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Chapter- 4 Introduction

Plants produce several secondary metabolites with varying characteristics like alkaloids, terpenoids, flavanoids, steroids, saponins, tannins etc. This helps plant for self-defense from herbivores and micro-organisms. These metabolites can be used in as a medicine, antioxidant/food preservative in food industry, pesticide industry for insect-pest control and as a disinfectant (Sabnis and Daniel, 1990; Daniel, 1991). In the present study, research was carried out to develop an eco-friendly herbal fungicide product for agriculture. An effort was directed to search for antifungal property from plants of medicinal, aromatic and conventional utility through microbiological assays. The positive results so obtained were evaluated for HPTLC based finger printing to develop a digital phyto-chemical profile (Cimpoiu, 2006). This study helps to identify active metabolites involved and as an analytical tool in quality control aspects of the developed product (Pattanaya et al, 2010). Each component in the sample is separated on TLC plate through selected solvent system considering the polar, non-polar or the intermediate nature of the metabolites. This is then followed by scanning, Rf value and λmax profile metabolites (Sharma and Patel, 2009). The HPTLC technique is helpful to compare profiles of the crude plant extracts prepared using various solvents for the maximum extraction of the desired metabolites in selected solvents. The comparative analysis with different solvent extracts would also serve for value addition of the product (Harborne, 1984). HPTLC - A modern quality control tool: TLC- Thin Layer Chromatography is a common analytical technique widely used for the analysis of phyto-constituents in plant extracts and also for plant identification. HPTLC is a modified and an advanced version of TLC technique (Daniel, 1991). HPTLC- High Performance Thin Layer Chromatography is a sophisticated, a powerful, reliable, efficient and automated form of TLC having the latest technical developments for quality assessment and evaluation of botanical materials (Cimpoiu, 2006; Khushboo et al, 2009; Saraswathy et al, 2010). The advancements include enhancement to the basic method of TLC to automate the different steps, increase the resolution, high sample throughput together with better analytical precision and accurate quantitative measurements with reduced consumption of mobile phase per sample (Khan et al, 2010). Automation in HPTLC is useful to avoid uncertainty in application size and position, when the sample is applied manually to the TLC plate. In addition, many samples of divergent nature can be run in a single analysis with simultaneous processing of sample and standard with varied analytical time (Harborne, 1984). The compounds can be detected

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by post-chromatographic derivatization. Moreover, it can provide chromatographic fingerprints generated at different wavelengths of light, which can be visualized and stored as electronic images. HPTLC is thus a valuable tool for reliable standardization of compounds (Cimpoiu, 2006). For the analysis, the aluminum coated silica gel TLC plates are activated (oven at 110 -120ºC for 15 -30 min) prior to sample application in the sample concentration range of 0.1-1µg/µl using automatic applicator Linomat IV. Principle: Chromatography is an important liquid-solid adsorption technique have stationary and mobile phase where the mobile phase ascends the thin layer of stationary phase by capillary action carrying compounds with it, coated on to a backing support or stationary phase and separate mixtures of two or more compounds based on differences in polarity (Sharma and Patel, 2009). Other factors such as Lewis acid- Lewis base interactions, hydrogen bonding interactions and Van der Waals interactions also affect a compound’s affinity for the stationary or liquid phase (Harborne, 1984). Role of solvent system: Depending on the polarity of the compounds in the extract, a compound travels at different distances up the plate. More polar compounds will stick to the polar silica gel and thus travel up to short distances on the plate. While non-polar substances will spend more time in the mobile solvent phase and travel larger distances on the plate (Sabnis and Daniel, 1990; Sharma and Patel, 2009). The measure of the distance a compound travels on the plate is called the Rf value. The identification of separated compounds in HPTLC is mainly done on the basis of retention factor parameter (Rf), colors of spots and the computerized fingerprints of samples. These HPTLC generated fingerprints can be saved as electronic images (Harborne, 1984; Cimpoiu, 2006). Rf value of each spot is calculated by calculating and dividing the distance traveled by the solute (compound) from the baseline upon distance traveled by the solvent from the baseline (solvent front). Different steps of HPTLC fingerprinting: a). Chamber saturation: Chromatographic chamber is filled with the solvent system 30 minutes prior to development of plate, to get uniform distribution of solvent vapours in the chamber. b). Application of sample and standard: Sample is spotted on the TLC plate with automatic applicator Linomat IV attached with the compressed nitrogen gas cylinder and operated with software winCATS.

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c). Selection of mobile phase: The solvent system is selected considering the nature of the components to be separated like polar or non-polar and also solubility, affinity and resolution, as the compound will follow the rule of ‘like dissolves like’ (Sharma and Patel, 2009). The polar compounds like alkaloids, saponins, curcuminoids, glycosides, flavanoids, flavones, catechins, phenols, tannins, carotenoids etc. are separated using solvent system made of polar solvents (Sabnis and Daniel, 1990; Daniel, 1991). While non-polar compounds viz. terpenoids, sterols, steroids, volatile and fixed oils etc. are separated using non-polar solvent system containing non-polar solvents. The desired mobile phase would provide the greatest solubility, while providing affinity for the sample on the stationary phase. Highly polar solvents are water, methanol, ethanol, acetone, diethyl ether, ethyl acetate, etc. while non-polar solvents are dichloromethane, toluene, chloroform, cyclohexane, petroleum ether, hexane etc (Harborne, 1984; Daniel, 1991). d). Chromatographic development and drying: Sorbent layer thickness of TLC plate is 100µm and due to this smaller particle size, separations are achieved at low distance route viz. at 3 to 5 cm. Because of this shorter migration distance, less amount of mobile phase is required and the analysis time is greatly reduced. After development, remove the plate and Dry in vacuum desiccator. e). Detection and visualization: Different compounds are distinguished by their retention factor, or Rf values generated. The Rf value determines the distance traveled of each individual compound within a mixture. Rf is the ratio of the distance traveled by the compound to the distance traveled by the solvent front. Detection under UV light is first choice non destructive method. Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length). When individual component does not respond to Uv - derivatisation required for detection. f). Quantification: After development of the chromatogram, it is scanned in Camag TLC scanner III having Uv/ visible/ fluorescence scanning facility. g). Documentation: The chromatogram is automatically recorded during photo documentation, having a facility of three lights options viz. visible, UV and florescent (Sabnis and Daniel, 1990; Daniel, 1991). Phyto-chemical profile generation and comparative analysis: Because of the simplicity of the sample preparation and the possibility of analyzing several samples of herbal products simultaneously in a short time, the HPTLC can be used as a rapid method to control the quality of raw plant materials and formulations (Bhise and Salunkhe, 2009; Pattanaya et al, 2010; Saraswathy et al, 2010). Thus HPTLC method is feasible for the comprehensive quality evaluation of herbal products and to detect substitution of plants by comparing the

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plant standard chemical fingerprint with chemical fingerprint of the product (Pawar et al, 2008; Kotagiri et al, 2009). Advantages of HPTLC: Nowadays HPTLC is a routine analytical technique and have very easy sample preparation along with here, multiple samples can be run together with the standards on a single plate in a same solvent and thereby saving a time (Khushboo et al, 2009). Thus simultaneous chromatographic analysis of samples and standards on the same plate under identical conditions gives results with better accuracy and precision (Paramasivam et al, 2008) Another advantage of HPTLC is that, the chromatogram can be scanned at broad range of wavelength and recorded in the computer for comparative analysis (Patra et al, 2010). Thus HPTLC is a modern standardized technique having a large applicability in the field of plant material analysis and stability tests of extracts and finished products (Harborne, 1984; Khushboo et al, 2009). HPTLC has many advantages, such as lower costs, simplicity of sample preparation, simultaneously multiple samples analysis, short time analysis, rapidity and the facility of analyzing multiple samples at the same time under the same chromatographic conditions, the possibility of multiple detection and specific derivatization on the same plate, etc (Gallo et al, 2008). More specifically, the same HPTLC plate can be visualized with and without derivatization using different light sources like UV, visible and florescent. Chemical derivatization is done by spraying or dipping into solution, of specific or non-specific reagents and then heating the plate in an oven for proper visualization (Sabnis and Daniel, 1990; Daniel, 1991; Cimpoiu, 2006). HPTLC technique also helps in the analysis of antioxidants. The natural antioxidants present in plant extracts play a key role and act as free radicals scavenging agents in biological systems. The separation of antioxidant compounds from each other and from other components of the complex crude plant extracts can be done by HPTLC developed techniques (Rathee et al, 2006). HPTLC has been used for the determination of individual antioxidant capacity of target compounds and it might be of interest to the routine chemical or biological screening and they can solve real analytical problems (Cimpoiu, 2006). HPTLC is an important tool used for the identification, evaluation, purity and stability testing, dissolution value and content uniformity testing of various raw materials like herbal extracts, tinctures, essential oils, fermentation mixtures, drug and excipients and formulated products of ayurvedic, pharmaceuticals, cosmaceuticals and nutraceuticals industry (Sharma and Patel, 2009; Khan et al, 2010). HPTLC is thus a method of choice in fingerprint analyses and comprehensive quality evaluation of herbal products (Gallo et al, 2008).

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Need for development of chromatographic fingerprints: The quality control and quality assurance is necessary in order to maintain the efficacy standards and dose optimization of the active ingredients of the herbal formulation to get a desired effect (Paramasivam et al, 2008). Many kinds of chemical fingerprint analysis methods used for herbal drugs quality control for the purpose of species authentication, evaluation of quality and ensuring the consistency and stability of herbal drugs and their related products are available such as Paper chromatography, Thin layer chromatography, Spectrophotometry, Column chromatography, Gas chromatography, Gas liquid chromatography, High performance liquid chromatography, High performance thin layer chromatography etc (Harborne, 1984; Daniel, 1991; Bhise and Salunkhe, 2009; Sanja et al, 2009; Pattanaya et al, 2010; Saraswathy et al, 2010). The entire pattern of chromatographic fingerprint of herbal drugs represents a comprehensive qualitative approach to determine the presence or absence of desired markers or active constituents and their quantity in the drug (Kotagiri et al, 2009; Sharma and Patel, 2009). Chromatographic fingerprint analysis for herbal medicines: A chromatographic fingerprint of herbal product represents a chromatographic pattern of pharmacologically active or chemically characteristic constituents present in the extract/ product, indicating the quality of herbal product (Bhise and Salunkhe, 2009; Sanja et al, 2009). The construction of HPTLC chromatographic fingerprints represent appropriately the ‘chemical integrities’ and thus help in the authentication, identification and quality control of herbal formulations and medicines (Patra et al, 2010; Pattanaya et al, 2010).

The analysis of herbals and herbal preparations is quite challenging as herbal extracts are extremely complex in order of diversity of components, belonging to different classes. The problem of quality assurance of herbal products has been solved to a great extent with help of HPTLC chromatographic fingerprint analysis, where variation and similarity between the peaks and regions in a set of chromatographic fingerprints and comparison with the standard chromatographic fingerprints are evaluated for the qualitative and quantitative information on the characteristic components of herbal product (Harborne, 1984; Bhise and Salunkhe, 2009; Pattanaya et al, 2010). Furthermore, pattern recognition in chromatographic fingerprint helps to distinguish different types and quality of samples of herbal product (Sanja et al, 2009; Saraswathy et al, 2010). HPTLC is thus an inexpensive, effective, valuable and automated technique of choice for the analysis and identification of plant materials, screening of plant extracts.  

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Chapter- 4 Literature Review

HPTLC- An advanced tool for phyto-chemical analysis: HPTLC (High Performance Thin Layer Chromatography) provides an efficient, fast and reliable technique for the quantitative and qualitative analysis of new and unknown phyo-chemicals in plant extracts and essential oils. Thus the technique is now widely used in phyto-chemistry, toxicology and forensic laboratory (Pawar et al, 2008). The literature included here, supports the importance of HPTLC technique. HPTLC chemical finger printing of various plant extracts: As with the arrival of modern chromatographic systems there is constantly increasing aim to develop easy, rapid and convenient methods for standardization of herbal drugs considering their bioactive phytoconstituents (Kotagiri et al, 2009). Quantification of solasodine glycoside from cultured hairy roots of Solanum surattense was investigated by Pawar et al (2008) using HPTLC and by comparing the percent peak area of test sample with standard sample at known concentration. The HPTLC plate developed in benzene: methanol (5: 1, v/v) solvent system and visualized by spraying anisaldehyde- sulfuric acid (H2SO4). In the study by Pedro et al (2002), transformed root cultures of Anethum graveolens and its essential oil were compared with the parent plant material for oil composition by TLC method. Here the essential oil from the roots of the parent plant was 0.06% (v/w), but only 0.02% (v/w) was found in the hairy root cultures. However, the essential oil composition did not change significantly during their growth and showed the presence of carvone, α-phellandrene and apiole. In another study, HPTLC estimation of curcuminoids from essential oil of Curcuma longa rhizome samples was carried out by Garg et al (1999). Samples and standard curcumin (1 mg/ml) were spotted on 60F254 silica gel TLC plates and developed in chloroform: methanol (95: 5, v/v) mobile phase. The spots were scanned at 366nm, where curcumin was found at Rf 0.69.

Raghavendra et al (2006) in their study, have performed TLC (silica gel G) separation of methanol extract of Acacia nilotica leaves using three mobile phases; acetic acid: chloroform (1: 9, v/v); benzene: chloroform (1: 1, v/v) and chloroform: methanol: acetic acid: water (170: 25: 25: 4, v/v). In one similar study, Cassia fistula leaves extracts was applied on the silica plate and developed using solvent chloroform: methanol (8.2: 1.8, v/v) and chloroform: hexane (5.4: 6.6, v/v) and it was used for the bioutographic study to found the antifungal activity by Panda et al (2010). Sanja et al (2009) have performed HPTLC

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fingerprinting of Portulaca oleracea methanolic extract in toulene: ethylacetate: diethylamine (7: 2: 1, v/v) solvent system. The developed plate was then scanned at 200- 400nm range. HPTLC quantification of phyto-chemicals in plant extracts: HPTLC technique is been used routinely in regular laboratory experiments for quantification of phyto-chemicals of use in the plant samples. A standard compouns is run together with the sample extract to be tested. The comparable evaluation helps to detect/quantifity the amount of compound present in the test sample. There are many reports available including the similar study. A simple, fast and precise quantitative HPTLC method has been developed by Misra et al (2009) for quantitative estimation of mangostin in fruit pericarp of Garcinia mangostana methanol and chloroform extracts. Plates were developed in solvent system chloroform: methanol (27: 3, v/v) and post-chromatographic derivatization was done using anisaldehyde- sulphuric acid reagent and scanning at 382nm in ultraviolet-visible mode. A similar rapid and quantitative HPTLC method has been developed by Pawar et al (2010) for the determination of andrographolide in alcoholic extract of Andrographis paniculata whole plant samples. The samples were separated using toluene: ethyle acetae: formic acid (5: 4.5: 0.5, v/v) solvent system, derivatized with the anisaldehyde- H2SO4 reagent and visualized at 235nm wavelength. Su and Horvat (1988) have performed TLC analysis of dill (Anethum graveolens) seed acetone extract and compared with the standard d-carvone in benzene: chloroform (90: 10, v/v) solvent system and examined under UV at 254nm.

An efficient HPTLC method has been developed by Pawar et al (2011) for the determination and quantification of catechin in different samples of Smilax perfoliata using mobile phase chloroform: methanol (8: 2, v/v), sprayed by anisaldehyde- H2SO4 reagent and viewed at 455nm, where catechin was found at 0.33 Rf. A similar sensitive and reliable HPTLC method has been developed by Rakesh et al (2009) for the quantification of gallic acid in the dried flowers of Nymphaea stellata and using chloroform: ethyl acetate: formic acid (7.5: 6: 0.5, v/v) as a mobile phase, where gallic acid was detected at 0.3 Rf. Similarly a simple HPTLC method has developed for a routine quality control analysis for quantitative determination of a polyphenolic compound gallic acid by Patel et al (2010). According to the report, chloroform, alcoholic and aqueous extracts of Alangium salvifolium dry root powder, was developed using toluene: ethyl acetate: formic acid: methanol (6: 6: 1.6: 0.4, v/v) as mobile phase on silica gel 60F254 HPTLC plate. Gallic acid was detected at 0.40 Rf with reference to the standard gallic acid and was quantified at 254nm wavelength during the scanning. The gallic acid content in the alcoholic extract was 0.07μg and in aqueous

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extract was 0.04μg; while it was absent in the chloroform extract of Alangium salvifolium roots. HPTLC quantification and quality control of herbal products: HPTLC has been investigated for simultaneous assay of several components in a multi-component formulation, as well for the authentication of various plant species, to evaluate the stability and consistency of their preparations from different manufactures (Patra et al, 2010). HPTLC is a valuable tool for the investigation of herbal products to obtain ‘Finger Print’ patterns of herbal formulations, quantification of active ingredients, detection of adulteration and purity check (Sanja et al, 2009). The main advantage of HPTLC is that it facilitates automated application and scanning in situ and repeated detection of the chromatogram considering various parameters. Moreover, HPTLC results are reported as peak data along with presented and communicated as images (Khushboo et al, 2009).

Different market samples of medicinally important Plumbago zeylanica roots were quantified through HPTLC method for the active principle plumbagin content by Sasikumar et al (2010) in mobile phase toluene: formic acid (9.9: 0.1, v/v). Chromatograms of all the samples were compared with the standard plumbagin having 0.44 Rf value. A rapid and quantitative HPTLC method has been developed for determination of plumbagin in the chloroform extract of Plumbago zeylanica root samples and its ayurvedic formulation ‘Kalmegh Navayas Loha’ with physico chemical standardization by Pawar et al (2011). The solvent system used was toluene: ethyl acetae (3: 1, v/v) derivatized with the anisaldehyde- H2SO4 reagent and quantified at 270nm wavelength by comparing with the standard of plumbagin having 0.84 Rf. A simple, precise and rapid HPTLC method has been developed by Jirge et al (2011) for the simultaneous evaluation of betasitosterol-D-glucoside and withaferin A in an ayurvedic formulation containing ashwagandha (Withania somnifera) using mobile phase chloroform: methanol (8: 2, v/v). A HPTLC densitometry method is been developed by Sullivan and Sherma (2005), for assay of glucosamine of different forms in nine commercial dietary supplement tablets and capsules. The mentioned method is highly suitable for routine use in nutritional supplement analysis for manufacturing quality control or governmental regulatory purposes. Similarly HPTLC quantification of wedelolactone in methanolic extracts of herbal formulations was performed by Wani et al (2010) and separated with mobile phase toluene: ethyl acetate: acetone: formic acid (6: 2: 1: 1, v/v).

A similar HPTLC densitometric analysis method for the quantification of curcumin in bulk drug/ formulations was developed and validated by Ansari et al (2005). In their

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study, TLC aluminum plates precoated with silica gel 60F254 were used as the stationary phase and chloroform: methanol (9.25: 0.75, v/v) as the solvent system, which separated curcumin at 0.48 Rf and detected at 430nm. Many ayurvedic formulations contain combination of turmeric (curcumin) and black pepper (piperine). In this contex, Vyas et al (2011) have reported a simple, sensitive and fast HPTLC method for simultaneous quantification of active principles curcumin and piperine in crude powder mixture of ayurvedic formulation and for their routine quality control. The separation was performed on silica gel G 60F254 plate using mobile phase chloroform: methanol (9.6: 0.4, v/v). The plate was subjected for the densitometric scanning at 373nm where, curcumin was separated at Rf 0.57 and piperine at Rf 0.82. Bhat et al (2007) in their study, analyzed emulsifying ointment and carbopol 934 gel formulations containing Azadirachta indica, Tridax procumbens and Curcuma longa extracts by HPTLC method to identify the active constituents present in extracts. The components curcumin separated at 0.50 Rf, azadirechtine at 0.08 Rf and procumbetine at 0.62 Rf. A turmeric cream (Curcuma longa rhizome extract emulsified in a cream base) was standardized for pharmaceutically active curcuminoids viz mixtures of curcumin, demethoxycurcumin, bisdemethoxycurcumin as well as volatile oils (turmerone and zingiberone) by HPTLC method using pure curcumin as a bioactive chemical marker by Khan et al (2010). Samples were spotted on silica gel 60F245 TLC plate and developed in chloroform: methanol: acetic acid (48: 2: 0.1, v/v) solvent system and scanned at 300nm. The three curcuminoids were separated curcumin at Rf 0.40, demethoxycurcumin at Rf 0.23 and bisdemethoxycurcumin at Rf 0.19. The method mentioned by Khan et al (2010), can be used as a sensitive routine evaluation of curcuminoids in extract and in finished product/phytomedicines containing turmeric as an ingredient. Another HPTLC method has been developed for the routine analysis of peppermint oil dosage as therapeutic delivery in chewable tablets by Sachan et al (2010). The solvent system used in the study was toluene: ethyl acetate (95: 5, v/v) and derivatized by spraying anisaldehyde- sulfuric acid reagent. Menthol was detected by comparing with the standard menthol Rf value 0.22 and at 610nm wavelength.

Quality assurance of herbal products is ensured by proper quality control of the herbal ingredients and by means of good manufacturing practice. Pattanaya et al (2010) have developed a simple HPTLC method for the standardization and authentication of a polyherbal formulation namely ‘Sulaharan Yoga’ using HPTLC. A comparative study of the individual ingredients, in-house formulation and marketed formulation (methanolic extracts) were carried out along with the different marker compounds of active principles to ensure their presence in the formulations. The HPTLC fingerprint profile also helps to decide the

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identity, purity and strength of the polyherbal formulation. Several polyherbal formulations containing senna (Cassia angustifolia) leaves are available in the Indian market for the treatment of constipation. The HPTLC quantification method of hydroxy-anthracene glycosides sennosides A and B in the commercial formulation containing senna has been developed by Aktar et al (2008). For this, methanol extract of the formulations was analyzed using silica gel G 60F254

HPTLC plates and spots were visualized under UV and scanned at 350nm in absorption/ reflection mode.

Various products named henna- natural dye (Lawsonia inermis) leaves in the market is often mixed with other plants leaves such as Cassia obovata leaves or Indigofera tinctoria which give a chestnut and a dark colouration respectively. To detect such adulteration and to recognize actual composition of market Heena products, HPTLC chromatograms and fingerprint pattern of market samples were compared with the standard Lawsonia inermis HPTLC chromatographic profile at UV 365nm by Gallo et al (2008). In the study, methanol extracts of Lawsonia inermis leaves gave major characteristic spots at about Rf 0.46, 0.48, 0.59 and 0.62. While methanol extracts of Cassia obovata leaves and Indigofera tinctoria leaves gave major significant spots at about Rf 0.28 and 0.36 and at about Rf 0.77, 0.84 and 0.89 respectively. Thus the fingerprint differences from this comparison help to detect possible adulteration with a foreign substance or substitution of original plants in the formulation. Likewise a simple, accurate and precise quality control HPTLC method for quantification of psoralen from Psoralea corylifolia seed powder, extract and its formulation was developed by Khushboo et al (2009). HPTLC of the samples was done on TLC aluminium plates precoated with silica gel 60F254 and developed in toluene: ethyl acetate (7.5: 2.5, v/v) as the mobile phase. Psoralen was detected at Rf 0.47 and scanned at 299nm.

HPTLC is thus a powerful analytical technique due to its qualities of reliability, simplicity, reproducibility and speed. HPTLC profiling often helps for quantification of active medicinal component from the product. In one study by Bhise and Salunkhe (2009) developed, a sensitive and precise HPTLC method for qualitative analysis of antioxidant gallic acid, present in every crude materials used in the in a nutraceutical health drink (containing ashwagandha, tulsi, mulethi, awala, shatavari, gokharu, arjun, giloy, safed musli, kalimirchi, haldi, jaiphal as an active ingredients and a natural sweetener Stevia rebaudiana). In the study, 50μg/ml gallic acid methanolic solution, was used as a chemical marker compound. Both standard and the sample solution were applied on silica aluminum plate 60F254 and the chromatograms were developed using toluene: ethyl acetate: formic acid: methanol (3: 3: 0.8: 0.2, v/v) and scanned at 254nm. Gallic acid standard was detected at Rf 0.59 and compared with the health drink sample.

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Chapter- 4 Materials and Methods

HPTLC instrumentation:

Quantitative and qualitative analysis was performed with the help of HPTLC instrument.

The HPTLC system (Camag, Muttenz, Switzerland) consists of (1) TLC Scanner connected

to a PC running WinCATS software under MS Windows NT; (2) Linomat V Sample

applicator, (3) Photo documentation system Camag, Reprostar III. The HPTLC analysis

needs sample and solvent preparation.

Step 1: Sample preparation:

All reagents used in this study were of analytical grade. Each extract was redissolved at the

concentration of 50 mg/ml in respective solvent in which they were extracted in narrow

glass vial and used for plate application.

Step 2: Plate activation:

Aluminum sheet back coated with silica gel 60F254 plates were used in the study. The plates

were activated in an oven at 50 °C (10 min) prior to use. This process helps to remove the

moisture and activates the active sites of silica gel for better separation.

Step 3: Sample application:

Camag Linomat V (Camag, Muttenz, Switzerland) was utilized for nitrogen gas-assisted and

controlled application of samples on to TLC plate. The sample extracts were streaked in

form of narrow bands on the precoated silica gel 60F254 aluminum TLC plate, at a constant

application rate of 250 µl/s and gas flow 10 s/µl employed with help of Camag 100 µl

syringe connected to a nitrogen tank; using a Camag Linomat V (Camag, Muttenz,

Switzerland). Samples were applied at the height of 10 mm from the base, having specific

band width and space between two bands.

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Step 4: Plate development and chromatographic conditions:

After sample application, the plates were subjected to linear ascending development, in

selected solvent system, up to a distance of about 70 mm. Twin trough glass chamber (with

10 min prior saturation with the solvent system) was used at room temperature.

Step 5: Scanning of plate:

Subsequent to the development, the TLC plates were dried in a current of air. Densitometric

scanning was carried out using Camag TLC Scanner III (Camag, Muttenz, Switzerland) in

the absorbance mode at 200- 450 nm wavelength with a scanning speed of 20 mm/s, data

resolution 100 µm/step and a specific slit dimension. The source of radiation utilized was

deuterium and tungsten lamp. All remaining measurement parameters were at default

settings. The chromatograms were integrated and regression analysis and statistical data

were generated using WinCATS evaluation software (Version 1.4.2.8121).

Step 6: Photo documentation of plate:

After scanning, images were taken at three different wavelengths i.e. 254 nm by UV lamp,

366 nm by mercuric lamp, 400- 800 nm with Photo documentation system Camag,

Reprostar III.

Step 7: Post chromatographic derivatization of TLC plate:

Post-chromatographic derivatization of developed TLC plates was also performed wherever

necessary. Various spray reagents were used to mark visible spots visible on the plate. This

step is optional and used for better resolution and spotting of separated compounds on the

plate.

 

 

 

 

 

 

 

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Chapter- 4 Results and Discussion

The plants investigated for the presence of antifungal and antioxidant activity in the present

investigation were also subjected to the HPTLC analysis. TLC is a standard technique that

separates low molecular weight organic natural compounds according to their polarity.

Various phyto-chemicals present in the crude extracts have specific λmax and can be seen in

visible light and/or in the UV range. Spray reagents are used to facilitate the calculation for

Rf value the separated compounds on TLC plate. The reagents react with the components

and spots can be easily seen in the visible light. Khan and Nasreen (2010), used various

derivatization reagents such as dregendroff's reagent, mayer's reagent, wagner's reagent,

iodine vapours, ammonium vapours etc.

In the present study, HPTLC profiles of selected plants were developed. The four

extracts of identical utility and medicinal plants were loaded on the same TLC plate to

obtain a comparative view of separated compounds. Utility and medicinal plants were

derivatized using anisaldehyde- sulphuric acid reagent and aromatic herbs/spices as well as

essential oils with vanillin- sulphuric acid reagents to locate the spots or bands in the visible

light. Various organic solvents used to prepare and test appropriate solvent system for

specific plants were, methanol, chloroform, toluene, ethyl acetate, formic acid, acetic acid

etc. Solvent system/mobile phase used in the study were standardized/ optimized for each

plant extracts and the system exhibiting superior results were subjected for their HPTLC

chemical profiling. The chromatogram represents the profile of plants used in the

experiment. Thus these profiles can be utilized for the future quality control of

formulation/product.

Utility plants: Chemical profiles of eleven utility plants (Table 1) namely Ailanthus excelsa,

Calotropis procera, Cassia tora, Citrus limon, Clerodendrum inerme, Lantana camara,

Ocimum canum, Petunia violacea, Polyalthia longifolia, Pongamia pinnata, Salvadora

persica were developed (Table 13).

Ailanthus excelsa shows maximum number of bands at 200nm for all four extracts

developed using solvent system- chloroform: methanol: toluene (30: 5: 5, v/v). Bands were

not detected for the water extract at 450nm wavelength. While chloroform extract exhibited

highest 7 bands in the visible light (Fig 60).

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A phyto-chemical study demonstrated by Yadav et al (2010) that Ailanthes excelsa,

Calotropis procera, Datura stramonium and Eucalyptus globules leaves have alkaloids,

flavanoids, triterpenoids, tannins and phenols (Yadav et al, 2010). Similarly, Kumar et al

(2010) conducted HPTLC analysis of medicinally important Ailanthus excelsa stem-bark

using chloroform: methanol (9.5: 0.5, v/v) as a mobile phase and detection was done at 254

and 366nm wavelengths.

For water extract of Calotropis procera bands were not detected at 400 and 450nm

wavelengths. Chloroform extract revealed maximum number of quench bands in the solvent

system toluene: ethyl acetate: methanol (45: 3.5: 1.5, v/v) and derivatization (anisaldehyde-

sulphuric acid reagent) (Fig 61).

The Chromatogram of Cassia tora was developed in solvent system toluene: ethyl

acetate: methanol (45: 3.5: 1.5, v/v). Chloroform extract followed by methanol and

petroleum ether showed 10 to 12 bands in all the wavelengths (200- 450nm). For water

extract bands were detected very well at 200nm. The separated compounds were derivatized

using anisaldehyde- sulphuric acid reagent (Fig 62).

In Citrus limon, methanol followed by water, petroleum ether and chloroform

extracts showed good quantity of defined bands detected at selected wavelengths. From

water extract, spots were not detected at 450nm with toluene: ethyl acetate: methanol (50:

45: 5, v/v) as mobile phase. Active principle citral was found at ~ 0.5 Rf (Retention factor)

(Fig 63).

For Clerodendrum inerme, chloroform extract followed by methanol and petroleum

ether showed ten to twelve bands in all the wavelengths (200- 450nm) with solvent system

toluene: ethyl acetate: methanol (35: 12.5: 2.5, v/v). Spray with anisaldehyde- sulphuric acid

reagent gave better visualization of spots on the chromatogram (Fig 64). Kothari et al (2006)

demonstrated, maximum resolution of Clerodendrum inerme compounds on TLC with

toluene: ethyl acetate: methanol at the ratio of 7: 2.5: 0.5, v/v solvent system. The TLC plate

showed twelve quench bands at 254nm, and on derivatization with anisaldehyde- H2SO4

reagent, revealed nineteen bands of varied colours.

The water extract of Lantana camara was poorly separated with solvent system

toluene: ethyl acetate: methanol (45: 3.5: 1.5, v/v). While other extracts demonstrated 9- 13

quench bands at all scanned wavelengths (Fig 65). A TLC of lipid extracts of leaves and

flowers of Lantana was performed to separate the polar and neutral lipids (Ganjewala et al,

2009).

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Table 13. Number of separated bands recorded for utility plants

Utility Plants Track Wavelength (nm)

200 250 300 350 400 450 Ailanthus excelsa

1 6 4 7 1 1 - 2 11 3 5 6 9 4 3 9 5 7 6 6 7 4 3 6 7 4 4 3

Calotropis procera

1 5 1 2 2 - - 2 7 7 6 7 9 8 3 9 7 8 8 9 10 4 6 6 4 7 6 2

Cassia tora

1 11 3 2 1 1 1 2 11 9 12 11 11 11 3 12 11 11 10 10 11 4 8 10 9 9 13 11

Clerodendrum inerme

1 7 4 5 1 1 - 2 16 11 10 10 10 7 3 10 11 9 7 9 8 4 15 12 10 9 10 11

Lantana camara

1 6 4 4 4 5 3 2 11 11 10 13 12 12 3 13 12 10 12 12 11 4 12 12 9 9 11 11

Ocimum canum

1 4 3 1 - - - 2 7 9 7 6 6 5 3 8 8 7 8 7 6 4 6 8 7 7 6 5

Petunia violacea

1 8 3 6 3 2 2 2 9 8 11 9 12 10 3 16 13 13 11 12 12 4 12 8 10 9 10 8

Polyalthia longifolia

1 4 2 - - - - 2 9 9 9 8 10 8 3 9 8 9 11 13 9 4 5 5 8 6 8 5

Pongamia pinnata

1 6 8 6 9 5 3 2 10 11 10 11 9 5 3 9 11 11 11 10 8 4 6 8 8 7 8 6

Salvadora persica

1 5 2 1 - - - 2 12 10 11 12 13 13 3 12 10 10 10 11 9 4 7 7 5 6 6 8

[Note: Track 1 =Water extract, Track 2 =Methanol extract, Track 3 =Chloroform extract, Track 4 =Petroleum ether extract]

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The lipids were detected by spraying with a 0.05% (w/v) solution of 8-anilino-4-napthalene-sulphonic acid prepared in methanol and viewed under a UV light. For neutral lipids, solvent systems used were toluene: hexane: formic acid (140: 60: 1, v/v) and hexane: diethyl ether: formic acid (60: 40: 1, v/v). The polar lipids were separated with solvent system containing chloroform: methanol: toluene (65: 30: 10: 6, v/v) ammonium hydroxide (28%) and chloroform: methanol: toluene: acetone: acetic acid: water (70: 30: 10: 5: 4: 1, v/v). Similarly the bioactive, secondary metabolites and essential oils of aerial parts of Lantana camara were extracted using two solvents of different polarities like dichloromethane and methanol. TLC of extracts was performed using silica gel 60F254 and solvent system n-hexane: dichloromethane (1: 1, v/v) and chloroform: methanol: water (1: 1: 1, v/v). The TLC showed number of UV and visible components indicating presence of tannins, triterpenes and cardioactive glycosides (Qaisar et al, 2009).

The water extract of Ocimum canum did not show any bands at 350- 450nm. While other extracts showed 5- 8 bands in the solvent system toluene: ethyl acetate (93: 7, v/v) (Fig 66).

The lowest number of bands were found for water extract of Petunia violacea. While other extracts demonstrated 8- 13 quench bands with toluene: ethyl acetate: methanol (45: 3.5: 1.5, v/v). The compounds were derivatized using anisaldehyde-sulphuric acid reagent (Fig 67).

For Polyalthia longifolia, water extract was found poorly separated in the system toluene: ethyl acetate (93: 7, v/v). Other extracts demonstrated 5- 11 bands with maximum number of bands in the chloroform extract (Fig 68).

All four extracts of Pongamia pinnata demonstrated good separation of components in the solvent system toluene: ethyl acetate: methanol (45: 3.5: 1.5, v/v). Methanol and chloroform extract exhibited 9- 11 bands (Fig 69).

The water extract of Salvadora persica demonstrated 5 bands in the UV range while bands were not detected at 350- 450nm wavelengths. Chloroform and petroleum ether extracts showed 9- 13 quench bands with good separation in toluene: ethyl acetate: formic acid (70: 30: 2, v/v) solvent system (Fig 70). Medicinal plants: High performance thin layer chromatography analysis of medicinal plants is important for quality control and batch-to-batch reproducibility of botanical products (Sharma and Patel, 2009). The phytochemical constituents in a plant material form a characteristic fingerprint, representing quantity of the active constituents. Moreover, this

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helps to standardize the mixture unlike formulated herbal drug and market samples (Paramasivam et al, 2008).

In the present study, chemical profiles of eleven medicinal plants (Table 1) namely

Acacia nilotica, Azadirachta indica, Camellia sinensis, Cassia fistula, Curcuma longa,

Datura stramonium, Lawsonia inermis, Moringa oleifera, Murraya koenigii, Piper betel and

Sphaeranthus indicus were developed (Table 14).

Chromatogram of Acacia nilotica developed in ethyl acetate: toluene: methanol (30:

20: 10, v/v) exhibited maximum number of bands in methanol extract in UV and visible

range. Gallic acid was detected at 0.40 Rf at 200nm wavelength. This component was also

present in the methanol extract (Fig 71). TLC of crude water extract of Acacia nilotica bark

(Singh and Arora, 2009) showed, two spots at Rf 0.48 and 0.64 in solvent system of toluene:

ethyl acetate: formic acid (45: 45: 10, v/v). Among the compounds, the band at Rf 0.48 was

comparable to the standard gallic acid.

Methanol and chloroform extracts of Azadirachta indica were detected very well in

the entire scanned wavelength with maximum number of bands compared to water and

petroleum ether extracts. Among all four extracts, water extracts had shown lowest number

of bands in the solvent system toluene: ethyl acetate: methanol (45: 3.5: 1: 5, v/v) (Fig 72).

According to the studies of Ghimeray et al (2009), had quantified azadirachtin and

nimbin content in Azadirachta indica leaf extracts using HPLC (High pressure liquid

chromatography) by matching their RT (Retention Times) and spectra with the standards

and the quantitative data were calculated on the basis of the peak area of each compound.

They found 17μg/g dw azadirachtin content in water fraction and 112μg/g dw nimbin

content in hexane fraction of leaf. A semi quantitative estimation and identification of active

principles of the Azadirachta indica crude ethanolic leaf extracts was performed by Mondali

et al (2009) using TLC method and hexane: ethyl acetate (1: 1, v/v) as a solvent system. The

plate observed under UV light showed, triterpenoide nimbin at Rf 0.91 compared to the

standard.

Camellia sinensis water extract bands were not detected at 400 and 450nm

wavelengths and showed lowest number of quench bands, followed by methanol extract in

the solvent system toluene: ethyl acetate: methanol (45: 3.5: 6, v/v). Chloroform and

petroleum ether extracts revealed good number of bands, detected at entire scanned

wavelengths. A spray of anisaldehyde- sulphuric acid reagent used after scanning the plate

to visualized the spots in visible light (Fig 73).

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Table 14. Number of separated bands recorded for medicinal plants Medicinal Plants Track Wavelength (nm)

200 250 300 350 400 450

Acacia nilotica

1 2 1 2 2 2 - 2 3 3 1 2 3 2

Gallic acid 2 1 2 1 1 1

Azadirachta indica

1 3 4 4 3 2 3 2 13 13 12 12 12 13 3 14 13 12 11 15 11 4 11 10 10 7 9 8

Camellia sinensis

1 4 4 4 1 - - 2 8 5 7 5 6 3 3 8 11 12 11 13 10 4 11 10 9 10 11 9

Cassia fistula

1 3 5 4 2 1 1 2 4 4 2 2 1 1 3 9 9 9 9 10 8 4 7 5 4 4 4 2

Citrus limon

1 6 4 6 7 3 - 2 9 7 7 7 6 4 3 4 4 4 4 5 3 4 4 5 5 5 5 1

Curcuma longa

1 3 3 5 5 3 3 2 6 6 6 6 7 5 3 5 5 3 6 4 4 4 8 4 2 2 2 1

Datura stramonium

1 4 - 1 - - - 2 7 5 7 7 7 9 3 4 4 5 5 6 5 4 6 5 4 4 5 5

Lawsonia inermis

1 5 3 3 4 4 2 2 7 3 4 4 4 2 3 6 1 1 2 2 3 4 4 1 2 2 1 1

Moringa oleifera

1 7 3 - - - - 2 8 10 10 11 10 8 3 9 8 8 7 9 8 4 7 8 7 6 8 5

Murraya koenigii

1 7 5 5 2 2 2 2 13 16 16 15 10 13 3 12 14 15 13 10 10 4 10 12 12 14 9 11

Piper betel

1 4 3 3 2 3 2 2 2 4 5 3 5 4 3 4 7 7 7 8 8 4 6 1 3 4 5 3

Sphaeranthus indicus

1 8 2 2 2 1 1 2 9 7 7 4 4 6 3 7 3 3 3 3 3 4 5 3 3 3 4 4

[Note: Track 1 =Water extract, Track 2 =Methanol extract, Track 3 =Chloroform extract, Track 4 =Petroleum ether extract]

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Cassia fistula plate developed in toluene: ethyl acetate: methanol: butanol (36: 4: 8:

1, v/v) where chloroform extract revealed maximum numbers of bands, followed by

petroleum ether, methanol and water extracts. 200- 300nm wavelength detected good

amount of bands. Spray treatment of plate with anisaldehyde- sulphuric acid reagent gave

better visualization of spots on the chromatogram (Fig 74).

All four extracts of Curcuma longa demonstrated good separated bands in the

solvent system- chloroform: methanol: acetic acid (94: 5: 1, v/v) (Fig 75). According to

Wagner and Bladt (2007), the solvent system of chloroform: ethanol: glacial acetic acid (95:

5: 1) used for Curcuma rhizome oil showed, 7- 8 blue, red or violet- blue zones in the Rf

range 0.3 to the solvent front with a prominent sesquiterpene (Rf ~ 0.8) and solvent front.

The methanol extract of Curcuma in UV exhibited 5 yellow- white fluorescent zones

(365nm) with curcumin at Rf ~0.6, demethoxycurcumin below Rf 0.5- 0.55 and

bismethoxycurcumin at Rf ~ 0.3 respectively.

Zhao et al (2010) have separated sesquiterpenoids like curzerene, furanodiene, α-

turmerone, β-turmerone and β-sesquiphellandrene from four species of Curcuma by TLC

method in solvent system of petroleum ether: ethyl acetate (90: 10, v/v). A direct HPTLC

assay to determine total curcuminoids content and three individual curcuminoids namely,

curcumin, demethoxycurcumin and bisdemethoxycurcumin was developed earlier

(Sotanaphun et al, 2007; Pozharitskaya et al, 2008 and Paramasivam et al, 2009). A simple,

rapid and high precision HPTLC method for the determination and quantification of the

pharmacologically important active phenolic compound curcuminoids from commercial

turmeric Curcuma longa powder was similarly developed (Paramasivam et al, 2008).

Methanol extract of rhizome powder and standard compounds curcumin,

demethoxycurcumin and bisdemethoxycurcumin of known concentrations were spotted on

glass-backed silica gel 60GF254 HPTLC layers (300μm thick) and developed in mobile phase

of chloroform: methanol (48: 2, v/v). The curcuminoids were visualized at λmax 425nm with

curcumin at Rf 0.67, demethoxycurcumin at Rf 0.47 and bisdemethoxycurcumin at Rf 0.29.

These compounds were quantified by superimposing the UV spectra of samples and

standards within the same Rf window.

In the present study, Datura stramonium water extract was poorly separated in the

solvent system toluene: ethyl acetate (7: 3, v/v) while other extracts had demonstrated

average number of quench bands about 4- 7 in number. Spray with anisaldehyde -sulphuric

acid reagent gave better results (Fig 76). Sharma and Patel (2009) performed a TLC analysis

of Datura stramonium acetone extract using ethyl acetate: methanol: water (81: 11: 8, v/v)

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solvent system and spots were visualized by spraying anisaldehyde- sulfuric acid spray

reagent (465ml of ethanol, 5ml of glacial acetic acid, 13ml of p-anisaldehyde and 13ml of

sulfuric acid mixed in order) and heating the plate at 110°C for 10 min. The leaves of

Datura stramonium were found to contain the flavonoids, chrysin, liquiritigenin, naringenin,

kaempferol, quercetin, withanolide and withastramonolide.

Lawsonia inermis chloroform and petroleum ether extracts had demonstrated low

amount of bands compared to methanol and water extracts in the solvent system toluene:

ethyl acetate: formic acid (27.5: 20: 2.5, v/v). The plate was derivatized using anisaldehyde -

sulphuric acid reagent (Fig 77). Khan and Nasreen (2010) in their study, used eight different

solvent systems for TLC, like, chloroform: methanol (1: 1, v/v), toluene: ethyl acetate (1: 1,

v/v), ethyl acetate: acetic acid: formic acid: water (50: 5.5: 5.5: 13, v/v), ethyl acetate:

methanol: water (40: 5.4: 5, v/v), butanol: ethanol: water (5: 1: 2, v/v), chloroform: mathanol

(95: 5, v/v), n-butanol: acetic acid: water (4: 1: 5, v/v) and chloroform: methanol: water (64:

50: 10, v/v). Where, solvent system toluene: ethyl acetate (1: 1, v/v) showed higher band

separation in all methanol extracts of Datura metal leaves, Datura stramonium leaves and

Lowsonia inermis leaves.

Moringa oleifera water extracts in the present study, were only detected at 200 and

250nm wavelengths during the scan. While other extracts had shown 7- 10 number of

quench bands, detected at all scanned wavelengths from 200- 450nm with toluene: ethyl

acetate: methanol (43: 4: 3, v/v) solvent system. The plate was derivatized using

anisaldehyde- sulphuric acid reagent (Fig 78). HPTLC analysis of Moringa oleifera seed

extracts was performed by Elango and Jadhav, 2010 showed presence of large number of

bioactive compounds. Alkaloids were detected with toluene: ethyl acetate: diethylamine (7:

2: 1, v/v), essential oil with toluene: ethyl acetate (9.3: 0.7, v/v), cardiac glycosides using

ethyl acetate: methanol: water (10: 1.5: 1, v/v) and saponin by chloroform: acetic acid:

methanol: water (6.4: 3.2: 1.2: 0.8, v/v) solvent system from Moringa oleifera seed extract.

Murraya koenigii water extract in the present study, was found poorly separated with

lowest amount of bands in the solvent system toluene: ethyl acetate: methanol (45: 3.5: 1.5,

v/v). Other extracts were having excellent bands about 9- 16 in number. Anisaldehyde-

sulphuric acid reagent treatment of plate had revealed spots in visible light. Pande et al,

2009 in TLC study using solvent system toluene: ethyl acetate (93: 7, v/v) and ethyl acetate:

methanol: water (76.5: 13.5: 10, v/v) revealed that, alkaloid was prominently found in

benzene, chloroform, acetone and methanol extract of Murraya koenigii leaves. While the

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petroleum ether extract was found to contain volatile oil and benzene extract contain fixed

oil.

Piper betel all four extracts in the present study, showed 3- 7 quenching bands on the

chromatogram developed using solvent system toluene: ethyl acetate: methanol (43: 4: 3,

v/v) and were detected at 200- 450nm wavelengths. The plate was latter derivatized using

anisaldehyde- sulphuric acid reagent (Fig 80). Nalina and Rahim, 2007 analyzed the crude

aqueous extract of Piper betel by TLC method using solvent system chloroform: methanol

(90: 10, v/v) and tannic acid as a reference for phenolic compound. The spots were

visualized by UV irradiation (254 and 366nm) and 25% folin ciocalteu phenol reagent spay,

where phenolic compounds were recorded at Rf values between 0.82 to 0.91. According to

Rathee et al (2006) the HPTLC analyses of the ethanol extracts of three Piper betel varieties

(Bangla, Sweet, and Mysore) and found presence of antioxidant phenols like chevibetol and

allylpyrocatechol, separated in ethyl acetate: hexane (2: 8, v/v). While Piper betel glycosides

were separated using toluene: acetone: formic acid (3: 3: 4, v/v) solvent system.

In the present study, Sphaeranthus indicus chromatogram developed using toluene:

ethyl acetate: formic acid (25: 22.5: 2.5, v/v) solvent system. This revealed nice separation

of the compounds of all four extracts. Maximum number of quench bands was detected at

200 nm range. The plate was derivatized using anisaldehyde- sulphuric acid reagent (Fig

81).

Aromatic herbs/spices: In the present study, HPTLC profiles of aromatic herbs/spices

(Table 1) Anethun sowa, Cinnamomum tamala, Cinnamomum zeylanicum, Citrus sinensis,

Coriandrum sativum, Cuminum cyminum, Cymbopogon caesius, Elettaria cardamomum,

Foeniculum vulgare, Illicium verum, Mentha piperita, Myristica fragrans, Ocimum sanctum,

Santalum album and Trachyspermum ammi hexane extracts were developed (Table 15).

In the present study, monoterpenes, sesquiterpenes, phenols etc are the major

constituents of the aromatic herbs/ spices. They are non-polar in nature and thus can be

isolated in non-polar solvents like petroleum ether, toluene, hexane, benzene etc. in the

present study, all aromatic spices were extracted using hexane and chromatographed

(Wagner and Bladt, 2007) in the solvent system toluene: ethyl acetate (93: 7, v/v). The

components separated on the TLC plate were very clearly visualized in the florescent and

UV light. In the visible light they were marked with vanillin- sulphuric acid spray and heat

treatment in an oven (5- 10 min).

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The hexane extracts of aromatic herbs/spices were developed in the solvent system-

toluene: ethyl acetate (93: 7, v/v). After UV scanning, the plates were sprayed with 10%

ethanolic sulphuric acid and latter with 1% ethanolic vanillin reagent for clear band in the

visible light (Wagner and Bladt, 2007; Gende et al, 2008). Maximum bands (10- 12) were

detected with Myristica fragrans and Santalum album (Table 15). Cymbopogon caesius,

Cuminum cyminum, Elettaria cardamomum and Citrus sinensis extracts demonstrated upto 9

bands. Better separation was observed for all plants extracts in the range of 250- 350nm

wavelength.

Table 15. Number of separated bands recorded for aromatic herbs/spices Aromatic herbs/spices Wavelength (nm)

200 250 300 350 400 450 Anethun sowa 5 6 6 7 8 1

Cinnamomum tamala 3 5 7 7 7 7

Cinnamomum zeylanicum 3 4 3 6 5 2

Citrus sinensis 9 9 7 5 3 1

Coriandrum sativum 3 5 7 7 7 5

Cuminum cyminum 6 5 9 8 7 4

Cymbopogon caesius 6 5 9 8 8 9

Elettaria cardamomum 7 8 5 1 - -

Foeniculum vulgare 5 7 6 8 4 2

Illicium verum 7 7 7 2 - -

Mentha piperita 5 6 6 7 8 8

Myristica fragrans 10 12 9 6 8 6

Ocimum sanctum 5 7 6 8 9 7

Santalum album 10 12 9 6 3 1

Trachyspermum ammi 5 2 4 1 2 1

According to Dighe et al (2005), eugenol present in Cinnamomum tamala leaves

used for flavor in the food industry, has a variety of biological activity, and can serve as a

biomarker. TLC plates with extract and standard were developed in toluene: ethyl acetate:

formic acid (90: 10: 1, v/v) mobile phase. Detection and quantification was achieved at

280nm (λmax). The eugenol was resolved at Rf 0.60.

HPTLC finger-print with leaf extract (hexane) of Cinnamomum species was also

developed by Saraswathy et al (2010) using mobile phase of toluene: ethyl acetate (8: 1, v/v)

and for authentication of various species. The chromatogram was scanned under UV at

254nm and followed by a dip in vanillin-sulphuric acid reagent and heating (105°C).

In the present study, Cinnamomum zeylanicum extract was characterized by

cinnamic aldehyde, seen as major grey-blue zone at (Rf ~ 0.5). Eugenol as a brown zone was

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found in the lower Rf range, followed by violet-blue zone (Rf ~ 0.2), blue zone of linalool

(Rf ~ 0.4) and the terpene ester (Rf ~ 0.65). The phenyl propane derivatives as well as

coumarin were seen in UV at 254 nm as prominent quenching zones (Rf 0.45- 0.55) (Fig

83).

The chromatogram of cinnamon oil developed in toluene: ethyl acetate (93: 7, v/v)

solvent system and sprayed with sulphuric- vanillin, followed by heating exhibited

cinnamaldehyde (3-phenyl-2-propenal) at Rf 0.67 and eugenol (2-methoxy-4-propenyl-

phenol) at Rf 0.35 (Gende et al, 2008). El-Baroty et al (2010) characterized essential oil of

Cinnamomum zeylanicum bark by TLC using toluene: ethyl acetate (95: 5, v/v) and DPPH

or β-carotene/linoleic acid spray with, cinnamaldehyde at Rf 0.56, eugenol at 0.43 Rf and

methyl eugenol at 0.37 Rf respectively. Joshi et al (2010) had Cinnamomum zeylanicum bark

powder extracted in 50% ethanol was characterized by HPTLC using toluene: ethyl acetate

(9: 1.5, v/v) as solvent system. The cinnamaldehyde was confirmed found at 0.6 Rf as major

active principle.

A blue zone of linalool was found as the major compound of Coriandrum sativum

extract in the present study. Also geraniol at Rf 0.2 and geranyl acetate at Rf 0.7 were

detected as grey zones (Fig 82). Coriander seeds extracted with chloroform: methanol (2: 1,

v/v) was developed on silica gel 60F254 plates with n-hexane: diethyl ether: acetic acid (60:

40: 1, v/v), air dried and stained by rhodamine in ethanol (0.5 g/l). Under UV, individual

bands were visualized, and identified with the references. The major fatty acid was

petroselinic acid and linoleic acid (Ramadan and Morsel, 2002).

In the present study, Cuminum cyminum extract was characterized by the intense

raspberry-red zone of d-carvone at Rf ~ 0.5, terpene alcohol like carveol in the Rf range 0.2 -

0.25 and dihydrocuminyl alcohol between 0.2- 0.3 Rf. The terpene esters like

dihydrocuminyl acetate found at Rf 0.7. The major compound of Foeniculum vulgare was

found anethol as brown zone and fenchone as orange- red zone after spraying (Fig 82).

The chemical composition of volatile oils of five different plant essential oils of

Ocimum sanctum leaves, Eucalyptus globulus leaves, Mentha arvensis leaves, Citrus lemon

fruit epicarp and Citrus maxima fruit epicarp was determined by HPTLC analysis. HPTLC

analysis was performed using 60F254 precoated silica gel plates and the solvent system

toluene: ethyl acetate (93: 7, v/v). Ocimum sanctum oil is found to consist of eugenol

56.07%, Eucalyptus globulus 1, 8-cineole 17.34%, Mentha arvensis menthol 43.45%, Citrus

lemon limonene 78.28% and Citrus maxima limonene 49.66% as the major chemical

constituents (Pandey et al, 2010).

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In the present study, Elettaria cardamomum extract showed the prominent blue zone

of α -terpinyl acetate (Rf ~ 0.74), cineole (Rf 0.5) and three minor terpene alcohols such as

borneol, terpineol (Rf 0.2- 0.25), linalool (Rf ~ 0.35) and limonene at the solvent front (Fig

83). Major constituent anethol of Illicium verum appear red-violet to brown-violet (Rf 0.9-

0.95) after spraying. Also star anise was found to contain small amount of isomer

methylchavicol. Ocimum sanctum components methylchavicol was found (Rf 0.9- 0.95) as

dark brown zone and safrole as a black zone near the solvent front in the present study (Fig

83).

Mentha piperita leaf extract found to contain, beside L-carvone as red-violet zone

(Rf ~ 0.5), higher amounts of terpene alcohols like dihydrocuminyl alcohol (Rf range 0.2-

0.3) and terpene esters like dihydrocuminyl acetate (Rf 0.7). Menthol was seen at low Rf

range ~ 0.30 as a dark blue zone, piperitone at Rf ~ 0.35 as orange zone, cineole at Rf ~ 0.40

as a blue zone, pulegone at Rf ~ 0.48 as blue zone, menthine /isomenthone at ~ 0.55 as blue-

green zone, methyl acetate were found at high Rf range ~ 0.75 as a blue -purple zone and

menthofuran as a red-violet zone after a spray (Fig 84). According to the Wagner and Bladt

(2007) in Mentha piperita, terpenes such as methyl acetate produces intense, uniform blue-

black coloured zones with PMA reagent (20% ethanolic solution of phosphomolybdic acid).

While in Mentha arvensis the prominent terpenes I-VII were detected with anisaldehyde-

sulphuric acid spray reagent. Menthofuran was detected in freshly distilled peppermint oil.

In the present study, all phenylpropane derivatives of Myristica fragrans were seen

as quenching zones in UV 254nm and as brown to red-brown coloured zones with vanillin -

sulphuric acid reagent in visible light. While the major zone of myristicin (Rf 0.80), smaller

amounts of safrole (Rf 0.95) directly above, traces of eugenol (Rf 0.55) and 2-3 zones of

terpene alcohols (Rf 0.15- 0.25) in Myristica extract (Fig 84). According to Dighe and

Charegaonkar (2009), HPTLC analysis of the Myristica fragrans dried ripe seed methanolic

extract was performed with standards and developed with toluene as mobile phase. The

plates were scanned at 210nm for myristicin and 290nm for safrole. Myristicin was

separated at 0.47 Rf detected at 210nm and safrole at 0.67 Rf detected at 290nm. The λmax for

myristicin was 210nm and for safrole was found 290nm.

The essential oil of Trachyspermum ammi fruits hexane extract contains

predominantly thymol, seen as a characteristic bright red-pink zone at Rf ~ 0.5 (Fig 83).

Kaur and Arora (2009) had identified major phytoconstituents of Foeniculum vulgare and

Trachyspermum ammi aqueous, hexane, ethyl acetate, acetone and ethanol extracts by using

standard markers such as atropine, rutin, catechin, glycyrrhizic acid and lanatoside C were

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co-chromatographed for alkaloids, flavonoids, tannins, saponins and cardiac glycosides

respectively. TLC plates were developed in ethyl acetate: methanol: water (100: 13.5: 10,

v/v) solvent system, where Foeniculum vulgare alkaloids were detected at 0.22 Rf,

flavanoids at 0.25, 0.28, 0.37 Rf, tannins at 0.31 Rf and saponins at 0.32, 0.37, 0.48, 0.56 Rf.

While Trachyspermum ammi alkaloids were detected at 0.77 Rf, flavanoids at 0.15, 0.24,

0.37, 0.45, 0.68, 0.8 Rf, tannins at 0.29 Rf, saponins at 0.37, 0.48, 0.54 Rf and cardiac

glycosides at 0.79 Rf (Kaur and Arora, 2009).

Essential oils: Chemical profiling of four essential oils, Cymbopogon citrates, Eucalyptus

globules, Gaultheria procumbens and Syzygium aromaticum essential oils (Table 1) were

developed using the same solvent system, toluene: ethyl acetate (93: 7, v/v), as used for the

aromatic plants (Table 16). The separated compounds were derivatized using vanillin-

sulphuric acid reagent (pre-spray with 10% ethanolic sulphuric acid and post spray with

10% ethanolic sulphuric acid) (Wagner and Bladt, 2007).

Table 16. Number of separated bands recorded for essential oils

Essential oils Wavelength (nm)

200 250 300 350 400 450 Cymbopogon citrates 2 2 3 5 - -

Syzygium aromaticum 4 6 5 5 4 2

Eucalyptus globules 6 8 5 2 - -

Gaultheria procumbens 4 4 1 - 2 1

In the present study, the essential oils, Syzygium aromaticum and Eucalyptus globules

demonstrated maximum components. The lemongrass oil (Cymbopogon citrates) was

characterized by the presence of citral (Rf ~ 0.5). While Eucalyptus globules oil was

characterized by the major zone of cineole (Rf ~ 0.5), two minor zones of terpene alcohols

(Rf ~ 0.25- 0.35) (Fig 85).

Earlier the lemongrass oil constituents were separated (Paranagama et al, 2003) in

chloroform: toluene (3: 1, v/v). An analysis of clove oil (Park et al, 2007) in the solvent

system hexane: acetone (4: 1, v/v), revealed band of eugenol (Rf 0.64~ 0.42).