Mechanism of enhanced antiosteoporosis effect of circinal–icaritin by self-assembled nanomicelles in vivo with suet oil and sodium deoxycholate

Figures & data

Figure 1 Chemical structures of CIT and IT.

Abbreviation: CIT, circinal–icaritin.

Table 1 BMD and other parameters of trabecular bone structures

Parameters	Sham	OVX	SO	EV	CIT-H	CIT-M	CIT-L	CIT-SO-H	CIT-SO-M	CIT-SO-L
BMD (mg/cm^2)	282.93±9.33b	199.79±5.23	194.88±6.06	245.65±8.19a	227.23±2.59b	220.85±4.53a	212.04±5.59	240.45±3.23b,d	231.66±5.07a,c	212.34±5.66
BMC (mg)	263.84±5.25b	147.03±6.47	148.03±5.96	246.15±10.02b	222.08±3.39b	204.09±7.36b	182.172±4.39b	233.06±4.06b,f	212.64±5.96b	192.82±4.11b,e
TMC (mg)	245.16±4.57b	131.83±3.33	130.49±4.29	227.08±3.17b	201.52±3.16b	180.23±8.08b	163.56±6.53b	213.25±4.29b,f	196.07±5.06b,c	182.26±6.83b,e
TMD (mg/cm^3)	587.37±2.92b	488.14±4.62	483.15±1.87	579.00±4.77b	554.44±7.37b	515.86±5.49b	509.87±0.10b	576.74±4.27b,d	535.26±4.89b,c	523.01±4.22b,g
VOB (mm^3)	448.08±16.78b	241.25±17.07	237.26±15.03	415.75±12.94b	382.53±8.98b	358.19±10.16b	312.46±17.31b	409.60±13.06b,d	389.99±10.59b,c	347.79±11.05b,e
CalibTbTh3D (mm)	0.76±0.04b	0.44±0.04	0.42±0.05	0.67±0.01b	0.61±0.02a	0.57±0.01a	0.50±0.01	0.64±0.01b,d	0.59±0.01b,h	0.55±0.01a,g
CalibTbSp3D (mm)	0.22±0.01b	0.58±0.01	0.60±0.02	0.30±0.01b	0.35±0.02b	0.42±0.01b	0.53±0.01b	0.31±0.01b,d	0.36±0.02b,h	0.49±0.01b,g
BS/BV (1/mm)	2.71±0.09a	5.44±0.11	5.49±0.14	3.41±0.05a	3.77±0.07a	4.06±0.08a	4.55±0.09a	3.68±0.06a,d	3.87±0.04a,c	4.19±0.07a,g
BV/TV (%)	53.62±1.10a	38.56±1.02	38.13±1.27	50.98±1.59a	46.11±0.84a	44.36±1.16a	39.20±1.22a	49.56±0.56a,f	46.66±0.78a,c	42.95±1.04a,e
TbSp (mm)	0.24±0.01a	0.69±0.03	0.70±0.02	0.32±0.01a	0.41±0.02a	0.45±0.01a	0.61±0.01a	0.34±0.01a,d	0.40±0.01a,c	0.54±0.02a
TbTh (mm)	0.71±0.02a	0.33±0.02	0.31±0.01	0.64±0.03a	0.47±0.02a	0.44±0.01a	0.36±0.01a	0.59±0.05a,d	0.48±0.01a,c	0.37±0.01a
TbN (mm)	6.11±0.91a	1.68±0.06	1.72±0.10	4.13±0.32a	3.65±0.17a	2.74±0.12a	2.01±0.13a	3.98±0.10a,d	3.09±0.16a,c	2.14±0.18a

Notes:

a P<0.05 compared to OVX;

b P<0.01 compared to OVX;

c P<0.05 compared to CIT-M;

d P<0.05 compared to CIT-H;

e P<0.05 compared to CIT-L;

f P<0.01 compared to CIT-H;

g P<0.01 compared to CIT-L;

h P<0.01 compared to CIT-M. Data expressed as means ± standard deviation.

Abbreviations: BMD, bone mineral density; OVX, ovariectomized; SO, suet oil; EV, estradiol valerate; CIT, circinal–icaritin; H, high dosage; M, medium dosage; L, low dosage; BMC, BM content; TMC, tissue mineral content; TMD, TM density; VOB, volume of bone; CalibTbTh3D, calibration of trabecular thickness – 3-D; Sp, separation; BS/BV, bone surface over bone volume; TV, total volume; N, number.

Figure 2 Representative micro-computed tomography images of trabecular bone microarchitecture in the distal femurs.

Notes: The OVX rats presented notable reductions in trabecular number and trabecular area compared with the sham rats. CIT, CIT-SO, and EV partially prevented OVX-induced trabecular bone loss and significantly improved trabecular bone mass and microarchitecture. CIT-SO significantly improved trabecular bone microarchitecture compared with CIT.

Abbreviations: OVX, ovariectomized; SO, suet oil; EV, estradiol valerate; CIT, circinal–icaritin; H, high dosage; M, medium dosage; L, low dosage.

Table 2 Bone biomechanical parameters by mechanical testing

Parameters	Sham	OVX	SO	EV	CIT-H	CIT-M	CIT-L	CIT-SO-H	CIT-SO-M	CIT-SO-L
Ultimate load (N)	94.66±3.99b	63.10567±2. 69	60.11±5.67	88.76±1.72b	81.47±1.94a,e	76.10±1.57 a,d	68.38±1.73 a	87.26±2.91b,e	80.80±2.01b,d	72.16±1.56b,c
Stiffness (N/mm)	247.27±17.20b	146.29±12.33	143.62±9.68	224.02±6.38b	196.25±7.03b,e	177.48±8.53a	164.37±10.87	218.47±11.45b,e	197.08±7.65b,d	176.85±6.58a
Energy to failure (mJ)	23.37±2.02b	11.51±1.16	11.26±0.83	21.07±0.62b	18.92±0.47b,e	16.94±0.98b,d	13.99±1.00a	20.90±1.04b,e	19.16±0.83b,d	14.36±0.66a

Notes:

a P<0.05 compared to OVX;

b P<0.01 compared to OVX;

c P<0.05 compared to CIT-L;

d P<0.05 compared to CIT-M;

e P<0.05 compared to CIT-H. Data expressed as means ± standard deviation.

Abbreviations: OVX, ovariectomized; SO, suet oil; EV, estradiol valerate; CIT, circinal–icaritin; H, high dosage; M, medium dosage; L, low dosage.

Figure 3 Serum parameters changes in the OVX model of osteoporosis.

Notes: ^aP<0.05 compared to OVX; ^bP<0.01 compared to OVX; ^cP<0.05 compared to CIT-L; ^dP<0.01 compared to CIT-L; ^eP<0.05 compared to CIT-M; ^fP<0.01 compared to CIT-M; ^gP<0.05 compared to CIT-H; ^hP<0.01 compared to CIT-H.

Abbreviations: OVX, ovariectomized; SO, suet oil; EV, estradiol valerate; CIT, circinal–icaritin; H, high dosage; M, medium dosage; L, low dosage; HOP, hydroxyproline; ALP, alkaline phosphatase; OPG, osteoprotegerin; OCN, osteocalcin; RANKL, receptor activator of nuclear factor-κB ligand; TRACP-5b, tartrate-resistant acid phosphatase 5b.

Figure 4 Related protein and gene expressions in osteoporotic rat femurs.

Notes: (A) OPG and RANKL protein expressions in femurs of OVX rats. Effects of 12-week treatment with EV, CIT-H, CIT-M, CIT-L, CIT-SO-H, CIT-SO-M, and CIT-SO-L on protein expressions of OPG and RANKL. (B) OPG and RANKL mRNA expressions in femur of OVX rats treated with EV, CIT-H, CIT-M, CIT-L, CIT-SO-H, CIT-SO-M, and CIT-SO-L for 12 weeks. Total RNA was isolated, and reverse-transcription polymerase chain reaction was performed to determine OPG and RANKL mRNA expressions. Values are expressed as means ± standard deviation (n=8); ^aP<0.05 compared to OVX, ^bP<0.01 compared to OVX, ^cP<0.05 compared to CIT-L, ^dP<0.05 compared to CIT-M, ^eP<0.05 compared to CIT-H, ^fP<0.01 compared to CIT-H.

Abbreviations: OVX, ovariectomized; SO, suet oil; EV, estradiol valerate; CIT, circinal–icaritin; H, high dosage; M, medium dosage; L, low dosage; RANKL, receptor activator of nuclear factor-κB ligand; OPG, osteoprotegerin; mRNA, messenger ribonucleic acid; OCN, osteocalcin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Table 3 Pharmacokinetic parameters of CIT and CIT-SO administered orally to rats (n=3)

Parameters	CIT-H	CIT-M	CIT-L	CIT-SO-H	CIT-SO-M	CIT-SO-L
AUC0–t (mg/L per hour)	607.75±3.16	279.54±7.46	151.44±19.65	751.54±32.90**	335.55±9.16##	204.31±3.56Δ
AUC0–∞ (mg/L per hour)	680.40±2.61	313.20±8.00	171.34±22.98	865.31±38.98**	379.09±10.13##	229.56±1.90Δ
MRT0–t (hours)	12.11±0.17	12.23±0.05	12.23±0.03	12.83±0.23*	12.20±0.05	12.16±0.04
MRT0–∞ (hours)	16.79±0.49	16.90±0.09	17.43±0.38	18.63±0.31**	17.35±0.13##	17.00±0.54
t½ (hours)	12.92±0.42	12.88±0.15	13.55±0.42	13.56±0.53**	13.54±0.13##	13.15±0.63
Cl (L/h/kg)	59.03±0.90	127.77±3.31	236.08±2.94	46.29±2.14**	105.57±2.85##	174.26±1.45Δ
Cmax (μg/L)	56.72±0.62	26.11±0.53	14.16±1.90	64.38±0.43**	31.56±0.86##	18.98±0.42Δ

Notes:

* P<0.05,

** P<0.01 compared with CIT-H group;

## P<0.01 compared with CIT-M group;

Δ P<0.05 compared with CIT-L group. Data expressed as means ± standard deviation.

Abbreviations: CIT, circinal–icaritin; SO, suet oil; H, high dosage; M, medium dosage; L, low dosage; AUC, area under the curve; MRT, mean residence time; t_½, half-life; Cl, clearance; C_max, maximum concentration.

Figure 5 The plasma concentration–time curve of CIT in rats after oral administration of CIT and CIT-SO.

Notes: Dosage 40 mg/kg for CIT-H and CIT-SO-H, 20 mg/kg for CIT-M and CIT-SO-M, and 10 mg/kg for CIT-L and CIT-SO-L.

Abbreviations: CIT, circinal–icaritin; SO, suet oil; H, high dosage; M, medium dosage; L, low dosage.

Table 4 Physicochemical properties of CIT with or without SO

Groups	Average size (nm)	Polydispersity
CIT-DOC	204.77±6.81	0.36±0.02
CIT-DOC-SO	100.80±7.21a	0.30±0.02b

Notes:

a P<0.01 compared with average size of CIT-DOC group;

b P<0.05 compared with polydispersity of CIT-DOC group. Data expressed as means ± standard deviation.

Abbreviations: CIT, circinal–icaritin; SO, suet oil; DOC, sodium deoxycholate.

Figure 6 Particle-size distribution and transmission electron microscopy of CIT-DOC and IT-SO-DOC.

Notes: (A) Particle-size distribution of CIT-DOC; (B) particle-size distribution of CIT-SO-DOC; (C) transmission electron microscopy of CIT-DOC; (D) transmission electron microscopy of CIT-SO-DOC. The average size of CIT-SO-DOC micelle was 100.80±7.21 nm, which was significantly lower than the 204.77±6.81 nm of the CIT-DOC group. The CIT-SO-DOC self-assembled nanomicelles seemed to be monodisperse spherical particles with smooth surfaces.

Abbreviations: CIT, circinal–icaritin; DOC, sodium deoxycholate; SO, suet oil.

Figure 7 The permeability-coefficient (P*eff) values of CIT and CIT-SO in the duodenum (Duo), jejunum (Jej), ileum (Ileu), and colon.

Notes: ^aP<0.01 compared with CIT-DOC group. Data expressed as means ± standard deviation (n=4).

Abbreviations: CIT, circinal–icaritin; SO, suet oil.