Monet 200 9, 231-20 i hey enecay 2510106 tng 0105.05, aa oe en ORIGINAL PAPER Extreme homeopathic dilutions retain starting materials: A nanoparticulate perspective Prashant Satish Chikramane', Akkihebbal K Suresh*, Jayesh Ramesh Bellare! * and Shantaram Govind Kane!* "Department of Chemical Enginering, Indian Instat of Technology (ID), Bombay, Adi Shankaracharya Mare, Pow Mumbai 400 076, Maharashra, India ’Deparimen of Biosciences and Bioengineering, Indian Institute of Technology (HT), Bombay, Adi Shankaracharya Marg Pov, Mumbai 400 076, Maharashra India Homeopathy is controversial because medicines in high potencies such as 30¢ and 2002 involve huge dilution factors (10! and 10% respectively) which are many orders of magnitude greater than Avogadro's number, so that theoretically there should be no measurable remnants of the starting materials. No hypothesis which predicts the retention of properties of starting materials has been proposed nor has any physical entity been shown to exist in these high potency medicines. Using market samples of metal derived medicines from reputable manufacturers, we have demonstrated for the first time by Transmission Electron Microscopy {TEM}, electron diffraction and chemical analysis by Inductively Coupled Plasma-Atomic Emission Spectroscopy {ICP-AES}, the presence of physical entities in these extreme dilutions, in the form of nanoparticles of the starting metals and their aggregates. Homeopathy (2010) 99, 231-242. Keywords: Homeopathy; Nanoparticles; Nanocrystalline materials; Transmission Electron Microscopy Introduction Homeopathy, a mode of therapy, was established in the Jate 18th century by German physician, Samuel Hahne- ‘mana, Hahnemann, during his experiments, prepared mel- ines from a wide variety of natural products, He discerned thatthe infinite dilutions of these substances carted out in steps and accompanied by vigorous shaking ‘suecussion ogether known as poteatizaten) at each dilution step. elicited some kind of a potent activity o these solutions. "= In spite of the vanous controversies and frequent challenges by the scientific community regarding ite elicacy, this mode of treatment has stood the test of time, fand is stil being wsed in many countries for treatment of vatious chronic conditions with medicines being prepared fiom a vatety of hebal, animal, metal and other mineral tt Technology i) Bombay, Aa Shenk ‘Mumba a0 076, shares, Inde. Becehad 6 Novarbe 200; read 2 Api 2046; scepted 22 However, a major lacuna hasbeen the lack of evidence of physica existence ofthe starting material. The main di fculy in arivng aa rational explanation stems from the {act that homeopathic medicines are used in exueme die tions, including diuion factors exceoding Avogadro's number by several orders of magnitude, in which one ‘would not expect any measurable remnant of the starting material to Be presen. In elinicat practice, homeopathic potencies of 30° and 200¢ having dition factors of 10° and 10" respectively, fa beyond Avogado's number of 6023 x 10" molecules in one male, are routinely used. “Many hypathesss have hoon postulated ta justi and elie ‘idte their mechanisms of acten. While some hypotbses sich a the theory of water memcy.** formation of Clatrates® and epitaxy” ae conjectural in nature, others such as those based on the quantum physical aspects of the sokaions"? have not been sufficiently tested, either ‘ue to complenity in validating the hypothesis or de to non-repreducible resus, Th ‘silica hypothesis" isthe nly model that proposes the presence of physical enities Such as slloxanes ot sileates raul fot leseting fom the gas containers. Fellowing a deat of eel ad test- able hypotheses to identity any physical entity responsible {or medicinal activiy, most madera sient conliaue (0 believe that homeopathy a best provides «placebo elect.Roimee Fara Despite the extreme dilations in 30e and 200e potencies, ‘ous approach has een to test forthe presence of the starting ‘materials inthe form of nanoparticles. Medicines selected ‘were motal-based, and were so chosen tha the metals would ot arse either a impusities or at contaminants, The six ‘metals and their respective homeopathic medicines were {ld (Aurum metallicim of Aurum met), copper (Cuprum ‘metalicwm or Cupram met), tin (Stanniam metallicun or Stannum met, zine Zincum metallicum oF Zincum met, Silver (Argentim metallcum or Argentum met) and platinum (Platinum metalicum ot Plasinum met), Thee potencies 6c, 30c, and 200 were selected, The dilution factor for 6e is 10" which is less than Avogadro's number, whereas the lution factors for 30 and 200e are well above, Market samples of these medicines in 90%viv ethanol were foblained from two reputable manufacturers: SBL. India and Dr. Willmar Schwabe India (WSI) Private Limited ‘We examined the following physico-chemical aspects: The presence of the physical entities in nanoparticle form and their size by ‘Transmission Electron Microscopy (TEM) by brightfleld and dark-field imaging b. Their identification by matching the Selected Area ‘SAED) pattems against literature standards for the corresponding known crystals. «Estimation ofthe levels of starting metals by a SO0-fold concentration of medicines, followed by chemical analysis using Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). Materials andmethods Materi “The homeopathic medications used for the purpose of re search were obtained commercially fom authorized distib- ‘tors ofa leading homeopathic drug manufacturer in India {SBL) and an Indian subsidiary of a multi-national homeo- pathic company viz. Dr. Willmar Schwabe India Pvt. Lid Random batch number samples were purchased from the ‘market and no special effort was made to get samples from the company. Since we purchased these medicines ‘rom the market, only in certain cases were we able to obtcin them fom a single manufacturing batch. leo no special et forts were made to obtain the drugs from a batch, The High performance liquid chromatography (HPI.C) grade ethanol ‘used forthe purpose of ICP-AES analyses was procured rom Commercial Alcohols Tne, Canada. The TEM grids ‘blaine from Pacific Grid-Tech (USA) were 200 mesh cop- per gride coated with carbon—formvar ‘Methods ‘Nanoparticle characterisation by TEM/SAED: The TEM analyses were performed on Teenai G2 120 kV Cxyo-TEM (FEL, Hillsboro, USA). All samples were viewed at 120 KY. ‘The TEM analyses were pecformed for the medicines by placing adop ofthe eriginal solution (without pre-concen- {ration) on the cazbon—formvar coated copper TEM grids in a clean environment. The drop of the solution was emo then allowed to dry completely after which another drop was added. The usual drying time for each drop was ap proximately 3060 min in air at room temperature. This procedure was repeated 5 times, After airdrying the sam- ple for further 30—60 min, the grid was kept under an TR lamp for approximately 20 min to ensure complete drying of the sample and thereby preventing the possibility of solvent molecules from adhering to the particles on the ‘grid. The SAED pattems of the particles were taken and the d-spacings were calculated using the camera length (Galibrated daly using « standard gold colloid), ‘The dark-fetd images were aio taken by selecting three spats from two inner tings on the SAED pattern, The d-spacing values from SAED patterns and the crystallite sizes from the dark-feld images were calculated using the Image-I software, Elemental composition by ICP-AES: Te determination of the starting clements in ulta-tace concentrations was performed on Ultima 2, (ohin Yvon Horiba, Japan). The ‘operating parameters forthe ICP-AES instrument were as follows: plasma gas flow rate (Argon gas): 12 Vii; auxie fary gas low rae: 0.2 Vin; sample uptake: 25 ml/min, Integration time: 5.0 s, Spray Chamber: cyclonic chamb The limit of detection of the instrument wat 10 ppb. For the purpose of ICP-AES analyses, the samples were pre- pared by pre-concentrating the solutions (6c, 30c, and 200¢ potencies) SOOfold in a vacuum rotary evaporator Roteva Model #8706R (Bquicon, India) at 45°C and 100 spm speed. “The homecpathic medicines that we purchased were ine- ther 100 mil or 500 mi capacity bottles. Most of the SBL homeopathic medicine bottles were of 500 mI capacity with a few of 100 ml capacity, while those obtained from Willmae Schwabe India (WSD Pvt, Lid, were ll 100 ml bot- es, Inthe cate of medicines obtained a 500 mal bots, so- lutions from 4 botles of the same medicine and potency were pooled together for concentration, whereas for mei- cines which were marketed as 100 ml bottles, solutions from 20 botles of each medicine at the same potency ‘were pooled, The concenration was caried out ina SO mi clean round bottom flask on a rotary vacttm evaporator. ‘The flask was filled with the solution (approximately 30-35 ml at a time) and the solvent was evaporated ‘Upon complete evaporation of the slveat, the ask was re- filled with fresh homeopathic solution and the process was repeated til the entize volume of 2000 ml was evaporated Only ene botle was opened at time to maintain the integ- rity ofthe purchased medicines. To prevent contamination, tunder no circumstances was the soltion in the botle kept exposed. The residues of Cuprim met, Stamum met, and Zincum met were acidified to solubilize the particle oftheir respective starting metals by addition of concentrated nitic sci, Similarly, aqua epia (concentrated nite acid and con- centrated hydrochloric acid in the ratio 13) was added to resides of Aurum met, Argentum met, and Plainam met A L:Lratio of water: acl was maintained forall he concen twated samples, The amount of acid and water was adjusted tha the final volume was 4m, hus, amounting Wo & con- centration by a faclor of SOQ. The samples were Siltredtyough Whasman 40 filter paper toremave the residual mat- ‘er prior to analysis, The SBI. samples were analyzedin cip- licate and samples from WST were analyzed in duplicate. As a negative control, 90% ethanol samples were also pre= pared using HPLC grade ethanol and Mili-Q water. These cthanolic solutions were also conceniated in the manner simlar to that employed for the medicines. “The emission lines selected for measuring the concenti- tion ofthe metals are a follows: Gold: 242.795 nm, Copper 324.754 nm, Tin: 283.999 nm, Zinc: 213.856 nm, Silver: 328.068 nm, Platinum: 265.945 nra, The instrument re sponse was calibrated using standards prior to analyses of the samples, Results and discussion Determination of size and morphology by TEM Zincuom met, Aurum met, Stannum met and Cuprum met 30¢ and 200c were analyzed by TEM. The results are given ‘as photomicrographs (Figure 1(@)~(p), which clearly dem- onstrate the presence of nanoparticles and thir aggregates Dae to extreme dilution often only a single nanoparticle or a larg aggregate is seen Hereafter, the term ‘particles’ col- Tectvelytefets to the nanopatticles and ther aggregates, ‘We noted a high polydispersity of the particles i the so- lutions with respect to their shapes and sizes for various medicines and potencies, A scrupulous examination of the entire manufacturing process of these medicines sug- gested that two key processes played vital role in impart- ing the high polydispersity. 1. The dlusion steps in the solid phase (Gil 6x potency) in- volved trituration ofthe raw materials with lactose. Such ‘ comminution process i expected to generate particles of varied shapes and sizes. The physical characteristics ofthese particles are dependent onthe typeof raw mate- rial and the shearing force applied. 2, During liquid dilutions, the succussion process at each potentization step played a vital role, The suocussions fiven tothe liquid mass ae expected to produce particles fof varied shapes and sizes due to three factors including shearing forces generated during the pounding of the lig- uid container against an elastic stop, the properties ofthe raw materials involved, and variations during pounding ofthe container, between individual. The permutations and combinations of the above-men- Woned factors and the possible subtle differences in the smanufacturing processes employed by various manufac- turers can explain the findings regarding polydispersity between different medicines and manufacteers, We also made another prominent observation regarding the presence of surface asperties on the particles which were clearly evident from the differences in contrast on sut- faces of these particles along with a substantial difference ‘in their size between different stating metals. Ths, larger faggregates were found in Zincum met (Figure 1(a)—(0) and Stannum mer Figure 1@)—() as compared to those trig mata intamenai meres TH observed in Aurum mer (Figure 1(@)—()) and Cuprum ‘met (Figure (zm)=(p) atthe seme potencies The mechanism of cavitation oF generation of vapor bub- bles caused by ulta-sound irradiation (acoustic cavitation) inthe entire liquid mass during manufacturing may explain the observations noted above. We suggest that the process of succussion isthe cause of cavitation, As sol oul na later section, the extant theories of cavitation''—"* can, in principle, provide an explanation of our findings. The aggregation behavior of the particles seems to bed pendent on the physical property ofthe starting metal, spe~ ‘tically its melting point. We observed that the aggregates fof zine in Zincum met and tn in Stanmume met were tela lively larger as compared to the smaller aggregates of gold and copper found in Aurum met and Cuprum met spectively. The bulk melting points of tin and zine are '~505K and ~692 K respectively as compared to the higher melting points of gold and copper (~1337 K and '~ 1357 K respectively). A decrease in melting points of metallic and semiconductor particles with decreasing size hha also been well characterized." A combination of ex- ‘uemely high surface temperatures along with a decrease inthe melting point of these particles could facilitate the formation of aggregates that we found is probable tha during the succussion process, the col- lisions of the particles induce surface temperatures well above the melting points of in and zine, thereby facia ing their aggregation. However, the melting points gold and copper being much higher, the occurrence melting and fasion ofthese particles would be relatively lege frequent than for tin and zine (Overall, our data for bright-field TEM do not indicate A major difference inthe size or nature of the particles in 4 patticular medicine as we increase potency fom 30e (0 200e. Therefore, the individual erysalite sizes were deter- mined by dark-feld TEM (as shown for Zincum met for both manufactures in Figure 2(a)—(@). We observed that the aggregates of all the metas tested had maximum crystal- lites (40-50%) inthe size range of S—10 nm, and th 70-95% of ll the crystallites were below 1S nm (igure $1 — Supplementary information). Thus, in the case of dari-field TEM also, there was no major potency dependent difference in size distibution of crystallites, Confirmation of elemental composition of particles by SAED The nanoparticles and aggregates identified in TEM were analyzed by SAED for confirmation of the elemental compo- sition. We took multiple SAED pattems ofthe same particle st varying intensities s0 a8 to focus onthe inner and oxter Tings for calculation ofthe dspacings ofthe respective el rents. The SAED pattems ofthe nanopaticles and thet ag- xegates found in the metal-based homeopathic medicines ae represented in Figure 3(9)—(. SAED analyses ofall samples showed pattems consis tent with the starting materials In particular, Auriom met and Cyprum met from both suppliers (SBL and WSD in- dexed to gold and copper respectively. Table | shows the values of the d-spacings calculated from the diameters of Foren