Mhaveer Singh

Development and validation of HPLC method for simultaneous estimation of piperine and guggulsterones in compound Unani formulation (tablets) and a nanoreservoir system

An attempt has been made to develop and validate a simultaneous HPLC method for novel approach of drug release via oil-in-water (o/w) nanoemulsion formulation and Habb-e-Khardal Unani tablet containing piperine and guggul sterones E and Z as main ingredients. Nanoemulsion was prepared by titration method using sefsol-218 as an oily phase, cremophor-EL as a surfactant, transcutol as a co-surfactant and distilled water as an aqueous phase. The formulation was optimized on the basis of thermodynamic stability and dispersibilty test. The nanoformulation was evaluated for particle size, surface morphology, electrical conductivity and viscosity determination. The in vitro dissolution was carried out by dialysis bag method. Drugs were quantified using an HPLC method developed in-house with a C(18) column as stationary phase and acetonitrile and water as mobile phase at λ(max) of 240 nm. The optimized formulation showed higher drug release, lower droplet size and less viscosity as compared with the conventional Habb-e-Khardal Unani tablet. The present study illustrated the potential of nanoemulsion dosage form in improving biopharmaceutic performance of piperine and guggul sterone. The HPLC method was also found to be quite sufficient for the routine quality control of formulations containing piperine and guggul sterone E and Z as ingredients and also for in vitro drug release studies.

Development and validation of a stability-indicating HPTLC method for analysis of arjunolic acid in a herbal formulation

The stem bark of Terminalia arjuna Linn. (family: Combretaceae), commonly known as Arjuna in Indian systems of medicine, is an important drug widely used in the preparation of Ayurvedic and Unani formulations used in cardioprotection [1, 2]. T. arjuna stem bark is reported to contain different groups of chemical constituents, for example hydrolyzable tannins [2, 3], triterpene acids, flavanoids, phenolics, and phyto sterols [3]. Important triterpene acids are arjunetin, arjunic acid, arjunolic acid, and arjungenin [4–6]. Arjunolic acid (2,3,23-trihydroxyolean-12-en-28-oic acid; Figure 1) is used for its hypotensive effect and as an antioxidant, antiallergic, and antiasthmatic [6]. HPTLC and HPLC methods have been reported for analysis of arjunolic acid in the crude form [7–9] but the methods lack proper validation. Because no stability-indicating analytical method had been reported for quantification of arjunolic acid in pharmaceutical or herbal dosage forms it was thought worthwhile to develop a stability-indicating HPTLC method. The proposed method will be useful for standardization and quality control of formulations which contain arjunolic acid or arjuna as an ingredient.

Caspase mediated synergistic effect of Boswellia serrata extract in combination with doxorubicin against human hepatocellular carcinoma.

The study investigated the growth-inhibiting and apoptosis mediating effects of B. serrata extract as monotherapy and combination therapy with DOX against hepatocellular carcinoma cell lines. Boswellic acid rich fraction of B. serrata extract was prepared. MTT assay on HepG2 and Hep3B cells was carried out using B. serrata alone and in combination with DOX. Further, caspase-3 activity, TNF-α level, and IL-6 level were estimated. Isobolographic analysis was carried out to evaluate the effect of combination therapy. Additionally, protective effect of B. serrata extract on DOX induced hepatic toxicity was also evaluated in Wistar rats. B. serrata extract inhibited growth of HepG2 (IC50 value of 21.21 ± 0.92 μg/mL) as well as HepG2 (IC50 value of 18.65 ± 0.71 μg/mL). DOX inhibited growth in HepG2 and Hep3B cells with an IC50 of 1.06 ± 0.04 μg/mL and 1.92 ± 0.09 μg/mL. Isobolographic analysis showed combination index (CI) of DOX and B. serrata extract of 0.53 ± 0.03 to 0.79 ± 0.02 suggesting synergistic behavior against the two cell lines. B. serrata extract also caused dose dependent increase in caspase-3 activity, TNF-α level, and IL-6 level which was higher (P < 0.001) with DOX (1 μM) and B. serrata extract (20 μg/mL) combination. B. serrata extract also protected Wistar rats against DOX induced hepatic toxicity.

Alternative approach for mitigation of doxorubicin-induced cardiotoxicity using herbal agents.

Doxorubicin (DOX) is an effective and frequently used chemotherapeutic agent for various malignancies. However, its clinical use is hampered due to the development of cardiotoxicity. Investigations have proved that DOX-induced cardiotoxicity occurs through mechanisms other than those mediating its antitumor effect. This theory sheds light on the development of strategies for cardioprotection without altering therapeutic effectiveness of DOX. Bioactive plant constituents of dietary supplements, traditional herbs and foods with potential health benefits can play an important role in therapeutics. This manuscript is an exhaustive review and prospect of herbal and botanical agents against DOX-induced cardiotoxicity with their proposed mechanisms. The activity of herbs evaluated against DOX-induced cardiotoxicity has shown number of mechanisms including apoptosis, antioxidant potential, effect on mitochondria and calcium ion regulation etc. The manuscript reveals that most of the herbal drugs studied are effective through antioxidant mechanism and only few through other major pathways such as apoptosis and iron mediated pathways in DOX-induced cardiotoxicity. Only limited reports are available for the prevention of DOX-induced drug resistance using botanicals. Manuscript reports a number of constituents with evident potential in prevention of DOX cardiotoxicity e.g. proanthocyanidins, epigallocatechin-3-gallate, S-allylcysteine, reseveratrol, rutoside etc. In the present communication, several herbal drugs have also been discussed, which can act through mechanisms other than antioxidant and may be evaluated as a combination therapy for prevention of DOX-induced cardiotoxicity in future.

Rapid RP-HPLC Method for the Quantification of Glabridin in Crude Drug and in Polyherbal Formulation

A simple, economic, selective, precise and robust method has been developed and validated for the analysis of glabridin in crude drugs and polyherbal formulations. Reversed-phase chromatography is performed on a C18 column with water and acetonitrile as mobile phase in gradient elution method at a flow rate of 1 mL/min. Detection is performed at 230 nm and a sharp peak is obtained for glabridin at a retention time of 14.9 ± 0.02 min. Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 1-500 µg/mL; the regression coefficient is 0.9992 and the linear regression equation is y = 26.683x - 142.17. The method is validated for accuracy, precision, reproducibility, robustness and detection and quantification limits, in accordance with International Conference on Harmonization guidelines. Statistical analysis proved that the method is precise, reproducible, selective and accurate for the analysis of glabridin. The proposed, developed and validated high-performance liquid chromatography method for the quantification of glabridin can be used for the quality control and standardization of licorice (Glycyrrhiza glabra Linn.) and different herbal formulations in which licorice is present as a constituent.

Scientific validation of cardioprotective attribute by standardized extract of Bombyx mori against doxorubicin-induced cardiotoxicity in murine model.

Doxorubicin (DOX) is an excellent antineoplastic agent used for the treatment of hematological and solid malignancies. The aqueous extract of Bombyx mori (BMAE) contains amino acids and some flavonoids with obvious cardioprotective effect. The aim of this study was to investigate the possible protective effect of BMAE against DOX-induced cardiotoxicity and its underlying mechanisms on murine model. The metabolic profiling of BMAE was carried out by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and the amino acid profiling by HPLC method using fluorescence detector (HPLC-FLD). The biochemical parameter like caspase-3, tumor necrosis factor–alpha (TNF-α), interleukin -6 (IL-6), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and malondialdehyde (MDA) were studied. Tissue damage was further evaluated by histopathological studies. The metabolic profiling of BMAE exhibited presence of quercetin 7-O-ß-D-glucoside, kaempferol 7-O-ß-D-glucopyranoside, coumaric acid glucoside, 2-hydroxy-nonadecanoic acid and 9,12-dihydroxy stearic acid as important constituents. The amino acid profile by HPLC-FLD showed presence of 17 amino acids. The BMAE showed prominent free radical scavenging activity when assessed by the H2O2 and super-oxide method. The results of present investigation showed protection against DOX-induced oxidative stress (lipid peroxidation), by reverting activities of apoptotic markers (caspase-3 and TNF-α), cardiac markers (CK-MB and LDH activities) as well as pro-inflammatory marker IL-6 followed by oral administration of BMAE. In addition, results of histopathology also supported well the above results. It was observed that BMAE protects DOX-induced cardiotoxicity by virtue of its antioxidants possibly by flavonoids and amino acids.

Metabolic diversity in Coleus forskohlii Briq. of Indian subcontinent

New economic, easy, specific, accurate, robust, validated high performance thin layer chromatography (HPTLC) and high performance liquid chromatography methods with good range of linearity and sensitivity were developed for quantification of forskolin in ten samples collected from different regions of Indian subcontinent, which showed a large variation among samples (0.074–0.282%, w/w). Metabolic diversity of all the samples using HPTLC fingerprint method showed a total of 16 well separated spots. There is no significant metabolic diversity among the samples collected from different locations of Indian subcontinent, which was obtained from HPTLC fingerprinting. The results of locational variation showed highest content of forskolin in Bengaluru sample by both analytical methods. The validated quantification methods and fingerprint profile together can act as a good authentication tool for coleus as well as for other medicinal plants.

Glabridin, a stable flavonoid of Glycyrrhiza glabra: HPTLC analysis of the traditional formulation

A novel HPTLC method has been developed for the estimation of glabridin in Licorice rhizome and its Unani polyherbal formulation (Qurs-e-Gul). Separation was achieved on silica using toluene, dichloromethane, and ethyl acetate in equal ratios. A compact, well resolved peak of glabridin with RF value 0.56 ± 0.02 was observed. Calibration curve revealed a good linear relationship with r2 value of 0.993 between the peak area and concentration in the range of 25–500 ng spot−1. The proposed method was validated as per the International Conference on Harmonization (ICH) guidelines. The stability assessment was carried out by studying degradation of glabridin stressed by acid, base, oxidation, thermal, and humidity. Photodegradation was also carried out after keeping the drug in sunlight, dark, and in UV lights. The method proposed can be used for routine determination of glabridin in crude drugs and in herbal formulations containing Licorice as one of the ingredients, for quality control as well as for stability testing with high precision, accuracy and a wide range of linearity.

SIMULTANEOUS ESTIMATION OF GALLIC ACID, ELLAGIC ACID, AND ASCORBIC ACID IN EMBLICA OFFICINALIS AND IN UNANI POLYHERBAL FORMULATIONS BY VALIDATED HPLC METHOD

A simple, rapid, and economic simultaneous HPLC method was developed and validated for the quantification of Gallic acid (GA), Ellagic acid (EA), and Ascorbic acid (AA), in Emblica officinalis Linn. (aamla) and in two poly herbal Unani formulations, containing aamla as an ingredient. Separation was achieved on a reverse phase C18 (250 × 4.6 mm, 5 µm) column with a mobile phase of 0.1% orthophosphoric acid and acetonitrile in gradient elution. The flow rate was kept as 1 mL min−1 and detection was carried out at 254 nm. The proposed method was validated as per the ICH guidelines for accuracy, precision, robustness, LOD, and LOQ. The proposed method was linear over a wide range of concentration with a good regression coefficient of more than 0.99. The LOD of method was 0.035 µg mL−1, 0.31 µg mL−1, and 0.38 µg mL−1, whereas LOQ was 0.1 µg mL−1, 1.0 µg mL−1 and 1.3 µg mL−1 for ascorbic acid, ellagic acid, and gallic acid, respectively. The results demonstrated that the proposed method is a simple, reproducible, accurate, economic, and suitable method for the quality control of aamla, its formulations, as well as a large population of plants with anti-oxidant property.

Standardization and in vitro antioxidant activity of jatamansi rhizome

Background: Nardostachys jatamansi Linn. commonly known as jatamansi is a well notorious drug in Indian systems of medicines having various health-related benefits and employed in various herbal formulations due to the presence of high levels of valuable phenolic constituents. The present study was aimed to quality assessment of Jatamansi rhizome by studying macro- and micro-scopic characters along with physicochemical tests, chemo-profiling using thin layer chromatography (TLC), and gas chromatography–mass spectrometry (GC-MS), in vitro antioxidant activity. Materials and Methods: Standardization was carried out as per the pharmacopeial guidelines and contaminant estimation was carried out by analyzing the samples for the determination of heavy metals, pesticides, and aflatoxins. Chemo-profiling was done with TLC by optimizing the mobile phase for different extracts. The GC-MS chemo-profiling was also carried out by using hexane soluble fraction of the hydroalcoholic extract. The drug is well known for a protective role in the human body as an antioxidant, so total phenolic contents and in vitro antioxidant efficacy was also determined by using established methods. Results: The results of quality control and anatomical studies were very much useful for its identification, whereas significant antioxidant efficacy was also observed. The drug was found free of contaminants when analyzed for pesticides and aflatoxins, whereas heavy metals were found under the pharmacopeial limit. Conclusion: The findings of the present research can be utilized for the identification and quality control of the jatamansi rhizome.