The rats were also given intramuscular injection of Baytril 5% (Bayer Health Care, Thailand) for 5 days. The sham-operated rats underwent a similar procedure except that the adrenal glands were not removed. The treatment was started two weeks after adrenalectomy. Dexamethasone (Sigma, USA) was dissolved in olive oil (Bertolli, Italy) and administered

IM (120 µg/kg/day) six days-a-week for two minths. The dose and duration of treatment was determined by a pilot study. The Piper sarmentosum leaves extract was provided by the Forest Research Institute of Malaysia (FRIM). Piper sarmentosum and GCA (Sigma, Inhibitors,research,lifescience,medical USA) were dissolved in normal saline and administered for two months. The sham-operated rats were administered equivalent Inhibitors,research,lifescience,medical volumes of vehicle (olive oil) intramuscularly and vehicle (normal saline) by oral gavage. The dexamethasone-treated adrenalectomized rats (G3) were also administered with vehicle (normal saline, 0.1 ml/100 g) by oral gavage. The administrations of Piper sarmentosum leaves water extract, GCA and dexamethasone were started simultaneously two weeks after the adrenalectomy. The treatment was given for two months. The animals were kept in clean cages under natural sunlight during daytime and darkness at night. They

had free access to rat pellets (Gold Coin, Malaysia). The sham-operated Inhibitors,research,lifescience,medical animals had free access to tap water, while the adrenalectomized animals had free access to normal saline instead of tap water to replace the salt loss due to post-adrenalectomy mineralocorticoid deficiency. The activity and expression of 11β-HSD1 in femoral bone were measured at the end of two months Inhibitors,research,lifescience,medical of treatment. Sample Collection The right femoral bones were cleared of surrounding tissues, wrapped in a piece of gauze and aluminium foil, and frozen at -70°C until analyzed. The left femoral bones were cut at the mid shaft with Inhibitors,research,lifescience,medical a rotary blade (Black & Decker) to separate

the distal and proximal parts. The distal part was cut longitudinally to separate the bones into medial and lateral parts. The lateral part of the bones was then subjected to decalcification BTK inhibitor process in a mixture of ethylenediamintetraacetate (EDTA) and 10% formalin. check Assay for 11β-HSD1 Dehydrogenase Activity The activity of 11β-HSD1 dehydrogenase was measured using the modified technique of Cooper et al.13 The right femoral bones were dissected, cleared from soft tissues and washed extensively in phosphate-buffered saline to reduce the fat content. They were ground into small pieces before being suspended in Krebs-Ringer bicarbonate buffer and homogenized overnight at 4ºC. The bone homogenate was centrifuged at 12,100 g for 20 min at 4°C, and the supernatant was decanted. The total protein content was estimated calorimetrically (Bio-Rad, Hercules, CA, USA).