Traditional systems of medicines in India include Ayurveda, Yoga, Naturopathy, Unani, and Siddha. Among them Ayurveda, Siddha, Unani systems of medicines use plants, minerals, and animal products as main drug for treatment of various diseases. Herbs and plants are used in many formulations to cure diseases and maintenance of health. The growing demand for plant based medicine, health products and pharmaceutical etc. depletion of plant resource. Hence immediate focus on conservation and sustainable use of medicinal plants is required.
Ayurveda is a highly progressed and organized system of life and health science based on its own unique original concepts and fundamental principle. In Ayurveda almost all medicinal preparations are derived from plants. Herbs and plants are not only effective or valuable for their active ingredients but also for their minerals, vitamins, volatile oils, glycosides, alkaloids, acids, alcohols, esters etc.1 Ayurveda emphasises selection of genuine and quality drugs for therapeutic uses. The important advantages claimed for therapeutic uses of medicinal plants in various ailments are their safety besides economical, effective and their easy availability, 2 so to ensure quality of medicinal plant products by using modern techniques is become a need.2
In Ayurveda and siddha medicinal systems most of the preparations are based on plants, Bhallataka is one of the most powerful and fast acting Ayurvedic herb.3 Bhallataka is identified botanically as Semecarpus anacardium Linn. comes under the family Anacardiaceae. It is distributed in the sub Himalayan region, tropical and central part of india.1, 3, 5It has been freely used all over India.1 The word of semecarpus is derived from greek word simeion meaning marking or tracing and carpus meaning nut,4 Anacardium means cardium heart shaped marking nut,1, 4 so it is commonly known as ‘marking nut’3, 4,5. It is one of the best versatile and most commonly used herb as household remedy.1 In Ayurveda it is also known as ‘Ardha-Vaidya’3. Bhallataka means sharp like spear. Bhallataka or marking nut is known to world since ancient times4 where fruits, gum, nut, stems, oil is used for their medicinal proprtises3, 4 such as alleviates skin diseases, fever, asthma, also acts as antitumor, ant allergic, antineoplastic, cardiac depressant, antioxidant, analgesic, anticancer. Maharashi Charaka has categorised Bhallataka as an appetizer, accumulation breaking herb, anti-diuretic and anti dermatosis, it is drug of choice in the treatment of piles of vata and kapha types.1 It is classified in Ayurveda under the category of toxic plants, 4 it contain chemical constituents such as phenols minerals vitamins, flavonoids, fixed oils, bilwanoals, semecarpol, semecarpetine, urushenol, anacardol, oleic acid, linoleic acid stearic acid. The fruits contain tarry oil which causes contact dermatitis.6 They are always associated with several side effects, if used unpurified.
It is stated that, Bhallataka is one of the Upavisha dravya (Semi poisionous drug).6 Upavisha are the group of drugs which were less toxic in nature and not so lethal but produce certain toxic symptoms on consumption or administration. The symptoms produced in the body due to Upavisha are less toxic, less severe, usually not life threatening and their toxicity can be controlled by therapeutic measures.7
Bhallataka mentioned under the list of Poisonous substances under the Ayurvedic includuing Siddha, Unani Systems of Medicines, Ayurveda advocates Ballataka after shodhana. Bhallataka must be purified before administrating to patients.
There are different purification methods (Shodhana) mentioned in Ayurveda, The Shodhana process described in various Ayurvedic Classics is not simply a process of separation or detoxification rather it increases the therapeutic efficacy of the drug and also the changes takes place in the purified drugs, which may be beneficial for therapeutic purposes,7 Shodhana is a process of separation by which physical and chemical impurities get separated from the substances by treatment with various drugs.7
In Ayurveda, a series of pharmaceutical procedures which converts a poisonous drug into a therapeutically very effective medicine for various ailments is termed as Shodhana. It is a process by which blemishes are separated from the substance by means of pharmaceutical processing like Swedana (Boiling under liquid bath), Bharjana (Frying), Nimajjana (Dipping), Prakshalana (Washing), Prithakikarana (Separation), Atapa Soshana (Drying), Mardana (Trituration), Bhavana (Levigation), Abhishek (Sprinkling), Nirvapa (Heating and Quenching), Dhalana (Melting and Quenching), Galana (Melting and Straining), Achushana (Absorption), Nirjalikarana (Evaporation of water), Patana (Sublimation), Parishravana (Straining), Vilayana (Elutriation).7
Shodhana of drugs are usually performed with a view to accomplish the objective such as,7
‘ To reduce toxicity of the material.
‘ To remove physical and chemical impurities.
‘ To Transfer hard and non-homogeneous material to soft, brittle, ductile and homogeneous material.
‘ Potentiation of therapeutic efficacy of the drug material.
‘ Induction of desired qualities.
‘ Conversion of the material in suitable form for further processing.
‘ To lead unique and suitable physico-chemical changes.
‘ For the direct therapeutic uses in some cases.7
Various Medias are used for purification process (Shodhana) specific media is used for Shodhana of particular substances. The media used for Shodhana also play a crucial role in either breaking down or transforming the toxic chemical constituents in to their relatively nontoxic derivatives. Sometimes media acts like solvent and separate the substances from insoluble impurities or it removes toxic alkaloids from the drug completely or partially. Following are some of the examples of media from various sources (animal, plant, mineral etc.) frequently used for Shodhana of certain plant drugs.7
‘ Gomutra (cow urine)
‘ Goghrita (cow ghee)
‘ Gomaya (cow dung)
‘ Godugdha (cow milk)
‘ Aja dugdha (goat’s milk)
‘ Kanji (sour gruel)
‘ Eranda taila (castor oil)
‘ Ardraka swarasa (ginger juice)
‘ Narikela udaka (coconut water)
‘ Triphala kwatha (decoction of Triphala)
‘ Panchapallava Kwatha
‘ Apamarga kwatha.
Mineral source 7
‘ Ushnodaka (hot water)
‘ Istika choorna (brick powder)
‘ Churnodaka (lime water)
‘ Multani mitti.
The change that takes place during the Shodhana process can be explored by modern analytical methods. Among the modern Analytical tools HPTLC is a powerful analytical method equally suitable for qualitative and quantitative analytical tasks.9, 11 HPTLC is playing an important role in today analytical world, not in competition to HPLC but as a complementary method. High Performance Thin layer Chromatography (HPTLC) is a sophisticated and automated form of the thin-layer chromatography (TLC) with better and advanced separation efficiency and detection limits.9
Fig No 1- HPTLC Equipment
Advantages of HPTLC include9, 10, 11
‘ In HPTLC, the sample preparation is very simple.10, 11
‘ Lower analysis time and less cost per analysis.10,11
‘ Better accuracy and sensitivity.9, 11
‘ Samples in minute quantities like in nano-gram range.9
‘ Minimum handling and human errors due to automation9
‘ There is very less need of Internal Standard.10
‘ Simple to learn and the instrument is easy to operate.10
‘ It involves very low maintenance cost.10
‘ Solvents used in HPTLC needs no prior treatment like filtration and degassing.10, 11
‘ The annular grade solvents are suitable.10
‘ The mobile phase ingesting for sample is extremely low.10,11
‘ The use of corrosive and UV absorbing mobile phases possible. 10
‘ The system equilibrium time is lesser.10
‘ In HPTLC, the stationary phase is usually not susceptible to sample poisoning.10
Analytical study is one of the advanced fields for investigation in order to establish facts and analyse their significance by which drugs can be understood well and vividly interpreted in the light of technology. HPTLC has been utilized to separate new promising pharmaceutical therapeutants which could be used in pharmaceutical industries
Steps Involved In HPTLC 9, 10
Selection of HPTLC plates10
Hand plates were available which are made up of cellulose and other materials which are not used much now-a-days. Pre coated plates are used which have different support materials such as glass, aluminium sheet (0.1mm) and polyester sheet (0.2mm thick)and sorbent layers with different format and thickness. 20 X 20 cm, 10 X 20 cm, 5 X 10 cm, 5 X 7.5 cm these are different size of plates used for HPTLC. A good cut edge of sheets is important to obtain constant Rf values.
Pre washing of pre coated plates: Pre washing is required because sorbents with large surface area absorb not only water vapours and other impurities from atmosphere but also other volatile substances which often condense particularly after the packing has been opened and exposed to laboratory atmosphere for a long time. Such impurities include elutable components of the binder usually give dirty zones and fail to give reproducible results. So all the times pre-coated plates are always packed with glass or foil side upward. Pre washing of pre coated plates carried out by using Different methods such Ascending method, Dipping method, Continuous methods. Methanol, Chloroform: Methanol, Ammonia solution these solvents are used for pre washing.
Proper sample preparation is an important pre-requisite for success of TLC separation. For normal chromatography non-polar and volatile solvents are used. For reversed chromatography, Polar solvent is used for dissolving the sample. Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones.
Application of Sample10
It is the most critical step for obtaining good resolution for quantification by HPTLC. Some applicators used for spotting such as Capillary tubes, micro bulb pipettes, micro syringes, automatic sample applicator. The major criteria are that they shouldn’t damage the surface. Sample spotting should not be excess or not low. If sample is in small increments, previously applied zones get chromatographed on such subsequent application leading for the formation of horse shoe shaped developed spot. When overloaded it may not be absorbed uniformly throughout the layer, as a result tailing of zones and poor resolution is observed. Problem from overloading can be overcome by applying the sample as band.
Mobile phase which is used should be of high graded. Chemical properties and analytes and sorbent layer factors should be considered while selection of mobile phase.
Chromatographic Development 10
For chromatographic development Different types of chambers are used such as, Twin trough chamber, Rectangular chamber, V-shaped chamber, Sandwich chamber, Horizontal development chamber, Automatic development chamber
Detection of Spots10
One of the characteristic features of HPTLC is the possibility to utilize post-chromatographic off line derivatization.
Detection is of two types:
HPTLC is routinely used for qualitative analysis of raw materials, finished products, plant extracts etc. It involves the identification of unknown sample mixture by comparing the Rf values of the sample components with the standards.
Quantitative of the chromatogram by HPTLC basically involves direct and indirect methods. Indirect method involves removal of analyte from the plate followed by quantitation. Eg; Scrapping and elution
‘ It is an instrumental measurement of visible, UV absorbance, fluorescence quenching directly on the layer without resorting to scrapping and elution.
‘ It involves resolving of a compound on thin layer plate, visualizing the spots and measuring the optical density of each spot / band directly on the plate.
‘ The amount of material / compound in the unknown is measured by comparing them to a standard curve from reference standard chromatographed with the same condition.
‘ Chromatographic zones remit a lower light intensity than the environment around it. Absorption spectra can be directly determined on the plate by comparison with substance free area of sorbent layer.
‘ Measurements are usually made by reflection from the plate using single beam, double beam or single-beam dual wavelength operation of scanning instrument.
‘ The scanner present converts the spot / band on the Layer into chromatogram consisting peaks.
‘ Position of scanned peaks on recorder chart is related to Rf values of the spots on the layer and peak height or area is related to the concentration of the substance on spot.
‘ Signals which are measured represent the adsorption of transmitted or reflected light passes through the spot compared to blank portion of sorbent layer.
Advantages of densitometer/Scanner
1. The purpose of scanner is to convert the spot /band on the layer into densitogram consisting of peaks similar in appearance to HPLC.
2. The position of the scanned peaks on recorder chart is related to Rf values.
3. Peak height/area is related to the concentration of the substance in the spot.
4. Quantitation is faster, reliable, accurate and reproducible.
1 Documentation is important because labelling every single chromatogram can avoid mistake in respect of order of application.
2 Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded.
3 To assist the analysts and researchers E-Merck has introduced HPTLC pre-coated +6plates with an imprinted identification codes.
4 Supplier’s name, item number, batch number, individual plate number is imprinted near upper edge of pre-coated plates. This will not only help in traceability of analytical data, but will also avoid manipulation of data at any stage as coding will automatically get recorded using photo-documentation.
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