1. INTRODUCTION:

Homeopathic system of medicine is a medical discipline whose primary prominence is on therapeutics. It is a low-cost system which is employed for non-toxic drugs. The word Homeopathy is derived from the two Greek words Homoios (like) and Pathos (treatment). This system of medicine is a comprehensive approach that takes into consideration the whole person and the relation of lifestyle to disease. Its aim is to bring back the lost equilibrium of the sick individual by stimulating and strengthening the immune response. Homeopathy emerged as an important therapeutic technique during the next half of the nineteenth century and has undergone periods of expansion and then decline. It has been serving the humanity for over two centuries, having passed through sudden change of time and has emerged as a time-tested therapy. In India, the importance of homeopathy has become increasingly essential due to the safety of its medicine and gentleness of its cure. For more than a century and a half, homeopathy is now practiced in India ¹.

2. REGULATION OF HOMEOPATHIC REMEDIES:

Homeopathic remedies have been in use in the United States since 1835, but these remedies were not included in the Food, Drug and Cosmetic act of 1906 ². This was clarified when the act was revised in 1938. At present, more than 2500 substances from plant, animal and mineral sources are used in preparing homeopathic remedies ³. The FDA regulates homeopathic remedies in significantly different ways. Homeopathic remedies delayed to submit new drug application to the FDA and their manufacturing processes are released from good manufacturing practice requirements for pharmaceutical companies ⁴.

3. THUJA

Thuja is commonly known as eastern arbor vitae or white cedar is indigenous to North Europe as an ornamental tree in parks and churchyards ^{5, 6}.

3.1. Biological source

Thuja koraiensis Nakai - Korean thuja - Jilin, Korea

Thuja occidentalis L. - Eastern arborvitae, northern whitecedar - E Canada (Manitoba to Nova Scotia), E United States (primarily Northeast, Great Lakes, Appalachians)

Thuja plicata Donn ex D.Don - Western redcedar - from Alaska to Mendocino Country in California

Thuja standishii (Gordon) Carrière - Japanese thuja - Honshu, Shikoku

Thuja sutchuenensis (Gordon) Carrière - Sichuan thuja - Sichuan, Chongqing China almost extinct in the wild

Formerly placed here

Austrocedrus chilensis (D.Don) Pic.Serm. and Bizzarri (as T. chilensis D.Don)

Callitris rhomboidea R.Br. ex Rich. (as T. australis Poir.)

Cupressus nootkatensis D.Don (as T. excelsa Bong.)

Glyptostrobus pensilis (Staunton ex D.Don) K.Koch (as T. pensilis Staunton ex D.Don)

Libocedrus plumosa (D.Don) Sarg. (as T. doniana Hook.)

Platycladus orientalis (L.) Franco (as T. orientalis L.)

Podocarpus javanicus (Burm.f.) Merr. (as T. javanica Burm.f.)

Tamarix aphylla (L.) H.Karst. (as T. aphylla L.)

Tetraclinis articulata (Vahl) Mast. (as T. articulata Vahl)

Thujopsis dolabrata (Thunb. ex L.f.) Siebold and Zucc. (as T. dolabrata Thunb. ex L.f.)

Widdringtonia nodiflora (L.) Powrie (as T. cupressoides L.) ^7,8,9

The leaves of Thuja occidentalis is shown in the figure 1 ¹⁰.

Figure 1. Image of Thuja occidentalis leaves

3.2. General Information

Scientific name: Thuja occidentalis

Pronunciation: THOO-yuh ock-sih-den-TAY-liss

Common name(s): White cedar, arborvitae, Northern white cedar

Family: Cupressaceae

Uses: Screen; hedge; specimen; reclamation; highway median

Description: Thuja is a slow-growing tree reaches height from 25 to 40 feet and spreads to about 10 to 12 feet wide, preferring a wet or moist, rich soil. Transplanting is moderately superficial if plants are root-pruned and either balled and burlapped or potted. White cedar is tolerant to harsher climate conditions (like high humidity, wet soils and some drought). The foliage turns brownish in winter, especially on cultivars with colored foliage and on exposed sites open to the wind ¹¹. Leaves are usually 1-10 mm long, needle like in first year, and become scale like in later. These leaves are arranged in alternate decussate pairs in four rows laterally the twigs. The flower is monoecious (individual flowers are either male or female, but both sexes can be found on the same plant) and are pollinated by wind. The male and female flowers are usually borne on separate twigs or branchlets. They are tiny, terminal, cone-like bodies. The male cones rounded and reddish or yellowish, the female very small and green or tinged with purple. Mature cones are solitary, egg shaped or oblong, 8 to 16 millimeters (about 1/2 inch) long, with 4 to 6 (but sometimes 3 or as many as 10) pairs of thin, versatile scales that terminate in thickened ridges processes. Seed flattened, ovoid 5-7mm and 3-4 mm wingless. It is extensively cultivated as an ornamental tree in cool and moist places for its attractive dense foliage and bush like habit of growth. It is also grown as a hedge plant in India ¹².

3.3. Active constituents

The plant thuja consists of different chemical constituents like diterpenes (dehydroabietane, neothujic acids III and IV), lignans [(-)- matairesinol, (-)- thujaplicatin methyl ether, (-)- wikstromol, epi-pinoresinol], monoterpenes (R-thujone, α-thujone, fenchone) and a sesquiterpene alcohol [(+)-occidentalol] ¹³, α-Phellandrene, α-Pinene, β-Phellandren, β-Myrcene, D-Limonene, Linalool, 4-Terpineol, Citronellic acid, Carvone, Caryophyllene, γ-Elemene, Caryophylene oxide. Essential oils are natural products, volatile, with terpenic structure and are mostly found in plant leaves and flowers. They are secondary metabolites playing significant functions such as protection of the plants against bacteria, viruses, fungus, insects and herbivores. Due to the wide range of pharmaceutical activities and having the fragrance property, Essential oils have been used extensively in food, drug and perfumery industries ¹⁴.

3.4. Pharmacological Uses

It is used for the treatment of several diseases such as rheumatism, gout, dermatitis, diarrhoea. The plant has been exhibited various biological properties including antiepileptic, antiinflammatory, hair growth promoting, antiviral, antiallergic, antibacterial, antioxidant, molluscicidal, antifungal activities ¹⁴.

3.5. Homeopathic use

It is mainly used in homeopathy as mother tincture or dilution. Homeopathic uses are androgenic, alopecia, hemostatic activity, antimicrobial activity, neurotoxicity, cytotoxicity, antibacterial, antioxidant, hepatoprotective activity. Antipsychotic homeopathic drug used mainly for wart like excrescenses upon mucus and cutaneous surface, vegetative condylomata, and spongy tumours ¹⁵.

4. ANALYTICAL CHARACTERISATION OF HOMEOPATHIC MOTHER TINCTURES:

As described in homeopathic monograph, quality of homeopathic mother tinctures established by the starting material, the manufacturing process and the analytical characteristics. An analytical characterisation of mother tincture comprises of appearance, odour, identity, density and dry residue ¹⁶.

The most widely used methods for quantitative determination of plant marker includes Gas Chromatography (GC), High-performance liquid chromatography (HPLC), Thin layer chromatography (TLC), NMR, GC-MS, LC-MS, LC-MS-MS is used for structure identification.

4.1. Chromatography

Chromatography, although primarily a separation technique, is mostly employed in chemical analysis. It is particularly used for isolation of relatively small amount of materials that have comparatively high intrinsic value. Chromatography is maybe the foremost powerful and versatile technique offered. It can separate a mixture into its individual components and simultaneously provide quantitative estimate of each constituents. Samples may be gaseous, solid, or liquid in nature and can range complexity from a single blend of two enantiomers to a multi component mixture containing widely different chemical species ¹⁷. In this a liquid stream over stationary phase, conveying with its solutes that have varying degree of affinity for the stationary phase. Different flow rate is thus produced for each solute and leading to the separation of the components as they flow out the column. Various methods of chromatography are paper, thin layer, column, gas chromatography etc. The two phases are stationary and mobile phase. Chromatographic methods can be classified according to the nature of the stationary and mobile phase. The difference between adsorption and partition chromatography can be based on the distribution of solutes between the two phases ¹⁸.

4.2. High Performance Liquid Chromatography

High Performance Liquid Chromatography is one of the mostly used analytical techniques. In HPLC, utilizes liquid mobile phase to separate the components of a mixture. The stationary phase can be a liquid or a solid phase. These components are first dissolved in solvent, and then forced to flow through a chromatographic column under a high pressure. The stationary phase is defined as immobile packing material in the column. The components of sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles ¹⁸. HPLC-UV Diode array detection (DAD) and HPLC-MS techniques make use of chromatography as a separation method and DAD or MS as identification and quantification methods. The HPLC equipment consists of a high-pressure solvent delivery system, a sample, auto injector, a separation column, a detector (UV or DAD), a computer to manage the system and display results ¹⁹.

4.3. Mass Spectrometry

Mass Spectrometry (MS) methods are applicable to a wide range of compounds of pharmaceutical interest, sensitivity, selectivity and speed of analysis. It is easier to use and more reliable instruments. Analytical technique by which chemical substances are identified by sorting gaseous ions by mass using electric and magnetic fields. A mass spectrometer uses electrical means to detect the sorted ions, while a mass spectrograph uses photographic or other non-electrical means and that device is known as mass spectroscope. The process is widely used to measure masses and relative abundances of various isotopes, to analyze products of a separation by liquid or gas chromatography and to test vacuum integrity in high-vacuum equipment ¹⁸. A mass spectrometer generates multiple ions from the sample, then it separates them as per their specific mass to charge ratio and then record the relative abundance of each ion type. Mass spectrometry has both qualitative and quantitative uses. MS is now in very common use in analytical laboratories that study physical, chemical or biological properties of a great variety of compounds ²⁰.

4.4. Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) spectrometry is basically another form of absorption spectrometry related to IR or UV spectrometry. NMR spectroscopy has been developed to be the most powerful analytical method. Under appropriate condition in a magnetic field, a sample can absorb electromagnetic radiation in the radio frequency (rf) region at frequencies governed by the characteristics of the sample. Absorption is a component of specific nuclei in the molecule. A plot of the frequencies of absorption peaks versus peak intensities constitutes an NMR spectrum ²¹. NMR is nondestructive and gives molar response that allows structure elucidation and quantification simultaneously. Magnetic interactions between NMR-active nuclei along covalent bindings result in spin-spin couplings. Within the group of spectroscopic methods, the NMR spectroscopy uses the lowest irradiation energy for excitation. Owing to the low-energy level excitation, relaxation and sensitivity of NMR spectroscopy are specifically different from other spectroscopic methods ^22,23. Aside from X-ray crystallography, NMR crystallography is the most important tool for identifying the structure of both pure compounds and mixtures as either solids or liquids ²⁴.

4.5. Gas Chromatography-Mass Spectrometry

Gas Chromatography-Mass Spectrometry (GC-MS) is the most widely used hyphenated technique. It is relevant to the coupling of the GC to the MS. The interfacing of the GC outlet to the MS inlet usually requires some type of selective carrier gas removal. Although direct connection of the GC to the MS is feasible (if large enough vacuum pumps are used), this is rarely done. This is because the vacuum at the outlet of the column can affect the separation efficiency, making most calculations of column retention parameter or efficiency calculations impossible, and the MS system must be shut down for column switching. The large excess of carrier gas is inherently not compatible with the vacuum needed for MS ²⁵.

5. METHODS OF ANALYSIS OF HOMEOPATHIC MEDICATION:

5.1. Physical evaluation

5.1.1. Determination of ash

3g of accurately weighed drug in a tared platinum or silica dish previously ignited and weighed. The ground drug is dispersed on the bottom of the dish in a sharp thin layer. Inflame by gradually increasing the heat is not exceeding until free carbon produced cool and weighed. If the residue obtained not carbon free, then drain the charred mass with hot water, collect the residue on ashless filter paper, inflammable residue and filter paper, add the filtrate, evaporate to dryness and ignite at a low temp. Calculate the % of ash with respect to dry drug ²⁶.

5.1.2. Determination of sulphated ash

2 or 3 g of the drug was accurately weighed, moistened with sulphuric acid, ignited gently, again moistened with sulphuric acid re-ignited, cool and weighed. Calculate the % of the sulphated ash with reference to the air-dried drug ²⁶.

5.1.3. Determination of Water-Soluble Ash

Boil the ash with 25ml of water for 5 minutes. Collect the insoluble matter in a crucible or an ashless filter. Wash with hot water and ignite to constant weight at a low temp. Subtract the weight of insoluble matter from the weight of ash, the difference in the weight shows the water-soluble ash. Calculate the % of water-soluble ash with reference to the air-dried drug ²⁶.

5.1.4. Determination of Moisture content

5.1.4.1. Gravimetric Method:

(a) Loss on drying

2g of sample was accurately weighed. If the particle size is large, it can be reduced by crushing to 2mm. Take a weighing bottle that has been dried for 30 min under the same conditions. Sample was put into the bottle, replace the cover and the bottle was weighed. Distribute the sample evenly to a depth of 5mm by gentle shaking. Place the bottle into the drying chamber, removing the stopper and leaving it also in the chamber and dry the sample at the temperature and time specified in monograph. Upon opening the chamber close the bottle and allow it to the room temperature before weighing. If the substance melts at a lower temp than that specified for the determination of loss on drying, expose the bottle for 1 to 2 hours at temp 5-10°C below the melting temp, then dry at the specified temp ²⁶.

(b) Determination of Alcohol-Soluble Extractive

5g of the drug was coarsely powdered and macerate with 100ml alcohol of the specified strength in a flask for 24hours shaking frequently during 6 hours and allowed to stand for 18 hours. Filter the solution and evaporate it to dryness at 105°C and then weighed ²⁶.

(c) Determination of Water-soluble extractive

This procedure is started with use of chloroform water instead of alcohol as given for the determined of alcohol soluble extracts. Then water was added into the flask. Shake well and allow to stand for 10 mins; cool to 15°C and add 2g Kieselghur filter. Transfer filtrate to a evaporating basin, evaporate the solvent on water bath, continue drying for half an hour, finally dry in a steam oven for 2 hours and weigh the residue ²⁶.

5.1.5. Determination of Saponification value

The saponification value is the number of mg of potassium hydroxide required to neutralize the fatty acids, resulting from the complete hydrolysis of 1g of the oil or fat, when determined by the method. Dissolve potassium hydroxide in water, and add sufficient alcohol to make upto the volume of 1000ml. The substance was allowed it to stand overnight in an oven at 30°C and poured off the clear liquor. 2g of substance was accurately weighed in a flask, 25 ml of alcoholic solution of potassium hydroxide was added, attach a reflux condenser and boil on water-bath for one hour, contents of the flask to cool and add phenolphthalein solution and filtrate the excess of alkali with HCl acid.

(b-a) ×0.02805×1.000

Saponification value= -----------------------------------

Where, W is the weight in g of the substance taken

Note the number of ml required (a) repeat the experiment with the same quantities of the same reagents in the manner omitting the substance, (b) Calculate the saponification value from the above formula mentioned ²⁶.

5.1.6. Determination of Acid value

The acid value is the number of mg potassium hydroxide required to neutralize the free acid in 1 g of the substance, when determined by the method. 10g of substance was accurately weighed and transferred into flask and add 50ml mixture of equal volumes of alcohol and solvent ether and add 1ml of phenolphthalein. Gently heat on a water bath until the substance has completely melted, titrate with 0.1 N Potassium hydroxide and shaking constantly until a pink colour is obtained.

a ×0.00561×1000

Acid value=-------------------------

Where ‘a’ is the number of ml of 0.1 N potassium hydroxide required and ‘w’ is the weight in g of the substance taken

Note the number of ml required. Calculate the acid value from the above-mentioned method ²⁶.

5.1.7. Method for determination of alcohol content in Mother Tincture

The amount of alcohol content in homeopathic mother tincture can be determined by the following method:

The mother tincture was taken into the distillation flask, dilute the solution with water and add few pieces of pumice. Distilled the solution and collect the distillate into the volumetric flask. Adjust the temperature and dilute with water. Determine the specific gravity at 25° with pycnometer. When the distillate contains the volatile substance, it will turbid. If the acids are present in the solution by adding alkaline solution, it will get alkaline and phenolphthalein is used as an indicator ²⁶.

5.2. Preliminary phytochemical analysis

The phytochemical examinations were carried out with the standard procedures for all the extracts. Qualitative phytochemical analysis for the plant extract was carried out for the identification of secondary metabolites. The plant parts were subjected to macerate for 7 days. The extract was filtered and filtrate was collected. The filtrate was subjected to preliminary or qualitative screening for the identification of various active constituents by using standard methods ^{27, 28, 29}.

Several tests were applied for certain compounds. Positive results indicate the presence of the compounds. Positive tests were denoted as (+) and absent was (-). The following active compounds observed in plants are alkaloids, tannins, steroids, flavonoids, phlobatannins, glycosides, saponins, terpenoids, etc.

5.2.1. Detection of Alkaloids

Extracts were dissolved individually in dilute hydrochloric acid and filtered. Each filtrate was tested with the following reagents:

Dragendroff’s test: Few drops of Dragendroff’s reagent (solution of potassium bismuth iodide) were added to each filtrate and observed for the formation of orange yellow precipitate which may indicate the presence of alkaloids.

Mayer’s test: Few drops of Mayer’s reagent (Potassium mercuric iodide solution) were added to each filtrate and observed for the formation of white or cream colour precipitate which may indicate the presence of alkaloids.

Hager’s test: Few drops of Hager’s reagent (saturated aqueous solution of picric acid) were added to each filtrate and observed for the formation of yellow precipitate which may indicate the presence of alkaloids.

Wagner’s test: To the first portion, few drops of Wagner’s reagent (solution of iodine in potassium iodide) were added; occurrence of reddish-brown precipitate was taken as positive ³⁰.

5.2.2. Tests for Flavonoids

Shinoda test: In this process, the concentrated HCl and the pieces of magnesium ribbon were mixed with crude plant extract and after few minutes pink colored scarlet appeared that indicated the presence of flavonoids.

Alkaline reagent test: A solution of 2 ml of 2% NaOH solution was mixed with plant crude extract, intensive yellow color was formed, which turned into colorless when added 2 drops of diluted acid to solution, this result indicated the presence of flavonoids³¹.

Lead acetate Test: The extract was treated with few drops of lead acetate solution; the formation of yellow precipitate reveals the presence of flavonoids ³².

5.2.3. Test for Anthraquinones

Powdered extracts were shaken with 10 mL of benzene. The solution was filtered and 5 mL of 10 % NH₄OH solution was added to the filtrate. A pink, red or violet color in the ammonical (lower) phase indicated the presence of anthraquinones ³⁰.

5.2.4. Test for Glycosides:

5.2.4.1. Test for Cardiac glycosides:

Keller Killani test (Deoxy sugar): Leaf and bark mixture extract were treated with chloroform and evaporate it to dryness. Separately 0.4 ml of glacial acetic acid containing a trace amount of ferric chloride was added and transferred to a small test tube added with carefully 0.5 ml of concentrated Sulphuric acid by the side of the test tube, blue colour appears in the acetic acid layer indicating the presence of glycosides ³³.

5.2.4.2. Anthraquinone glycosides (Borntrager’s test):

The crude extract was mixed with 2 mL of dilute sulphuric acid and 2 mL of 5 % aqueous ferric chloride solution, boiled for 5 minutes which lead to oxidation to anthraquinones, indicating the presence of glycosides ³⁰.

5.2.5. Tests for tannins and phenolic compounds:

Ferric chloride test: Small amount of leaf and bark extract was agitated with water individually and warm. Then about 2 ml of 5% ferric chloride solution was added and observed for the formation of green or blue colour which may indicate the presence of phenols.

Gelatin test: 1% gelatin solution containing 10% sodium chloride was added to each leaf and bark extract, precipitate was formed which shows the presence of tannins and phenolic compounds.

Iodine test: Leaf and bark extract were treated with diluted iodine solution separately. Appearance of transient red colour indicated the presence of tannins and phenolic compounds.

Nitric acid test: Leaf and bark extract were treated with dilute nitric acid separately. Formation of reddish to yellowish colour indicated the presence of tannins and phenolic compounds ³³.

5.2.6. Test for Steroids: (Salkowski test)

The crude plant extract was mixed with chloroform and few drops of conc. H₂SO₄, agitated well and allowed it to stand for some time, formation of red color at the lower layer indicated the presence of Steroids ³⁰.

5.2.7. Tests for Saponins: (Frothing test and Emulsion test)

Small quantity of powdered extract was boiled in 10 mL of distilled water for 5 minute and decanted while still hot. The filtrate was used for the following test.

(a) Frothing test: 1 mL of filtrate was diluted with 4 mL of distilled water and mixture was shaken vigorously and observed for persistent foam which lasted for at least 15 minutes.

(b) Emulsion test: This was performed by adding 2 drops of olive oil to the frothing solution and shaken vigorously. Formation of an emulsion indicated a positive test ³³.

5.2.8. Test for Phlobatannins

Deposition of a red precipitate when an aqueous extract was boiled with 1% aqueous hydrochloric acid indicated the presence of phlobatannins ³⁰.

5.2.9. Test for Terpenoids

5 mL of aqueous extract is mixed with 2 mL of CHCl₃ in a test tube, 3 mL of concentrated H₂SO₄ is carefully added to the mixture to form a layer. An interface was formed with a reddish-brown coloration if presence of terpenoids ³⁰.

5.2.10. Test for Carbohydrate:

A small quantity of the extracts was dissolved separately in 5 ml distilled water and filtered. The filtrates were subjected to the following tests to detect the presence of carbohydrates.

Molisch’s test: The filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube separately and 2 ml of concentrated sulphuric acid was added carefully along the sides of the test tubes. Formation of violet ring at the junction may indicate the presence of carbohydrates ³³.

5.2.11. Test for reducing sugar:

Fehling’s test: The equal volumes of filtrates were treated with 1ml Fehling A and 1ml Fehling B solutions, boiled for one minute separately. The mixtures were boiled for 5-10 minutes on water bath. Reddish brown colour was obtained due to formation of cuprous oxide which indicated the presence of reducing sugar.

Benedict’s test: The filtrates were treated with equal volumes of Benedict’s reagent in test tubes separately. The mixtures were boiled for 5-10 minutes on water bath. Solution appeared green, yellow or red depending on amount of reducing sugar present in each filtrate ³³.

5.2.12. Tests for Amino acid and Protein:

Biuret test: Leaf and bark extracts were treated with 1 ml of 10% sodium hydroxide solution separately and heated and 0.7% copper sulphate solution dropwise added to the above mixtures. The formation of purplish violet colour may indicate the presence of proteins.

Million’s test (for proteins): 3 ml test solutions were mixed with 5 ml of Million’s reagent separately. White precipitate was formed which on heating turned to brick red. It may indicate the presence of amino acids ³³.

5.2.13. Tests for Sterols and Triterpenoids:

Liebermann-Burchard test: Leaf and bark extracts were treated with few drops of acetic anhydride separately. It was boiled and then cooled and concentrated sulphuric acid was added from the side of the test tubes. A brown ring at the junction of two layer and the upper layer turning green which indicated the presence of sterols while formation of deep red colour indicated the presence of triterpenoids.

Salkowski’s test: Leaf and bark extract were treated in chloroform separately with few drops of concentrated sulphuric acid, shaken well and allowed to stand for some time, red colour appeared in the lower layer indicated the presence of sterols while formation of yellow coloured lower layer indicated the presence of triterpenoids ³³.

5.3. Method Development

5.3.1. Analytical Method Development

An analytical procedure is developed to check a specified characteristic of the drug substance or drug product against established acceptance criteria for that characteristic ³⁴.

5.3.2. Method validation

“Validation of an analytical method is the process by which it is established by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application ³⁵”.

The methods were validated according to International Conference on Harmonization (ICH) guidelines ³⁵ for validation of analytical procedures. Validation is essential for any new or amended method to make sure that it's capable of giving reproducible and consistent results, when used by different operators employing the same equipment in the same or different laboratories. The type of validation program required depends completely on the particular method and its proposed applications.

Typical analytical parameters used in assay validation include:

• Accuracy

• Precision

• Specificity

• Detection Limit

• Quantitation Limit

• Linearity

• Range

• Robustness

Accuracy: Accuracy is a measure of closeness between the measured and real value ³⁶.

Precision: Precision of an analytical procedure reveals the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions of repeatability, intermediate precision reproducibility ³⁷.

Specificity: Specificity is the ability to measure the desired analyte in the presence of components which may be expected to be present. Typically, these might include impurities, degradants, matrix, etc. ³⁵.

Detection limit: The detection limit of an analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. It can be determined by visually, signal to noise ratio, standard deviation of the response and the slope ³⁸.

Quantitation limit: The quantitation limit of an analytical procedure is that the lowest quantity of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is employed mainly for the determination of impurities and/or degradation products ³⁵.

Linearity: The linearity of an analytical procedure is its capability to obtain test results which are directly proportional to the concentration of analyte in the sample. Test results should be estimated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares ³⁷.

Range: The range is the interval between the upper and lower concentration of analyte in the sample for which it has been confirmed that the analytical procedure has a suitable level of precision, accuracy and linearity ³⁸.

Robustness: The robustness is an amount of its capacity to remain unaffected by small, but speculate variations in method parameters and provides an indication of its reliability during normal usage ³⁵.

Only specificity is needed for an identification test. However, the full range of specificity, accuracy, linearity, range, limit of detection, limit of quantitation, precision, and robustness testing is needed for more-complex methods such as quantitative impurity methods ³⁸.

6. CONCLUSION:

It is evident from the available literature that leaves and root of Thuja occidentalis are the most widely used parts of the plant. The plant is mainly used as anti-diarrhoeal, antioxidant, anti-ulcer, anti-inflammatory activity. It can also be used to cure CNS, neurological disorders, bronchial catarrh, enuresis, cystitis, psoriasis, uterine carcinomas, amenorrhea and rheumatism. The main constituents were monoterpene ketones, α and β-thujone, fenchone and sabinene as well as the diterpenes, beyerene and rimuene. As the plant validates nearly all the traditional uses, clinical trials and formulation development could be taken as future directions along with the mechanistic approach for these studies.

7. CONFLICT OF INTEREST:

Authors declare no conflict of interest.

8. ACKNOWLEDGMENTS:

Authors are grateful to Director, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur for providing opportunity to do this work.

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Received on 10.04.2019 Modified on 22.06.2019

Research J. Pharm. and Tech 2019; 12(9):4523-4530.

DOI: 10.5958/0974-360X.2019.00779.0