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. 2022 Sep 15;13:937956. doi: 10.3389/fendo.2022.937956

Table 3.

Alliaceae-derived OSCs: effects on in vivo models of bone loss.

Molecule tested Experimental in vivo model description Mode of administration, dose and duration Main effect Specific outcomes Authors Ref
Ethanol extracts of Allium macrostemon bulbs Female, 25-day-old, Sprague–Dawley rats
(adolescent mice)		 Gavage, 100 and 300 mg/kg, twice daily for 10 days Increase tibial longitudinal bone growth

  • Increase tibial longitudinal bone growth (fluorescence photomicrograph after tetracycline hydrochloride)

  • ↑ IGF-1 and BMP-2 in the proliferative and hypertrophic zones of growth plate (immunohistochemistry)

 Kim et al. (85)
Hot-water extracts of Allium hookeri roots Female, 3-week-old, Sprague-Dawley rats Oral treatment, 500 mg/kg, single daily dose, for 6 weeks Improved bone formation

  • ↑ serum levels of OCN (ELISA)

  • ↑ BMD, BV, BV/TV, Tb.Th, Tb.N;

  • ↓ Tb.Sp, BS/BV (microCT in proximal tibia)

 Park et al. (48)
Wheat bread added with Allium sativum L. Male weaning Wistar rats Oral administration, 3 g per 100 g wheat flour, for 60 days Increase in BMD

  • ↑ total skeleton BMC and BMD, femur BMD, tibia BMD

  • Spine (S-BMD) and proximal tibia (T-BMD) was not affected (DEXA)

  • ↑ femur calcium

 Weisstaub et al. (88)
Ethanolic extracts of Allium cepa L. bulbs Male, 9-week-old, Wistar Hanlbm rats Orally given, one gram, daily treatment, for 10 days Inhibition of bone resorption ↓ bone resorption (urinary excretion of tritium) Wetli et al. (41)
Homogenized of Allium sativum L. Hypercholesterolemic rat model (Pregnant albinorat Wistar fed with hypercholesterolemic diet,
and their offspring)		 Intragastrical injection, 100 mg/kg, a week prior to onset of feeding with hypercholesterolemic diet Improved endochondral ossification ↑ ossification in mandibular, humerus, radio-ulna, femur, tibio-fibula, scapula and ilium (Alizarin red S for
ossified skeletal bones in fixed offspring)		 El-Sayyad  et al. (89)
Water Allium sativum L. extract In vivo implants in bicortical rat distal femur defects (Sprague–Dawley rats) 3D-printed calcium phosphate scaffolds designed with a bimodal pore distribution releasing ginger and garlic extract, implanted for 4-10 weeks Increase in osteoinductivity

  • ↑ osteoid tissue formation, mineralization (masson-goldner trichrome assay)

  • ↑ bone area, osteocytes (haematoxylin and eosin)

  • ↑ Col I (Col I staining)

 Bose et al. (82)
Aqueous and ethanolic extracts of Allium fistulosum CDD mice - Mice model of bone loss due to nutritional
deficiency (Male, 4-week-old, C57BL/6 mice, fed with a calcium- and vitamin D-deficient diet for 5 weeks)		 Oral treatment, 150 and 450 mg/kg, ad libitum feeding for 4 weeks Prevention nutritional deficiency-induced bone loss and retarded bone growth

  • ↑ serum calcium, OC and Col I vs CDD mice (ELISA)

  • ↑ serum ALP, OCN and Col I vs normal control mice (ELISA)

  • ↑ femoral and tibial BMC and BMD vs CDD mice and similar to normal control (DEXA)

  • Thicker growth plates vs CDD mice and similar to normal control (measured after hematoxylin and eosin stain)

 Ryuk et al. (80)
Water extract of Allium fistulosum root Rat model of OP and osteoarthritis (Female, 8-week-old, Sprague–Dawley rats, ovariectomy and MIA-induced OA) Within rice porridge, 250 and 750 mg/kg, food supply was replaced every two
days for 8 weeks		 Prevention of bone loss

  • ↑ BMD in lumbar bone spine, OA leg and control leg (DEXA)

  • ↓ serum ALP activity (ELISA)

 Yang et al. (47)
Oil extract of Allium sativum L. from raw cloves Rat model of OP (Female albinorats, ovariectomy) Gavage, 100 mg/kg body wt/day, single evening dose for 30 days Prevention of bone loss

  • ↓ serum ALP activity (pNPP measurements) and TRAP activity (commercial kit)

  • ↑ BMD of femur, thoracic rib, thoracic vertebra and lumbar vertebra (measured by Archimedes’ principle)

 Mukherjee et al. (90) (91)
↑ calcium and phosphate content in femur, lumbar vertebra, thoracic vertebra, thoracic rib (method of Adeniyi et al. (1993) and Lowry and Lopez
(1946))		 Mukherjee et al. (91) (92)

  • ↑ tensile strength of the femur (method of Shapiro and Heaney (2003)

  • ↑ serum estradiol levels (ELISA)

  • serum PTH levels is not affected (ELISA)

 Mukherjee et al. (92)
Oil extract of Allium sativum L. from raw cloves Rat model of OP (Female Wistar, ovariectomy) Gavage, 100 mg/kg body wt/day, single evening dose for 30 days Increase in bone strength and inhibition of bone resorption

  • ↑ tensile strength of the femurs (method of Shapiro and Heaney (2003)

  • ↓ serum TRAP activity (commercial kit)

 Mukherjee et al. (93)
Allium cepa L. powder Rat model of OP (Female, 14-week-old, Wistar rats) treated or not with 1 mg/kg/day alendronate Dietary administration, diet containing 3%, 7% and 14% (wt/wt) Allium cepa L. powder, for 6 weeks Prevention of Ovx-induced bone loss and deterioration of biomechanical properties (efficacy was slightly inferior to that of alendronate)

  • ↓ serum calcium (measured with an automatic chemistry analyzer)

  • ↑ serum OCN (ELISA)

  • ↑ BV/TV, Tb.N, ↓ Tb.Sp (histomorphometry on histological specimen)

  • ↓ osteoclasts (TRAP staining on histological specimen)

  • ↑ loading force to maximal load and tissue fracture, ↑ stiffness (three-point bending test)

 Huang et al. (94)
Diallyl disulfide (DADS) * A mouse calvarial osteolysis model (Female, 6-wk-old, C57BL/6 mice, LPS treatment 5 mg/kg) Subcutaneous injections, 20-40 mg/kg DADS, every alternate day for 14 days Inhibition of LPS-induced osteolysis

  • ↓ bone erosion as compared to LPS, ↑ BV/TV, ↓ porosity (microCT)

  • ↓ osteoclasts (histologic and histomorphometric analysis TRAP staining)

 Yang et al. (86)
Allyl sulfide (AS) * Age-associated OP mouse model (Female, 20-months-old (aged), C57BL/6 J mice) Oral gavage, 200 mg/kg, 3-months Restored osteogenesis and bone density

  • ↑ plasma levels of P1NP and CTX-I

  • ↑ bone density in the femur’s metaphyseal area (X-ray in vivo imaging)

 Behera et al. (83)
Allicin * Mice model of lead-induced bone loss (Male, 3-weeks-old, C57BL/6 J mice, 0.2% lead acetate in drinking
water ad libitum for 12 weeks)		 Intraperitoneally injection, 10 mg/kg, in the last 4 weeks Prevention lead-induced bone loss

  • ↑ BMD, BVF, Tb.N, Tb.Th, ↓ Tb.Sp (microCT)

  • ↑ CAT, SOD, reduced GSH; ↑ MDA on femur homogenates (commercial kits)

  • ↓ TRAP, CTSK, NFATc1, MMP-9 mRNA in femur (RT-PCR)

  • ↑ SIRT1 and ↓ of acetylated FOXO1 on femur homogenates (western blot)

 Li et al. (95)
Allicin * Mice model of aging rats (Male, 13 months-old, F344 rats) Intragastric administration, 4-8-16 mg/kg, once daily for 8 months Reverse aging-associated bone loss and frailty

  • ↑ femoral, spinal, tibial BMD (DEXA)

  • ↑ elastic load and maximum load in femur - ↑ bone strength (Three-Point Bending Test)

  • ↑ serum P1NP, ↑ serum CTX-I (ELISA)

 Liu et al. (96)

Most in vivo studies were conducted by using water or ethanol extracts of Allium edible plants (11 studies; Allium macrostemon, Allium hookeri, Allium fistulosum, Allium sativum L., Allium cepa L.). A few studies used Allium-derived OSCs (4 studies; diallyl sulfide, allyl sulfide, allicin). Most studies were performed in normal control mice showing improved bone formation and inhibited bone resorption; and in osteoporosis mice showing prevention of bone loss. Notably, only the effects of purified OSCs (labeled with * in the table) can be attributable entirely to OSCs. Markers of bone formation in serum: procollagen 1 intact N-terminal propeptide (P1NP); osteocalcin (OCN); collagen I (Col I), alkaline phosphatase (ALP), parathormone (PTH). Markers of bone resorption in serum: serum type I collagen breakdown product (CTX-I). Markers of bone resorption in urine: urinary excretion of tritium. Bone microstructural parameters analyzed by microCT analysis: BMD (bone mineral density), bone volume fraction (BVF), spine BMD (s-SMD), tibia BMD (t-BMD), BMC (bone mineral content), bone volume (BV), bone volume/total volume (BV/TV), bone surface/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular space (Tb.Sp), bone volume fraction (BVF). Bone mineral density analyzed by dual-energy X-ray absorptiometry (DEXA). Markers of bone formation in histological specimen: ALP, Col I. Osteoid tissue detection by masson-goldner trichrome assay. Markers of osteoclasts/bone resorption in histological specimen: tartrate-resistant acid phosphatase (TRAP), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), cathepsin K (CTSK). Markers of redox stress response: catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA). Measurements of bone strength: method of Shapiro and Heaney (2003); three-Point Bending Test. Other abbreviations: insulin-like growth factor 1 (IGF-1), bone morphogenetic protein 2 (BMP-2), lipopolysaccharide (LPS), sirtuin (SIRT); forkhead box O (FOXO). ↑ means up-regulation; ↓ means down-regulation.