Synthesis and promotion of the osteoblast proliferation effect of morroniside derivatives

Article abstract of DOI:10.3390/molecules23061412

Sambucus williamsii Hance has been used in fractures for thousands of years, but research on its active components, such as morroniside, until now had not been carried out. In this study, morroniside was taken as the leading compound, and fourteen derivatives were synthesized. The promotion of osteoblast proliferation effect of the derivatives was evaluated on MC3T3-E1 cells. Five derivatives (2, 3, 4, 5, and 14) showed a good proliferation effect on MC3T3-E1 cells, and their promoted expression effects on OC (Osteocalcin) and ALP (Alkaline phosphatase) in MC3T3-E1 cells were measured. Compound 3 was shown to have the strongest proliferation effect (EC₅₀ = 14.78 ± 1.17 μg/mL) and to significantly promote the expression of OC and ALP.

Full text of DOI:10.3390/molecules23061412

molecules
Article
Synthesis and Promotion of the Osteoblast
Proliferation Effect of Morroniside Derivatives
Hua Han, ZhengQing Li, Na Qu, Si Chen and PeiLiang Dong *
Key Laboratory of Chinese Materia Medica, Ministry of Education, Heilongjiang University of Chinese
Medicine, Harbin 150040, China; hh7551@163.com (H.H.); lizhengqing1125@163.com (Z.L.);
Qu399127818@163.com (N.Q.); m13945033486@163.com (S.C.)
* Correspondence: peiliang_D@163.com
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 April 2018; Accepted: 7 June 2018; Published: 11 June 2018
Abstract: Sambucus williamsii Hance has been used in fractures for thousands of years, but research on
its active components, such as morroniside, until now had not been carried out. In this study,
morroniside was taken as the leading compound, and fourteen derivatives were synthesized.
The promotion of osteoblast proliferation effect of the derivatives was evaluated on MC3T3-E1
cells. Five derivatives (
and their promoted expression effects on OC (Osteocalcin) and ALP (Alkaline phosphatase) in
MC3T3-E1 cells were measured. Compound was shown to have the strongest proliferation effect
2, 3, 4, 5, and 14) showed a good proliferation effect on MC3T3-E1 cells,
3
(EC50 = 14.78 ± 1.17 µg/mL) and to significantly promote the expression of OC and ALP.
Keywords: morroniside; derivatization; osteoblast proliferation effect; MC3T3-E1
1. Introduction
Fracture is one of the most common clinical diseases and often causes substantial damage to
a patient’s quality of life [
to the patient [ ]. In recent years, with the increase in the elderly population, the incidence of
osteoporosis has become the seventh most common affliction in the world, and about 80% of elderly
people have different degrees of osteoporosis [ ]. Due to osteoporosis in patients with osteopenia,
bone strength decreases, bone fragility increases, and the probability of fracture greatly increases [ ].
1]. Without proper care and recovery, it can cause irreversible damage
2
3,4
5
Moreover, the healing process of osteoporosis fracture is long, and clinical efficacy is extremely low,
seriously affecting the daily life of the elderly [6].
Currently, conservative treatment of osteoporotic fracture in the elderly has become one of the
most commonly used clinical methods [
7]. The main drugs for the treatment of fractures in clinics are
anti-inflammatory drugs, analgesics, and drugs that promote fracture healing [8,9
]. Sambucus williamsii
Hance has been used in the clinical treatment of various types of fractures for thousands of years,
with short course, rapid recovery, and high efficacy [10]. Previous studies have shown that morroniside
is the main component of S. williamsii Hance [11
deposition of calcium salts, promote collagen synthesis, improve the quality of callus, and has great
potential for clinical applications [13 15]. However, our previous studies have shown that morroniside,
when used alone, weakly promotes the proliferation of MC3T3-E1 cells (the proliferation rate is
about 20% in 24 h with 62.5 g/mL morroniside) [15 16]. In addition, the poor solubility, the rapid
,12], which can lower serum calcium, promote the
–
µ
,
metabolism in the body, and the low bioavailability of morroniside has limited its clinical application,
so morroniside cannot be developed as a clinical drug [17].
In this study, we used morroniside as a leading compound to obtain different derivatives to obtain
a compound with more value and a strong promoting effect on fracture healing. Two of the derivatives
are new compounds (12 and 13).
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2. Results and Discussions
2.1. Preparations of Morroniside Derivatives
Compound
1 was synthesized by hydrolyzing morroniside in a 20% hydrochloric acid aqueous
solution at 50 ◦C for 1 h, in a yield of 35% (Figure 1).
◦
Figure 1. Synthesis of Compound 1. (1) 20% HCl, 50 C, 1 h.
Compounds
2–
9
were synthesized by reacting morroniside with different alcohols at room
temperature in the presence of concentrated nitric acid as a catalyst for 30 min, for yields of 23%, 55%,
26%, 51%, 24%, 48%, 24%, and 49%, respectively (Figure 2).
Figure 2. Synthesis of Compounds 2–9. (2) RH, concentrated nitric acid, r.t., 30 min.
Compounds 10–12 were synthesized by reacting morroniside with acetic anhydride at different
temperatures in methanol for 1 h, for yields of 22%, 40%, and 45%, respectively (Figure 3).
Figure 3. Synthesis of Compounds 10
acetic anhydride, 200 C, 1 h.
–
12. (3) MeOH, acetic anhydride, 80 ◦C, 1 h. (4) MeOH,
◦

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Compounds 13 and 14 were synthesized as monoterpene alkaloids derivatives with different
primary amines, in yields of 32% and 46%, respectively (Figure 4).
◦
Figure 4. Synthesis of Compounds 13 and 14. (5) Methylamine ethanol solution, 10 C, 3 h. (6) C2H5OH,
◦
HOAc-NaAc buffer solution, β-glucosidase, 50 C, 3 h.
2.2. Cell Viability Assay
The MTT assay was used to evaluate the proliferation effect of the morroniside derivatives on
MC3T3-E1 cells, and their values EC50 are shown in Table 1. Five compounds (
promoted proliferation on MC3T3-E1 cells (EC50 < 500) more strongly than did the other derivatives.
Among these five compounds, Compound showed the strongest proliferation effect on MC3T3-E1
2, 3, 4, 5, and 14)
3
cells, Compound 14 showed the weakest, and Compounds 2, 5, and 4 showed a medium effect.
Table 1. EC50 values of derivatives promoting proliferation on MC3T3-E1 cells (mean ± SD), n = 5.
Compound
EC50 (µg/mL)
Compound
EC50 (µg/mL)
1
2
3
4
5
6
7
2042 ± 3.310
118.2 ± 2.073
14.78 ± 1.170
434.2 ± 2.638
164.4 ± 2.216
1342 ± 3.128
1028 ± 3.012
8
9
10
11
12
13
14
1068 ± 3.029
656.0 ± 2.817
961.6 ± 2.983
1022 ± 3.009
927.0 ± 2.967
637.2 ± 2.804
307.8 ± 2.488
The activity of morroniside is conspicuously decreased when morroniside is hydrolyzed into
aglycone (Compound ). In addition, iridoids cannot keep in a form of prototype drug in the human
body due to rapid metabolism [18], possibly resulting in low activity of morroniside in vivo.
In the case of acetylation into Compounds 10 11, and 12, morroniside gains less bioactivity.
1
,
Nonetheless, the acetyl group can be removed through metabolism of enzymes, thereby releasing
the morroniside and postponing its metabolism, consequently prolonging drug action time [19].
Therefore, the bioactivity of acetylated morroniside still needs to be further researched.
The activity of morroniside, to some extent, is likely to be enhanced when morroniside is alkylated
at C-7 position, and enhancement only occurs when substituted with methyl or ethyl. Research also
shows that the activity of morroniside will be gradually decreased with the increasing number of carbon
atoms on the alkyl chain. When the number of carbon atoms on the alkyl chain exceeds 3, the activity
of alkylated morroniside will be far lower than that of morroniside itself. Moreover, the activity will be
tremendously enhanced if the substituent is in β configuration rather than α configuration.
Research on alkaloids has been extensively carried out owing to the diverse structural
characteristics and pharmacological actions of such substance [20]. In this study, when aglycone
is transformed into pyridine monoterpene alkaloids derivatives (Compound 13), the pharmacological
activity is significantly enhanced, which proves that alkaloids have massive pharmacological potential.

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Besides, the activity of morroniside is conspicuously stimulated when its mother nuclear structure is
broken, which transforms morroniside into Compound 14. Furthermore, the stability of alkaloids is
stronger than that of morroniside because glycosidic linkage and hemiacetal structure are absent in
Compounds 13 and 14. It is speculated that the metabolism of alkaloids probably takes much longer
time than that of morroniside and its action efficacy is likely to be further extended as a consequence.
2.3. Activities of OC and ALP Assay
Osteocalcin (OC) and alkaline phosphatase (ALP) are two substances secreted by osteoblasts [21,22]
and can promote the deposition of bone salt. The expression of OC and ALP can reflect the rate of
fracture healing [23]. Therefore, in this study, we measured the effects of five compounds on the
expressions of OC and ALP in osteoblasts, and the results are shown in Figure 5.
Figure 5. Promotion effect of 90
cells: ( ) OC levels; and ( ) ALP activities. All values are represented as mean
versus the control. *** p < 0.001 versus the control.
µ
g/mL of
2
,
3
,
4
,
5
, and 14 on OC and ALP expression in MC3T3-E1
A
B
±
SD, n = 5. ** p < 0.01
As can be seen in the figure, the above-mentioned five derivatives are able to elevate the expression
OC and ALP, in significant contrast to the blank group (p < 0.05). Among the five derivatives,
Compound 3 stands out as possessing remarkable efficacy superior to the other four (p < 0.05). It is
shown that Compound 3 has the best activity in elevating the expression OC and ALP.
3. Materials and Methods
3.1. General
NMR spectra were performed on a Bruker DRX-400 NMR spectrometer at 400 MHz using CD3OD
as solvent in Zhengzhou University. The purity of morroniside and the derivatives were checked
on a Waters e2695 liquid chromatograph equipped with 2424 ELS detector and 2998 PDA detector.
GF254 silica plate was purchased from Qingdao Haiyang Chemical Co. Ltd. (Qingdao, China).
The Elisa kits of OC (H152) and ALP (A059-2) were purchased from Nanjing Jiancheng Bioengineering
Institute (Nanjing, China). All the chemical reagents were purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). The morroniside was extracted and purified in the medicinal
chemistry department, Heilongjiang University of Chinese Medicine.
3.2. Synthesis
3.2.1. Synthesis of Compound 1
An amount of 0.1 g of morroniside was dissolved with 2 mL of a 20% hydrochloric acid solution,
◦
and the mixture was heated for 1 h at 50 C. After this mixture was cooled to room temperature,
saturated sodium bicarbonate was added to remove the hydrochloric acid, the solvent was recovered
in vacuum, and the residue was purified by silica gel column chromatography and eluted with
petroleum ether-ethyl acetate (5:1) to obtain Compound 1 (21.1 mg, 35% yield).

Molecules 2018, 23, 1412
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The 1H-NMR and 13C-NMR results of Compound
1
are consistent with
β-morroniside aglycone,
so it was concluded that Compound 1 is β-morroniside aglycone [24].
3.2.2. Synthesis of Compounds 2–9
An amount of 0.1 g of morroniside was dissolved with 2 mL of different alcohols (methanol,
ethanol, isopropanol, and n-butanol), and 0.1 mL of concentrated nitric acid was added. This mixture
was stirred for 30 min at room temperature. After the reaction, moderate saturated sodium bicarbonate
was added to remove the concentrated nitric acid, the solvent was recovered in vacuum, and the
residue was purified by silica gel column chromatography and eluted with dichloromethane-methanol
(16:1) to obtain Compounds
(54.5 mg, 51% yield), (26.5 mg, 24% yield),
and 9 (55.8 mg, 49% yield).
The 1H-NMR and 13C-NMR of Compounds
2–9 are consistent with those reported in the previous
2
(23.8 mg, 23% yield),
3
(56.9 mg, 55% yield), (27.8 mg, 26% yield),
4
5
6
7
(53.0 mg, 48% yield), 8 (27.3 mg, 24% yield),
study. However, it is worth noting that, if the configuration at C-7 is
more than 30, and C-8 is more than 70; if the configuration at C-7 is
between 25 and 29, and C-8 is between 62 and 66 [25].
α
, the chemical shift of C-5 is
β, the chemical shift of C-5 is
3.2.3. Synthesis of Compounds 10 and 11
An amount of 0.1 g of morroniside was dissolved with 2 mL of methyl alcohol, and 2 mL of
acetic anhydride was added. The mixture was refluxed for 1 h at 80 ◦C. The solvent was recovered in
vacuum, and the residue was purified by silica gel column chromatography and eluted with petroleum
ether-ethyl acetate (3:1) to obtain Compounds 10 (31.9 mg, 22% yield) and 11 (58.0 mg, 40% yield).
1
The H-NMR and 13C-NMR of Compounds 10 and 11 are consistent with those reported in
the previous study, so it was concluded that Compounds 10 and 11 are 7α-methoxy tetraacethyl
morroniside and 7β-methoxy tetraacethyl morroniside, respectively [26].
3.2.4. Synthesis of Compound 12
An amount of 0.1 g of morroniside was dissolved with 2 mL◦of methyl alcohol, and 2 mL of
acetic anhydride was added. The mixture was refluxed for 1 h at 200 C. The solvent was recovered in
vacuum, and the residue was purified by silica gel column chromatography and eluted with petroleum
ether-ethyl acetate (3:1) to obtain Compound 12 (61.6 mg, 45% yield).
1
6,7-double bond tetraacethyl morroniside (12): white powder; H-NMR (400 MHz, CD3OD)
δ: 1.44
(3H, d, J = 6.9 Hz, H-10), 1.97 (3H, s, 60-CH3), 2.0 (1H, m, H-9), 2.01, 2.01, 2.04 (each 3H, s, 30, 40 and
20-CH3), 3.41 (1H, m, H-5), 3.71 (3H, s, H-12), 3.92 (1H, m, H-20), 4.14 (1H, dd, J = 2.5 Hz, 12.3 Hz, H-60a),
4.18 (1H, o, H-50), 4.31 (1H, dd, J = 4.9 Hz, 17.3 Hz, H-60b), 4.69 (1H, dt, J = 1.6 Hz, 6.2 Hz,
H-6), 4.96 (1H, dd, J = 8.1 Hz, 9.7 Hz, H-40), 5.03 (1H, t, J = 6.9 Hz, H-8), 5.15 (1H, d, J = 8.1 Hz, H-10),
5.33 (1H, dd, J = 9.5 Hz, 19.0 Hz, H-30), 5.40 (1H, d, J = 8.6 Hz, H-1), 6.30 (1H, dd, J = 2.3 Hz, 6.1 Hz, H-7),
7.48 (1H, s, H-3); 13C NMR (100 MHz, CD3OD)
δ:19.5, 20.5, 20.6, 20.6, 20.6, 31.0, 39.0, 51.9, 62.9, 69.9,
72.5, 73.1, 74.1, 74.8, 96.5, 98.7, 103.8, 110.3, 144.1, 153.2, 168.7, 171.1, 171.3, 171.6, 172.3; ESI-MS m/z
557 [M + H]+ (calcd for C25H32O14).
3.2.5. Synthesis of Compound 13
An amount of 0.1 g of morroniside was dissolved with 2 mL of a methylamine alcohol solution,
and the mixture was stirred for 3 h at 10 ◦C. The solvent was recovered in vacuum, and the residue
was purified by silica gel column chromatography and eluted with petroleum ether-ethyl acetate (1:1)
to obtain Compound 13 (20.3 mg, 32% yield).
2-N-Methyl morroniside aglycone (13): white powder; 1H-NMR (400 MHz, CD3OD)
δ: 1.30
(3H, d, J = 6.5 Hz, H-10), 1.50 (1H, m, H-9), 2.40 (2H, m, H-6), 2.93 (1H, m, H-5), 3.12 (3H, s, N-CH3),
3.67 (3H, s, H-12), 4.16 (1H, dd, J = 6.5 Hz, 13.9 Hz, H-8),4.72 (1H, d, J = 2.2 Hz, H-7), 5.10 (1H, s, H-1),

Molecules 2018, 23, 1412
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7.29 (1H, s, H-3); 13C-NMR (100 MHz, CD3OD)
δ
: 19.4, 24.9, 34.2, 39.2, 40.8, 51.4, 71.0, 80.4, 87.5, 104.7,
144.1, 169.9; ESI-MS m/z 258 [M + H]+ (calcd for C12H19NO5).
3.2.6. Synthesis of Compound 14
An amount of 0.1 g of morroniside was dissolved with 0.5 mL of an ethanol solution, and 5 mL
of HOAc–NaOAc buffer solution (pH 5) and 0.1 g of β-glucosidase were added. The mixture was
reacted for 3 h at 50 ◦C. The solvent was recovered in vacuum, and the residue was purified by silica
gel column chromatography and eluted with petroleum ether-ethyl acetate (5:1) to obtain Compound
14 (20.5 mg, 46% yield).
The 1H-NMR and 13C-NMR of Compound 14 are consistent with those reported in the previous
study, so it was concluded that Compound 14 is 5-(10-hydroxyethyl) nicotinic acid methyl ester [27].
3.3. Biological Assays
The MC3T3-E1 cell line was obtained from Procell (Wuhan, China) and was grown in DMEM
medium supplemented with 10% fetal bovine serum and a 1% penicillin–streptomycin solution.
The cell viability was evaluated using an MTT assay. The cells were plated in a ◦96-well plate
104 cells/well) and then incubated for 24 h at 37 C. The cells
with 100
µL in each well (1
×
were then exposed to different concentrations (100, 10, 1, and 0.1
incubated for another 24 h, and 20 L MTT regents (5 mg/mL) were subsequently added to each
well, which was followed by incubation for 4 h. The medium was removed, and 100 L of DMSO
µg/mL) of the derivatives and
µ
µ
was added to each well, which were then shaken for 10 min. The absorbance was measured by a
microplate reader at a wavelength of 490 nm. Control wells received only the medium without the
derivatives. Each derivative concentration had five replicates. The cell viability was calculated using
the following formula:
cell viability (%) = (ASample/AControl − 1) × 100%.
All results are expressed as mean
Shenzhen, China). Statistical analysis was carried out with SPSS 24 software (STRONG-VINDA,
Beijing, China).
±
SD. All figures were illustrated using Prism 7 (SOFTHEAD,
3.4. Activities of OC and ALP Assays
The Elisa kits of OC (H152) and ALP (A059-2) were purchased from Nanjing Jiancheng
Bioengineering Institute (Nanjing, China).
The cells were plated on a 12-well plate with 1000
then incubated for 24 h at 37 C. The cells were then exposed to compound
µ
L in each well (1
×
104 cells/well) and
, and 14 in
◦
2,
3,
4,
5
90
1.5 mL centrifuge tube, frozen and thawed three times, then placed in an ice box, and broken by an
ultrasonic protein breaker. According to the ELISA kit instructions, 5 L of double distilled water
(blank), 5 L of a 0.1 mg/mL phenol standard application solution (standard), and 5 L of derivative
solution (measurement) were added. Afterwards, 50 L of buffer and matrix solution each were
added, and the mixture was then placed in a 37 ◦C water bath for 15 min. An amount of 150
L of a
µg/mL amounts, and incubated for three days. The cells and the medium were transferred to a
µ
µ
µ
µ
µ
chromogenic agent was added, and the OD (optical density) values were measured at 520 nm.
4. Conclusions
Serial morroniside derivatives were synthesized, and their proliferation effects on MC3T3-E1 cells
were evaluated in vitro. From the above studies, we can draw the following conclusions: (1) A series
of products with alkylation at the C-7 position was synthesized by a new method. Compared with
previous iodine catalysis [22], this synthetic method is more secure and economical and simplified the
post-processing program. (2) Among the morroniside derivatives, Compounds 2, 3, 4, 5, and 14 had

Molecules 2018, 23, 1412
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strong proliferation effects on MC3T3-E1 cells. Among them, Compound
3
exhibited the strongest
effect. (3) The introduction of methyl, such as in Compounds
2 and 3, at Position 7 improved the
proliferation effect on MC3T3-E1 cells. However, with the extension of the carbon chain, the activity
decreases gradually. (4) With substitution with beta configuration at Position 7, the effect of the
proliferation is higher than the alpha configuration substitution, such as Compounds 2, 3, 4, and 5.
(5) Acetylation in morroniside did not affect the proliferation effect on MC3T3-E1. (6) Transformation
into the N-containing derivative (Compound 14) slightly increases the effect, but this effect is still less
substantial than the alkylation derivatives (Compounds 2 and 3).
Author Contributions: H.H. and P.D. conceived and designed the experiments; N.Q. and S.C. performed the
experiments; Z.L. analyzed the data; H.H. contributed reagents/materials/analysis tools and Z.L. wrote the paper.
Funding: This research was funded by [Education Department of Heilongjiang Province] grant number [1153G038]
and [Heilongjiang University of Chinese Medicine] grant number [B201003].
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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