMR spectra for the solutions of the compounds in CD3ОD were recorded on the Bruker AV-600 spectrometer [600.30 (1Н), 150.95 МHz (13С)]. Chemical shifts were reported in ppm (δ) relative to internal TMS for all the signs that could be identified with certainty.
Coupling constants (J) were measured in Hz. The NMR signals were determined by using various 1H-1H and 1H-13C correlation spectroscopy experiments (COSY, HMBC, COLOC, COXH, and ROESY). The specific
rotation values [α]25
D were determined on a PolAAr 3005 polarimeter, and expressed in (deg×mL)/(g×dm), while
concentration was expressed in g per 100 mL of solution. The melting points were determined
on a Stuart SMF-38 melting point apparatus and are uncorrected. UV spectra were obtained
on an HP 8453 UV-Vis spectrometer (Hewlett-Packard) in EtOH solutions (10-4 mol/L). CHN analysis was carried out on a Carlo Erba 1106 elemental analyzer.
Plant material
Leaves of S. controversa were collected in the region of Irkutsk, Russia, during the flowering phase in July
of 2013 and were air-dried. The plants were collected by Prof. A. A. Semenov and identified
by Prof. M. N. Shurupova. A voucher specimen (No.TK-004605) has been deposited at
the Herbarium of Tomsk State University (Tomsk, Russia).
Extraction and isolation
Raw materials (600 g) were extracted with hot 40% EtOH (3×6000 mL, 80+°C, 1 h each).
The extract was filtered and evaporated until it became an aqueous residue, which
was treated sequentially in a separating funnel with CHCl3 (3 × 200 mL), ethyl acetate (6×200 mL), and n-butanol (10×200 mL). After removal of the solvents, a 0.72-g (0.12% on air-dried
raw material) CHCl3 fraction and a 4.38-g (0.73%) ethyl acetate fraction were obtained. When butanol
was removed, yellow amorphous powder easily precipitated and was separated by decantation
and dried; its weight was 2.0 g. After the residue was dried, a butanol faction with
a yield of 11.00 g (1.83%) was obtained.
Through chromatography of the butanol precipitate (1.5 g) on silica gel (2.5 × 70 cm;
Lachema L 100/250) using ethyl acetate with a gradual increase in the content of EtOH
(5-80%), three flavonoids were successively isolated: 1 (yield 0.080 g), 2 (yield 0.468 g), and 3 (yield 0.570 g). In the following rechromatography, fractions of the butanol precipitate
on microcrystalline cellulose (2.3 × 60 cm; Lachema) using water with a gradual increase
in the content of EtOH (5-90%), two other flavonoids were successively obtained: 4 (yield 0.067 g) and 5 (yield 0.216 g).
Biological experiments
Biological experiments were carried out on 24 male Wistar rats, aged 3 months and
weighing 300-350 g (Laboratory of Biological Modeling at Siberian State Medical University,
Tomsk). Animals were kept in standard conditions with free access to water and food.
The experimental studies were governed by the principles set out in the European Community
directives (86/609/EEC) and the Helsinki Declaration, and were approved by the local
ethical committee of Siberian State Medical University (No. 4316 from 9 November 2015).
The rats were divided into four groups: intact (group 1), experimental osteomyelitis
(group 2), experimental osteomyelitis treated with Cefazolin (group 3), and experimental
osteomyelitis treated with SCFG (group 4). Experimental osteomyelitis of the right
femur was simulated in the rats of groups 2-4 [16]. The complex of SCFG in the proportional relationship of 2 (1): 8 (2): 10 (3): 1 (4): 4 (5) was administered intragastrically in a water suspension at a dose of 10 mg/kg in
a volume of 2 mL for 28 days. The antibiotic Cefazolin (purity 99%; Rusfarm), which
is used as the standard treatment of osteomyelitis, was administered intramuscularly
in a dose of 50 mg/kg for 5 days.
After euthanasia by CO2 asphyxia on day 36 of the experiment, the condition of bone marrow hematopoiesis
in the rats was examined by counting the TNM on the femur (106/femur) and myelogram on smears prepared from the homogenate of fragment myeloid tissue
taken from a segment of the sternum and autologous serum (1:1), stained using Nocht’s
azure II-eosin. The percentage of separate cell forms when counting myelograms of
the rats was translated into absolute numbers of cells on the femur × 106.
For the histological examination, the right femur was decalcified using Grip’s method
[17], dehydrated in EtOH, and embedded in paraffin. Sections 7 µm thick were deparaffinized
and stained with hematoxylin and eosin.
Statistical analysis
Statistical processing of the results was carried out using the Statistica 8.0 statistical
analysis software package. To evaluate the significance of differences between samples,
we used nonparametric Mann-Whitney criteria with a calculation of the arithmetic mean
M and its standard error. Statistically significant differences were considered at
p<0.05.
Quercetin 7-О-α-L-rhamnoside-3-О-β-D-glucoside (1): light yellow, amorphous powder (MeOH-CHCl3); m.p. 188-190°С; [α]25
D +48 (c 0.28, EtOH); UV (MeOH) λ
max 290, 355 nm [11].
Quercetin 3-О-β-D-diglucoside-О-α-L-rhamnoside (2): light yellow, amorphous powder (MeOH-CHCl3); m.p. 175-176+°C; [α]25
D +66 (c 0.42, EtOH); UV (MeOH) λ
max 292, 357 nm; anal C 50.66, H 5.05, O 44.29, calcd. for С33Н40О18, 724 [12].
Quercetin 7-О-α-L-rhamnoside-3-О-β-D-xyloside (4): yellow, amorphous powder (MeOH-CHCl3); m.p. 183-185+°C; [α]25
D +78 (c 0.26, EtOH); UV (MeOH) λ
max 294, 334 nm; 1H NMR (CD3ОD, 600 MHz) δ 5.14 (d, 8.0, C-1///), 3,46 (d, 8.0, C-2///), 3.50 (d, 8.0, C-3///), 3.57 (m, C-4///), 3.42, 3.82 (d, 10.0, C-5///); 4.53 (1Н, s), 3.26-3.40 (4Н, m), 1.18 (3Н, d, 6.0); 8.06 (3/-OH), 8.11 (4/-OH), 12.26 (5-ОН); 13C NMR (CD3ОD, 150 MHz) d 105.33 (C-1///), 74.90 (C-2///), 77.62 (C-3///), 68.93 (C-4///), 67.91 (C-5///); 101.76 (C-1//), 71.66 (C-2//), 70.33 (C-3//), 71.28 (C-4//), 73.45 (C-5//), 17.61 (C-6//).
Quercetin 7-О-β-D-glucoside-3-О-α-L-rhamnoside-О-β-D-glucoside (5): yellow, amorphous powder (MeOH-CHCl3); m.p. 197-199+°C; [α]25
D +72 (c 0.36, EtOH); UV (MeOH) λ
max 293, 326 nm; 1H NMR (CD3ОD, 600 MHz) δ 5.00 (1Н, d, 7.5), 3.40-3.68 (8Н, m), 3.60 (2Н, dd, 9.0, 2.0), 3.81 (2Н, d, 10.0);
4.51 (1Н, s), 3.39-3.66 (4Н, m), 1.17 (3Н, d, 6.0); 8.08 (3/-OH), 8.10 (4/-OH), 12.28 (5-OH); 13C NMR (CD3ОD, 150 MHz) d 105.03 (C-1///), 105.09 (C-1////), 75.23, 76.67, 71.63, 77.82, 68.16 (C-6///), 68.19 (C-6////); 102.02 (C-1//), 71.88 (C-2//), 70.77 (C-3//), 69.26 (C-4//), 73.63 (C-5//), 17.74 (C-6//).
Supporting information
Microscopic sections of the affected area of the femurs of rats before and after treatment,
tables with the NMR data for compounds 1-5, spectra for 1H NMR, 13C NMR, COLOC, and COXH for compound 5, and key COSY correlations for compound 5 are available as Supporting Information.
