Quality Assessment of Serially Ultradiluted and Agitated Drug Digitalis purpurea by Emission Spectroscopy and Clinical Analysis of Its Effect on the Heart Rate of Indian Bufo melanostictus

The investigation of ultradiluted (homeopathic) drugs is extremely interesting and challenging, and from that point of view this study shows novelty. A study of in vivo changes in heart rate of the Indian Bufo melanostictus caused by commercially available serially ultra-diluted and agitated extract of Digitalis purpurea has been tried in order to understand their pharmacological role. RR interval (of ECG) was compared after intraperitoneal administration of serially diluted and agitated Digitalis purpurea extract, diluent rectified spirit, and Digoxin in anesthetized animals. The study revealed statistically significant changes in the heart rate after application of these drugs except in case of Digoxin and the 200th serial dilution of Digitalis purpurea. The duration of RR intervals after application of the drugs was corroborative of the effect of Digoxin and Digitalis purpurea extract up to 30th dilution. Emission spectra were obtained for the experimental ultra-diluted Digitalis purpurea extract and Digoxin to identify and characterize them. The observed RR pattern and emission spectra show an association. The quality assessment of the commercial ultra-diluted organic drugs obtained from natural products may be initiated by monitoring in vivo studies on animal models.


Introduction
Exploration of in vivo effect of commercially available serially ultradiluted (with recti�ed spirit) and agitated drugs (SAD) is required [1], to understand their pharmacological role. Although "drug proving" studies of the effect of ultradiluted drugs on humans have been done, these studies did not deliver enough supporting laboratory evidence to show its relationship with ultradiluted drug. Myths regarding ultradiluted drugs continue. Myths are that homeopathic drugs available readily across the counter, specially the ultradiluted ones, are placebo and do not cause physiological and toxicological changes. e medical and scienti�c fraternity cast aspersions on the role of homeopathic medicine because of paradigmatic differences [1]. Hence, the assessments of the quality of SAD in terms of their physiological and toxicological effectiveness are aspects that need attention. e investigation of homeopathic drugs is extremely interesting and challenging. is motivated us to take up the study, which was conducted in two parts: �rst, an in vivo observation of the cardiac effect produced by the serially ultradiluted and agitated Digitalis purpurea extract and the purest known comparable drug Digoxin and,second, the �uoroscopic analysis of these drugs by identi�cation of medicinally active ingredients in them. Small animal, the Indian Bufo melanostictus (IBM), was chosen as the subject in which the changes in the heart rate (HR) were recorded through digital ECG signals produced aer application of intraperitoneal injection of the natural cardiotropic SAD Digitalis purpurea, Digoxin, and recti�ed spirit (91.4%). Electrocardiograph signal was 2 Journal of Pharmaceutics acquired by using special electrodes placed on limbs of the IBM. e in vivo study was considered unique for qualitative assessment of the role of level of serial dilution of Digitalis purpurea extract in producing changes in the heart rate (RR). e emission spectroscopy of SAD Digitalis purpurea and Digoxin added analytical dimension to the work. RR represents the ventricular interbeat interval, recordable in vivo [2]. No work showing ECG recording of an IBM is available. is study would be important in drug research of complementary and alternative medicine (CAM), experimental drug, veterinary science, toxicology, and cardiology.
Practitioners of different systems of medicine with paradigmatic differences have used Digitalis purpurea and the like in varying concentration dilution, to treat patients for clinical conditions having manifestation contrary to each other. In modern medicine "Digitalization" is used to reduce the heart rate, while practitioners of homeopathy use it in ultradiluted doses in cases having low heart rate. Details of use of ultra-dilution of Digitalis purpurea extract in clinical conditions with low heart rate are given in Homeopathic Materia Medica Pura written around 1833, by founder of homeopathy Hahnemann and Lectures on Homoeopathic Materia Medica by James Tyler Kent in 1905, e Study of Materia Medica by C.M. Boger in 1908 and many more of such literature. William Withering started using decoction of Digitalis purpurea for "Dropsy" since December 8, 1775 and undertook many such applications thereaer.
Anything more diluted than microvolume (10 6 ), that is, as a concentration of one microliter or microgram of solute in one liter of solvent, is considered as "ultra-dilution. " In SAD, plant extracts of drugs, say Digitalis purpurea, indicated by " " or "Q" are diluted serially to 1: 99 with recti�ed spirit (91.�%) followed by giving ten strokes/jerks (succussion) to the container. is level of diluted attenuation is called 1st potency, denoted by 1 or 1c. In this dilution scale, the �rst potency should contain 1/100th part of the original drug. Next potency 2c will contain 1/100th part of the 1st potency, and so on. Digitalis purpurea "1c" is diluted to the level of 1x ; the dilution "30" contains about 1x 60 of ingredients, and so on. SAD dilutions in other scales have not been used in this work, and for brevity, they are not described here. In biological �uids, it is not yet possible to measure Digitalis purpurea in 6, 30, and 200 or other ultradilutions. Table 1 shows computed level of dilution of SAD drugs.
e objectives of the study were (a) to observe the effect of commercially available SAD (homeopathic drug) Digitalis purpurea (in different dilutions) and Digoxin on the heart rate of small animal Indian Bufo melanostictus, (b) to compare the effects produced by (homeopathic drug) Digitalis purpurea in different dilutions and Digoxin, and (c) to identify and characterize the experimental drugs by �uorescence spectroscopy.
IBM is not an endangered species and serves as food for some tribes in West Bengal, India. A preliminary comparison between HR of IBM, rabbits, and albino rats was made practically to �nd out the suitability of the animal species for the present research. Data related to albino rats and rabbits are not produced in this paper for brevity. In the study, IBM was found to be susceptible to Digitalis purpurea. It has a normal heart rate that may be comparable to humans, does not require sacri�ce for this experimentation, and can be anesthetized easily, and the IBM recovers well aer the experiment. Isolation of IBM heart and additional temperature control for maintenance of heart are not required for recording its ECG in a noninvasive method. ese conditions allow near-natural and comparable parameters with the humans. In order to avoid impact of climate and weather change, a complete set of studies were conducted in one rainy season. During rainy season, IBMs are healthy, active, their metabolic function is at its best, and they have consistent HR.

Methodology
e Institutional Ethical Committee for Research on Humans and Animals (IEC) of Indian Institute of Technology, Kharagpur, India, provided necessary ethical clearance and infrastructural support to conduct the experiment as per the prevalent guidelines for animal experimentation in laboratory. Dedicated electrical earthing was installed in the animal house and in the entire laboratory. A veterinary surgeon was engaged to give guidance on all the aspects of animal facility, care, and for conducting research. SAD drugs (prepared as per Indian Homeopathic pharmacopoeia) and contamination-free double-distilled Recti�ed Spirit (91.�%) were procured from an authorized manufacturer, M/s Hahnemann Publishing Company, Kolkata, India. Digitalis purpurea Q or was of batch no. 6725 of September 2005, Digitalis purpurea 6, Digitalis purpurea 30, and Digitalis purpurea 200, all from the same batch of June 2005. Doubledistilled water was used in the experiment. Merck make Diethyl ether was used for inducing general anesthesia. Inj Digoxin, 25 mg/mL, manufactured by Samarth Pharma was used. Electrocardiogram and tailor-made soware of Recorders and Medicare System, India, was used to record and measure digital cardiac signals. Good conducting electrodes, suitable for the limbs of IBM, were specially designed and manufactured for this experiment. Animals, procured from authorized vendors, were screened for general health conditions and injury marks. Cardiac condition of the IBM was assessed using highly sensitive cardiophonic stethoscope. Simultaneously, neural re�exes were monitored. Stabilized healthy IBMs, of medium size, weighing ∼ 120-200 grams, were randomly selected for each set of experiments. Stabilized IBMs were acclimatized for 2 weeks. IBMs were kept exclusively in very spacious natural surroundings, with natural diet. ey were handled gently to familiarize with human interaction, equipment, and accessories in the animal house and laboratory. Entry of outsiders was prohibited, to restrict disturbance to the animals. High standard of personal hygiene and cleanliness was strictly maintained throughout for conducting the research. Prior to the experiment, the IBMs were given a wash with double-distilled water. Each IBM was given general ether inhalation anesthesia till complete �accidity of the limbs was reached, and stimulus failed to evoke any re�ex. Subsequently, on different sets of ether anesthetized IBM 0.5 mL of intraperitoneal injection of either of the drugs, (only one test drug for one animal), Digoxin, Digitalis purpurea Q or , Digitalis purpurea 6, Digitalis purpurea 30, and Digitalis purpurea 200, and also the diluent Recti�ed spirit (91.4%), were administered. e same experiment was repeated by inhalation of ether for half a minute more aer the complete �accidity of limbs, to monitor the effect of prolonged use of ether. Highly conducting electrode jelly was applied on the limbs (at the site of attachment of electrodes) of the anesthetized IBM. Electrodes, as per the markings, for example, LA for le front limb, RF for right hind leg, and so on, were put on all the limbs for real-time continuous monitoring and recording digital cardiac signals. ECG was obtained for each case as mentioned previously. Normal ECG signals and ECG showing variations were especially frozen and saved. Each screen could hold and display 10 seconds of recorded ECG signals. e bass �lter, frequency for acquiring signal, magni�cations, noise reduction, base line correction, calibration, and optimization were set as per recommendation of the soware for obtaining digital cardiac signals.
Ten IBMs died during the experiment. Out of this ten, four animals died during deep anesthesia, four animals died aer Digoxin was given, one died aer getting a dose of Digitalis purpurea , and one died aer getting a dose of Digitalis purpurea 30. Aer the experiments the animals were kept under observation for the next three days. When normal activities were noticed, the animals were released safely in the jungles.
From the obtained ECG, RR (in milliseconds) was manually and electronically calculated. To avoid bias, the entire statistical analysis was carried out by a biostatistician and also by an epidemiologist, who were not involved with the laboratory work. Excel, origin, and SPSS were used for the analysis.
e �uorescence spectra of the drugs were measured using a Shimadzu (model no UV-1601) spectrophotometer and a Spex-Fluorolog-3 Spectro�uorimeter (model no. FL3-11). e �uorescence spectroscopy was done to detect Digoxin or Digitalis-like ingredient in the drugs used and to note its relevance, if any, with the obtained RR. We have taken the �uorescence spectra of the compound at different dilutions. e obtained emission spectra are shown in Figure 1.

Discussion
RR data obtained were measured and veri�ed, and appropriations were carried out electronically. e entire readings were crosschecked thrice. Because of soware / hardware problem, a few on-screen signals could not be retrieved, and those entries were kept blank. e data which could not be retrieved during veri�cation, as well as the outliers (> ±3 standard deviations), were excluded from the data analysis, as they were thought to be biologically implausible values which have shown idiosyncrasies or because of other unknown factors. RR interval data found faulty due to technical and soware snag were not considered for statistical analyses. Unequal sample size has appeared in various test groups as it appears in Table 2 because of reasons mentioned previously, malfunctions, and death of animals.
It was also a target to use minimum number of animals for this pilot study. e ANOVA, , and values obtained are given in Table 3.

Descriptive Data
Since mean is the best estimate of the true value of an experimental quantity, the mean RRs of the IBMs injected with experimental drugs varied as shown in Table 2. e highest mean RR was found to be in the case of Deep Ether Anesthesia (766.22), followed by Digitalis purpurea 30 (722.18) and Ether Anesthesia (701.97). e lowest mean was found to be for Digitalis purpurea 200 (582.46) whose standard error (9.83), standard deviation (51.07), and sample variance (2608.39) have been the lowest.

ANOVA
e one-way ANOVA, given in Table 3, was used in the study to test the hypothesis about the mean (average) of the dependent variable, the RR. Since eight types of drugs were used, the one-way ANOVA was used to �nd out the differences between (inter) and within (intra) groups. e null hypothesis for ANOVA is that the mean (average value of the dependent variable) is the same for all groups. e alternative or research hypothesis is that the average is not the same for all groups. However, there are exceptions to the rule as is stated in the next section under hypothesis testing.

Hypothesis Testing
Ho. e null hypothesis: application of different drugs has the same effect in RR changes.
H1. e alternate or research hypothesis: application of different drugs does not have the same effect in RR changes: Ho: 1 = 2 = 3 =… (Null hypothesis: means of groups are equal), H1: 1 <or> (Alternative or research: means of groups are not equal).
Since the value 8.02 is greater than 2.07, the results are signi�cant at the 5% signi�cance level. e null hypothesis is rejected, as there is strong evidence that the expected values in the eight groups differ though the value for this test is higher than 0.05. It is concluded that the average of the dependent variable is not the same for all groups.

Results with Generalized Estimation Equation (GEE)
e effect of different medicinal preparations on the RR intervals (in milliseconds) was seen in the Indian Bufo melanostictus. Outliers and omitted readings were not considered in this analysis. e medicines used were analyzed groupwise as given as follows: ether (general inhalation) anesthesia (group = Imedicine_0), ether with double-distilled contamination-free recti�ed spirit (group = Imedicine_1), ether (producing deep anesthesia) (group = Imedicine_2), ether with Digitalis purpurea (group = Imedicine_3), ether with Digitalis purpurea 6 (group = Imedicine_4), ether with Digitalis purpurea 30 (group = Imedicine_5), ether with Digitalis purpurea 200 (group = Imedicine_6), and ether with Digoxin (group = Imedicine_7). Linear regression analysis was carried out to see the effect of the drugs on RR interval aer adjusting for IBM as a covariate in the equation. e RR interval was treated as a continuous variable with normal frequency distribution. Considering that repeated measurements were made on the same animal aer giving the drug, the second analysis was performed considering the data as panel data. e results of linear regression analysis and generalized estimation equation (GEE) are given in Tables 4 and 5, respectively.
Further, considering that repeated measurements were made on a set of animals aer giving the drug, the data was treated as panel data set and a GEE was settled. e GEE results are given in Table 5. In both equations, the experimental animal was included as a covariate to nullify in�uence of interanimal differences.
For the GEE, animals treated with ether anesthesia only were considered as the reference groups. ). With Digoxin, the coefficient was 6.46, and it was not statistically signi�cant. In both groups, the variance was very wide, which may be due to technical difficulty in recording the observations and/or smaller number of observations particularly in case of IBMs injected with Digoxin. However, to establish the effect of Digoxin and that of Digitalis purpurea 200, further experiments have to be conducted in the future. It has also been observed that the type of medication explains only 16% of variance in frequency of RR, thus suggesting that other in�uencing (confounding) variables should also be taken into consideration to improve explanation of variance in RR. From the previous equation, it may be concluded that with increasing potency up to 30 of Digitalis purpurea, the coefficient of variance of RR is increasing; however, with Digitalis purpurea 200, the coefficient of variance of RR is like coefficient of variance of Digoxin, thereby suggesting that RR is signi�cantly in�uenced by the Digitalis purpurea. e observations are in conformity with the equation using linear regression method.

Emission Spectra
e emission spectra of Digitalis purpurea , 6, 12, 30, and 200 and Digoxin were obtained to detect any plausible correlation with the RR results. e primary aim of this work is to detect the presence of Digoxin or Digitalis-like medicinally active ingredient in the serial ultradilutions of Digitalis purpurea using �uorescence spectroscopy, for comparison of the components of the drug which is having intrinsic �uorescence. e �uorescence is likely to be due to , -unsaturated lactone present in the compound. e �uorescence spectrum of the compound is taken at different dilutions in aqueous ethanol [3]. In all the emission studies, the samples were excited at 255 nm [4]. e emission spectra of different dilutions are shown as C, D, and E, and purest medicinally active ingredient Digoxin is shown as B in Figure 1. ese �uorescence spectra revealed that Digoxin component shows a structureless �uorescence with emission maxima at 318 nm. e emission spectra of Digitalis purpurea 6 are shown in Figure 1 as C6.In this case the structureless emission is found to be modi�ed with a partially structured emission with maxima at 357 nm and 374 nm, and further a shoulder at about 318 nm remained which could be due to the original B (Digoxin).e appearance of additional bands at 357 and 374 nm may be ascribed to reduction of self-quenching or dissociation of component substance due to serial dilution. ese bands showed an increase in intensity with subsequent dilutions up to a certain level for 30 [5].e serial sequential dilution of the natural product at 6c, 30c, and 200c shows emission maxima at 318 nm rather than the structureless emission as observed with purest Digoxin. is could arise possibly due to the interference caused by other inclusions in the samples. Although, with the dilution, the peak intensity of the compounds should gradually increase or decrease, the gradual increase in emission with dilution is due to reduction of self-quenching of �uorophores as a result of concomitant dilution [6].
e decrease of emission with further dilution is caused probably by simple dilution effect. is is in accordance with the variance in RR observed by use of different sets of drug on IBM. Since the medicinally active ingredients in serial dilution of drug are not detectable by the common analytical methods in vogue, the results cannot be compared with usual standard methodologies. us, �uorescence data seem to have some correlation with the results from other techniques [4].

Conclusion
It is revealed from the emission spectra that Digoxin or Digoxin-like product is present in the commercially available serially ultradiluted Digitalis purpurea up to 200. With increasing potency up to 30 of Digitalis purpurea extract, the coefficient of variance of RR increases as also the intensity of emission spectra. Intensity of absorption in emission spectra decreases on further serial dilution of Digitalis purpurea extract to 200. e result shows a similarity in pattern of variance of RR produced by the experimental drugs and emission spectra obtained from these experimental drugs. Quality assessment of the commercially available ultradiluted organic drugs obtained from natural products may be initiated by monitoring physiological and toxicological studies on animal models. An advance level of analytical procedure may help in identi�cation of medicinally active ingredient in organic drugs derived from natural plant products.

Limitations of the Study
Our study has several limitations. It is prospective in nature, and, therefore, it has all the problems associated with such analysis. ere are some other components, which are naturally present with Digitalis purpurea and may be responsible for its different activities. We cannot exclude the presence of other important predictors of heart rate and variability analysis. Only one statistical method of interpretation that represents this work in true spirit is included. Other than RR, ECG-associated parameters though studied are not included owing to the con�nes of the paper. Medicinally active ingredients of SAD are difficult to detect analytically in laboratory. SAD is not detectable in biological specimen, bringing limits to the design of the research. e drug Digitalis purpurea was not diluted for improved physicochemical analytical studies; such dilutions are not found in commercial circulation. e aspects dealing with dilution factor have not been included. e statistical analyses with respect to the toxicological �nding/death of animals have not been given.

Future Scope
It remains to be seen if any change appears in con�guration, dilution, and effectiveness due to ultra-dilution and agitation of drug, which may be directly linked to �uorescence data.
Further analysis of �uorophores from the drug Digitalis purpurea and Digoxin may provide in-depth understanding of the components that can bring changes in heart rate. In turn, �ne tuning of the heart rate may become possible without any side effect of the drugs like Digitalis or similar ones. e diluted drug Digitalis purpurea may be tried for improved physicochemical and analytical studies. is opens up a vast research area for exploration of in vivo study of SAD/ultradiluted drugs.

�on�ict of �nterests
ere is no known con�ict of interests. AYUSH had partially provided equipment and materials for monitoring ECG; IIT Kharagpur had provided other infrastructures.