Standardization of Homoeopathic Mother Tincture of Strychnous nux vomica with the help of High-Performance Thin Layer Chromatography and correlation of its alkaloid markers with its Toxicological action

 

Dr. Dharmendra B. Sharma¹, Dr. Parth Aphale², Dr. Vineet Sinnarkar³,

Prof. Dr. Sohan S. Chitlange⁴, Dr. (Mrs.) Asha Thomas⁵

¹Principal, Professor and H.O.D., Department of Forensic Medicine and Toxicology, Dr. D.Y. Patil Homoeopathic Medical College and Research Centre, Dr. D.Y. Patil Vidyapeeth, Pimpri, Pune, India.

²Associate Professor and H.O.D, Department of Homoeopathic Pharmacy, Dr. D.Y. Patil Homoeopathic Medical College and Research Centre, Dr. D.Y. Patil Vidyapeeth, Pimpri, Pune, India.

³Associate Professor, Department of Forensic Medicine and Toxicology, Dr. D.Y. Patil Homoeopathic Medical College and Research Centre, Dr. D.Y. Patil Vidyapeeth, Pimpri, Pune, India.

⁴Principal, Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research,

Dr. D.Y. Patil Unitech Society, Pimpri, Pune, India.

⁵Professor and H.O.D, Department of Pharmaceutical Chemistry, Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Dr. D.Y. Patil Unitech Society, Pimpri, Pune, India.

 

ABSTRACT:

Background: Chromatography is one of the important laboratory technique in which the components of a mixture are separated on an adsorbent in order to analyze, identify, purify and quantify a mixture. Thin Layer Chromatography (TLC)is used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound. High Performance Thin Layer Chromatography is a sophisticated and automated form of Thin Layer Chromatography (TLC). The procedure simultaneously processes the sample and standard that results in better analytical precision and accuracy at a faster pace. Pharmacological/ Toxicological action of Nux Vomica is because of its active principles present in the seeds namely strychnine, brucine etc. This research paper aims to corelate the active principles present in Nux Vomica with the toxicological action of the same. Materials and Methods: 1. Standard Nux Vomica mother tincture was tested for its alkaloid markers and its correlation with the toxicological action was studied. 2. Analysis of the mother tincture was done using High Performance Thin Layer Chromatography. 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: Colours seen on the HPTLC Plates of samples are greenwhich corresponds to strychnine, dark blue which corresponds to brucine, orange to alkaloids fluorescent green to sterols and pink to fatty acids which are evident on the chromatogram. Conclusion: Therapeutic action of Nux Vomica as noted in Homoeopathic Materia Medica is because of the active principles like strychnine, brucine, alkaloids, sterols, fatty acids present in it which is evident from the chromatogram.

 

 

 

 

INTRODUCTION:

Chromatography means “Colour Writing”. The principle behind this technique is differential adsorption of various components of the mother tincture between two phases especially the stationary phase and the mobile phase. The active principles present in the homoeopathic mother tinctures get separated by distributing their component molecules between the two phases by adsorption based on the structure, size, shape, and molecular weight.¹

 

Chromatography can be used for both, qualitative as well as quantitative analysis of homoeopathic mother tinctures.

 

Advantages of Thin Layer Chromatography is that it is quick, sensitive, reliable, and simple procedure which does not require a large and expensive set up and decreases the time of analysis of mother tinctures. Another important advantage of Thin Layer Chromatography is that mother tincture analysis can be conducted accurately by using just a few micrograms of the mother tincture.¹

 

High Performance Thin Layer Chromatography (HPTLC) is a more sophisticated technique implemented for qualitative as well as quantitative analysis of mother tinctures with respect to the alkaloids, glycosides etc. present in the respective homoeopathic mother tinctures which form the basis of the pharmacological action of those mother tinctures.

 

HPTLC has been proved to a forerunner in standardization of homoeopathic mother tinctures based on their fingerprint characteristics and corelating their pharmacological action with the active principles present in the mother tinctures.¹

 

Strychnine and brucine present in Nux vomica can easily be segregated with the help of High-Performance Thin Layer Chromatography. Strychnine antagonizes the inhibitory neurotransmitter amino acid glycine at postsynaptic receptors. As a result, the ongoing neural excitability is increased resulting in powerful muscular contractions and reflexes which is the main pharmacological action of Nux Vomica and is also evident in strychnine/ Nux Vomica poisoning. In this manner, the pharmacological/toxicological action of Nux Vomica can be corelated with the action of its active principles by employing the procedure of HPTLC.

 

Aim:

To correlate its alkaloid markers with the toxicological action of Homoeopathic Mother tincture of Strychnous nux vomica with the help of HPTLC.

 

Objectives:

1.     To analyze the homoeopathic mother tincture of Nux Vomica and to study the co-relation of its alkaloid markers to the toxicological action mainly Strychnine.

2.     To analyze the homoeopathic mother tincture of Nux Vomica and to study the co-relation of its alkaloid markers to the pharmacological action mainly Strychnine.

 

MATERIALS AND METHODS²:

A. Chemicals and Materials:

Authentic parts of plant were used to prepare the mother tincture as specified in HPI and other official pharmacopoeias. Nux Vomica mother tincture of a renowned homoeopathic 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.²

 

B. Instrument:

Camag HPTLC System, consisting of Linomat V Spotting device and scanner-III with Win cats 4 software from Pharmaceutical Chemistry lab of Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-18.

 

C. Stationary phase:

TLC Aluminium sheets, silica gel 60 F253 pre-coated layer (20cm x 10cm), thickness - 0.2mm, no. of tracks-18, band length-6mm²

 

D. Mobile Phase:

Chloroform: Methanol (9.5:0.5)

Development Chamber: Twin through chamber (20 x 10) Distance run: 75mm

Scanning wavelength: 366 nm slit dimension- 6 x 0.45mm, Micro-measurement mode- Absorbance 2

 

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)²

 

Reagents used to spray on the plates for different active principles were 0.2% Ninhydrinin acetone for amino acids, antimony trichloride for triterpenoids, Dragendorff’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.²

 

 

Table 1: Differentiation of Active Principles³

Sr. No.	Active Principle	Colour
1	Strychnine	Green
2	Brucine	Dark Blue
3	Alkaloids	Orange
4	Sterols	Flourescent Green
5	Fatty acids	Pink

 

Observations:

 

Figure 1: Chromatogram of Standard

 

 

Figure 2: Chromatogram of Nux Vomica Mother tincture

 

Figure 3: Overtly Spectra Comparison

 

DISCUSSION AND CONCLUSION:

As seen in the graphs above, following are the findings

1.     The peaks observed in the chromatogram of the standard match with the peaks and Rf values of the Nux Vomica Mother tincture under study.

2.     Colours seen on the HPTLC Plates of samples are green which corresponds to strychnine, dark blue which corresponds to brucine, orange to alkaloids fluorescent green to sterols and pink to fatty acids.

3.     The spectra analysis of the sample shows peak/rise and fall in the spectra at equivalent wavelengths.

4.     It has been described in the textbooks of Homoeopathic Materia Medica as well as in the official homoeopathic pharmacopoeias and source books on toxicology that the chief action of Nux Vomica is due to the presence of strychnine and brucine.

5.     Strychnine and brucine are potent stimulators of the spinal cord. Thus, we can see their chief action as making the CNS sensitive to afferent stimuli. This is also evident in the pharmacological action as well as toxicological action of Nux Vomica.⁴

6.     Strychnine and Brucine boost the immune system, have an anti-convulsant effect, anti-acid effect and antioxidant effect, and promote growth of granulation tissue⁴.

7.     Alkaloids are proved to be potent analgesics, vasoconstrictors. They have been proved to possess anti-tumour activity as well as muscle relaxation and vasodilatation activity.

8.     Sterols are effective to lower cholesterol and reduce the risk of heart disease which is also evident in the pharmacological action as well as toxicological action of Nua Vomica.⁴

 

If we were to correlate the pharmacological action of Nux Vomica, Nux Vomica produces an irritable, nervous system, hypersensitive and over-impressionable, which Nux will do much to soothe and calm. Especially adapted to digestive disturbances, portal congestion, and hypochondrical states depending thereon. Convulsions, with consciousness; worse, touch, moving. Zealous fiery temperament. Nux patients are easily chilled, avoid open air, etc. Nux always seems to be out of tune, inharmonious spasmodic action.

 

Thus, it will be very right to say that the pharmacological/toxicological action of nix Vomica is because of the actions of the active principles present in Nua Vomica namely strychnine.⁵

 

FURTHER SCOPE:

Study needs to be carried out to explore the phytochemical activity of a single active principle by its isolation and detailed pharmacological action.

 

Also, in-vitro and in-vivo study of Nux vomica Homoeopathic mother tincture needs to be carried out to establish the pharmacological or toxicological action of Nux Vomica and direct correlation with the action of its active principle namely Strychnine.

 

ACKNOWLEDGEMENT:

Firstly, we would like to thank Hon’ble Chancellor 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 and 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 and Research, Pimpri, Pune for their valuable time and help.

 

CONFLICT OF INTEREST:

There is no conflict of interest amongst the authors.

 

REFERENCES:

1.      Mandal P. and Mandal B., A Textbook of Homoeopathic Pharmacy, thoroughly revised and enlarged 3^rd Edition 2012, Page. 95-103: 346

2.      Dharmendra. B. Sharma et.al. Qualitative Analysis of Calendula officinalis Homeopathic Mother Tincture with the help of High Performance Thin Layer Chromatography. Research J. Pharm. and Tech 2020; 13(3): 1113-1116.

3.      Abid Kamal et.al. Simultaneous HPTLC determination of strychnine and brucine in Strychnous nux vomica seed, J Pharm Bioallied Sci, 2012; 4(2): 134-139

4.      Bardale R, Principles of Forensic Medicine and Toxicology, 1^st Edition 2011, J.P. Publication, Page-535-537

5.      Boricke W, New Manual of Homoeopathic Materia Medica and Repertory, Augmented edition based on 9^th edition, page 463, 464

 

 

Received on 03.08.2020           Modified on 18.09.2020

Accepted on 21.10.2020         © RJPT All right reserved