Dr. Dharmendra. B. Sharma1, Dr. Parth Aphale2, Dr.Vipul Gandhi3, Prof. Dr. Sohan S. Chitlange4, Dr. (Mrs.) Asha Thomas5
1Principal, Professor & H.O.D., Department of Forensic Medicine & Toxicology, Dr. D.Y. Patil Homoeopathic Medical College & Research Centre, Dr. D.Y. Patil Vidyapeeth , Pimpri, Pune,
2Associate Professor & H.O.D, Department of Homoeopathic Pharmacy, Dr. D.Y. Patil Homoeopathic Medical College & Research Centre, Dr. D.Y. Patil Vidyapeeth, Pimpri, Pune
3Professor & H.O.D, Department of Practice of Medicine, Dr. D.Y. Patil Homoeopathic Medical College & Research Centre, Dr. D.Y. Patil Vidyapeeth, Pimpri, Pune
4Principal, Dr. D.Y. Patil Institute of Pharmaceutical Sciences & Research, Dr. D.Y. Patil Unitech Society, Pimpri, Pune
5Professor & H.O.D, Department of Pharmaceutical Chemistry, Dr. D.Y. Patil Institute of Pharmaceutical Sciences & Research, Dr. D.Y. Patil Unitech Society, Pimpri, Pune
Chromatography is useful in qualitative and quantitative analysis of drugs especially mother tinctures and lower potencies. Thin layer chromatography (TLC) and paper chromatography (PC) are usually employed in the H.P.I. assays and tests. Thin layer chromatography (TLC) is a simple, quick, and inexpensive procedure that gives a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf of a known compound (preferably both run on the same TLC plate). Materials & Methods: 1Standard Calendula Officinalis mother tincture was tested against a pharmaceutical preparation of mother tincture of the same. 2 It was analysed for its components using HPTLC. 3 Stationary phase consisted of TLC Aluminium sheets with silica gel 60 F253 pre-coated layer (20cm x 10cm), thickness-0.2mm, no. of tracks-18, band length-6mm. 4 Mobile Phase consisted of Chloroform:Methanol (9.5:0.5). 5 The plate was developed in developing chamber and observed under U.V. Light. Results: 1 Colours seen on the HPTLC Plates of samples are pink/violet which corresponds to amino acids, green which corresponds to sterols, dark blue which corresponds to saponins, purple which corresponds to triterpenoids and orange which corresponds to alkaloids. 2 The spectra analysis of standard and sample shows equivalent peak/rise and fall in the spectra at exactly equivalent wavelengths. Conclusion: Therapeutic action of Calendula Officinalis as noted in Homoeopathic Materia Medica is because of the active principles like saponins, alkaloids, triterpenoudsaponins, sterols and amino acids present it which is evident from the chromatogram.
Chromatography was invented by Russian Botanist Mikhail Semyonovich Tsvet in 1901. It means “Colour Writing”. This method of standardization of plant drug substances has come a long way since its invention with several advancements being made in the process.1
It is one of the most important method of standardization of Homoeopathic mother tincture based on the principles of analysis, identification, purification and quantification of components of a mother tincture.1
It can be used both for qualitative as well as quantitative analysis of mother tinctures to test their purity and find any adulterants if present.1
This method is based on the very principle of adsorption of components between 2 phases namely mobile phase and the stationary phase.1
As the terms suggest, stationary phase is fixed whereas the mobile phase flows over the stationary phase and separation of components occurs by adsorption.1
Advantages of chromatography:
One of the most important advantage of chromatography technique is that it is sensitive, reliable and separation of components of a complex mixture occurs by using only few micrograms of the sample.2
It is also less time consuming and requires less complex set up as compared to other analytical techniques which makes it the preferred method to separate the components of a complex mixture when standard is known.2
High Performance Thin layer chromatography (HPTLC):
HPTLC is an automated form of chromatography which is more sensitive, less time consuming more accurate form of chromatography. It is possible to analyse more than 2 samples at the same time with the help of High Performance Thin Layer Chromatography (HPTLC) which again makes it the most preferred method to analyse homoeopathic mother tincture for their standardization.2
Qualitative analysis of Mother Tincture of Calendula Officinalis manufactured by a Homoeopathic Pharmaceutical industry with the help of High Performance Thin Layer Chromatography.
1. To analyse Calendula Officinalis Mother Tincture marketed by a homoeopathic pharmaceutical company using its fingerprint characteristics and to further analyse them with specific active principle of the known fraction.
2. To determine the identity of mixture, or components based on known components.
3. To purify i.e. separate the components in order to isolate one of interest for further study.
4. To analyze i.e. examine a mixture, its components and their co-relation to the pharmacological action of the mother tincture under study.
MATERIALS AND METHODS:
A. Chemicals and Materials:
Authentic parts of plant were used to prepare the mother tincture as specified in HPI and other official pharmacopoeias. Calendula Officinalis mother tincture of a pharmaceutical industry was studied.
The solvents like 99.9% absolute ethanol, HPLC water, toluene, ethyl acetate, diethyl amine etc. based upon the different mobile and stationary phases specified for mother tinctures were used
B. Preparation of Standard Mother Tincture: Standard mother tincture was prepared as specified in HPI and other official Homoeopathic Pharmacopoeias and was used in this investigation
Camag HPTLC System, consisting of Linomat V Spotting device and scannerIII with Win cats 4 software from Pharmaceutical Chemistry lab of Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri,Pune-18
TLC Aluminium sheets, silica gel 60 F253 pre-coated layer (20cm x 10cm), thickness-0.2mm, no. of tracks-18, band length-6mm
Development Chamber: Twin through chamber (20 x 10) Distance run:75mm
366 nm slit dimension- 6 x 0.45mm, Micro-measurement mode- Absorbance
The plate was developed in CAMAG twin horizontal developing chamber (20 x 10 cm) saturated with solvent (ethyl acetate: glacial acetic acid: formic acid: water 100:11:11:25 v/v)
Reagents used to spray on the plates for different active principles were 0.2% Ninhydrinin acetone for amino acids, antimony trichloride for triterpenoids, Dragendroff’s reagent for alkaloids, 5% sulphuric acid and 1% vanillin reagent for saponins, alcoholic sulphuric acid reagent for sterols and potassium permanganate solution in alcohol for fatty acids.3
Table.1- Differentiation of Active Principles:3
Violet to pink colour
Dark bluish to black
Dark brown spots
Figure.1 MOBILE PHASE: ETHYL ACETATE: GA: FA WATER (10 1.1 1.1 2.5)
Figure. 2 MOBILE PHASE: ETHYL ACETATE: GA: FA WATER (10 1.1 1.1 2.5)
Figure.3- Spectra Comparison
Figure.4-Overly spectra std+sample
As seen in the graphs above, following are the findings:
1. Colours seen on the HPTLC Plates of samples are pink/violet which corresponds to amino acids, green which corresponds to sterols, dark blue which corresponds to saponins, purple which corresponds to triterpenoids and orange which corresponds to alkaloids.
2. The spectra analysis of standard and sample shows equivalent peak/rise and fall in the spectra at exactly equivalent wavelengths.
3. The wavelengths and Rf.Values of standard match with those of the sample which shows that the sample mother tincture of Calendula Officinalis contains the same active principles as that of the standard Calendula Officinalis mother tincture,
4. Alkaloids are proved to be useful as analgesics, vasoconstriction, anti-tumor properties, muscle relaxant, stimulant, vasodilation etc.
5. Saponins boost the immune system, have an antioxidant effect, and also promote growth of granulation tissue. Saponins are also expectorant, anti-inflammatory, hepaprotective, combats cholesterol build up and antifungal.
6. Triterpenoidsaponins have the following medicinal properties- Wound healing, anti-scarring. They can cause the destruction of red blood cells if injected causing anaemia.
7. All the above medicinal effects of the above active principles are observed in the Homoeopathic Materiamedica of Calendula Officinalis.
8. As quoted in Homoeopathic MateriaMedica by Dr.Wiiliam Boricke-
Calendula Officinalis has wide healing properties when used in external applications. It is used for healing of all types of wounds especially open wounds, non-healing ulcers etc. It promotes faster healing by rapid granulation tissue formation and healing by 1st intention. It acts as a great haemostatic agent. Also lessens the pain in areas where it is out of proportion to the extent of injury. Also used as inter-current in cases of cancer.4
Thus we can conclude that the therapeutic action of Calendula Officinalis as noted in Homoeopathic MateriaMedica is because of the active principles like saponins, alkaloids, triterpenoudsaponins, sterols and amino acids present it.
Further isolation of individual active principles is possible from the data analysed and can be further explored for single phytochemical activity.
Also in-vitro and in-vivo study to support the above findings can be done and will act as a feather in cap to prove that Homoeopathy is a evidence based system of medicine.
Firstly, we would like to thank Hon’ble Cancellor of Dr. D.Y. Patil Vidyapeeth, Pune, Dr. P.D. Patil Sir for giving us the opportunity to take up this research project. We would also like to thank the Vice-Chancellor of Dr. D.Y. Patil Vidyapeeth, Pune, Prof. Dr. N.J. Pawar Sir for his constant support & guidance. We would also like to thank the registrar of Dr. D.Y. Patil Vidyapeeth, Dr. A.N. Suryakar Sir for his support and guidance throughout this research project.
We would also like to thank the Ethics committee of Dr. D.Y. Patil Vidyapeeth, Pimpri, Pune for approving this research project.
Last but not the least, we would also like to thank all the technical staff of pharmaceutical chemistry laboratory of Dr. D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune for their valuable time and help.
CONFLICT OF INTEREST:
There is no conflict of interest amongst the authors
1. Mandal P. & Mandal B., A Textbook of Homoeopathic Pharmacy, Thoroughly revised and enlarged 3rd Edition 2012, Page.95-101
2. Goel S., Art and Science of Homoeopathic Pharmacy, 2nd Enlarged and revised edition, 2007, page.414-417
3. Chakraborthy G.S. et.al, International Journal of Research in Ayurveda and Pharmacy, 2010,1(1): 131-134
4. Boricke W, New Manual of Homoeopathic MateriaMedica& Repertory, Augmented edition based on 9th edition, page 153,154
Accepted on 22.11.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2020; 13(3):1113-1116.