Synthesis and Promotion of the Osteoblast Proliferation Effect of Morroniside Derivatives

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.


Introduction
Fracture is one of the most common clinical diseases and often causes substantial damage to a patient's quality of life [1]. Without proper care and recovery, it can cause irreversible damage to the patient [2]. 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 [3,4]. Due to osteoporosis in patients with osteopenia, bone strength decreases, bone fragility increases, and the probability of fracture greatly increases [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 [11,12], which can lower serum calcium, promote the deposition of calcium salts, promote collagen synthesis, improve the quality of callus, and has great potential for clinical applications [13][14][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 µg/mL morroniside) [15,16]. In addition, the poor solubility, the rapid 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).
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).

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). 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).   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). 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).

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). 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).   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).

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 (2, 3, 4, 5, and 14) promoted proliferation on MC3T3-E1 cells (EC50 < 500) more strongly than did the other derivatives. Among these five compounds, Compound 3 showed the strongest proliferation effect on MC3T3-E1 cells, Compound 14 showed the weakest, and Compounds 2, 5, and 4 showed a medium effect.
The activity of morroniside is conspicuously decreased when morroniside is hydrolyzed into aglycone (Compound 1). 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. 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. 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

Cell Viability Assay
The MTT assay was used to evaluate the proliferation effect of the morroniside derivatives on MC3T3-E1 cells, and their values EC 50 are shown in Table 1. Five compounds (2, 3, 4, 5, and 14) promoted proliferation on MC3T3-E1 cells (EC 50 < 500) more strongly than did the other derivatives. Among these five compounds, Compound 3 showed the strongest proliferation effect on MC3T3-E1 cells, Compound 14 showed the weakest, and Compounds 2, 5, and 4 showed a medium effect. The activity of morroniside is conspicuously decreased when morroniside is hydrolyzed into aglycone (Compound 1). 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. 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.
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.

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.
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.

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.
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.

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.

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).
The 1 H-NMR and 13 C-NMR results of Compound 1 are consistent with β-morroniside aglycone, so it was concluded that Compound 1 is β-morroniside aglycone [24]. 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.

General
NMR spectra were performed on a Bruker DRX-400 NMR spectrometer at 400 MHz using CD 3 OD 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.

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).
The 1 H-NMR and 13 C-NMR of Compounds 2-9 are consistent with those reported in the previous study. However, it is worth noting that, if the configuration at C-7 is α, the chemical shift of C-5 is more than 30, and C-8 is more than 70; if the configuration at C-7 is β, the chemical shift of C-5 is between 25 and 29, and C-8 is between 62 and 66 [25].

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).
The 1 H-NMR and 13 C-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].

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).

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).

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 1 H-NMR and 13 C-NMR of Compound 14 are consistent with those reported in the previous study, so it was concluded that Compound 14 is 5-(1 -hydroxyethyl) nicotinic acid methyl ester [27].

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 with 100 µL in each well (1 × 10 4 cells/well) and then incubated for 24 h at 37 • C. The cells were then exposed to different concentrations (100, 10, 1, and 0.1 µg/mL) of the derivatives and incubated for another 24 h, and 20 µL 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 µL of DMSO 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 (%) = (A Sample /A Control − 1) × 100%.
All results are expressed as mean ± SD. All figures were illustrated using Prism 7 (SOFTHEAD, Shenzhen, China). Statistical analysis was carried out with SPSS 24 software (STRONG-VINDA, Beijing, China).

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 µL in each well (1 × 10 4 cells/well) and then incubated for 24 h at 37 • C. The cells were then exposed to compound 2, 3, 4, 5, and 14 in 90 µg/mL amounts, and incubated for three days. The cells and the medium were transferred to a 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 µL of double distilled water (blank), 5 µL of a 0.1 mg/mL phenol standard application solution (standard), and 5 µL of derivative solution (measurement) were added. Afterwards, 50 µL 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 µL of a chromogenic agent was added, and the OD (optical density) values were measured at 520 nm.

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