New Methyl Threonolactones and Pyroglutamates of Spilanthes acmella (L.) L. and Their Bone Formation Activities

In our continuing research for bioactive constituents from natural resources, a new methyl threonolactone glucopyranoside (1), a new methyl threonolactone fructofuranoside (2), 2 new pyroglutamates (3 and 4), and 10 known compounds (5–14) were isolated from the whole plant of Spilanthes acmella (L.) L. The structures of these compounds were determined based on various spectroscopic and chemical analyses. All of the isolated compounds were evaluated on bone formation parameters, such as ALP (alkaline phosphatase) and mineralization stimulatory activities of MC3T3-E1 cell lines. The results showed that the new compound, 1,3-butanediol 3-pyroglutamate (4), 2-deoxy-d-ribono-1,4-lactone (6), methyl pyroglutamate (7), ampelopsisionoside (10), icariside B1 (11), and benzyl α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside (12) stimulated both ALP and mineralization activities.


Introduction
Osteoporosis is an age-related chronic disease characterized by a decrease of bone mineral density and an increased risk of bone fracture. As the population in the world ages, osteoporosis is becoming an important social problem. In the world, more than 200 million people are suffering from osteoporosis. Approximately 50% of women over 50 years old will have an osteoporosis-related fracture in their lifetime. An imbalance of bone remodeling causes osteoporosis through bone resorption by osteoclasts and bone formation by osteoblasts. Insufficient bone formation is an essential cause of osteoporosis. Mesenchymal stem cells differentiate to osteoblasts with activation of alkaline phosphatase (ALP) and bone mineralization, which are regulated by a variety of molecules such as Runt-related transcription factor 2 (Runx2), bone morphogenetic proteins (BMPs), and estrogen [1].
The main constituents from the whole aerial parts, flower heads, and roots of this plant are spilanthol and acmellonate used to reduce toothaches, to induce saliva secretion [4,6,9], as powerful insecticides [10,11], and as local anesthetics [3]. It is also an important source of highly valuable bioactive compounds, such as phenolics, coumarins, triterpenoids [12], and flavonoids [13].
In our previous study, a combination of 70% ethanol extract of this plant and physical exercise increased testosterone level and osteoblast cell differentiation against glucocorticoid-induced osteoporotic male mice [14]. In addition, the 1-butanol and water layers of a 70% ethanol extract of this plant stimulated an osteoblast cell marker, ALP, of MC3T3-E1 osteoblast-like cells (126% and 127%, respectively) [15]. Based on these results, this plant extract seems to have the potential to be used as osteoporotic therapy by increasing bone formation. Therefore, it is important to know which compounds support these activities to understand the molecular basis and future development of the anti-osteoporotic remedy.
The absolute stereochemistry of compounds 3 and 4 remains to be elucidated because of the insufficient amount for further analysis.

Osteoblast Activity
Osteoblasts are the most important cells in bone tissue and are critical for bone formation through proliferation and differentiation. During osteoblast differentiation, BMPs induce the expression of osteoblastic markers, such as ALP. Proliferating osteoblasts show ALP activity, which is greatly enhanced during in vitro bone formation. ALP is a membrane-bound enzyme that is often used as a marker for osteogenic differentiation. 17β-estradiol has a significant impact on bone mineral metabolisms. It affects osteoblast proliferation through modulating the release of several local
The absolute stereochemistry of compounds 3 and 4 remains to be elucidated because of the insufficient amount for further analysis.

Osteoblast Activity
Osteoblasts are the most important cells in bone tissue and are critical for bone formation through proliferation and differentiation. During osteoblast differentiation, BMPs induce the expression of osteoblastic markers, such as ALP. Proliferating osteoblasts show ALP activity, which is greatly enhanced during in vitro bone formation. ALP is a membrane-bound enzyme that is often used as a marker for osteogenic differentiation. 17β-estradiol has a significant impact on bone mineral metabolisms. It affects osteoblast proliferation through modulating the release of several local regulators of bone turnover from monocytes and enhanced BMP-4 induced osteoblastic marker expression and mineralization [28].
To evaluate the effects of 1-14 on osteoblast function, ALP activity, which is related to the osteoid formation and initiation of the deposition of minerals, was evaluated. In this study, it was found that 4, 6, 7, 10, 11, and 12 stimulated ALP activity, which markedly increased osteoblast growth and differentiation of osteoblastic MC3T3-E1 cells. Compounds 7 and 11 did not show concentration dependency, probably due to the toxicity at higher concentrations. At concentrations of 25 µM, 6, 10, and 12 stimulated ALP activity up to 112% comparable to the positive control, 17β-estradiol at 0.02 and 0.01 µM (Figure 4).
Molecules 2020, xx, x FOR PEER REVIEW 6 of 11 regulators of bone turnover from monocytes and enhanced BMP-4 induced osteoblastic marker expression and mineralization [28].
To evaluate the effects of 1-14 on osteoblast function, ALP activity, which is related to the osteoid formation and initiation of the deposition of minerals, was evaluated. In this study, it was found that 4, 6, 7, 10, 11, and 12 stimulated ALP activity, which markedly increased osteoblast growth and differentiation of osteoblastic MC3T3-E1 cells. Compounds 7 and 11 did not show concentration dependency, probably due to the toxicity at higher concentrations. At concentrations of 25 µM, 6, 10, and 12 stimulated ALP activity up to 112% comparable to the positive control, 17β-estradiol at 0.02 and 0.01 µM (Figure 4). Osteoblasts can be induced to produce vast extracellular calcium deposition in vitro. This process is called mineralization. Calcium deposition is an indication of successful in vitro bone formation and can specifically be stained bright orange-red using Alizarin Red S. The effects of 1-14 were then examined by measuring the calcium deposition by Alizarin Red staining. As was found for the ALP activity study above, 4, 6, 7, 10, 11, and 12 showed stimulatory effects on mineralization. Compounds 6, 10, and 12 stimulated the mineralization up to 112% at 25 μM, comparable to that of the positive control, 17β-estradiol, at 0.02 and 0.01 µM ( Figure 5). In bone formation, osteoblasts are key cells in bone matrix formation and calcification. Osteogenesis starts with osteoblast production and secretion of type I collagen, which makes up about 90% of the organic bone matrix or the osteoid. Osteoblast also becomes high in alkaline phosphatase. Alkaline phosphatase is released into the osteoid to initiate the deposition of minerals.  Osteoblasts can be induced to produce vast extracellular calcium deposition in vitro. This process is called mineralization. Calcium deposition is an indication of successful in vitro bone formation and can specifically be stained bright orange-red using Alizarin Red S. The effects of 1-14 were then examined by measuring the calcium deposition by Alizarin Red staining. As was found for the ALP activity study above, 4, 6, 7, 10, 11, and 12 showed stimulatory effects on mineralization. Compounds 6, 10, and 12 stimulated the mineralization up to 112% at 25 µM, comparable to that of the positive control, 17β-estradiol, at 0.02 and 0.01 µM ( Figure 5).
Molecules 2020, xx, x FOR PEER REVIEW 6 of 11 regulators of bone turnover from monocytes and enhanced BMP-4 induced osteoblastic marker expression and mineralization [28].
To evaluate the effects of 1-14 on osteoblast function, ALP activity, which is related to the osteoid formation and initiation of the deposition of minerals, was evaluated. In this study, it was found that 4, 6, 7, 10, 11, and 12 stimulated ALP activity, which markedly increased osteoblast growth and differentiation of osteoblastic MC3T3-E1 cells. Compounds 7 and 11 did not show concentration dependency, probably due to the toxicity at higher concentrations. At concentrations of 25 µM, 6, 10, and 12 stimulated ALP activity up to 112% comparable to the positive control, 17β-estradiol at 0.02 and 0.01 µM (Figure 4). Osteoblasts can be induced to produce vast extracellular calcium deposition in vitro. This process is called mineralization. Calcium deposition is an indication of successful in vitro bone formation and can specifically be stained bright orange-red using Alizarin Red S. The effects of 1-14 were then examined by measuring the calcium deposition by Alizarin Red staining. As was found for the ALP activity study above, 4, 6, 7, 10, 11, and 12 showed stimulatory effects on mineralization. Compounds 6, 10, and 12 stimulated the mineralization up to 112% at 25 μM, comparable to that of the positive control, 17β-estradiol, at 0.02 and 0.01 µM ( Figure 5). In bone formation, osteoblasts are key cells in bone matrix formation and calcification. Osteogenesis starts with osteoblast production and secretion of type I collagen, which makes up about 90% of the organic bone matrix or the osteoid. Osteoblast also becomes high in alkaline phosphatase. Alkaline phosphatase is released into the osteoid to initiate the deposition of minerals.  In bone formation, osteoblasts are key cells in bone matrix formation and calcification. Osteogenesis starts with osteoblast production and secretion of type I collagen, which makes up about 90% of the organic bone matrix or the osteoid. Osteoblast also becomes high in alkaline phosphatase. Alkaline phosphatase is released into the osteoid to initiate the deposition of minerals. After mineralization, the bone becomes hard and rigid with necessary mechanical properties to withstand the external forces to support the body and protect the internal organs. Our study demonstrated that 4, 6, 7, 10, 11, and 12 stimulated both ALP activity and calcium deposition in osteoblastic MC3T3-E1 cell in vitro, which suggests that the extract of S. acmella and these compounds have potential to be a remedy for osteoporosis as osteoblastic bone formation stimulant.

General Experimental Procedures
1 H and 13 C-NMR spectra were taken on a Bruker Ultrashield Avance 600 spectrometer at 600 MHz and 150 MHz, respectively, with TMS as an internal standard. IR and UV spectra were measured on a HORIBA FT-720 FT-IR spectrophotometer and JASCO V-520 UV-vis spectrophotometer, respectively. Optical rotation was measured on a JASCO P-1030 digital polarimeter. Positive ion HR-ESI-MS was recorded using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Silica gel open column chromatography (CC) and reversed-phase (ODS) CC were performed on silica gel 60 (E. Merck, Darmstadt, Germany), and Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan; Φ = 35 mm, L = 350 mm), respectively. HPLC was performed on an ODS column (Inertsil ODS-3, GL Science, Tokyo, Japan; Φ = 6 mm, L = 250 mm, 1.5 mL/min), and the eluate was monitored with a JASCO RI-930 intelligent detector and a JASCO PU-1580 intelligent pump unless otherwise specified.

Extraction and Isolation
The air-dried plants (2.0 kg) were extracted with methanol (MeOH, 10.0 l × 3). The methanol solution was concentrated and adjusted to 95% aq. methanol by the addition of water and then partitioned with n-hexane (1.0 l × 3, 23.5 g). The remaining aqueous methanol layer was evaporated and resuspended in 0.5 l of water and then partitioned with ethyl acetate (1.0 l × 3, 40.4 g) and 1-butanol (1.0 l × 3, 47.5 g), successively.