Sankaradoss Nirmala1*,
Velayutham Ravichandiran2, A Vijayalakshmi1,
P. Nadanasabapathi3
1Research Scholar, School of Pharmaceutical Sciences, Vistas, Vels University, Chennai
2National Institute of Pharmaceutical Education and
Research, Kolkata.
3Aizant
Drug Research Solutions, Hyderabad.
*Corresponding Author E-mail: cognosy@gmail.com
ABSTRACT:
Objective: To synthesis
chitosan reduced gold particle by using gymnemic acid isolated from Gymnema
sylvestre leaves and to evaluate the antidiabetic activity against
streptozocin and highfat diet treated rats. Methods: In this research paper,
the synthesis gold particle by using gymnemic acid isolated from Gymnema
sylvestre leaves with chitosan reducer. The synthesized AuNps were
determined by UV–vis spectrum, high resolution transmission electron
microscopy, X-ray diffraction, and Fourier transmission infrared spectroscopy
analysis. The synthesized gymnemic acid coated chitosan reduced gold
nanoparticles is subjected to evaluate its antidiabetic activity using high fat
diet fed streptozotocin induced type 2 diabetes mellitus rats. Results: UV–vis
spectrum showed a peak at 520 nm due to excitation of surface Plasmon
vibrations. Fourier transmission infrared spectroscopy showed that
nanoparticles were coated with plant secondary metabolites. HR-TEM and AFM
images of synthesized AuNPS are spherical in shape and HR-TEM pictures
evidently confirms the prepared AuNPS have the uniformed spherical shape with
diameter of 14±3.69 nm in size respectively. The result of antidiabetic
activity, chitosan reduced gold nano particles shows significant in both invitro
and invivo models in high fat diet fed streptozotocin induced type 2
diabetes mellitus
KEYWORDS: Gymnemic acid,
Gold Nanoparticles, GLUT IV, PPar γ and diabetes.
1. INTRODUCTION:
Nanomaterials have innumerable
applications in variety of industries1. In general, noble metal
nanoparticles are utilized for various biomedical applications and significant
results have been obtained in the recent past2. Particularly, gold
nanoparticles (AuNPs) are employed for the treatment of various diseases3 due
to their unique optical, chemical and biological properties4. Though
variety of protocols is in practice5, preparation of AuNPs by
biosynthesis process has been the most preferred methodology for its facile and
environment-friendly approach.
However, a major
drawback of biosynthesis includes agglomeration, uncontrolled size, shape,
instability etc.
Currently employed
physical, chemical and biological methods have associated with certain problems
like toxicity, uncontrolled nucleation and polydispersity; therefore, a proper
stabilizing as well as reducing agent is indispensable for the synthesis of
monodispersive nanoparticles which is a major challenge to attain desired
relevance in the field of Nanomedicine. Consequently AuNPs formation using
biopolymers and biomolecules have attracted much attention and recently emerged
as an exciting area of research in nanotechnology field.
Gymnema sylvestre R. Br is a woody,
climbing plant that grows in the tropical forests of central and southern India
and in parts of Asia6. It is a pubescent shrub with young stems and
branches, and has a distichous phyllotactic opposite arrangement pattern, which
are 2.5-6 cm long and are usually ovate or elliptical, the flowers are small,
yellow, in umbellate cymes and follicles are terete, lanceolate, up to 3 inches
in length.
Gymnema sylvestre has been used in
the treatment of diabetes since ages in folk, ayurvedic and homeopathic systems
of medicine. It is also used in the treatment of asthma, eye complaints, family
planning, snakebite, urinary complaints, stomach ailments, piles, chronic
cough, breathing troubles, colic pain, cardiopathy, constipation, dyspepsia and
hemorrhoids, hepato splenomegally. In addition, it also possesses
antimicrobial, antitumor, obesity, anti-Inflammatory, and Antihyperglycemic
Activity7. It has been extensively studied for its
antihyperglycemic effect contains several active compounds useful for reducing
the blood glucose levels in the case of diabetes mellitus. Gymnemic triacetate
isolated from G. sylvestre was tested for its antidiabetic activity on
streptozotocin-induced diabetic model. Similarly, extensive studies have been
carried out for antidiabetes and its related complications using various
medicinal plants. In the present investigation, biosynthesis of chitosan
reduced AuNPs using antidiabetic potent gymnemic acid fraction from G.sylvestre
R. Br. and its effect on high fat diet fed and streptozotocin induced type 2
model is discussed.
2. MATERIALS AND
METHODS:
2.1 Collection and
authentication:
Gymnema sylvestre leaves were
collected from Anna Herbal Garden, Chennai, Tamil Nadu. It was identified by
Botanist, Plant Anatomy Research Centre (PARC), Tambaram, Chennai, Tamil Nadu
and specimen voucher (PARC/2012/1279) are kept at Department of
Pharmacognoscy, Vels College of Pharmacy, Chennai. The leaves of Gymnema
sylvestre were shade dried grounded and stored dry until extraction.
2.2 Extraction of Gymnemic
acid by Hoopers method8:
Step 1: Extraction with
petroleum ether (Defatting process):
I kg of dried Gymnema
sylvestre dry leaf powder was packed into a clean soxhlet extraction unit.
Seven litres of petroleum ether (60-80°C) was added and extracted for 24-36 hrs
till all components are soluble in petroleum. petroleum ether extract is
collected and distilled. Then a net of 240gm of petroleum extracts was
obtained. petroleum ether extracts was obtained.
Step 2: Extraction with 90%
methanol:
The plant material is then
extracted with 90% methanol. 90% methanol is added to the marc and the
extraction was continued up to 24-36hrs till total methanol soluble extracts
was obtained. Then methanol soluble extract was distilled and finally 185gm of
thick paste were obtained.
Step 3: Isolation of pure
gymnemic acid from methanol extract:
175gm thick paste of methanol
soluble extract was dissolved in 1% aq.KOH solution on continuously stirring
for 45 min to 1hr.The solution is then filtered through filter paper to
separate the undissolved particles. Diluted HCL was added slowly under constant
stirring, during which the gymnemic acids were precipitated. Precipitated
solution was filtered under suction and precipitate was dried. The pure
gymnemic acid was obtained. Crude gymnemic acid fraction toal yield was found
to be 29.6%. The isolated gymnemic acid fraction was subjected to qualitative
chemical test and thin layer studies and positive tests for steroids,
terpenoids and glycosides. For further studies the gymnemic acid fraction was
dissolved in ethanol and used.
2.3 Chemicals:
Gold (III) chloride
trihydrate (HAuCl4.3H2O, 99.99%), Gymnemic acid, nitric acid (HNO3)
and hydrochloric acid (HCL) were purchased from Sigma-Aldrich. DMEM without FCS
medium, penicillin/streptomycin and glutamine, Foetal calf serum was
purchased from sigma aldrich. Streptozotocin from Sigma, USA. Cholic acid is
purchased from Loba Chemie. Pvt. Ltd’ Mumbai. Cholesterol was purchased from
SISCO Research Laboratories Pvt Ltd, Mumbai, India. Egg yolk powder was
purchased from Himedia laboratories Pvt. Ltd. Mumbai., Adrenaline bi tartrate
was purchased from Sisco Research Laboratories Pvt Ltd, Mumbai. All chemicals
were of analytical grade and used as received without any further purification.
All glassware and magnetic
stir bars used in the synthesis and storage of AUNPs were thoroughly washed in
aqua regia (HCL: HNO3 [3:1] v/v) to dissolve any residual metallic
particles, that may interfere with the synthesis followed by rinsed in Milli-Q
water and allowed to oven dry, to avoid unwanted nucleation and aggregation
during the synthesis and storage. Before the preparation of AUNPs, working
solution of chitosan were prepared by dissolving a definite amount of stock
chitosan (v/v).
AuNPS were synthesized by
well-known wet-chemical reduction method using chitosan biopolymer with some
changes as described earlier.16 Before the preparation of gold nanoparticles,
acetic acid was diluted to 1% aqueous solution and the stock solutions of
medium molecular weight chitosan biopolymer were prepared by dissolving various
amount of chitosan (0.2–1.0%) in 1% acetic acid solution (v/v). Due to the condensed
solubility of polymer chain of chitosan, the mixture was sonicated about 30 min
and allowed to retain for about 1 week at room temperature to obtain clear
solution. Followed by 3 ml of HAuCl4 and 1 ml of 1.0% chitosan solutions were
taken then the mixture was allowed to boil in water bath at 80°C for 1 h under
reflux. After synthesis, AuNPs was confirmed by the color change from pale
yellow to dark ruby red color. The synthesized AuNPs were further puri- fied by
centrifugation at 13, 000 rpm for 15 min at room temperature and redispersed in
Milli-Q water for further analysis.
Loading of gymnemic acid
fraction on to chitosan reduced gold nanoparticles:
A calculated amount of
gymnemic acid fraction was added to dispersion of gold nanoparticles reduced
using 1% chitosan to yield an gymnemic acid fraction concentration of 10mg/ml
in solution. The dispersion was incubated for 16hrs at 2-8°C followed by
ultracentrifugation at 30, 000 rpm for 30mts. The pellet thus obtained was
separated from the supernatant solution and redispersed in MilliQ water prior
to further characterization.
Loading Efficiency:
ϕ%
= ([GA]Tot – [GA] free ) / [GA] Tot x 100 =
60±4.9 %
2.5 Characterization of
gymnemic acid coated chitosan reduced gold nanoparticles:
The size and
surface morphology of as synthesized AuNPs were analyzed using high-resolution
transmission electron microscopy (H-TEM, JOEL 2010) operated at 200 kV. The
number of AuNS per Liter was calculated according to the number of atoms
present in each AuNPs. The size and surface charge of the synthesized AuNPs at
different concentrations were examined using (Malvern Zetasizer, Nano ZS90)
instrument (5 mW HeNe laser _ = 632 nm). The sample was taken in a
cuvette of 1 cm path length with an equilibration time of 60 S at 25 _C.
Particle size of the sample was measured as such without dilution. The optical
absorption spectrum in the wavelength of 200–800 nm was measured using
spectrophotometer in a 2 ml glass cuvette.
2.6 Cell culture:
L6 cells (from
ATCC) were grown in DMEM (4.5 g/liter glucose) supplemented with 10% (vol/vol)
fetal bovine serum, 10 mM HEPES buffer, 2 mM L-Glutamine, 50 U/ml penicillin
and 50 µg/ml streptomycin at 37°C with 8% CO2. Upon reaching
confluence, differentiation was induced by media containing 2% (v/v) fetal
bovine serum for 7 days.
The cells were
grown in monolayers at 37 °C and 5% CO2. For individual experiments,
cells were seeded into six-well plates at a density of 1×105 cells
per well for L6 cells in complete DMEM media for 24 h. After incubation, remove
the medium from the wells for MTT assay. In each well wash with MEM (w/o) FCS.
And add 200µl of MTT concentration of (5mg/ml). And incubate for 6-7hrs in 5%
CO2 incubator. After incubation 1ml of DMSO was added in each well
and mix by pipette and leave for 45 seconds and it shows the purple color
formation. The suspension is transferred in to the cuvette of spectrophotometer
and O.D values is read at 595nm and % of cell viability was calculated using
the formula.10,11,12
Graph was plotted
using the % of cell viability at Y-axis and concentration of the sample in
X-axis. Cell control and sample control was included in each assay to compare
the full cell viability in cytotoxicity assessments.
(OD of sample/OD of cell control)*100 = %cell viability
Quantification of
cell death:
Routine measurement
of cell viability was determined by the ability of the cells to exclude trypan
blue. After incubation, all cells (adhered and detached) were collected,
centrifuged at 200 g for 5 min and resuspended in complete DMEM
containing trypan blue (0.4% in PBS) in 1:1 ratio. Viable and dead cells were
counted using a haemocytometer and the dead cells were expressed as the percentage
of the total cells for each condition. Over the course of the present
experiments, the extent of cell death recorded in untreated L6 cells averaged
9.8±1.1%.
Caspase-3 activity
measurement:
Caspase-3 activity
was measured using a western blotting method. Cell and islet extracts were separated on NuPage SDS-PAGE
gels (Invitrogen) and transferred to polyvinylidine difluoride membranes. Equal
loading of protein between lanes was confirmed by Coomassie staining and
subsequent β-actin immunoblots.
Glucose uptake
assay13:
Cells were cultured
on 6 well plates and incubated for 48 h at 37°C in a CO2 incubator.
When semi confluent monolayer was formed, the culture was renewed with serum
free DMEM containing 0.2% BSA and incubated for 18 h at 37°C in the CO2
incubator. After 18 h, the media was discarded and cells were washed with KRP
buffer once. The cells were treated with Insulin, standard drug and plant
extract and added glucose (1M) and incubated for half an hour. The supernatant
was collected for glucose estimation and glucose uptake was terminated by
washing the cells thrice with 1 ml ice-cold KRP buffer. Cells were subsequently
lysed by freezing and thawing thrice. Cell lysate was collected for glucose
estimation.
Glucose uptake was
calculated as the difference between the initial and final glucose content in
the incubated medium by GOD-POD method as follows:
Mix 10 μl of
sample and 1 ml of reagent, incubate for 25 min at 15-25°C or 10 min at 37°C.
Measure the absorbance of the standard (Astandard) and the sample (Asample)
against the reagent blank within 60 min, the time interval from sample addition
to read time must be exactly the same for standard/control and sample.
PPAR gamma was measured by
using PPAR gamma Transcription Factor Assay Kit
2.7 Animals:
Male wistar rats (150-200g)
were used in this investigation. Animals were maintained under standard
environmental conditions and had free access to feed and water ad libitum.
Experimentation on animals has approved by CPCSEA and Institutional Animals
Ethics Committee (IAEC). The approval no is XV/VELS/PCOG/04/2000/CPCSEA/IAEC/30.10.13.
2.8 Induction
hyperlipedemia with Type-2 diabetes:
Animals are treated with modified
high fat diet every day for 30 days. The high fat diet is freshly prepared
every day and the method of preparation was described earlier by Devi et
al., (2004). Control animals are provided with normal pellet chow (Lipton,
India) and water ad libitum. After 1 week on high fat diet, animals were
fasted overnight and Diabetes was induced by injecting streptozotocin (STZ)
(Subdiabetogenic dose – 30mg/kg in 0.1mol/L citrate buffered saline, pH 4.5) in
to the tail vein via (Yong et al., 2005). 14
2.9 Animal
Grouping and drug administration:
Animals are divided in five
groups. Group 1 (n=6) served as control animal treated with 0.9% saline. Group
2 (n=6) served as high fat diet fed diabetic animal treated with 0.9% saline.
Group 3 (n=6) served as high fat diet fed diabetic animals treated with
Metformin 200mg/kg. Group 4(n=6) served as high fat diet fed diabetic animals
treated with GA-AUNPs 0.25mg/kg in normal saline. Group 5 (n=6) served as high
fat diet fed diabetic animals treated with GA-AUNPs 0.5mg/kg in normal saline.
2.10
Biochemical estimation:
2.10.1
Estimation of blood glucose, insulin and lipid profiles:
Blood glucose level was
determined by one touch horizon blood glucose meter (One Touch, Johnson &
Johnson Ltd, Mumbai) using one drop of blood collected from tail vein. At the
end of 30th day animals (n=3) were sacrificed by carbon dioxide
euthanasia and blood was collected. Lipid profiles, total cholesterol and
lactate dehydrogenase (LDH) are determined by using standard bio chemical Kit (Auto
analyser). Insulin was measured by Radio immuno assay method.
2.10.2
Estimation of oxidative stress markers and antioxidants in vital organs:
At the end of 30th
day animals (n=3) were sacrificed by carbon dioxide euthanasia and organ like
heart, and liver were isolated, weighed and homogenized with ice cold phosphate
buffer (pH 7.2) in Teflon glass homogenizer. The homogenate was centrifuged at
1000 rpm 4◦C for 15 min. Protein was estimated by the method Lowry et
al., (1951).15 The supernatant was used for estimation of
oxidative stress markers, and antioxidants.
2.10.3 Estimation of super oxide dismutase (SOD):
Super oxide dismutase measurement was done based on the ability of SOD to inhibit spontaneous oxidation of adrenaline to
adrenochrome. 2.78ml of sodium carbonate
(0.05mM) buffer (pH 10.2), 100μl of EDTA (1.0mM) and 20μl of tissue homogenate or sucrose (blank) at 30°C for 45 min. After 45 min absorbance was
adjusted to zero to sample. Thereafter, reaction was initiated by adding 100 μl of adrenaline solution (9.0mM). The change in the absorbance was recorded at 480 nm for 8-12 min. Throughout the assay procedure temperature was maintained at
30°C. Similarly, SOD calibration curve was prepared by taking 10 units/ml of standard solution. One unit of SOD produced
approximately 50% inhibition of auto oxidation of adrenaline. Percent inhibition of the sample was calculated by calculating dy/dx of the straight line portion of both blank and sample. The results are expressed as units (U) of SOD activity/mg protein (Saggu et al., 1989)16.
2.10.4 Estimation of Catalase (CAT):
Catalase measurement was done based on the ability of CAT to oxidize hydrogen peroxide (H 2O2). 2.25 ml potassium phosphate buffer (65mM, pH 7.8) and 100 μl of the tissue homogenate or sucrose (0.32M) were incubated at 25°C for 30 min H2O2 (7.5mM) 650 μl was added to initiate the reaction. The change in absorption at 240 nm was measured for 2 to 3 min. dy/dx for every minute for each assay was calculate and the result are expressed as CAT µM/min/g of protein (Beers and Sizer, 1952)17.
2.10.5 Thiobarbituric acid reactive substance (TBAR):
LPO end product malondialdehyde (MDA) was measured by the method Okhawa et al. (1979). 0.1 ml of tissue homogenate was treated with 20% of 1.5 ml of acetic acid (pH 3.5), 1.5 ml thio barbituric acid and 0.2 ml sodium dodecyl sulphate (8.1 %). The mixture was then heated at 100°C for 60 min. The mixture was cooled with tap water and 5 ml of n- butanol-pyridine mixture (15:1 % v/v) followed by 1 ml of distilled water was added. The mixture was shaken vigorously. After centrifugation of the mixture at 4000 rpm for 10 min, the organic layer was withdrawn and absorbance was measure at 532 nm. The concentration of MDA formed is expressed as n mole/mg of protein.18
2.11 Histopathological examination:
After 30 days of STZ and STZ+ GA-AUNPs treated animals were euthanized. The pancreas was dissected out quickly, fixed in 10% formalin and 10-μm thick sections were taken. The sections were processed and stained in 0.1% Hematoxylin and Eosin. The stained sections were observed under a binocular light microscope and photographed. Quantitative scoring of histopathological examination was performed according to (Block and Schwarz (1996)) method with slight modifications. Scoring of damaged pancreas induced by STZ was done by assessing the histological picture as follows: 0–10% = 1 (no morphological signs of damage and few dark stained cells); 11–30% = 2 (edema or inflammation); 31–50% = 3 (Per vascular infiltrate) and 51–100% = 4 (Acinar necrosis). A total histological score of the pancreatic area was calculated by adding all the regional scores and then expressed based on their respective percentage of damage.
2.12 Statistical Analysis:
For in-vivo experiments values are represented by mean ± SEM. The mean values are analyzed by one way ANOVA followed by Dunnets test. The p<0.05 was considered as statistically significant.
3. RESULTS AND
DISCUSSION:
3.1 Synthesis and
Characterization of AuNPS:
The physicochemical
properties like particle size, charge and shape highly influence the
biodistribution, clearance and metabolism of nanoparticles in vivo.20
Hence the synthesis and characterization of nanoparticles is the crucial step
to determine its potential as a therapeutic drug carrier at in vivo level.16
In this method chitosan monolayer protected gymnemic acid coated gold
nanoparticles were successfully synthesized using previously described wet-chemical
reduction method using chitosan biopolymer with required modification. During
synthesis, a chitosan concentration appears to be very crucial for controlling
the particle size of the AuNPs. Hence tetrachloroauric acid concentration was
kept constant (1 mM) and varied concentrations of chitosan were used (0.2–1.0%)
to get the desired monodispersed nanoparticles (Table 5.1 and Table 5.2).
Finally, the optimal concentration for the synthesis was determined. Chitosan
monolayer protected GA-AuNPs were synthesized rapidly and confirmed by
UV-visible spectrophotometer with defined characteristic narrow plasmon
resonance peaks in the visible region at 520 nm shown in Figure 6.1.The FTIR
and HR-TEM analysis of the synthesized GA-AuNPs shows monodispersive spherical
nanoparticles shown in Figure 6.2(a&b), Figure 6.3(a&b), ranging
average particle size of 64nm±1.99 nm in diameter with uniform
distribution. Shows SAED pattern of GA-AuNPs that shows planar atom arrangement
corresponding to the lattice parameters (FCC) from inner to outer layer at 111,
200, 220 and 311 and EDX spectrum confirms the presence of GA-AuNPs shown in
Figure 2(d). Fig 6.4 The DLS analysis of synthesized GA-AuNPs shows
hydrodynamic size of 29.62± 0.32 d.nm in diameters and zeta potential of about
(+) 50.77 mV with low polydispersive index 0.382±0.00. This positive surface
charge of GA-AuNPs shows the higher colloidal stability with no agglomeration
for several months. Interestingly, synthesized nanoparticles aid the tissue
uptake and no capping agent requires for maintaining its stability. The
concentration of synthesized AuNPs was determined by ICP-OES analysis and found
to be 77mg/l.
3.2 INVITRO
STUDIES:
The ability of the
cells to survive a toxic insult has been the basis of most cytotoxicity assays.
It depends both on the number of viable cells and on the mitochondrial activity
of cells. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)
assay is based on the assumption that dead cells or their products do not
reduce tetrazolium. Tetrazolium salts are reduced only by metabolically active
cells. Thus MTT can be reduced to a blue coloured formazan by mitochondrial
enzyme succinate dehydrogenase. The amount of formazan produced is directly
proportional to the number of active cells. GA-AUNPs toxicity was tested in
vitro in L6-GLUT4 myc cell following MTT as described in the materials and
methods. Test concentrations that kept at least 90% cell viability were
considered as safe. Gymnemic acid fraction (Fig. 6.) was found to be safe up to
50µl concentration. Accordingly, all the efficacy studies were performed at
safe concentrations.
Hence, in this
study L6 cell lines are used to determine the glucose uptake activity of
GA-AUNPs and the results are presented in Table 1. The glucose utilization in
L6 cell lines showed that the GA-AUNPs were found to be prominent over control.
The L6 cell lines enhance caspase and the glucose uptake by 70.19±1.72at
500 μg/ml concentration (Fig. 6.7 and Fig 6.8). These results were
compared with insulin and metformin, which were used as the standard
antidiabetic drugs. Insulin at a concentration of 1IU/ml and metformin at a
concentration of 100 μg/ml were found to enhance the glucose uptake over
control.
3.3 Anti- diabetic
effect of GA-AUNPs in high fat meal treated diabetic rats:
The anti-diabetic effect of GA-AUNPs has shown in table 5.3. HFM and STZ treated diabetic rats treated with two different GA-AUNPs had significant (P<0.05) decrease in reducing blood glucose levels at 7th Day as compared with saline treated HFM + STZ rats. The similar significant anti-hyperglycemic effect was noted at 14th day (as well as at the end of the experiment at 30th day as compared with HFM + STZ treated diabetic control. The anti diabetic effect of GA-AUNPs in reducing the blood glucose level at 30th day was comparable to that of metformin (111.0 ± 4.41).(Table 1)
3.4
Anti-hyperlipedimic effect GA-AUNPs in high fat meal treated diabetic rats:
Fig 6.9 depicts the effect of GA-AUNPs on total cholesterol and lipid profiles of the high fat meal fed diabetic rats. Administration
of HFM significantly (P<0.01) increase the serum TC, LDL, Triglycerides (TG) with significant decrease (P < 0.01) in HDL level compared with normal rats. Administration GA-AUNPs significantly (P<0.05) protected the rats against HFM induced hyperlipidemia as observed by decrease in the
TC and LDL level. In addition GA-AUNPs significantly increased the HDL level
respectively. However, the effects of tannins on TG levels were insignificant.
3.5 Effect of
GA-AUNPs on serum CPK LDH and Uric acid:
Fig 6.10 Depicts
the effect of GA-AUNPs on CPK and LDH the high fat diet treated diabetic rats.
There is a significant increase in CPK and LDH level was observed in HFM
treated diabetic rats compared with normal rats. Attenuation of CPK and LDH
level was observed in HFM treated diabetic rats fed GA-AUNPs.
3.6 Effect of
GA-AUNPs on SOD level of heart, liver and kidney of HFM fed diabetic rats:
It is observed from
the Fig 6.11 that significant (P<0.01) depletion in superoxide dismutase
level (SOD) in heart, Kidney and liver of GA-AUNPs fed diabetic rats as
compared with non diabetic control rats. Per oral administration of GA-AUNPs
significantly (P <0.05) increased the SOD levels in heart, liver and Kidney
of the HFD treated diabetic rats as compared with vehicle treated
hyperlipedemic diabetic rats. The effect was dose dependent.
3.7 Effect of
GA-AUNPs in catalase (CAT) in Heart, liver and kidney of HFM fed diabetic rats:
Fig 6.12 shows the
significant (P< 0.01) decrease in the catalase (CAT) level was observed in
heart, liver and kidney of HFM fed diabetic rats as compared with non –
diabetic control rats. GA-AUNPs at two different doses significantly
(P<0.05) increase the catalase level of the insulin dependent liver tissue
and non-Insulin dependent tissue kidney and heart.
3.8 Effect of GA-AUNPs in TBARS level
of heart, liver and kidney of HFM fed diabetic rats:
Fig 6.13 depicts the effect of
TF on TBARS levels in vital organs of the rats fed with HFM and streptozotocin.
Significant (P<0.05) increase in the TBARS was observed. High fat diet
treated diabetic rats heart, liver and kidney has compared with non-diabetic control
a animal group. Administration of insulin, different doses of GA-AUNPs are
significantly (P<0.05) decrease the elevated TBARS level in insulin
dependant liver and non insulin dependant kidney of the high fat diet treated
diabetic rats compared with saline treated high fat treated diabetic animals.
3.9 Histopathology:
The effect of GA-AUNPs at high
dose on histopathlogical findings on the pancreas shown in Fig 6.14(A-E). It is
observed that diabetogenic agent Streptozotocin produced lesion in the pancreatic
islets as viewed by very scanty islets with acinar tissue. Treatment with
insulin has decreased the degree of lesions as indicated by partial intact
pancreatic cells with acini. However attenuation of pancreatic degeneration was
observed in high fat diet treated diabetic animals treated with GA-AUNPs
0.5mg/kg.
T
able 5. 1. Effect of chitosan concentration on the physicochemical
characteristics of synthesized AuNPs.
|
S.No |
Physicochemical characteristics of AuNPs |
Chitosan Concentration 1 (%) (w/v) |
|
1 |
Hydrodynamic Size (d.nm) |
29.62± 0.32 |
|
2 |
PDI |
0.382± 0.00 |
|
3 |
Zeta Potential (mV) |
50.88 ±0.16 |
|
4 |
Zeta Deviation |
4.43 |
|
5 |
Conductivity (mS/cm) |
1.73 |
Ratio (3:1, HAuCl3 : Chitosan)
Table5. 2. Physicochemical characteristics of GA- AuNPs
|
S.No |
Physicochemical characteristics of AuNPs |
GA-Chitosan Concentration 1 (%) (w/v)
|
|
1 |
Hydrodynamic Size (d.nm) |
71.78±0.99 |
|
2 |
PDI |
0.27±0.01 |
|
3 |
Zeta Potential (mV) |
+46.4±0.78 |
|
4 |
AuNPS |
+50.6± 1.15 |
Figure:6.1 UV-visible absorption Spectrum of Synthesized Gold
Nanoparticles and GA coated Gold nanoparticles
Figure 6.2 a&b FTIR Spectrum of Synthesized gymnemic acid and GA
coated chitosan reduced Gold nanoparticles
Fig.6.3. a&b. HR-TEM images of Synthesized Gold Nanoparticles
.
a) Size distribution of AuNPs mean particle size was found to be around
64 nm in dm. (Image J Software)b. Size distribution of AuNPs mean particle size
was found to be around 64nm in dm
Figure 6.4 Hydrodynamic Size distribution of GA- AuNPs
Mean particle size & PDI was found to be 29.62± 0.32 d.nm. &
PDI=0.382±0.00 (b) Zeta potential of synthesized AuNPs was 50.77 mV.
Fig.6.5. AFM images of Synthesized gymnemic acid coated chitosan
reduced Gold Nanoparticles
Fig 6.6 Invitro Cytotoxicity effect of gymnemic acid coated
chitosan reduced AUNPs sample on skeletal muscle cell on (L6
myotubes).
Normal L-6 cell
lines
GAF-AUNPS 12.5µg/mL
GAF-AUNPS
25.00µg/mL
Fig 6.7 Caspase studies of gymnemic acid fraction and
gymnemic acid coated chitosan reduced AUNPs sample on skeletal muscle cell
on
|
Fig 6.8 Glucose uptake assay of gymnemic acid coated chitosan reduced AUNPs sample on skeletal muscle cell on |
Figure 6.9 Effect of GA-AuNPs in lipid profile levels of the high fat treated Diabetic rats |
Table: 5.3 Effect of GA-AuNPs in lipid profile levels of the high fat
treated Diabetic rats
|
Groups |
Fasting Blood glucose levels |
|
Control |
76.03 ± 7.6 |
|
HFD+Diabetic |
217 ± 6.1a |
|
Metformin 100mg |
72.98 ± 3.2 |
|
GA-AUNPs 0.25mg |
85.98 ± 4.2 |
|
GA-AUNPs 0.5mg |
81.65 ± 3.4 |
a indicates p<0.01 Control Vs diabetic; b, c &d
indicates P<0.05 Treatment Vs diabetic; .05 Treatment Vs
diabetic
Figure 6.10 Effect of GA-AuNPs on total protein insulin and LDH Levels
of the High fat treated diabetic rats
a indicates p<0.01 Control Vs diabetic; b, c indicates P<0.05
Treatment Vs diabetic ; .05 Treatment Vs diabetic
Figure 6.11 Effect of GA-AuNPs
in SOD level of the heart,liver and kidney of the high fat diet treated
diabetic rats
a indicates p<0.01 Control Vs diabetic; b indicates
P<0.05 Treatment Vs diabetic ; .05 Treatment Vs diabetic
Figure 6.12Effect of GA-AuNPs
in CAT level of the heart,liver,kidney of the High fat diet treated diabetic
rats
a indicates p<0.01 Control Vs diabetic; b indicates
P<0.05 Treatment Vs diabetic ; .05 Treatment Vs diabetic
Figure 6.13 Effect of GA-AuNPs
in TBARS level of the heart, liver,kidney of the High fat diet Treated
diabetic rats
a indicates p<0.01 Control Vs diabetic; b indicates
P<0.05 Treatment Vs diabetic ; .05 Treatment Vs diabetic
Figure 6.13
|
A)Histopathological Section of Control Pancreas (Magnification 400x) |
B) Histopathological Section of high fat diet fed Streptozotocin treated Pancreas (Magnification 400x) |
Figure C: Histopathological Section of high fat diet fed Streptozotocin + Metformin 100mg/kg treated Pancreas(Magnification 400x) |
|
Figure D: Histopathological Section of high fat diet fed Streptozotocin + GA-AUNPs 0.25 mg/kg treated Pancreas (Magnification 400x ) |
Figure E: Hstopathological Section of high fat diet fed Streptozotocin + GA-AUNPs 0.5 mg/kg treated Pancreas (Magnification 400x )
|
|
Figure 6.15(A-E)
4. DISCUSSION:
In the present study
emphasized the protective effect of biocompatible nontoxic gymnemic acid
coated- chitosan reduced gold nanoparticles (GA-AUNPs) synthesized from the
gymnemic acid fraction isolated from Gymnema sylvestre leaves in high
fat diet /streptozotocin treated type II diabetic rats. The present study is
the first biochemical inspection to shows the anti diabetic effect of GA-AUNPs
present in Gymnema sylvestre in animal model of type II diabetes
associated with hyperlipidemia. Metallic nanostructures play a substantial role
in nanotechnology. Tremendous efforts have been devoted toward developing new
methods of preparing metal nanoparticles with different topologies, such as
spheres, tetrapots and wires. Based on the interesting chemical and physical
properties, gold nanoparticles used for catalysis, biological labeling and
sensing. A number of methods have been used to synthesise the gold nano
particles in both organic and aqueous media. Ever-growing epidemiological and
recent clinical reports suggest that the high prevalence of cardiovascular
diseases like coronary artery disease (CAD) is associated with
hypercholesterolemia and diabetes19,20,2122. Treatment of rats with
high fat diet and STZ is an established model for Type II. Diabetes is
associated with profound alterations in the plasma lipid and lipoprotein
profile and with an increased risk of coronary heart disease 23, 24, 25.
Many medicinal
herbs from Indian system of medicine have been shown to have hypoglycemic and
hypolipidemic properties26,27. In this present study the well known
anti diabetic plant Gymnema sylvestre is examined for its efficacy in type II
diabetes. The main chemical constituent of of Gymnema sylvestre is gymnemic
acid. The gymnemic acid chemically triterpenoidal saponin glycosides. The
bioavailability of gymnemic acid is very low. So the present research designed
to improve the bioavailability of gymnemic acid, by converting in to metallic
nanoparticles and investigate its antidiabetic action.
While metal
nanoparticles are being increasingly used in many sectors of the economy, there
is growing interest in the biological and environmental safety of their production.
The main methods for nanoparticle production are chemical and physical
approaches that are often costly and potentially harmful to the environment.
Type II diabetes are an increasingly serious threat to human health. It is
necessary to overcome it with the help of nature. Therefore, there is an
increase in the investigation of plants as a source of human illness management
28, 29 Medicinal plants have been a potential source of therapeutic
agents for thousands of years. An impressive number of modern drugs have been
derived from natural sources like plants which have been recognized as a part
of the improvement of human healthcare for thousands of years 30.In
addition, within the past decade, it has been demonstrated that many biological
systems like plants, can transform metal ions into metal nanoparticles via the
reductive capacities of metabolites present in these organisms 31.
Gold has been widely employed for many years in human history. Gold nanoparticles interaction with light is strongly
dictated by their environment, size and physical dimensions. GA-AUNPs
have been demonstrated to exhibit antidiabetic properties against high fat diet
fed STZ induced type II diabetes with close attachment of the nanoparticles
themselves to the anti diabetic activity being size dependent 32.
Fundamental studies showed that GA-AUNPs exhibit a rare combination of valuable
properties, namely, unique optical properties associated with the surface
plasmon resonance, catalytic activity, high electrical double layer capacitance,
etc. In this regard an effective and versatile method was performed for the
synthesis of gymnemic acid coated chitosan reduced gold nanoparticles. A
monodisperse AuNPs were successfully synthesized by wet chemical reduction
method. An optimal ratio were found for the synthesis of stable
uniformed size AuNPs. Analytical results of UV-Visible spec, HR-TEM, AFM
confirmed that formed AuNPs are spherical in shape and having size around 64nm.
Zeta potential measurement of AuNPs shows high positive charge +50.77mV that
confirms the high stability of AuNPs. The amount of Au in the nanoparticle
solution was measured by ICP-OES through acid digestion method and it was found
to be 77mg L-. FTIR spectra of Aurocholic acid shows the presence of the
respective peaks and gymnemic acid shows the presence of triterpenoids, saponin
and glycosides. The synthesized AuNPs were found to be stable for more than 6
months in Milli-Q water without any agglomeration.
We compared antidiabetic
properties of standard drug metforminis compared with the biosynthesized
GA-AUNPs. Antidiabetic effects of GA-AUNPs have been evaluated in several
studies. These studies shows 90% of cell viability in MTT- cytoprotective assay
in insulinoma cell lines(L6 cells). The glucose utilization in L6 cell lines
showed that the GA-AUNPs were found to be prominent over control. The L6 cell
lines enhance the glucose uptake by 70.19±1.72at 500 μg/ml concentration
Evidence is presented that the
gymnemic acid coated chitosan reduced gold nanoparticles shows antidiabetic
activity in addition lipid lowering action in diabetic animals. It is observed
from the biochemical datas that elevated blood glucose level, increase in
plasma cholesterol and lipid profile level was observed in high fat diet treated
STZ induced diabetic rats. Treatment of GA-AUNPs normalized the blood glucose
level, triglyceride, LDL levels in plasma possibly by increasing glucose
metabolism and controlling the hydrolysis of lipoproteins, their selective
uptake and metabolism by different tissues. The effect of GA-AUNPs on
controlled mobilization of serum triglycerides, total cholesterol and lipid
profiles. The strong anti hyperlipidemic property of GA-AUNPs evidenced by
control of hyperglycemia and control of total cholesterol and low density
protein levels.
In this biological
investigation shows protective effect of oxidative damage in high fat diet and
STZ induced diabetic rats. Superoxide dismutase and catalase are the enzymes
that protects our tissues from the effects of free radicals and lipid
peroxides, both increased in diabetic animals because the animals affected free
radical mediated injury and lipid peroxidation.
In conclusion, the
results of this study show that 2 doses of GA-AUNPs had a favorable effect on
plasma glucose and insulin concentrations. It also had an influence on
attenuating oxidative stress in diabetic rats. In addition, larger amounts of
GA-AUNPs supplementation may have beneficial effects on reducing plasma TC,
triglyceride and LDL levels. Further characterization of active GA-AUNPs is
warranted and studies are in progress to target the molecular levels.
5. CONFLICT OF
INTEREST STATEMENT:
We declare that we
have no conflict of interest.
6. ACKNOWLEDGMENTS:
We are greatly
thankful to the School of pharmaceutical sciences, Vels University for
providing platform to complete the research.
7. REFERENCES:
1.
C.
Santhosh, P. Kollu, S. Doshi, M. Sharma, D. Bahadur, M.T. Vanchinathan, et al.,
Adsorption, photodegradation and antibacterial study of
graphene–Fe3O4nanocomposite for multipurpose water purification application, RSC
Adv. 4(2014) 28300–28308.
2.
F.
Arockiya Aarthi Rajathi, C. Parthiban, V. Ganesh Kumar, P. Anantharaman,
Biosynthesis of antibacterial gold nanoparticles using brown alga, Stoechosper-mum
marginatum (kützing), Spectrochim. Acta A Mol. Biomol. Spectrosc.
99(2012) 166–173.
3.
T.S.
Dhas, V.G. Kumar, L.S. Abraham, V. Karthick, K. Govindaraju, T. Stalin Dhas, et
al., Sargassum myriocystum mediated biosynthesis of gold nanoparticles,
Spectrochim. Acta A Mol. Biomol. Spectrosc. 99 (2012) 97–101.
4.
V.G.
Kumar, S.D. Gokavarapu, A. Rajeswari, T.S. Dhas, V. Karthick, Z. Kapadia, et
al., Facile green synthesis of gold nanoparticles using leaf extract of
antidia-betic potent Cassia auriculata, Colloid Surf. B Biointerf.
87 (2011) 159–163.
5.
Nirmala
Grace, K. Pandian, One pot synthesis of polymer protected Pt, Pd, Ag and Ru
nanoparticles and nanoprisms under reflux and microwave modeof heating in
glycerol—a comparative study, Mater. Chem. Phys. 104 (2007)191–198.
6. R. Balamurali
krishna, Sujitha r. reddy, Harika javangula, D. Swapna, K. Jagadeeswara reddy
An easy and simple method of isolation and purification of genomic DNA from the
leaves of Gymnema sylvestre-an anti-diabetic plant., International
journal of life sciences and pharma research, 2012; 2( 1): 15-20.
7. Sankaradoss Nirmala,
Velayutham Ravichandiran, A Vijayalakshmi In Vitro Cytotoxicity and
Glucose Uptake Activity of Gymnemic Acid Fraction of Gymnema Sylvestre
Leaves. International Journal of Pharma Research and Health Sciences Volume
4 (2), 2016, Page-1104-1109
8. Hooper, D. Isolation
and antiviral activity of gymnemic acid. Pharm. J. Trans. 1887; 17: 867-
868.
9. G. Sonavane, K.
Tomoda, and K. Makino, Colloids and Surfaces B: Biointerfaces 66, 274
(2008).
10. Mosmann, T. (1983).
Rapid colorimetric assay for cellular growth and survival: Application
toproliferation and cytotoxicity assays. Journal of Immunological Methods
1983; 65: 55–63.
11. Saad, B., Zaid, H.,
& Said, O. (2013). Tradition and perspectives of diabetes treatment in
grecoarab and Islamic medicine. In R. R. Watson & V. R. Preedy (Eds.),
Bioactive food as dietary interventions for diabetes. San Diego: Academic
Press
12. Asokan A, Thangavel
M, In vitro cytotoxic studies of crude methanolic extract of Saraca
indica bark extract. IOSR Journal of Pharmacy and Biological Sciences
2014; 9(4): 26-30.
13. Mathews AM, Sujith
K, Pillai S, Christina AJM, Study of glucose uptake activity of Solanum xantohocarpum
in L-6 cell lines. European Journal of Biological Sciences 2013;
14. Devi R, Sharma DK.
Hypolipidemic effect of different extracts of clerodendron colebrookinumwalp in
normal and high fat diet fed rats. J Ethnopharmacol 2004; 90(1): 63-68.
15. Lowry OH, Rose
brough NJ, Farr AL. Proteine measurement with the Folin phenol reagent. J
Boil Chem 1951; 193: 265-275.
16. Saggu H, Cookey J,
Dexter DA. A selective increases in particulate superoxide dismutase activity
in parkinsonism. J Neurochem 1989; 53: 629-697.
17. Beer RF, Seizer TW.
A spectrophotometric method for measuring breakdown of hydrogen peroxide by
catalase. J Biol Chem 1952; 195: 133-140
18. Okhawa H, Ohishi N,
Yagi K. Assay for lipid peroxides in animal tissues by TBA reaction. Ann
Clin Biochem 1979; 95: 351-358.
19. Srinivasan K,
Ramarao P. Animal models in type 2 diabetes research: An overview. Indian J
Med Res 2007; 125: 451-472.
20. Gupta R, Singh AK,
Basira R, Gupta N, Kanodia A, Gupta KD. Influence of total cholesterol levels
on long-term mortality in coronary heart disease: a reappraisal. Indian
Heart J 2000; 52(1): 23-28.
21. Bhardwaj R, Kandoria
A, Marwah R, Vaidya P, Dhima P. Coronary heart disease in rural population of
himachal - a population based study. J Assoc Physicians India 2009; 57:
505-507.
22. Brown WV.
Lipoprotein disorders in diabetes mellitus. Med Clin N Am 1994; 78:
143-161.
23. Saravanan S,
Srikumar R, Manikandan S, Jeya Parthasarathy N, Sheela Devi R. Hypolipidemic
effect of Triphala in experimentally induced hypercholesterolic rats Yakugaku
Zasshi. BMC Complementary Altern Med 2007; 127(2): 385-388.
24. Kalaiarasi P,
Kaviarasan K, Pugalendi KV. Hypolipidemic activity of 18 -glycyrrhetinic acid
on streptozotocin-induced diabetic rats. Eur J Pharmacol 2009; 612(1-3):
93-97
25. Zhang LY, Keung W,
Samokhvalov V, Wang W, Lopaschuk GD. Role of fatty acid uptake and fatty acid 毬-oxidation in mediating insulin resistance in heart
and skeletal muscle. Biochim Biophys Acta 2010; 1801(1): 1-22.
26. Handa SS, Rajesh MS,
Satyaprakash RJ, Shivananda TN. Plants used against diabetes mellitus. Biomed
2006; 1: 1-21.
27. Mukherjee PK, Maiti
K, Mukherjee K, Houghton PJ. Leads from Indian medicinal plants with
hypoglycemic potentials. J Ethnopharmacol 2006; 106: 1-28
28. Mickymaray S, Saleh
Al Aboody M, Kumar Rath P, Annamalai P, Nooruddin T. Screening and
antibacterial efficacy of selected Indian medicinal plants. Asian Pac J Trop
Biomed 2016; 6: 185-91.
29. Mary EJ, Inbathamizh
L. Green synthesis and characterization of nano silver using leaf extract of Morinda
pubescens. Asian J Pharm Clin Res 2012; 5(Suppl 1): 159-62.
30. Sarker S, Nahar L.
Chemistry for pharmacy students: general, organic and natural product
chemistry. New Jersey: John Wiley & Sons, Inc.; 2007.
31. Makarov VV, Love AJ,
Sinitsyna OV, Makarova SS, Yaminsky IV, Taliansky ME, et al. “Green” nanotechnologies:
synthesis of metal nanoparticles using plants. Acta Naturae 2014; 6:
35-44.
32. Huang J, Li Q, Sun
D, Lu Y, Su Y, Yang X, et al. Biosynthesis of silver and gold nanoparticles by
novel sundried Cinnamomum camphora leaf. Nanotechnology 2007; 18:
105104-14.
Received on 02.11.2017
Modified on 07.12.2017
Accepted on 20.01.2018
© RJPT All right reserved
Research J. Pharm. and Tech.
2018; 11(3): 1193-1206.
DOI: 10.5958/0974-360X.2018.00222.6