Homeopathic medicine is a traditional system of medicine whose primary emphasis is on therapeutics. The word homeopathy is originated from two Greek words (Homoios means similar and Pathos means treatment). This system is a holistic approach. It works on the principle of “Similia Similibus Currentur”, given by German physician Samuel Hahnemann in 1796. This simplifies that substances are capable of causing issues in healthy subjects are utilized as medicines to treat comparable examples of disorder experienced by sick individuals. ^[1] More than 2000 years ago, Hippocrates composed that there were approaches to heal the similar homeopathic medicines (like cures like) but it was formally established by Hahnemann. ^[2]It stimulates auto-regulatory and self-healing processes. Homeopathy chooses substances by matching a patient’s symptoms with symptoms produced by the substances in healthy individuals.^[3]

Hahnemann believed that if a patient had an illness, it can be cured by giving a medicine to a healthy person would produce similar symptoms of that same illness but to a slighter degree. This process is called proving.^[4]Homeopathy is now practiced widely in Germany, Britain, France, India, South Africa and South America, with rising activity in most developed countries. Homeopathy is complementary to other medical disciplines and can be applied as an adjunct to surgery.^[5]Homeopathy has its very own rationality and its therapeutics depends on certain fundamental principles for example, Law of Similia, Law of Simplex, Law of Minimum, Doctrine of Drug Proving, Theory of Chronic Disease, Theory of Vital Force, and Doctrine of Drug-Dynamization. ^[6] The analytical methods are used for the quantification of markers present in plant. The analytical method development is a major challenge for quantitative analysis of marker/ bioactive compounds. Pharmacognostical analysis of medicinal herbs remains challenging issues for analytical chemists, as herbs are a complicated system of mixtures. Analytical separation techniques such as Column Chromatography, High performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS), High Performance Thin Layer Chromatography (HPTLC) etc. among the most popular methods of choice used for quality control of raw material and finished herbal product. ^[7]

1.1. Mother Tincture

Homeopathic mother tinctures are introduced from the whole plants, leaves, fruits, etc. Mother tincture is prepared on the basis of symptom similarity. However, the result of utilization of mother tincture is to control the manifestations or troubling stage. Mother tinctures cannot cure any ailment. It can just offer a palliative treatment. ^{[8], [9]}Homoeopathic remedies are not mere dilutions, succession is required after each step of dilution ^[10] The mother tinctures are clear fluids and its colour ranging from pale straw to dark brown or dark red. The mother tinctures can be governed to a qualitative and quantitative analysis that assesses the identity, potency, purity, and stability of the preparation. ^[11]The mother tincture additionally prepared the potencies by serial dilution. The mother tincture is composed of the lowest possible potency of any particular homeopathic medicaments. It is considered as base of a homeopathic remedy. It is denoted as ‘Q’. Mother tinctures are prepared with alcohol so it is readily absorbed in stomach and intestine.

1.2. Method of Preparation of homeopathic mother tincture

The mother tincture is prepared by maceration or percolation in a way that the alcoholic strength varies widely from 25% to 95% according to the general technique for preparation of mother tincture. It consists of leaving the fresh plants by 8-10 days in contact with total volume of the proper alcohol. For mother tinctures whose monographs determine the specified marking content, a concentration adjustment of this marker may be performed by adding ethanol with the same content as the one used for the preparation of the mother tincture. ^[12]

2. CHALLENGES/ ISSUES FOR THE ESTIMATION OF MARKERS:

There are various challenges for the estimation of markers in plants as follows:

i. In mostly cases the active constituents or markers are unknown.

ii. Selective analytical methods or reference compounds may not be accessible commercially.

iii. Plant materials are chemically and naturally variable.

iv. The source and nature of the plant material are variable.^{[13, 14]}

v. Chemical standardization of marker compounds or active ingredients in herbal preparations.

vi. As sample preparation is the most important step in the development of analytical methods for the analysis of constituents present in herbal preparations.

vii. The strength, weakness and applicability of various separation tools like HPLC, HPTLC etc.

viii. Procedures for the identification of marker or active compounds in plant extracts, using analytical techniques also should be known. ^[7]

3. NEED FOR ANALYTICAL PROCEDURES:

The advancement of new methods to look at the homeopathic plants at the same time and the fundamental need is without interferences in the analytical method. In this manner, it becomes necessary to develop new analytical methods for such homeopathic plants for which there is no analytical method till now available for estimation. ^{[15, 16]}

Newer approaches for utilization of analytical methods in homeopathy, for example,

i. Plants contain a few hundred constituents and some of them are present at very low concentrations. An appropriate standardization and quality control of crude material and the homeopathic preparations should be carried out.

ii. If active principles are present in plants are unknown then identification of marker substances for analytical procedures are required.^[17]

iii. The marker compound is a chemical constituent of botanical drug products or profiles that is used for identification and quality control purposes.

iv. Chemical standardization involves chemical identification by spectroscopic or other chromatographic methods such as such as HPLC, HPTLC, HPLC/MS and HPLC/MS/MS etc. and chemical assay for estimation of markers present in plant. ^[7]

v. Quantitative estimation of chemical markers of each ingredient in the poly herbal preparation required ideal separation technique. For herbal preparations (including polyherbal), there is a need for validation with chemical standardization procedures, biological assays, HPTLC thus offers major advantages over other commonly available conventional chromatographic technique. ^{[18, 19]}

4. ANALYTICAL TECHNIQUES:

Different analytical techniques have been utilized for the estimation of marker present in homeopathic drug, for example, Chromatographic techniques like Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), High Performance Thin Layer Chromatography (HPTLC), Mass Spectrometry (MS), Ultraviolet-Visible (UV) spectrophotometry, Infrared Spectrometry (IR), Nuclear Magnetic Resonance (NMR), Gas Chromatography (GC), and HPLC Coupling with various spectroscopic techniques like LC/MS, GC/MS, LC/MS/MS.

4.1. Column Chromatography

Column Chromatography is an analytical method of separation in which the components to be separated are distributed between two phases, one of which is stationary phase while the other is mobile phase moves in a definite direction. The molecules in the sample will have different affinities and interactions with the stationary support, leading to separation of molecules. Sample components that display stronger interactions with the stationary phase will move more slowly through the column than components with weaker interactions. Different compounds can be separated from each other as they move through the column. ^[20]

4.2. Thin layer Chromatography

Thin layer chromatography is one of the most important analytical technique for qualitative and semi quantitative analysis of plants. ^[21]TLC can give an indication whether the active ingredient is present and its level of content, and, therefore, if the product is qualified or authorized. Some related substances may also be detected and quantified. However, TLC will not detect counterfeits that have wrong active or inactive ingredients if they are not visualized by the detection method being used for the correct active drug. ^{[22, 23]} TLC determination has been developed for the quantitative estimation of the active constituents in the plant and in ayurvedic and homeopathic drugs. ^{[24, 25]} TLC offers many advantages over paper chromatography, which is limited to the use of cellulose as a stationary phase. TLC utilizes a range of sorbent layers that offer superior resolution, speed, and sensitivity. The main advantages of TLC are its low cost and the relative speed of analysis. The materials needed to perform TLC are minimal. They include a development chamber, chromatographic plates, solvents, detection reagents, and reference materials. Also, TLC can be applied to the detection and identification of a wide range of materials, like those found in binding media. Disadvantages of TLC analysis include the need for a larger sample size and its lower sensitivity in comparison with other methods, such as HPLC or GC. ^[26]

4.3. High Performance Thin Layer Chromatography

High performance thin layer chromatography (HPTLC) is a sophisticated instrumental technique of the full abilities of thin layer chromatography. The advantages of automation, scanning, full optimization, selective detection principle, minimum sample preparation, hyphenation, and so forth empower it to be an intense diagnostic instrument for chromatographic information of complex blends of inorganic, organic, and biomolecules. HPTLC is a valuable device for reliable identification because it can provide chromatographic fingerprints that can be pictured and saved as electronic images.^{[27, 28]}The different literature survey has been used for the determination of bioactive markers present in plants by HPTLC methods and for quantification of markers and finger printing of the in-housed mother tincture considered here to be a standard with that of different marketed samples available from manufacturers of homoeopathic medicines in India.^[29-31]Patel VR and Patel RK, 2012 described about the development and validation of methods for quantification of some of the important marker compounds viz. Curcumin and Ellagic acid in Dhatrinisha churna. It deals with the advances in chromatographic techniques made it possible to quantify the chemical constituents in a mixture with comparatively little clean-up using high performance thin layer chromatography. ^[32]HPTLC may be an improvement over conventional TLC for the analysis of crude plant extracts. The HPTLC is becoming a routine analytical techniques due to its advantages of low operatig cost, high throughput and need for minimum sample clean-up. Nile and Park, 2015 studied to develop ad quantification of quercetin, rutin, luteolin and vitexin in Asparagus racemosus, Withania somnifera, Vitex negundo, Plumbago zylenica, Butea monosperma, and Tephrosia purpurea extracts using HPTLC. ^[33]

4.4. High Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is a separation technique that can be used for the analysis of organic molecules and ions. HPLC is based on mechanisms of adsorption, partition and ion exchange, depending on the type of stationary phase used. HPLC involves a solid stationary phase, normally packed inside a stainless-steel column, and a liquid mobile phase. Separation of the components of a solution results from the difference in the relative distribution ratios of the solutes between the two phases. HPLC can be used to assess the purity and/or determine the content of many pharmaceutical substances. It can also be used to determine enantiomeric composition, using suitably modified mobile phases or chiral stationary phases. Individual separation mechanisms of adsorption, partition and ion exchange rarely occur in isolation since several principles act to a certain degree simultaneously.^[34-36] For quality evaluation of plant there is a need to develop a rapid, reliable and reproducible analytical method based on the bio-active marker. HPLC has been used identification and quantification of several herbal formulations. HPLC provides selective, specific, sensitive and quantitative results often with reduced sample preparation and analysis time when compared to other techniques commonly employed. ^[37-45] HPLC is used in phytochemical and analytical chemistry to identify, quantify and purify the individual components of the mixture. Because of its advantages and popularization, HPLC fingerprint analysis has been regarded as the first choice. The fingerprint analysis shows only the result of similarity calculated based on the relative value; retention time, with the selected marker compound as reference standard.

4.5. Ultraviolet Spectrophotometry

The UV/Vis spectrophotometric determination is one of the most widely used methods for quantification of total flavonoids in raw plant materials due to its simplicity, low cost of implementation and wide availability in laboratories for quality control. Marques et al 2013 discussed comparative evaluation of UV/VIS and HPLC analytical methodologies applied for quantification of flavonoids from leaves of Bauhinia forficata. ^[46]Patil and Salunkhe 2012 reported simultaneous estimation of curcumin and quercetin in Ayurvedic medicine by UV Spectrophotometry. ^[47]

4.6. Mass Spectrometry

Mass Spectrometry is a powerful analytical techniques used to evaluate known compounds, to identify unknown compounds within a sample, and to elucidate structure and chemical properties of different molecules. This process involves the conversion of sample compounds into gaseous ions, with or without fragmentation, which are then characterized by mass to charge ratios and relative abundances. A mass spectrometer generates multiple ions from the sample, it then separates into mass to charge ratio (m/z). ^[48]It is routine that mass spectrometer coupled with the chromatographic instrument, such as gas chromatography (GC-MS) or a liquid-chromatography (LC-MS). The mass spectrometer finds widespread use in the analysis of compounds whose mass spectrum is known and in the analysis of completely unknown compounds. In general, the method of ionization is independent of the method of ion separation and vice versa. Some of the ionization methods depend on a specific chromatographic front end (e.g., LC-MS) while others are used for introduction of samples (e.g., FAB and MALDI). ^[49]

4.6.1. Liquid Chromatography Mass Spectrometry (LC-MS)

LC-MS method developed and validated method as per international conference on harmonization guidelines using ultra high performance liquid chromatography with mass spectrometry operated in the multiple reaction monitoring modes for bioactive compounds. ^[50-52], Tine et al., 2017 designed to validate a highly sensitive and selective method for the qualitative and quantitative determination of these components in the fruit essential oils and solvent extracts (fruits, leaves, and barks) of Senegalese Z. zanthoxyloides species by LC/MS/MS techniques. ^[53]

4.6.2. Gas Chromatography Mass spectrometry (GC-MS)

Gas Chromatography has transformed into a main technique for the separation and examination of volatile compounds. It has been utilized to analyze gases, liquids and solids superseded by dissolve in volatile solvents. Both organic and inorganic materials can be examined, and molecular weights can range from 2 to over 1000 Daltons. In Gas chromatography, the gas is used as a moving phase.^[54]Gas chromatography is an analytical technique utilized for the product identification (under exceptionally controlled conditions) and should be particularly coupled to a mass spectrometer. The bombardment of the sample component with high-speed electrons takes place and the examination relies on nature and dispersion of molecular fragments. ^[49] It gives information other than comparative fingerprint (pyrogram) is required, for instance, positive identification of peaks on the chromatogram. The GC-MS analyzed for the presence of biologically active constituents. ^[55-56] Proestos and Komaitis, 2013 described Analysis of Naturally Occurring Phenolic Compounds in Aromatic Plants by RP-HPLC Coupled to Diode Array Detector (DAD) and GC-MS after Silylation. A number of analytical methods have been proposed for the separation and determination of phenolic compounds mainly based on a high performance liquid chromatography (HPLC) technique with UV spectrophotometry because derivatization is not required prior to analysis. However, compared to mass spectrometry (MS), the UV-Vis spectrum does not supply sufficient identifying power. Hence, gas chromatography coupled with mass spectometry (GC-MS) can provide more accurate results. Both methods were used for the analysis of plant extracts. Analysis of the non volatile and thermolabile phenolic compounds by GC-MS presupposes their conversion into volatile and thermotolerant ones by chemical derivatization. ^[57]

4.7. Infrared Spectrometry

Infrared spectrometry is a standout amongst the most fundamental analytical techniques accessible to the present research. The most critical advances in infrared spectrometry are the introduction of Fourier-transform spectrometers. Fourier-transform infrared (FTIR) spectrometry has drastically enhanced the nature of infrared spectra and limited the time required to acquire information. Infrared spectrometry is a method is view on the vibrations of the atoms of a molecule. An infrared spectrum is commonly obtained by passing infrared radiation through a sample and figuring out what division of the occurrence radiation is consumed at a particular energy. The energy at which any peak in an absorption spectrum appears resemble to the frequency of a vibration of a part of a sample molecule.^[58] Infrared radiation refers extensively to the part of the electromagnetic spectrum between the visible and microwave regions. It ranges between 4000 and 400 cm-¹. The near-IR region ranges from 14290- 4000 cm-¹ and far-IR regions range from 700-200 cm^-1.The technique seems to be useful in the detection and identification of compounds/chemical groups present in plants were analysed using FT-IR spectroscopy. ^[59-61]

4.8. Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy has been produced to be the most effective analytical method. NMR permits the visualization of single atoms and molecules in various media in solution likewise in solid state. NMR is a nondestructive technique and gives molar reaction that permits structure elucidation and quantification simultaneously. Chemical shift has become a tool for structure elucidation.^[62] NMR gives information about the quantity of magnetically distinct atoms of the type being studied.^[63] It recognizes the structure of both pure compounds and mixture considering solids or liquids. The procedure includes investigations to conclude the molecular structure from the magnetic properties of the atomic nuclei and the surrounding elements. ^[64] Anick DJ 2004 studied homeopathic remedies made in water via high sensitivity proton nuclear magnetic resonance spectroscopy. ^[65]

5. METHOD OF ANALYSIS OF PLANT MARKER:

There are various methods for analysis of markers present in plant. Some examples are as listed in table-1.

6. DISCUSSION:

In this context, number of methods was developed for the development and determination of active constituents or markers present in different plants and homeopathic mother tincture. There are several methods for the identification of markers present in plant such as UV spectrophotometry, Infrared spectroscopy, High Performance Liquid Chromatography, High Performance Thin Layer Chromatography; Mass Spectroscopy, Nuclear Magnetic Resonance Spectroscopy, etc. It also describes the challenges and issues for the determination of markers present in plant.

7. CONCLUSIONS:

In spite of the fact that the scientific evaluation of the homeopathic preparation is a novel approaches, modern analytical method permit the qualitative and quantitative approach of highly diluted homeopathic products. This review highlights the issues related with estimation of markers present in plants used in homeopathy and various analytical instruments available like Chromatography, Ultraviolet Spectroscopy, High Performance Liquid Chromatography, High Performance Thin Layer Chromatography, Infrared spectroscopy, Nuclear Magnetic Resonance, Mass Spectrometry etc. In this way it can be effectively applied for routine analysis in research and quality control departments. It can likewise be utilized to determine the purity of drugs by identifying the related impurities.

Table-1: Methods for analysis of markers present in plant

S. No.	Plant marker/ constituents	Plant sources	Method of analysis	Homeopathic use	References
1	Quercetin	Tribulus terrestris, Aganosma dichotoma [Roth] K. Schum, Hypericum perforatum, Camelliae sinensis, Elaeocarpus ganitrus Roxb. seeds, Calendula officinalis	HPLC, HPTLC, TLC	Diuretic, mood enhancer, antiseptic, anti-inflammatory agent, antitumour, antifungal, anti-cancer, antispasmodic	[66], [67], [68], [69], [70], [71]
2	Aloe emodin and Aloin A	Aloe plants	Gas chromatography-mass spectrometry	Gastrointestinal disorders, skin burns, insect bites, laxative properties	[72]
3	Strychnine and brucine	Strychnos nux vomica	NMR, HPLC	Treatment of nervous diseases, vomiting, arthritic, traumatic pains	[73]
4	Reserpine and ajmalicine	Rauwolfia serpentina and Rauwolfia tetraphylla	HPTLC	Anticancer, antimalarial, antimicrobial, central nervous system depressant, antihypertensive	[74]
5	Quinine, Quinidine, Cinchonine, Cinchonidine	Cinchona and Remijia species	TLC, HPTLC	Pharmaceutical, as bitter flavourings in the food industry and as chiral reagents in organic synthesis	[75]

8. CONFLICTS OF INTEREST:

All authors in this manuscript did not have any conflicts of interest to declare.

9. ACKNOLEDGMENTS:

The authors are so grateful to the University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G.) for this study.

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Received on 25.05.2019 Modified on 14.06.2019

Asian J. Research Chem. 2019; 12(4):231-237.

DOI: 10.5958/0974-4150.2019.00044.0