INTRODUCTION

Bone is made up of four different types of cells: Osteocytes, osteoblasts, bone lining cells, and osteoclasts [1]. Quiescent, revitalizing, resorption, creation, and mineralization are the five stages of bone’s remodeling [2]. Bone matrix products are produced by osteoblasts as their main function (collagen types I and V, proteoglycans sialoprotein and osteopontin) [3] and osteoclasts are enormous cells that arise from hematopoietic stem cells in bone marrow in response to the number of stimuli, including macrophage colony-stimulating factor (M-CSF) [4] which osteocytes and osteoblasts both release, as well as receptor activator of nuclear factor kappa-B (RANK) ligand [5]. M-CSF stimulates proliferation and inhibits apoptosis via attaching to the osteoclast's tor receptor. RANKL is a significant element that binds to its receptor RANK and promotes osteoclastogenesis in osteoclast precursors [6]. By binding to RANKL and blocking the RANK/RANKL interaction, osteoprotegerin (OPG), a substance made by osteoblasts, inhibits osteoclastogenesis [7]. Consequently, the RANKL/RANK/OPG system is a crucial osteoclastogenesis regulator [4].

Numerous medications can impact bone metabolism. Heparin, warfarin, cyclosporine, and glucocorticoids are a few examples, the anticoagulant drug heparin is currently the most widely used worldwide [8]. Anticoagulants are widely used to treat thromboembolic cases and to prevent stroke. Some of the information on their effects on bone health is scarce, but in the case of others (heparins), it is strong enough to advise caution while using them in those who are at risk of osteoporosis. Osteoprotegerin (OPG), a RANKL decoy receptor released by osteoblasts, inhibits osteoclast production and activation, which are both triggered by the receptor activator of NFkappaB ligand (RANKL) on osteoblasts [9].

Taraxacum officinale is a plant in the Asteraceae Compositae family [7]. Indeed, It includes a wide range of phytochemicals, whose biological effects are being researched in several fields related to human health. Numerous biological effects are produced by Taraxacum officinale and its components, which contain antioxidant and anti-inflammatory characteristics [10]. To prevent osteoporosis and lower the risk of fractures, non-pharmacological management is also important for optimal osteoporosis treatment, even though pharmaceutical treatment is the most popular. The herb Taraxacum officinale is high in minerals such as iron, magnesium, sodium, calcium, silicon, copper, phosphorus, and zinc as well as vitamins (A, C, D, E, and B), inositol, and lecithin [11], which also contain inulin and fructooligosaccharides, can increase calcium absorption, limit bone resorption later in life, and suppress bone loss during growth [12].

Vitamin C (VC) has a significant impact on the skeletal system; its potential effects on gene transcription, deoxyribonucleic acid (DNA), and osteoblast maturation range from suppressing osteoclast activity and encouraging osteoblast maturation by enhancing collagen type I production, and histone methylation. Along with treating the condition, effective osteoporosis prevention is crucial to halting the disease's progression and reducing related fractures. However, according to some data, vitamin C may help prevent osteoporosis and fractures [13]. VC is an important cofactor for lysine and proline hydroxylation, which are required for collagen fibril cross-linking in bone. Increases the activation of alkaline phosphatase, which is an indicator of osteoblast formation [14]. Is implicated in osteoblastogenesis through PPAR-g expression and osteoclastogenesis through RANKL expression [15].

Vitamin K (liposoluble vitamin) contains a variety of bone-specific modes of action [16]. Gamma-carboxyglutamic acid is formed when the enzyme gamma-glutamyl carboxylase carboxylates glutamic acid (Glu) residues in vitamin K-dependent proteins (GIa). Matrix G1a protein, periostin, and osteocalcin are examples of vitamin K-dependent proteins found in bone (or bone Gla protein). During the mineralization phase of bone, osteoblasts produce osteocalcin, which binds to calcium ions and hydroxyapatite crystals to govern their size and structure [17]. Vitamin K is crucial for bone health through methods other than gamma-carboxylation. In addition to inhibiting bone production, it can control the transcription of osteoblastic markers' genes [18].

The goal of the current study was to determine whether a natural plant called Taraxacum officinale, which is available in the Kingdom of Saudi Arabia, could prevent osteoporosis and its complications, such as oxidative stress, inflammation, and apoptosis, in female rats being treated with heparin. Vitamins C and K were also compared to the potential prophylactic effects of this plant.

MATERIALS AND METHODS

Chemicals

Heparinol, heparin sodium 5,000 I.U./ml injectable or infusion solution concentrate, Ain Medicare Sdn Bhd, Malaysia, Vitamin C (ascorbic acid), manufactured for Doctor’s Best, Inc. Tustin, California,92780 USA, Vitamin K (phytonadione) tablets, 500 mcg by Source Naturals and Taraxacum officinale plant collected in spring from southern Saudi Arabia were used in this study. Kits from MyBioSource were obtained to quantitatively determine various characteristics (San Diego, United States).

Animals and Treatment

In this study, fifty adult female albino rats (180-200gm) were used, from the Faculty of Pharmacy at King Abdulaziz University. Animals were housed under controlled conditions (18-23oC, humidity 40-60%, and the usual cycle were 14 hours of light and 10 hours of darkness). Making sure there were no lights on and that neither scientists nor technicians visit the mouse room while it is dark). Diet: The fat content ranges between 4% and 11%. Water: Water should always be available. Rat handling was done following the expectations of the pharmacy faculty at King Abdulaziz University. Seven days were given to the animals to acclimate. The creatures were split into five groups of ten rats apiece:

• Group I: Normal rats that will be treated orally with D.W. only for 30 days.
• Group II: Rats will be treated by subcutaneous injection of unfractionated heparin (0.001 mg/ kg) 30-day period [19].
• Group III: Rats will be co-administered orally with Taraxacum officinale water extract dose (2.4 mg/ kg) along with heparin injection for 30 days [20].
• Group IV: Rats will be co-administered orally with vitamin K (50 mg/kg) [21] and vitamin C (200 mg/ kg) [22] with heparin injection for 30 days.
• Group V: For 30 days, rats will also get heparin and the oral mixture of Taraxacum officinale, vitamins C and K.

Extraction of Taraxacum officinale leaves, collected fresh leaves of Taraxacum officinale were, frozen for ten hours at -80 °C, then moved to a lyophilizer (vacuum, freeze at -65 °C), where they were dried completely for two days. A plastic bag was used to crush the dried plant, which was then pulverized, sieved, and weighed. High-performance liquid chromatography (HPLC) was used to separate the weight of 6.37 g of the powdered dry plant from 150 ml of methanol, 150 ml of methanol, 150 ml of methanol, stopped, shacked for 5 minutes, and then filtered over a 0.45 Nylon membrane while under vacuum. The solvent was eliminated using a rotary evaporator at 35 °C, and the clear filtrate was then transferred to another clean, dry, 250 ml round flask. The residue that was collected weighed 0.728 g and was stored at -20 °C until use.

[20]

Biochemical Analysis

Serum Analysis

Serum bone formation markers bone-specific alkaline phosphatase (ALP), osteocalcin (OC), and bone resorption markers deoxypyridinoline (DPD) and tartrate-resistant phosphatase (TRAP) using an automated analyzer, were assessed as biomarkers of bone osteoporosis damage.

Histopathological Studies

The histopathological changes of bone in different experimental heparin groups were examined using bone tissues. Samples of bone tissue were taken, and they were fixed in 4% formaldehyde for 24 hours. They were then gradually dehydrated in ethyl alcohol, cleaned in xylene, and set in paraffin. For staining, paraffin blocks were mounted on glass slides after being sliced with a microtone at 4-5 micrometers. Hematoxylin and eosin were used to stain the sections (H&E) [23].

Statistical Analysis

To statistically assess the data, the results for the various experimental groups were compared to the values of the individual normal groups. The outcomes are presented as mean + SD. Significant differences between groups were investigated using one-way analysis of variance (ONE-WAY ANOVA) and post-Hoc least significant difference (LSD). For an analysis of variance (ANOVA), 0.05 was the threshold for significance. Version 23 of the statistical package for the social sciences (SPSS) was used to conduct the statistical analysis.

RESULTS AND DISCUSSION

Effect of Taraxacum Officinale and Vitamins (C, K) on Bone Formation Indices in Heparin-Treated Female Rats

Table 1 and Figures 1 and 2 respectively, show the serum levels of bone on osteoporosis parameters, Bone-specific alkaline phosphatase (ALP), and Osteocalcin (OC). The impact of the combination of Taraxacum officinale, Vitamin C, and K with heparin-treated rats was the most potential prophylactic impact on the levels in relation to the treatment with each agent lonely.

As compared to the normal group (G1) after 30 days. A very highly significant decrease was found in serum levels of ALP (u/l), while a very highly significant increase in OC (ng/ml) in G2 (P≤0.001). Non-Significant decrease ALP (u/l)), a highly significant decrease of OC (ng/ml) ((P≤0.01) in G3. A very highly significant decrease was found in serum levels of ALP (u/l), but a very highly significant increase in G4 (P≤0.001). Was found a non-significant increase in serum levels of ALP (u/l), but a non-significant decrease of OC (ng/ml) in G5.   

When compared to heparin treated group (G2) in 30 days, A very highly significant increase was found in serum levels of ALP (u/l), but a very highly significant decrease of OC (ng/ml) (P≤0.001)   in G 3,4,5 (P≤0.001).

When comparing the group co-administered heparin and orally with Taraxacum officinale water extract (G3) for 30 days. A very highly significant increase was found in serum levels of ALP (u/l), but a very highly significant decrease of OC (ng/ml) (P≤0.001)   in G 4. Was found non- a significant decrease in serum levels of ALP (u/l), and a significant increase of OC (ng/ml) (P<0.05) in G5.

When compared to the combination group (G5) co-administered heparin and orally with Taraxacum officinale water extract and vitamin C and K for 30 days, A very highly significant decrease was found in serum levels of ALP (u/l), but a very highly significant increase in serum levels of OC (ng/ml) (P≤0.001) in G4.

Values are expressed as mean ±SD of 10 rats. ^a***P ≤0.001, ^a**P≤0.01, compared with the control group(G1). ^b***P≤0.001, compared with the heparin-treated group(G2). ^c***P≤0.001, ^c*P<0.05compared with the heparin+ taraxacum (G3). ^d***P≤0.001,  compared with the heparin+ Vit (C, K) (G4).

Effect of Taraxacum Officinale and Vitamins (C, K) on Bone Resorption Indices in Heparin-Treated Female Rats

Table 2 and Figures 3 and 4 respectively, show the serum levels of bone on osteoporosis parameters deoxypyridinoline (DPD), and tartrate-resistant acid phosphatase (TRAP). The impact of the combination of Taraxacum officinale, vitamin C, and K with heparin-treated rats was the most potential prophylactic impact on the levels in relation to the treatment with each agent lonely.

As compared to the normal group (G1) after 30 days. A very highly significant decrease was found in serum levels of DPD (nM) and TRAP (ng/ml) in G2 (P≤0.001). Significant decrease of DPD (nM) (P<0.05), while a non-significant decrease of TRAP (ng/ml) in G3. A very highly significant decrease was found in serum levels of DPD (nM) and TRAP (ng/ml) in G4 (P≤0.001). Was found a non-significant increase in serum levels of DPD (nM) and   TRAP (ng/ml) in G5. 

When compared to heparin treated group (G2) in 30 days, A very highly significant increase was found in serum levels of DPD (nM) and   TRAP (ng/ml) in G 3,4,5 (P≤0.001).

When comparing the group co-administered heparin and orally with Taraxacum officinale water extract (G3) for 30 days A very highly significant increase was found in serum level of DPD (nM) and   TRAP (ng/ml) (P≤0.001) in G4. Significant decrease of DPD (nM) (P<0.05), while a non-significant decrease of TRAP (ng/ml) in G5.

When compared to the combination group (G5) co-administered heparin and orally with Taraxacum officinale water extract and vitamin C and K for 30 days, A very highly significant decrease was found in serum levels of DPD (nM) and   TRAP (ng/ml) (P≤0.001) in G4.

Values are expressed as mean ±SD of 10 rats. a***P ≤0.001, a*P<0.05, compared with the control group (G1)

b***P≤0.001, compared with the heparin-treated group (G2). c***P≤0.001, c*P<0.05compared with the heparin+ Taraxacum officinale (G3).d***P≤0.001, compared with the heparin+ Vit (C, K) (G4).

Histopathological Examination of Bone Tissue

In Figure 5, a light microscopic examination of hematoxylin and eosin (H&E) stained sections of rat bone in the control group showed the normal histological structure of bone with normal osteocytes. The bone cells most found in the bone tissue generally have protoplasmic projections and lacunae or small chambers. Cancellous bone Provides structural support and flexibility without the weight of compact bone. The medullary cavity is the hollow part of the bone that contains bone marrow and is made up of small, needle-like pieces of bone arranged like a honeycomb. In the group, negative control showed normal cancellous bone and normal spaces of medullary a.

Showed loss of cancellous bone, enlargement of the medullary spaces, and cortical porosity in bone sections of rats treated with heparin showed disfiguration of bone architecture, which was evident b. Bone sections of rats treated with heparin in concurrent with either dandelion extract only showed moderate loss of cancellous bone, enlargement of the medullary spaces, and cortical porosity c. As well as in bone sections of rats treated with vitamin (C, K) simultaneously with heparin, showed slight, of the medullary spaces, and cortical porosity d. Bone sections of rats treated with heparin simultaneously with dandelion extract and vitamins (C, K) showed normal cancellous bone and normal spaces of the medullary e.