Ash of silver is prepared by mixing pure silver leaves with equal quantity of sulphur by weight and one half quantity of arsenic trisulphide soaked in lemon juice and subjected to calcination process in sealed earthen containers at a temperature of 300°C.[1,7] The material is scrapped after cooling, triturated with lemon juice, pulverized, and calcined again. It undergoes the same process repeatedly (trituration with lemon juice, pulverization, and calcination) for 14 times to procure the ash of silver.[2–5,7] This process is chemical reduction of silver converting the silver molecule into uniform nano-particles, spherical in shape and with size of 16 nm without change in morphology of silver. Nanosize of silver particle is probably responsible for improving the penetration of silver in brain; hence, ash of silver has been used in past for the treatment of various pain and neurological conditions.[1,4,8,9]

Group 1: Ash of silver (50 mg/kg) suspended in 1% solution of gum acacia, administered p.o. 20 min before exposure to noxious stimuli.

Group 2: Control, received gum acacia 1% solution p.o.

Group 3(a): Morphine hydrochloride (5 mg/kg) i.p. was administered 20 min before exposure to noxious stimuli.

Group 3(b): Aspirin (100 mg/kg) p.o was administered 20 min before exposure to noxious stimuli.

Once analgesic activity of ash of silver (50mg/kg) p.o was confirmed, further experiments were conducted to know its effect on opioidergic receptors as in group 4.

Group 4: Naloxone (1 mg/kg) i.p. was administered 30 min prior to ash of silver to study the mechanism and site of action of ash of silver (50 mg/kg) p.o. The responses of all drugs were assessed at 0, 30, 60, 90, and 120 min using analgesiometer in Tail flick test, Eddy's hot plate method, Haffner's tail clip method, and 0.6% acetic acid induced writhing methods, respectively.[10,11]

Ash of silver (50 mg/kg p.o.) has shown to exhibit significant analgesic activity at 60 min (P<0.01) compared to control and significantly less than standard drug morphine (5 mg/kg i.p.) in Eddy's hot plate, Haffner tail clip, Tail flick methods in central pain producing models and comparable to aspirin (100 mg/kg p.o.) in peripheral pain producing method viz writhing episodes induced by using 0.6% acetic acid.[10,11] Ash of silver exhibited maximum analgesic effect at 60 min in contrast to morphine which exhibited maximum analgesic effect between 60 to 90 min AMK [Tables ​[Tables11–3] after which the effects started declining. Analgesic effect was observed in the form of increase in the reaction time of mice as paw licking or jump response, dislodging or biting of clip and tail flicking response in Eddy's hot plate, Haffner's tail clip and tail flick methods, respectively. Peripheral analgesic effect was tested by using 0.6% acetic acid [Table 4]. Number of writhing episodes before and after drugs administration was observed and percentage inhibition of writhing using ash of silver was compared with standard (aspirin 100mg/kg p.o.) and control (gum acacia) in the initial 1 hour. Percentage inhibition of writhing using ash of silver (42.1%) was comparable to that of the standard drug aspirin (48.8%), but not with control (gum acacia 6.25%) indicating that gum acacia does not have analgesic action of its own and does not potentiate the action of ash of silver in which it is suspended. The reduction in writhing episodes compared to basal number of writhings in first 60 min indicates an analgesic response.

To find the nature and site of action of analgesic action of ash of silver, we tried to block opioid receptors by pre treatment of mice with naloxone (1 mg/kg i.p.) in all central methods of nociception (Eddy's hot plate, Tail flick, and Tail clip methods) followed by the administration of ash of silver (50 mg/kg p.o.), morphine (5 mg/kg i.p.), and gum acacia (1% p.o.). There was significant reduction in analgesic responses elicited by drug ash of silver.

Ash of silver exhibited potent analgesic effect against thermal and mechanical noxious stimuli indicating that it as a predominant central analgesic, but also inhibited peripheral pain components as shown in writhing method. This is further supported by the fact that when we blocked opioidergic system using naloxone in central pain producing methods, the analgesic effect was reduced significantly. Any injury or tissue damage is associated with pain. Analgesics can act on peripheral or central nervous system to block pain perception. Peripherally acting analgesics act by inhibiting PGs and bradykinins at the site of pain, thereby suppressing the generation of impulses at chemoreceptor site of pain as shown in chemical method, while centrally acting analgesics not only raise the threshold of pain, but also alter the physiological response to pain by acting on CNS and suppress patient anxiety and apprehension.[12–14] Further, it can be hypothesized that ash of silver may be having an inhibitory action on excitatory neuro-transmitters glutamate, aspartate, or N-methyl-D-aspartic acid (NMDA) receptors which needs to be evaluated. As far as traditional system of medicine is concerned, ash of silver is being used for treating pain and inflammatory conditions since ancient times,[15] It might be having inhibitory action on certain pain and inflammatory mediators e.g. PGs, substance-p, bradykinin, LTs etc.

The previous studies by Khanna et al. suggest moderate to marked analgesic activity against chemical and electrical pain, but not against mechanical pain in contrast to our study which has shown moderate analgesic effect against mechanical pain which is also centrally mediated. The authors suggested a role of opioidergic system in pain suppression by silver. One of the studies has proposed that ashes of heavy metals used in traditional system of medicine act as catalyzer by their presence in intestine, plasma and blood, thereby acting as free radical scavengers.[16] Ash particles of heavy metals (gold, silver) in calcined form, being insoluble, exist as nanoparticles (16 nm) which are very tiny particles and found biocompatible, therefore, can cross blood brain barrier to produce various central actions viz analgesic, anti-inflammatory, anti-anxiety, cognitive, antidepressant, neuroleptic, and antiepileptic for which they are used in traditional Indian system of medicine.[1,3,4,17]

Use of metals in medicine is associated with toxicity.[18] The use of metal based medicines can be attributed to various causes including a need to revive a rich tradition, the dependency of 80% population of India on these drugs, their easy availability, comparatively low cost, and therefore, increasing world wide use. Calcined form of silver has been found to be non-toxic and exhibiting free radical scavenging activity by virtue of their antioxidant activity as shown in some studies. The quantitative analysis of ash of silver by spectroscopy has detected traces of heavy metals (in parts per million) like arsenic, lead, chromium, and iron. Ashes are associated with organic macromolecules show increased superoxide dismutase and catalase activity which reduce free radical concentration.[1–3] Nitrate based salt of silver has found to be toxic at 50 mg/kg p.o. in rats and mice leading to hepatic necrosis and deaths.[19–21] As far as safety of ash of silver is concerned, in our study, three mice died on day 11. Rest of mice did not show any weight loss or any morbidity. Ash of silver has been found to be safe up to 1.5 g/kg p.o. which is its maximum tolerated dose (MTD). Four among 18 mice showed characteristic bluish black pigmented patches over the skin. Autopsy reports of skin, spleen, liver, and lymph nodes from two dead mice have shown deposits of silver in these sites owing to their very small particle size. LD50 was found to be 2 g/kg p.o. The characteristic bluish black pigmentation is known as argyria is due to deposition of silver particles and melanocyte stimulating effect of silver.[22] Few studies have shown cyanocobalamin, vitamin E, and selenium antagonize the toxic effects of silver.[23,24] Such reports indicate the possibility that silver as such is not harmful in humans, but may be toxic in those deficient in vitamins and trace minerals.[25] A correlation between silver and vitamins like vitamin E and trace minerals need to be explored.