anti-diabetic activity and safety assessment of ayurvedic...

Indian Journal of Experimental Biology

Vol. 51, October 2013, pp. 811-822

Anti-diabetic activity and safety assessment of Ayurvedic medicine, Jasada

bhasma (zinc ash) in rats

Rinku D Umrani, Durgashankar S Agrawal1 & Kishore M Paknikar*

Centre for Nanobioscience, Agharkar Research Institute, G. G. Agarkar Road, Pune 411004, India

Received 21 March 2013; revised 21 June 2013

Jasada bhasma (zinc ash) is an extensively used Ayurvedic medicine for treating diabetes mellitus. The present

communication presents yet unavailable comprehensive scientific data on its physico-chemical nature vis-à-vis anti-diabetic

activity and toxicity profile.Zinc ash prepared by traditional method was found to consist of 200-500 nm sized particles,

predominantly zinc oxide with hexagonal wurtzite crystal structure.The effective dose range of zinc ash in oral glucose

tolerance tests performed using normoglycemic Wistar rats was found to be 3-30 mg/kg. Subsequently anti-diabetic activity

was assessed in streptozotocin induced type 1 and type 2 diabetic rats. Four weeks treatment with zinc ash (1, 3, 10 mg/kg)

resulted in improved glucose tolerance (16-19%), lowered blood glucose levels (20-33%) and reduced serum insulin levels

(27-32%). Systemic absorption was assessed by single dose pharmacokinetic study where serum zinc levels were found to

be elevated (3.5 folds) after oral administration of zinc ash. Acute and sub-acute toxicity tests demonstrated safety of zinc

ash up to 300 mg/kg doseie. 100 times the efficacy dose in rats.These findings, the first of their kind, provide concrete

scientific evidence that justifies usage of zinc ash in diabetes treatment.

Keywords: Ayurveda, Diabetes, Jasada bhasma, Pharmacokinetics, Rats, Streptozotocin, Toxicity, Zinc ash

Indian system of medicine, Ayurveda uses several

metal based preparations (bhasmas) for the treatment

of diseases like anemia, jaundice, skin diseases,

tuberculosis, sexual disorders, urinary disorders,

tumors, colitis, osteoporosis, ischemia, arthritis and

diabetes1. In the texts of Rasashastra (a branch of

Ayurveda dealing with metallic medicines), bhasmas

of Mandura (iron), Vanga (tin), Naga (lead), Tamra

(copper) and Jasada (zinc) have been mentioned for

the treatment of diabetes2. Jasada bhasma is cited for

use in several other conditions including, anemia,

neuromuscular diseases, eye diseasesand as a wound

healing, anti-microbial and anti-aging agent3-5

.

However, very few studies investigating the anti-

diabetic effects of Jasada bhasma are reported. An

early anecdotal studyshowed anti-diabetic activity of

Jasada bhasma in diabetic patients6. There are sporadic

reports on reduction of fasted glucose levels7, improved

glucose tolerance8, anti-diabetic activity

9 in rats treated

with Jasada bhasma. In fact a modern version of

Jasada bhasma, viz. zinc oxide nanoparticles have

been thoroughly investigated for their anti-diabetic

effect10

. Taking an inspiration from this work, it was

thought worthwhile to investigate Jasada bhasmafor its

anti-diabetic activity and toxicity assessment.

Several researchers have synthesized bhasmas and

employed modern imaging techniques to determine

the particle sizes11-15

. These studies suggested that

bhasmas, when prepared by traditional methods,

contain sub-micronic particles.Traditionally, Jasada

bhasmais administered orally with different vehicles

such as honey, milk or ghee for a prolonged period of

time to attain therapeutic effect. The lack of

bioavailability data has resulted in the belief that it

acts through local effects at the intestine, which needs

to be validated experimentally. Presence of heavy

metals in Ayurvedic medicines has been a cause of

serious concerndespite their traditional and

widespread use16-18

. It is necessary to prove their

safety at therapeutic doses on the basis of well-

established toxicological tests and models.

Clearly, there is a need for undertaking a systematic

study on the physico-chemical characterization,anti-

diabetic efficacy, oral bioavailability, and toxicity of

Jasada bhasma. This communication describes

detailed studies on a Jasada bhasma sample

synthesized by traditional documented method. The

study confirms sub-micronic nature of the bhasma and

____________

Present address: 1Mrutyunjaya Clinic, 78/2 BiyabaniDhar Road, Indore 452 002, India

*Correspondent author

Telephone: 020-25653680

Fax: 020-25651542

E-mail: [email protected]

INDIAN J EXP BIOL, OCTOBER 2013

812

provides pharmacological evidence of its anti-diabetic

activity in both type1 and type 2 diabetic rats. Also,

pharmacokinetic studies show systemic absorption of

orally administered Jasada bhasma. Toxicological

studies establishing safety up to 100 times the

therapeutic dose are also described.

Materials and Methods Synthesis of Jasada bhasmasample—Zinc metal was

subjected to the traditional method of shodhan

(purification) and maaran (incineration). For shodhan,

zinc was melted and poured in sesame oil

(Sesamumindicum). On cooling, the mixture was

filtered and the residue again melted and poured in oil.

This procedure was repeated seven times. Similarly,

seven cycles each were carried out using butter milk,

cow urine and kulathi (aqueous extract of

Dolichosbiflorus, horse gram seeds). Finally, the

molten zinc was poured in lime water in an earthen pot

and kept for 3 h. Purified zinc so obtained was ground

with equal parts of mercury until converted to pishtee

form (amalgam). Lime juice was then added and

further ground. Once, paste was converted to black

colour, it was thoroughly washed with warm water and

air dried. Paste was then ground with equal amount of

pure sulfur and converted to powder. For maaran, the

powder was transferred to earthen crucibles and sealed

with clay. Calcination was then carried out by heating

up to 500 °C in kukkut put (a pit in the ground, 9 × 9 ×

9 inches in size). After overnight calcination, the

product was taken out, ground and again calcined. This

cycle was repeated 30 times to get the final product19,20

.

The bhasma sample so obtained (zinc ash) was

analyzed for size, shape and elemental composition

using the following techniques.

Evaluation by classical tests of Ayurveda—The

synthesized zinc ash was subjected to several tests

mentioned in Ayurveda that evaluate the particle size,

density and chemical stability of bhasmas. These tests

include (a) Rekhaapoornatwa (particles in bhasma

should be fine enough to lodge itself between the fine

lines of fingers when rubbed), (b) Vaaritaratva

(bhasma when sprinkled should float on water and not

settle down), (c) Nischandratva (should not shine in

sunlight), (d) Nirutthatva (should not form an alloy

when burned on a thin silver sheet) and (e)

Apunarbhawatwa (on heating with ghee, honey and

borax; should not lead to reappearance of the source

material).

Scanning electron microscopy (SEM) coupled with

electron dispersive spectroscopy (EDS)—Powder

suspensions of zinc ash(1 mg/mL) were spin coated

on glass cover slips and imaged using Scanning

Electron Microscope (Model JSM-7001, JEOL,

Tokyo, Japan) after gold sputtering. Spot EDS of the

particles was performed to analyze the elemental

composition.

High resolution transmission electron microscopy

(HRTEM) coupled with selective area electron

diffraction (SAED)—Powder sample was sprinkled on

adhesive carbon tape, placed on copper grid and

visualized using Transmission Electron Microscope

(Model Tecnai-G2, FEI, Hillsboro, USA). SAED was

carried out to gather information on crystal structure.

Xray diffraction (XRD)—Additional information

on morphology and chemical composition was

obtained from XRD studies. Powder XRD was

performed using desktop X Ray diffractometer

(Model MiniFlex II, Rigaku, Tokyo, Japan) with Cu

Kα radiation (K value 0.9 and 1.5405 A°) and 2θ

ranging from 10-80°.

Atomic absorption spectroscopy (AAS)—Zinc ash

sample was acid treated and its zinc content estimated

using Atomic Absorption Spectrometer (Model

AAnalyst 800, Perkin Elmer, Waltham USA).

Other chemicals—Streptozotocin, glibenclamide

and pioglitazone were purchased from Sigma Aldrich

Corporation, St. Louis, USA. Carboxymethyl

cellulose (CMC) used for preparation of zinc ash

suspensions was purchased from Merck, Darmstadt,

Germany. Glucose used in oral glucose tolerance test

(OGTT)was procured from HiMedia Laboratories

Limited, Mumbai, India.

Experimental animals—All animal experiments

were performed after the approval of the Institutional

Animal Ethics Committee. Adult Wistar rats of both

sexes (~ 8 weeks old, weighing 200-250 g) were used

in the experiments. In case of studies in type 2

diabetic rats, five day old pups that were injected

streptozotocin were allowed to grow until 12 weeks of

age and then included in the study. Rats of different

groups and sexes were housed in different

polypropylene cages. Standard laboratory pellet feed

and purified drinking water were provided ad libitum.

Temperature of the experimental room was

maintained at 22±3 °C. Relative humidity was

controlled between 30-70% and 12:12 h L:Dcycle was

maintained. Oral dosing was performed on body

weight basis using an 18 gauge oral feeding needle.

Single dose OGTT study in normoglycemic rats—

Four groups of rats (n=5) were fasted for 18 h.

UMRANI et al.: ANTI-DIABETIC ACTIVITY OF ZINC ASH

813

Subsequently, zinc ash suspension made in 0.5%

aqueous solution of CMC was administered to treatment

group rats (final dosage 30, 100 and

300 mg/kg). Untreated ‘control’ group rats were dosed

withCMC solution (0.5%, 1 mL/kg). Blood samples (~ 5

µL) were drawn from retro-orbital plexus under ether

anesthesia, after 1 h and glucose levels were checked

using glucose monitor (Accuchek Active, Roche

Diagnostics, Manheim, Germany). Each of the rats was

then given an oral glucose load of 1.5 g/kg (time of

administration being noted as ‘0’ min). Blood samples

(~ 5 µL) were then drawn at 30, 60 and 120 min

intervals and glucose levels recorded.

Repeated dose study in normoglycemic rats—Daily

oral administration of CMC (1 mL/kg) and zinc ash (3,

10, and 30 mg/kg) was carried out to control and

treatment group rats (n=5) for 14 days and blood glucose

levels were estimated on 14th day. The rats were then

fasted for 18 h after which zinc ash was administered

and OGTT performed (as described above).

Induction of diabetes in rats—Type 1 diabetes was

induced by injecting streptozotocin (45 mg/kg)in the tail

vein of adult rats21

. For induction of type 2 diabetes,

streptozotocin (90 mg/kg) was injected intraperitoneally

to 5 day old rat pups22

. Blood glucose levels were

checked one week later (type 1 model) or at 12 weeks of

age (type 2 model).Animals showing hyperglycemia

were selected and grouped (n=6, uniformly distributed

over glucose levels ~250-500 mg/dL). Glibenclamideat

the dose of 5 mg/kg23

and pioglitazone at the dose of 30

mg/kg24

were used as positive controls in studies using

type 1 and type 2 diabetic rats, respectively.

Studies in diabetic rats—Rats of non-diabetic and

diabetic control groups were administered CMC solution

(0.5%, 1 mL/kg). Treatment group rats received zinc ash

at different doses of 1, 3 and 10 mg/kg. Positive control

group rats received either glibenclamide (5 mg/kg) or

pioglitazone (30 mg/kg). Rats were dosed orally on body

weight basis,once daily for 4 weeks. On day 28, blood

samples (~ 500 µL) were drawn from non-fasted rats

and glucose levels estimated. Serum was separated by

centrifugation, stored and analyzed later for insulin

levels using rat insulin ELISA kit (Millipore

Corporation, Billerica, USA). Next day, fasted rats (18

h) were subjected to OGTT(as described above). Blood

glucose levels were recorded at all the time points.Blood

samples (~ 500 µL) drawn at 0 min time point were

centrifuged, serum separated and stored until analysis of

insulin levels. Serum triglycerides (TG)and non-

esterified fatty acids (NEFA) levels were later estimated

using standard kits (Randox Laboratories, Crumlin,

UK).Next day, rats were euthanized using ether over

anesthesia. Liver, kidney, spleen, heart, muscle and

pancreas were collected and stored in 10% formal saline

for further processing. Later, sections (~5 µm thick)

were cut, fixed on slides, stained using hematoxylin and

eosin, and observed under the microscope for

histopathological changes.

Dialysability study—Zinc ash was suspended in

simulated gastric buffer (0.1 N HCl, pH 1.6) or

intestinal buffer (phosphate buffered saline, pH

6.8)and dispersion transferred to a dialysis membrane

bag. The bag was placed in a beaker containing

simulated blood buffer (0.9% saline, pH 7.4) and

shaken for 30 min or 2 h. The concentrations of zinc

in the beaker as well as dialysis bag i.e. the dialyzed

and undialyzed fraction respectively, were measured

by AAS. Percent dialysability was calculated by the

formula:

D (%) = [dialyzed/(dialyzed + undialyzed)] ×100

Single dose pharmacokinetic study—Adult Wistar

rats were divided into 2 groups of 3 male and 3 female

rats each. Control and treated rats were orally

administered CMC solution (0.5%, 1 mL/kg) and zinc

ash (300 mg/kg) respectively. Blood samples (~ 800

µL) were drawn through retro-orbital plexus under

ether anesthesia at 0, 0.5, 1, 2, 4, 8 and 24 h time points

after dose administration. Serum was separated and

analyzed for zinc levels by AAS. After 24 h time point

blood collection, anesthetized animals were

immediately euthanized and tissues (liver, kidney,

heart, spleen, pancreas, muscle, adipose and brain)

were collected. Tissue samples were acid digested and

processed to estimate zinc content by AAS.

Acute toxicity study—Wistar rats were distributed

into groups of five animals each. Zinc ash was

administered orally (30 and 300 mg/kg doses) to

treatment group rats, while control rats received CMC

solution (0.5%, 1 mL/kg). Behaviour, clinical signs and

mortality were monitored at 0.5, 1, 2, 4 and 8 h

intervals post dosing on the first day. Subsequently, all

parameters were monitored at least once a day for 14

days. Any signs of abnormal behaviour or toxicity seen

were scored (on a scale of 0 to 5) and recorded. Body

weight was recorded on days 1, 7, 14 and 15 after

dosing. On day 15, rats were euthanized and major

organs viz. liver, kidney, spleen and heart were

observed for any gross pathological changes. Organs

were then collected, their weights recorded; and stored


814

in formal saline to be processed later for

histopathological examination.

Sub-acute toxicity study—Adult Wistar rats were

distributed into groups of five animals each. CMC

solution (0.5%, 1 mL/kg) or zinc ash (30 and 300

mg/kg) was orally administered, once daily to rats for

28 days. On day 28 of treatment, blood samples

(~ 500 µL) were drawn from retro-orbital plexus and

serum collected. Biochemical parameters viz. serum

glutamic oxaloacetic transaminase (SGOT), serum

glutamic pyruvic transaminase (SGPT), creatinine and

urea were measured using standard diagnostic kits

(Ranbaxy Laboratories, Gurgaon, India). On day 29,

animals were euthanized, major organs collected,

weighed and stored in formal saline for later

histopathological examination.

Statistical analyses—Data are presented and

analyzed using GraphPad Prism software. The mean

and standard error of mean (S.E.M.) values of each

group are shown in the tables and graphs. Area Under

Curve (AUC) valueswere calculated from OGTT

graphs.Statistical significance was tested by One Way

Analysis of Variance (ANOVA) followed by

Dunnett’s Multiple Comparison Test.

Results

Evaluation by classical tests of Ayurveda—

Compliance to tests evaluating the particle size,

density and chemical stability of bhasmas as

mentioned in standard Ayurvedic texts indicated

complete conversion of metal to oxide form and

desired size reduction.

Scanning electron microscopy (SEM) coupled with

electron dispersive spectroscopy (EDS)—SEM of

zinc ash showed 200 to 500 nm sized particles. EDS

data showed 29.8% zinc content in the sample. Other

elements viz. oxygen (34.3%), sodium (15.2%), sulfur

(13.3%) and potassium (7.5%) were also present.

Toxic metals viz. mercury, lead, arsenic, aluminium,

antimony and bismuth were not detected.

High resolution transmission electron microscopy

(HRTEM) coupled with selective area electron

diffraction (SAED)—TEM image (Fig. 1a) of zinc ash

also showed 200 to 500 nm sized particles, more or

Fig. 1Physicochemical characterization of zinc ash.(a) transmission electron microscopy image; (b) high resolution transmission electron

microscopy image; (c) fast fourier transform image; (d) X ray diffraction pattern.


815

less spherical. HRTEM image (Fig. 1b) clearly

showed the lattice arrangement of atoms.Fast Fourier

Transformation (FFT)of the HRTEM image (Fig. 1c)

and SAED showed atoms (brighter spots) arranged in

hexagonal pattern suggesting wurtzite structure of

zinc oxide.

X ray diffraction (XRD)—The XRD pattern of zinc

ash is presented in Fig. 1D. Zinc ash showed four

peaks at 31.9, 36.4, 47.7 and 56.5; in agreement with

standard zinc oxide (JCPDS data no. 75-0576);

indicating (100), (101), (102) and (110) phase

structure. Three other peaks seen at 27.0, 28.7 and

30.7 can be attributed to the presence of hexagonal

zinc sulfide (JCPDS data no 75-1547).

Atomic absorption spectroscopy (AAS)—Zinc ash

sample was found to contain 70.7% zinc,

corresponding to 88% zinc oxide.

Single dose OGTT study in normoglycemic rats—

Results obtained showed that treatment with zinc ash

(100, 300 mg/kg) caused significant suppression of

glucose levels at 30, 60 and 120 min time points

(Fig. 2a) suggesting improved glucose tolerance.

Reduction in AUC values was also seen; statistically

significant at 100 and 300 mg/kg dose (Fig. 2b).

Repeated dose study in normoglycemic rats—Zinc

ash treatment (at all the doses) resulted in significant

reduction of nonfasted blood glucose levels as

compared to control group (Fig. 2c). Fasted glucose

levels were not altered with treatment. Lack of effects

on fasted glucose levels in normoglycemic condition

suggested no risk of hypoglycemia. In OGTT

conducted at the end of treatment, zinc ash showed

modest reduction of AUC values (Fig. 2d).

Studies in type 1 diabetic rats—Zinc ash treatment

showed suppression of glucose levels in OGTT and

reduction of AUC values (~16%, Fig. 3a) suggesting

improved glucose tolerance. Glibenclamide treatment

also showed suppression of glucose peaks in OGTT.

Type 1 diabetic rats exhibited hyperglycemia under

fasted as well as non-fasted conditions. Treatment

Fig. 2Range finding studies in normoglycemic rats. [(a) baseline corrected blood glucose levelsand; (b) baseline corrected AUC values of

OGTT performed after single administration of zinc ash (ZA) to normoglycemic rats; (c) Nonfasted blood glucose levels; (d) AUC values of

OGTT after 2 weeks zinc ash(ZA) treatment to normoglycemic rats. P values: *<0.05 and **<0.001 when compared with control, as

analyzed by One Way ANOVA followed by Dunnett’s test]


816

with zinc ash resulted in dose dependent reduction of

nonfasted glucose levels (~ 20% at 10 mg/kg dose,

Fig. 3b). Glibenclamide treatment resulted in modest

reduction of nonfasted blood glucose levels. A

significant reduction in the fasted circulating glucose

levels (~ 33%) was seen at 3 and 10 mg/kgdoses of

zinc ash (Fig. 3c). Nonfasted serum insulin levels

decreased with zinc ashtreatment (~ 32% at 10 mg/kg

dose, Fig. 3d) as compared to diabetic control group.

No effect was seen on fasted insulin levels after zinc

ash treatment. Glibenclamide showedmodest increase

in nonfasted as well as fasted insulin levels, as is

expected in the case of type 1 diabetic rats.

Body weight, feed intake and lipid parameters of

type 1 diabetic rats are shown in Table 1. Diabetic

control rats displayed polyphagiaand polydipsiaas

compared to nondiabetic rats. Treatment with zinc

ashor glibenclamidedid not affect feed intake in

diabetic rats. Nondiabetic rats gained ~ 3 times more

body weight than diabetic rats. Body weight was

reduced in zinc ash (10 mg/kg) group, albeit not

statistically significant. Glibenclamide treatment

showed body weight neutrality. In case of lipid

parameters, no significant changes were noticed in

serum TG levels of diabetic control and zinc ash

treated rats. Also, the escalated NEFA levels in

diabetic rats could not be normalized by zinc ash

treatment. As expected, neither TG nor NEFA levels

were reduced by glibenclamide treatment.

Microscopic examination of tissue sections revealed

kidney damage and pancreatic islet necrosis in

diabetic control group as compared to nondiabetic

control group. Tissue sections of zinc ashtreated

groupsdid not reveal any further histopathological

changes.

Studies in type 2 diabetic rats—Treatment with

zinc ash resulted in suppression of glucose peaks in

OGTT suggesting improved glucose tolerance.

Significant reduction of AUC values (~19%) was seen

in zinc ash (3 mg/kg) group (Fig. 4a). A significant

reduction in nonfastedand fasted glucose levels

(~20% and ~25% respectively at 10 mg/kg dose,

Fig. 4b and 4c) was also seen after zinc ash treatment,

comparable to pioglitazone (~21% and ~18%

Fig. 3Anti-hyperglycemic activity of zinc ash (ZA) in type 1 diabetic rats. NDC, DC and Glb indicate nondiabetic control, diabetic control

and glibenclamide. [(a) AUC values of OGTT; (b) nonfasted blood glucose levels; (c) fasted blood glucose levels; (d) nonfasted serum

insulin levels. P value: **< 0.001 when compared with diabetic control, as analyzed by One Way ANOVA followed by Dunnett’s test]


817

respectively). Further, zinc ash treatment showed

trend towards decrease in nonfasted insulin levels

(~27% at 10 mg/kg dose, Fig. 4d), similar to

pioglitazone. Fasted insulin levels were not affected

by zinc ash treatment. These results suggested

possible insulin sensitizing effects of zinc ash in

diabetic rats.

As can be seen in Table 1, type 2 diabetic rats were

dyslipidemic as compared to non-diabetic rats. Zinc

ash treatment (10 mg/kg) resulted in modest reduction

Table 1Body weight, feed intake and lipid parameters of type 1 (A) and type 2 (B) diabetic Wistar rats.

[Values are mean ± SE]

Treatment group Body weight gain (g) Feed intake (g/day) Serum triglycerides (mg/dL) Serum NEFA (mmol/L)

Non-diabetic Control A 43.8 ± 5.6 19.7 ± 0.8** 61.0 ± 1.2 0.35 ± 0.0

B 36.2 ± 3.0** 18.3 ± 1.8 25.5 ± 4.0 0.35 ± 0.0*

Diabetic Control A 14.8 ± 7.4 27.8 ± 0.9 59.7 ± 6.3 0.60 ± 0.1

B 10.2 ± 4.8 23.9 ± 4.1 37.6 ± 2.4 0.55 ± 0.1

Zinc ash (1 mg/kg) A 17.7 ± 15.3 29.2 ± 1.6 65.5 ± 9.4 0.58 ± 0.1

B 5.0 ± 4.9 21.2 ± 3.9 37.2 ± 3.8 0.67 ± 0.1

Zinc ash (3 mg/kg) A 16.6 ± 11.0 26.8 ± 1.2 57.7 ± 5.3 0.56 ± 0.1

B 13.0 ± 3.3 23.3 ± 4.1 40.8 ± 1.4 0.72 ± 0.1

Zinc ash (10 mg/kg) A -11.2 ± 6.5 27.1 ± 1.2 63.6 ± 9.6 0.50 ± 0.1

B 8.5 ± 4.0 22.3 ± 1.8 31.3 ± 3.4 0.70 ± 0.0

Glibenclamide (5 mg/kg) A 18.0 ± 4.7 26.0 ± 1.1 56.6 ± 3.0 0.54 ± 0.1

Pioglitazone (30 mg/kg) B 29.7 ± 5.5* 23.3 ± 2.3 24.7 ± 2.1* 0.64 ± 0.0

ZA = zinc ash, NEFA = non esterified fatty acids

P values: *< 0.05, **< 0.001, when compared to diabetic control, as analyzed by One Way ANOVA followed by Dunnett’s test.

Fig. 4Anti-hyperglycemic activity of zinc ash (ZA) in type 2 diabetic rats. NDC, DC and Pio indicate nondiabetic control, diabetic control

and pioglitazone. [(a) AUC values of OGTT; (b) nonfasted blood glucose levels; (c) fasted blood glucose levels; (d) nonfasted serum insulin

levels. P values: *< 0.05 and **< 0.001 when compared with diabetic control, as analyzed by One Way ANOVA followed by Dunnett’s test]


818

of TG levels. Pioglitazonetreatment, as expected,

resulted in significantTG reduction. Neither zinc ash

nor pioglitazone could result in reduction of NEFA

levels. Type 2 diabetic rats too displayed lower body

weight gain despite polyphagia as compared to non-

diabetic rats. Pioglitazone treatment significantly

increased the body weight gain (known side effect of

thiazolidinediones). Zinc ash treatment did not result

in any significant effect on body weight gain and feed

intake. Further, zinc ash treatment did not result in

any histopathological changes as compared to diabetic

control group.

Dialysability study—The cumulative release (under

acidic and basic conditions) of zinc ions from zinc ash

was to the tune of 30%. The undialyzable component

of zinc ash was >50% suggesting that under in vivo

conditions, zinc ashsplits into two phases, the soluble

or ionic zinc and the insoluble or particulate zinc.

Single dose pharmacokinetic study—Zinc ash

treatment resulted in higher serum zinc concentration

(~ 3.5 mg/L) as compared to control (~ 1 mg/L,

Fig. 5). Maximum concentration (Cmax) was attained

at 2 h time point, after which zinc levels declined and

came back to basal values within 4 h. No significant

change was seen in tissue zinc levels of control and

zinc ash treated groups.

Acute toxicity study—No abnormal behaviour,

clinical signs or mortality was seen in any of the

treated rats indicating safety after single oral

administration of high dose of zinc ash. There was no

significant difference in body weight gain of zinc ash

treated group rats as compared to control group rats.

No significant effect on relative organ weights was

seen in any of the treatment groups as compared to

control group. No gross pathological changes were

seen after single dose administration of zinc ash.

Major organs (liver. kidney, heart and spleen)

appeared normal in rats of zinc ash treated groups

after histological examination.

Sub-acute toxicity study—No significant effect was

seen on body weight gain or relative organ weights of

rats treated with 30 and 300 mg/kg doses of zinc ash.

No significant effect was seen on SGOT or SGPT

activity (Fig. 6a and 6b) suggesting no major

hepatotoxicity or cardiotoxicity. Serum creatinine

(Fig. 6c) and urea levels were also not altered after

four weeks treatment with zinc ash.No major

histopathological changes were observed (Fig. 7) in

zinc ash treated groups as compared to control group.

These results indicate no risk of organ damage after

multiple administrations of high doses of zinc ash.

Discussion Although bhasmas have proved their clinical

efficacy in Ayurveda, there are several issues that

limit their acceptability in modern medicine. Firstly,

Fig. 5Effect of single oral dose of zinc ash (ZA, 300 mg/kg) on

serum zinc levels at different time points.

Fig. 6Effect of 4 weeks zinc ash (ZA) treatment on (a) SGOT

activity; (b) SGPT activity; (c) serum creatinine levels of Wistar rats.


819

the synthesis procedures are laborious, time

consuming and often difficult to interpret from

ancient texts. Secondly, commercial preparations may

not be authentic as standards for manufacture and

quality control are not yet properly defined. These

lacunae tend to permit sale/availability of sub-

standardproducts in the market (with impurities such

as heavy metals) often causing undesirable toxicity

effects upon prolonged use25

.

In the present work Jasada bhasma (zinc ash) was

synthesized as per standard procedures mentioned in

Ayurvedaand subjected to classical tests26,27

. Besides

these tests detailed characterization of the preparation

was carried out using modern analytical and imaging

techniques to determine its physical and chemical

nature and to detect heavy metal contaminants, if any.

SEM of the zinc ash sample used in the present study

revealed 200 to 500 nm particles in the preparation,

confirming its sub-micronic nature. HRTEM analysis

also established the sub-micronic nature of zinc ash.

The FFT image showed well defined spots,

confirming the single crystalline nature of zinc ash.

The spot pattern can be assigned to the hexagonal

phase of zinc oxide28

. Bhowmick et al.29

reportedzinc

oxide as nanoparticles in the filtered fraction of

Jasada bhasma whereas the unfiltered fraction

contained micron sized particles. However,

nanoparticles could not be detected in the present zinc

ash sample. EDS of zinc ash sampleused in the

present study revealed the presence of zinc and

oxygen suggesting formation of zinc oxide. Detection

of sulfur in the sample indicated the possibility of zinc

sulfide formation. No traces of mercury were

detected. The EDS results were in agreement with

XRD data that showed peaks corresponding to zinc

oxide and zinc sulfide. Nonetheless, lesser amount of

sulfur as compared to oxygen suggested zinc oxide as

the predominant component. Several researchers have

used atomic absorption spectroscopy (AAS) to

estimate metal content in bhasma samples14,30

. Zinc

oxide content in the present sample worked out to

88% (estimated as zinc by AAS) which is higher than

reported 69.5% by Shubha and Hiremath31

. These

results in conjunction with imaging and EDS data

suggest optimal oxidization as a result of incineration

process confirming that the zinc ashpreparation

consisted of sub-micronic particles, predominantly of

zinc oxide.

During the synthesis process of zinc ash,

ingredients such as sesame oil (Sesamumindicum),

butter milk, cow urine and kulathi (aqueous extract of

Dolichosbiflorus, horse gram seeds) were employed.

Some of these ingredients, viz. sesame oil, cow urine,

and horse gram seedsper sehave been reported to

possess anti-diabetic activity32-34

. It must be

mentioned, however, that any organic remnants of

these ingredientswould be oxidized during the

incineration process and hence are not likely to

contribute to any biological activity.

Preliminary range finding studies of zinc ash were

carried out in normoglycemic rats. Based on the dose

used in Ayurveda practice, 30 mg/kg was used as the

Fig. 7Photomicrographs of liver (L), kidney (K) and heart(H) of Wistar rats following administration of zinc ash (300 mg/kg). [Upper

panel: control, lower panel: experimental. H & E, 200X]


820

starting dose. To evaluate dose dependent effects, two

higher doses (100 and 300 mg/kg) were also tested.

Results of single dose OGTT study demonstrated

similar improvement of glucose tolerance at 100 and

300 mg/kg doseof zinc ash, suggesting saturation of

efficacy. Therefore, repeated dose study was carried

out at lower doses of 3, 10, 30 mg/kg. Modest

improvement in glucose tolerance at lower doses

suggested beneficial effects on multiple dosing.

After completing therange finding studies in

normoglycemic rats, zinc ash was evaluated in type 1

as well as type 2 diabetic rats (models of insulin

deficiency and insulin resistance, respectively). The

observed improved glucose clearance in OGTT can be

attributed to two major known mechanisms. Firstly,

zinc ashcouldinhibit intestinal alpha-glucosidase

enzyme and thereby reduce glucose absorption as

reported by Ueda et al.35

for anorgano-zinc complex.

Secondly, increased glucose-stimulated insulin

secretionby zinc could result in better glucose disposal.

Zinc accumulates in pancreatic beta cells due to beta

cell specific zinc transporter 8and results in increased

glucose stimulated insulin secretion36,37

. Similar

mechanisms may be operative in the case of zinc ash.

Further, improved glucose clearance in OGTT after

multiple dosing,and the fact that these results were

comparable to pioglitazone treatment suggest insulin

sensitization as a possible mechanism. In the present

study, a reduction in serum insulin levels was also seen

after zinc ash treatment, similar to pioglitazone38

.

These results also suggest that zinc ash possibly works

as an insulin sensitizer. Reduction in nonfasted as well

as fasted glucose levels was seen in zinc ash treated

type 1 (insulin deficient) and type 2 (insulin resistant)

diabetic rats, suggesting that multiple mechanismsmay

be involved. Zinc per seis known to regulate glucose

metabolism; enhance insulinhalf-life; and play a role in

insulin secretion39,40

. Further, zinc ions are reported to

enhance insulin signaling and action41

that might have

contributed to control of nonfasted hyperglycemia.

Zinc is also reported to regulate glucagon secretion

from pancreatic alpha cells42,43

. As a result, glucagon

stimulated hepatic pathways viz. glycogenolysis and

gluconeogenesis would be suppressed in fasting state

that might have contributed to reduced fasted blood

glucose levels observed in the present study. However,

further experimentation is necessary to identify the

targets of action.

It has been argued that the process of bhasmikaran

(synthesis of bhasma) results in size reduction thereby

facilitating absorption and assimilation in the body11,44,45

.

Particle solubility especially plays an important role in

bioavailability. Solubility of zinc ash was evaluated by

using a simple dialysis experiment. Researchers have not

used this technique for bhasmas although reports on

nanoparticle dissolution exist46

. It is expected that only

ions can pass through a dialysis membrane of 12 KD

cut-off value. In the present study, detection of zinc in

the dialyzed fraction indicates that zinc ash gets ionized

in aqueous media. It was found that only about 30% of

the total zinc could get dialyzed suggesting that under in

vivo conditions zinc ash is encountered as zinc ions as

well as particulates, after oral administration and hence

the extent of their uptake in the stomach and intestine

would be crucial.It is highly unlikely that zinc, in any

form, would be absorbed from the stomach47

. The

primary site of exogenous zinc absorption is the

intestine48,49

, where it occurs through the apical surface

of enterocytes50

. The soluble fraction of zinc ash could

get immediately absorbed from the intestine and enter

circulation through specific transporters (e.g. zinc

transporter 1) that are abundant along basolateral

membranes of enterocytes51

. This could result in the

initial spurt of zinc in blood as observed in the present

study. The uptake of particles from the GI tract is

governed by both their size and surface characteristics.

Thus smaller, typically nanometricparticles have been

shown to exhibit increased uptake52

. In the present study

serum zinc levels plummeted rapidly after 2 h of oral

administration of zinc ash. These results clearly suggest

that a major portion of the particulate fraction of zinc ash

is not taken up through the intestine and is probably

excreted in the feces. Nevertheless, serum zinc levels

were elevated in pharmacokinetic study, confirmingoral

absorption of zinc ash.

It is widely believed that metals are toxic limiting

their use in medicine. However, amongst the several

trace elements, zinc is probably the safest; virtually

nontoxic to living organisms. It is the only element

that isneither cytotoxicnor systemically toxic,

carcinogenic, mutagenic, or teratogenic53

. The

reported oral LD50 of zinc salts is 237-623 mg/kgin

rats54

. In the present study, toxic effects were not seen

at doses that were 10 and 100 times the optimal

therapeutic dose (3 mg/kg) of zinc ash, clearly

indicating its safety.

Several zinc complexes55-57

havebeen recognized as

potential candidate drugs by modern medicine.

However, poor oral bioavailability and toxicity

concerns associated with their usage remain as


821

formidable problems yet to be resolved satisfactorily.

Thus development of zincbased anti-diabetic drugs

has lagged behind, despite their tremendous potential.

Over this background, Jasadabhasma (zinc ash)

appears to be a clever way invented by Ayurveda for

delivering zinc to treat diabetes and other diseases.

Conclusions

Ayurvedic medicine Jasada bhasma (zinc ash)

comprises of sub-micronic particles, predominantly of

zinc oxide. Anti-diabetic activity of Jasada bhasma has

been pharmacologically validated using both type 1

and type 2 diabetes rat models. Even though the

preliminary findings suggest that the anti-diabetic

activity of zinc ash might be due to insulin sensitizing

effects, further studies to identify the targets of action

need to be carried out. Pharmacokinetic data confirm

systemic absorption of zinc ash after oral

administration. The studyalso suggests that the

preparation is free of heavy metals and is safe for use

even at 100 times the efficacy dose. To the best of our

knowledge, this is the first comprehensive study of its

kind validating/endorsing the extensive use of zinc ash

preparationin Ayurveda for the treatment of diabetes.

Acknowledgement

The authors thank Dr. Jyutika Rajwade for help

with XRD studies and Dr. MayurTemgire for

HRTEM imaging. RU gratefully acknowledges award

of senior research fellowship of the Indian Council of

Medical Research (ICMR), New Delhi, India.

References 1 Galib, Barve M, Mashru M, Jagtap C, Patgiri BJ&Prajapati

PK, Therapeutic potentials of metals in ancient India: A

review through Charaka Samhita, J Ayurveda Integr Med, 2

(2011) 55.

2 Dole VA&Paranjpe P, A textbook of Rasashastra

(Chaukhambha Sanskrit Pratishthan, New Delhi) 2004, 317.

3 Datta HS, Mitra SK&Patwardhan B, Wound healing activity

of topical application forms based on Ayurveda, Evid Based

Complement Alternat Med, 2011 (2011) Article ID 134378.

4 Shah DP, Sathaye S&Korde A, Pharmacological evaluation

of wound healing potential of Jasad bhasma using wistar

rats: a mechanistic approach, Pharmacology Online, 2 (2009)

1269.

5 Bhojashettar S, Poornima BT&Jadar PG, Evaluation of

market samples of “Yashada bhasma” using “Namburi

Phased Spot Test”, J Ayurveda Integr Med, 2(2011) 69.

6 Sathe RV, Talwalkar NG&Ajgaonkar SS, Treatment of

diabetes with an oral Ayurvedic preparation-Jasada bhasma,

J Assoc Physicians India, 8 (1960) 331.

7 Bharati, Singh RH&Chansouria JPM, Hypoglycemic

property of Shilajeet and Yashada bhasma, Ancient Sci Life,

16 (1996), 118.

8 Prasad CM&Sharma AV, Yashada bhasma: an effective

hypoglycemic drug, Ancient SciLife, 9 (1989) 69.

9 Nayagam JA, KumaraswamyA&Pemaiah B, Anti-diabetic

activity of traditional Ayurvedic formulation-Jasad bhasma,

J Pharm Res, 3 (2010) 2916.

10 Umrani R D & Paknikar K M, Zinc oxide nanoparticles

show antidiabetic activity in streptozotocin-induced

Types 1 and 2 diabetes in rats, Nanomedicine, 2013

doi:10.2217/NNM.12.205.

11 Wadekar MP, Rode CV, Bendale YN, Patil KR, Gaikwad

AB&Prabhune AA, Effect of calcination cycles on the

preparation of tin oxide based traditional drug: studies on its

formation and characterization, J Pharm Biomed Anal, 41

(2006) 1473.

12 Brown CL, Bushell G, WhitehouseMW, Agrawal DS, Tupe

SG, Paknikar KM&Tiekink ERT, Nanogold pharmaceutics

(i) The use of colloidal gold to treat experimentally-induced

arthritis in rat models; (ii) Characterization of the gold in

Swarna bhasma, a microparticulate used in traditional Indian

medicine, Gold Bull, 40 (2007) 245.

13 MohapatraS&Jha CB, Physicochemical characterization of

Ayurvedic bhasma (Swarna makshika bhasma): An approach

to standardization, Int J Ayurveda Res, 1 (2010) 82.

14 SinghN&Reddy KRC, Particle size estimation and elemental

analysis of Lauha bhasma, Int J Res Ayurveda Pharm, 2

(2011) 30.

15 UmraniR D &Paknikar KM, Ayurvedic medicine zinc

bhasma: Physicochemical evaluation, anti-diabetic activity

and safety assessment, J Biomed Nanotech, 7 (2011) 148.

16 Saper RB, Kales SN, PaquinJ, Burns MJ, Eisenberg DM,

Davis RB&Phillips RS, Heavy metal content of Ayurvedic

herbal medicine products, J Am Med Assoc, 292 (2004) 2868.

17 Saper RB, Phillips RS, SehgalA, Khouri N, Davis RB,

Paquin J, Thuppil V&Kales SN, Lead, Mercury, and Arsenic

in US- and Indian-manufactured Ayurvedic medicines sold

via the internet, J Am Med Assoc, 300 (2008) 915.

18 Dargan PI, Gawarammana IB, Archer JRH, House IM, Shaw

D&Wood D M, Heavy metal poisoning from Ayurvedic

traditional medicines: an emerging problem, Int J Env

Health, 2 (2008) 463.

19 Kulkarni DA, Rasratnasamucchaya – vijgyanprabodhini

commentary(Mehar Chand Lachhmandas Publications, New

Delhi) 1998.

20 Sharma S, Rastarangini(MotilalBanarsidas Publisher, New

Delhi) 2004.

21 Rodrigues B, Poucheret P, Battell ML&McNeill JH,

Streptozotocin induced diabetes: Induction, mechanism(s)

and dose dependency, inExperimental models of diabetes,

edited by McNeill JH (CRC Press, New York) 1999, 3.

22 Schaffer S W &Mozaffari M, The neonatal STZ model of

diabetes, inExperimental models of diabetes, edited by

McNeill JH (CRC Press, New York) 1999, 231.

23 Altan N, Yigit S, Elmali E, Malhatun E, Rota S&Kilic N,

Effect of the sulfonylurea glyburide on superoxide dismutase

in streptozotocin induced diabetic rat muscle, Gen Pharmac,

28 (1997) 795.

24 Kubota N, Terauchi Y, Kubota T, Kumagai H, Itoh S, Satoh

H, Yano W, Ogata H, Tokuyama K, Takamoto I, Mineyama

T, Ishikawa M, Moroi M, Sugi K, Yamauchi T, Ueki K,

Tobe K, Noda T, Nagai R &Kadowaki T, Pioglitazone


822

ameliorates insulin resistance and diabetes by both

adiponectin-dependent and -independent pathways, J

BiolChem, 281 (2006) 8748.

25 Pattanaik N, Singh AV,PandeyRS, Singh BK, Kumar M,

Dixit SK& Tripathi YB, Toxicology and free radical

scavenging property of Tamra bhasma, Indian J

ClinBiochem, 18 (2003) 181.

26 Savrikar S & Ravishankar B, Introduction to “Rasashaastra”-

the iatrochemistry of Ayurveda, Afr J Tradit Complement

Altern Med, 8 (2011) 66.

27 Devanathan R, Concept of bhasmikarana, Int J Res Ayurveda

Pharm, 2 (2011) 18.

28 Kumari L&Li WZ, Synthesis, structure and optical properties

of zinc oxide hexagonal microprisms, Cryst Res Tech, 45

(2010) 311.

29 Bhowmick TK, Suresh AK, Kane SG, Joshi AC&Bellare JR,

Physicochemical characterization of an Indian traditional

medicine, Jasada bhasma: detection of nanoparticles

containing non-stoichiometric zinc oxide, J Nanopart Res, 11

(2009) 655.

30 Pandit S, Biswas TK, DebnathPK, Saha AV, Chowdhury U,

Shaw B P, Sen S &Mukheerjee B, Chemical and

pharmacological evaluation of different Ayurvedic

preparations of iron, J Ethnopharm, 65 (1999) 149.

31 ShubhaH S &Hiremath RS, Evaluation of antimicrobial

activity of Rasaka bhasma, AYU, 31 (2010) 260.

32 Bigoniya P, Nishad R&Singh CS, Preventive effect of

sesame seed cake on hyperglycemia and obesity against high

fructose-diet induced type 2 diabetes in rats, Food Chem,

133(2012) 1355.

33 Jarald EE, Edwin S, Tiwari V, Garg R&Toppo E, Anti-

diabetic activity of cow urine and a herbal preparation

prepared using cow urine, Pharm Biol, 46 (2008) 789.

34 Pant MC, Uddin I, Bhardwaj UR&Tewari RD, Blood sugar

and total cholesterol lowering effect of Glycine soja (Sieb

and Zucc.), Mucunapruriens (D. C.) and Dolichosbiflorus

(Linn.) seed diets in normal fasting albino rats, Indian J Med

Res, 56 (1968) 1808.

35 Ueda E, Yoshikawa Y, Sakurai H, Kojima Y&Kajiwara NM,

In vitroalpha-glucosidase inhibitory effect of Zn(II) complex

with 6-methyl-2-picolinmethylamide, Chem Pharm Bull, 53

(2005) 451.

36 Chimienti F, Devergnas S, Pattou F, Schuit F, Garcia-Cuenca

R, Vandewalle B, Kerr-Conte J, Lomme LV, Grumwald D,

Favier A&Seve M, In vivo expression and functional

characterization of the zinc transporter ZnT8 in glucose-

induced insulin secretion, J Cell Sci, 119 (2006) 4199.

37 Richards-Williams C, Contreras JL, Berecek

KH&Schwiebert EM, Extracellular ATP and zinc are co-

secreted with insulin and activate multiple P2X purinergic

receptor channels expressed by islet beta-cells to potentiate

insulin secretion, Purinergic Signal, 4 (2008) 393.

38 Ding SY, Shen ZF, Chen YT, Sun SJ, Liu Q&Xie MZ,

Pioglitazone can ameliorate insulin resistance in low-dose

streptozotocin and high sucrose-fat diet induced obese rats,

Acta Pharmacol Sin, 26 (2005) 575.

39 Chausmer AB, Zinc, insulin and diabetes, J Am CollNutr, 17

(1998) 109.

40 Mason P, Physiological and medicinal zinc, The Pharm J,

276 (2006) 271.

41 Haase H&Maret W, Fluctuations of cellular, available zinc

modulate insulin signaling via inhibition of protein tyrosine

phosphatases, J Trace Elem Med Biol, 19 (2005) 37.

42 Franklin I, Gromada J, Gjinovci A, Theander S&Wollheim

CB, β-cell secretory products activate α-cell ATP-dependent

potassium channels to inhibit glucagon release, Diabetes, 54

(2005) 1808.

43 Egefjord L, Petersen AB, Bak AM& Rungby J, Zinc, alpha

cells and glucagon secretion, Curr Diabetes Rev, 6 (2010)

52.

44 Sarkar P K &ChaudharyAK, Ayurvedic bhasma: the most

ancient application of nanomedicine, J Sci Ind Res, 69(2010)

901.

45 Krishnamachary B, Rajendran N, PemiahB, Krishnaswamy

S, Krishnan UM, Sethuraman S&Sekar RK, Scientific

validation of the different purification steps involved in the

preparation of an Indian Ayurvedic medicine, Lauha bhasma,

J Ethnopharm, 142 (2012) 98.

46 Franklin NM, Rogers NJ, Apte SC, Batley GE, Gadd GE&

Casey PS, Comparitive toxicity of nanoparticulate ZnO, bulk

ZnO, and ZnCl2 to a freshwater microalga

(Pseudokirchneriellasubcapitata): The importance of particle

solubility, EnvironSciTechnol, 41 (2007) 8484.

47 Andersson KE, Bratt L, Dencker H&Lanner E, Some aspects

of the intestinal absorption of zinc in man, Eur J Clin

Pharmacol, 9 (1976) 423.

48 Barceloux DG, Zinc, ClinToxicol, 37 (1999) 279.

49 Krebs NF, Overview of zinc absorption and excretion in the

human gastrointestinal tract, J Nutr, 130(2000) 1374S.

50 Moltedo O, Verde C, CapassoA, Parisi E, Remondelli P,

Bonatti S, Alvarez-Hernandez X, Glass J, Alvino CG&

Leone A, Zinc transport and metallothionein secretion in the

intestinal human cell line Caco-2, J BiolChem, 275(2000)

31819.

51 Lichten LA&Cousins RJ, Mammalian zinc transporters:

Nutritional and physiologic regulation, Annu Rev Nutr, 29

(2009) 153.

52 Stern ST&McNeil SE, Nanotechnology safety concerns

revisited, ToxicolSci, 101 (2008) 4.

53 Stefanidou M, Maravelias C, DonaA&Spiliopoulou C, Zinc:

A multipurpose trace element, Arch Toxicol, 80(2006) 1.

54 Maret W&Sandstead HH, Zinc requirements and the risks

and benefits of zinc supplementation, J Trace Elem Med

Biol, 20 (2006) 3.

55 Kojima Y, Yoshikawa Y, Ueda E, Ueda R, Yamamoto S,

Kumekawa K, Yanagihara N&Sakurai H, Insulinomimetic

zinc(II) complexes with natural products: in vitro evaluation

and blood glucose lowering effect in KK-Ay mice with type 2

diabetes mellitus, Chem Pharm Bull, 51 (2003) 1006.

56 Sakurai H &Adachi Y, The pharmacology of the

insulinomimetic effect of zinc complexes, Biometals, 18

(2005) 319.

57 Nakayama A, Hiromura M, Adachi Y&Sakurai H, Molecular

mechanism of anti-diabetic zinc-allixin complexes:

regulations of glucose utilization and lipid metabolism, J

BiolInorg Chem, 13 (2008) 675.

anti-diabetic activity and safety assessment of ayurvedic...

Documents