Osteogenic Potential and Bioactive Profiles of Piper sarmentosum Ethanolic Extract-Treated Stem Cells

Abstract

Piper sarmentosum is a well-known traditional herbal plant in various diseases treatments. Multiple scientific studies have also reported various biological activities exhibited by the plant’s extract, such as antimicrobial, anticarcinogenic and antihyperglycemic activities, and, in addition, a bone protective effect in ovariectomized rats has been reported. However, no known Piper sarmentosum extract is involved in osteoblast differentiation using stem cells. Our study aims to identify the potential of P. sarmentosum ethanolic extract to induce osteoblast differentiation of human peripheral blood stem cells. Prior to the assay, the proliferation ability of the cells was observed for 14 days and the presence of hematopoietic stem cells in the culture was determined by the expression of SLAMF1 and CD34 genes. During the differentiation assay, the cells were treated with P. sarmentosum ethanolic extract for 14 days. Osteoblast differentiation was examined using an (alkaline phosphatase) ALP assay, by monitoring the expression of osteogenic gene markers and by von Kossa staining. The untreated cells served as the negative control, while cells treated with 50 µg/mL ascorbic acid and 10 mM β-glycerophosphate acted as the positive control. Finally, the determination of the compound profile was performed using a gas chromatography-mass spectrometry (GC-MS) analysis. The isolated cells were able to proliferate for 14 days during the proliferation assay. The expression of hematopoietic stem cell markers was also upregulated during the 14 days assay. Following the differentiation induction, the ALP activity exhibited a significant increase (p < 0.05) from day 3 of the differentiation assay. A molecular analysis also showed that the osteogenic markers ALP, RUNX2, OPN and OCN were upregulated compared to the positive control. The presence of mineralized cells with a brownish-stained morphology was observed, indicating the mineralization process increased in a time-dependent manner regardless of the concentration used. There were 54 compounds observed in the GC-MS analysis, including β-asarones, carvacrol and phytol, which have been shown to possess osteoinductive capacities. Our results demonstrate that the ethanolic extract of P. sarmentosum can induce osteoblast differentiation of peripheral blood stem cells. The extract contains potent compounds which can potentially induce the differentiation of bone cells, i.e., osteoblasts.

Evaluation of in vitro osteoblast and osteoclast differentiation from stem cell: a systematic review of morphological assays and staining techniques

“…A total of 14 articles related to the morphological approach, published between 2013 and 2023 were selected. The human cultured stem cells used for the in-vitro studies include both peripheral blood mononuclear cells (PBMSC) ( Kleinhans et al, 2015 ; Maria et al, 2018 ; Steller et al, 2020 ; Grünherz et al., 2020 ; Zainol Abidin et al., 2023 ) and mesenchymal stem cells (MSc) involved 5 studies ( Maria et al, 2017 ; Hashimoto et al, 2018 ; Liu et al, 2018 ; Ganguly et al, 2020 ; Nugraha et al, 2023 ) whereas periodontal ligament stem cells (PDLSC) ( Di Vito et al, 2020 ; Frasheri et al, 2023 ) and dental pulp stem cells (DPSC) ( Escobar et al, 2020 ; Escobar et al, 2023 ) involved two studies ( Table 3 ). The outcomes of the risk of bias assessment in Table 4 reflect the quality assessment analysis.…”

“…Seven studies focus solely on the staining of osteoblast differentiation ( Escobar et al, 2020 ; Escobar et al, 2023 ; Ganguly et al, 2020 ; Di Vito et al, 2020 ; Frasheri et al, 2023 ; Zainol Abidin et al., 2023 ; Nugraha et al, 2023 ), three studies only on morphological evaluation methods for osteoclast differentiation ( Kleinhans et al, 2015 ; Steller et al, 2020 ; Grünherz et al., 2020 ) and another four articles involved qualification analysis of both osteoblast and osteoclast differentiation ( Hashimoto et al, 2018 ; Maria et al, 2018 ; Liu et al, 2018 ) ( Table 3 ). All the staining techniques were proven to be successful in evaluating the presence of osteoblast and osteoclast differentiation in cells.…”

“…Therefore, von Kossa staining can be a preliminary indicator of mineralization and requires confirmation with other approaches due to potential non-specificity. The von Kossa staining method, although less commonly utilized in osteoblast differentiation, was identified in three articles ( Hashimoto et al, 2018 ; Frasheri et al, 2023 ; Zainol Abidin et al., 2023 ) as detailed in Table 3 and Fig. 2 .…”

Analysis Of Antioxidant and Anticancer Properties in Methanolic Extract of Piper Sarmentosum Roxb. Against Mcf-7 Cells

“…In The antioxidant and anticancer properties of the methanolic extract of P. sarmentosum can be attributed to the synergistic effects of its diverse array of compounds. Phenolic compounds, flavonoids, alkaloids, and terpenoids such as myricetin, quercetin, apigenin, naringenin, rutin and vitexin, collectively contribute to the observed effects, highlighting the potential of this natural extract as a source of bioactive compounds with therapeutic implications (Ismail et al, 2018;Zainol Abidin et al, 2023). The relationship between the total phenolic content, antioxidant activity, and…”

In silico identification of compounds from Piper sarmentosum Roxb leaf fractionated extract inhibit interleukin-6 to prevent rheumatoid arthritis

“…The leaves and aerial parts of the plant have been particularly significant in these compound discoveries. Several pharmacological agents, including antiosteoporosis ( Suhana Mohd Ramli et al, 2012 ; Ekeuku et al, 2023 ; Zainol Abidin et al, 2023 ), anti-inflammatory agents ( Zakaria et al, 2010 ; Makchuchit et al, 2017 ; Akmal et al, 2022 ), antioxidants ( Ugusman et al, 2011 ; Wang et al, 2017 ), antimalarial agents ( Najib Nik A Rahman et al, 1999 ), antimicrobial agents ( Masuda et al, 1991 ), antihypertensive agents ( Mohd Zainudin et al, 2015 ; Othman et al, 2022 ), antidiabetic agents ( Othman et al, 2022 ; Nguyen et al, 2023 ), anticancer agents ( Zainal Ariffin et al, 2009 ; Hematpoor et al, 2018 ) and neuroprotective ( Ridtitid et al, 1998 ; Yeo et al, 2018 )agents, have been found.…”