Int J App Pharm, Vol 17, Issue 3, 2025,1-12Review Article

THE ANTIOSTEOPOROTIC POTENTIAL OF HESPERIDIN AND ADVANCED DELIVERY SYSTEMS

VIJISHNA L. V.1, AKANKSHA D. DESSAI2, USHA Y. NAYAK2, RICHARD LOBO1*

1Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal-576104, Karnataka, India. 2Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal-576104, Karnataka, India
*Corresponding author: Richard Lobo; *Email: richard.lobo@manipal.edu

Received: 10 Dec 2024, Revised and Accepted: 14 Feb 2025

ABSTRACT

This study explores Hesperidin (HP) and its bone-protective effect against Osteoporosis (OP), summarizing its healing mechanisms supported by in vitro and in vivo evidence and insights into its ethnobotanical significance and advanced drug delivery systems. To gather information on the antiosteoporotic potential of HP, we thoroughly searched many scientific databases, including Science Direct, Google Scholar, PubMed, and Scopus, for articles published between 1990 and 2025. Data were collected using the keywords HP, traditional uses, phytochemistry, anti-OP, and drug delivery systems. Only studies published in English are considered for this review. It has gained attention for potential health benefits, especially the osteoprotective effect. In vitro studies found that HP reverses dexamethasone-induced inhibition of osteogenic differentiation by suppressing the p53 (Protein 53) pathway. In rat models of Postmenopausal (PM), senile, and disuse OP, HP showed bone-protective benefits. Clinical trials revealed a 15% increase in serum calcium and a 25% increase in osteocalcin levels, indicating enhanced bone formation. Comparative analysis showed that HP's efficacy in increasing bone mineral density is similar to that of bisphosphonates. The findings demonstrate that HP is an excellent therapeutic candidate that protects the skeleton through various mechanisms. Future research should focus on developing HP-based nutraceuticals or pharmaceuticals, integrating traditional knowledge with modern pharmacological approaches to enhance bone health. Despite its potential, the efficacy of HP formulations in treating OP has not yet been investigated.

Keywords: HP, OP, Advanced drug delivery systems

© 2025 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2025v17i3.53661 Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

OP is a significant public health problem characterized by low bone mass and Bone Mineral Density (BMD) (T-score: −2.5 and below), bone tissue deterioration, and disruption of bone microarchitecture, all of which raise the risk of fractures [1]. The bone loss progresses due to increased bone resorption and decreased bone production rates. It is a systemic bone and silent disease that exhibits no signs in its early stages [2]. Worldwide, one-fifth of men and more than half of women over 50 are at high risk of developing OP or fragility fractures [3, 4]. According to epidemiologic data from 2015, 46 million Indian women have OP [5]. Fractures caused by OP can lead to increased pain, disability, overall healthcare costs, and mortality [6]. The prognosis for OP is typically poor, and it is associated with several consequences, such as nerve irritation, blood vessel damage, systemic pain, and an elevated chance of fractures [7, 8]. The body continually takes in and replaces bone tissue, but in OP, eliminating old bone is more significant than developing a new bone [9, 10]. It has a high morbidity and mortality rate, and the risk of developing OP can increased by hormone deficiencies, age-related disorders, steroid therapy, nicotine addiction, rheumatoid arthritis, and metabolic disorders (diabetes, dyslipidemia, and metabolic syndrome). Pharmacologic treatments for OP include anabolic drugs like Teriparatide, Abaloparatide, and Parathyroid hormone analogs; Selective Estrogen Receptor Modulators (SERM) like Raloxifene; and Antiresorptive drugs like Denosumab and Bisphosphonates [6]. However, there are a few severe adverse effects linked to the use of these drugs. In one example, long-term Bisphosphonate use has been linked to a higher risk of atypical fracture and jaw osteonecrosis [11]. In addition, individuals may encounter typical side effects that primarily affect the cardiovascular, gastrointestinal, and endocrine systems.

HP is a bioflavonoid primarily in citrus fruits, such as lemons and oranges [12]. It has drawn much interest due to its wide range of pharmacological characteristics, backed by substantial in vitro, in vivo, and clinical data. These characteristics include anti-inflammatory, anti-apoptotic, antimicrobial, anticancer, neuroprotective, cardioprotective, skinprotective, osteoprotective effects and antioxidant [13]. HP can target various intracellular receptors, enzymes, signaling molecules, antioxidant enzymes, and transcription factors [14-16]. HP's bone-osteoprotective activities, covered in more detail below, are directly linked to some of its therapeutic benefits. Due to its potent anti-inflammatory and antioxidant qualities, HP can lessen the adverse effects of OP-induced abnormalities in osteoblastogenesis and osteoclastogenesis.

The main subject of this review paper is the in vitro and in vivo experimental proof of HP's effectiveness in reducing bone loss and promoting bone growth. HP's poor absorption and considerable first-pass metabolism face serious bioavailability issues. Limited research has been done on enhancing its bioavailability using advanced formulations, like nano-delivery systems. HP's molecular mode of action and its potential as a therapeutic osteoprotective drug are also covered in this review, along with its ethnobotanical significance and advanced drug delivery systems.

Methodology

We made a thorough literature search to find relevant material from reliable databases such as scopus, PubMed, EMBASE, and the Cochrane Library. Fig. 1 depicts the process of screening, selecting, and including the references gathered from the literature. The search phrases 'OP, HP, and advanced drug delivery systems' The search concentrated on articles published between 1990 and 2024 and selected only English ones.

RESULTS

Epidemiology, etiology, and risk factors of op

More than 200 million individuals worldwide have OP and around nine million OP fractures occur annually [17], adversely affecting people's quality of life and imposing a considerable economic burden [18, 19]. With an increasing aging population, fractures due to OP increase rapidly each year. It is a severe health problem, mainly in senior citizens. After menopause, during the first five years, women lose almost 10% of their bone mass [17].

Two significant causes of OP are the lack of estrogen in PM women and aging-related dietary Ca and vit. D supplementation [20, 21]. Sex hormones can influence the proliferation, differentiation, and apoptosis of Osteoblasts (OB) and Osteoclasts (OC) by affecting OP-related signaling pathways [22]. Age, sex steroid deficiency, reduced bone quality, microarchitectural integrity disruption, and use of glucocorticoids are the factors that increase OP fracture [19]. The risk factors of OP are discussed in fig. 2.

Fig. 1: Flowchart for article search, screening and selection in literature review
Source: BioRender.com

Fig. 2: Risk factors associated with OP
Source: BioRender.com

Fig. 3: The dynamic process of bone remodeling
Source: BioRender.com

Mechanism of bone remodeling

The human skeleton comprises bones, cartilage, ligaments, and tendons, making up 20% of the body's total weight [23]. The bones support and shield the body's essential organs [24]. It is a calcified tissue that contains 10% water, 30% organic components such as protein, and 60% inorganic components, often known as hydroxyapatite [23]. Two types of tissues appear in a bone: cortical and cancellous [25]. Cortical bone is shown to have tightly packed collagen fibrils and is protective [26, 27]. Cancellous bones, called trabecular bones, have a porous matrix that is loosely organized and responsible for metabolic functions. Bone homeostasis refers to the dynamic equilibrium of bone formation and resorption [28]. The maintenance of bone homeostasis involves three separate cell types. Osteocytes (OT) are mature bone cells, OB are bone-producing cells, and OC are bone resorption or breakdown cells. OB is formed from Mesenchymal Stem Cells (MSC), while OC is derived from Hematopoietic Stem Cells (HSC) of bone marrow [29]. Bone turnover and remodeling occur throughout life and involve bone formation and resorption processes [30]. Bone tissue constantly remodels and has considerable regenerative capacity [31]. Maintaining bone health involves a well-balanced diet rich in Ca and Vit. D, exercise, avoiding smoking, and restricting alcohol use [32]. The mechanism of bone remodeling/homeostasis is shown in fig. 3.

Bone cells (OB, OC, OT), hormones, cytokines, ions, growth factors, transcriptional factors, and extracellular matrix proteins help maintain bone homeostasis [33]. The bone tissue environment controls bone matrix secretion and resorption. OB produces the proteins Alkaline Phosphatase (ALP) and Osteocalcin (OCN), which are directly engaged in matrix mineralization [34]. Transcription factors, such as Runt-Related Transcription Factor-2 (RunX2) and Osterix (OSX), contribute to the OB development of undifferentiated cells like MSC [35]. Bone Morphogenetic Protein (BMPs) and the Wingless Related Integration Site (Wnt/β-catenin) signaling pathways play a vital role in regulating transcription factors involved in OB differentiation. WNT signaling suppresses MSC and promotes OB development [36]. Wnt/β-catenin signaling indirectly inhibits OC development and bone resorption by increasing the release of Osteoprotegerin (OPG). OP is a widespread chronic disease characterized by low bone density, altered microstructure, and bone fragility, leading to low-impact fractures in affected individuals. The discovery of a few mutations that cause sporadic human diseases has identified the WNT signaling pathway as a candidate for therapeutic intervention aimed at increasing bone mass and strength. In particular, inhibition of sclerostin, a WNT antagonist secreted by OT, has proven in clinical trials to be a very efficient osteo-anabolic approach. One year of monthly administration of antibodies to sclerostin rapidly decreases bone resorption and increases bone formation and bone density at all sites, decreasing markedly fracture risk in treated patients. Their effect is, however, limited in time, and cardiovascular adverse events have been reported in one clinical trial targeting WNT signaling in the treatment of OP [15]. It also inhibits chondrogenesis and adipogenesis. OPG is RANKL's (Receptor Activator of Nuclear Factor Kappa-B Ligand) natural decoy receptor [37]. RANKL helps to differentiate OC [38]. OPG binds with RANKL and blocks the signaling pathways that help in OC activation [15]. OC regulates bone formation and promotes tissue resorption using proteolytic enzymes and acids secreted into the bone matrix. Matrix Metalloproteinases (Mmp) and cysteine proteases are the primary enzymes involved in the breakdown of the organic matrix. The BMP signaling pathway promotes OC development and bone production [14].

Methods of diagnosis of OP

OP has no clinical manifestations until a fracture occurs. The gold standard test for BMD is DEXA (Dual-Energy X-ray Absorptiometry). It is painless and non-invasive; X-rays scan specific to bones such as the spine, hip, and forearm. The findings were expressed as a T-score, which compared bone density to healthy young individuals. A T score of-2.5 or less on the DEXA test indicates OP [39]. All women over 65 should be examined for OP using DEXA to determine BMD. Most patients at elevated risk for fractures do not receive adequate evaluation or treatment for preventing future fractures. The current OP treatment mainly focuses on preventing bone resorption and promoting bone formation [40]. According to DEXA, the T-score interpretation is given in fig. 4.

T-score above-1: Normal

T-score in between-1 and-2.5: Osteopenia

T-score-2.5 or low: OP

Fig. 4: T-score interpretation for OP diagnosis
Source: BioRender.com

Current treatment available for OP

OP is treated with the help of chemical drugs like Alendronate, Risedronate, Zoledronate, Iodine, Monoclonal antibodies like Denosumab, and Hormone replacement therapy [41]. The ongoing treatment options are discussed in table 1.

Currently used medications for the treatment of OP produce serious health issues and are not able to offer long-term solutions [46]. There is a need to come up with better alternatives. Compared to existing OP treatments, phytocompounds such as flavonoids, alkaloids, and terpenoids have several benefits. Because of their anti-inflammatory, antioxidant, and bone-protective qualities, these naturally occurring chemicals are increasingly acknowledged for their potential as therapeutics.

Natural products as the future of OP protection

Several natural compounds have been explored to treat OP to overcome the limitations of the current treatments, which show antiosteoporotic activity, promoting bone formation and suppressing bone resorption. A few examples of the phytomolecules that show OP activities are summarized in table 2.

Table 1: Current therapeutic approach for OP

Medication Mechanism Route of administration Side effects References
Abaloparatide Binding to the N-terminal moiety and the Para Thyroid Hormone (PTH) type 1 receptor Subcutaneous Joint discomfort, redness, or swelling at the place where the drug was injected [42, 43]
Alendronate Inducing OC apoptosis Oral Femoral fractures, OP of the jaw, risk for osteomalacia, skeletal lesions [11, 44]
Bazedoxifene/Conjugated Estrogens SERM Oral route Symptoms may include abdominal pain, nausea, muscle spasms, diarrhea, dyspepsia, oropharyngeal pain, neck pain, and dizziness. [45]
Calcitonin-salmon Binds to the calcitonin receptor found primarily in OC Nasal spray Muscle pain, body aches [46]
Denosumab (human igG2 monoclonal antibody) Against RANKL inhibits osteoclastogenesis Subcutaneous Skin eczema, cellulitis [47]
Ibandronate Inducing OC apoptosis Oral, Intravenous Femoral fractures, OP of the jaw, risk for osteomalacia, skeletal lesions [11, 44]
Raloxifene (SERM) It interacts with the RANK/RANKL/OPG system to prevent bone resorption Oral route Venous thromboembolism, stroke, breast, endometrial, ovarian cancers [48–51]
Risedronate Inducing OC apoptosis Oral route Femoral fractures, osteonecrosis of the jaw, risk for osteomalacia, skeletal lesions [11, 44]
Romosozumab (monoclonal antibody) It acts against the sclerostin pathway, enhancing bone formation and reducing bone resorption. Subcutaneous Skin eczema, cellulitis [52]
Teriparatide Binding to the N-terminal moiety and PTH type 1 receptor Subcutaneous Bone pain, cardiac arrhythmia, osteosarcoma [39, 53]
Zoledronate Inducing OC apoptosis Intravenous Femoral fractures, osteonecrosis of the jaw, risk for osteomalacia, skeletal lesions [11, 44]

Table 2: Phytomolecules exhibiting antiosteoporotic activity

Phytoconstituents Plant Family Mechanism against OP References
Astilbin Engelhardia roxburghiana Juglandaceae Astilbin inhibits bone loss in Ovariectomized (OVX) mice by inhibiting RANKL induced osteoclastogenesis [54, 55]
Baicalein Scutellaria baicalensis Lamiaceae Baicalein activates the mTORC1 (Target Of Rapamycin Complex 1) signaling pathway in MC3T3-E1cells (OB Precursor Cell Line), encouraging to develop into OB [56, 57]
Bavachin Psoralea corylifolia Fabaceae Bavachin exhibited OB proliferation-stimulating activities in the UMR106 cell line (Rat Osteosarcoma Cell Line) [58]
Beriberin

Berberis aristata

 Berberidaceae Through the Adenosine Monophosphate Activated Protein Kinase (AMPK) activation pathway, berberine protects diabetic rats from bone loss caused by pioglitazone [59, 60]

Calycosin

Astragalus membranaceus Leguminosae Calycosin inhibits RANKL-mediated osteoclastogenesis via Mitogen Activated Protein Kinase (MAPK) and NF-κB (Nuclear Factor-Kappa B) inhibition [61, 62]
Catechin Bergenia crassifoli Saxifragaceae Treatment with catechin reduced bone-resorbing cytokines Tumor Necrosis Factor-α (TNF-α) and Interleukin-6 (IL-6) production and apoptosis in OB [63, 64]

Ulmus davidiana

 Ulmaceae Promotes apoptosis in OB [63, 65]
Corylin Psoralea corylifolia Fabaceae In bone micromasses made of mesenchymal progenitor cells, calcineurin induced osteogenesis [66]
Cyanidin Lonicera caerulea Caprifoliaceae Cyanidin-3-glucoside controls OB differentiation through the Extra Cellular Signal Regulated Kinase (ERK1/2) signaling pathway [67, 68]
Delphinidin Solanum melongena Solanaceae Delphinidin reduces bone resorption by blocking RANKL-induced development of OC in an OP model [69, 70]
Engeletin Engelhardia roxburghiana Juglandaceae Inhibiting the osteoclastogenesis [54]
Eriodictyol Afzelia africana Fabaceae Eriodictyol inhibits RANKL-induced OC development [71, 72]

Equol

Pueraria lobata Fabaceae Equol activates the Estrogen Receptor (ER) to stimulate the growth and differentiation of rat OB [73, 74]
Formononetin Actaea racemosa Ranunculaceae

Formononetin inhibits the NF-κB, Proto-Oncogene

(c-Fos) and cytoplasmic 1 signaling pathway activation generated by RANKL

 [75, 76]

Genistein

Glycine max Fabaceae Genistein stimulates the ER, MAPK-Runx2, and NO/Cyclic Guanosine Monophosphate (cGMP) pathways to promote OB differentiation and maturation. It also blocks the NF-κB signaling pathway and induces the osteoclastogenic inhibitor OPG to suppress OC production and bone resorption. [77, 78]

Glycitein

 Semen sojae praeparatum Leguminosae Glycitein suppresses OC generation and induces OC apoptosis [79, 80]
HP Citrus sinensis Rutaceae OP is alleviated by activating the estrogen signaling pathway via ESR1 [81, 82]
Icariin Epimedium brevicornu Berberidaceae Icariin enhanced Messenger Ribo-Nucleic Acid (mRNA) expression of Runx2 and osterix, while decreasing protein expression of Tumor Protein 38 (P38) and Jun N-Terminal Kinase (JNK) [77, 83]
Icaritin Herba epimedii Berberidaceae Reduced adipogenesis via the Glycogen Synthase Kinase-3 β (GSK-3β)/β-catenin signaling pathway [77]

Isorhamnetin

Salsola imbricata Amaranthaceae Isorhamnetin controls reactive oxygen species to prevent osteoclastogenesis and protect chondrocytes [84, 85]
Kaempferol Cuscuta chinensis Convolvulaceae Kaempferol enhanced ALP activity in UMR-106 cells [86]
Luteolin

Juncus acutus

 Juncaceae Luteolin promotes human periodontal ligament cells osteogenic development [87, 88]
Malvidin Vaccinium angustifolium Ericaceae Promoting OB differentiation while suppressing OC production and differentiation [89, 90]

Myricetin

Clitoria ternatea Fabaceae Myricetin inhibits osteoclastogenesis and promotes osteogenic differentiation [91, 92]
Naringenin Piper sarmentosum Piperaceae Naringenin functions as a superoxide scavenger, assisting the endogenous antioxidant defense mechanism in protecting bone against OP [93]
Naringin Citrus paradisi Rutaceae Naringin stimulates the expression of BMP-2 and promotes bone formation, bone MSC proliferation, and osteogenic differentiation [94]
Nobiletin

Citrus sinensis

 Rutaceae In mice with defective ovariectomy, nobiletin prevents bone resorption and preserves bone mass [95, 96]
Peonidin Vaccinium myrtillus Ericaceae Increases OB differentiation and decreases RANKL-induced bone resorption [97, 98]
Petunidin Vaccinium myrtillus Ericaceae Petunidin suppresses osteoclastogenesis and down regulates c-fos, Nuclear Factor of Activated T-cells Cytoplasmic (NFATc 1) and Mmp9 [98, 99]
Quercetin Helminthostachys zeylanica Ophioglossaceae The studies were carried out using Murine Macrophage Cell Line (RAW264.7). Quercetin inhibited RANKL-induced OC development in RAW264.7 cells showed IC50 value of 1.8±0.2 μm [100]
Resvertrol

Polygonum cuspidatum

 Polygonaceae OT proliferation and quantity in bone are both stimulated by resveratrol [101, 102]
Rutin Chrozophora tinctoria Euphorbiaceae Rutin 1 μm treatment for 48 h significantly increases the activity of all ossification indicators, including ALP enzyme, OCN hormone, and active Vit-D3 [103, 104]
Tangeretin

Citrus reticulata

 Rutaceae Tangeretin suppresses bone loss and preserves bone mass in estrogen-deficient OVX rats [96, 105]
Taxifolin Larix olgensis Pinaceae Taxifolin increases osteogenic development of human bone marrow MSC through the NF-κB pathway [106]

Among the many phytomolecules, HP shows significant benefits in terms of OP treatment. It is a polyphenolic bioflavonoid flavanone glycoside compound of citrus fruits such as grapefruit (Citrus paradise), lime (Citrus aurantifolia), tangerine (Citrus reticulata), lemon (Citrus limon) and orange (Citrus sinensis) belongs to the family Rutaceae. HP exists in the skin of fruits and vegetables and was previously famous as vitamin P because of its vitamin-like action. It is also found in small amounts of mint plant extracts, honey, and aromatic teas. It was initially separated from citrus peel by the French chemist "Lebreton" in 1828 [107, 108]. It possesses anti-aging, anti-inflammatory, anticancer, and antimicrobial properties [109–111]. Citrus peels contain more HP than the fruit itself, though this may vary from fruit to fruit. HP is abundant in citrus fruits' white, delicate inner layer and bright outer peel layers [112].

HP is a pale yellow hair-like needle, primarily found in the form of flavonoids, and is an odorless, tasteless compound obtained by alkaline hydrolysis or acid hydrolysis [113]. Chemically, HP is 3, 5, 7-trihydroxy flavanone or 7-rhamnoglucoside or hesperetin-7-O-rutino-side has a molecular weight of 610.57 with a molecular formula C28H34O15 and is composed of aglycone hesperetin and disaccharide rutinose [109]. Naturally, it resembles long, tan, or pale-yellow, hair-like needles. It melts at a temperature of 258-260 °C. At 25 °C, it has a saturation solubility of 4.93±0.99 μg/ml, indicating a slight solubility in water [114]. Its solubility varies with pH; at pH 9.11 (8.93±0.73 μg/ml) it is the most soluble, while at pH 1.21 (4.15±0.34 μg/ml) it is the least soluble. For pure HP, Differential Scanning Calorimetry (DSC) thermal analysis reveals a distinctive endothermic peak at 259 °C, corresponding to its melting point [115]. Using Fourier-Transform Infra-Red (FTIR) spectroscopy, the hesperidin spectrum shows a prominent absorption band around 1644 cm−1, associated with its carbonyl stretching vibration. HP's stability varies depending on the chemical and environmental circumstances [116]. The structure of HP is shown in the fig. 5.

Fig. 5: Structure of HP
Source: ChemSketch

HP is poorly absorbed in the gastrointestinal system and has a low bioavailability. Substances such as bile salts can improve its absorption. HP is dispersed throughout the body after absorption. It has been discovered in a number of tissues, such as the brain, kidneys, and liver. In the liver, HP is extensively metabolized. The gut microbiota and enzymes like CYP3A and P-glycoprotein are mainly responsible for its metabolism to produce the more bioactive hesperetin and excreted through urine [117].

HP has been used in traditional medicine for decades, especially in areas with many citrus fruits. Its medicinal qualities have made it highly valued, particularly for promoting digestive and cardiovascular health. Improving circulation and treating ailments like varicose veins and weak blood flow are two of HP's main traditional uses. Current studies highlight its vasoprotective benefits, showing that teas or extracts from citrus peels frequently strengthen blood vessels and reduce inflammation [118]. Besides offering circulatory advantages, HP often promotes digestive well-being. People have long used citrus peel infusions to calm the stomach and ease the symptoms of ulcers, gastritis, and indigestion. Recent studies indicate that HP's anti-inflammatory properties may help reduce gastrointestinal distress, supporting this consistency [119]. Citrus fruits prevent or lessen colds and flu, and HP is also employed in traditional medicines to boost immune function. HP's antioxidants and antibacterial qualities probably helped produce these immune-stimulating effects [120].

HP possesses several biological and medicinal qualities, such as neuroprotective, anti-inflammatory, anti-allergic, antimicrobial, antiviral, and antioxidant activities [120]. HP can be utilized as a lead chemical or as a cheap anti-inflammatory drug, particularly for people who are allergic to the commonly used non-steroidal anti-inflammatory drugs. With Diosmin, HP exhibits a notable protective effect against inflammatory illnesses in vitro and in vivo, potentially via a mechanism involving antioxidant free radical scavenger activity and/or suppression of eicosanoid production [121]. Cytokines like IL-1β (Interleukin-1β) and inflammatory mediators like TNF-α, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase, Inducible Nitric Oxide Synthase (iNOS), and Nitric Oxide (NO) were all reduced when HP administered. HP treatment reduces the adverse effects of long-term exposure to chlorpyrifos on hepato-renal damage by inhibiting inflammatory factors such as NF-κB, IL-1β, TNF-α, and iNOS. It is a promising natural substance for treating and preventing OP because it has shown strong osteoprotective properties [96]. Its advantages come from the capacity to control bone metabolism, reduce inflammation, and combat oxidative stress, which are all important aspects of bone health. Therefore, HP is studied more in terms of bone health and repair. This review aims to summarize the protective benefits of HP on bone as demonstrated by the currently available research. HP may also improve bone health through particular mechanisms, which will be covered in the current review.

Molecular mechanisms of HP in OP prevention

A growing amount of data indicates that HP, through various mechanisms, increases bone density. It is essential in improving skeletal disorders and boosting bone health in general, including OP. These processes reduce oxidative stress and inflammatory responses, promoting OB development and OC differentiation. The mechanism of action of HP for the treatment of OP is given in fig. 6.

HP activates the Wnt/β-catenin signaling pathway, enhances the estrogen signaling pathway, inhibits RANKL to reduce bone resorption, and suppresses Macrophage-Colony Stimulating Factor (M-CSF) to decrease OC differentiation.

Fig. 6: Therapeutic mechanism of HP in combating OP
Source: BioRender.com

MSC-Mesenchymal Stem Cells, HSC-Hematopoietic Stem Cells, ER-Estrogen Receptor, Wnt/β-Wingless related integration site, RANK-Receptor Activator of Nuclear factor Kappa, RANKL-Receptor Activator of Nuclear factor Kappa-B Ligand.

HSC and MSC are essential for bone remodeling and regeneration. HSCs generate blood cells involved in bone remodeling, and MSCs have the ability to develop into a variety of cell types, including OB [34]. HP stimulates the activity and development of OB, the cells that build bones. On the other hand, it prevents OC, which helps in bone resorption. Through a variety of mechanisms, HP exhibits strong antiosteoporotic potential. It alters the ER signaling pathway, which is crucial for preserving bone density, especially in women who have gone through menopause. HP has been found to increase ALP activity by 25% and boost OB proliferation by 30%. Furthermore, by blocking the cathepsin K enzyme, it lowers OC activity by 40% [81]. HP also stimulates OB development and bone production through the Wnt/β-catenin signaling pathway and reduces the breakdown of bone tissue by blocking the action of the enzyme cathepsin K, which contributes to bone resorption [122]. Studies show that HP has a 50% rise in β-catenin levels and a 35% increase in Wnt target genes such as Axin2. This process is essential for the development and maintenance of bones [37]. In order to preserve bone integrity, HP controls MMPs, which are involved in bone matrix turnover. To promote osteoclastogenesis, the production of OC, which are cells in charge of bone resorption, RANKL attaches itself to RANK on OC precursors via the RANKL/RANK/OPG pathway [123]. HP modifies this mechanism to balance bone growth and resorption and affects M-CSF, which is involved in OC differentiation [81].

Antiosteoporotic potential of HP: evidence from preclinical and clinical studies

Evidence from preclinical studies

According to preclinical research, HP increases the ALP activity and OB proliferation, both of which are essential for bone formation. Additionally, it alters estrogen signaling pathways, which are vital for healthy bones. We have summarized the activity of HP through cellular and animal experiments, which are illustrated in table 3.

Table 3: Cellular level observations of HP for the prevention and treatment of OP

Type of cell/Animal model

Treatment

(HP concentration)

 Findings Statistical difference Reference
Bone marrow MSC induced by dexamethasone 10 µM Activation of p53

Significantly increases ALP staining

P<0.001 and Runx2 expression P<0.0001

 [124]
Healthy human alveolar OB 10 μmol/l Activation of Wnt/β-Catenin pathway, expressions of β-Catenin and CyclinD1↑

Significantly enhances ALP activity

p<0.05 and Runx2 expression P<0.01

 [122]
MC3T3-E1 induced by dexamethasone 10 μg/ml

Cell proliferation, ALP, ESR1↑

oxidative stress ↓

Significantly increases ALP activity

p<0.05, ALP staining p<0.05 and MC3T3-E1 cell proliferation p<0.05

 [81]
MC3T3-E1 pre-osteoblastic cells 500 µM RunX2, OSX, bone sialoprotein, Collagen Type I Alpha 2 Chain (CO L1A2)↑ At 1 µm significantly elevated osteogenic markers in pre-OB cells on day 7and 14 P<0.05 [3]
Critically sized defect rat mandible model 100 µM Mature collagen fibers, matrix mineralization ↑ BMP and HP together significantly increase bone repair P<0.05 [3]
OVX ddY mice 0.5 g/100 g Number of OC, serum, hepatic lipids ↓ Hepatic lipid and serum level significantly lower P<0.05 [125]
OVX female Wistar rats 5g/Kg

Improved BMD,

femoral load improved

 Significantly reduce serum and hepatic lipid levels P<0.05 [126]
OVX Sprague-Dawley rats 20 mg/kg OC, ALP, ACP, β-isomerized C-terminal telopeptides ↑ Significantly enhances bone density, biomechanical properties P<0.001 [127]
Prednisolone induced Zebrafish OP model 1.25-10 μg/ml Promotes osteogenesis, oxidative stress ↓, Estrogen signaling pathway activation through ESR1

Fluorescence staining results showed lower oxidative stress

P<0.01

 [81]

Clinical evidence

Studies have looked into how HP affects PM women, a crucial demographic for studying OP. Research has examined how HP affected PM's bone health both with and without a calcium supplement called Calcilock. Twelve healthy PMs participated in the double-blind, placebo-controlled, randomized-order crossover study. To assess bone calcium retention, the researchers measured the rare isotope 41Ca's excretion in the urine. A statistically significant p-value of less than 0.04 indicated that the combination of calcium and HP increased bone calcium retention by 5.5%. However, there was no discernible improvement in bone calcium retention with HP alone. This study found that HP and calcium supplements (Calcilock) can effectively maintain bone health in PM, indicating the possible synergistic benefits of both substances. According to these results, HP by itself might not be enough to improve bone health, but when combined with calcium, it presents a viable method of lowering PM's risk of OP [128].

Drug delivery system for the treatment of OP

Nanoparticles are able to overcome the constraints of traditional therapies because of their unique physicochemical qualities, which include their small size, huge surface area-to-volume ratio, and customizable surface characteristics. Enhanced medication stability, controlled release kinetics, targeted bone tissue delivery, and better drug bioavailability are the benefits of nanoparticles. Specific physicochemical characteristics of nanoparticles, including silica, polymeric, solid lipid, and metallic nanoparticles, make them viable drug carriers for the treatment of OP [129]. Due to its porosity, silica nanoparticles can be used for imaging and medication delivery [130]. Targeted and prolonged medication release is made possible by the adaptability and customization of polymeric nanoparticles. Solid lipid nanoparticles can be used in a variety of medicinal applications because of their enhanced drug stability, controlled release, and biocompatibility. For imaging, medication delivery, and therapeutic uses, metallic nanoparticles like gold nanoparticles have unique optical qualities. The effectiveness of OP treatment is improved by their enhancement of medication bioavailability, stability, and targeted delivery [131]. HP has a few limitations that can be addressed through such drug delivery systems.

The main drawbacks of HP are its low water solubility and bioavailability. Because the gut microbiota must change into its more accessible form, hesperetin, its absorption in the gastrointestinal system is limited. HP's hydrophobic properties further limit its ability to dissolve in water, reducing its therapeutic effectiveness and absorption efficiency [117].

Several strategies can be used to address HP solubility and bioavailability problems. Solid Lipid Nanoparticles (SLNs) and liposomes are examples of nanotechnology that improves solubility and absorption. While Self-Emulsifying Drug Delivery Systems (SEDDS) increase bioavailability by creating emulsions in aqueous settings, co-formulation with cyclodextrin improves dissolution rates. Enhancers of bioavailability, such as piperine (found in black pepper), improve absorption by blocking metabolic enzymes. HP is protected and released under regulated conditions by microencapsulation. Its hydrophilicity can also be increased by chemical changes like glycosylation, increasing its solubility and bioavailability and boosting its therapeutic efficacy in diseases like OP [132]. Researchers have tried to make HP-encapsulated nanoparticles to improve their properties, and an overview of such drug delivery systems is illustrated in table 4, demonstrating the enhanced functions.

Table 4: Hesperidin drug delivery systems across various diseases

Drug delivery system Disease Property Study type Key findings Reference
Self nano-emulsifying drug delivery system Wound-healing Enhanced solubility and absorption Wistar rats Effective Co-delivery [133]
HP loaded Polylactic-Co-Glycolic Acid (PLGA) nanoparticles Colorectal cancer Enhanced bioavailability, reducing survival rate of cancer cells HCT116 colorectal cancer cell line Ensuring consistent drug delivery [134]
Nanotheranostic carrier system Cancer Controlled drug release Balb/c mice and HeLa cell line Targeted drug delivery [135]
Chitosan/HP nanoparticles Cancer Enhance the bioavailability and solubility of hesperidin MDA-MB-231 cell line Targeted drug delivery [136]
Extracellular vesicle nanodrugs Malignant glioma Enhance the efficacy Balb/c mice Enhanced drug delivery [137]
Magnetic gelatin-HP microrobots Diabetic foot ulcers Enhance the bioavailability and solubility of HP Human dermal fibroblasts Enhanced drug delivery [138]
HP-loaded Polyvinyl alcohol/alginate hydrogel Skin injuries and wound healing Enhance the bioavailability and solubility of HP Human dermal fibroblasts Controlled drug release [139]
HP-conjugated gold nanoparticles Diabetes-induced cognitive impairment Enhance the bioavailability and solubility of HP Sprague-Dawley rats Targeted delivery [131]
HP-loaded lipid-polymer hybrid nanoparticles Wound healing Controlled drug release Human dermal fibroblasts Enhanced drug delivery [140]
Nanostructured lipid carrier Helicobacter pylori infection Enhances the delivery and efficacy of HP against Helicobacter pylori Gastric epithelial cells Targeted action [141]

Toxicity profile of HP

In a 2019 study, Li et al. extracted HP from orange peel and conducted both acute and sub-chronic oral toxicity tests using Dawley rats. For the acute toxicity study, ten rats were administered HP orally at various doses (55, 175, 550, 1750, and 5000 mg/kg). The animals were monitored for 14 d for any signs of illness or mortality. The results showed no indications of acute toxicity.

For the sub-chronic toxicity trial, 15 rats were given oral doses of HP at 0, 250, 500, and 1000 mg/kg for 13 w. The study found no signs of sub-chronic toxicity, indicating that HP is safe at these dosage levels [142].

In another study by Hu et al., zebrafish larvae were used for toxicity tests. The larvae were treated with varying concentrations of HP (0, 1.25, 2.5, 5, and 10μg/ml). The results demonstrated that HP was not toxic to zebrafish larvae [143].

Comparative research has demonstrated that HP's anti-inflammatory and antioxidant properties are similar to those of other flavonoids, such as rutin, quercetin, and naringin. A metabolite of HP, hesperetin, exhibits more decisive antibacterial and radical scavenging action. Rutin, quercetin, naringin, and hesperetin have low toxicity. Studies show they are generally safe at typical dietary or supplemental levels, but high doses may cause adverse effects like nausea and diarrhea.

In contrast to Bisphosphonates and SERMs, HP exhibits the potential for increasing bone formation and decreasing resorption without increasing the risk of atypical femoral fractures and Osteonecrosis of the jaw. HP's safety has been validated by toxicity tests conducted on Dawley rats and zebrafish larvae.

DISCUSSION

HP, a bioflavonoid in citrus fruits, offers advantages over standard OP treatments. In contrast to traditional treatments, it targets the p53 signaling pathway, which is essential for osteogenic differentiation. The usefulness of HP is suggested by the fact that it can reverse the suppression of osteogenic differentiation caused by dexamethasone by decreasing p53 activation. Additionally, it increases the microarchitecture and mineral density of bones, strengthening the skeleton and lowering the risk of fracture. Because of this all-encompassing strategy, HP is a viable substitute for conventional therapies, offering specific advantages for the treatment of OP.

Various study designs, dosages, populations, formulations, and techniques can result in variations in the effectiveness of hesperidin. Reviewing these is essential to get a thorough understanding. As an osteoporosis treatment and prevention medication, hesperidin exhibits great promise and presents a viable substitute with fewer adverse effects.

CONCLUSION

Several studies have reported the protective effect of HP against OP. By controlling bone production and metabolism, HP inhibits activated p53, which may help to treat or prevent OP. It regulates cell differentiation through the Wnt/β-catenin signaling pathway and affects the mineralization process by upregulating the expression of the osteogenic genes (ALP, OCN, Osx, and Runx2) in human alveolar OB. By encouraging osteoblast genesis and bone production, HP has antiosteoporotic, antioxidant, and anti-inflammatory properties.

By maintaining intestinal absorption of calcium, HP also boosts endogenous antioxidants, especially those found in the intestines. HP has anti-inflammatory qualities because it inhibits proinflammatory cytokines like Tumor necrosis factor-α, Interleukin-1β, and Interleukin-6, as well as NF-κB.

In vitro studies established its anti-OC activity by inhibiting osteoclastic markers like RANKL, cathepsin K, TRAP, and proinflammatory cytokines such as Interleukin-1β, Interleukin-6, Interleukin-8, TNF-α, and Matrix metalloproteinases-3, 9, 13, as well as by reducing Reactive Oxygen Species (ROS). It has advantages for mineralized tissue due to its anti-collagenolytic activity.

Based on research done on animal models, HP lowers bone loss and enhances the regeneration of bone tissue. In vivo, evidence indicates that HP's antiosteoporotic activity reduces trabecular bone loss and increases bone mineral density.

Emerging nanotechnology-based Drug Delivery Systems (DDS) offer promising advancements in the targeted treatment of OP. These systems can deliver HP more precisely and effectively to specific body locations, reducing adverse effects and improving patient outcomes.

FUTURE DIRECTIONS

Future directions include conducting clinical trials to further validate the efficacy and safety of HP in treating OP and exploring formulation innovations to enhance HP's bioavailability and therapeutic effect. Still, more research is needed to minimize the limitations of HP. Continued research and innovation in this area hold great promise for developing more effective OP therapies.

ACKNOWLEDGMENT

Authors Vijishna. L. V and Akanksha D Dessai are thankful to the Manipal Academy of Higher Education (MAHE) for providing Dr. T. M. A. Pai Doctoral Fellowship. The authors also acknowledge with thanks to Manipal College of Pharmaceutical Sciences for providing facilities for this work. The authors also thank BioRender.com and ChemSketch.

FUNDING

This research received no external funding.

AUTHORS CONTRIBUTIONS

VIJISHNA L V: Conceptualization, data curation, writing original draft, writing-review and editing.

Akanksha D Dessai: Writing original draft, writing-review and editing.

Usha Y Nayak: Investigation, Supervision.

Richard Lobo: Investigation, Supervision.

CONFLICT OF INTERESTS

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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