09_Chapter 3

CHAPTER 3 : MATERIALS AND METHODS
COLLECTION AND CULTIVATION OF SAMPLES:
Market samples were procured from-
1. Saharanpur - M/S Talwar traders
2. Delhi
Kharibaoli market
3. Amritsar
4. Meerut
Masala mandi
Masala mandi
In these markets, turmeric (Haldi) varieties are procured from
Western and Southern India, especially from Nanded (Maharashtra),
Erode (Tamil Nadu) and Nizamabad (Andhra Pradesh), these varieties
are consistently available in the market.
Some unnamed mixed varieties grown in Punjab, Haryana, U.P.
and Uttarakhand are also available in the market. These varieties are
unnamed and availability is not consistent.
Following named forms and varieties were procured and
analyzed because their availability was consistent for three years.
Table- 9
S.No
Place of Supply
Name of commercial Form
1
Nanded
a)
Bulb
b)
Finger
c)
Splits
a)
Bulb
b)
Finger
c)
Splits
a)
Bulb
b)
Splits
c)
Fingers
2
3
4
Erode
Nizamabad
Warangal
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Besides these commercially available varieties Some 26 named
varieties of Curcuma longa were collected and 21 were grown. Places of
collections are as followsSardar Vallabh Bhai Patel University Meerut
Government Garden Saharanpur
Cultivation was done as recommended by Dr SK Upadhayaya
(Upadhyaya SK etal. 2000)
Plots of 10x10 m were prepared at Delhi Road in an agriculture
field for field cultivation. All the Haldi varieties were also cultivated in 18
inch (45 centimeters) diameter earthen pots.
At Horticulture research and training centre, Saharanpur, Beribagh
garden Nearly 21 varieties are in cultivation.
This germplasm has been maintained for last 10 years. Sample
cultivated in this institute were also procured and analyzed.
All the studied varieties are recorded in the following table
Table- 10
1
CL-8
14
CEL-324
2
CL-67
15
NH-5
3
CL-68
16
NDH-18
4
CL-69
17
ALLEYPPE
5
CL-70
18
VALLABH PRIYA
6
CL-72
19
KEDARAM
7
CL-73
20
MANGO GINGER
8
CL-315
21
PRABHA
9
CL-320
22
ROMA
10
CL-321
23
ROMA PAHARI
11
CLS-16
24
SONIA RAJINDER
12
CEL-6
25
SUVARNA
13
CEL-318
26
VAYAMA
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STUDY OF COMMONLY AVAILABLE MARKET SAMPLES:
Different commonly available market varieties of Nanded, Erode,
Nizamabad
and
Warangal were taken from 4 different markets-
Saharanpur, Delhi, Amritsar and Meerut. Theses were analysed to
confirm and quantify the Curcumin contents in them.
POST HARVEST TREATMENTS OF FRESH SAMPLES
Boiling and Curing TechniquesTo improve the colour of Curcuma (Haldi) processing is done by
boiling in some mild alkali like Sajji or NaHCO 3. Experiments were
performed to study the effect of various treatments on Curcumin and
curcuminoid contents. Prior to phytochemical analysis some material was
boiled in 1% NaHCO3. Samples were also boiled in pure water till they
are tender and emit some specific aroma. Obviously aroma is of the
aromatic oils present in it. This process results in loss of aromatic oil but
catabolic enzymes denatured, so during drying of rhizome weight loss is
significantly less in comparison to when drying is done directly in sun
without boiling.
Drying TechniquesTo study the effect of drying methods different drying techniques
were used to prepare the samples for analysis. The samples were
prepared by drying in sun light, microwave oven and under Infra red
light. Fresh samples of rhizomes were also analysed to compare the
quantity
of
Curcumin
and
curcuminoids.
Shade
drying
was
not
considered for analyses as it detoriates the shape and heavy loss in dry
weight was also noted during preliminary studies.
Drying is the major issue in the processing of Curcuma for markets as,
1. Direct sun and shade drying result in loss of weight and also shape.
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2. Boiling maintained weight and shape but may result in loss of some
phytochemicals
Drying
for
commercial
production
of
phytochemicals
some
advance scientific technique may be beneficial so we tried infrared and
microwave oven drying.
STUDY ON PHYTOASSOCIATES-
In UP, Haryana and Punjab Curcuma is cultivated as intercrop.
Since last few years farmers are cultivating turmeric as a prime crop
also. As Curcuma is cultivated in orchard and under commercial
plantation,
so
Curcuma
samples
were
collected
to
quantify
the
phytochemicals and impact of phytoassociates.
Samples collectedTable- 11
S.No
Curcuma Variety
Phytoassociate
1
Roma
Mango
2
Roma
Litchi
3
Roma
Guava
4
Roma
Neem
5
Roma
Jatropha
6
Vallabh priya
Mango
7
Vallabh priya
litchi
8
Vallabh priya
Guava
9
Vallabh priya
Neem
10
Vallabh priya
Jatropha
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QUANTIFICATION OF PHYTOCHEMICALS IN ANATOMICAL ZONES-
Anatomically bulbs and fingers are divided in three zones, Outer
peel/skin of the rhizomes which is very clear and is named as cover. It
includes epidermis and hypodermis. Other two zones are cortex and pith
of the rhizome.
This was done because during commercial processing epidermis,
hypodermis and a part of cortex is removed. This process is known as
polishing. This study was done to know whether this process may results
in loss of some phytochemicals.
QUANTIFICATION IN MORPHOLOGICAL FORMS
There are two main forms available in market, fingers and bulbs.
Nukara or splits are small fine branches which are not so commonly
available.
During our cultivation experiments, no variety produced Nukara
i.e. splits.
Thus, bulb and finger samples of rhizomes were analyzed for
Curcumin and curcuminoids content.
PREPARATION OF EXTRACT OF AUTHENTIC SAMPLES
84
1. Fresh samples
50 mg of the fresh sample were taken and extracted with
methanol (15x3) on water bath. Extracts were filtered through filter
paper. Filtrates were collected. The volume was made up to 50 ml with
methanol. These samples were applied for HPTLC analysis.
2. Sundry SamplesCurcuma longa materials were dried under sunlight and grounded
to powder. 50 mg of powder sample of each variety was taken. Extract
was made with methanol (15x3) on water bath. Samples were filtered
through filter paper. The volume was made up to 50 ml with methanol.
Now these prepared samples were used for HPTLC analysis.
3. Boiled samplesTurmeric rhizomes of each variety were boiled for 20 minutes in
water. These were dried in sunlight; similar samples were made to dry in
infrared (IR) and microwave conditions also. Samples were grounded to
powder. 50 mg of the powdered sample was taken, extracted with
methanol (15x3) on water bath and filtered through fine filter paper.
The volume was made up to 50 ml with methanol. These prepared
samples were used for HPTLC analysis.
4. Samples boiled with NaHCO3Turmeric rhizomes were boiled for 20 minutes in water with
baking soda. These were dried in two conditions- firstly under infrared
light and similarly under microwave conditions. Dried samples were
grounded to powder. 50 mg of the powdered sample was taken,
extracted with methanol (15x3) on water bath and filtered through the
fine filter paper. The volume was made up to 50 ml with methanol.
These
prepared
samples
were
used
for
HPTLC
analysis.
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PHYTO CHEMICAL ANALYSIS
Chemical used were of ANALAR grade obtained from e-Merck, BDH
and Ranbaxy.
Instruments used are
I.
HPLC
II.
HPTLC
III.
GC/MS
IV.
Plasma separator
V.
UV Viewer
VI.
Silica Plates
All glass apparatuses were of corning glass.
1.
2.
Thin film evaporater of Buchhi
Soxhelet apparatus etc.
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC)
In this, the desired sample is applied on TLC plate using capillary
or microprocessor based application device such as Linomat IVR where
known quantity of test material can be spotted in the form of band or
spot. The plate is then developed in TLC chambers using suitable
solvents. Once the separation of compounds takes place then the plates
were scanned using TLC scanner.
The plates were scanned in the wavelength range of 190
800 nm
covering both visible and ultraviolet range. The instrument has the
facilities of scanning in situ reflectance spectrum. This provides useful
information about the identification of a particular compound along with
the purity of peak. Any plant product can be analyzed on this qualitative
and quantitative parameters.
86
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
The basic instrument consists of a pump, injector, column,
detector
and
the
software
which
converts
the
signal
to readily
processible data. Using this instrument, we can qualitatively and
quantitatively analyze any herb or herbal product. Using this instrument
also we can scan the UV spectrum which provides useful information on
the identity of isolated compound. Here the sample is injected which is
elucidated into different compounds in the column with the help of
suitable mobile phase under pressure and the signals are converted into
peaks giving the height and area which can be computed. Different
columns and detectors can be used to analyze different phytochemicals.
Following columns are normally used.
Normal phase silica column
Reverse phase silica column
CN column
Amino column
Carbohydrate column etc.
Similarly following detectors are in column use
1. UV detector (UVD)
2. Photodiode array detector (PAD)
3. Electrochemical detector (ECD)
4. Refractive index detector (RID)
5. Pukseamperiometeric detector (PAMD)
ANALYSIS OF HALDI SAMPLES
87
Physical and other parameters for stationary and mobile phase
during analysis of market samples are indicated in table 12 & 13.
Table- 12
Table- 13
88
Standard Preparation-
89
A 10mg/100ml solution of standard Curcumin in methanol was
prepared.
Chromatographic conditionsAC
and twin trough development chamber with cats 4.05 evaluation
software was used.
Procedure
Application of 30 microlitre of test samples along with 1, 2, 5, 10
(1,2,3,4,5)microlitre of standard Curcumin on TLC aluminum plate
coated with silica gel 60F254 was made (E. Merck, Cat. No.5554).
Development of the chromatogram was done in chloroform: methanol:
acetic acid=95:05:01, solvent system up to 80 mm. Plates were air dried
and scanned using the TLC scanner 3 at mercury 360nm, in absorbance
mode. Rf values of curcuminoids are approximately 0.30, 0.38 & 0.45
respectively.
Calculation
To calculate the amount of Curcumin & total curcuminoids linear
regression equation was plotted between concentrations of sample and
area of different standard concentration using following formulae.
%of Curcumin = conc. of std. (microgram) x Area of test
sample/
x 100
Area of std.xconc of test sample
%of total curcuminoids = Conc. Of total curcuminoids in std.
sample x Area of test sample
x 100
/ Area of std. x conc. Of test sample
QUANTIFICATION OF BIOAVAILABILITY OF CURCUMIN IN BLOOD
90
Studies were made on five volunteers to estimate the Curcumin in
their blood samples after 1.5 hours of Curcuma powder ingestion.
In
first phase each volunteer was ingested 5oomg of Curcuma powder.
After 1.5 hours 5ml blood sample was obtained and 100 microlitre
heparin (Heparin is a highly sulphated glycosaminoglycan and is very
good anti-coagulant) added to this blood sample. This resulted in
precipitation of the sample. This blood sample was ultrasonicated (In
biological applications sonication disrupts biological material. It is often
used to disrupt cell membrane and release cellular content. So that all
the contents come in plasma), centrifuged to separate the plasma and
corpuscles. Collected 1ml plasma was extracted with methanol to make
volume up to 1.5 ml.
20 microlitre of extracted plasma was used to analyze & quantify
Curcumin by HPLC apparatus.
The peaks were also obtained by injecting pure Curcumin. In
plasma Curcumin peaks were observed at 14 minutes which is standard
for Curcumin.
In second phase after 4 days same volunteers were given 500mg
of Curcuma powder along with 125mg of piperine obtained from Piper
nigrum.
Blood samples were obtained and processed in the similar manner
as described above and analyzed by HPLC apparatus
HPLC Conditions:
510 Solvent
Array Detector), evaluation with Millennium 2010 chromatography work
station V 2.10, using LichosorbR 100 RP-18 endcapped (5 micrometre)
column(4.9x250nm) with a RP C18 Guard column.
Mobile Phase
:
Methanol: Water =70:30 (v/v)
91
Flow Rate
:
1.0ml/min
Scanning wavelength
:
420nm
Name of volunteers Dr AVM Murganandam- incharge R&D*
Dr SK Upadhayaya- guide
Dr Saini- microbiologist*
Mr Pankaj- research associate*
Ms Richa Atreya- research scholar
*Associated with Indian Herbs Research and Supply Company Ltd,
Saharanpur.
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