Correlation of Pharmacological action of Ignatia Amara Homoeopathic Mother tincture to its active principles employing High Performance Thin Layer Chromatography

 

Parth Aphale¹*, Dharmendra B. Sharma², Sohan S. Chitlange³, Asha Thomas³

¹Professor and HOD, Department of Homoeopathic Pharmacy, Dr. D. Y. Patil Homoeopathic Medical College and Research Centre, Dr. D.Y. Patil Vidyapeeth (Deemed to be University), Pimpri, Pune, Maharashtra India.

²Professor and HOD, Department of Forensic Medicine and Toxicology, Dr D. Y. Patil Homoeopathic Medical College and Research Centre, Dr. D.Y. Patil Vidyapeeth (Deemed to be University), Pimpri,

Pune, Maharashtra India.

³Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Sant Tukaram Nagar, Pimpri,

 

ABSTRACT:

Ignatia contains a considerable amount of strychnine and Brucine, more indeed in proportion than Nux Vomica. It is these alkaloids that it owes much of its energy. In poisonous quantities, Ignatia causes tetanic spasms while in small doses, it cures the same symptoms. Ignatia exalts the impressionability of the incident nerves all over the body. It causes reflex excitation- twitching, constrictions, and spasms. This action is not deep and long lasting. Alternate set of symptoms are numbness, torpor. Ignatia acts specially on the Medulla oblongata and spinal nervous system giving rise to these tetanic convulsions. The entire nervous system gets affected making the system sensitive so much that there is intense acuteness of the nerves of sensation and of special senses and increased susceptibility of external impressions, mental and physical. If we were to correlate this with the pharmacological action of Strychnine and Brucine, all the actions of Ignatia can aptly be assigned to the pharmacological action of these two active principles. This can be verified by High Performance Thin Layer Chromatography which would help to segregate and identify the constituents present in the Ignatia Amra mother tincture and correlate their action with the pharmacological action of Ignatia Amra. Materials and Methods: Standard Ignatia Amara mother tincture was tested for its alkaloid markers and its correlation with the pharmacological action was studied. High Performance Thin Layer Chromatography was used for qualitative analysis if Ignatia Amra homoeopathic mother tincture. TLC Aluminium sheets with silica gel 60 F253 pre-coated layer (20cm x 10cm), thickness-0.2mm, no. of tracks-18, band length-6mm constituted the stationary phase. Chloroform: Methanol (9.5:0.5) constituted the mobile phase. The plate was developed in developing chamber and observed under U.V. Light. Results: Rf. Values of the Ignatia Amara homoeopathic mother tincture were observed at 0.67 and 0.70 which corresponds to the Rf. Value of Brucine which is 0.69. Superimposed UV Spectra of brucine standard shows y-max at 306nm whereas superimposed UV Spectra of strychnine standard shows y-max at 259nm which matches with the y-max shown by Ignatia Amra mother tincture one at 260nm and other at 306nm. Conclusion: Thus, it is evident from the chromatogram that Ignatia Amara homoeopathic Mother tincture contains both Brucine and Strychnine which is supported by evidence in the source books. This helps correlate the action of Ignatia Amara homoeopathic mother tincture with its active principles namely Brucine and Strychnine.

 

INTRODUCTION:

Ignatia is one of the drugs which was fully proved by Dr. Samuel Hahnemann himself. It is indigenous to southern Philippines islands and was naturalised in China. The beans (seeds) are used for preparation of mother tincture. Ignatia Amra contains Strychnine and Brucine as its active principles. It is without doubt that the action of Ignatia Amra homoeopathic mother tincture can be attributed to these two active principles.¹

 

If we were to compare this with the pharmacological action of Ignatia Amara Homoeopathic mother tincture, in large doses, Ignatia produces tetanic spasms and can even cause death by dyspnoea. Ignatia exalts the impressionability of the incident nerves all over the body. It causes reflex excitation- twitching, constrictions, and spasms. This action is not deep and long lasting. Alternate set of symptoms are numbness, torpor. Ignatia acts specially on the Medulla oblongata and spinal nervous system giving rise to these tetanic convulsions. The entire nervous system gets affected making the system sensitive so much that there is intense acuteness of the nerves of sensation and of special senses and increased susceptibility of external impressions, mental and physical^1,2

 

Strychnine is a neurotoxin which majorly affects the spinal cord. It has primary action on the motor nerve fibres which control and co-ordinate the muscular contractions. It causes seizures, cramps, stiffness, hypervigilance, and agitation of all senses.

 

Brucine on the other hand has a wide range of pharmacological activity such as anti-tumour, anti-inflammatory, analgesic. It also has action on cardiovascular and nervous system. It has got main action on the central nervous system, wherein the therapeutic window is quite narrow. Brucine has also got paralytic effect on the nerve endings.³

 

Thus, it would be right to correlate the pharmacological action of Ignatia Amara Homoeopathic mother tincture with its active principles namely brucine and strychnine. And this is possible with the help of High-Performance Thin Layer Chromatography. HPTLC is a quick, sensitive, and reliable technique which helps in qualitative analysis of mixtures of a component based on adsorption. It is less time consuming, more accurate and less expensive procedure which can analyse many mother tinctures at one time.

 

Thus, this study aims to corelate the pharmacological action of Ignatia Amara Homoeopathic mother tincture to the active principles present in it namely strychnine and brucine by employing High Performance Thin Layer Chromatography.

 

AIM:

To correlate the pharmacological action of Ignatia Amara Homoeopathic Mother tincture to the active principles present in it namely strychnine and brucine by employing High Performance Thin Layer Chromatography

 

OBJECTIVES:

1.     To analyse the homoeopathic mother tincture of Ignatia Amara based on the active principles present in it.

2.     To identify the components of Ignatia Amara Homoeopathic Mother tincture based on the Rf values of the standards and their fingerprinting characteristics.

 

MATERIALS AND METHODS^4-13

A. Chemicals and Materials: Authentic parts of plant were used to prepare the mother tincture as specified in HPI and other official pharmacopoeias. Ignatia Amara homoeopathic mother tincture of a renowned homoeopathic pharmaceutical industry was studied.

 

The solvents like 99.9% absolute ethanol, HPLC water, toluene, ethyl acetate, diethyl amine based upon the different mobile and stationary phases specified for mother tinctures were used.⁴

 

B. Instrument:

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.^4-6

 

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-6mm.^7-9

 

D. Mobile Phase:^10-13

Chloroform:Methanol (9.5:0.5)

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

Scanning wavelength:

366nm slit dimension- 6 x 0.45mm, Micro-measurement mode- Absorbance

 

The plate was developed in CAMAG twin horizontal developing chamber (20 x 10cm) saturated with solvent (ethyl acetate: glacial acetic acid: formic acid: water 100:11:11:25 v/v)

 

RESULTS:

 

Figure-1: Chromatogram of Standard

 

Figure-2: Chromatogram of Ignatia Amara homoeopathic Mother tincture under study

 

Table.1- Area Travelled by unknown substance

Peak	Start Position	Start Height	Max Position	Max Height	Max Percent	End Position	End Height	Area	Area Percent	Assigned Substance
1	-0.04Rf	6.5AU	-0.02Rf	100.7AU	8.06%	-0.01Rf	9.4AU	1149.9AU	4.98%	Unknown
2	0.01Rf	19.2AU	0.03Rf	106.6AU	8.53%	0.06Rf	0.4AU	1873.9AU	8.11%	Unknown
3	0.07Rf	0.0AU	0.10Rf	182.1AU	14.57%	0.14Rf	0.3AU	3699.1AU	16.02%	Unknown
4	0.14Rf	0.7AU	0.15Rf	13.8AU	1.11%	0.17Rf	2.7AU	126.3AU	0.55%	Unknown
5	0.18Rf	6.9AU	0.21Rf	16.3AU	1.30%	0.22Rf	14.9AU	394.7AU	1.71%	Unknown
6	0.22Rf	14.7AU	0.26Rf	34.7AU	2.78%	0.28Rf	31.3AU	1068.0AU	4.62%	Unknown
7	0.30Rf	32.4AU	0.34Rf	40.4AU	3.23%	0.38Rf	1.8AU	1580.3AU	6.84%	Unknown
8	0.60Rf	9.6AU	0.64Rf	330.3AU	26.44%	0.65Rf	30.8AU	6179.1AU	26.75%	Unknown
9	0.65Rf	300.9AU	0.66Rf	338.5AU	27.09%	0.68Rf	20.4AU	4818.7AU	20.86%	Unknown
10	0.69Rf	20.8AU	0.70Rf	22.0AU	1.76%	0.71Rf	9.9AU	343.9AU	1.49%	Unknown
11	0.72Rf	10.7AU	0.74Rf	15.0AU	1.20%	0.77Rf	1.3AU	368.5AU	1.60%	Unknown
12	0.78Rf	0.0AU	0.82Rf	17.6AU	1.41%	0.82Rf	15.9AU	363.2AU	1.57%	Unknown
13	0.82Rf	16.0AU	0.87Rf	31.4AU	2.52%	0.90Rf	2.9AU	1130.7AU	4.90%	Unknown

 

Table:2- Area travelled by Iganatia Amara Homoeopathic Mother tincture

Peak	Start Position	Start Height	Max Position	Max Height	Max Percent	End Position	End Height	Area	Area Percent	Assigned Substance
1	-0.04Rf	4.7AU	-0.03Rf	97.5AU	5.21%	-0.01Rf	4.1AU	1100.3AU	2.16%	Unknown
2	0.02Rf	0.9AU	0.04Rf	227.5AU	12.14%	0.06Rf	71.7AU	3512.4AU	6.91%	Unknown
3	0.06Rf	71.7AU	0.10Rf	263.6AU	14.07%	0.15Rf	5.6AU	8574.2AU	16.86%	Unknown
4	0.15Rf	6.1AU	0.20Rf	80.3AU	4.29%	0.24Rf	12.6AU	2057.4AU	4.05%	Unknown
5	0.24Rf	12.6AU	0.26Rf	23.3AU	1.25%	0.28Rf	20.1AU	521.9AU	1.03%	Unknown
6	0.28Rf	20.3AU	0.31Rf	97.1AU	5.18%	0.34Rf	30.7AU	2215.3AU	4.36%	Unknown
7	0.34Rf	31.2AU	0.36Rf	48.1AU	2.57%	0.40Rf	1.7AU	1293.5AU	2.54%	Unknown
8	0.46Rf	0.1AU	0.51Rf	58.1AU	3.10%	0.54Rf	5.1AU	1572.2AU	3.09%	Unknown
9	0.55Rf	4.3AU	0.57Rf	20.7AU	1.11%	0.61Rf	2.0AU	517.4AU	1.02%	Unknown
10	0.66Rf	1.4AU	0.72Rf	734.8AU	39.22%	0.76Rf	35.3AU	24472.0AU	48.12%	Unknown
11	0.76Rf	86.3AU	0.77Rf	115.9AU	6.18%	0.80Rf	75.2AU	2842.5AU	5.59%	Unknown
12	0.80Rf	75.5AU	0.80Rf	80.2AU	4.28%	0.85Rf	8.9AU	1608.2AU	3.16%	Unknown
13	0.85Rf	9.3AU	0.88Rf	26.5AU	1.41%	0.90Rf	0.0AU	565.7AU	1.11%	Unknown

 

Table: 3- Value and Max position of standard of Ignatia Amara

Peak	Start Position	Start Height	Max Position	Max Height	Max Percent	End Position	End Height	Area	Area Percent	Assigned Substance
1	0.67Rf	0.1AU	0.71Rf	686.8AU	100%	0.76Rf	0.3AU	21517.6AU	100%	Unknown

Table.4- Rf Value and Max position of homoeopathic mother tincture of Ignatia Amara under study

Peak	Start Position	Start Height	Max Position	Max Height	Max Percent	End Position	End Height	Area	Area Percent	Assigned Substance
1	0.63Rf	0.2AU	0.66Rf	241.2AU	100%	0.68Rf	0.7AU	3444.3AU	100%	Unknown

 

 

Figure-3: Overtly Spectra Comparison

 

Figure-4: Rf Value and Max position of standard of Ignatia Amara

 

Figure-5: Rf Value and Max position of homoeopathic mother tincture of Ignatia Amara under study

As seen in the chromatograms and evident from the tables, following are the findings

1.     Figure-1, chromatogram of standard, shows the maximum peak at Rf value 0.72 and continues till 0.76 Rf with total area of 48 percent.

2.     Figure-2, chromatogram of Ignatia Amara homoeopathic mother tincture under study shows the maximum peak at 0.73 which continues till 0.78 with total area of 26.04 percent.

3.     Figure-3: Superimposed UV Spectra of brucine standard shows y-max at 306nm whereas superimposed UV Spectra of strychnine standard shows y-max at 259nm which matches with the y-max shown by Ignatia Amara mother tincture one at 260nm and other at 306nm.

4.     Figure-4: The chromatogram of the standard shows maximum position at 0.71 Rf. with total area of 100 percent. It begins at 0.67Rf and ends at 0.76 Rf.

5.     Figure-5: The chromatogram of Ignatia Amara homoeopathic mother tincture shows the maximum area/ peak at Rf value 0.73 with a total area of 100%. It begins at 0.70 Rf and ends at 0.78 Rf.

6.     Hence it is evident from the chromatogram that the peak value in the standard corresponds to that in the mother tincture.

7.     Thus, it is evident from the chromatograms that Ignatia Amara homoeopathic mother tincture contains brucine and some amount of strychnine as its active principles.

 

DISCUSSION:

If we were to correlate the pharmacological action of Ignatia Amara, Ignatia produces a marked hyperæsthesia of all the senses, and a tendency to clonic spasms. Mentally, the emotional element is uppermost, and co-ordination of function is interfered with. Hence, it is one of the chief remedies for hysteria.⁵ It is especially adapted to the nervous temperament-women of sensitive, easily excited nature, dark, mild disposition, quick to perceive, rapid in execution. Rapid change of mental and physical condition, opposite to each other. Ignatia is a great nervous remedy. It acts on the spine as like Nux Vomica affecting both motor and sensory nerves. It is one of the best remedies for convulsions as well as spasmodic affections.¹⁴ This action can be attributed to the pharmacological action of both brucine and strychnine. Ignatia has twitching all over the body documented in the homoeopathic Materia medica and is one of the best leading remedies in chorea which again can be attributed to its active principles namely brucine and strychnine.

FURTHER SCOPE:

More research needs to be undertaken in the direction of studying the action of Ignatia Amara homoeopathic mother tincture in-vitro and in-vivo to correlate and establish direct evidence of the action of Ignatia Amara homoeopathic mother tincture to its active principles.

 

CONCLUSION:

From the chromatograms, it is evident that the Ignatia Amara homoeopathic mother tincture contains Brucine and Strychnine as its active principle. Also, the pharmacological action of Ignatia Amara homoeopathic Mother tincture as recorded in the official homoeopathic Materia Medica can be correlated with the pharmacological action of Brucine and Strychnine present in the mother tincture.

 

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.

 

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Accepted on 01.04.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5383-5387.