2019 Biswas J Ethnopharm Suvarna Bhasma
2019 Biswas J Ethnopharm Suvarna Bhasma
2019 Biswas J Ethnopharm Suvarna Bhasma
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Keywords: Ethnopharmacological relevance: Suvarna Bhasma is a gold-based Ayurved medicine that has a wide range of
Gold nanoparticle therapeutic indications like tuberculosis, diabetes mellitus, rheumatoid arthritis and nervous diseases. Suvarna
Swarna Bhasma Bhasma is also used in Suvarnaprashana, an Ayurved advocated therapy being practised to improve immunity in
Anxiolytic effects children.
Zebrafish behaviour
Aim of the study: To augment traditional understanding, here we present an evidence-based study on Suvarna
Novel tank experiment
Bhasma regarding its physicochemical properties, toxicity and efficacy.
Suvarnaprashana
Materials and methods: Suvarna Bhasma was characterised by physicochemical characterization techniques such
as scanning electron microscope (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and
atomic emission spectroscopy (ICP-AES). Toxicity of Suvarna Bhasma was studied in Holtzman rats with daily
oral dose from 3 mg/kg (therapeutic dose, TD) up to 30 mg/kg (10 TD) body weight for 90 days. Behavioural
study, such as motor and geotactic behaviour were examined in zebrafish model to find out any sign of neu-
rotoxicity or behavioural changes due to Suvarna Bhasma administration.
Results: Suvarna Bhasma has two types of gold particles, large ones (~60 μm) having irregular shapes, and nano-
sized spherical particles (starting from ~10 nm), the latter coated with Fe, Si, O, P and Na. XRD study revealed
that all the peaks of Suvarna Bhasma match well with pure gold (face centred cube) with crystallites size
45 ± 2.8 nm. In rat studies, some change in biochemical parameters such as urea, creatinine and alanine
aminotransferase (ALT) was observed mainly at the higher therapeutic dose; however, those parameters were
within the normal range. There were no significant macroscopic as well as microscopic treatment-related al-
teration observed, in any of the organs and tissues evaluated. In zebrafish behavioural study, the motor para-
meters of Suvarna Bhasma treated fish showed normal behaviour analogous to the vehicle control group.
Interestingly, the geotactic behaviour showed anxiolytic effects of Suvarna Bhasma as evidenced by the time
spent in the upper zone, and average swimming height. The anxiolytic effects persisted for more than 30 days
after withdrawing the Suvarna Bhasma treatment.
Conclusions: Suvarna Bhasma contained spherical gold nanoparticles. It was nontoxic in rat model at the does
tested. Suvarna Bhasma has anxiolytic effects in zebrafish behavioural model.
1. Introduction and V Shah, 2013). In Suvarna Bhasma, gold in elemental form is the
major element (> 98%) and it is the active ingredient. Suvarna Bhasma,
Suvarna Bhasma (also spelt as Swarna Bhasma) is a gold-based along with honey, was also prescribed as a tonic for rejuvenation
Ayurved medicine. It is used in the treatment of diseases such as (Williamson, 2004). Suvarnaprashana is widely advocated by Ayurved
asthma, rheumatoid arthritis tuberculosis, diabetes mellitus, immune practitioners and is gaining popularity as an Ayurved therapy to im-
and nervous disorder (Singh, 2014; Yadav and Chaudhary, 2015; Patel prove immunity of the child (Rao et al., 2012; Samant and Patil, 2014).
∗
Corresponding author. Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, India.
∗∗
Corresponding author.
E-mail addresses: geetavanage@gmail.com (G. Vanage), jb@iitb.ac.in (J. Bellare).
https://doi.org/10.1016/j.jep.2019.112388
Received 25 March 2019; Received in revised form 12 July 2019; Accepted 11 November 2019
0378-8741/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Snehasis Biswas, et al., Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2019.112388
S. Biswas, et al. Journal of Ethnopharmacology xxx (xxxx) xxxx
In this samskara (ritual), Suvarna Bhasma is administered and some- toxicity study was carried out with three different doses, a low dose
times administered with pure honey and medicated ghee processed in (3 mg/kg), a medium dose (15 mg/kg) and a high dose (30 mg/kg) of
Medhya (nootropic) and Rasayana (rejuvenating/anti-ageing) herbs as Suvarna Bhasma was administered orally for consecutive 90 days to
per the preference of the practitioners. Kashyap Samhita describes it to male and female rat separately. Various haematological, biochemical
improve cognitive functions, digestive capacity, strength and longevity and histopathological analysis was conducted after 45 and 90 days of
(Jyothy et al., 2014a). Likewise, in recent times, gold nanoparticles are Suvarna Bhasma treatment.
attracting researchers, especially in the biomedical field, as these na- Additionally, Suvarna Bhasma was administered in a zebrafish
noparticles are biocompatible and having immense therapeutic and model to observe behavioural changes. The behaviour of zebrafish is
diagnostic applications. Suvarna Bhasma, comprising of nanogold par- robust; abnormality in behaviour can be evoked by drug-induced
ticles, renewed the interest of the scientific community for finding its toxicity (V Kalueff et al., 2013). With the aid of video tracking meth-
applicability in chronic diseases such as diabetes and cancer (Das et al., odology, the behaviour of zebrafish can be analysed with high accu-
2012; Kumar Pal, 2015). racy. In this study, zebrafish were efficiently tracked in the novel tank
Suvarna Bhasma is considered as one of the most prominent metal with the automated video tracking process. With the aid of indigenously
based medicine in Ayurved, which has both protective and curative build MatLab code, various motor and phenotype behaviour were ac-
properties on numerous health problems (Jyothy et al., 2014a). Gold curately evaluated.
based Ayurved medicines are traditionally used for centuries, however,
a scientific evidence-based study is extremely required to find out the 2. Materials and methods
safety and efficacy of Suvarna Bhasma in the animal models. Therefore,
this study was focused on a comprehensive assessment of Suvarna 2.1. Chemicals
Bhasma by exploring its safety in rat model and efficacy in zebrafish
behaviour model along with the physicochemical characterization of The preparation of the Suvarna Bhasma (Batch no: P110600209)
Suvarna Bhasma particles. used in this study was carried out by Shree Dhootapapeshwar Limited,
Interestingly, the physicochemical properties of Suvarna Bhasma Mumbai, according to the classical Ayurved text (Bharat Bhaishajya
may differ from manufacturer to manufacturer. This may happen be- Ratnakar 5/8357, Fig. 1). Suvarna Bhasma was prepared (Fig. 1) from
cause the traditional pharmacopoeia (texts) describe different pre- gold bar (99.99% purity), following a rigorous Ayurved process con-
paration protocols for the same medicine. For example, approved sisting of Shodhana and Marana (special procedures meant for the
Ayurved scripture, Rasatarangini has described five different methods conversion of metals into Bhasma). The gold bar was converted in the
for the preparation of Suvarna Bhasma. Also, in the 5th volume of Bharat form of thin gold ribbons by passing through a machine by exerting
Bhaishajya Ratnakar (A compilation of Ayurved formulations from pressure and these were further cut into small pieces. These gold pieces
various Ayurved texts) has described 20 different methods for the were then purified by quenching them in Taila (sesame oil), Takra
preparation of Suvarna Bhasma. (Suvarna Bhasma used in these studies (butter milk), Gomutra (Cow's urine), Kanji (fermented rice water),
was manufactured using the method described in Bharat Bhaishajya Kulattha kwatha (decoction of Dolichos biflorus seeds) and Kanchanara
Ratnakar 5/8357). Particles size, shape, and gold concentration vary kwatha (decoction of Bauhinia variegata stem bark), for 7 times in each
largely for Suvarna Bhasma from a different origin. For example, liquid. Then the purified gold pieces were amalgamated with Hg at
Beaudet et al. (2017) showed that Au present in Suvarna Bhasma was 1:2 wt ratio (Au:Hg). The Au–Hg amalgamate was then covered with S
approximately 57 wt%, Brown et al. Brown et al. (2007) reported 92 wt powder in an earthen pot and heated slowly up to 750 °C. After cooling
% Au in Suvarna Bhasma, whereas Thakur et al. (2017) found 98% gold down of the earthen pot, the intermediate product was found in a
in Suvarna Bhasma. Similarly, the Suvarna Bhasma crystallite size also powder form. The heating process of the intermediate product with S
varies largely from 23 nm (Brown et al., 2007) to 60 nm (Beaudet et al., powder was repeated for 13 times (total 14 times) which resulted in the
2017) according to the two different research articles. Therefore, before final form of Suvarna Bhasma in the form of fine powder. The manu-
in-vivo studies, Suvarna Bhasma used in this work was physicochemi- facturing process of Suvarna Bhasma took about 21 days. A quantitative
cally characterised. Here, the physicochemical study revealed some detail for a typical Suvarna Bhasma batch (1 kg) has been provided in
unique features about Suvarna Bhasma which were not reported in the Supplementary File S2.
literature. Additionally, it was found that for this particular Suvarna
Bhasma, batch to batch variation was not significantly observed. 2.2. Physicochemical characterization of Suvarna Bhasma
In the Suvarna Bhasma preparation process, metallic Hg is very
frequently used, which is a common neurotoxin and Au is also not an Suvarna Bhasma was characterised to understand its physicochem-
essential element (Bhattacharya et al., 2016; Broussard et al., 2002), ical properties. The crystallographic details of Suvarna Bhasma were
though a dose of 15–30 mg for an adult human is normal and the dose analysed by powder X-ray diffraction (XRD, Smart Lab, Rigaku, Japan)
may be prescribed for a longer duration. Therefore, besides its tradi- and peaks were compared with ICDD (International Centre for
tional belief, a proper toxicological study was carried out in this work. Diffraction Data) database. Transmission electron microscope (TEM,
In recent literature, some in-vitro and in-vivo toxicological studies were JEOL 2100, Japan) attached with energy dispersive spectroscopy
been reported, which concluded that Suvarna Bhasma was nontoxic (EDAX, OXFORD instrument, United Kingdom) was used to observe the
(Beaudet et al., 2017; Paul and Sharma, 2011; Mitra et al., 2002; morphology and elemental mapping of Suvarna Bhasma particles. TEM
Khedekar and Priya, 2016; Jamadagni et al., 2015). The genotoxicity of analysis was carried out after suspending Suvarna Bhasma particles in
Suvarna Bhasma was studied by Selkar et al. (2016) and they reported it water; then the suspension was sonicated for 10 min and allowed to
is safe in terms of genotoxic and mutagenic activity. However, most of settle down for 5 min. After settling down, the lightweight suspended
the in-vivo toxicity studies reported short term effects of Suvarna Suvarna Bhasma particles from the upper water level were pipetted out
Bhasma in the rodent, for example, Khedekar et al. (Khedekar and Priya, and fixed on the carbon coated copper grid for TEM analysis (Brown
2016) reported the effect of Suvarna Bhasma in albino rats for 10 days et al., 2007). The backscatter imaging of Suvarna Bhasma was grabbed
treatments. On the other hand, Khan et al. (2018) administered Suvarna by a scanning electron microscope (FEGSEM, JEOL, Japan). Elemental
Bhasma for 20 days in albino rats. Jamadagni et al. (2015) did a 90 days analysis of Suvarna Bhasma was carried out using inductively coupled
toxicity study in Wistar rats with a maximum Suvarna Bhasma dose of plasma atomic emission spectroscopy (ICP-AES, ARCOS, SPECTRO
13.5 mg/kg. However, this group (Jamadagni et al., 2015) reported Analytical Instrument, Germany) after digesting Suvarna Bhasma in
data only for the alteration of body/organ weight and histopathology of aqua regia. The surface elemental analysis was conducted by high-re-
Suvarna Bhasma treated rats. In this present study, a comprehensive solution X-ray photoelectron spectroscopy (HRXPS, Kratos Analytical,
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Fig. 1. Flow chart of Suvarna Bhasma preparation starting from gold bar. (For interpretation of the references to colour in this figure legend, the reader is referred to
the Web version of this article.)
Japan) equipped with a monochromatic X-ray source of 1486.6 eV. 2.4. Toxicity assessment of Suvarna Bhasma in rat-model
Thermogravimetric analysis was carried out with TGA analyser
(PERKIN ELMER, USA). Details of physicochemical analysis carried out Toxicological study of Suvarna Bhasma was conducted in the
are enlisted in Table 1. Holtzman rat model following OECD guidelines for sub-chronic toxi-
city. Total 90 rats (45 males and 45 females) of 9–10 weeks of age were
randomly selected and assigned to the control and the treatment groups
2.3. Animal ethics
(Table 2) after the acclimatization of five days prior to the start of the
study. The weight variation of animals used did not exceed ± 20% of
Ethical clearance for the use of animals in the study was obtained
the mean weight of each sex. The animals were kept in polypropylene
from the Institutional Animal Ethics Committee of National Institute for
cages and maintained under controlled temperature (23 ± 1 °C), hu-
Research in Reproductive Health (NIRRH), Parel (Study number 09/11)
midity (55 ± 5%), and in a 14 h light/10 h dark cycle. Maximum three
prior to the initiation of the study. The experiments were performed in
animals were housed in a single cage. All animals were acclimatized for
accordance with the guidelines of the Committee for the Purpose of
five days prior to exposure of the test items.
Control and Supervision of Experimental Animals (CPCSEA), India.
Table 1
Details of physicochemical analysis carried out in this study.
Analysis Instrument Operation details Sample Preparation
X-ray diffraction (XRD) XRD, Smart Lab, Rigaku, Source: Cu K-α, wavelength = 1.5406 Å Powder sample was placed on a glass plate and put for XRD
Japan Scanning range 2theta = 10–90° scanning
Transmission electron microscope JEOL 2100, JEOL, Japan Electron source 200 KV Powder gold particles (Suvarna Bhasma) were suspended in
(TEM) water and sonicated for 10 min, after settling down, the
particles from upper water layer were pipetted out and put on
the carbon coated grid
Scanning electron microscope (SEM) JSM-7600F, JEOL, Japan Field emission gun Suvarna Bhasma powder was fixed on carbon tape, then the
tape was placed on a sample holder.
Energy dispersive spectroscopy OXFORD instrument, UK, Mapping in scanning transmission electron Same as TEM sample preparation
(EDS) attached with TEM microscope (STEM) mode
Atomic emission spectroscopy (ICP- SPECTRO Analytical Wavelength Range: 130 nm–770 nm. Suvarna Bhasma (~10 mg) was digested in aqua regia and then
AES) Instruments GmbH, Detector: Charge Coupled Devices (CCD) diluted with MiliQ water
Germany
Thermogravimetry (TGA) PERKIN ELMER, From room temperature to 1250 °C at Powder sample used directly
USA 10 °C/min rate
High-resolution X-ray photoelectron Kratos Analytical, Japan X-ray source of 1486.6 eV Suvarna Bhasma powder fixed on the carbon tape and placed
spectroscopy, (HRXPS) on the sample holder
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Table 2 Positive Control (PC, 50 mg/L caffeine) groups. For VC and SB groups, a
Grouping of rats and dose level of Suvarna Bhasma. total of 40 fish/group was allotted, whereas CC and PC group had 20
Group No Groups Group Dose (mg/kg No of Males No of fish each. For the CC group, alcohol (1.5 vol %) and for PC group,
Name body weight) Females caffeine (50 mg/L) was given for 30 min (n = 20) to fish individually
before behaviour tracking. In each experimental set, the male to female
I Control VC Vehicle 10 10
ratio was the same for VC and SB groups. Zebrafish from each group
II Low SB3 3 mg/kg (TD) 10 10
III Mid SB15 15 mg/kg 10 10
were kept as a cohort of 10 fish separately in a tank; therefore beha-
(5 × TD) viour experiment was independently run in quadruplicate for VC and
IV High SB30 30 mg/kg (10 × TD) 10 10 SB (Total n = 40) groups, whereas behavioural experiment was done in
a
V Recovery SB30R 30 mg/kg (10 × TD) 5 5 duplicate for CC and PC groups with total n = 20 fish/group.
a All fish were given dry food twice a day (Tetra, Germany). The
Recovery group was sacrificed 15 days after the completion of treatment.
Suvarna Bhasma was orally given mixed with dry granular food (Micro
TD = therapeutic dose.
Wafers, Hikari Tropical, Japan) to the SB group daily for 15 consecutive
days at the afternoon time. After the completion of Suvarna Bhasma
Suvarna Bhasma was administered orally to the animals with a
treatment, on the 16th day at the afternoon time fish were placed in-
syringe once daily for a period of 90 days. Animals from the control
dividually in a novel tank (dimension:
group were treated with vehicle alone (xanthine gum, 2% w/v). Dose-
L × H × W = 200 mm × 170 mm × 130 mm) and the video was re-
volume was adjusted based on the weekly body weight of the individual
corded from the top and side views with two webcams (Logitech B525).
animal. Except for the treatment with the test item, animals in the
For CC and PC group, fish was treated with alcohol and caffeine re-
control group were handled in an identical manner to those in the test
spectively and behaviour was recorded in the same novel tank.
groups. The dose volume administered to each animal was calculated
Comparative control (CC) fish were individually exposed to 1.5 vol %
based on a constant factor of 2 ml/kg. Dose levels of 3, 15 and 30 mg/kg
alcohol in water for 30 min in a two-litter tank before novel tank
of Suvarna Bhasma were given for this study. Suvarna Bhasma was
tracking which has anxiolytic effects on zebrafish (Hamilton et al.,
weighed and formulated with xanthine gum (2% w/v) freshly on each
2017). Caffeine, an anxiogenic to zebrafish behaviour, was similarly
day of dosing. The concentration of Suvarna Bhasma was adjusted at
treated for 30 min at a concentration of 50 mg/L water to the PC group
1.5, 7.5 and 15 mg/ml to administer the doses of 3, 15 and 30 mg/kg
(Egan et al., 2009; Collier, 2017).
body weight. Body weight and feed intake of all the animals were
Trajectories of all fish from individual videos were extracted with
monitored throughout study period.
the help of open source MatLab software: idTracker (Pérez-Escudero
Blood was collected from retro-orbital plexus under light anaes-
et al., 2014). Each video was grabbed at a speed of 30 frames/sec.
thesia (3–4% isoflurane) in two separate vials, one for haematology
Videos of individual tracking were processed from frame number 1800
(EDTA used as anticoagulant) and other for serum biochemistry ana-
to 10800 (total 5 min/fish). From the trajectories, various motor para-
lysis. The haematological analysis was performed on freshly collected
meters (speed, meander, and freeze point) and geotaxis phenotype
blood samples by using haematology analyser (Abacus, Diatron,
behaviour (preference of being in the upper or lower zone) were cal-
Hungary) and, the serum samples separated after incubation at 37 °C of
culated by indigenously developed MatLab code. To observe the after
whole blood were stored at −20 °C for further analysis. The serum
effect of Suvarna Bhasma, 30 fish from each SB and VC groups was kept
biochemistry analysis was performed by using fully automated serum
in housing system and again tracked after 30 days (without any drug
biochemistry analyser (EM 200, ERBA).
treatment) in the novel tank similarly as described above.
At scheduled terminal necropsy, all surviving animals were huma-
The oral drug dose for zebrafish was prepared by mixing Suvarna
nely euthanized by CO2 asphyxiation and subjected to complete gross
Bhasma with dry granular food and COD liver oil as sticking substance
pathological examination. All collected organs were fixed in 10%
for 15 min so that the Suvarna Bhasma stuck to outside of the granular
neutral buffered formalin until processing. Organ weights of tissues
food. After ICP-AES quantification of the gold per gram of dry
were taken immediately after collection. These tissues were processed
food + Suvarna Bhasma, the exact amount dose (dry food + Suvarna
in an automatic tissue processor (ASP300, Leica, Germany) and em-
Bhasma) was given to the fish of SB group at a dose of 60 mg/kg fish
bedded in the paraffin wax using a tissue embedding system (EG
weight. The calculated dose of the granules was added to the feed tank.
1150H, Leica, Germany). The embedded tissues were further trimmed
Since Suvarna Bhasma is not soluble in water, granules retained their
with the help of an automatic microtome (RM 2255, Leica, Germany)
identity until consumed. The control groups were also provided dry
and sections were cut at 5 μm thickness and taken on a clean, grease-
food mixed with COD liver oil (without Suvarna Bhasma).
free slide for further staining with Haematoxylin and Eosin with auto-
As drug dose was given in the water, there was always a chance of
matic tissue stainer (Autostainer XL, Leica, Germany).
elution of Suvarna Bhasma. However, from ICP-AES analysis, it was
Histopathological examination was performed on the specified list of
confirmed that elution of Suvarna Bhasma particles was not more than
tissues including all macroscopically abnormal tissues of all control and
10% even after 5 min immersion in water. On the other hand, as the
high dose group animals sacrificed at termination.
oral dose was given to the fish cohort, so there is always a possibility of
unequal distribution of drug dose. One could separately feed those fish,
2.5. Behavioural experiments on zebrafish but as zebrafish is a social animal, separating them could lead to un-
natural behaviour in them. Also, we observed that in isolation, attrac-
Adult wild-type zebrafish (Danio rerio) of 5–6 months of ages were tion towards food in zebrafish decreases. The cohort was given dose
purchased from an authenticate zebrafish supplier (Vikrant which contains approximately 40–50 food granules (with Suvarna
Aquaculture, Mumbai, India). The zebrafish were kept in a lab-cos- Bhasma), and within 2 min the whole bowl of the dose was consumed.
tumed housing system (Biswas et al., 2018), having biological, chemical There was no avoidance towards Suvarna Bhasma dose observed in the
and mechanical filtration facility to maintain the water quality pre- fish cohort. Therefore, it was concluded that the amount of Suvarna
ferred for zebrafish. The water temperature of zebrafish housing was Bhasma dose consumed by fish was proportional to fish's body weight.
kept consistent throughout the experimentation at 25–27 °C. The fish
were maintained in 14 h light and 10 h dark cyclic period. The ex- 2.6. Statistical analysis
periment was carried out after two months of acclimatization periods.
Zebrafish were divided into four groups as Vehicle Control (VC), Statistical analysis was performed using GraphPad Prism and Origin
Suvarna Bhasma (SB), Comparative Control (CC, 1.5% alcohol) and software. Data were analysed for dose wise comparison. One-way-
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Fig. 2. Physicochemical characterization of Suvarna Bhasma. A) SEM backscatter image, B) TEM image of big Suvarna Bhasma particles, C) XRD profile of Suvarna
Bhasma, D) TEM image and EDAX mapping of small Suvarna Bhasma particles, E) TGA profile of Suvarna Bhasma, and F) HRXPS (gold 4f region). XRD profile of
Suvarna Bhasma perfectly matches with pure gold. Suvarna Bhasma contained encapsulate spherical nanoparticles starting from 10 nm size. EDAX mapping of small
Suvarna Bhasma particles shows peaks of Au, Si, O, P, Fe, Ca etc. From TGA profile, it was observed that organic carbon may not present in Suvarna Bhasma. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Au 98.2 ± 1.82
3.1. Physicochemical characterization of Suvarna Bhasma Si 0.06
Fe 0.19
Physicochemical study of Suvarna Bhasma revealed some new in- Ca 0.13
teresting features about it. Suvarna Bhasma contained very big particles Cu 0.01
Zn 0.05
as well as nanosize gold particles (Fig. 2A, B, and 2D). The nanosized
Pb ND
gold particles were spherical, starting from ~10 nm in size (Fig. 2D). As ND
The spherical gold nanoparticles were encapsulated by an envelope of Hg ND
Si, O, P and Fe coating (Fig. 2D). These whole encapsulate gold particles
were again embedded in the big gold particles (Fig. 2A). The large
particles were agglomerated and irregular in shape having size up to represented in Fig. 3 for male (Fig. 3A, C) and female (Fig. 3B, D) rats
60 μm. The XRD peaks of Suvarna Bhasma matched with that of pure separately. There was no significant alteration observed in Suvarna
gold with no other extra peaks (Fig. 2C). The crystal size of Suvarna Bhasma treated groups after 45 days (Fig. 3A and B) of treatment among
Bhasma, calculated using Sherrer's equation, was approximately any of the haematological parameters such as, haemoglobin level,
45 ± 2.8 nm. However, EDAX (Fig. 2D) and ICP-AES analysis show counts of blood cells, mean corpuscular haemoglobin concentration
presence other elements as well, such as Fe, Ca, Si etc. Further analysis (MCHC) etc. when compared with the vehicle control group (Details of
of Suvarna Bhasma using ICP-AES and ICP-AAS revealed that it contains other haematological parameters are enlisted in the Supplementary file
approximately 98% Au (Table 3). S1-Table 3). Similarly, there were no significant alterations in any of
Up on thermal analysis (TGA), it was noticed that only 0.05% the haematological parameters performed at the termination day (after
weight loss occur up to 350 °C and 0.16% weight loss up to ~1050 °C. 90 days) except percentage of red blood cells PCV % in SB3 group in
HRXPS found only gold state (Au0), other gold peaks (such as gold salt) female rats (Fig. 3A and B and Table 3d in Supplementary file S1).
was not present in the Suvarna Bhasma.
3.2.2. Clinical chemistry
3.2. Toxicological assessment in rat model Serum biochemistry was similarly analysed at the two time points in
rats (Fig. 4). After 45 days of treatment, significant alteration in some
3.2.1. Haematology serum biochemical parameters was witnessed in the treated groups
The effects of Suvarna Bhasma on the haematological parameters are when compared with the control group. The significant variation in
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Fig. 3. Effects of Suvarna Bhasma on haematological parameters in rats. A) Male rats after 45 days of treatment, B) Female rats after 45 days of treatment, C) Male rats
after 90 days of treatment, and D) Female rats after 90 days of treatment. Values are expressed as mean ± SEM (standard error of the mean) with *p < 0.05 vs
vehicle control [n = 10 animal/group, except Group V (n = 5)]. No significant alteration observed among any of the haematological parameters. Neutrophil counts
for Suvarna Bhasma treated groups was increased for male rat after 90 days treatment without any statistical significance (p > 0.05).
males included increased creatinine and triglyceride levels in the mid- (Supplementary file S1-Table 5).
dose group (SB15) (Fig. 4A). The significant variations in females
(Fig. 4B) included decreased creatinine and AST in the high dose group
3.2.4. Gross pathology
(SB30), increased glucose and DBIL (direct bilirubin) in the recovery
There were no gross pathology observations recorded during the
group (SB30R), increased glucose level of the recovery group (SB30R),
terminal necropsy examination of all animals including control and
increased TBIL (total bilirubin) in the SB3 group, and increased phos-
treatment groups. The only gross pathology observation recorded
phorus levels in the groups SB30 and SB30R as compared to vehicle
during the study was with the found dead animal (from SB30, Animal
control females (Supplementary file S1-Table 4). All the above varia-
No. 459F, Datafile 1), it was suppuration in meninges and brain. This
tions were well within the normal range, hence do not carry any tox-
gross pathology observation was not related to treatment, but it was
icological significance.
due to infection in meninges and brain.
Likewise, in the serum biochemistry performed at the termination
(after 90 days), many biologically as well as toxicologically insignif-
icant parameters either increasing or decreasing mere trends were ob- 3.2.5. Histopathology
served (Fig. 4C and D). Those statistical significant variations in males Histopathological examination was performed on the specified list
included increased urea in mid and high dose (SB15, SB30 and SB30R), of tissues including macroscopically abnormal tissues of all control and
increased creatinine in high dose group (SB30), decreased TBIL at high high dose group animals sacrificed at termination. Tissues collected
dose (SB30), decreased creatinine and cholesterol in recovery dose from a found dead animal (animal no. 459 F) were also processed for
group (SB30R) when compared to the vehicle control group (Fig. 4C histopathology examination.
and Supplementary file S1-Table 4c). Similar significant variations in Four step grading system of minimal (+), mild (++), moderate (+
females (Fig. 4D and Supplementary file S1-Table 4d) included de- ++) and severe (++++) were used to rank microscopic findings for
creasing trends AST and ALT in mid (SB15) and high (SB30) dose comparison among the groups. The histopathology lesions observed are
groups, increasing DBIL in SB15 and SB30, decreasing uric acid and summarized in the following table (Table 4) separately for males and
increasing in glucose level in SB30 as compared to the female control females:
group. However, all the above variations were well within the normal There were no significant macroscopic as well as microscopic
range, hence do not carry any toxicological significance. treatment-related alteration observed, in any of the organs and tissues
evaluated (Fig. 5). All above histopathological alterations that were
3.2.3. Organ weight observed were all considered as either spontaneous or known back-
No treatment-related adverse effects in absolute organ weight and ground findings that are usually observed in laboratory rats of this
relative organ weight were noticed during the study period strain and age under present experimental conditions.
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Fig. 4. Effects of Suvarna Bhasma on serum biochemistry in rats. A) Male rats after 45 days of treatment, B) Female rats after 45 days of treatment, C) Male rats after
90 days of treatment, and D) Female rats after 90 days of treatment. *p < 0.05 vs vehicle control [n = 10 animal/group, except Group V (n = 5)]. Alteration
observed for few parameters such as urea, ALT, AST etc. However, the alterations were not consistent, for example, after 90 days treatment, urea and creatinine were
significantly changed for male rats (SB15 and SB30) whereas for female rats the changes were not significant statistically for urea and creatinine parameters.
3.3. Zebrafish behavioural study kept for 30 days in the housing tank without Suvarna Bhasma treatment.
After 30 days without treatment, i.e. on day-46, the behaviour tracking
3.3.1. Fish behaviour after 15-day Suvarna Bhasma treatment was repeated to study recovery from treatment. The SB and VC groups
Fig. 6 shows the behaviour alteration of various treatment. The showed analogous motor behaviour (speed, meander and freeze point,
zebrafish motor behaviours such as speed (Fig. 6A), meander (Fig. 6B) Fig. 8) to each other without any significant variation on day-46.
and freeze points (Fig. 6C) for Suvarna Bhasma treated (SB) group was However, here also the SB group preferred the upper zone significantly
similar to vehicle control group (VC) and no significant alteration ob- (spent 2.4 min) as compared to VC group (1.49 min, p < 0.05).
served between these two groups. Whereas, the alcohol-treated group
(CC) showed a significant increase in speed and meander (p < 0.05). 4. Discussions
Caffeine treated (PC) group showed lower speed, meander and en-
hanced number of freeze points (p < 0.05). In geotaxis behaviour, SB Suvarna Bhasma is one of the most expensive medicine in Ayurved
group preferred the upper zone as compared to the VC group, similar to yet frequently prescribed by Ayurved practitioners for health improve-
the CC group (1.5% alcohol), although the motor behaviour of SB group ment. According to the traditional Ayurved practitioners, to ensure
was not similar to the alcohol-treated group. In contrary, caffeine- safety and efficacy, textual manufacturing process needs to be followed
treated group (PC) preferred the upper zone least in the novel tank (see stringently. However, due to the larger production urges and mal-
Fig. 7 for representative track plot). From the upper-lower zone tran- practices, many Ayurved drug manufacturers do not follow the tradi-
sitions, it was observed that for CC and PC group, the zone transition tional ways to manufacture Suvarna Bhasma, which could lead to toxic
was lowest (Fig. 6E). The average height of swimming (Fig. 6F) for effects instead of its benefits. Variation of physicochemical properties of
various groups showed that alcohol group swam at the maximum such metallic based medicines can lead to serious ill-effects in patients.
height throughout the tracking periods indicating anxiolytic effect, Modern science has also established the vital role of physicochemical
whereas, caffeine-treated anxious fish swam at the minimum height. properties such as size, shape and chemical composition of the nano-
Suvarna Bhasma treated group swam at more height than the control medicines for its biological effects. (Liu et al., 2013). In view of this, this
group, which indicates the anxiolytic behaviour. in-depth study was done.
The Suvarna Bhasma manufacturing process, which resembles the
3.3.2. Behaviour of zebrafish 30 days after the treatment completion top-down method of modern nanoparticle preparation, the gold bar is
The treatment regime was daily dosing for 15 days. After behaviour reduced to gold particles, size varying from 10 nm to 60 μm. During
tracking on Day 16, 30 fish from each VC and SB groups were further rigorous annealing steps (14 heating and cooling cycles) the nanogold
7
S. Biswas, et al. Journal of Ethnopharmacology xxx (xxxx) xxxx
Table 4 female rats at 15 mg/kg, and 30 mg/kg dose level after 90 days of
Grading of histopathological observation in the vehicle control group (VC) and treatment, however, such alteration was not observed in male rats.
30 mg/kg Suvarna Bhasma treated group (SB30). These liver enzymes are elevated only during cell damage. Urea and
Tissue and Histopathological Male (numbers) Female (numbers) creatinine were significantly increased in the male rats after 90 days for
Observations higher dose Suvarna Bhasma treatment. In female rats such effects were
VC SB30 VC SB30 not seen. At the low dose (3 mg/kg, therapeutic dose) treatment, both
haematology and biochemical parameters did not change except bilir-
(VC) (30 mg/kg) (VC) (30 mg/kg)
ubin in the female rat after 45 days of treatment (Supplementary file
Number of animals examined 10 10 10 10 S1-Table 4). Most importantly, the changes in the biochemical para-
Lungs meters were well within the normal range and acceptable limits. The
Alveolar histiocytosis, focal (+) 1 0 1 0
histopathological alterations observed at a dose of 30 mg/kg are usually
Increased size of BALT (+) to (++) 1 1 1 1
Leukocytic infiltration in and around 2 0 0 1 observed in the laboratory rats of this strain and age under present
bronchiolar lumen (+++) to experimental conditions. The feed intake, body and organ weights were
(++++) not changed due to the Suvarna Bhasma treatment. Therefore, in sum-
Liver mary, from the rat study, it can be concluded that Suvarna Bhasma is
Hepatocellular hypertrophy, focal (+) 0 1 1 1
Spleen
safe up to a dose of 30 mg/kg (10 times the therapeutic dose).
Increased EMH, focal (+) 1 1 1 1 Similar to our work, a previous study also reported no observed
Kidney adverse effect level of Suvarna Bhasma up to 13.5 mg/kg dose in Wistar
Dilated medullary tubules, focal (+) 0 1 0 1 rats (Jamadagni et al., 2015). Whereas, several beneficial effects of
Vacuolar degeneration in cortical 1 0 1 0
Suvarna Bhasma are also reported in literature especially as an im-
tubules, focal (+)
Mesenteric LN munomodulator: In an experimental study in mice, Suvarna Bhasma
Increased histiocytes in medullary 1 0 significantly (P < 0.001) increased counts of peritoneal macrophages
sinuses (+) and stimulated phagocytic index of macrophages indicating its im-
Colon munostimulant effect (Bajaj and Ahmad, 2001). Suvarnamalini vasanta a
Enlarged GALT (+) 1 2 1 0
Suvarna Bhasma containing generic preparation exhibited im-
Testes
Atrophy of seminiferous tubules near 1 0 munomodulatory potential as evidenced by an increase in percent
vasa recta (++)- unilateral phagocytosis and protection against E. coli induced peritonitis in mice
Atrophy of seminiferous tubules, 0 1 (Sangle et al., 2004). Madhu-Ghrita-Swarna-Vacha combination showed
multifocal (++)- unilateral
a significant effect on humoral antibody formation in neonates and it
Cervical LN
Increased histiocytes in medullary 1 0 acted on the immunological system, which was evident by triggering
sinuses (+) the response of immunological system by a rise in the total proteins and
serum IgG levels (Jyothy et al., 2014b). Suvarna bhasma was evaluated
Key: (+) = Minimal, (++) = Mild, (+++) = Moderate and (++++ in a global and focal model of ischaemia in albino rats (Shah and
+) = Severe; MNC = Mononuclear Cells, LN = Lymph Node, GALT = Gut Vohora, 2002). Enzymatic parameters (lipid peroxidase, reduced glu-
Associated Lymphoid Tissue, BALT = Bronchiole Associated Lymphoid Tissue, tathione, catalase, glutathione reductase, glutathione‐S‐transferase,
EMH = Extra Medullary Haematopoiesis.
glutathione peroxidase, superoxide dismutase, and glucose‐6‐phosphate
dehydrogenase) were used to assess the ischaemic brain damage and its
particles formed in Suvarna Bhasma were in spherical shape. EDAX
modulation by using Suvarna Bhasma. Suvarna Bhasma (25 mg/kg, or-
mapping demonstrated that the nano-sized gold particles were sur-
ally for 10 days) significantly restored the altered values to near normal
rounded by Si, O, P, Fe and Ca. The entrapment of nano-gold particles
levels suggesting its potential in cerebrovascular diseases.
might happen in the heating steps, SiO2 mainly come from the earthen
On the other hand, zebrafish behavioural assessment is able to
pot used during the preparation process. ICP-AES study revealed that
screen various toxic or neurotoxic drugs. Drug-induced anxiogenic or
Suvarna Bhasma used in this study contained approximately 98% Au
anxiolytic behaviours are well defined in the literature for zebrafish (V
along with Si, Fe, Na, and Ca. It is important to note that from a tox-
Kalueff et al., 2013). Motor behaviour, scototaxis, geotaxis and cohort
icological viewpoint that despite being used in the manufacturing
behaviour of zebrafish are studied extensively in recent literature
process, Hg was not found in the finished product even after using three
(Maximino et al., 2010). In this study, we find that the motor behaviour
different elemental analytical methods, and this is in agreement with
of zebrafish did not alter significantly due to the action of Suvarna
the toxicological data presented above. This can be explained as fol-
Bhasma treatment. Average speed, meander, freezing points etc. for
lows: In the manufacturing process, the mercury in the Hg–Au amal-
Suvarna Bhasma treated and control fish were similar. However, geo-
gamate was extracted and eliminated with the help of S powder suc-
taxis behaviour such as swimming height and preference of height zone
cessively in each of the 14 cyclic heating and cooling steps. Due to the
was significantly changed (p < 0.05) in Suvarna Bhasma treated fish
strong affinity between Hg and S and the repetitive heating and cooling
compared to the vehicle control group. The SB and CC groups spent
steps, Hg was completely eliminated.
more time in the upper zone (> 2 min) as opposed to the VC and PC
The toxicity assessment of Suvarna Bhasma in the rat model was
groups (less than 2 min). Although, the motor behaviour of CC group
conducted with the dose up to 30 mg/kg which was approximately 10
differ from SB group. Usually, zebrafish in the novel tank initially prefer
times higher as compared to the therapeutic dose in human. In hae-
the bottom of the tank due to protective intuition. Preference to upper
matological parameters, after 90 days, neutrophil counts in the male
zone in the novel tank for Suvarna Bhasma treated groups indicated
rats of all Suvarna Bhasma treated groups was increased compared to
anxiolytic-like behaviour similar to alcohol-treated groups (CC) which
the control group. However, this alteration was not statistically sig-
is anxiolytic to zebrafish (Tran et al., 2016). Anxiogenic caffeine-treated
nificant. Moreover, neutrophil counts with similar dose did not causes
fish, in contrast, preferred lower zone.
similar changes in Suvarna Bhasma treated female rats. Therefore,
There is always a chance that toxicity of any drug may appear long
haematology parameters in rats did not indicate any adverse effect of
after completion of the drug treatment. To address this issue, in this
Suvarna Bhasma, even at a considerably high dose as compared to the
study, zebrafish were tracked 30 days after completion of the drug
therapeutic dose in human.
treatment, which resulted in no unnatural motor behaviour in the fish.
A few alterations in the serum biochemical parameters were ob-
However, similar to the previous novel tank experiment, Suvarna
served in rats. ALT, AST parameters decreased significantly in the
Bhasma treated fish preferred upper zone compared to the VC group.
8
S. Biswas, et al. Journal of Ethnopharmacology xxx (xxxx) xxxx
Fig. 5. Histopathology of vehicle control (VC) and 30 mg/kg SB treated rat (SB30). Various organs were imaged, such as A) Liver B) Kidney C) Heart, D) Lungs, E)
Testis, and F) Epididymis. No treatment related alterations in tissue morphology were observed.
Fig. 6. Behavioural study of Suvarna Bhasma treated zebrafish. The fish were tracked a day after 15 days of Suvarna Bhasma treatment. A) Speed, B) Meander, C)
Freeze points, D) Time spent in the upper zone, E) Number of transition between upper zone and lower zone, and F) Average swimming height from the bottom of the
novel tank. VC = vehicle control group, SB = Suvarna Bhasma treated group, CC = comparative control, and PC = positive control group. Values are expressed as
mean ± SEM with *p < 0.05 vs VC group, #p < 0.05 vs SB group and, @p < 0.05 vs CC group. No statistical variation was observed between VC and SB groups in
motor parameters. However, geotactic behaviour such as the preference of swimming zone significantly varied between SB and VC groups.
9
S. Biswas, et al. Journal of Ethnopharmacology xxx (xxxx) xxxx
Fig. 8. Zebrafish behaviour of Suvarna Bhasma treated groups 30 days after completion of the drug treatment. A) Average speed, B) Meander, C) Freeze points, D)
Time spent in the upper zone, E) Number of transition between upper zone and lower zone, and F) Average swimming height from the bottom of the novel tank.
VC = vehicle control group, and SB = Suvarna Bhasma treated group. Values are expressed as mean ± SEM (n = 30 animal/group) with *p < 0.05. All motor
parameters of Suvarna Bhasma treated fish were normal and analogues with the vehicle control group. However, here also SB group preferred upper zone compared to
the VC group.
For zebrafish, preference towards the bottom of the tank is in response implants mediate antiapoptotic, anti-inflammatory and neuroprotective
to the novel environment. However, for SB treated fish, it swims at a effects (Østergaard et al., 2010). Ionic gold inhibits of proinflammatory
higher level in compared to VC group. Therefore, it can be inferred that mediators such as tumor necrosis factor alpha (TNFα), interleukin-1
Suvarna Bhasma has an anxiolytic effect on zebrafish behaviour model and interleukin-6, leukotrienes, prostaglandins, nitrogen oxide and ly-
and this anxiolytic effect presumed long after completion of the drug sosomal proteases (Østergaard et al., 2010). Gold ion also heals injured
treatment. neurons by increasing neurotrophin (NT-4), transforming growth
Gold, in modern medicine, is used mainly in the therapy of rheu- factor-beta 3 (TGF-β3), leukemia inhibitory factor (LIF), and me-
matoid arthritis. Additionally, bio-liberated gold ions from gold tallothionein (MT-I + II) which may be the reason for the
10
S. Biswas, et al. Journal of Ethnopharmacology xxx (xxxx) xxxx
neuroprotective effect of gold. Interestingly, in Ayurveda Suvarna and Technology (DST), India and Indian Council of Medical Research
Bhasma is also used as a nerve tonic and to treat various neuronal (ICMR), India for infrastructure. Authors are thankful for technical and
diseases. In recent studies, several neurons related beneficial effect has animal experimentation assistance of Mr. P. Salunkhe, Mr. J. Tare, Mr.
been reported in the literature, such as neuroprotective against cogni- M. Mali and Mr. S. Lokhande in National Institute for Research in
tive impairment or as anxiolytic and antidepressant agent (Bajaj and Reproductive Health (NIRRH).
Vohora, 2000). Although Suvarna Bhasma contains gold particles (Au 0),
it can release ionic gold in biological medium (e.g. gastric fluid). This Appendix A. Supplementary data
bio-released gold ion may contribute its neuroprotection.
Anxiety, depression and various neurodegenerative diseases are Supplementary data to this article can be found online at https://
widely prevalent in the modern stressful lifestyle. Anxiety is a part of doi.org/10.1016/j.jep.2019.112388.
the normal behavioural repertoire of defence mechanism to deal with
novel environment. GABAergic neurotransmission in the amygdala al- References
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Jamadagni, P.S., Jamadagni, S.B., Singh, A., Singh, R.K., 2015. Toxicity study of swarna
This project (grant no: DO/2018-SDLP001) was funded by Shree bhasma, an ayurvedic medicine containing gold. Wistar Rats, Toxicol. Int 11–17.
https://doi.org/10.22506/ti/2015/v22/i3/137618.
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Conceptualization: Snehasis Biswas (SB1), GV, JB. 158978.
Methodology: SB1, RD, NS, Sharad Bhagat (SB2). Khan, A.Y., Sheikh, A.A., Tenpe, C.R., Patole, A., Biyani, K.R., 2018. Neuroprotective
efficacy of swarna bhasma on sleep deprived induced cognitive impairment in rats.
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Supervision: GV, JB. Khedekar, S., Priya, A., B, P., M, N., PK, P., 2016. immunomodulatory activity of swarna
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of gold nanoparticles with both phagocytic and nonphagocytic cells. Langmuir 29,
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