Methanolic Sulphuric Acid Research Articles

Quality Control of Herbal Drug (Paxherbal Bitter Tea) Via Thin-Layer Chromatography and Phytoconstituent Analysis

The effectiveness of any herbal medication depends on the presence or absence of pharmacologically active phytocompounds. These ingredients are significantly affected by the quality control system espoused during and after manufacturing processes and the operation of such pharmaceuticals. Thin-layer chromatography (TLC) is a quality control parameter suitable to identify and codify substances predicated on their distinctiveness and uniformity, giving an identity for such substances. Paxherbal Bitter Tea is a polyherbal medicine harnessed as an anti-diabetes medicine. This study assessed the distinguishable phytoconstituents present and evolved attribute profiles (TLC) for the herbal (Paxherbal Bitter Tea) to serve as a quality control check during manufacturing for consistency and market identity. Qualitative phytochemical and chromatographic assays were carried out employing standard methodologies. The qualitative test revealed the presence of saponin, steroids, reducing sugar, cardiac glycoside, and terpenoids, and the finger-print chromatograms after progression with n-hexane: ethyl acetate (9:1) flaunted three dissimilar elements under ultraviolet light at 365 nm and five spots, when dotted with 20% methanolic sulphuric acid under visible light. In this study, we developed an identity profile for Paxherbal Bitter Tea via its unique chemical biographies, which can be exercised in verifying the quality and consistency of the herbal product.

Bioactive composition and TLC profile data on Pax Herbal Malatreat Tea

The biological/pharmacological activities of any herbal drug are determined by the bioactive components contained in the plant(s) used in its formulation, these substances are greatly affected by the quality control system adopted during and after production processes and handling of such product. Thin Layer Chromatography is one of such quality control parameters that demonstrate uniqueness and uniformity between various substances, thus serving as an identity for such products. Pax Herbal Malatreat tea is a polyherbal drug used in the management and treatment of malaria symptoms. This study evaluated the different phytoconstituents present and developed fingerprint profiles (TLC) for the drug (Pax Herbal Malatreat Tea) to serve as a quality control check during the production consistency and market uniqueness. Qualitative phytochemical and chromatographic analyses were carried out using standard methods. The qualitative test revealed the presence of Tannin, Flavonoid, Saponin, Steroids, Reducing sugar, and Terpenoids, and the finger-print chromatograms after development with chloroform-ethyl acetate (9.5:05) showed five distinct components at 365 nm and four spots when sprayed with 20% methanolic sulphuric acid and viewed under visible light. From this present report, an identity card has been developed for the drug (Pax Herbal Malatreat Tea) via bioactive composition and TLC profiles which can be used in accessing the quality and consistency of the product.

Quantification of tocochromanols in vitamin E dietary supplements by instrumental thin-layer chromatography

A variety of vitamin E dietary supplement capsules (DSC) based on different natural oils or synthesis products are currently found on the market whose vitamin contents need to be controlled before and after marketing. Here, we present an instrumental thin-layer chromatography (TLC) method which allows a direct determination of all tocopherols (T) and tocotrienols (T3) as well as α-tocopherol acetate simultaneously in one run with short analysis time. For this purpose, contents of the DSC were extracted, applied on silica gel 60 plates, and developed with n-hexane/ethyl acetate/acetic acid, 90:10:2 (v/v/v) as mobile phase. The UV scan of the plate at 293 nm was used for quantification based on the peak height. Following the scan, the plate was treated with 10% sulphuric acid in methanol which led to characteristic yellow-to-brown colouring of the tocochromanol spots which allowed to distinguish tocochromanols from matrix components with similar Rf values. In most cases, determined vitamin E contents matched well with the information listed on the label of the investigated DSC samples. The method is fast, easy to perform and gently treats the analytes as it requires no thermal treatment prior to quantification, which makes it suitable as a screening method.

ESTIMATION OF TADALAFIL IN HUMAN PLASMA BY SPECTROFLUOROPHOTOMETRY

A new, specific, selective, simple, rapid and inexpensive spectrofluorophotometric method has been developed for the determination of tadalafil in spiked human plasma. The fluorescence spectrum of tadalafil in 0.1M methanolic sulphuric acid showed excitation wavelength at 315 nm and emission wave-length at 332 nm. The method for tadalafil was found to be linear over the concentration range of 10-50 ng/mL with a correlation coefficient of 0.9991. Limit of detection and limit of quantification were found to be 0.235 ng/mL and 0.701 ng/mL, respectively. The method was validated and found to be suitable for the estimation of tadalafil from human plasma. Satisfactory recovery of tadalafil from the human plasma suggests no interference of any debris present into human plasma. This method can be used to deter-mine plasma tadalafil concentration in drug monitoring or pharmacokinetic investigation.

Screening the elite chemotypes of Gloriosa superba L. in India for the production of anticancer colchicine: simultaneous microwave-assisted extraction and HPTLC studies

BackgroundGloriosa superba L. (Colchicaceae) is a high-value medicinal plant indigenous to Africa and Southeast Asia. Its therapeutic benefits are well-established in traditional medicines including Ayurveda. It is well known for its natural bioactive compound colchicine which exhibits a wide range of pharmacological activities i.e. rheumatism, gout and was also introduced into clinical practices. The increasing demand as well as its illegal harvesting has brought this valuable plant under threatened category.MethodsThe present investigation describes a microwave assisted extraction (MAE) strategy coupled with a densitometric-high performance thin layer chromatographic (HPTLC) methodology for the analysis of colchicine from 32 different populations of G. superba. A Box-Behnken statistical design (3 level factor) has been employed to optimize MAE, in which power of microwave, time of irradiation, aqueous ethanol and pH were used as independent variables whereas colchicine was used as the dependent variables. Chromatography was carried out on Silica gel 60 F254 TLC plates with toluene: methanol, 85:15 (v/v) being used as solvent system. Densitometric measurement was performed at λ=254 nm following post-derivatization (10% methanolic sulphuric acid).ResultsOptimal conditions for extraction to obtain the maximum colchicine yield was found to be 7.51 mg g− 1 which was very close to be predicted response 7.48 mg g− 1 by maintaining microwave power (460 W), irradiation time (6.4 min), aqueous ethanol-30, pH -3. Colchicine content ranged between 2.12–7.58 mg g− 1 among 32 G. superba populations in which only three chemotypes viz. GS- 1, GS- 3, and GS- 2 collected from West Bengal and Sikkim, respectively exhibited maximum yield of colchicine.ConclusionTherefore, this newly developed optimized MAE coupled with HPTLC densitometry methodology not only quantifies colchicine in order to identify elite chemotypes of G. superba, but it also encourages in selecting high yielding populations of the plants for industrial use and economic boost for the farmers. This validated, simple and reproducible HPTLC protocol is being used for the first time to estimate colchicine from natural populations of G. superba obtained from 32 different geographical regions of India.Graphical abstract

Outdoor cultivation of Chlorella pyrenoidosa in paddy-soaked wastewater and a feasibility study on biodiesel production from wet algal biomass through in-situ transesterification

Sustainable resources management, incorporating energy markets and resources such as electricity, fossil fuels, renewable and sustainable energy capital is essential for society to understand production and conversion of various forms of energy, their current as well as future supply. Waste-to-energy (WTE) or energy-from-waste (EFW) is a well-identified transitional technology which could prevent complete depletion of renewable resources. In our present study, the selected microalgae (Chlorella pyrenoidosa) was cultured in paddy-soaked wastewater (PWW) using outdoor raceway ponds of 50 L capacity where biotransformation of nutrients (NH3–N removal: 75.89 ± 0.69%; PO4–P removal: 73.71 ± 0.75%; yield co-efficient YN: 6.12 mg biomass/mg of N; YP: 7.77 mg biomass/mg P) has occurred with better growth and biochemical composition (dry biomass weight: 1.56 ± 0.11 g/L; chlorophyll: 15.57 ± 0.14 mg/L; specific growth rate (SGR): 0.42/d; lipids: 27.47 ± 1.41% biomass; carbohydrates: 23.77 ± 1.00% and protein: 46.12 ± 3.55%). Further, the obtained algal lipid was identified for a wide range of fatty acid methyl esters (FAME) and consequently brought forward to in-situ single-step transesterification by optimizing reaction conditions. Central composite design (CCD) of response surface methodology (RSM) has given optimized conditions of sample amount: 2 g (wet); methanol sulphuric acid volume: 3 mL; and hexane volume: 4 mL, under the reaction temperature of 90 °C for maximum biodiesel conversion (46.54% of algal lipids). The outcome of our current research may add value to the application and development of WTE technology for sustainable energy conservation.

HPTLC Fingerprint Profile of Leaf and Stem Methanolic Extract of Asclepias Curassavica L.

Asclepias curassavica, a member of the family Asclepiadaceae, is useful for a variety of health conditions such as abdominal pain, hemorrhage, asthma, and piles. It has a wide range of chemical constituents including flavonols, flavonol glycosides, triterpenes, cardenolides and many other biologically active compounds. Here we attempted to characterize the HPTLC profile for Leaf and Stem of methanolic extracts of Asclepias Curassavica L. Asclepias Curassavica L. family Asclepiadaceae, is important medicinal plant and traditionally used in indigenous system of medicine Extract of Asclepias Curassavica L. Methanolic extracts were prepared using sonication. The mobile phase for triterpenes and saponins was used in different ratio for better separation. The fingerprint showed better resolution after derivatization in methanolic sulphuric acid reagent. The fingerprint has revealed a distinct and repetitive pattern of bands. In conclusions, the HPTLC profile of the plant will be helpful for crude drug identification, serve to check the adulteration and act as biomarker for this plant in the Pharmaceutical industry.

GC-MS ANALYSIS, HPTLC FINGERPRINT PROFILE AND DPPH FREE RADICAL SCAVENGING ASSAY OF METHANOL EXTRACT OF MARTYNIA ANNUA LINN SEEDS

Objective: To investigate the photochemical constituents present in methanol extract of martynia annua seeds using Gas Chromatography-Mass Spectroscopy(GC-MS), High-Performance Thin–Layer Chromatography(HPTLC) analysis and study antioxidant activity.
 Methods: Methanol extract of Martynia annua seeds were subjected to GC-MS and HPTLC analysis. HPTLC analysis was carried out using GAMAG system with a linomate5 applicator, system mobile phase (Toluene: Chloroform: Ethanol (4:4:1 V/V/V)), two different volume of extract was applied 2 µl and 5 µl. GC-MS analysis was carried out on JEOL GC MATE ΙΙ, column HP 5 MS and Quadruple double focusing mass analyzer. Antioxidant activity was determined by DPPH assay.
 Results: GC-MS analysis provided 17 peaks indicating the presence of seventeen different phytochemicals in methanol extract of martynia annua seeds. HPTLC fingerprint showed 6 peaks at both size 2 µl and 5 µl at 254 nm whereas 4 peaks, 9 peaks were detected at 366 nm for 2 µl and 5 µl respectively. After derivatization with 10 % methanolic sulphuric acid, 8 peaks, 11 peaks were detected for 2 µl and 5 µl respectively when the derivatized plate was scanned at 540 nm. DPPH free radical scavenging result showed EC50 value of 44.1±1.1 µg/ml.
 Conclusion: The GC-MS analysis showed the presence of fatty acids, ester, aldehydes and ketones whereas in HPTLC different peaks at different UV-lights before and after derivatization were observed. Maximum percentage inhibition using DPPH assay was found 74 at concentration of 50 µg/ml.

PHARMACOGNOSTIC STANDARDISATION AND CHROMATOGRAPHIC FINGERPRINTING OF LEAVES AND FRUITS OF ZANTHOXYLUM RHETSA

Objective: To conduct pharmacognostic standardisation and chromatographic fingerprinting of leaves and fruits of Zanthoxylum rhetsa.Methods: The macroscopic, microscopic and physicochemical evaluation of the crude drugs were conducted as per I. P and WHO guidelines. The chromatographic fingerprint was also developed for the leaves and fruits.Results: The microscopic characteristics of leaves exhibited anomocytic stomata, fibrovascular tissue, lignfied pericyclic fibers and pitted xylem vessels. The fruits exhibited pericarp, oil cells, stone cells, xylem and endosperm. The physicochemical analysis was also conducted. The leaf and fruit powders complied with WHO prescribed limits for microbial load NMT 1× 105 CFU/ml and were found to be free from pathogenic organisms. The HPTLC fingerprint was established for methanol extract of leaves and fruits using Dichloromethane: chloroform: ethanol (4:4:1) as solvent and 10% methanolic sulphuric acid as spray reagent.Conclusion: The present work provides referential information for the correct identification and standardisation of the plant.

A Specific Thin Layer Chromatography Method for the Identification and Separation of Medium Chain Acylglycerols.

Medium chain fatty acids (MCFAs), have gained nutritional relevance in the past few years. They are continuously used in obtaining structured lipids like medium chain acylglycerols (MCAs) for various purposes. However, because of their chemical structure pertaining carbon chain length and the presence of saturated and unsaturated fatty acids, sensitive detection techniques are required for their correct identification and separation. In the present work, a specific thin layer chromatography (TLC) method for MCAs was developed. The proposed method consisted of the use of a mixture of hexane: acetone (70:30 v/v) as mobile phase, since it proved effectiveness for the separation of compounds of interest (MCAs) as well as having the necessary sensitivity to separate different species of monoacylglycerols (MAGs), diacylglycerols (DAGs) and triacylglycerols (TAGs) of MCFAs. For observation of the compounds, a single oxidizing agent was not sufficient, thus a combination of visualization reagents was used (first a 10 % solution of sulphuric acid in methanol followed by a 10 % solution of phosphomolybdic acid in methanol) achieving the correct visualization of the desired compounds.

A Rapid Densitometric Comparison Method For The Quantification of Corosolic Acid In Lagerstroemia Speciosa Leaves Using HPTLC

Background:Banaba (Lagerstroemia speciosa, Lythraceae, Pride of India) is a medicinal plant that grows in the Philippines, Japan, India, Southeast Asia, etc. Traditionally, the leaves are used to treat diabetes and hyperglycemia. This effect is attributed to its chemical constituents belonging to the groups of terpenoids, tannins, and flavanoids. The objectives of this paper are to present a new method of identification and quantification of corosolic acid, a terpenoid, using HPTLC.Material and methods:The hydroalcoholic extract of leaves was chromatographed on aluminum plates coated with silica gel 60F254, with Toluene: Acetone: Formic acid (5:2:1 v/v) as the mobile phase. Densitometric quantification of corosolic acid was performed after derivatization with 10 % methanolic sulphuric acid in fluorescence mode at 366 nm.Results:Amounts of corosolic acid (at 366 nm, Rf value = 7.2) in the sample was calculated using regression equations (R2 = 0.9959 (366 nm) of calibration plots, which showed there was a good polynomial relationship between peak area and amount of corosolic acid in the range 250-3000 ng/band. The limits of detection (LOD) and limits of quantitation (LOQ) were 70 and 245.6 ng/band, respectively. The method was validated for precision and accuracy. Recovery was determined by spiking the extract with corosolic acid standard and found to be in the range 94.4–96.5%.Conclusion:The corosolic acid content found in the leaves was 1.5% w/w at 366 nm.

Phytochemical Analysis and HPTLC Finger Printing of Nyctanthes arbor-tristis Linn. Leaf

Herbal medicine also known as botanical medicine refers to using plants seeds, flowers, roots for medicinal purpose. Nyctanthes arbor-tristis Linn is a large shrub or small tree, found throughout in India as well as chitrakoot region. It is commonly known as Harshingar, night jasmine, and parijat. It is used for its antibacterial, anthelmintic, anti-inflammatory, hepato-protective, immune potential, antipyretic, antioxidant and antifungal activity. The present investigation deals with preliminary phytochemical screening and High Performance Thin layer Chromatography (HPTLC) fingerprints profile of Parijat –leaf. For Phytochemical screening two various extracts (ethanolic extract and aqueous extract) of leaf were used and found the Alkaloids, Resins Saponins, Tannins, Terpinoids and Cardiac glycoside present. HPTLC finger printing were carried out on Toluene: Ethyl acetate Original Research Article Prajapati et al.; IJBCRR, 11(4): 1-7, 2016; Article no.IJBCRR.25118 2 (7:3); as a mobile phase. The plate ware developed over a distance of 9 cm in a saturated development chamber Twin through chamber 10X10 cm with SS lid and visualized under at 254nm, 366nm, under ultraviolet before and after derivatization. After spraying with 5% methanolic sulphuric acid followed by heating at 105°C for 5-10 minutes. Rf values were noted with colour before and after derivatization. Rf values are before derivatization at 366 nm 0.05, 0.27, 0.31, 0.37, 0.44, 0.51, 0.69, 0.85, 0.92, 0.96 (all red); under ultraviolet: 0.36, 0.41, 0.50, 0.55, 0.95 (light yellow). After derivatization at 366 nm 0.09 (faint white), 0.31, 0.38, 0.49 (orange), 0.58 (yellow), 0.54, 0.84 (both violet), 0.90 (light pink), 0.96(white); under ultraviolet 0.36, 0.51 (both light brown), 0.58 (light black), 0.98 (brown).

HPTLC Densitogram Profile of Aqueous Distillate (Arka) from Roots of Boerhaavia diffusa

The aqueous distillate (arka) of Boerhaavia diffusa was prepared and investigated qualitatively for TLC. The following work constitutes the enrichment of constituents present in the aqueous distillate (arka) by partition technique using various solvents such as toluene and ethyl acetate in the ratio of (7:3), toluene:acetone:acetic acid (8.5:1:0.5) chloroform:methanol (9:1) Enrichment was determined by Thin Layer Chromatography and HPTLC. Hence it was concluded that aqueous distillate showed the better result for TLC in different solvent systems tried, but best solvent system selected for TLC was toluene: acetone: acetic acid in the ratio of (8: 0.5:1.5) using 5% methanol-sulphuric acid solution as detecting reagent.

Fatty acids in female’s gonads of the Red Sea fish Rhabdosargus sarba during the spawning season

Objective: To determine the fatty acids profiles in female fish, Rhabdosargus sarba (R. sarba) from the Red Sea during the spawning season. Methods: Monthly individual R. sarba were obtained from Bangalah market in Jeddah, Red Sea and transported to the laboratory in ice aquarium. The total length, standard length and weight were measured, fishes were dissected. Ovaries were removed, weighed and 10 mL of concentrated hydrochloric acid were added to 10 g of the ovary in a conical flask and immersed in boiling water until the sample was dissolved and the fat was seen to collect on the surface. The conical was cooled and the fat was extracted by shaking with 30 mL of diethyl ether. The extract was bowled after allowing the layers to separate into a weighed flask. The extraction was repeated three times more and distilled off the solvent then the fat dried at 100 °C, cooled and weighed. Then 50 mg of lipid was put in a tube, 5 mL of methanolic sulphuric acid was added and 2 mL of benzene, the tube well closed and placed in water bath at 90 °C for an hour and a half. After cooling, 8 mL water and 5 mL petroleum were added and shaked strongly and the ethereal layer was separated in a dry tube, evaporated to dryness. The fatty acid methyl esters were analyzed by using a Hewlett Packard (HP 6890) chromatography, a split/splitless injector and flame ionization detector. Results: In female R. sarba, a total of 29 fatty acids were detected in ovaries throughout the spawning season. The main fatty acid group in total lipid was saturated fatty acid (SFA, 28.9%), followed by 23.5% of polyunsaturated fatty acids (PUFA) and 12.9% of monounsaturated fatty acids (MUFA). The dominant SFA were palmitic and stearic, the major MUFA were palmitoleic and oleic, and the major PUFA were C18:2 and C22:2. During spawning stages, there were no significant differences in total SFA, MUFA and PUFA. The highest value of SFA was in late spawning (36.78%). However, the highest value of MUFA and PUFA was in spawning (16.70% and 24.96% respectively). During spawning season there were significant differences in total SFA between March (late spawning stage) and December (nearly ripe stage), P<0.05. Conclusions: From the present study it has been shown that 29 fatty acids were detected in ovaries of R. sarba throughout the spawning season. The main fatty acid group in total lipid was SFA followed by PUFA and MUFA. The dominant SFA were palmitic and stearic, the major MUFA were palmitoleic and oleic, and the major PUFA were C18:2 and C22:2. The highest value of SFA was in late spawning. However, the highest value of MUFA and PUFA was in spawning.

Isomerization of octadecapentaenoic acid (18:5n-3) in algal lipid samples under derivatization for GC and GC-MS analysis.

During gas chromatography (GC) analysis of fatty acid (FA) composition of the dinoflagellate Gymnodinium kowalevskii, we found unex-pectedly low and irreproducible content of all-cis-3,6,9,12,15-octadecapentaenoic acid (18:5n-3), which is an important chemotaxonomic marker of several classes of microalgae. We compared chromatographic behavior of 18:5n-3 methyl ester and other GC derivatives obtained using different conventional methods of derivatization. The use of methods based on saponification or base-catalyzed transesterification resulted in a mixture of double-bond positional isomers of 18:5. On a SUPELCOWAX 10 column, the equivalent chain length (ECL) value for authentic 18:5n-3 methyl ester was 20.22, whereas the main component after base-catalyzed methylation had ECL 20.88. Attempts to prepare N-acyl pyrrolidides or 4,4-dimethyloxazoline (DMOX) derivatives of 18:5n-3 also gave inadequate results. These derivatives also showed a main peak corresponding to isomerized 18:5. Mass spectra for both DMOX and pyrrolidide derivatives of this compound showed the base peak at m/z 139, probably corresponding to 2,6,9,12,15-18:5 acid. Of all methods tested for methylation, only derivatization with 5% HCl or 1% sulphuric acid in methanol gave satisfactory results. Therefore, GC or GC-mass spectrometry analyses of algal lipids containing 18:5n-3 may be inaccurate when base-catalyzed methods of FA derivatization are applied. The best and simplest way to avoid incorrect GC results is to use standard acid-catalyzed methylation.

Simultaneous estimation of three triterpenoids‑ursolic acid, b‑sitosterol and lupeol from flowers, leaves and formulations of Rhododendron arboreum Smith. using validated HPTLC method

Background: This paper enfolds a rapid and sensitive high‑performance thin‑layer chromatographic (HPTLC) method for the simultaneous estimation of three triterpenoids namely ursolic acid, β‑sitosterol and lupeol from the leaves, flowers and herbal formulations of Rhododendron arboreum Smith., an ethnomedicinal Himalayan tree. All the three phytoconstituents have high therapeutic value. Aims and Objectives: The main aim is to separate, resolve and simultaneously quantitate the three markers‑ursolic acid, β‑sitosterol and lupeol from R. arboreum using normal phase HPTLC. Materials and Methods: Separation was performed on TLC aluminium plates precoated with silica 60 F254 followed by detection of ursolic acid, β‑sitosterol and lupeol carried out by derivatizing the plate with 10% methanolic sulphuric acid reagent followed by heating at 110°C for 7 min. Camag TLC scanner 4 equipped with winCATS software was used for densitrometric scanning at 366 nm. The proposed method was further validated in terms of linearity, precision, accuracy and sensitivity as per the International Conference on Harmonisation (ICH) guidelines. Results: A good linear relationship was obtained for the calibration plots with r2 = 0.999, 0.993 and 0.995 for ursolic acid, β‑sitosterol and lupeol, respectively. Accuracy of the method was checked by recovery study conducted at three different levels with the average recovery between 95% and 98% for all the three markers. Conclusion: The developed method can be used for the assessment of the quality of botanicals in terms of bioactive content. Key words: Ethnomedicinal, Rhododendron arboreum, simultaeneous estimation, triterpenoids

Development and validation of HPTLC method for quantification of the antidiabetic compound α-amyrin acetate inStreblus asperLour

The bioactive molecule, α-amyrin acetate, was isolated from the nhexane extract Streblus asper stem bark by column chromatography. The structure of the compound was characterized with the help of physical and spectroscopic data. A high-performance thin-layer chromatography (HPTLC) method has been developed and validated for its quantification in S. asper leaf, stem bark, and root. Chromatographic separation of the compound was achieved on high-performance precoated TLC plates by using optimized binary mobile phase consisting of n-hexane-ethyl acetate (9.6:0.4, v/v). HPTLC scanning was performed at 472 nm after derivatization of the plate with methanol-sulphuric acid reagent in reflection/absorption mode. The method was validated according to International Conference on Harmonization (ICH) protocol for specificity, sensitivity, linearity, accuracy, precision, repeatability, and robustness. The developed method is found to be very simple, rapid, precise, sensitive, and accurate for the quantification of α-a...

Development and Validation of a Stability-Indicating Method for Determination of Free Sterols in the Asian Medicinal Leech Hirudo manillensis

A rapid, simple, sensitive, selective, precise and robust thin-layer chromatography densitometric method for the determination of free sterols in leech was developed and validated on silica gel layer using carbon tetrachloride-methanol-formic acid (9.5:1.5:0.55, v/v/v). Spectrodensitometric scanning was carried using a Camag TLC scanner III at 366 nm after spraying 2% methanolic sulphuric acid, which gave compact spots for cholesterol (R(F) = 0.35 ± 0.02). The regression analysis data for calibration plot implied a good linear relationship (r(2) = 0.99958) between response and concentration over the range 100-600 ng per spot with respect to peak area. The limits of detection and quantification were found to be 13.8 ± 0.51 and 45.01 ± 1.29 ng per spot, respectively. Validation was in accordance to the International Conference on Harmonization guidelines. Cholesterol was subjected to forced stress conditions of oxidation, hydrolysis and heat. Degradation products resulting from the forced stress did not interfere with detection because the degradant peaks were well separated from the cholesterol peak. The densitometric method can be regarded as stability-indicating and can be used for quality control assay of cholesterol in leech extract.

Development and validation of HPTLC method for the estimation of diosgenin inIn Vitroculture and rhizome ofDioscorea deltoidea

Summary A new simple, accurate, selective, precise, economical and stability-indicating high-performance thin layer chromatographic method for the analysis of diosgenin in callus and rhizome of Dioscorea deltoidea was developed and validated. The method was developed on TLC aluminium plates precoated with silica gel 60F254 using solvent system petroleum ether-isopropanol (12:1, v/v), which gives a compact spot of diosgenin (RF value 0.76 ± 0.02). Densitometric analysis of diosgenin was carried out in the absorbance mode at 366 nm after spraying with methanolic sulphuric acid. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.991 and 0.995 for diosgenin with respect to peak height and peak area, respectively, in the concentration range of 100–1000 ng per spot. The limits of detection and quantification for diosgenin were 16.58 and 50.25 ng per spot. The proposed method was applied for determination of diosgenin in rhizome of D. deltoidea (0.047% w/w) ...

Production of biodiesel from Terminalia catappa seed oil with high free fatty acids using an acid-catalyzed pretreatment step

Biodiesel was produced from the underutilized seed oil of Terminalia catappa using a two-step reaction system. The first step is a pretreatment process, which involved the use of 2% sulphuric acid in methanol, and the second step is a transesterification reaction using KOH as the catalyst. The results of the applied method showed a conversion that gave an ester content of above 97% with phosphorus content below 1 ppm in the biodiesel. The biodiesel produced from the oil of T. catappa exhibited properties that are in agreement with the recommended European standards (EN 14214) and American standards (ASTM D 6751-07b). This study supports the use of crude T. catappa as a viable and valuable raw feedstock for biodiesel production.