Bioactive fraction from Lagerstroemia speciosa leaves (DLBS3733) reduces fat droplet by inhibiting adipogenesis and lipogenesis.

Background: Obesity has become a risk factor for metabolic diseases. One of the cellular characteristics of obesity is the occurrence of adipose cells hyperplasia. Lagerstroemia speciosa is a plant which has been used for the treatment of diabetes. Furthermore, some studies also indicated that L. speciosa possesses antiobesity activity. Its antiobesity activity was examined in the present study through adipogenesis, lipogenesis, and lipolysis pathways. Aim: DLBS3733, a bioactive fraction of L. speciosa, was explored for its potential benefits to alter obesity through adipogenesis and lipogenesis inhibition and lipolysis induction activity. Materials and methods: This study was performed using 3T3-L1 cells. mRNA level and protein expressions related to adipogenesis, lipogenesis, and lipolysis pathways were assayed in this study. Results: Antiadipogenic effects of DLBS3733 (15 µg/mL) were found to be mediated by a significant downregulation of mRNA level of multicomponents involved in adipogenesis which include C/EBPα (CCAAT/enhancer-binding protein alpha) and PPAR-γ (peroxisome proliferator-activated receptor gamma) by 75% and 80.1% (p<0.05), respectively. DLBS3733 was found to inhibit lipogenesis, as shown by the significant reductions of adiponectin excretion and mRNA level of fatty acid synthase, SREBP (sterol regulatory element-binding protein), and ACC-ß (Acetyl-CoA carboxylase) by 44.7%, 70.9%, and 83.1%, respectively (p<0.05). In addition, DLBS3733 was found to inhibit fat droplets accumulation in the cells in a dose-dependent manner through Oil-Red O staining. pAMPK protein was upregulated by 75% and ACC-ß was downregulated by 88% (p<0.05) which indicates the reduction of lipid synthesis. Meanwhile, DLBS3733 showed an insignificant effect on adipose triglyceride lipase, hormone-sensitive lipase, and carnitine palmitoyl-CoA transferase-1 which indicate that DLBS3733 does not induce lipolysis. Conclusion: These results demonstrate the inhibitory activity of DLBS3733 on adipogenesis and lipogenesis. DLBS3733 may provide an effective and potential benefit in the prevention of obesity.

Upregulation of endogenous erythropoietin expression by DLBS6747, a bioactive fraction of Ipomoea batatas L. leaves, via increasing HIF1α transcription factor in HEK293 kidney cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ipomoea batatas L., locally known as ubi jalar, is widely used in Indonesia and other countries as a folk remedy for various chronic diseases, including anemia-associated chronic kidney disease by increasing hematological parameters such as packed cell volume, white blood cells and platelet counts. AIM OF THE STUDY: The aim of this study is to evaluate the effect of DLBS6747, a bioactive fraction of I. batatas L. leaves, on increasing EPO expression through the upregulation of HIF1α. MATERIALS AND METHODS: Effect of DLBS6747 on EPO expression and its transcription factor, HIFs, was evaluated in normoxia and hypoxia conditions. Effect of DLBS6747 on several genes involved in EPO expression were evaluated in a time-course manner using conventional and real-time PCR, while the protein level were revealed using western blot and ELISA. The involvement of HIF1α was also confirmed by HIF1α siRNA. RESULTS: Administration of DLBS6747 increased transcriptional activity of EPO through the regulation of its transcriptional factors, which include HIF1α, HIF2α and NFᴋB. The effect was found to be dependent on oxygen availability, wherein DLBS6747-increased EPO expression was found to be more significant in hypoxic condition. In normoxia and hypoxia, 40 µg/mL DLBS6747 increased HIF1α and HIF2α expressions at mRNA level, wherein the peak appeared in 12 h treatment (up to 7.9- and 8.6-folds, respectively). On the other hand, increased protein level was only found in hypoxia, where the highest HIF1α expression was observed at 6 h (7.5-folds increase) and started to decrease after the hours, while HIF2α was found to be increased time-dependently (up to 13.8-folds in 24 h). The mechanism of action of DLBS6747 as erythropoietin stimulating agent is more likely to affect the regulation of HIF1α, as confirmed by HIF1α siRNA which showed that DLBS6747 failed to increase EPO expression during co-incubation with HIF1α siRNA. DLBS6747 treatment also decreased NFᴋB time-dependently in normoxia, while no NFᴋB was detected in hypoxia, which revealed mimicking hypoxia activity of DLBS6747 to increase EPO expression. CONCLUSION: These findings showed convincing evidences that DLBS6747 increases endogenous EPO production primarily via upregulation of its transcription factors, especially HIF1α, in human embryonic kidney HEK293 cells. This is the first molecular report that reveals the mechanism of action of natural-based erythropenia drug in different oxygen availability.

Bioconjugation of Captopril-Light Subunit of Agaricus bisporus Mushroom Tyrosinase: Characterization and Potential Use as a Drug Carrier for Oral Delivery.

We show that a lectin like protein from the mushroom Agaricus bisporus (LSMT) is capable to permeate the epithelial monolayer barrier of the intestine ex vivo. The protein is not toxic or immunogenic upon prolonged administration and elevated dose in mice. Thus, it could be a candidate as a drug carrier for oral administration. However, its permeability should be tested after the protein has been modified, mimicking the condition in which it is used as a drug carrier. The protein was conjugated to captopril, the selected model of a Biopharmaceutical Classification System (BCS) class III drug, with high solubility but poor permeability. The drug was conjugated to LSMT that had been modified with 4-succinimidyloxycarbonyl-alpha-methyl-2-pyridyldithiotoluene (SMPT) as a linker. The success of LSMT modification was confirmed with TLC and MS; the latter also indicated the amount of captopril molecule linked. The modified LSMT could permeate through the intestinal monolayer barrier, and thus could be absorbed in the intestine after modification. The modified protein appears to remain stable after incubation in simulated gastrointestinal fluids. This pioneering work provides an essential basis for further development of the protein as a drug carrier for oral administration.

The In Vitro-In Vivo Safety Confirmation of PEG-40 Hydrogenated Castor Oil as a Surfactant for Oral Nanoemulsion Formulation.

Evaluation on the safety use of high concentration of polyoxyl 40 (PEG-40) hydrogenated castor oil as a surfactant for oral nanoemulsion was performed in Webster mice. As previously reported, nearly 20% of PEG-40 hydrogenated castor oil was used to emulsify the glyceryl monooleate (GMO) as an oil to the aqueous phase. Thermodynamically stable and spontaneous nanoemulsion was formed by the presence of co-surfactant polyethylene glycol 400 (PEG-400). Standard parameters were analyzed for nanoemulsion including particle size and particle size distribution, the surface charge of nanoemulsion, and morphology. To ensure the safety of this nanoemulsion, several cell lines were used for cytotoxicity study. In addition, 5000 mg/kg body weight (BW) of the blank nanoemulsion was given orally to Webster mice once a day for 14 days. Several parameters such as gross anatomy, body weight, and main organs histopathology were observed. In particular, by considering the in vivo data, it is suggested that nanoemulsion composed with a high amount of PEG-40 hydrogenated castor oil is acceptable for oral delivery of active compounds.

Molecular effects of bioactive fraction of Curcuma mangga (DLBS4847) as a downregulator of 5α-reductase activity pathways in prostatic epithelial cells.

DLBS4847 is a standardized bioactive fraction of Curcuma mangga. In this study, we used prostate cancer (PC)-3 as the cell line to study the effects of DLBS4847 on prostatic cell viability, as well as related molecular changes associated with the decreased cell number. The observation revealed that DLBS4847 inhibited the growth of PC3 cells through downregulation of the 5α-reductase (5AR) pathway. At the transcription level, 5AR1 and androgen-receptor gene expressions were downregulated in a dose-dependent manner. Furthermore, 5AR-1 and dihydrotestosterone expression were also downregulated at the protein level. A microarray study was also performed to see the effects of DLBS4847 on differential gene expressions in prostate cancer 3 cells. Among others, DLBS4847 downregulated genes related to prostate growth and hypertrophy. Our results suggested that DLBS4847 could potentially become an alternative treatment for prostate disorders, such as benign prostatic hyperplasia. In this regard, DLBS4847 exerts its growth inhibition partially through downregulation of the 5AR pathway.

Glucose-lowering effect of DLBS3233 is mediated through phosphorylation of tyrosine and upregulation of PPARγ and GLUT4 expression.

BACKGROUND: DLBS3233 is a standardized extract combination containing Lagerstroemia speciosa and Cinnamomum burmannii. The effect of DLBS3233 on glucose uptake, adiponectin secretion, and insulin signaling was examined in this study. METHODS: 3T3 Swiss albino preadipocytes and adipocytes were used to investigate gene expression detected using the reverse transcription polymerase chain reaction method. Immunoblotting assay and in vitro glucose uptake assay were also carried out in the experiment. RESULTS: DLBS3233 was seen to increase phosphorylation at the tyrosine residue of the insulin receptor substrate. DLBS3233 was also found to enhance the expression of genes associated with increased insulin signaling and sensitivity, such as peroxisome proliferator-activated receptor gamma, phosphatidylinositol-3 kinase, Akt, and glucose transporter 4. In addition, glucose transporter 4 protein levels were seen to increase as a result of DLBS3233 administration. The combination of extracts also increased glucose uptake and adiponectin secretion, and decreased resistin secretion significantly relative to control cells. Moreover, DLBS3233 administered to insulin-resistant Wistar rats showed an ability to control blood sugar, insulin levels, and other lipoproteins, including high-density lipoprotein, low-density lipoprotein, triglycerides, and total cholesterol. CONCLUSION: DLBS3233, as a combination of herbal extracts, holds promise in the treatment of type 2 diabetes, and possibly also in prevention of the disease.