HPLC Coupled with Chemometric Analysis and LC-MS Studies of Three Flavonoids in Tephrosia purpurea (L.) Pers Revealed Impact of Chemodiversity on the Quest for the Chemical Markers.

Globally, Tephrosia purpurea (L.) Pers is used as an important component in herbal drug formulations for liver health. The present study is aimed to develop a suitable analytical approach for simultaneous analysis of three flavonoids (rutin, deguelin and rotenone) to establish quality control methods for plant. A novel High-performance liquid chromatography photodiode array detector (HPLC-PDA) method has been developed to quantify these flavonoids in T. purpurea. The method was validated, and data were subjected to chemometric analysis to select most optimal marker compound. The method that was found linear with R2 values ranges from 0.996 to 0.998 with good recoveries. Intra- and inter-day precision values were <2. HPLC analysis revealed high level of chemodiversity. Quantity of all the three chemical markers was found significantly disparate in samples from different locations. Deguelin was detectable only in three out of total eight samples. However, liquid chromatography-mass spectrometry analysis was found sufficiently sensitive to detect all the compounds in all samples. Thus, results suggest to apply combination of approaches to enhance confidence in chromatographic methods for quality control of herbal drugs. Principal component analysis ranked the markers as Rutin>Rotenone>Deguelin. This comprehensive approach employing multichromatography platforms can be successfully utilized in analysis of these bioactive markers and routine standardization of herbal material and formulations containing T. purpurea.

Investigation on apposite chemical marker for quality control of Tephrosia purpurea (L.) Pers. by means of HPTLC-chemometric analysis.

Application of phytochemicals as the markers for quality assurance of the herbal medicinal material is one of the prominently used approach. In the present study, six major chemical compounds i.e. rutin, quercetin, lupeol, ß-sitosterol, rotenone, deguelin of therapeutically important plant Tephrosia purpurea were tested for their significance to be used as chemical markers in analytical methods. Plants were collected from different locations. Extraction procedures as well as HPTLC analytical methods have been optimized for each compound. All these methods have been validated in terms of linearity, intraday-interday precision, LOD, LOQ, accuracy and repeatability. Metabolites have been quantified and quantitative data has been subjected to chemometric analysis. Results revealed that Rf values of all the compounds are between 0.3 and 0.4. Rutin, lupeol and ß-sitosterol gave desirable response and other three compounds were found undetectable in HPTLC. Content of rutin, beta-sitosterol and lupeol ranged from 0.095 to 0.84, 0.043 to 0.125, 0.023 to 0.045 w/w % respectively. Among them, rutin content was highest in all samples. Quantitative measurements combined with chemometric analysis displayed chemodiversity in the samples. In addition, principal component analysis ranked the marker as in order of their significance rutin > ß-sitosterol > lupeol. Results indicate rutin to be most preferable chemical marker for the Tephrosia. Furthermore, application of all the three compounds combined as the multimarker system should be preferred for standardization of T. purpurea.

In vitro - In vivo metabolism and pharmacokinetics of picroside I and II using LC-ESI-MS method.

Picroside I and II, iridoid glycosides, are the major active markers of roots and rhizomes of Picrorhiza kurroa (family: Scrophulariaceae). The rhizomes of P. kurroa have been traditionally used to treat worms, constipation, low fever, scorpion sting, asthma and ailments affecting the liver. Various Ayurvedic and herbal preparations are available in the market which contains P. kurroa e.g. Arogyavadhini vati, Tiktadi kwath, Picrolax capsules and suspension. These preparations are used without any significant pharmacokinetics data. Previously, we have reported that oral bioavailability of picroside I and II is low. Most of the iridoid glycosides are primarily metabolized by intestinal microbial flora. So, it is necessary to determine the metabolic profile of picroside I and II and check the correlation with lower bioavailability. Therefore, this study was designed to check metabolic (in vitro and in vivo) profile along with pharmacokinetic profile of picroside I and II. For this, a sensitive and selective LC-ESI-MS method was developed and validated for simultaneous determination of picroside I and II in rat plasma. Chromatographic separations were performed on C18 column. The mobile phase consisted of acetonitrile: 10 mM ammonium acetate buffer [90:10 v/v], pH 3.5. In-vitro Metabolic study was performed on rat liver microsomes and primary hepatocytes. In-vivo pharmacokinetic and metabolic profile of picroside I and II was generated after oral administration of Kutkin (mixture of picroside I and II) to Sprague-Dawley rats. Various pharmacokinetic parameters viz. Cmax, Tmax, AUC(0-t) were determined. In metabolic study, eight metabolites of picroside I and six metabolites of picroside II were identified in vitro, out of which four metabolites for each picroside I and picroside II were identified in vivo.

Anti-inflammatory, Anti-estrogenic, and Anti-implantation Activity of Bergia suffruticosa (Delile) Fenzl.

BACKGROUND: Bergia suffruticosa (Delile) Fenzl (Syn. Bergia odorata Edgew) (Elatinaceae family) is used traditionally to repair bones and is applied as a poultice on sores. It is also used for stomach troubles and as an antidote to scorpion stings. So far, very little scientific work has been reported to validate its ethnomedical uses in the alleviation of pain, bone repair, etc. OBJECTIVE: This study was designed to explore the anti-inflammatory and anti-implantation potential of n-hexane extract of B. suffruticosa whole plant in mice along with identification of its chemical constituents. MATERIALS AND METHODS: n-Hexane extract of B. suffruticosa whole plant was screened for acute and chronic anti-inflammatory activity followed by an anti-estrogenic activity. Eventually, n-hexane extract was tested for anti-implantation activity by exploiting markers of uterine receptivity, lipid peroxidation, and superoxide enzyme activity. The extract was administered orally at a dose of 100 mg/kg body weight in each study. RESULTS: Thin layer chromatography fingerprint profile of n-hexane extract revealed the presence of lupeol and ß-sitosterol. The n-hexane extract reduced the edema by 80% in acute inflammation, whereas it reduced edema to 75% on the 5(th) day in chronic inflammation. The n-hexane extract reduced elevated malonaldehyde level from 6 to 2.5 nmol/g × 10(-5) and increased superoxide dismutase enzyme activity from 0 to 350 units/g in treated animals on the 5(th) day of pregnancy. Moreover, extract decreased uterine weight from 0.33 to 0.2 g in estradiol treated animals. CONCLUSION: These results indicate that n-hexane extract of B. suffruticosa is having potent anti-inflammatory, anti-estrogenic, and anti-implantation activity. This is the first report of all the pharmacological activities of B. suffruticosa mentioned above. SUMMARY: TLC fingerprint profile of n-hexane extract of Bergia suffruticosa whole plant revealed the presence of lupeol and ß-sitosteroln-Hexane extract showed in vivo anti-inflammatory activity in both acute and chronic model of inflammation in ratsn-Hexane extract possess significant anti-estrogenic activityn-Hexane extract altered the levels superoxide anion radical and superoxide dismutase enzyme activity during the blastocyst implantationAnti-implantation activity of n-hexane extract is attributed to its anti-inflammatory and anti-estrogenic potential. Abbreviations used: TLC: Thin layer chromatography; LPO: Lipid peroxidation; SOD: Superoxide dismutase; B. suffruticosa: Bergia suffruticosa; TNF-α: Tumor necrosis factor-α; NO: Nitric oxide; IL-1: Interleukin-1; LIF: Leukemia inhibitory factor; CSF-1: Colony-stimulating factor; COX: Cyclooxygenase; SDS: Sodium dodecyl sulfate; IAEC: Animal House Ethics Committee; CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; HBSS: Hank's balanced salt solution; MDA: Malonaldehyde; and TBA: Thiobarbituric acid.

Antiestrogenic and Anti-Inflammatory Potential of n-Hexane Fraction of Vitex negundo Linn Leaf Extract: A Probable Mechanism for Blastocyst Implantation Failure in Mus musculus.

The anti-implantation potential of different fractions of Vitex negundo Linn leaf extract was evaluated in female Swiss Albino mice. Animals from different groups were dosed orally either with 0.2% agar (vehicle) or with fractions of V. negundo leaf extract (n-hexane, chloroform, n-butanol, and remnant fractions) at 10:00 a.m., from day 1 to day 6 of pregnancy. The pregnant females from each group were sacrificed on different days of pregnancy (n = 6), and uterus was excised and used for estimation of lipid peroxidation and assay of superoxide dismutase activity as a marker for blastocyst implantation. Animals treated with n-hexane fraction showed altered level of superoxide anion radical and superoxide dismutase activity as compared to control animals. The probable mechanism by which this extract exhibits inhibition of blastocyst implantation is through the anti-inflammatory and antiestrogenic potential.

Comparative pharmacokinetic profiles of picrosides I and II from kutkin, Picrorhiza kurroa extract and its formulation in rats.

Picrosides I and II are the active chemical constituents, present in the roots and rhizomes of Picrorhiza kurroa Royle (family: Scrophulariaceae). The plant is ethnomedically claimed for the treatment of liver and upper respiratory tract infection, fever, dyspepsia and scorpion sting. This study attempts to determine the in vivo pharmacokinetic profile of picrosides I and II in rats after oral administration of three different preparations namely, kutkin (a mixture of picrosides I and II), P. kurroa extract and Picrolax® capsule (marketed formulation). A simple, precise, specific and sensitive method was developed for simultaneous quantification of picrosides I and II in rat plasma and was applied for the pharmacokinetic study. Pharmacokinetic parameters were calculated from the observed plasma concentration of picrosides I and II. The results showed a significant difference (p≤0.05) in oral bioavailability of picrosides I and II from different preparations. Both the compounds were found to be more bioavailable from P. kurroa extract followed by Picrolax® capsule and kutkin. Moreover, we also developed a novel method for isolation of kutkin from roots of P. kurroa with a high yield of 2.4% w/w. The information gained from this study provides a meaningful basis for clinical application and mechanistic study of such phytochemicals.

The hydroalcoholic extract of Cassia alata (Linn.) leaves and its major compound rhein exhibits antiallergic activity via mast cell stabilization and lipoxygenase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves of Cassia alata (family: Caesalpiniaceae) are ethnomedically claimed as anti-asthmatic. In the current study we aimed to investigate the anti-allergic activities of hydro-methanolic extract of Cassia alata (Linn.) and its constituents rhein and kaempferol on triple antigen/sheep serum-induced mast-cell degranulation in rats. MATERIALS AND METHODS: Antiallergic activity of hydroalcoholic extract of Cassia alata along with its two components rhein and kaempferol was evaluated using in vivo mast cell stabilization assay. Inhibitory effect on lipoxygenase (LOX) enzyme was also evaluated in vitro. Further chemical standardization of Cassia alata extract was done using rhein and kaempferol by HPTLC-densitometric method. RESULTS: The hydroalcoholic extract of Cassia alata significantly inhibited mast cell degranulation at 200mg/kg dose. Both chemical constituents rhein and kaempferol also showed potent (>76%) inhibition of mast-cell degranulation at 5mg/kg. Extract and rhein inhibited LOX enzyme with IC(50) values of 90.2 and 3.9µg/mL, respectively, whereas kaempferol was inactive. CONCLUSION: Our results suggest that Cassia alata exhibit anti-allergic activity through mast cell stabilization and LOX inhibition. Thus, Cassia alata or its active constituents could be potential alternative treatment for allergic diseases.

Simultaneous quantification of berberine and lysergol by HPLC-UV: evidence that lysergol enhances the oral bioavailability of berberine in rats.

A sensitive and simple HPLC method was developed for the simultaneous quantification of berberine and lysergol in rat plasma. The chromatographic separation was achieved on a C(18) column using isocratic elution with methanol-acetonitrile-0.1% ortho-phosphoric acid (25:20:55, v/v/v), pH adjusted to 6.5 with triethylamine and detected at a UV wavelength of 230 nm. The extraction of the berberine and lysergol from the rat plasma with methylene chloride resulted in their high recoveries (82.62 and 90.17%). HPLC calibration curves for both berberine and lysergol based on the extracts from the rat plasma were linear over a broad concentration range of 50-1000 ng/mL. The limit of quantification was 50 ng/mL. Intra- and inter-day precisions were <15% and accuracy was 87.12-92.55% for berberine and 87.01-92.26% for lysergol. Stability studies showed that berberine and lysergol were stable in rat plasma for short- and long-term period for sample preparation and analysis. The described method was successfully applied to study the pharmacokinetics of berberine as well as lysergol following oral administration in Sprague-Dawley rats. The results of the study inferred that lysergol improved the oral bioavailability of berberine.

Formulation and pharmacokinetic evaluation of hard gelatin capsule encapsulating lyophilized Vasa Swaras for improved stability and oral bioavailability of vasicine.

The oral bioavailability of vasicine (1) was investigated in hard gelatin capsules of lyophilized Vasa Swaras (aqueous extract of Adhatoda vasica Nees.,Fam.: Acanthaceae) The rat pharmacokinetic profile of lyophilized Vasa Swaras, Vasa Swaras, vasicine (1) (chief marker compounds of A. vasica) and a marketed capsule formulation of A. vasica were compared. Vasicine (1) was found to be more orally bioavailable from lyophilized Vasa Swaras, with an overall minor conversion to vasicinone (2).

Effect of hydrolysis on the yield of hederagenin and High-performance thin-layer chromatography densitometric quantification of hederagenin in fruit pericarp of Sapindus spp.

Fruit pericarp of Sapindus species are reported to contain glycosides with hederagenin as an aglycone. To free the aglycone from the glycosides, they need to be hydrolyzed, and the commonly used method is hydrolysis with either hydrochloric or sulfuric acid. In the present work, we studied the effect of hydrolysis on the yield of hederagenin from the fruit pericarp of 3 species of Sapindus, viz., S. mukorossi, S. laurifolius, and S. emarginatus. A high-performance thin-layer chromatography densitometric method for the quantification of hederagenin was developed and validated. It involved automated application of samples as bands onto silica gel 60F254 plates, development with toluene-ethyl acetate-formic acid (7 + 3 + 5, v/v/v) mobile phase, detection with anisaldehyde-sulfuric acid reagent, and scanning at 595 nm. The yield of hederagenin ranged from 0.035 to 1.29% (w/w) with different methods of hydrolysis. Hydrolysis with 3.5 M aqueous sulfuric acid under reflux for 6 h gave the maximum yield of hederagenin in all 3 species, with the highest amount in S. emarginatus (1.29%, w/w).

Evaluation of free radical scavenging activity of an ayurvedic formulation, panchvalkala.

We report the free radical scavenging activity of an Ayurvedic preparation Panchvalkala and its individual components (stem bark of Ficus benghalensis, F. glomerata, F. religiosa, F. virens and Thespesia populnea). Being stem barks, these samples contain phenolics (ranging from 3.5% to 10.8% w/w) and tannins (1.6% to 7.0% w/w). This prompted us to study the free radical scavenging activity of Panchvalkala and its components which was evaluated in three in vitro models viz. 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, superoxide radical scavenging activity and reducing power assay. Panchvalkala and its individual components showed significant antiradical activity by bleaching 1,1-diphenyl-2-picrylhydrazyl radical (EC(50) ranging from 7.27 to 12.08 microg) which was comparable to pyrogallol (EC(50) 4.85 microg). Thin layer chromatography of the methanol extracts when sprayed with 0.2% 1,1-diphenyl-2-picrylhydrazyl in methanol revealed several bands with antiradical activity as seen by bleaching of 1,1-diphenyl-2-picrylhydrazyl. All the samples showed good superoxide scavenging potential (EC(50) ranging from 41.55 to 73.56 microg) comparable to ascorbic acid (EC(50) 45.39 microg) in a dose-dependent manner. The reduction ability, Fe(3+) to Fe(2+) transformation was found to increase with increasing concentrations of all the sample extracts.

Validation of Different Methods of Preparation of Adhatoda vasica Leaf Juice by Quantification of Total Alkaloids and Vasicine.

Leaf of Adhatoda vasica (Vasaka) is an important drug of Ayurveda, prescribed as an expectorant. Quinazoline alkaloids present in the leaves are established as active principles. In Ayurveda, its leaf juice (Vasa swarasa) is incorporated in many formulations. Classical method for extracting the juice (swarasa) from the leaf is an elaborate process, which involves subjecting a bolus of crushed fresh leaf to heat followed by squeezing out the juice. Commercially, to prepare the juice of Vasaka, manufacturers have been adopting different methods other than the traditional method. In an effort to evaluate these modified processes phytochemically to identify the process which gives juice of the quality that is obtained by traditional method, in terms of its alkaloid content, we prepared the leaf juice by traditional Ayurvedic method, its modification by steaming of leaf to simulate the traditional method and other methods adopted by some manufacturers. These juice samples were evaluated for the total alkaloid content by spectrophotometric method and vasicine content by thin layer chromatography densitometric method using high performance thin layer chromatography. The high performance thin layer chromatography method was validated for precision, repeatability and accuracy. The total alkaloid content varied from 0.3 mg/ml to 5.93 mg/ml and that of vasicine content varied from 0.2 mg/ml to 5.64 mg/ml in the juice samples prepared by different methods. The present study revealed that steaming of fresh leaves under 15 lb pressure yielded same quantity of juice as the traditional bolus method (25 ml/100 g leaf) and its total alkaloid content and vasicine content (4.05+/-0.12 and 3.46+/-0.06 mg/ml, respectively) were very high when compared to the other methods, though the traditional method was found to give the best quality juice with highest amount of total alkaloids (5.93+/-0.55 mg/ml) and vasicine (5.64+/-0.10 mg/ml) content.

Quantification of eugenol, luteolin, ursolic acid, and oleanolic acid in black (Krishna Tulasi) and green (Sri Tulasi) varieties of Ocimum sanctum Linn. using high-performance thin-layer chromatography.

Ocimum sanctum (family Lamiaceae) is a reputed drug of Ayurveda, commonly known as Tulasi. In the present work, we quantified 4 marker compounds, viz., eugenol, luteolin, ursolic acid, and oleanolic acid, from the leaf of green and black varieties of O. sanctum using high-performance thin-layer chromatography (HPTLC) with densitometry. The methods were found to be precise, with relative standard deviation (RSD) values for intraday analyses in the range of 0.52 to 0.91%, 0.77 to 1.29%, 0.11 to 0.16%, and 0.34 to 0.42% and for interday analyses in the range of 0.73 to 0.96%, 1.02 to 2.08%, 0.11 to 0.12%, and 0.39 to 0.64% for different concentrations of eugenol, luteolin, ursolic acid, and oleanolic acid, respectively. Instrumental RSD values were 0.24, 0.39, 0.21, and 0.18% for eugenol, luteolin, ursolic acid, and oleanolic acid, respectively. Accuracy of the methods was checked by conducting a recovery study at 3 different levels for the 4 compounds, and the average recoveries were found to be 99.73, 99.3, 100.58, and 100.57%, respectively. Eugenol content ranged from 0.175 to 0.362% (w/w) and luteolin from 0.019 to 0.046% (w/w) in the samples analyzed. Green variety was found to contain higher amounts of ursolic acid [0.478 and 0.348% (w/w), from Sources 1 and 2, respectively] than the black variety [0.252 and 0.264% (w/w) from Sources 1 and 2, respectively]. Black variety had 0.174 and 0.218% (w/w) of oleanolic acid from Sources 1 and 2, respectively, while it was not detected in the green variety. Ursolic acid and oleanolic acid ran at the same Rf value and could not be resolved in several solvent systems tried. However, we observed that only ursolic acid gave yellow fluorescence under 366 nm ultraviolet light after derivatization with anisaldehyde-sulfuric acid reagent. The HPTLC-densitometry methods for the quantification of the 4 markers in O. sanctum leaf will have the applicability in quality control.