Ayurveda-based Botanicals as Therapeutic Adjuvants in Paclitaxel-induced Myelosuppression.

Chemotherapy-induced myelosuppression is one of the major challenges in cancer treatment. Ayurveda-based immunomodulatory botanicals Asparagus racemosus Willd (AR/Shatavari) and Withania somnifera (L.). Dunal (WS/Ashwagandha) have potential role to manage myelosuppression. We have developed a method to study the effects of AR and WS as therapeutic adjuvants to counter paclitaxel (PTX)-induced myelosuppression. Sixty female BALB/c mice were divided into six groups-vehicle control (VC), PTX alone, PTX with aqueous and hydroalcoholic extracts of AR (ARA, ARH) and WS (WSA, WSH). The myelosuppression was induced in mice by intraperitoneal administration of PTX at 25 mg/kg dose for three consecutive days. The extracts were orally administered with a dose of 100 mg/kg for 15 days prior to the induction with PTX administration. The mice were observed daily for morbidity parameters and were bled from retro-orbital plexus after 2 days of PTX dosing. The morbidity parameters simulate clinical adverse effects of PTX that include activity (extreme tiredness due to fatigue), behavior (numbness and weakness due to peripheral neuropathy), body posture (pain in muscles and joints), fur aspect and huddling (hair loss). The collected samples were used for blood cell count analysis and cytokine profiling using Bio-Plex assay. The PTX alone group showed a reduction in total leukocyte and neutrophil counts (4,800 ± 606; 893 ± 82) when compared with a VC group (9,183 ± 1,043; 1,612 ± 100) respectively. Pre-administration of ARA, ARH, WSA, and WSH extracts normalized leukocyte counts (10,000 ± 707; 9,166 ± 1,076; 10,333 ± 1,189; 9,066 ± 697) and neutrophil counts (1,482 ± 61; 1,251 ± 71; 1,467 ± 121; 1,219 ± 134) respectively. Additionally, higher morbidity score in PTX group (7.4 ± 0.7) was significantly restricted by ARA (4.8 ± 1.1), ARH (5.1 ± 0.6), WSA (4.5 ± 0.7), and WSH (5 ± 0.8). (Data represented in mean ± SD). The extracts also significantly modulated 20 cytokines to evade PTX-induced leukopenia, neutropenia, and morbidity. The AR and WS extracts significantly prevented PTX-induced myelosuppression (p < 0.0001) and morbidity signs (p < 0.05) by modulating associated cytokines. The results indicate AR and WS as therapeutic adjuvants in cancer management.

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.

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.

Development and application of HPLC-RI and HPLC-MS/MS based methods for quantification of residual deoxycholate levels in pneumococcal polysaccharides.

The analysis of residual sodium deoxycholate (DOC); a detergent of biological origin used in manufacturing of polysaccharide vaccines is challenging due to complex sample matrices and the lack of suitable methods. Here we report, rapid and sensitive high-performance liquid chromatography-refractive index (HPLC-RI) and tandem mass spectrometry (HPLC-MS/MS) methods for estimation of residual DOC in pneumococcal polysaccharides. For HPLC-RI method, separation was achieved using Luna C18 column and mobile phase compositions of acetonitrile: methanol: 20 mM sodium acetate (60:05:35% v/v). For HPLC-MS/MS method, separation was achieved using a Hypersil BDS C18 column with gradient elution of methanol and water (0.1% formic acid). MS/MS method showed linearity (r2 = 0.997) over the range of 10-320 ng/mL with limits of detection (LOD) and lower limit of quantitation (LOQ) of 3 and 10 ng/mL respectively. Precision (% RSD) and accuracy (% recovery) for both methods were in the range of 0.74-8.29% and 82.33-117.86% respectively. Sample matrices interferences were addressed following novel sample clean-up method based on liquid-liquid extraction. Both methods enabled traceable quantitation of DOC in intermediate and purified pneumococcal polysaccharides of serotypes: 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F.

HPLC/tandem mass spectrometric studies on steroidal saponins: an example of quantitative determination of Shatavarin IV from dietary supplements containing Asparagus racemosus.

Asparagus racemosus (AR) is a popular botanical present in several Ayurvedic medicines and nutritional and dietary supplements with immunomodulatory, galactogogue, and anticancer activity. A steroidal saponin known as shatavarin IV is one of the active constituents of AR. A new, selective, and rapid HPLC/MSIMS method has been developed and validated for quantitative estimation of shatavarin IV in crude, processed, and marketed samples of AR. The analytes were separated on a Luna C18 column using simple isocratic elution with water (0.1% acetic acid)-acetonitrie;(0.1% acetic acid; 70 + 30, vIv) at a flow rate of 0.8 mLlmin. The analytes were detected by electrospray ionization (ESI)-MS/MS and quantified using multiple reaction monitoring techniques in the positive ion mode. The method showed excellent linearity (r2 > 0.998) over the concentration range of 7.5 to 254 ng/mL with LOD of 2.5 ng/mL. Precision (RSD) and accuracy (recovery) were found in the ranges of 2.00 to 5.15 and 102 to 110%, respectively. The validated HPLC/ESI-MS/MS method was successfully applied to the quantification of shatavarin IV in crude, processed, and marketed (single or multiherb) AR samples. Therefore, this method could be used for QC and standardization of pharmaceutical or nutritional products containing AR.

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 RACEMOSU: (root aqueous extract; ARE), WITHANIA SOMNIFER: (root aqueous extract; WSE), and TINOSPORA CORDIFOLI: (stem aqueous extract, TCE) on human CYP3A4 isoenzyme, responsible for metabolism of several chemotherapy agents. EXPERIMENTAL DESIG: . Testosterone 6-ß 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.

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.

Development and biological evaluation of Gymnema sylvestre extract-loaded nonionic surfactant-based niosomes.

AIM: To develop and characterize Gymnema sylvestre extract-loaded niosomes using nonionic surfactants, and to evaluate their antihyperglycemic efficacy in comparison with the parent extract. MATERIALS & METHODS: Nonionic surfactant-based G. sylvestre extract-loaded niosomes were prepared using the thin-film hydration method. The optimized formulation was screened for entrapment efficiency of the constituents, as well as other parameters such as release kinetics, vesicle size, zeta-potential and stability studies. The parent extract and optimized niosomal formulation were evaluated for their antihyperglycemic potential in an alloxan-induced diabetic animal model. RESULTS: Niosomes prepared using Span™ 40 (SD Fine Chemicals Ltd, Mumbai, India) provided sterically stable vesicles 229.5 nm in size with zeta-potential and entrapment efficiency of 150.86 mV and 85.3 ± 4.5%, respectively. The surface morphology of vesicles was confirmed to be spherical by scanning electron microscopy studies. An in vitro release study demonstrated 77.4% of phytoconstituents release within 24 h. The niosome formulation demonstrated significant blood glucose level reduction in an oral glucose tolerance test, and increased antihyperglycemic activity compared with the parent extract in an alloxan-induced diabetic model. CONCLUSION: This study reveals the merits of G. sylvestre extract-loaded niosomes, and justifies the potential of niosomes for improving the efficacy of G. sylvestre extract as antidiabetic. Original submitted 30 March 2012; Revised submitted 29 August 2012; Published online 24 December 2012.

Determination of gymnemagenin in rat plasma using high-performance liquid chromatography-tandem mass spectrometry: application to pharmacokinetics after oral administration of Gymnema sylvestre extract.

A sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for the determination of gymnemagenin (GMG), a triterpene sapogenin from Gymnema sylvestre, in rat plasma using withaferin A as the internal standard (IS). Plasma samples were simply extracted using liquid-liquid extraction with tetra-butyl methyl ether. Chromatographic separation was performed on Luna C(18) column using gradient elution of water and methanol (with 0.1% formic acid and 0.3% ammonia) at a flow rate of 0.8 mL/min. GMG and IS were eluted at 4.64 and 4.36 min, ionized in negative and positive mode, respectively, and quantitatively estimated using multiple reaction monitoring (MRM) mode. Two MRM transitions were selected at m/z 505.70 → 455.5 and m/z 471.50 → 281.3 for GMG and IS, respectively. The assay was linear over the concentration range of 5.280-300.920 ng/mL. The mean plasma extraction recoveries for GMG and IS were found to be 80.92 ± 8.70 and 55.63 ± 0.76%, respectively. The method was successfully applied for the determination of pharmacokinetic parameters of GMG after oral administration of G. sylvestre extract.

Quantitative estimation of gymnemagenin in Gymnema sylvestre extract and its marketed formulations using the HPLC-ESI-MS/MS method.

INTRODUCTION: Gymnema sylvestre, with gymnemic acids as active pharmacological constituents, is a popular ayurvedic herb and has been used to treat diabetes, as a remedy for cough and as a diuretic. However, very few analytical methods are available for quality control of this herb and its marketed formulations. OBJECTIVES: To develop and validate a new, rapid, sensitive and selective HPLC-ESI (electrospray ionisation)-MS/MS method for quantitative estimation of gymnemagenin in G. sylvestre and its marketed formulations. METHOD: HPLC-ESI-MS/MS method using a multiple reactions monitoring mode was used for quantitation of gymnemagenin. Separation was carried out on a Luna C-18 column using gradient elution of water and methanol (with 0.1% formic acid and 0.3% ammonia). RESULTS: The developed method was validated as per International Conference on Harmonisation Guideline ICH-Q2B and found to be accurate, precise and linear over a relatively wide range of concentrations (5.280-305.920 ng/mL). Gymnemagenin contents were found from 0.056 ± 0.002 to 4.77 ± 0.59% w/w in G. sylvestre and its marketed formulations. CONCLUSION: The method established is simple, rapid, with high sample throughput, and can be used as a tool for quality control of G. sylvestre and its formulations.

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.

High-performance liquid chromatographic fingerprint for quality control of Terminalia arjuna containing Ayurvedic churna formulation.

Because Ayurvedic herbal preparations contain a myriad of compounds in complex matrixes, it is difficult to establish quality control standards for raw materials and to standardize finished Ayurvedic drugs. A novel, accurate, and valid fingerprint method was developed using HPLC for quality control of a traditional Ayurvedic Arjuna churna formulation, which is used as a cardiotonic drug. Comprehensive comparison of chromatograms of standardized formulation of Arjuna churna and marketed formulations revealed eight characteristic peaks in chromatograms, which unambiguously confirmed the presence of authentic raw material used in the formulation on the basis of their retention time values and UV data. An HPLC fingerprint was also developed for total sapogenins present in Terminalia arjuna. The six common peaks observed in chromatograms of isolated sapogenins, standardized formulations, and marketed formulations can serve as a quality control tool for qualitative estimation of total saponin glycosides present in an Arjuna churna formulation.

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.