INTRODUCTION:

Periodontium, the fundamental tooth-supporting structures is comprised of the gingiva, the cementum, the periodontal ligament and the alveolar bone. It is most commonly affected by inflammatory disease called the periodontitis. It results in loss of integrity and health of periodontal tissues. In the current standard of care, therapy for periodontitis focuses on removal of the main etiology- that is the plaque, followed by local inflammation control.¹ This may involve scaling and root planing. Surgical intervention is needed in certain advanced cases.^2-5 However, in certain cases, elimination of etiology alone may not be sufficient and may require regeneration of lost tissues, especially the osseous component. Major regenerative therapies are guided tissue regeneration (GTR) and bone grafting, where both these tehcniques may offer variable and unpredicatable results.^6-9 Hence, a search for better technologies is warrented.¹⁰

In this direction, for grafting bone various autogenous and allogenous substances are tried.¹¹ Of them, Hydroxyapatite (HA) takes the center stage due to its presence in natural bone.^12,13 It has been used in combination with various natural and synthetic polymers. By virtue of its outstanding biocompatibility, bioactivity and osteo-conductivity, HA is considered to be an ideal material for the purpose.¹⁴ It is chemically a bioceramic material that can be stored in room temperature for long periods of time.¹⁵ Further, when natural source is not available or is expensive, synthetic sources are used with good results.^16,17 Biologically active substances like Bone morphogenetic proteins are used to enhance the growth. Or in other words, Hydroxyapatite is used as a vehicle for such active molecules.¹⁸ However, it these biologically derived growth factors are expensive and require special storage requirements. In order to alleviate these problems, naturally occurring phytochemicals can be used.¹⁹

Phytochemicals are chemical molecules that occur in plants. These may possess biological activity in animal physiological systems and for the basis of herbal medicines around the world. By combining traditional wisdom with modern knowledge, better and affordable standard of care can be offered to the patients. In this context, osteogenic phytochemicals are reported in the literature.²⁰ However, there is a lacuna in the literature in comparative efficacy of these phytochemicals. In this study three phytochemicals are compared – viz.β-sitosterol, Genistein and Emodin.

β-sitosterol is a phytochemical reported from cissus quadrangularis, an edible plant from south asia, belonging to grape family.¹⁹ Emodin is a phytochemical found in Cassia Occidentalis, an osteogenic herb widely used in Indian State of Andhra Pradesh.²¹ Genistein is reported to be osteogenic by activating Bone morphogenetic protein pathways and influencing major histocompatibility complexes.^22-23

In order to understand the interaction of phytochemicals with periodontium it is necessary to explore the biological pathways of periodontium and areas where these phytochemicals can act to restore the periodontium.

Major Biological Pathways Involved in Periodontal Regeneration:

Considered to be a powerful mediator of periodontal tissue regeneration is platelet-derived growth factor.²⁴ In the initial stages of wound healing after surgery, the combination of PDGF-B and IGF-I can significantly improve the formation of periodontal attachment apparatus.²⁵ Fibroblast growth factor has a significant impact on the recovery of periodontal soft tissue and bone. Human endothelial and periodontal ligament cell migration and proliferation were shown to be stimulated by concentrations of -FGF, according to Terranova et al. (1989).²⁶ Undifferentiated pluripotent cells can differentiate into cartilage and bone-forming cells with the aid of bone morphogenetic protein-2.²⁷ Along with -FGF, it promotes bone formation by enhancing angiogenesis and alkaline phosphatase activity. According to Sigurdsson (1995), rhBMP-2 treated sites displayed higher alveolar bone levels in comparison to the control sites.²⁸ In vitro, the insulin-like growth factor (IGF) I has a significant impact on the protein synthesis and mitogenesis of periodontal ligament fibroblasts. It encourages cementogenesis and osteogenesis.²⁹ IGF-I was shown to have mitogenic effects on periodontal ligament fibroblastic cells by Matsuda et al. in 1992.³⁰ IGF-I may induce the generation of DNA in periodontal ligament fibroblasts, according to a 1992 study by Soren Blom et al. This is probably due to IGF-I's ability to bind to high affinity cell surface receptors.³¹ TGF- selectively stimulates the proliferation of periodontal ligament fibroblasts while inhibiting the production of metalloproteinases and plasminogen activator. TGF-A also seems to prevent the growth of cells similar to osteoclasts.³²

Periodontal Ligament Derived Growth Factor is highly specific and potent autocrine chemotactic agent for human periodontal ligament cells involved in the regeneration of periodontal ligament. In addition to these molecules, many other molecules are known to affect regeneration. Some of the examples include Vit D³³, hormones, inflammatory mediators³⁴ and so on.^35-37

In light of these findings, in connection to use of phytochemicals, if they can interact with these pathways/recpetors, they can work to regenerate the periodontium. Therefore, in this review, 3 such major phytochemicals are analysed for their potential use in periodontal regeneration.

β-Sitosterol:

Beta-sitosterol, as name suggests is a phytosterol found in numerous plants. It is one of the active principles found in many bone regenerating plants such as cissus quadrangularis.^38,39 It structurally resembles cholesterol, however, beta-sitosterol cannot be converted to testosterone.⁴⁰ It has affinity towards estrogen receptor beta of osteoblasts.⁴¹ It has numerous benefits reported with its consumption. It acts with various molecular pathways to express pharmacological activities such as anti-tumor, anti-inflammatory, antioxidant and anti-osteoporotic effects etc.⁴² However, the concern of current paper is with its activity on bone, as it is a major part of periodontium. Since it is reported to regulate bone turnover and metabolism effectively, it is included in the review.

Bone loss is a major consequence of periodontal destruction leading to permanent periodontal deformity. In regenerative periodontics, any molecule acting to inhibit osteoclast and promote osteoblasts can be regarded as an advantageous molecule. While bone regeneration is orchestrated through multiple pathways, by far estrogen receptor β mediated signalling of bone growth has been well reported in the literature. Binding to the estrogen receptor beta in bone leads to activation the ERK/MAPK pathway. Its downstream signal transduction leads to BMP-2 levels and increased Runx2 expression which in turn leads to osteoblastogenesis.⁴³

The sources of β-sitosterol like Cissus quadrangularis have been shown to increase bone growth clinically.⁴⁴ However, the pathways of action need to be studied in cell lines. BMP-2/Smad/Runx2/Osterix is the pathway by which osteoblast differentiation takes place.⁴⁵ According to Nguyen et al., (2022), β-sitosterol was able to up-regulate multiple marker genes associated with osteoblast differentiation such as runx2, osx and col I by multiple times. Further, p38 and ERK protein expressions that cause mineralization also increased.⁴⁶ By far this is the only paper that shows the molecular pathways of action beta sitosterol while other papers illustrate osteogenic effects of herbs containing beta sitosterol.^47-50

Due to such potent osteogenic activity, the herbs have been used in osteo-regenerative scaffolds.⁵¹ In this direction, since bone regeneration is a major task in periodontal regeneration, introduction of beta sitosterol in the vicinity of periodontal defects can potentially stimulate bone growth leading to correction of periodontium.

Currently, beta sitosterol for periodontal use as drug or as scaffold is not commercially available in the market. However, it is to be learnt that clinical trials are on the way.⁵² Possible ways of delivery of beta sitosterol to periodontal bone include incorporation with grafts, scaffolds, ointments, injections and by systemic consumption. Summarily, beta sitosterol is confirmed to have osteogenic activity that can be tapped for use in periodontal regeneration. It has great potential for regenerating periodontal bone.

Genistein:

Genistein is a phytoestrogen present abundantly in legumes and soybean products. It has resemblance with estrogen and can bind to estrogen receptor (ER).^53-55 Genistein could prevent effectively act as estrogen in ovariectomy-induced osteoporosis in rats.^56,57 It is a milder agonist compared to estrogen.⁵⁸ Hence, Genistein can possibly substitute estrogen to exert osteogenic action. Of the pathways explained above, Genistein has been reported to enhance osteogenic gene expressions and its consequent osteogenic differentiation.⁵⁹

In a study by Dai et al. (2013), they have clearly elucidated the osteogenic properties of genestein at molecular level (Table 1).²² In addition, Kim at al., (2018) has shown that that two genes, Ereg and Efcab2 were upregulated and they enhanced osteoblastic cell differentiation.⁶⁰ In addition, three genes were down-regulated, viz. Hrc, Gli, and Ifitm5, which inhibits the differentiation. In addition, King et al., (2015) have indicated that Genistein can inhibit methotrexate-induced osteoclastogenesis.⁶¹ Numerous molecular pathways involved in promoting osteogenic bone formation and preventing osteoclastic bone resorption are impacted by genistein. Genistein stimulates osteogenesis by activating the p38MAPK-Runx2 pathway, which results in the induction of ALP, bone sialoprotein (BSP), OCN, and osteopontin (OPN) expression. Through the nitric oxide/cGMP pathway, genistein also promotes osteoblastic differentiation and proliferation. Tyrosine kinase inhibition by genistein prevents osteoclastic bone resorption. The actions of RANKL (receptor activator of NF-B ligand), RAN (receptor activator of NF-B), and OPG (osteoprotegerin) are likely how genistein influences the NF-B pathway, which encourages osteoclast formation.⁶²

Therefore, having sufficient information on effect of genestein on bone, it is now prudent to explore the possibility of using Genestein in periodontal regeneration. It has been reported that Genestein has anti-inflammatory and antimicrobial properties, especially in periodontium.⁶³ It downregulated the production of inflammatory mediators and osteoclast activators. It also reduced the mitochondrial oxidative damage. Genistein significantly reduced the amount of iNOS-derived NO and IL-6 that P. intermedia LPS-induced production of in RAW264.7 cells, according to Choi et al.'s (2016) research.⁶⁴ Additionally, genistein significantly inhibited the reductions in alveolar bone height and bone volume fraction brought on by the placement of ligatures in rats. Additionally, genistein treatment prevented changes in the microstructural characteristics of trabecular bone, such as changes in trabecular thickness, trabecular separation, bone mineral density, and structure model index, caused by ligature.

Therefore, modalities of delivering genitesin to periodontsl tissues can be explored. Numerous articles have reported preparation of genestein incorporated scaffolds, however use in periodontal regeneration is yet in its infancy and needs thorough evaluation in future.^65-66 In this regard there is a lacuna that has to be addressed.

Emodin:

Emodin is an anthraquinone derivative phytochemical that can isolated from numerous plants like cassia occidentalis, espically used as antibacterial agent and for bone healing.^67-68 It has wide range of pharmacological actions such as antineoplastic, anti-inflammatory, antioxidant and antimicrobial activities (including bacteria and viruses).⁶⁹ In the in vitro level, it promoted both the differentiation and the mineralization in MC3T3-E1 cells. At RNA level, emodin significantly induces the mRNA corresponding to BMP-9. Furthermore, it activates the BMP-Smad signaling axis and p38 MAPK pathway. In the in vivo level, The in vivo level, in ovariectomized rats, it acted as an adjuvent to estrogen.⁷⁰

Yang et al., (2014) have reported that emodin encourages osteogenic differenciation and supresses adipogenesis in stems cells.⁷¹They studied the molecular mechanisms of emodin on the processes of osteogenesis and adipogenesis in ovariectomized mouse and bone marrow mesenchymal stem cells.

In another study, emodin suppressed RANKL-induced osteoclast differentiation of macrophages in bone marrow. (RANKL-induced NF-κB, c-Fos, and NFATc1 expression). Further, emodin also raised the ALP, gene markers (Runx2 and osteocalcin) and the Alizarin Red-mineralization activity. Lee et al., (2008) showed that low concentrations of emodin could promote the osteoblast differentiation by inducing of the BMP‐2 gene by activating the PI3K‐Akt and/or MAP kinase–NF‐κB signalling pathways.⁷² There was a marked attenuation of LPS-induced bone erosion.⁷³ Therefore, there is a clear evidence to show that emodin possesses osteogenic potential at cellular level. It is prudent to evaluate the biomaterial applications of emodin.

In this regard, emodin loaded polycaprolactone Hydroxyapatite scaffolds are reported to have good osteogenic properties. In vitro studies showed promising proliferation and mineralization of surrounding bone.⁷⁴ Despite its proof in the in vitro and in vivo studies, reports regarding its use in bone scaffolds is scanty. Forementioned paper is such a rare report pertaining to applied form of emodin use. This opens new vistas for use of emodin inscaffolds.

With respect to its delivery periodontium, sustained delivery can be achieved by incorporation in to bone grafts. Emodin loaded liposomal nanoparticles were prepared by Gupta et al., (2011).⁷⁵ It was tested for anticancer activity, however, the feasibility of using such materials can be extended to periodontal region also. Preferably, emodin can possibly be loaded on to resorbable scaffolds of various composites and delivered to periodontal region. No such study has been reported in the literature and it’s a great vista to be studied.

Table 1: Summary of the review of major contributing articles.

Study	Analyses	Observation	Conclusion
Nguyen et al (2022)⁴⁶	They used MC3T3-E1 pre-osteoblasts. They analysed Viability, Proliferation, alkaline phosphatase activity, Mineralization assay, RTPCR for several marker genes	· β--sitosterol was non-toxic and significantly increased alkaline phosphatase activity, mineralization activity, and up-regulated several osteoblast differentiation marker genes. (runx2, osterex, COLI, II, p38, ERK)	From their results, β—sitosterol upregulated the osteogenic genes and can be a good chemical for increasing bone growth
Dai et al., (2013)²²	They differentiated Human Bone marrow cells using 1) Osteogenic medium 2) Adipogenic medium 3) Genestein They used · Alizarin red, Oil Red O stains, · Flow cytometry, · BrDU incorporation assay, · Quantitative realtime RTPCR (BMP2, SMAD5, RUNX2/CBFA1, Alkaline phosphatase and OSTEOCALCIN), · Small interfering RNA and western blot analysis for SMAD5 and BMP-2 · Western blot analysis for β-ACTIN, GAPDH, BMP2, SMAD5, RUNX2, ALP and Osteocalcin	· Genistein increased cell proliferation and osteoblastic differentiation but had no effects on cell apoptosis. · Genestein induced changes the expression of 29 genes, where those of BMP/SMAD signaling pathway had the strongest enhancement. · Osteogenic genes were upregulated by 1.5 times and its antagonists were downregulated 1.5 times.	From their results, it was seen that genestein downregulated pleuripotency and differentiated cells towards osteogenic pathway.
Chen et al (2017)⁷¹	They used murine pre-osteoblastic MC3T3-E1 cell lines, cultured in the presence of 0 to 10 μM emodin for 7 days. They analysed · BMP-9 gene expression (qRT-PCR for GAPDH, P38, ERK1, JNK, BMP-9, BMP-2, ALK1, Smad1, Smad9, Msx2 and Osterix), · Alkaline phosphatase activity, · Cell proliferation assay, · ALP staining assay, · alizarin red staining. · In vivo studies in Sprague-Dawley rats for · serum concentrations osteocalcin and TRACP 5b, · The trabecular microarchitecture of the L4 vertebra (microCT) · Mechanical properties of L3 vertibra.	· Emodin has no effect on cell growth. · Emodin increases differentiation and mineralization. · Emodin increased ALP activity. · Emodin stimulates the expression of osteogenic genes. · Emodin stimulates the BMPR-Smad signalling pathway. · Smad1/5/8 and p38 MAPK are turned on. · Emodin inhibits the overectomy-induced bone turnover.	Emodin is a good phytochemical for inducing bone growth
Yang et al., (2014)⁷¹	They have analysed in Mice bone marrow mesenchymal stem cells: Alkaline phosphatase assay Oil Red O Staining real time RT-PCR (Runx2, ALP, osterix, collagen type I, osteocalcin) Western blot (Runx2 and PPARγ protein) In vivo studies on female ICR rat · HandE · immunohistochemical assay · Micro-CT.	· Emodin has the ability to stimulate cell proliferation and osteogenic differentiation. · Emodin increased mRNA expression of osteogenic markers like BMP4, ColI, OC, Runx2, and osterix. · Emodin increased the number of osteoblasts and improved cancellous bone density. · In immunohistochemistry, emodin treatment increased the density of Runx2 positive staining and decreased the density of PPAR. · Emodin prevented bone loss, according to microCT.	Emodin is a good phytochemical for inducing bone growth

CONCLUSION:

Having proven by the previous investigators that β-sitosterol, Genitein and Emodin have effective osteogenic action when applied locally, it would be prudent to analyse the synergistic action for better osteogenesis. In principle, synergistic action is advantageous for both intensity and duration of action. As these molecules act at cellular level and have action on wide range of cells, it is necessary to deliver them locally with a proper scaffold or instrument. With periodontal regeneration as current focus, delivery through graft materials or suture threads can be though about, as these are established means of delivery to said region. Effective combinations of these molecules can be incorporated with periodontal regenerative materials (GTR) to shift the balance towards regeneration of periodontium. In that direction, most commonly used periodontal graft is some form of hydroxyapatite.⁷⁶ The market is flooded with various forms of hydroxyapatite from various sources viz. natural and synthetic, biomimetic, nanosized, porous blocks and so on. It has been added to with various polymers also for enhanced properties.

For artificial bone grafts, hydroxyapatite has been widely used as a bone replacement material.⁷⁷ Due to its structural and chemical similarities to naturally mineralized bone, it has been used to regenerate bone. Once inserted, hydroxyapatite-based BGs may chemically bond with bone to create an initial bone matrix that may consist of a network of organised collagen fibres or globular deposits. Addition of these phytochemicals to various bone grafts can enhance the periodontal regeneration.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

Centre of Medical and Bio-Allied Health Sciences and Research, Ajman University, Ajman P.O. Box 346, United Arab Emirates.

This research study was supported by Ajman University Internal Research Grant No. [DGSR Ref. 2022-IRG-DEN-6].

REFERENCES:

1. Health U.S.D.o., Human S. NIH Publication; 2000. Oral Health in America : A Report of the Surgeon General; pp. 155–188.

2. Deas D.E. Scaling and root planing vs. conservative surgery in the treatment of chronic periodontitis. Periodontol. 2000;71(1):128–139. 2016.

3. Graziani F. Nonsurgical and surgical treatment of periodontitis: how many options for one disease? Periodontol. 2000;75(1):152–188. 2017.

4. Smiley C.J. Evidence-based clinical practice guideline on the nonsurgical treatment of chronic periodontitis by means of scaling and root planing with or without adjuncts. J. Am. Dent. Assoc. 2015;146(7):525–535.

5. John M.T. Network meta-analysis of studies included in the Clinical Practice Guideline on the nonsurgical treatment of chronic periodontitis. J. Clin. Periodontol. 2017;44(6):603–611.

6. Villar C.C., Cochran D.L. Regeneration of periodontal tissues: guided tissue regeneration. Dent. Clin. 2010;54(1):73–92.

7. Kao R.T., Nares S., Reynolds M.A. Periodontal regeneration - intrabony defects: a systematic review from the AAP Regeneration Workshop. J. Periodontol. 2015;86(2 Suppl):S77–S104.

8. Lin Z., Rios H.F., Cochran D.L. Emerging regenerative approaches for periodontal reconstruction: a systematic review from the AAP Regeneration Workshop. J. Periodontol. 2015;86(2 Suppl):S134–S152.

9. Li F. Evaluation of recombinant human FGF-2 and PDGF-BB in periodontal regeneration: a systematic review and meta-analysis. Sci. Rep. 2017;7(1):65.

10. Liang Y, Luan X, Liu X. Recent advances in periodontal regeneration: A biomaterial perspective. Bioact Mater. 2020 Feb 28;5(2):297-308.

11. Zhao R, Yang R, Cooper PR, Khurshid Z, Shavandi A, Ratnayake J. Bone Grafts and Substitutes in Dentistry: A Review of Current Trends and Developments. Molecules. 2021 May 18;26(10):3007.

12. Singh S, Pal A, Mohanty S. Nano Structure of Hydroxyapatite and its modern approach in Pharmaceutical Science. Research Journal of Pharmacy and Technology. 2019;12(3):1463-72. doi: 10.5958/0974-360X.2019.00243.9

13. Girija C, Sivakumar MN. Amalgamation and characterization of hydroxyapatite powders from eggshell for functional biomedical application. Research Journal of Pharmacy and Technology. 2018;11(10):4242-4. doi: 10.5958/0974-360X.2018.00777.1

14. Ali F, Radvar M, Shayesteh E, Mohammadipour HS. Clinical assessment of a synthetic biomaterial containing hydroxyapatite and beta tricalcium phosphate in socket preservation. Research Journal of Pharmacy and Technology. 2022;15(11):5126-31. doi: 10.52711/0974-360X.2022.00862

15. Fang CH, Lin YW, Lin FH, Sun JS, Chao YH, Lin HY, Chang ZC. Biomimetic Synthesis of Nanocrystalline Hydroxyapatite Composites: Therapeutic Potential and Effects on Bone Regeneration. Int J Mol Sci. 2019 Nov 28;20(23):6002.

16. Kamadjaja MJK, Salim S, Subiakto BDS. Application of Hydroxyapatite scaffold from Portunus pelagicus on OPG and RANKL expression after tooth extraction of Cavia cobaya. Research Journal of Pharmacy and Technology. 2021; 14(9):4647-1. doi: 10.52711/0974-360X.2021.00807

17. Ratnayake JTB, Mucalo M, Dias GJ. Substituted hydroxyapatites for bone regeneration: A review of current trends. J Biomed Mater Res B Appl Biomater. 2017 Jul;105(5):1285-1299.

18. Kolk A, Handschel J, Drescher W, Rothamel D, Kloss F, Blessmann M, Heiland M, Wolff KD, Smeets R. Current trends and future perspectives of bone substitute materials–from space holders to innovative biomaterials. Journal of Cranio-Maxillofacial Surgery. 2012 Dec 1;40(8):706-18.

19. Raghavan RN, Somanathan N, Sastry TP. Evaluation of phytochemical-incorporated porous polymeric sponges for bone tissue engineering: a novel perspective. Proc Inst Mech Eng H. 2013 Aug;227(8):859-65.

20. Raghavan RN, Vignesh G, Kumar BS, Selvaraj R, Dare BJ. Phytochemicals: do they hold the future in stem cell differentiation. Int J Res Pharma. 2015;6(3):379-81.

21. Pal S, Kumar P, Ramakrishna E, Kumar S, Porwal K, Kumar B, Arya KR, Maurya R, Chattopadhyay N. Extract and fraction of Cassia occidentalis L. (a synonym of Senna occidentalis) have osteogenic effect and prevent glucocorticoid-induced osteopenia. J Ethnopharmacol. 2019 May 10;235:8-18.

22. Dai J, Li Y, Zhou H, Chen J, Chen M, Xiao Z. Genistein promotion of osteogenic differentiation through BMP2/SMAD5/RUNX2 signaling. Int J Biol Sci. 2013 Nov 21;9(10):1089-98.

23. Agil M, Laswati H, Kuncoro H, Ma'arif B. Effect of ethyl acetate fraction of Marsilea crenata Presl. Leaf extract on major histocompatibility complex class II expression in microglial HMC3 cell lines. Research Journal of Pharmacy and Technology. 2021;14(12):6374-8. doi: 10.52711/0974-360X.2021.01102

24. Dabra S, Chhina K, Soni N, Bhatnagar R. Tissue engineering in periodontal regeneration: A brief review. Dent Res J (Isfahan). 2012 Nov;9(6):671-80.

25. Lynch SE, Buser D, Hernandez RA, Weber HP, Stich H, Fox CH, et al. Effects of platelet derived growth factor/insulin like growth factor-1 combination on bone regeneration around titanium implants. Results of a pilot study on beagle dogs. J Periodontol. 1991;62:710–6.

26. Terranova VP, Odziemiec C, Tweden KS, Spadone DP. Repopulation of dentin surfaces by periodontal ligament cells and endothelial cells. Effect of basic fibroblast growth factor. J Periodontol. 1989;60:293–301.

27. Wozney JM, Rosen V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop Relat Res. 1998;346:26–37.

28. Sigurdsson TJ, Lee MB, Kubota Kl. Periodontal repair in dogs: Recombinant bone morphogenetic protein 2 significantly enhances periodontal regeneration. J Periodontol. 1995;66:131–8.

29. Bennett NT, Schultz GS. Growth factors and wound healing: Biochemical properties of growth factors and their receptors. Am J Surg. 1993;165:728–37.

30. Matsuda N, Lin WL, Kumar NM, Cho MI, Genco RJ. Mitogenic, chemotactic and synthetic response of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro. J Periodontol. 1992;63:515–25.

31. Blom S, Holmstrup P, Dabelsteen E. The effect of insulin like growth factor 1 and human growth hormone on periodontal ligament fibroblast morphology, growth pattern, DNA synthesis and receptor binding. J Periodontol. 1992;63:960–8.

32. Position Paper. The potential role of growth and differentiation factors in periodontal regeneration. J Periodontol. 1996;67:545–53.

33. van de Peppel J, van Leeuwen JP. Vitamin D and gene networks in human osteoblasts. Front Physiol. 2014 Apr 9;5:137. doi: 10.3389/fphys.2014.00137.

34. Sugiatno, E. , Herminajeng, E. and Sosroseno, W. (2020) The Role of Prostaglandin E2 on Osteoblast Proliferation Induced by Hydroxyapatite. Journal of Biosciences and Medicines, 8, 42-55. doi: 10.4236/jbm.2020.81006.

35. Plotkin LI, Bruzzaniti A. Molecular signaling in bone cells: Regulation of cell differentiation and survival. Adv Protein Chem Struct Biol. 2019;116:237-281. doi: 10.1016/bs.apcsb.2019.01.002.

36. Kurnia S, Prahasanti C, Hendro OV, Siki YO, Riawan W, Bargowo L. OPG and RANKL Signal Transduction in Osteoblast Regulation Post Application Extract Collagen in Osteogenesis. group. 2022 Jun 28;15:1-92261. doi: 10.52711/0974-360X.2022.00442

37. Prahasanti C, Perdana S. The Roles of Insulin Growth Factors-1 (IGF-1) in Bone Graft to increase Osteogenesis. Research Journal of Pharmacy and Technology. 2022; 15(4):1737-2. doi: 10.52711/0974-360X.2022.00291

38. Jain M, Nivedhitha MS, Deepak S, Rajesh KS. A Novel Natural Product for Bone Regeneration in Dentistry--A Review. Journal of Evolution of Medical and Dental Sciences. 2020 Sep 21;9(38):2833-9.

39. Mohammed M, Mahdi MF, Talib B, Abaas IS. Identification and Isolation of lupeol and β-sitosterol from Iraqi Bauhinia variegata and determination the cytotoxic activity of the hexane extract of its leaves, stems and flowers. Research Journal of Pharmacy and Technology. 2021; 14(11):5703-8. doi: 10.52711/0974-360X.2021.00991

40. Benign Prostatic Hyperplasia. In: Rakel D. Integrative Medicine (Fourth Edition). Elsevier; 2018.

41. Young H. Ju, Laura M. Clausen, Kimberly F. Allred, Anthony L. Almada, William G. Helferich, β-Sitosterol, β-Sitosterol Glucoside, and a Mixture of β-Sitosterol and β-Sitosterol Glucoside Modulate the Growth of Estrogen-Responsive Breast Cancer Cells In Vitro and in Ovariectomized Athymic Mice, The Journal of Nutrition, Volume 134, Issue 5, May 2004, Pages 1145–1151, https://doi.org/10.1093/jn/134.5.1145

42. Bao X, Zhang Y, Zhang H, Xia L. Molecular Mechanism of β-Sitosterol and its Derivatives in Tumor Progression. Front Oncol. 2022 Jun 8;12:926975. doi: 10.3389/fonc.2022.926975.

43. Singh P, Gupta A, Qayoom I, Singh S, Kumar A. Orthobiologics with phytobioactive cues: A paradigm in bone regeneration. Biomedicine and Pharmacotherapy. 2020 Oct 1;130:110754.

44. Brahmkshatriya HR, Shah KA, Ananthkumar GB, Brahmkshatriya MH. Clinical evaluation of Cissus quadrangularis as osteogenic agent in maxillofacial fracture: A pilot study. Ayu. 2015 Apr-Jun;36(2):169-73. doi: 10.4103/0974-8520.175542.

45. Ruangsuriya J, Charumanee S, Jiranusornkul S, Sirisa-Ard P, Sirithunyalug B, Sirithunyalug J, et al. Depletion of β-sitosterol and enrichment of quercetin and rutin in Cissus quadrangularis Linn fraction enhanced osteogenic but reduced osteoclastogenic marker expression. BMC Complementary Medicine and Therapies., 20 (2020), 10.1186/s12906-020-02892-w

46. Nguyen HT, Ngo QV, Le DT, Nguyen MT, Nguyen PT. β-sitosterol from Clinacanthus nutans Lindau enhances osteoblastogenic activity via upregulation of differentiation related genes and proteins. Bioscience, Biotechnology, and Biochemistry. 2022 Dec;86(12):1615-22.

47. Toor RH, Malik S, Qamar H, Batool F, Tariq M, Nasir Z, Tassaduq R, Lian JB, Stein JL, Stein GS, Shakoori AR. Osteogenic potential of hexane and dichloromethane fraction of Cissus quadrangularis on murine preosteoblast cell line MC3T3-E1 (subclone 4). J Cell Physiol. 2019 Dec;234(12):23082-23096. doi: 10.1002/jcp.28869..

48. Potu BK, Nampurath GK, Rao MS, Bhat KM. Effect of Cissus quadrangularis Linn on the development of osteopenia induced by ovariectomy in rats. Clin Ter. 2011;162(4):307-12.

49. Singh V, Singh N, Pal US, Dhasmana S, Mohammad S, Singh N. Clinical evaluation of cissus quadrangularis and moringa oleifera and osteoseal as osteogenic agents in mandibular fracture. Natl J Maxillofac Surg. 2011 Jul;2(2):132-6. doi: 10.4103/0975-5950.94466.

50. Nayak T, R K. An Assessment of the Osteogenic Potential of Cissus quadrangularis in Mandibular Fractures: A Pilot Study. J Maxillofac Oral Surg. 2020 Mar;19(1):106-112. doi: 10.1007/s12663-019-01230-z.

51. Sairaman S, Nivedhitha MS, Shrivastava D, Al Onazi MA, Algarni HA, Mustafa M, Alqahtani AR, AlQahtani N, Teja KV, Janani K, Eswaramoorthy R, Sudhakar MP, Alam MK, Srivastava KC. Biocompatibility and antioxidant activity of a novel carrageenan based injectable hydrogel scaffold incorporated with Cissus quadrangularis: an in vitro study. BMC Oral Health. 2022 Sep 5;22(1):377. doi: 10.1186/s12903-022-02409-6.

52. A clinical trial of YH14642 in patients with chronic periodontal disease - full text view [Internet

53. Rickard DJ, Monroe DG, Ruesink TJ. et al. Phytoestrogen genistein acts as an estrogen agonist on human osteoblastic cells through estrogen receptors alpha and beta. J Cell Biochem. 2003;89(3):633–46.

54. Wang TT, Sathyamoorthy N, Phang JM. Molecular effects of genistein on estrogen receptor mediated pathways. Carcinogenesis. 1996;17(2):271–5.

55. Wang H-J, Murphy PA. Isoflavone Composition of American and Japanese Soybeans in Iowa: Effects of Variety, Crop Year, and Location. Journal of Agricultural and Food Chemistry. 1994;42(8):1674–77.

56. Arjmandi BH, Alekel L, Hollis BW. et al. Dietary soybean protein prevents bone loss in an ovariectomized rat model of osteoporosis. Journal of Nutrition. 1996;126(1):161–67.

57. Uesugi T, Toda T, Tsuji K, Ishida H. Comparative study on reduction of bone loss and lipid metabolism abnormality in ovariectomized rats by soy isoflavones, daidzin, genistin, and glycitin. Biol Pharm Bull. 2001;24(4):368–72.

58. D'Anna R, Cannata ML, Marini H. et al. Effects of the phytoestrogen genistein on hot flushes, endometrium, and vaginal epithelium in postmenopausal women: a 2-year randomized, double-blind, placebo-controlled study. Menopause. 2009;16(2):301–6.

59. Liao QC, Xiao ZS, Qin YF, Zhou HH. Genistein stimulates osteoblastic differentiation via p38 MAPK-Cbfa1 pathway in bone marrow culture. Acta Pharmacol Sin. 2007;28(10):1597–602.

60. Kim M, Lim J, Lee JH, Lee KM, Kim S, Park KW, Nho CW, Cho YS. Understanding the functional role of genistein in the bone differentiation in mouse osteoblastic cell line MC3T3-E1 by RNA-seq analysis. Sci Rep. 2018 Feb 19;8(1):3257. doi: 10.1038/s41598-018-21601-9. Erratum in: Sci Rep. 2018 Apr 18;8(1):6429.

61. King TJ, Shandala T, Lee AM, Foster BK, Chen KM, Howe PR, Xian CJ. Potential Effects of Phytoestrogen Genistein in Modulating Acute Methotrexate Chemotherapy-Induced Osteoclastogenesis and Bone Damage in Rats. Int J Mol Sci. 2015 Aug 6;16(8):18293-311. doi: 10.3390/ijms160818293.

62. Ming LG, Chen KM, Xian CJ. Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling. Journal of Cellular Physiology. 2013;228:513–521.

63. Bhattarai G, Poudel SB, Kook SH, Lee JC. Anti-inflammatory, anti-osteoclastic, and antioxidant activities of genistein protect against alveolar bone loss and periodontal tissue degradation in a mouse model of periodontitis. J Biomed Mater Res A. 2017 Sep;105(9):2510-2521. doi: 10.1002/jbm.a.36109.

64. Choi EY, Bae SH, Ha MH, Choe SH, Hyeon JY, Choi JI, Choi IS, Kim SJ. Genistein suppresses Prevotella intermedia lipopolysaccharide-induced inflammatory response in macrophages and attenuates alveolar bone loss in ligature-induced periodontitis. Arch Oral Biol. 2016 Feb;62:70-9. doi: 10.1016/j.archoralbio.2015.11.019.

65. Luo S, Wang P, Ma M, Pan Z, Lu L, Yin F, Cai J. Genistein loaded into microporous surface of nano tantalum/PEEK composite with antibacterial effect regulating cellular response in vitro, and promoting osseointegration in vivo. Journal of the Mechanical Behavior of Biomedical Materials. 2022 Jan 1;125:104972.

66. Sarkar N, Bose S. Controlled release of soy isoflavones from multifunctional 3D printed bone tissue engineering scaffolds. Acta Biomater. 2020 Sep 15;114:407-420. doi: 10.1016/j.actbio.2020.07.006. Epub 2020 Jul 8.

67. Manisha Arora Nagpal, Navneet Nagpal, Sandeep Rahar, Gaurav Swami, Reni Kapoor. Pharmacological and Phytochemical Review on Cassia fistula. Research J. Pharm. and Tech. 4(2): February 2011; Page 214-222.

68. Chukwujekwu JC, Coombes PH, Mulholland DA, Van Staden J. Emodin, an antibacterial anthraquinone from the roots of Cassia occidentalis. South African Journal of Botany. 2006 May 1;72(2):295-7.

69. Dong X, Fu J, Yin X, Cao S, Li X, Lin L; Huyiligeqi; Ni J. Emodin: A Review of its Pharmacology, Toxicity and Pharmacokinetics. Phytother Res. 2016 Aug;30(8):1207-18. doi: 10.1002/ptr.5631.

70. Chen X, Zhang S, Chen X, Hu Y, Wu J, Chen S, Chang J, Wang G, Gao Y. Emodin promotes the osteogenesis of MC3T3-E1 cells via BMP-9/Smad pathway and exerts a preventive effect in ovariectomized rats. Acta Biochim Biophys Sin (Shanghai). 2017 Oct 1;49(10):867-878. doi: 10.1093/abbs/gmx087.

71. Yang F, Yuan PW, Hao YQ, Lu ZM. Emodin enhances osteogenesis and inhibits adipogenesis. BMC complementary and alternative medicine. 2014 Dec;14(1):1-9.

72. Lee SU, Shin HK, Min YK, Kim SH. 2008. Emodin accelerates osteoblast differentiation through phosphatidylinositol 3‐kinase activation and bone morphogenetic protein‐2 gene expression. Int Immunopharmacol 8: 741–747.

73. Kim JY, Cheon YH, Kwak SC, Baek JM, Yoon KH, Lee MS, Oh J. Emodin regulates bone remodeling by inhibiting osteoclastogenesis and stimulating osteoblast formation. J Bone Miner Res. 2014 Jul;29(7):1541-53. doi: 10.1002/jbmr.2183.

74. Pan Y, He M, Chen S, Meng Y, Wang C, Ni X. Preparation and Osteogenic Efficacy of Emodin-loaded Hydroxyapatite Electrospun Fibers. Journal of Bionic Engineering. 2022 Dec 24:1-2.

75. Gupta V, Mun GH, Choi B, Aseh A, Mildred L, Patel A, Zhang Q, Price JE, Chang D, Robb G, Mathur AB. Repair and reconstruction of a resected tumor defect using a composite of tissue flap–nanotherapeutic–silk fibroin and chitosan scaffold. Annals of biomedical engineering. 2011 Sep;39(9):2374-87.

76. Keerthic Aswin S, Jothishwar S, Visvavela Chellaih Nayagam P, G. Priya. Scaffolds for Biomolecule Delivery and Controlled Release–A Review. Research J. Pharm. and Tech 2018; 11(10): 4719-4730. doi: 10.5958/0974-360X.2018.00861.2

77. Shaikh MS, Zafar MS, Alnazzawi A, Javed F. Nanocrystalline hydroxyapatite in regeneration of periodontal intrabony defects: A systematic review and meta-analysis. Annals of Anatomy-Anatomischer Anzeiger. 2022 Feb 1;240:151877.

Received on 18.05.2023 Modified on 11.08.2023

Research J. Pharm. and Tech 2024; 17(2):686-692.

DOI: 10.52711/0974-360X.2024.00106