Chemical profiling and identification of anti-osteoporosis chemical-markers of Cinnamomum cassia (L.) presl extracts using GC-MS and spectrum-activity analyses

Abstract Cinnamomum cassia (L.) Presl (cinnamon), an important folk medicine is widely used to prevent osteoporosis for long time in China. Our study aimed to investigate the anti-osteoporosis activity and mechanisms of cinnamon extracts obtained by supercritical CO2 extraction (SFE) and identify activity associated chemical components by gas chromatography-mass spectrometry. The cinnamon SFE exhibited superior anti-osteoporosis efficacy in an ovariectomised mice model to common alcohol extracts. It could induce calcified nodules and ALP activity, upregulate the mRNA expression of ALP, BMP-2, and RUNX2 in MC3T3-E1 cells. The major chemical classes of cinnamon extracts were alcohol esters (28.2%), and terpenes (16.1%). The spectrum-activity analysis indicated that the potential chemical-markers of extracts could be (E)-Cinnamaldehyde, γ-Sitosterol, and (Z, Z)-9,12-Octadecadienoic acid, which could induce the proliferation and ALP activity in MC3T3-E1 cells. Our study revealed the promising applications of the cinnamon SFE in prevention of osteoporosis, and identified its anti-osteoporosis associated compounds. Graphical Abstract


Introduction
Osteoporosis, the most common systemic bone disease, leads to a dramatic increase in bone fragility and fracture risk (Rizzoli 2018). Severe osteoporosis causes extremely high risk of fracture and is associated with high mortality and morbidity (Miller 2016). The nature products extracts possessing significant anti-osteoporosis activities are good alternatives to bisphosphonates (Putnam et al. 2007). Nature herbal medicine extracts based-anti-osteoporosis functional natural products are urgently needed to be developed.
Cinnamomum cassia (L.) Presl (Cinnamon) is an important Chinese herbal medicine, which is widely used in the prevention of a variety of orthopaedic diseases such as osteoporosis and osteoarthritis (Chen et al. 2022). Preclinical studies indicated that cinnamon extracts could direct stimulate bone formation, and rescue bone mass loss and metabolic disorder in postmenopausal osteoporosis rat models (Hong et al. 2022). Even the chemical components of cinnamon are complex and have not been identified yet. The volatile oils and lipids are considered the main active components of herbal medicine (Lv et al. 2021). Because the common alcohol extraction method may cause oxidation and loss of the active lipophilic components of cinnamon, supercritical fluid CO 2 extraction (SFE) could bring more advantages in the enrichment of volatile and other lipophilic components from herbal products (Garc ıa-P erez et al. 2016).
In this study, the anti-osteoporosis activity of cinnamon extracts obtained by supercritical CO 2 extraction has less been investigated. The anti-osteoporosis efficacy of the cinnamon SFE was evaluated in an ovariectomised (OVX) model and the underlying molecular mechanisms were further investigated. The chemical components of cinnamon extracts were analysed by Gas chromatography-automated mass spectral deconvolution and recognition system-mass spectrometry (GC-AMDIS-MS). Finally, Antiosteoporosis biochemical-markers were explored by spectrum-activity analyses and were validated experimentally in vitro.

Results and discussion
2.1. The extracts of cinnamon showed anti-osteoporosis effect in a murine OVX model In current study, the BMD of animals in the model group significantly decreased after ovariectomy surgery (p < 0.05) compared with the sham group ( Figure S1A). The SFE extracts of cinnamon could rescue BMD loss in the lumbar spine and femurs of mice, and demonstrated a superior therapeutic effect compared with the ALC extracts (Figures S1B-D and S5A). Compared with the model group and the ALC group, Masson staining showed that the morphological structure of the intervertebral disc of the mice in the SFE group was completed and clear, and an obvious collagen fiber network could be observed ( Figure S5B). Serum pharmacological study showed that SFE extracts containing serum could significantly induce ALP activity (p < 0.05) ( Figure  S1E) and calcified nodules (p < 0.05) (Figures S1I and S8A) increasing in MC3T3-E1 cells. As results shown in Figure S1F-H, SFE extracts containing serum exposure could remarkably upregulate the mRNA expression of ALP (2.38-12.18 folds, p < 0.05), BMP-2 (8.81-52.32 folds, p < 0.05), and RUNX2 (7.02-40.03 folds, p < 0.05) compared with blank serum and followed a dose-response relationship in MC3T3-E1 cells.

Chemical profiling of the extracts of Cinnamomum cassia (L.) presl
A total of 47 compounds with a wide range of peak intensities were detected and identified in the SFE and ALC extract samples ( Figure S6 and Table S1). Unfortunately, there is one phthalate among the identified components (entry 26, Table S1). It was previously reported that phthalate is a contaminant rather than a natural product (Bianco et al. 2014;Venditti 2020). The mass-to-charge ratio of each identified compound was corrected by AMDIS ( Figure S7). A total of 43 compounds were from SFE and only 13 compounds were from ALC ( Figure S2A). In terms of composition classification ( Figure S2B), the SFE extracts were mainly dominated by the class of fatty acids (28.3%), alcohol esters (28.2%), terpenes (16.1%), alkenes (7.2%), and aldehyde (7.1%). Meanwhile the major components of ALC extracts belong to fatty acids (23.2%), aldehyde (22.6%), naphthalenes (15.8%), alcohol esters (15.1%) and terpenes (7.6%).

Spectrum-activity association analyses
Spectrum-activity analysis was used to identify activity-related chemical markers from the complex ingredients of natural products due to its reliability, efficiency, and simplicity (Wu et al. 2021). Pearson correlation is one of the correlation analyses methods, which is often used in medical and bioinformatics analysis because of its low computational complexity and high prediction accuracy (Chang et al. 2021). For better interpretation of the obtained compound data, we performed a principal component (PCA) and hierarchical clustering analyses (HCA) ( Figure S2C-D). PCA and HCA classified samples from different extraction methods into two subtypes and distinguished compound profiles of SFE and ALC. The Pearson correlation coefficient was used to assess the spectrum-activity relationships between the areas of 47 peaks in the fingerprint and the main osteoporosis parameters (BMD of lumbar spine, right femur and left femur) (Table S2). We screened out the key compounds with high correlation with pharmacodynamic through the PCC model ( Figure S3A-C and Table S3). Meanwhile, the high correlation compounds in different tissue sites are intersected to obtain the main ingredients. The functional activity of SFE extracts on lumbar vertebrae, right femur, and left femur showed a close positive correlation with 16 ingredients ( Figure  S3D). The biological effects of the ALC extracts correlated positively with 11 ingredients ( Figure S3D). In addition, we found six compounds that were commonly positively related to the anti-osteoporosis effect parameters ( Figure S3E and Table S3). Then we performed grey correlation analysis (GRA) analyses to further confirm the activity correlation (correlation coefficient > 0.6) of these potential biochemicalmarkers of Cinnamon extracts ( Figure S4A). Then we selected three compounds with a total relative amount of peak intensity over 2.0% (Table S4) including (E)-cinnamaldehyde, c-sitosterol, and (Z, Z)-9,12-octadecadienoic acid as chemical-markers of Cinnamomum cassia (L.) Presl extracts.
Preclinical studies reported that (E)-cinnamaldehyde could promote osteogenesis by increasing osteoblast differentiation and inhibiting osteoclast formation ) and active multiple bone formation and differentiation pathways including WNT/CTNNB1, BMP/SMAD, MAPK pathway, and RANKL/OPG . Sitosterol was reported to improve bone formation through the upregulation of osteoblast-specific chemical markers (ALP, BSP, OCN, OPN, COL I), transcription factors (RUNX2, CBFA1, SP7), local factors (reactive oxygen species, nitric oxide) and the OPG/ RANKL signalling . Previous animal study also indicated that Octadecadienoic acids could increase bone mineral density and minimise femoral bone loss in OVX rats (Watkins et al. 2006).

The activity validation of chemical-markers of cinnamon extracts
As shown in Figure S4B, after culture in the medium containing each compound respectively, the MC3T3-E1 cell colony number remarkably increased compared with the blank control group (p < 0.05), suggesting that all selected chemical markers could promote the proliferation of osteoblast precursor cells ( Figure S8B). In addition, the results of alkaline phosphatase staining of MC3T3-E1 cells are shown in Figure S4C, all the three selected key chemical markers can significantly induce the increase of ALP content in osteoblast precursor cells (p < 0.05) ( Figure S8C).

Experimental
For the details of this section, please refer to supplementary material.

Conclusion
In conclusion the supercritical CO 2 extracts from Cinnamomum cassia (L.) Presl showed potential anti-osteoporosis efficacy and the mechanisms may at least related to enhance the proliferation and differentiation of osteoblast cells through upregulating intercellular ALP activity and gene expression of ALP, BMP-2, and RUNX2. The spectrum-activity analysis indicated that potential anti-osteoporosis-oriented chemicalmarkers could be (E)-Cinnamaldehyde, c-Sitosterol, and (Z, Z)-9,12-Octadecadienoic acid, which could significantly induce the proliferation and ALP activity in MC3T3-E1 cells. The study can be considered a first step in revealing the anti-osteoporosis activity of Cinnamon extracts which is compatible with its folk uses in the prevention of osteoporosis disorders.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This research was supported by Natural Science Foundation of Zhejiang Province grants (LY19H280001); the Traditional Chinese Medicine Science and Technology Plan of Zhejiang