Varsha Gupta

Kaempferol has osteogenic effect in ovariectomized adult Sprague–Dawley rats

Kaempferol (K), a flavonol, is known to have anti-osteoclastogenic effect. We here show that K, from 0.2 to 5.0 microM, increased mineralized nodules in rat primary osteoblasts. K also significantly attenuated adipocyte formation from bone marrow cells (BMCs). A single oral dose of 1 mg/kg body weight of K in Sprague-Dawley (180-200 g) rats resulted in a peak serum level of 2.04+/-0.8 nM in 30 min (Tmax), suggesting its rapid absorption. The Cmax of K in bone marrow was 0.684 nM after 90 min. Rats were ovariectomized (OVx) along with sham-operated rats and left for 4 weeks. Daily oral administration of K (5 mg/kg body weight) was then started to one group of OVx rats, and continued for 10 weeks. K levels were found to be 0.311 and 0.838 nM at the end of 4 and 10 weeks, respectively. K exhibited no estrogenicity at the uterine level. The K-treated group exhibited significantly higher bone mineral density (BMD) in the trabecular regions (femur neck, proximal tibia and vertebrae) and lower serum ALP (bone turnover marker) compared with the OVx rats. The compressive energy of the vertebrae was significantly higher in the OVx+K-treated group compared with the OVx group. K treatment of OVx rats resulted in the increase in osteoprogenitor cells as well as inhibition of adipocyte differentiation from BMCs compared with the OVx group. Together we show that K is non-estrogenic in vivo and exerts bone anabolic activity with attendant inhibition of bone marrow adipogenesis.

Effects of Egb 761 on bone mineral density, bone microstructure, and osteoblast function: Possible roles of quercetin and kaempferol.

The effects of standardized and concentrated extract of Ginkgo biloba, Egb 761, were studied on estrogen deficiency-induced bone loss in ovariectomized (OVx) rats rendered osteopenic. Upon osteopenia development, Egb 761 was administered at a dose of 100mgkg(?1)day(?1) by oral gavage to OVx rats whereas control group received vehicle. Following 5 weeks of treatment, the OVx+Egb 761 group (n=12) of rats exhibited significantly higher whole body BMD and lower bone turnover markers (serum alkaline phosphatase and osteocalcin) than OVx rats that were given vehicle (n=12). BMD levels in excised bones were also found to be higher in both trabecular (most robustly in lumbar vertebrae) and cortical bones of OVx+Egb 761 compared with OVx+vehicle group. Egb 761 did not exhibit estrogen agonistic activity at the uterine level. Microcomputed tomography demonstrated that OVx+Egb 761 group had better bone microarchitectural parameters compared with OVx+vehicle group. Moreover, OVx+Egb 761 group had higher femoral mRNA levels of osterix, type I collagen and osteocalcin compared with OVx+vehicle group. Determination of levels of three flavonoids of Egb 761, that are known to have bone conserving property, in serum and bone marrow suggests that kaempferol and quercetin, and not rutin, likely mediate the beneficial actions observed with Egb 761 treatment. These results show for the first time that oral administration of Egb 761 restores bone mass in aged OVx rats.

A naturally occurring naringenin derivative exerts potent bone anabolic effects by mimicking oestrogen action on osteoblasts

BACKGROUND AND PURPOSE Naringenin and its derivatives have been assessed in bone health for their oestrogen‐‘like’ effects but low bioavailability impedes clinical potential. This study was aimed at finding a potent form of naringenin with osteogenic action.

In vivo efficacy studies of layer-by-layer nano-matrix bearing kaempferol for the conditions of osteoporosis: A study in ovariectomized rat model

A prototype formulation based on layer-by-layer (LbL) nano-matrix was developed to increase bioavailability of kaempferol with improved retention in bone marrow to achieve enhanced bone formation. The layer-by-layer nano-matrix was prepared by sequential adsorption of biocompatible polyelectrolytes over the preformed kaempferol-loaded CaCO(3) template. The system was pharmaceutically characterized and evaluated for osteogenic activity in ovariectomized (OVx) rats. Data have been compared to the standard osteogenic agent parathyroid hormone (PTH). Single oral dose of kaempferol loaded LbL nano-matrix formulation increased bioavailability significantly compared to unformulated kaempferol. Three months of Formulated kaempferol administration to osteopenic rats increased plasma and bone marrow Kaempferol levels by 2.8- and 1.75-fold, respectively, compared to free Kaempferol. Formulated Kaempferol increased bone marrow osteoprogenitor cells, osteogenic genes in femur, bone formation rate, and improved trabecular micro-architecture. Withdrawal of Formulated kaempferol-in OVx rats resulted in the maintenance of bone micro-architecture up to 30days, whereas micro-architectural deterioration was readily observed in OVx rats treated with unformulated kaempferol-within 15days of withdrawal. The developed novel formulation has enhanced anabolic effect in osteopenic rats through increased stimulatory effect in osteoblasts. Treatment post-withdrawal sustenance of formulated kaempferol could become a strategy to enhance bioavailability of flavanoids.

Osteogenic efficacy enhancement of kaempferol through an engineered layer-by-layer matrix: a study in ovariectomized rats

AIM A layer-by-layer matrix (LBL) comprising kaempferol (LBL-KEM) was prepared for improved osteogenic action. MATERIALS & METHODS The LBL-KEM consisted of alternate layers of sodium alginate and protamine sulfate, which were sequentially deposited on the preformed kaempferol (KEM)-loaded CaCO3 core (CaCO3-KEM) by LBL self-assembly. The LBL matrix developed was evaluated for layer growth by ζ-potential and size alterations after self-assembly of each layer. Its physicochemical properties and intestinal absorption pattern were characterized and its pharmacokinetic behavior, mineralization of bone marrow cells, bone mineral density, bone strength, microcrack formation and estrogenicity were evaluated after oral administration. RESULTS The entrapment efficiency of KEM was 94 ± 2% and the cumulative %KEM released from LBL-KEM was 19.2 and 63.5% at pH 1.4 and 7.4, respectively, after 24 h. Stepwise polyelectrolyte assembly onto initially positively charged particles (+21.2 mV) resulted in alterations between -28.5 and +10.9 mV. A final ζ-potential of -8.9 mV was obtained after terminal surface modification with sodium deoxycholate. Fluorescein isothiocyanate-labeled LBL matrix was diffused into the basolateral lacteal region upon oral administration to rats. The area under the KEM serum concentration curve following oral administration of LBL-KEM to rats was 2479 ± 682 ng·h/ml, nearly twofold higher than free KEM. The concentration-time profile in bone marrow indicated improved penetration and retention of KEM on administration of LBL-KEM. Treatment with LBL-KEM restored bone mineralization, bone mineral density, microcrack formation and empty osteocyte lacunae density in ovariectomized (OVx) rats, which was significantly (p < 0.05) improved in femoral diaphysea, tibial head and vertebrae compared with free KEM treatment. Administration of LBL-KEM to growing female rats for 4 weeks resulted in no estrogenicity when compared with OVx rats. CONCLUSION The data suggests that LBL matrix enhanced drug delivery, improved pharmacokinetics and maintained better bone quality under OVx conditions.

Effect of polydimethylsiloxane and ethylcellulose on in vitro permeation of centchroman from its transdermal patches

Abstract This research aims at the development of controlled release contraceptive transdermal patches of centchroman using ethylcellulose (EC) as film-forming polymer, polydimethylsiloxane (PDMS) as pressure sensitive adhesive with propylene glycol and Di-n-butyl-phthalate for their penetration enhancer and plasticizing properties, respectively. The physicochemical compatibility of the drug and the polymers was performed by differential scanning calorimetry and Fourier transform infrared (FTIR) spectroscopic technique. Effects of EC and PDMS ratios on moisture uptake, moisture content, tensile strength (TS), Young’s modulus, adhesive strength, water vapor transmission rate (WVTR) and in vitro permeation of centchroman through Sprague–Dawley rats abdominal skin using Franz’s diffusion cell were evaluated. A 32 full factorial design was employed to observe the effect of independent variables; concentration of ethyl cellulose and PDMS on drug permeated after 32 h, which was selected as dependent variable. Compatibility studies suggested that there were no significant interaction between the drug and polymers used. It was found that incorporation of only EC resulted in too hard patches and addition of PDMS produced patches with lower TS, increased percentage elongation, WVTR and Young’s modulus. Statistical analyses suggested that independent variables have a significant effect on the dependent variable. All formulation follows zero-order release kinetics with r2 > 0.990. In conclusion, drug in adhesive transdermal patches can be successfully fabricated for non-steroidal contraceptive centchroman to obtain a zero-order release systems.

Iron Profile and Glycaemic Control in Patients with Type 2 Diabetes Mellitus

Iron overload is increasingly being connected to insulin resistance in Type 2 Diabetes Mellitus (T2DM) patients. Free iron causes the assembly of reactive oxygen species that invariably steer the body’s homeostasis towards oxidative stress-mediated diabetic complications. This study aims to assess the serum iron, total iron binding capacity (TIBC), and percentage transferrin saturation (Tsat) of 150 subjects divided into three groups (I,II,III) of 50. Healthy individuals (controls) constituted Group I. Group II consisted of T2DM patients with optimal glycaemic control. T2DM patients with suboptimal glycaemic control formed group III. Mean serum free iron concentration was 105.34 ± 3.5, 107.33 ± 3.45, and 125.58 ± 3.45 μg/dL in Group I, Group II, and Group III, respectively. Mean serum TIBC concentration in Group I, Group II, and Group III was 311.39 ± 5.47, 309.63 ± 6.1, and 284.2 ± 3.18μg/dL, respectively. Mean serum transferrin saturation (%) in Group I, Group II, and Group III was 34.17 ± 1.21, 35.02 ± 1.2, and 44.39 ± 1.07, respectively. The difference between TIBC, mean serum free iron concentration, and transferrin saturation between Group I and Group III (for all, p values <0.001), as well as between Group II and Group III (p values 0.0012, 0.0015, and <0.0001, respectively) was statistically significant. The fasting plasma glucose values of Groups II and III were significantly higher than those of Group I, (p < 0.0001). Glycated haemoglobin (HbA1c) values were also shown to increase from Group I to II and then III, and the increase was highly significant (all p values <0.0001). Thus, decreased glycaemic control and an increase in the glycation of haemoglobin was the key to elevation in serum iron values and alterations in other parameters. However, a significant correlation was absent between serum iron and HbA1c (r = 0.05) and transferrin saturation (r = 0.0496) in Group III.

Assay method for quality control and stability studies of a new anti-diabetic and anti-dyslipidemic flavone (S002-853)*

Background: Flavonoid-rich extract of the plant is long known for its anti-diabetic activities in traditional medicine. S002-853, a new flavone derivative synthesized by Central Drug Research Institute (CDRI) has been used for the present study. Objectives: The present study aimed at development of an assay method for quality control (QC) and stability studies of a new anti-diabetic and anti-dyslipidemic agent CDRI compound S002-853. Materials and Methods: A validated high-performance liquid chromatography analysis method for S002-853 was developed for in process QC and stability studies. The separation was achieved on a RP-C18 (25 cm × 0.4 cm, 5 μm, Phenomenex) at 240 nm with flow rate of 1.0 ml/min. This method was applied successfully in establishing forced degradation and drug-excipient testing protocols as per International Conference on Harmonization guidelines. Results: The result of estimation and stress testing studies indicated a high degree of selectivity of this method. S002-853 was most stable at pH 7 and under photolytic conditions. The temperature degradation pattern of S002-853 was found to follow the zero order degradation. Conclusion: The method described is easy and simple hence can be easily reproduced. This method can be very useful for bulk manufacture QC, and drug development process.

Chiral Separation of Ormeloxifene Hydrochloride, a Non-steroidal Contraceptive Agent

Ormeloxifene hydrochloride (Centchroman) is once-a-week non-steroidal oral contraceptive agent marketed in India and other countries. In this study, we report a validated isocratic high-performance liquid chromatographic (HPLC) method for chiral separation of D- and L-ormeloxifene hydrochloride. This method is capable of baseline separation of its D- and L-isomers. HPLC separation was achieved on a Lux 5µ cellulose-1 with a mobile phase comprising hexane, isopropanol, methanol and triethylamine (90:10:1:0.5). Validation parameters such as limit of detection, limit of quantitation, linearity, precision, accuracy, specificity and preformulation studies were conducted according to new guidelines of International Conference on Harmonization.

HISTOMORPHOLOGICAL STUDIES OF THE ACCESSORY ADRENAL NODULES IN PRENATAL GOATS

A study was conducted on 24 embryos/foeti of nondescript goats (Capra hircus) varying from day-old to 150 days of gestation. The embryos/foeti were assigned into three groups according to their gestational ages as, Gr- 1 (050 days), Gr-2(51–100 days) and Gr-3 (101- till term). The extra capsular accessory adrenal nodules were observed at 55, 60 and 76 days of gestation in Gr-1 while intra capsular accessory adrenal nodules were observed at 55, 60 and 91 days in Gr-II.