Dried pomegranate potentiates anti-osteoporotic and anti-obesity activities of red clover dry extracts in ovariectomized rats

Su Jin Kang, Beom Rak Choi, Seung Hee Kim, Hae Yeon Yi, Hye Rim Park, Dong Chul Kim, Seong Hun Choi, Chang Hyun Han, Soo Jin Park, Chang Hyun Song, Sae Kwang Ku, Young Joon Lee, Su Jin Kang, Beom Rak Choi, Seung Hee Kim, Hae Yeon Yi, Hye Rim Park, Dong Chul Kim, Seong Hun Choi, Chang Hyun Han, Soo Jin Park, Chang Hyun Song, Sae Kwang Ku, Young Joon Lee

Abstract

Red clover (RC) shows potential activity against menopausal symptoms and pomegranates have antioxidative and beneficial effects on postmenopausal symptoms; thus, we investigated whether the anti-climacteric activity of RC could be enhanced by the addition of dried pomegranate concentrate powder (PCP) extracts in ovariectomized (OVX) rats. Regarding the anti-osteoporotic effects, bone mineral density increased significantly in OVX induced rats treated with 60 and 120 mg/kg of an RC:PCP 2:1 mixture, respectively, compared with OVX control rats. Additionally, femoral, tibia, and L4 bone resorption was decreased in OVX induced control rats treated with the RC:PCP 2:1 mixture (60 and 120 mg/kg), respectively, compared with OVX control rats. Regarding anti-obesity effects, the OVX induced rats treated with 60 and 120 mg/kg of the RC:PCP 2:1 mixture showed a decrease in total fat pad thickness, the mean diameters of adipocytes and the body weights gain compared with OVX induced control rats. The estradiol and bone-specific alkaline phosphatase levels were significantly increased in OVX induced rats treated with the RC:PCP 2:1 mixture (120 mg/kg) compared with OVX induced control rats, also, the uterine atrophy was significantly inhibited in 60 and 120 mg/kg of the RC:PCP 2:1 mixture treatment compared with OVX control rats. In conclusion, our results indicate that PCP enhanced the anti-climacteric effects of RC in OVX rats. The RC:PCP 2:1 mixture used in this study may be a promising new potent and protective agent for relieving climacteric symptoms.

Figures

Figure 1
Figure 1
Significant (p < 0.01) increases of body weights were detected in all ovariectomized (OVX) rats as compared with sham control rats in this experiment (red arrow). However, significant (p < 0.01 or p < 0.05) decreases of body weights were demonstrated in mixture 120 mg/kg treated rats from 56 days after initial administration, from 70 days after initial treatment in red clover (RC), pomegranate concentrate powder (PCP), mixture 60 mg/kg and from 77 days after initial treatment in all treated group as compared with OVX control rats, respectively (arrows). Values are expressed mean ± S.D. of eight rats.
Figure 2
Figure 2
Significant changes of the serum estradiol levels were demonstrated in mixture 120 mg/kg treated rats as compared with those of single formula of PCP treated rats, respectively. Values are expressed mean ± S.D. of eight rats. ap < 0.01 as compared with sham control; bp < 0.01 and cp < 0.05 as compared with OVX control; dp < 0.05 as compared with PCP treated rats by Tamhane test.
Figure 3
Figure 3
Significant changes of the serum osteocalcin levels were demonstrated in mixture 60 and 120 mg/kg treated rats as compared with those of single formula of RC or/and PCP treated rats, respectively. Values are expressed mean ± S.D. of eight rats. ap < 0.01 and bp < 0.05 as compared with sham control; cp < 0.01 and dp < 0.05 as compared with OVX control; ep < 0.05 as compared with RC treated rats; fp < 0.01 and gp < 0.05 as compared with PCP treated rats by Tukey test.
Figure 4
Figure 4
Significant changes of the serum bALP levels were demonstrated in mixture 60 and 120 mg/kg treated rats as compared with those of single formula of RC or/and PCP treated rats, respectively. Values are expressed mean ± S.D. of eight rats. ap < 0.01 and bp < 0.05 as compared with sham control; cp < 0.01 as compared with OVX control; dp < 0.05 as compared with RC treated rats; ep < 0.01 and fp < 0.05 as compared with PCP treated rats by Tamhane test.
Figure 5
Figure 5
The strengths of femur mid-shaft regions in mixture 120 mg/kg treated rats were significantly decreased as compared with those of single formula of PCP treated rats, respectively. Values are expressed mean ± S.D. of eight rats. ap < 0.01 as compared with sham control; bp < 0.01 as compared with OVX control; cp < 0.05 as compared with PCP 20 mg/kg treated rats by Tukey test.
Figure 6
Figure 6
The strengths of tibia mid-shaft regions in mixture 60 and 120 mg/kg treated rats were significantly decreased as compared with those of single formula of RC or/and PCP treated rats, respectively. Values are expressed mean ± S.D. of eight rats. ap < 0.01 as compared with sham control; bp < 0.01 as compared with OVX control; cp < 0.01 as compared with RC 40 mg/kg treated rats; dp < 0.01 and ep < 0.05 as compared with PCP 20 mg/kg treated rats by Tamhane test.
Figure 7
Figure 7
Representative histological images of the adipocytes, taken from sham-operated or OVX rats abdominal fat pads deposited into left dorsal abdominal muscles. Obvious increases of mean adipocyte diameters, adipocyte hypertrophic changes, were demonstrated in OVX control rats, due to noticeable deposition of adipose tissues on the abdominal cavity. However, noticeable decreases of the mean diameters of adipocytes were detected in all test substance administrated rats as compared with OVX control rats, respectively. Especially, mixture 120 and 60 mg/kg treated rats also showed dramatic inhibitory activities on the hypertrophy of adipocytes in the abdominal cavity as compared with those of single formula of RC and PCP treated rats, respectively. (A); Shame control rat, (B); OVX control rat, (C); RC 40 mg/kg treated rat, (D); PCP 20 mg/kg treated rat, (E); RC:PCP 2:1 mixture (g/g) 120 mg/kg treated rat, (F); RC:PCP 2:1 mixture (g/g) 60 mg/kg treated rat, (G); RC:PCP 2:1 mixture (g/g) 30 mg/kg treated rat. All H&E stained. Scale bars = 240 μm.
Figure 8
Figure 8
Representative histological profiles of the femur and L4, taken from sham-operated or OVX rats. Although relatively well-developed trabecular and cortical bone were observed in the long bone-femur and the short bone-L4 of sham control rats, classical osteoporotic histological profiles were demonstrated in OVX control rats as dramatic decreases of trabecular and cortical bone masses, increase of connective tissues in periosteum of cortical bone results from resorption of osteoid tissues related to osteocalst activations in this study. However, dramatic increases of the bone mass and structures, the both trabecular and cortical bones were detected in all test substance administered rats including PCP as compared with OVX control rats, related to their inhibitory activities on osteoclast cell activities, respectively. Especially, mixture 120 and 60 mg/kg treated rats also showed noticeable inhibition of the bone losses and osteoclast cell activations as compared with those of single formula of RC and PCP treated rats, respectively. (A); Shame control rat, (B); OVX control rat, (C); RC 40 mg/kg treated rat, (D); PCP 20 mg/kg treated rat, (E); RC:PCP 2:1 mixture (g/g) 120 mg/kg treated rat, (F); RC:PCP 2:1 mixture (g/g) 60 mg/kg treated rat, (G); RC:PCP 2:1 mixture (g/g) 30 mg/kg treated rat. L4; Fourth lumbar vertebrae, Cb; cortical bone, Tb; trabecular bone, Bm; bone marrow, Gp; growth plate. All Safranin O stained. Scale bars = 480 μm.

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