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Comparative Study of Ashodhita and Shodhita Haridra
with respect to their Inhibitory Properties of
α-Amylase and α-Glucosidase
National Conference on Recent advances in
Ayurvedic Herbal Medicine - From source
to manufacturing at Dehradun
15-16 Sept.- 2017
Abstract ID-58
Institute for Post Graduate Teaching & Research in Ayurveda
GUJARAT AYURVEDA UNIVERSITY
Accredited ‘A’ grade by NAAC (CGPA 3.28)
Anuj Kumar*, J K Maji2, Prashant Bedarkar3, Harisha CR4,MB Nariya5
1
Turmeric (Curcuma longa Linn.) is one of the important herb for internal affections on the
liver disease and also found good antidiabetic effect1,2.
Ayurveda advocates sodhana (purification) a unique Pharmaceutical process which is used
to remove unwanted qualities (washable, soluble and volatile impurities) of a drug and it
will be made adequate for the administration of the body.
As per chemical point of view adding materials with purified drugs is nothing but a
impurities (reductionist approach) but therapeutical point of view may prove useful in
potentiating therapeutic effect (Synergistic approach) and neutralise the toxic effect
(Antagonistic approach).
Ayurvedic text books like Chakradatta, Bhaisajyaratnavali, Harmekhala recommended
purification processes for Haridra to its use numerous remedies.
In the above background, the present study was undertaken to investigate and establish a
justifiable identification system of various purification effect on turmeric samples based on
phyto-pharmacognostical, image processing (Nearest Neighbour classification),
physico-chemical (identity, purity) with multivariate technique (Principal component
analysis), high performance thin layer chromatography (HPTLC) and Enzymatic study of
methanolic extracts in context to Inhibition of α-amylase and α-glucosidase.
1. Khare CP. Indian medicinal plants. 1st ed. Spinger Science+buiseness media, LLC, New-York, USA; 2007.
2. Roman Adhikari, Jyothi Y, Deepika Bora, Vamsee Veena A. Combined effect of aqueous extract of Curcuma longa linn. with metformin
in diabetes induced neuroptahic pain in rats. Asian J Pharm Clin Res 2015;8:166-70.
INTRODUCTION
2
MATERIAL AND METHODS
3
Note: PCC stands for Parenchymatous cells, Ca. Ox. stands for calcium oxalate crystal
Pharmacognostical Study
Fig.-a Fig.-b
4
TR TT TW TGM TZ
Photomicrographs (a) and (b) at 40Xshowed powder characters of raw Turmeric and different
characters of purified samples. TR (raw) showed general characters of Turmeric powder (fig. 1.01 to
fig. 1.16), TT showed almost similar except fibre attached with starch gain (fig. 2.4).
TW also showed similar characters except for the decrease in the size of oleoresin. TGM showed the
character of media cow’s urine as Calcium oxalate crystal (fig. 4.5) and fibre attached with calcium
oxalate crystal (fig. 4.6). Characters of media can be seen in TZ also, fig. 5.07 showed the character
of cow’s urine, fig. 5.08 and fig. 5.09 showed characters of Alambusha and fig. 5.10 to 5.16 showed
characters of Panchapallava Kwatha (decoction).
It seems, Shodhana act as synergic and antagonist due decrease in the characters of Turmeric and
addition of the characters of media. Shape and size variation can be seen in the starch grain and
oleoresin after the process that directly shows the effect of media and process.
Scalariform vessel of TGM and annular vessel of TZ got burst due to boiling (heat treatment). Media
characters incorporated with the components of Turmeric in TGM as the crystal of cow’s urine
attached with the fibre and same in TZ with the characters of Alambusha and Panchapallava shows
the addition of extra chemical material those may alter the therapeutic efficacy also.
Result & Discussion of Pharmacognostical Study
5
The image was acquired using the Image
Acquisition Toolbox by Matlab 2016.In
brief L. a. b represent the lightness of the
color.
The nonlinear relations for L*, a* and b* are
intended to mimic the nonlinear response of
the eye.
Each of the pixels in a region is similar with
respect to some characteristic or computed
property such as color, intensity and texture.
L*a*b* model is developed to measure color
differences consistently with the perceived
color differences [fig. c].
In this way, this technique is fulfilled
automatic identification system without help
of lack of subject specialist.
In this way, identify different colors in the
image by analyzing the image color space to
build reference database for various turmeric
purifactory environment.
Result & Discussion of
Image Processing study
[fig. c]. Representative different image of coded
samples raw image and color space converted image
6
Fig. e: Developed plate under daylight,
UV 254 nm, 366 nm with standard,
Different purified samples
Fig. d: 3D densitogram of curcumin
standard with the blank
HPTLC STUDY
7
30, 1670.9
40, 2182.8
50, 2575.9
100, 4391.2
200, 8708
y = 40.899x + 470.22
R² = 0.9987
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
050 100 150 200 250
Area Under Curve (AUC)
Concentration (µg/ml)
Linearity curve of CurcuminLine…
Graph-1
The HPTLC separation was performed on precoated HPTLC plate using toluene, chloroform,
methanol (5:4:1, v/v/v) as a mobile phase and Densitometric analysis was performed at 430 nm.
A compact spot of Curcumin found at Rf value of 0.34 of standard curcumin diluted with methanol
(concentration of C1=30, C2=40, C3=50, C4=100, C5=200 (µg/ml), C0= Blank) and Turmeric
samples’ methanolic extract 10 µg/ml methanol
The plate was observed under visible light, UV254nm (short wavelength) and UV 366nm (long
wavelength)
Quantification of Curcumin was calculated by the regression equation, y = 40.899x + 470.22 with
R² = 0.9987 describe by calibration curve (graph-1)
Samples
Concentration
Prepared
(mg/ml)
CD
(µg)
% of C. in extract
(mg/100mg) M. E.
Value (%)
Powder
of drug
(gm)
M. Extract
(gm) EC. in
powder
drug
(µg)
TR
10.1
57.257
0.5669
10.639
5.142
0.547
3100.98
TT
10.3
56.907
0.5525
8.446
5.131
0.433
2392.34
TW
10.9
76.830
0.7048
8.877
5.172
0.459
3235.32
TGM
10.6
61.937
0.5843
10.442
5.146
0.537
3137.75
TZ
10.1
49.147
0.4866
9.411
5.021
0.472
2296.78
Table 1: Curcumin present in different treated turmeric powders
Result and Discussion of HPTLC
8
The amount of curcumin in the raw and purified samples was computed from the calibration
curves, which suggests that the highest reduction of curcumin was found in TZ and TT sample.
It might be due to the fact that prolonged contact of the turmeric rhizome with boiling specified
medias for TZ and takra (acidic medium) for TT may helped to extracted out some quantity of
curcumin and may also converted di-hydroxy curcumin3.
The curcumin aromatic ring systems are consisted polyphenol are connected by two α, β
unsaturated carbonyl group which is good Michael acceptor and undergo nucleophilic addition
along with its hydrophobic nature, the water treated methanolic extract is shown high-level
curcumin.
3. Ajay Kumar et al. Antioxidant efficacy and curcumin content of turmeric (Curcuma longa Linn.) flower. Int. J Curr. Pharm Res 2016;8(3):112-4
Sam
-
ples
pH
WSE
MSE
TA
AIA
TR
4.982
±
0.008
20.648
±
0.326
10.627
±
0.109
8.054
±
0.149
0.492
±
0.022
TT
4.964
±
0.005
15.797
±
0.147
8.458
±
0.049
7.994
±
0.072
0.716
±
0.042
TW
5.022
±
0.010
14.416
±
0.092
8.886
±
0.043
7.493
±
0.195
0.611
±
0.022
TGM
5.452
±
0.016
13.277
±
0.171
10.410
±
0.052
7.378
±
0.235
0.623
±
0.037
TZ
5.46
±
0.023
16.450
±
0.056
9.509
±
0.302
7.678
±
0.178
0.784
±
0.033
Table 2: Physicochemical constant result
expressed as % w/w except pH (n=5; ±SD)
Result & Discussion of PCA with Physichochemical Analysis
Fig. f: PCA score showing the distribution pattern, the ellipse
represents the Hotelling T2 with 95 % confidence in score plot Fig. g: Loading plot
Data operations (PCA) were performed using Unscrambler
and exported as 2D ASCII files
The samples (TT, TW, TZ) cluster indicating their similarity, and
TGM, TR dissimilarity due to the position of the respective
coordinate. (Fig-f)
From the loading plot in (fig. g) it appeared that the pH and WSE
were the physicochemical parameters contributing to the grouping
of turmeric samples and that these attributes corresponded to the
PC1which explained about 90%of the total variance.
It should be noted that TGM and TZ samples are differentiated
from other samples by their higher pH and water soluble extractive
value (WSE) as well as lower total ash (TA) content.
9
Alpha-amylase & Alpha-glucosidase
Enzymatic Study
Methods:
A total of 500μl of methanolic extract of test samples
(TR, TT, TW, TGW, TZ), distilled water for control
group and Acarbose as standard drug were used in the
concentration of (50, 100, 150,200μg/ml) in both
method. α-amylase 500μl (0.5mg/ml) and 1ml of α-
glucosidase enzyme (1U/ml) were used .
Procedure describe by Miller (Heidari R. et al,
Pakistan Journal of Nutrition, 2005) was followed
for α-amylase inhibitory activity and Glucose oxidase
peroxidase method (Andrade-Cetto A. et al Journal
of ethnopharmacology,2008) for α-glucosidase
inhibitory activity
Absorbance was measured at 540 nm.&510 nm.for
α-amylase & α-glucosidase inhibitory activity
respectively and finally, % of inhibition was calculated
as
10
Samples
Concentration
(in µg/ml)
Percentage of Inhibitions
α –
Amylase α –
Glucosidase
AC
(Standard)
50 47.53 49.81
100 53.01 53.47
150 60.2 57.59
200 62.5 62.58
TR
50 40 42.73
100 49.13 45.01
150 54.29 50.55
200 58.06 54.88
TT
50 36.4 31.02
100 42.23 40.56
150 49.65 45.37
200 54.11 51.28
TW
50 40.57 42.72
100 49.65 46.91
150 54.11 50.78
200 58.33 54.89
TGM
50 26.41 34.17
100 33.89 37.34
150 40.45 40.22
200 47.29 45.11
TZ
50 41.35 43.78
100 50.79 49.63
150 54.54 52.51
200 58.51 55.17
Table-3: α-Amylase & α-glucosidase inhibitory
activity of Test samples and Standard drug
Result & Discussion of α-amylase & α-glucosidase Enzymatic Study
Note: TGM trendline in the graph was
extended to 50 &80 points in α-Amylase &
α-glucosidase respectively to get the IC50.
Graph-2 Inhibition of α-Amylase enzyme
Graph-3 Inhibition of α-glucosidase enzyme
11
Graph-4: Comparison of IC50 dose for inhibition of α-
amylase & α-glucosidase enzymatic activity
A dose-dependent inhibitory effect of raw and Shodhita
Haridra on α-amylase and α-glucosidase enzyme can be
seen in graph-2 and graph-3.
Decreasing order of IC50 in α-amylase: TZ (113.72) <
TW (119.22) < TR (121.861) < TT (161.35) < TGM
(218.85)
Decreasing order in α-glucosidase:TZ (121.32) < TW
(139.48) < TR (145.29) < TT (185.51) < TGM (276.12).
So, TZ is effective at minimum dose levels than others,
shows significant therapeutic effect of Shodhana.
This study shows the addition of characters of medias drug with turmeric powders like
crystal, starch grain,pollen grain,epidermal cells, fibre.
Identify different perceivable colours in various processed turmeric by analysing the Lab
colour space as a image signature.
PC1and PC2explained (90 + 9) % total variance in score plot of respective purify turmeric
samples shown clear grouping in relation to physico-chemical constant.
HPTLC chromatographic fingerprinting reveals that samples treated water TW contained
maximum amount of curcumin with help of standard curve.
Enzymatic study proves TZ most effective than all other samples. TW and TR were also
close to TZ, but TZ is consider better due to it shows minimum curcumin level in analytical
section, that proves some special properties of media has increase the therapeutic property of
TZ, which proves the synergic approach of Shodhana.
This study proved that purification in Ayurveda not only refers to the elimination of toxin but
also transformation in the properties in the primary substance rendering it safe as well as
many desired qualities are imbibed in it.
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