Dada Patil

Immunomodulatory activity of Asparagus racemosus on systemic Th1/Th2 immunity: Implications for immunoadjuvant potential

ETHNOPHARMACOLOGICAL RELEVANCE Roots of Asparagus racemosus Willd (Shatavari in vernacular) are widely used in Ayurveda as Rasayana for immunostimulation, galactogogue as also in treatment of conditions like ulcers and cancer. Various studies have indicated immunomodulatory properties of Shatavari root extracts and formulations. AIM OF THE STUDY To study the effect of standardized Asparagus racemosus root aqueous extract (ARE) on systemic Th1/Th2 immunity of SRBC sensitized animals. MATERIALS AND METHODS We used HPTLC to quantify steroidal saponins (Shatavarin IV, Immunoside) and flow cytometry to study effects of ARE on Th1/Th2 immunity. SRBC specific antibody titres and DTH responses were also monitored as markers of Th2 and Th1 responses, respectively. We also studied lymphocyte proliferation. Cyclosporin, cyclophosphamide and levamisole were used as controls. RESULTS Treatment with ARE (100mg/(kg b.w.p.o.)) resulted in significant increase of CD3(+) and CD4/CD8(+) percentages suggesting its effect on T cell activation. ARE treated animals showed significant up-regulation of Th1 (IL-2, IFN-g) and Th2 (IL-4) cytokines suggesting its mixed Th1/Th2 adjuvant activity. Consistent to this, ARE also showed higher antibody titres and DTH responses. ARE, in combination with LPS, Con A or SRBC, produced a significant proliferation suggesting effect on activated lymphocytes. CONCLUSION The study suggests mixed Th1/Th2 activity of ARE supports its immunoadjuvant potential.

Determination of withaferin A and withanolide A in mice plasma using high-performance liquid chromatography-tandem mass spectrometry: application to pharmacokinetics after oral administration of Withania somnifera aqueous extract.

Withania somnifera (WS) is one of the popular botanical medicines widely used in Ayurveda. Withanolides such as withaferin A (WA) and withanolide A (WLD) are its bioactive constituents reported as promising drug candidates in cancer and neurological disorders respectively. A new, selective and rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for simultaneous determination of WA and WLD in mice plasma. Simple liquid-liquid extraction procedure was followed using ter-butyl methyl ether (TBME) for plasma sample pretreatment. Analytes were separated on Hypurity C18 column using methanol and ammonium acetate (95:5, v/v) as a mobile phase and detected by electrospray ionization in the multiple reaction monitoring (MRM) mode. The mass transition ion-pair was followed as m/z 471.3→281.2 for WA; m/z 437.2→292.2 for tianeptine (IS) and m/z 488.3→263.1 for WLD; m/z 315.9→270 for clonazepam (IS). The method showed excellent linearity (r(2)>0.997) over the concentration range of 0.484-117.880ng/ml for WA and from 0.476-116.050ng/ml for WLD. The lower limits of quantification (LLOQs) were found to be 0.484ng/ml and 0.476ng/ml for WA and WLD respectively. Precision (% CV) and accuracy (% bias) were found in the range of 3.7-14.3% and -14.4-4.0%, respectively. The validated method was successfully applied to a pharmacokinetic (PK) study for estimation of WA and WLD in mice plasma following oral administration of W. somnifera root aqueous extract (WSE). The PK study suggested rapid oral absorption of these withanolides. The PK study revealed that withaferin A has one and half times more relative bioavailability as compared to withanolide A.

Physicochemical Stability and Biological Activity of Withania somnifera Extract under Real-Time and Accelerated Storage Conditions

Stability testing at preformulation stages is a crucial part of drug development. We studied physicochemical stability and biological activity of Withania somnifera (ashwagandha) dried root aqueous extract during six months real-time and under accelerated storage conditions. The characteristic constituents of ashwagandha roots include withanolides such as withaferin A and withanolide A. We modified and validated the HPLC-DAD method for quantitative measurement of withanolides and fingerprint analysis. The results suggest a significant decline in withaferin A and withanolide A content under real and accelerated conditions. The HPLC fingerprint analysis showed significant changes in some peaks during real and accelerated storage (> 20 %). We also observed incidences of clump formation and moisture sensitivity (> 10 %) under real-time and accelerated storage conditions. These changes were concurrent with a significant decline in immunomodulatory activity (p < 0.01) during the third month of the accelerated storage. Thus, adequate control of temperature and humidity is important for WSE containing formulations. This study may help in proposing suitable guidance for storage conditions and shelf life of ashwagandha formulations.

Effect of Botanical Immunomodulators on Human CYP3A4 Inhibition: Implications for Concurrent Use as Adjuvants in Cancer Therapy

Purpose. Many botanical immunomodulators are used as adjuvants along with cancer chemotherapy. However, information on the impact of concurrent administration of such botanicals on pharmacokinetics of chemotherapy agents is inadequate. This study investigates inhibitory activities of 3 popular botanical adjuvants: Asparagus racemosus (root aqueous extract; ARE), Withania somnifera (root aqueous extract; WSE), and Tinospora cordifolia (stem aqueous extract, TCE) on human CYP3A4 isoenzyme, responsible for metabolism of several chemotherapy agents. Experimental design. Testosterone 6-β hydroxylation was monitored using high-performance liquid chromatography as an indicator of CYP3A4 catalytic activities. Ketoconazole (positive control) and extracts were studied at their in vivo–relevant concentrations. Results. TCE showed mild inhibition while no significant inhibitory activities were observed in WSE and ARE. TCE was further fractionated to obtain polar and nonpolar fractions. The nonpolar fraction showed significant CYP3A4 inhibition with IC50 13.06 ± 1.38 µg/mL. Major constituents of nonpolar fraction were identified using HPLC-DAD-MS profiling as berberine, jatrorrhizine, and palmatine, which showed IC50 values as 6.25 ± 0.30, 15.18 ± 1.59, and 15.53 ± 1.89 µg/mL, respectively. Conclusion. Our findings suggest that constituents of TCE extract especially protoberberine alkaloids have the potential to interact with cancer chemotherapy agents that are metabolized by CYP3A4 in vivo.

Anti-hyperglycemic and anti-hyperlipidaemic effect of Arjunarishta in high-fat fed animals

Background Arjunarishta (AA), a formulation used as cardiotonic is a hydroalcoholic formulation of Terminalia arjuna (Roxb.) Wight and Arn. (TA) belonging to family Combretaceae. Objective To evaluate the anti-hyperglycemic and anti-hyperlipidemic effect of Arjunarishta on high-fat diet fed animals. Materials and methods High-fat diet fed (HFD) Wistar rats were randomly divided into three groups and treated with phytochemically standardized Arjunarishta (1.8 ml/kg), and hydroalcoholic extract of T. arjuna (TAHA) (250 mg/kg) and rosuvastatin (10 mg/kg), for 3 months. Intraperitoneal glucose tolerance test, blood biochemistry, liver triglyceride and systolic blood pressure were performed in all the groups. Effect of these drugs on the expression of tumor necrosis factor-α (TNF-α) and insulin receptor substrate-1 (IRS-1) and peroxisome proliferators activated receptor γ coactivator 1-α (PGC-1α) were studied in liver tissue using Quantitative Real-time PCR. Results HFD increased fasting blood glucose, liver triglyceride, systolic blood pressure and gene expression of TNF-α, IRS-1 and PGC-1α. Treatment of AA and TAHA significantly reduced fasting blood glucose, systolic blood pressure, total cholesterol and triglyceride levels. These treatments significantly decreased gene expression of TNF-α (2.4, 2.2 and 2.6 fold change); increased IRS-1 (2.8, 2.9 and 2.8 fold change) and PGC-1α (2.9, 3.7 and 3.3 fold change) as compared to untreated HFD. Conclusion Anti-hyperglycemic, anti-hyperlipidemic effect of Arjunarishta may be mediated by decreased TNF-α and increased PGC-1α and IRS-1.

Chapter 9 – Proteomics

To accelerate the drug discovery process various omics tools including genomics, transcriptomics, epigenomics, and proteomics are used with a hope to address the issues pertaining to the tremendous dropout rate of lead drug candidates. Proteomics emerges as an area that promises translational research including biomarker discovery, early diagnosis of diseases, predicting disease prognosis, and identifying druggable targets for new therapeutics. Proteome bridges genotype to phenotype, translating genetic variability into phenotypic variations. Understanding the complex nature of limited genetic code translating into complex network of proteome and, in turn, phenotype remains a challenge. Proteomic technologies have been used to monitor the quality and to evaluate the pharmacological effects and mechanisms of action of traditional medicines. In this chapter, recent advances in proteomic technologies along with applications of proteomics in traditional herbal medicine research and development will be illustrated highlighting quality control, investigating pharmacodynamics, and toxicokinetics, with a special focus and discussion on herb–drug interactions. At the end, challenges and newer perspectives of proteomics in traditional herbal medicines research will be highlighted.

Herb-drug interaction of Nisha Amalaki and Curcuminoids with metformin in normal and diabetic condition: A disease system approach

Nisha Amalaki (NA), formulation with Curcuma longa Linn (Turmeric, Haridra, Nisha in Sanskrit; Family: Zingiberaceae) and Phyllanthus emblica Linn (Indian gooseberry, Amlaki in Sanskrit; Family: Phyllanthaceae) which is described for various diseases including diabetes in ayurvedic texts and Nighantus. The aim of the present study was to assess the pharmacokinetic (PK) and pharmacodynamic (PD) interactions of chemically standardized NA and Curcuminoids (CE) with metformin (MET) in normal and diabetic animals. Oral administration of NA (200 mg/kg) and CE (30 mg/kg) was carried out for seven days followed by co-administration of MET till fifteen days. MET plasma PK parameters including Cmax, AUC0-∞, t1/2, CL and Vd were measured on the eighth day. PD parameters including plasma glucose AUC followed by oral glucose tolerance test, high-density lipoproteins (HDL), total cholesterol (TC) and triglycerides (TG) were measured on the fifteenth day. In normal animals, co-administration of NA + MET and CE + MET resulted in significant increase (p < 0.05) in Cmax, AUC0-∞, t1/2, and reduction of CL and Vd. We report that co-administration of NA + MET and CE + MET significantly (p < 0.01, p < 0.001) reduced plasma glucose level, HDL level while a notable reduction in TG and TC level was observed. Interestingly, in diabetic condition, co-administration of NA + MET and CE + MET indicated a significant decrease (p < 0.05) in Cmax, AUC0-∞, t1/2 and enhanced CL and Vd. Hence, to conclude, co-administration of NA + MET and CE + MET resulted in beneficial PK and PD interactions leading to antihyperglycemic and antihyperlipidemic effects in both conditions. However, PK interaction was drastically different in diabetic and normal conditions.

Why and How Drugs Fail

Most of the drugs recalled or withdrawn over the last half a century are associated with toxic effects on different organs and systems. Among various effects leading to drug recall or withdrawal, hepatic toxicity ranks the highest followed by cardiotoxicity. Specificity to a particular target may not ensure that a drug has no effect on other targets. Systems biology and metabolic network studies with drugs such as thalidomide, rofecoxib, statins, and troglitazone indicate the involvement of on-target pharmacological and off-target nonspecific effects. Drug toxicity is primarily due to cumulative effect of target modulation and intrinsic property of drugs, widely known as “toxicity triangle effect” or “the butterfly effect.” Potential exists for chemical or botanical adjuvants like withanolides, reseveratrol, ginsenosides, and curcumin to alleviate drug-induced toxicities based on modulation of intrinsic properties.

CHEMICAL MODIFICATION OF CHITOSAN AND FORMULATION OF ITS NANOPARTICLES FOR PROTEIN DRUG DELIVERY SYSTEM

The most common route of administration of proteins has been parenter als using invasive ways of administration but this route has many side effects like lack of patient compliance, cost, high drug levels etc. The development of an oral dosage form that improves the absorption of pro tein drugs is the most desirable formulati on but one of the greatest challenges in the pharmaceutical field. The major barriers in developing oral formulations for pep tides and proteins include poor intrinsic permeability, luminal and cellular enzymatic degradation and chemical and conformational stability. A number of innovative oral drug delivery approaches have been recently developed, including the drug entrapment within polymer nanoparticles. The various advantages o f oral delivery of proteins are like ease of administration, can be used as cu re in primary s tages of disease eg. Thrombosis, no internal bleeding, patient compliance and economical Our formulation development approach is to develop the formulations targeted to bypass the stomach with an aim to release the dru g in the intestine; wit h extend ed residence time in the GIT and for this polymer like Chitosan is used. To improve the lipophi l lic property of chitosan , to modify biodegrada tion pattern of polymer and for oral delivery of proteins (Insulin and serratiopeptidase) , modification is done using lactic - acid and polyethyleneglycol by copolymerization.