Characterizations of rasa chenduram (Mercury based) and naga parpam (zinc based) preparations: physicochemical and in vitro blood compatibility studies.
From as early as 2500BC, the therapeutic benefits of metal herb preparations have been reported in Indian, Arabic and Chinese literature (1). Metal herb preparations are made by a process called bhasmikarana which converts the metal into its specifically desired non-toxic compound with necessary medicinal benefits (2). Naga bhasma (which includes lead and different herbs) is one of such metallic preparation used in various diseases such as diarrhea, spleen enlargement and diabetes (3). Similarly Naga Parpam (NP) a zinc-based preparation of Siddha medicine is prescribed in the treatment of a variety of diseases. In folklore practice, NP is added as an ingredient in medicaments to treat obesity, in prevention of the elevation of total cholesterol, triglycerides, and low-density lipoproteins. Though some research work has been carried out on the different curative applications of Naga Parpam but none of them give the detail on the elemental and structural composition of the drug which is an essential requirement to discuss its non-toxicity and therapeutic value. Mercury which is considered as a deadly poison is converted into a life saving medicine for treating intractable diseases using various processes in Siddha medicine. When properly purified and processed, mercury is not only used to cure innumerable diseases, but also to rejuvenate the body and promote longevity. The method of processing mercury for therapeutic purposes is known as rasa suthi in Siddha system of medicine. Rasa bhasma has been proved to be an effective haemopoitic drug and a good anabolic agent. Rasa chenduram is one of the Siddha preparations that is prescribed for lowering of cholesterol (4).
In recent years, there has been a renewed interest in drug discovery strategies where natural products and traditional medicines are re-emerging as attractive options (5) and hence renewed interests in agents like naga parpam and rasa chenduram. It is known that nanoparticles can be absorbed through sublingual route directly into the blood stream (6). Therefore, it can be presumed that some of these particles may also get absorbed through the sublingual route directly into the blood stream. This has not been experimentally proved for naga parpam or rasa chenduram. Naga parpam prevented the elevation of total cholesterol, triglycerides, low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL) in serum, liver and heart tissues in a dose-dependent manner when compared to control animals (7).
It has been reported that one out of five available Ayurvedic herbal products in Boston, US and 30% in England contained lead, mercury, and/or arsenic. Similarly in India 64% contained lead and mercury and 41% contained arsenic (8). The estimated daily heavy metal intakes for each herbal preparation were more than the maximum allowable as per regulatory standards (8). Although cellular internalization of herbal preparations and/or its uptake via paracellular pathway have not been established yet (9), uptake of nanoparticles can occur not only via micro-fold (M)-cells, the highly specialized epithelial cells in the Peyer's patches and isolated follicles of the gut associated lymphoid tissue (GALT), but also across the apical membrane of enterocytes (9). It has been demonstrated that uptake of gold nanoparticles occurred in the small intestine by absorption through single, degrading enterocytes in the process of being extruded from a villus and gold nanoparticles typically less than 58 nm in size ultimately reaches blood and various organs through blood (10). Similarly uptake of rasa chenduram and naga parpam can be expected as its crystallites sizes are less that 50nm. Therefore, compatibility with blood is an extremely important factor when these particles are absorbed into the blood stream, in addition to its nontoxicity. Blood compatible materials can be defined as those materials which do not damage blood components when they come in contact with blood (11). In vitro biological evaluations of bhasma preparations are also needed along with the physicochemical characterization and clinical evaluation for present day standardization of metallic bhasma preparations to meet the criteria that supports its use worldwide. Therefore, an attempt has been made to study the physicochemical characterization and blood compatibility of rasa chenduram and naga parpam.
Materials and Methods
Rasa chenduram and naga parpam were purchased from The Indian Medical Practitioners Co-Operative Pharmacy and Stores Limited, Chennai, India with batch nos. S2.70B and SII-081 respectively. Complement protein C3 kit was from Orion Diagnostica, Finland. Platelet factor (PF4) kit and Asserachrom PF4, was from Diagnostica Stago, France. All other chemicals and reagents used were of analytical reagent grade.
Particle Size and Zeta Potential Determination by Dynamic Light Scattering (DLS)
The particle sizes and the zeta potentials of rasa chenduram and naga parpam samples were analyzed by photon correlation spectroscopy and laser Doppler anemometry, respectively, with a Zetasizer, Nano ZS (Malvern Instruments Limited, UK) at 25[degrees]C (12).
X-Ray Diffraction (XRD) Analysis
The XRD powder diffraction pattern of rasa chenduram and naga parpam was recorded on X-ray diffractometer (Siemens D5005 Diffractometer) using CuKa radiation, l = 1.5406 [Angstrom] over the range 20.0-80.0[degrees].
Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDAX)
The morphology and elemental composition of the rasa chenduram and naga parpam samples were determined by Environmental SEM (FEI Quanta) with EDAX. A representative portion of each sample was sprinkled onto a double side carbon tape and mounted on aluminium stubs, in order to get a higher quality secondary electron image for SEM and EDAX examination.
In Vitro Cytotoxicity Studies
The L929 fibroblast cells were seeded in 24 well plates at a density of 5 x [10.sup.5] cells/well, cultured for 24 h in incubator at 37[degrees]C under 5% C[O.sub.2]. The medium was replaced with rasa chenduram and naga parpam particle suspension in the medium at a concentration of 5 mg/ml/ well and incubated for 20h. Medium alone was used as control. The particles were removed and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was done.
Blood Cell Aggregation and Haemolysis Studies
RBCs were separated by centrifuging fresh blood at 700 rpm. This was washed with saline and diluted in saline in a ratio of 1:4. WBCs were isolated by centrifuging the fresh blood after layering with histopaque for 20min at 700 rpm. Platelet rich plasma (PRP) was collected by centrifuging the fresh blood at 1000 rpm for 20 min layered on histopaque solution. To 1 mg each of rasa chenduram and naga parpam particles, 100[micro]l of the diluted RBC, WBC suspension or PRP were added and incubated for 30 minutes at 37[degrees]C. Polyethylene imine (PEI) and saline were taken as positive and negative controls respectively for all studies. Aggregations if any were observed through a phase contrast microscope (Leica DM IRB, Germany) at a magnification of 20X. Haemolysis assay was done on the particles as reported elsewhere (13). Normal saline was used as negative control (0% lysis) and distilled water as positive control (100% lysis). The absorbance was measured at 541 nm by UV-Vis spectrophotometer (Varian).
Protein Adsorption Studies
The plasma was separated by centrifugation of fresh blood at 700rpm. 10mg each of rasa chenduram and naga parpam particles were dispersed in 200[micro]l of saline. To this 200[micro]l of plasma was added and incubated for 1hr. After incubation the plasma was separated by centrifugation at 10,000 rpm and diluted with saline. The proteins in the plasma samples before and after incubation were separated by polyacrylamide gel electrophoresis (PAGE) using discontinuous native-PAGE method of Laemmli (14). A resolving gel of 12% and a stacking gel 4% were used. Electrophoresis was carried out at 100V for 90mins using Mini-PROTEAN II electrophoresis cell (Bio-Rad, CA, USA). The gel was digitalized using an image analyzer (LAS 4000, Fuji) and the densitometry scans were done with the software Multi Gauge V3.
Complement activations by rasa chenduram and naga parpam were determined by the turbidimetric method, assessing the depletion of complement protein C3 on incubation with the nanoparticles. The particle suspensions (100ml) were incubated for 1hr at 37[degrees]C with 100ml of citrated blood. The final concentration of the particles in the assay system was maintained at 10mg/ml of blood. The assay was done as per the protocol provided by the kit manufacturer.
Human blood (5 ml) was collected from consented voluntary donor in the morning hours after 25-minute rest with slight or no stasis. It was immedi-ately placed in the ice/water bath for 20 minutes. Platelet Rich Plasma (PRP) was collected by centrifugation at 700rpm for 20 min. 10mg each of rasa chenduram and naga parpam samples were incubated with the fresh PRP for 15 min. This was centrifuged at 2500g for 20min. Plasma supernatant was collected by aspiration and PF4 was assayed by enzyme-linked immunosorbent assay (ELISA) kit (Diagnostica Stago, France) according to manufacturer's instructions. Samples were assayed in duplicates. PF4 levels were expressed in IU/ml. Precision of the assay was [+ or -] 0.7 UI/ml in replicate determinations.
Visualization of Tight Junctions
Caco-2 cells were seeded (at 20,000 cells/well) onto four well cell culture plates (Nunc). The cells were maintained in an incubator at 37[degrees]C under 5% C[O.sub.2] and used for transport experiments 6 days post-seeding (15). Medium was replaced with Hank's buffered salt solution (HBSS) transport medium, and cells were equilibrated at least for 2 h before uptake experiments. Cells were treated with 500 il of rasa chenduram and naga parpam particle suspension at a concentration of 10 mg/ml for 1 h. The particles were removed by washing the cells three times with phosphate buffered saline (PBS). The cells were fixed with 250 il of 4% paraformaldehyde for 20 min at room temperature. Then the cells were permeabilised using 0.2% Triton X-100 in blocking solution, made of 1% (w/v) bovine serum albumin (BSA) in PBS, for 20 min, so as to make the cell wall permeable to the stain. The permeabilised cells were then washed twice with PBS and incubated with 250 il of 1% BSA for 30 min.
For actin filament visualization, the blocking solution was removed and cells were incubated with 200 il rhodamine phalloidin solution (0.2 ig/ml) for 20 min at room temperature. After removal of rhodamine phalloidin, the cells were treated with 1% BSA as before. The cells were washed with PBS, and dried overnight at 4[degrees]C. Images were obtained using Carl Zeiss LSM Meta 510 inverted confocal laser scanning microscope (Carl Zeiss, Germany), equipped with He/Ne laser 543. The visualization of rhodamine phalloidin was done using excitation and emission wavelengths of 543 and 605 nm respectively.
The particle size distributions of rasa chenduram and naga parpam particles evaluated by dynamic light scattering are shown in figure 1. Rasa chenduram had a mean particle diameter of 538nm and naga parpam had a mean particle diameter of 1030nm. The zeta potentials of nanoparticles at neutral pH (pH7.4) were found to be 35.7 [+ or -] 1.06 mV and -32.6 [+ or -] 0.46 mV respectively for rasa chenduram and naga parpam preparations as shown in table 1.
The XRD patterns of rasa chenduram and naga parpam are shown in figures 2a and 2b. The sizes of crystallites in these preparations were calculated from the XRD pattern using the Scherrer formula and determined to be 28-30nm for rasa chenduram and 32-24nm for naga parpam. Morphologies of these preparations by scanning electron microscopy are shown in figures 3a and 3b respectively. The elemental composition of the samples was analyzed by EDAX as shown in table 2.
In vitro cytotoxicity of these particles has been done with L929 fibroblast cells as per ISO standard (16). It has been confirmed by the in vitro cytotoxicity studies that the rasa chenduram is highly toxic and naga parpam is slightly toxic. As compared to control (medium) the rasa chenduram exhibited only 40 [+ or -] 5% cell viability and naga parpam exhibited 75 [+ or -] 5% cell viability as shown in table 1.
[FIGURE 1 OMITTED]
The aggregations of the blood cells on interaction with the nanoparticles are shown in figure 4 respectively for RBC, WBC and platelets. It revealed no gross aggregation of blood cells on incubation of naga parpam. However, with rasa chenduram there exhibited a tendency for initiation of RBC, WBC and platelet aggregation. Polyethyeleneimine (PEI) which was used as positive control showed aggregation whereas saline used as negative control did not show any aggregation. The same pattern was visible with the haemolytic property of the nanoparticles given in table 1. The hemolysis induced by rasa chenduram was high at 2.47% indicating its RBC disruption tendency. This was also visible in RBC aggregation studies where there is an initiation of aggregation. However, hemolysis potential for naga parpam was only 0.34% which was well within the acceptable limits of 1% (17).
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
The protein adsorption studies evaluated using native-PAGE electrophoresis demonstrated no significant adsorption of proteins occurring onto rasa chenduram and naga parpam as shown in the densitometry scan of the treated plasma (figure 5). The figure shows the peaks of albumin, globulin region and fibrinogen. Compared to the densitometry scan of control plasma, the peak heights of albumin, fibrinogen or globulins of plasma treated with rasa chenduram and naga parpam did not show any appreciable change indicating no significant adsorption of blood proteins.
Measuring C3a or C5a in blood or serum after contact with a material has been the most usual way of assessing complement activation. It has been claimed that a surface is biocompatible if these markers are not increased in the fluid phase (18). Since C3 is cleaved to C3a and C3b by the contact of the surface with blood, irrespective of whether the activation occurs via classical or alternative pathways, and also C3a could be adsorbed on to the material surface just like any other proteins, C3 depletion in the medium can be taken as an indirect measure of complement activation. The amount of C3 in blood (pre-incubation) was 127mg%. After incubation with rasa chenduram and naga parpam it was 125mg% and 126mg% respectively, indicating no significant changes in the complement protein level.
Platelet factor 4 (PF4) is as a platelet-specific protein secreted when platelet is activated and belongs to the CX-C chemokine family. Measurements of plasma levels of PF 4 have been shown to be the marker of platelet degranulation and increased level of PF4 is used to detect platelet activation of the circulating pool of platelets (19). On incubation with naga parpam, the level of platelet factor 4 in plasma did not change appreciably compared to control plasma. However, the PF4 significantly increased on incubation with rasa chenduram. The PF4 level in control plasma was 7.5 [+ or -] 0.50 IU/ml and after incubation with rasa chenduram for 15 min it was 18.4 [+ or -] 1 IU/ml and for naga parpam it was 7.6 [+ or -] 0.6 IU/ml. The platelets adhered onto the particles were observed through scanning electron microscopy after incubating rasa chenduram and naga parpam with platelet rich plasma. Platelets adhered onto rasa chenduram were found to be activated with pseudopod formation as shown in figure 6a. However, platelets adhered onto naga parpam retained its round shape indicating no activation of platelets (figure 6b). SEM micrographs show a direct correlation with the release of platelet factor 4.
The control cells stained with rhodamine phalloidin to visualize actin protein showed uniform staining pattern (figure 7a). Cells treated with rasa chenduram and particles showed disrupted staining pattern as seen from the figure 7b. Actin filaments were observed to be discontinuous and disrupted as evidenced from the staining pattern and the clumping. Tight junctions are composed of transmembrane proteins occludin, claudins and junctional adhesion molecules which intercalate with corresponding proteins from adjacent cells to form the intercellular barrier. These proteins associate with peripheral membrane proteins including the membrane proteins zonula occludens (ZO-1to3) which joins the transmembrane proteins to the actin cytoskeleton. ZO-1 and occluding phosphorylation are associated with stimulus-induced tight junction disassembly and paracellular permeability changes.
[FIGURE 5 OMITTED]
[FIGURE 6 OMITTED]
Although the particle sizes of different batches showed similarity, it seems that these particles are aggregates of much smaller particles. When dispersed in an aqueous medium, these preparations form a negatively charged hydrophobic particle suspension. This hydrophobicity gives these particles a tendency to aggregate together to form larger particles (20). Both rasa chenduram and naga parpam exhibited larger sizes and agglomeration of the particles. However, the crystallite size calculated from XRD was much smaller. Therefore, the comparatively larger size may be due to the agglomeration of the particles by repeated cycles of calcinations involved in preparation as reported earlier (21). Zeta potential has been suggested to play an important role in particle uptake because the surface of the intestinal mucosa is negatively charged owing to the presence of glycocalix. Particles with a high positive surface charge like chitosan are usually attracted by the intestinal mucosa which helps in increasing the intestinal absorption of the encapsulated drug. However, the strong electrostatic interaction between the positively charged particles and the negatively charged glycocalix may slow down the progression and penetration of these particles towards the epithelial cell surface reducing their uptake. Also it has been shown that non-ionized particles have a greater affinity for M cells than for ionized particles (22) and positively charged particles (23). It has been suggested that rasa chenduram and naga parpam with negative zeta potential and nanosize may be up taken by a similar manner.
The X-ray diffraction peaks of rasa chenduram were identical with those reported for the standard mercury metal (Hg) (JCPDS File No. 89-3711). No other major diffraction peaks were observed confirming that the rasa chenduram is composed of mainly mercury nanoparticles. Similarly the X-ray diffraction peaks of naga parpam were identical with those reported for the standard zinc metal (Zn) (JCPDS File No. 89-1397). No other major diffraction peaks were observed confirming that the naga parpam is composed of mainly zinc nanoparticles. The high intensity of XRD lines in the XRD pattern suggests its crystalline nature. It has been reported that nanoparticles exhibited a size dependent uptake from the intestine, and its passage via the mesentery lymph supply and lymph nodes to the liver (24,25), with significant absorption for particles less than 100nm. Therefore, uptake of rasa chenduram and naga parpam with a crystallite size of 28-30nm and 32-34nm respectively through the intestine can be expected.
[FIGURE 7 OMITTED]
From the EDAX results it was confirmed that 90% of rasa chenduram and 95% of naga parpam contains pure mercury and zinc respectively and is in correlation with XRD data. EDAX provide good estimate of the concentration of main elements in the sample in a significantly faster way and provides useful information on the distribution of the element forming the sample and their possible chemical form (26).
The Ayurvedic multi ingredient compounds are formulated in a way that the ingredients are capable of counter balancing toxic effects, if any, present in the herbs or metals (bhasma) (27). These particles pass through extensive processing before they are declared fit for internal use. The processes consist of Shodhan and Marana. The initial event when a material comes in contact with blood is the adsorption of proteins. The nature of protein and amount of protein adsorbed will directly influence the compatibility of the particles with the blood. Although there was no significant adsorption of proteins on to these preparations aggregation of cells were noted. Activation of platelets initiates the deformation of the cells with pseudopod formation and ends with blood coagulation or thrombus formation (28). In the present study platelets seem to be activating and adhering onto the rasa chenduram preparation , however, there was no activation of platelets with naga parpam and the very few platelets adhered were not activated as seen from their round shape. This is an indication of the very high platelet compatibility of naga parpam preparation. However, rasa chenduram was highly platelet activating.
One of the negative effects of the clinical application of various blood-contacting materials is the activation of the complement system induced by the foreign surface. The response of blood in contact with the material depends on phisico-chemical features such as surface area, surface charge, hydrophobicity/hydrophilicity etc. The response depends directly on the surface area. Adsorption of C3 triggers complement activation (28). It has been demonstrated in this study that the adsorptions of C3 on rasa chenduram and naga parpam preparations were insignificant indicating that these preparations do not induce any complement activation when it reaches the systemic circulation.
Pharmacological effects exerted by the therapeutic agents depend upon its ability to cross the biological membranes into the systemic circulation and reach the site of action. This is usually occurred by one of the two pathways; paracellular or transcellular. Most drugs are transported transcellularly depending on their physiocochemical properties; however the paracellular route is usually the main route of absorption for nanoparticles. This is governed by the tight junctions (TJs). TJs are a multiple unit structure composed of multiprotein complex that affiliates with the underlying apical actomyosin ring. TJ proteins identified include transmembrane proteins; occludin and claudin, and cytoplasmic plaque proteins; ZO-1, ZO-2, ZO-3, cingulin, and 7H6. Although the adaptive mechanisms and specific regulation of these tight junctions are areas of active investigation and remain incompletely understood, it is known that some polymers can promote their widening, facilitating absorption of the particles into the systemic circulation. It has been established in this study by the tight junction visualization studies that the rasa chenduram and naga parpam preparations are capable of opening tight junctions, thus facilitating these particles to be absorbed into the systemic circulation and comes in direct contact with blood. Thus these preparations should be highly compatible with blood.
Chenduram and parpam are Siddha metal based preparations made by many systematic processes with herbs, converting raw metal into its therapeutic form. Rasa chenduram a therapeutic form of mercury metal of nano sized particles found to be with a crystallite size of 28-30 nm and was 90% pure mercury as visible from X-ray diffraction and elemental analysis. Similarly naga parpam is a therapeutic form of zinc with a crystallite size of 32-34 nm and was 95% pure zinc as visible from XRD and EDAX. They had a negative zeta potential in a physiological pH. The naga parpam preparations did not induce any blood cell aggregation or any protein adsorption. Activation potential of these preparations towards complement system or platelets was negligible. However, this preparation was slightly cytotoxic. But rasa bhasma preparations were highly cytotoxic and was platelet activating which induced red blood cell lysis also. Caco-2 cell experiments on tight junction integrity in the presence of these preparations demonstrated their ability to open the tight junctions. It has been demonstrated in earlier studies that the uptake of gold nanoparticles occurred in the small intestine by absorption through single, degrading enterocytes in the process of being extruded from a villus and gold nanoparticles typically less than 58 nm in size reach various organs through blood (1), which suggests the importance of the blood compatibility studies for the standardization of Siddha and Ayurveda metal herbal preparations. Since crystallite size of these preparation are within 28-34nm, it can reach the affected site on oral administration via intestinal absorption and possibly can release ions in a sustained manner required for therapeutic action (29). An earlier study on similar grounds with gold bhasma has demonstrated its blood compatibility (30). Therefore, the present study reinforces the importance of biological testing protocols for screening bhasma, chenduram and parpam preparations to meet the criteria that supports its worldwide usage in clinical medicine from the safety point of view, particularly when studies have reported heavy metal toxicity for these preparations.
We are grateful to the Director and the Head BMT Wing of SCTIMST for providing facilities for the completion of this work. Authors have full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Authors have no potential conflict of interest, including specific financial interests and relationships. This work was supported by the Department of Science & Technology, Govt. of India through the project 'Facility for nano/microparticle based biomaterials--advanced drug delivery systems' #8013, under the Drugs & Pharmaceuticals Research Programme.
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Willi Paul and Chandra P. Sharma *
Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram 695012, India
* Corresponding author, (firstname.lastname@example.org) Dr. Sharma C.P.
Received 18 July 2011; Accepted 18 August 2011; Available online 8 September 2011
Table 1: Zeta Potential of and the cytotoxicity, hemolysis, C3 depletion and platelet factor 4 (PF4) on incubation with rasa chenduram and naga parpam Zeta potential Cell Viability Hemolysis (mV) (%) (%) Control -- -- -- Rasa chenduram -35.7 [+ or -] 1.06 40 [+ or -] 5 2.47 Naga parpam -32.6 [+ or -] 0.46 75 [+ or -] 5 0.34 C3 concentration PF4 concentration (mg%) (IU/ml) Control 127 7.5 [+ or -] 0.50 Rasa chenduram 125 18.4 [+ or -] 1 Naga parpam 126 7.5 [+ or -] 0.50 Table 2: Elemental analysis of rasa chenduram and naga parpam by EDAX Element Rasa chenduram Naga parpam Fe 11.10 4.68 Hg 88.91 0 Zn 0 95.32
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|Title Annotation:||Original Article|
|Author:||Paul, Willi; Sharma, Chandra P.|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Oct 1, 2011|
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