Milind Pande, Anupam Pathak
Physico-chemical evaluations of homeopathic mother tincture of Mimosa pudica Linn. (lajvanti)
Homeopathic formulations do not have much more of standardization in our country. Even the pharmacopoeial standards for mother tinctures are far from complete. The objective of this work was to provide some additional parameters to test the mother tincture so as to determine the identity and quality. For this purpose, the mother tincture of Mimosa pudica Linn (Mimosae) roots 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: Mimosa pudica, lajvanti, chuimui, mimosine, HPTLC, tubulin.
Mimosa pudica Family Mimosae known as sensitive plant in English and Lajvanti or Chuimui in local Hindi language. The plant is distributed through out in India in moist locality. A diffuse prickly under shrub, is about 45 – 90 cm in height. Leaves bipinnately compound, pinnate 2-4, delicately arranged with 10 -20 pairs of leaflets, rachis clothed with ascending bristles. Flowers pink, in globose heads, peduncles prickly, usually in auxiliary pairs all along the branches. Fruits bristly pods, flat, straw coloured, consisting of 3-5 one seeded segments. The roots and leaves are commonly used in treatment as bitter, astringent, acrid, cooling vulnerary, alexipharmic, diuretic, antispasmodic, emetic, constipating, and febrifuge.
They are useful in vitiated conditions of pitta, leucoderma, vaginopathy, metropathy, ulcers, dysentery, inflammations, burning sensation, haemorrhoids, jaundice, asthma, fistula, small pox, strangury, spasmodic, affections and fevers.1
The leaves are bitter, sudorific and tonic, and are useful in hydrocele, haemorrhoids, fistula, scrofula, conjunctivitis, cuts, wounds, and hemorrhages. The whole plant is used internally for vesicle calculi and externally for odema, rheumatism, myalgia and tumors of the uterus. 2
Literature survey on Mimosa pudica suggest various therapeutic use of plant reported such as urolithiasis3, ovulation4, vibriocidal5, antidepressant 6, estrogenic and antiestrogenic activities7, antiimplantation and antiestrogenic activity8, effects on oestrous cycle and ovulation 9, hyperglycemic10, anticonvulsant activity11, hyaluronidase and protease activities. 12 The biomolecules isolated from Mimosa pudica were isolation of tubulin13, isolation of C-glycosylflavones 14, phenolic ketone15, a novel buffadienolide 16, analysis of 27 aromatic amino acids 17, and chemical constituents of C-glycosylflavones. 18
The roots of Mimosa pudica Linn. were purchased from local drug supplier and their identity was confirmed by comparing with herbal specimens preserved in Department of Pharmacy, Barkatullah University (BUPH/4041 E). The roots were first dried in shade and then dried in an oven at 40-50oC for five hours.
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 Mimosa pudica 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
All mother tincture was subjected to TLC evaluation Ethyl acetate: Methanol: Water (70:25:5) and detection reagent as Dragendorff,s reagent. The TLC plat was observed for development for orange brown colour spot. The final solvent system was selected by optimization and best results and further subjected to HPTLC screening.
Part II: Quantitative work assay of mother tincture
1) The standard mother tincture (as per Homeopathic Pharmacopoeia) was supplied by Neulife Homeo Pharma, Bhopal
2. Marketed mother tincture
Characterization of sample was done by
1) Physical evaluation19, 20. 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). The all-volumetric measurement used in the study were calibrated and validated21.
2) Chemical evaluation: Samples of the mother tincture were subjected to various chemical tests22, 23 to confirm the nature of phytoconstituents as reported in literature.
Analytical method development
An accurate and sensitive HPTLC method was developed for estimation of mimosine present in mother tincture.
Standard preparation: The 5 mg of mimosine (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.
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 366 nm silica gel 60 (E. Merck).
i) Chemicals: Different developing systems were tried in the preliminary trials. Chemicals used as ethyl acetate, methanol, ethanol, (HPLC Merck grade).
ii) Final experiment with due validation: HPTLCF 366 Silica gel 60 (E Merck), analyzed by HPTLC for its chief phytoconstituents. The accurately weighed mother tincture was dissolved in sufficient solvent (10 mg / 5 ml of ethanol) and used as sample. (Graph I).
The plate was allowed to run in Ethyl acetate: Methanol: Water (70:25:5). The plate was sprayed with Dragendorff,s reagent of alkaloid compound confirmed again with orange brown colour spot. The reference standard and ethanolic mother tinctures were taken in known volume (50µl each spots) and response is measured in terms of peak area.
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 1005 in comparison to other manufacturers (1072, 1092, 1034, 1045, 1078).
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 in chemical tests and thin layer chromatographc evaluation showed presence of flavonoids, phytosterol, alkaloids, amino acids, tannins, bitter glucoside, fatty acids, were prominently observed.
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 Mimosine content in different samples of mother tincture confirms the fact that different varieties of Mimosa could be responsible for variations because they separate out at different Rf values. Mother tincture from manufacturer 2 had lower Mimosine content when compared with the other samples. Thus, the values of Mimosine content in different samples of mother tincture are in no 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. Further structural elucidation was not possible due to puer state compound and paucity of time. This study is suggested as future line up for research in this study.
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