Physical and Chromatographic Evaluations on Homeopathic Mother Tincture of Ocimum gratissimum (Ram Tulsi)
Homeopathic formulations do not have much more of standardization in our country. Even the pharmacopoeial standards for mother tinctures are far from complete evaluation. The objective of this work was to provide some additional parameters to test the mother tincture to determine the identity and quality. For this purpose the mother tincture of Ocimum gratissimum Linn (Labiatae) was selected as a model tincture and investigated for the following parameters. Viz. organoleptic properties, physical properties, and chemical properties, HPTLC studies and quantification of active constituents.
Key words: Ram tulsi, mother tincture, menstrum, Ocimum gratissimum, HPTLC
Ocimum gratissimum (Labiatae) is a shrubby, perennial 0.2-1.2 meter in height, much branched, woody, below stems and branches sub quadrangular. Flowers were in simple or branched rather short racemes, in tolerably close whorls, rachis quadrangular, softly pubescent; bracts sessile, longer than the calyx, softly pubescent. Calyx 3 mm long in flower, becoming twice as long as in fruit, pubescent and glandular; upper lip rounded, veined, scarcely mucronate, curved upwards in fruit, longer than lower; lower lip strongly nerved, the two central teeth short subulate, the lateral teeth shorter and broader, lanceolate. Leaves are 6.3-12.5 cm by 3.8-1.8 cm in size along with elliptic lanceolate shape. Leaf has acute apex, coarsely crenate serrate margin with more or less pubescent. Corolla 4 mm long, pale greenish yellow, pubescent outside; upper lip 3 mm broad with 4 rounded teeth; upper filaments with graded tooth at the base. This plant is distributed throughout India and often cultivated. It is popularly known as Ram tulsi in Hindi. The plant has pungent taste with some characteristic flavour . Eugenol isolated from leaf oil was found as chief chemical constituent along with beta caryophylene, germacrene, alpha copaene, humutene, beta elemene, beta bourbonene, gama murolene, identified as minor component in oil by GC-MS spectroscopy method.  A new sequiterpene gratissimine is also isolated from leaf. 
The plant is reported useful in alexiteric, vomiting, fits, “vata” and “kapha”, skin diseases, erysipelas, inflammations, strangury, causes insomnia according to Ayurvedic system of medicine. The plant has also used as carminative, aphrodisiac, and in diseases of brain, heart, liver and spleen; removes foul breath; strengthens the gums; good for griping and piles. Aromatic bath or fumigations prepared with the plant are advised in the treatment of rheumatism and paralysis. A strong decoction of leaves has been found effective in apathies of children, in cases of seminal weakness and as remedy for gonorrhea. The seeds are given in headache, neuralgia. Considered digestive and pectoral in La Reunion. On the Gold coast, the leaves are mashed and used in enema by newly delivered women. It is also used for young infants. Very popular remedy in Madagascar, it is considered as aromatic, digestive, tonic, antiemetic, antispasmodic, and neuralgic. Antileishmanial activity of Eugenol-rich essential oil is also reported from Ocimum gratissimum.  The leaf is also found useful in various pharmacological activities as antibacterial, antifungal,  hypoglycemic,  increase sexual behavioral activities in mice, antidiarrhoeal , and analgesic.  There is incomplete information available in Homeopathic Pharmacopoeia for all mother tincture and hence more data on standardization and validation of these tinctures is to be generated. 
Materials And Methods
The leaves of Ocimum gratissimum collected in and around Bhopal were identified in Department of Pharmacy, Barkatullah University, Bhopal. A voucher specimen (No BUPH/4041B) was deposited in the department. Simple percolation method was used for manufacturing of mother tincture in laboratory. This process was divided in three stages as
a) Imbibition: The dried plant material was then subjected to size reduction to obtain fine powder (Mesh size 80) using grinding mill. The imhibition of drug powder was carried out for four hours in closed vessel. The moistened drug was packed in percolator and sufficient quantity of menstrum ie. Ethanol. When liquid coming out from the outlet of percolator, the outlet was closed.
b) Maceration: The moistened drug was left in contact with menstrum for 24 hours. During this period, the menstrum was dissolved in the active constituent of drug and became almost saturated with it.
c) Percolation: This stage was marked by the downward displacement of the saturated solution formed in a maceration and extraction of the remaining active constituent present in the drug by slow passage of the menstrum through the column of the drug. After collecting ¾ th volume of the mother tincture, the mark was pressed. The expressed liquid and percolate was mixed together. (Batch A)
d) The alternative way of preparation of mother tincture was maceration method by using wide mouth bottle was kept separately for extraction process with shaking at least once a day for seven days (Batch B). Each time after extraction the mark was pressed for complete recovery of solvent and again washed with next lot of solvent.
The mark was pressed manually after complete extraction. After that complete mother tincture subjected for filtration and volume was adjusted with washings of fresh solvents. These mother tinctures of Ocimum gratissimum subjected for various evaluations.
Along with this mother tincture of other marketed brand were procured from market and used as mfr. 1(Ramakrishna Homeo Pharma, Calcutta), 2 (Father Muller Charitable Institution, Mangalore) and 3(Allen Homeo Pharma, Calcutta).
The experimental works was divided in two parts.
Part I: Qualitative work done on mother tincture
Part II: Quantitative work assay of mother tincture
1. Preparation of in house (standard) mother tincture
2. Marketed mother tincture
Characterization of sample was done by
1) Physical evaluation [12-14]: The parameters carried out on mother tincture samples included determination of viscosity, surface tension, specific conductance, optical activity, pH, alcohol content, specific gravity, refractive index, and total solids (Table I) . All the volumetric used in the study were calibrated and the instrument calibrated/validated.
2) Chemical evaluation: Samples of the mother tincture were subjected to various chemical tests [15-16] to confirm the nature of phytoconstituents as reported in literature. 
Analytical method development
An accurate and sensitive HPTLC method was developed quantitative estimation of Eugenol present in mother tincture.
i) Standard preparation: The leaf oil of tulsi viz Eugenol was first subjected to validation and confirmation of identity through UV, IR, DSC and GC/MS technique. To 5 mg of Eugenol (reference standard) was accurately weighed, dissolved in about 6 ml of ethanol in a 10 ml volumetric flask and the solution adjusted to volume to represent 500 µg/ ml of substance. One ml of this solution was then diluted to 10 ml with ethanol to make a solution of 50 µm/ml. This solution was then used for subsequent steps of analysis.
i) Equipment: Camag HPTLC system equipped with sample applicator Linomet IV, twin trough developing chamber, TLC scanner III and integration software system; Cats 3.0. The plate used was HPTLC 254 silica gel 60 (E. Merck).
ii) Chemicals: Different developing systems were tried in the preliminary trials. Chemicals used as toluene, ethyl acetate, methanol, ethanol, acetone (HPLC Merck grade).
Final experiment with due validation: HPTLCF 254 Silica gel 60 (E Merck), developing solvent being Toluene: Ethyl acetate (93:7) and Vanilin Sulphuric acid (2%) as detecting reagent.
The standard Eugenol was first validated with respect to the parameters given in official standard book. The validation parameters determined included limit of detection (LOD) 250 ng, limit of quantification (LOQ) 400 ng, linearity range 400-1200 ng (response measured in terms of peak area). Its concentration in different mother tincture sample was estimated.
Result and Discussion
From the qualitative work done on the samples of mother tincture, it was concluded that significant differences exist in sample of mother tincture. Considering the physical parameters, it was observed that pH and optical activity of mother tincture samples of manufacturer 2 and 3 differed considerably. In the case of total solids the values obtained for manufacturer 1 (both batches) were to be very variably from the values obtained for manufacturers 2, 3, 4 and standard mother tincture. It was also clearly observed that the values of physical parameter for manufacturer 3 and standard sample were similar. A drop in specific conductance implies a low content of inorganic mater in the sample. The standard sample exhibited specific conductance of 995 in comparison to other manufacturers (1076, 1192, 1232, 1045, 978).
Alcohol content influences the viscosity of the sample. It was observed that samples with higher alcohol content as in the case of standard mother tincture, exhibited high viscosity too. However manufacturer 1, in spite of low alcohol content, exhibited high viscosity, which could be attributed to its high total solid content. Study of inter batch variations with respect to manufacturer 1 was successful. Significant variation in viscosity and refractive index were observed between batches A and B of manufacturer 1. (Table 1) The chemical test also revealed some interesting results. The positive results obtained with various tests revealed that Ocimum gratissimum leaves contain alkaloid, phytosterol, fixed oil, flavonoid and volatile oil. (Table 2) Thus it may be concluded that manufacturer 1 (batch A), 3 and 4 are qualitatively matching and comparable. With reference to Graph I slight variation obtained on quantification of (Graph 1) Eugenol content in different samples of mother tincture confirms the fact that different varieties of Ocimum could be responsible for variations because they separate out at different Rf values. (Image 1) Mother tincture from manufacturer 2 had lower Eugenol content when compared with the other samples. Thus, the values of Eugenol content in different samples of mother tincture are in now way close to value obtained for standard mother tincture.
In conclusion it may be stated that the approach given for standardization of homeopathic mother tinctures including physical and chemical evaluation and comparison with the preparation developed in house as reference standards should be followed by standardization of all mother tinctures. For developing an analytical method pure reference standard of reported active ingredient may have be procured or isolated. Using these reference standards, it will be possible to quantitatively determine the active ingredient in the mother tincture.
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Milind Pande#, Dhansingh Chandel **, Anupam Pathak*
Email : firstname.lastname@example.org
#NRI Institute of Pharmaceutical Sciences, 3 Sajjansingh Nagar, Raisen Road, Bhopal, 462022
** President, Bhopal Homeo Chkitsak Sangh, MIG 107, 2A, Saket Nagar, Bhopal (MP)
*Department of Pharmacy, Barkatullah University, Hoshangabad Road, Bhopal (MP) 462026