The protective effects of Ninjin'yoeito against liver steatosis/fibrosis in a non-alcoholic steatohepatitis model mouse.

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic form of non-alcoholic fatty liver disease. Liver fibrosis leads to liver cancer and cirrhosis, and drug therapy for NASH remains lacking. Ninjin'yoeito (NYT) has shown antifibrotic effects in a model of liver fibrosis without steatosis but has not been studied for NASH. Therefore, we evaluated the efficacy of NYT in mice fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a NASH model. Compared with the normal diet group, mice fed CDAHFD showed decreased body weight and increased white adipose tissue, liver weight, and triglyceride content in the liver. Furthermore, a substantial increase in the hepatic concentration of hydroxyproline, expression of α-smooth muscle actin (α-SMA), and transforming growth factor-ß was observed in CDAHFD-fed mice. Masson's trichrome and Picro-Sirius red staining revealed a remarkable increase in collagen fiber compared with the normal diet group. Compared with mice that received CDAHFD alone, those supplemented with NYT exhibited reduced hepatic triglyceride and hydroxyproline levels and α-SMA expression. Additionally, compared with the group fed CDAHFD alone, the stained liver tissues of NYT-treated mice exhibited a reduction in Masson's trichrome- and Picro-Sirius red-positive areas. Locomotor activity was significantly reduced in the CDAHFD-fed group compared with the normal diet group. In the NYT-treated group, the CDAHFD-induced decrease in locomotor activity was significantly suppressed. The findings indicate that NYT inhibited fatty and fibrotic changes in the livers of NASH mice and alleviated the decrease in locomotor activity. Therefore, NYT may serve as a novel therapeutic approach for NASH.

Ninjin'yoeito suppressed the onset of arthritis, pain, and muscle atrophy in rheumatoid arthritis model mice.

Rheumatoid arthritis is one of the most common diseases in orthopedic surgery. The main symptoms are joint pain and systemic symptoms. In recent years, rheumatoid arthritis is known to cause sarcopenia. Ninjin'yoeito (NYT), a traditional Japanese medicine, has been prescribed for patients with post-illness or post-operative weakness, fatigue, loss of appetite, rash, cold limbs, and anemia. In addition to its traditional use, NYT has been prescribed for treating frailty in gastrointestinal, respiratory, and urinary functions. Further, NYT is known to be effective in suppressing muscle atrophy in the prior literature. The present study aimed to investigate whether NYT suppresses various symptoms of the Collagen-induced arthritis (CIA) model. Long-term administration of NYT inhibited the increases in arthritis scores, decreases pain threshold, and muscle atrophy in the CIA model. In addition, NYT inhibited the elevation of the plasma IL-6 level. These results suggest that NYT may have therapeutic effects on symptoms, muscle atrophy and increase in plasma IL-6 level caused by rheumatoid arthritis.

Disuse muscle atrophy-improving effect of ninjin'yoeito in a mouse model.

Disuse syndrome indicates psychosomatic hypofunction caused by excess rest and motionless and muscle atrophy is termed disuse muscle atrophy. Disuse muscle atrophy-induced muscle weakness and hypoactivity further induces muscle atrophy, leading to a vicious cycle, and this is considered a factor causing secondary sarcopenia and subsequently frailty. Since frailty finally leads to a bedridden state requiring nursing, in facing a super-aging society, intervention for a risk factor of frailty, disuse muscle atrophy, is important. However, the main treatment of disuse muscle atrophy is physical therapy and there are fewer effective preventive and therapeutic drugs. The objective of this study was to search for Kampo medicine with a disuse muscle atrophy-improving effect. Ninjin'yoeito is classified as a qi-blood sohozai (dual supplement) in Chinese herbal medicine, and it has an action supplementing the spleen related to muscle. In addition, improvement of muscle mass and muscle weakness by ninjin'yoeito in a clinical study has been reported. In this study, the effect of ninjin'yoeito on disuse muscle atrophy was investigated. A disuse muscle atrophy model was prepared using male ICR mice. After surgery applying a ring for tail suspension, a 1-week recovery period was set. Ninjin'yoeito was administered by mixing it in the diet for 1 week after the recovery period, followed by tail suspension for 14 days. Ninjin'yoeito administration was continued until autopsy including the hindlimb suspension period. The mice were euthanized and autopsied immediately after completion of tail suspension, and the hindlimb muscles were collected. The food and water intakes during the hindlimb unloaded period, wet weight of the collected muscle, and muscle synthesis and muscle degradation-related factors in blood and muscle were evaluated. Ingestion of ninjin'yoeito inhibited tail suspension-induced reduction of the soleus muscle wet weight. In addition, an increase in the blood level of a muscle synthesis-related factor, IGF-1, and promotion of phosphorylation of mTOR and 4E-BP1 in the soleus muscle were observed. It was suggested that ninjin'yoeito has a disuse muscle atrophy-improving action. Promotion of the muscle synthesis pathway was considered the action mechanism of this.

Anti-inflammatory and Antinociceptive Constituents of Atractylodes japonica Koidzumi.

The rhizomes of many Atractylodes species, including Atractylodes chinensis Koidzumi, Atractylodes macrocephala Koidzumi, and Atractylodes japonica Koidzumi, are collectively termed Atractylodis Rhizoma. We prepared n-hexane extracts of the three species and evaluated their anti-inflammatory effects on lipopolysaccharide (LPS)-induced RAW 264.7 cells. Among all n-hexane extracts, those of A. japonica most strongly inhibited nitric oxide (NO) production in LPS-induced RAW 264.7 cells; five sesquiterpenes, atractylon, atractylenolide I, atractylenolide II, atractylenolide III, and 8-epiasterolid, were isolated from A. japonica. The phytochemical content of A. japonica was similar to those of A. chinensis and A. macrocephala. Moreover, the atractylon concentration was higher in A. japonica than in A. chinensis and A. macrocephala. Atractylon significantly inhibited NO and prostaglandin E2 production as well as inducible NO synthase and cyclooxygenase-2 expression in LPS-induced RAW 264.7 cells. Atractylon (40 mg/kg) also significantly reduced the acetic-acid-induced writhing response, carrageenan-induced paw edema, and hot-plate latent pain response in mice. According to the results, A. japonica has anti-inflammatory and antinociceptive effects and atractylon is the major active component of A. japonica. Therefore, atractylon can be used as a bioactivity marker in A. japonica.

Puerarin exerted anti-osteoporotic action independent of estrogen receptor-mediated pathway.

Puerarin, a daidzein-8-C glucoside, is the major isoflavonoid in Kudzu (Pueraria lobata), and is unique in that it contains C-C conjugated glucose at position 8 of the isoflavonoid structure. A puerarin diet at a dose of 5 mg/kg b.w./d to fed ovariectomized mice for 2 mo diminished the urinary deoxypyridinoline, a typical bone-degradation product. Since the bone absorption marker, serum tartarate-resistant acid phosphatase (TRAP) activity of puerarin-fed mice decreased but the bone formation marker, osteocalcin level, did not alter, the puerarin diet was proved to specifically depress the bone absorption, but not the overall bone metabolism. In accordance with that results, the femur structure of puerarin-fed mice was restored compared with that of puerarin-free diet mice. The atrophied uterine due to low estrogen (E2) level after ovariectomy was not restored by the puerarin diet, suggesting that puerarin exerted the anti-osteoporotic action through a non estrogen receptor (ER) mediated-pathway, in vivo. The growth of an ER-positive human breast cancer cell, MCF-70, was not enhanced by puerarin, suggesting that puerarin did not show estrogen-like action on MCF-7 cells, even at a ten thousand times higher concentration than that of E2. Furthermore, ICI182,780 (ICI), an estrogen antagonist, suppressed the enhanced growth of MCF-7 cells by E2, but not that by puerarin. In an ER-binding assay, puerarin was proved not to bind to ERα or ß, or if all, extremely weakly, although daidzein, an aglycon of puerarin, showed a little stronger binding compared with puerarin. All these results strongly indicate that puerarin exerts its anti-osteoprotic action independently of the ER-mediated pathway.

Kudzu (Pueraria lobata) vine ethanol extracts improve ovariectomy-induced bone loss in female mice.

Bone-loss-improving action of kudzu vine ethanol extracts (PVEE) was clarified. PVEE was composed roughly of 80% fiber, 10% puerarin, 3.6% daidzin, 2.5% 6″-O-malonyldaidzin, and the other minor isoflavones. Ten-week-old ovariectomized (OVX) mice were fed diets containing PVEE (20 mg/kg body weight/day) for 8 weeks. The bone resorption markers (urinary deoxypyridinoline and tartrate-resistant acid phosphatase activity) was elevated in OVX mice and was significantly decreased in OVX mice that consumed PVEE for 8 weeks. Consistent with the decrease in the markers, the number of matured osteoclasts in the distal femur was diminished in OVX mice fed PVEE diets. PVEE diets also suppressed the decrease in femoral bone mineral density (BMD) by OVX. PVEE showed the affinity for estrogen receptor α and ß nearly 1/10000 weaker than 17ß-estradiol. No hypertrophy in the uterus by the PVEE diet was observed. These results suggest that PVEE could be a promising resource for a functional food that improves osteoporosis.

Effects of keishibukuryoganryokayokuinin (gui-zhi-fu-ling-wanliao-jia-yiyiren) on the epidermal pigment cells from DBA/2 mice exposed to ultraviolet B (UVB) and/or progesterone.

The production of melanin is not only activated by external factors such as sunlight or UV-exposure, but is also considered to be triggered by hormonal factors, particularly sex hormones such as ovarian hormones. Previously, keishibukuryoganryokayokuinin (KBY) was reported to increase the pigmentation and moisture content of dermis in women during the luteal phase of the menstrual cycle, thus suggesting that progesterone could play a critical role in the development of skin pigmentation. In the present study, female DBA/2 mice, a dilute brown strain, were used to examine the effects of KBY on the increase in epidermal pigment cells in mice exposed to ultraviolet B (UVB) radiation or progesterone in an attempt to elucidate its mechanism. An increase in epidermal pigment cells was observed in mice exposed to progesterone. To the best of our knowledge, this is the first study to demonstrate that progesterone causes pigmentation in vivo. Furthermore, administration of KBY to progesterone-exposed mice significantly reduced the number of epidermal pigment cells. However, KBY had no such effects on UVB-induced pigmentation. Another important finding was the gain in body weight in progesterone-exposed mice, while body weight gain was reduced by KBY. The body weight gain was believed to be due to sodium and fluid retention, a kind of adverse effect of progesterone, which may further affect the intracellular pH of melanosomes, which synthesize melanin, in turn, leading to melanin production because tyrosinase activity is linked to the intracellular pH environment. This may help explain the mechanism of the role of KBY in pigmentation.