Characterization of active
constituents in Turmeric powder and validation of method for curcumin in
samples
Mukund Nagarnaik, Arun Sarjoshi, Ajay Bodkhe, Bhupendra Khanal, Mayuri Pise,
Girish Pandya*
Research and Development
Division, Qualichem Laboratories, Near Gokulpeth Market,
Nagpur 440010, India.
*Corresponding Author E-mail: pandyagh@rediffmail.com
ABSTRACT:
Chemical analysis of turmeric (Curcuma longa L) cultivated in parts of the state of Maharashtra was
carried out. Quantitative characterization of turmeric samples was carried out
by determining moisture content, total ash, acid insoluble ash, starch,
curcumin and lead and chromate levels in all the samples. The total ash levels
were within limits of Agmark and indicated the purity of samples. About 51%
level of starch in the samples indicate its commercial applicability also. The
curcumin in samples varied from 3.34 to 9.45 g /100 g (dry basis). The UV-Vis
method for curcumin was also validated by carrying out repeatability studies
and determining the precision and accuracy in terms of LOD and LOQ
measurements.
KEYWORDS: Turmeric, Curcumin, Validation.
The rhizomes of turmeric (Curcuma
longa L., family Zingiberaceae) play an important role as a coloring agent
in foods, cosmetics and textiles. The main yellow bioactive substances in the
rhizomes are curcumin and two closely related demethoxy compounds, viz.,
demethoxycurcumin and bisdemethoxycurcumin. The rhizomes have long been used in
traditional medicine in India for multiple pharmacological activities including
anti-inflammatory, hepatoprotective, antitumour, antiviral, anticancer
remedies. They are also used to treat gastrointestinal and respiratory
disorders (1-3). Curcuma longa and other Curcuma species
are also widely used in the treatment of snakebite poisoning (4).
The anti-venom activity was due to a different component, viz., ar-turmerone.
The antivenin effect of Curcuma longa, reported by Ferreira et
al. (5) against the haemorrhagic activity of a venomous pit viper (Bothrops
jararaca) was also due to ar-turmerone.
Turmeric is a spice and of
golden yellow color and is used extensively in cooking in India. Today, India
is the primary exporter of turmeric (also known as Haldi). It has the ability
to preserve food through its antioxidant mechanism and gives color and taste to
the food. Its health promoting effects are well known .Most active component of
turmeric is curcumin. Curcumin is an orange –yellow powder with a structure C21H20O6.
In the present investigation a
number of turmeric samples from the market in the form of powder were collected
and analysed. Physico-chemical evaluation
of the powder was carried out to meet the requirements of tests proposed by
Agmark Grading and Marketing Rules(6)
MATERIALS
AND METHODS:
Preparation of standard solutions:
An individual stock solution
of standard compounds was prepared at a concentration of 100μg/mL in
ethanol.
Preparation of sample solutions:
About 500 mg of Curcuma
species
were sonicated in 2.5 mL of ethanol for 30 min followed by centrifugation for
15 min at 3300 rpm. The supernatant was transferred to a 10 mL volumetric
flask. The procedure was repeated thrice and respective supernatants combined.
The final volume was adjusted to 10.0 mL with ethanol and mixed thoroughly.
Preparation of extract
The samples were analysed for
moisture content, total ash, acid insoluble ash, starch, chromate, lead.
Curcumin content was also determined using UV method.
Sample analysis:
Moisture content:
The moisture content in the turmeric
sample is determined by Dean and Stark Toluene Distillation method. The
procedure is as per IS 1797:19857.
About 20 to 40 g of the sample is weighed accurately and placed in a
flask and covered with toluene. Assemble the apparatus and fill the receiving
tube with toluene through the top of the condenser. Heat the flask gently for
15mins and when the toluene boils, distill at the rate of about 100 drops per
minute until most of the water has distilled over, and then increase the rate
to 200 droplets per minute until no more water is collected. When the water has
apparently completely distilled over, the inside of the condenser is rinsed
.Continue the distillation for 5 mins. Stop heating and allow the receiving
tube to cool at 25°C for at least 15 minutes. After complete separation of
water and toluene in tube, the volume of water collected in tube is noted the moisture
content is calculated as follows:
Moisture Content ( % w/w) =
100 x volume of water x 0.997 / weight of sample
Total Ash value:
2.0 gram of turmeric powder was taken
in a tared silica crucible. Dry the crucible in electrical oven at 105o C. Moisten the sample with ethanol and heat it
over a small flame, till swelling ceases. Then ignite it at 550oC in
a muffle furnace. Allow it to cool at room temperature and weigh. Again place
the crucible in the muffle for 30 min. The
difference between the two weights should be less than (0.001 g). Record the
lowest mass. Retain the total ash for determining the water soluble ash and
acid insoluble ash. The % of ash on dried basis is calculated as
Total ash (%dry basis) =
Weight of ash x 100x100 / Weight of
sample ((100-M),
Where, M is the moisture content of
the sample in % W/W basis.
Acid Insoluble Ash:
The total ash obtained
from 2 grams of powder was boiled for 5
minutes with 25 ml of dilute
hydrochloric acid and the insoluble matter was collected on ashless filter
paper. It was washed with hot water, ignited
and weighed. The percentage of acid insoluble ash was calculated on dry
weight basis.
Transfer the ash content obtained in total ash in a
beaker; ensure that the complete ash is transferred, where water soluble or
insoluble ash is to be determined, take the residue of the water insoluble
ash. Add 25 ml of Hydrochloric Acid
Solution (2:5). Boil for 10 minutes by covering the beaker with a watch-glass
to prevent sputtering. Allow to cool and filter the content through ash less
filter paper. Wash the filter paper with hot water until the washings are free
from hydrochloric acid, as tested by silver nitrate solution. (No turbidity is
observed). Transfer the filter paper in the pre-weighed crucible. Dry the
crucible carefully in electric oven. Transfer the crucible in muffle furnace
and ignite it for 30 minutes.
The % Acid Insoluble ash on dry
basis =
Weight of ash x100x100 / Weight
of sample (100-M),
Where, M is the moisture
content.
Starch Value
The Starch is water insoluble polysaccharide and on hydrolysis with
hydrochloric acid or diastase converted to reducing monosaccharide. The
monosaccharide so obtained is determined by titration with Fehling’s solution
(Solution A + Solution B). If with
starchy food other reducing sugars are present the starch is calculated by
subtracting reducing sugar from total reducing sugar obtained after hydrolysis
or separating the starch by filtration, drying and then hydrolyzing the starch
to monosaccharide.
Weigh the substance according to the estimated value of Starch Content,
(for turmeric weigh accurately about 2 gm). Transfer the material on filter
paper, wash with 10 ml of solvent ether, and then wash with ethanol, transfer
the residue carefully with aid of water in a 500 ml round bottom flask fitted
with the water cool condenser.
Add 200 ml dilute hydrochloric acid and reflux for 2 ½ hrs, cool,
transfer in 250 ml volumetric flask, make up the volume and filter. Take 100 ml
filtrate in a beaker and neutralized with sodium carbonate using methyl orange
indicator, transferred to a 250 ml volumetric flask and make up the volume. The
total volume will be 250 x 2.5 = 625.
Take 10 ml of Fehling soln. (5ml+5ml) in porcelain dish and find out
the glucose factor . Take 10 ml of Fehling soln. in porcelain dish, pour the
prepared soln. into a 50 ml burette, and from burette add 20 ml of soln. into
the porcelain dish, heat the contents of the flask over a wire gauze and boil
for 15 seconds (copper brick red colour appears), add 1 ml of the methylene
blue indicator while continuing the boiling, boil for 1 min. and then add 2 ml
prepared soln. and continue boiling for 10 seconds, add the prepared soln. from
burette, 2 ml each time till the blue colour disappears. Note the burette
reading, this is the control reading. The total time required for the complete
titration should not be more than 3 minutes of boiling. Take again 10 ml of
Fehling’s soln. in porcelain dish and add 1 ml less than the control reading of
the prepared soln. into the porcelain dish, heat on wire gauze and boil for 30
seconds, add 1 ml of methylene blue indicator and heat for 1 min., Add 0.1 ml
prepared soln. each time as per the procedure described above, till the blue
colour disappears note the reading. Repeat this procedure till the two
consecutive readings are less than 0.1 ml. Record the lowest reading
Starch on dry weight basis (%
w/w) =
0.9 ×v ×100 ×100/ Weight of
sample (100-M),
Where M is the moisture content
Chromate Test:
Turmeric is coloured by poisonous lead chromate. To identify the
chromate, the sample is ignited to ash, ash is dissolved in dilute sulphuric
acid, and chromate gives violet colour with diphenyl carbazide solution
Take approximately 2 gm of sample in porcelain dish and heat to ash on
burner. Cool the crucible, and transfer the ash in a 100 ml beaker. Add 4-5 ml
of dilute sulphuric acid in a beaker and boil for 2 minutes. Filter and collect
the filtrate in test tube. Add 1 ml of diphenyl carbazide solution in the test
tube. If violet colour is produced indicate presence of chromate. Otherwise
chromate test is negative.
Curcumin
determination:
The official standard method for determination of curcuminoids or Curcuma-based products is UV-Vis
spectrophotometry which is relied on the direct measurements of sample in
certain solvents like ethyl alcohol.An intensive absorption intensity peak at
wavelength of 425nm is obtained. This is also termed as colour value. The
sample is prepared by grinding the sample and passing through 20 No. (850
micron sieve)
Weigh accurately 0.1gm of grounded turmeric powder into a flask fitted
with reflux condenser. Add 40ml Ethyl Alcohol and reflux for 2 hrs 30 minutes
on a water bath.
Cool this solution and filter in
a 100ml volumetric flask. Wash with
Ethyl Alcohol till free from yellow colour and make up the volume till the mark
with Ethyl Alcohol
(Solution A)
Pipette out 10ml of Solution A in 50ml Volumetric Flask and make up the
volume to the mark with Ethyl Alcohol (Solution B). Pipette out 10ml
Solution B in separate 50ml Volumetric Flask and make up the volume to mark
with Ethyl Alcohol ( Solution C).
Measure the absorbance of the Solution C at 425 nm in 1cm cells against
the alcohol as blank.
Curcumin is deteremined using visible spectroscopy in ethanol at λ
425 nm. When absorbance of 0.00025% standard solution of curcumin is measured in 1cm path cell at 425nm, an
absorbance of 0.42 is obtained which results in
E 1% of 1680.
Therefore, E 1% = 0.42
x 0.00025 = 1680
Curcumin on dry weight basis =
Where, M = Moisture
RESULTS AND DISCUSSION:
Validation procedure:
The
analytical method was validated in the laboratory by conducting experiments
with curcumine samples and standards that were similar to unknown samples
analyzed routinely. The experimental procedure meets all the criteria for
validation process. A good preparation work was carried out for efficient
experiment execution. All the reagents, reference standards, were accurately
weighed and checked for exact composition and purity as per specifications.
Other consumables such as glassware’s were calibrated to make sure that it
meets the functional and performance specifications as required for the
analytical method.
A sequence was developed based on our laboratory
experience with the UV spectrophotometer. The validation consisted of
Linearity, specificity, detection limits, and recovery of samples.
Linearity:
A stock solution of curcumine was prepared. The samples were analysed by UV
spectrophotometer at 425nm. A linear curve with coefficient of correlation of
0.9970 was obtained.
Selectivity/Specificity
For selectivity/specificity, analysis of blank samples of the
appropriate sample matrix was obtained. Each blank sample was tested for
interference, and selectivity was ensured at the lower limit of detection.
Blank sample analysis showed no interference.
Detection limits
Accuracy was determined by replicate analysis of samples containing
known amounts of the analyte. Accuracy was determined using a minimum of seven
determinations per concentration. The deviation of the mean from the true value
serves as the measure of accuracy.
Method detection limits (MDL) were also determined for curcumin sample
under this study. It provides a useful mechanism for illustrating the
capability of the analytical method. MDLs were calculated as follows:
The sample standard deviation is multiplied by the correct Student's
t-value from the statistical Tables.
It provides a useful mechanism for illustrating the capability of the
analytical method. MDLs were calculated for Curcumin as follows:
MDL= (s)(t-value)= 0.12. x 3.143= 0.40 ug/ ml.
Similarly, LOQs were subsequently established as 10 times the Standard
Deviation of the recovered curcumin..
The limit of quantitation was also calculated as :
LOQ= 10 x (s)= 10 x 0.12 = 1.2 ug/ml
The MDL and LOQ
were thus calculated for all the curcumin in turmeric under study and are
summarized in Table 1.
Characterization of Turmeric powder
The results obtained in the analysis of turmeric powder in various
samples is summarized in Table 2.The parameters determined are moisture, total
ash, acid insoluble ash, starch, curcumin, lead and chromate.
Moisture :
The determination of moisture content of spices is of importance for
many scientific reasons. Water occurs in foods essentially in two forms, as
bound water and as available free water. Bound water includes water molecules
chemically or hydrogen bonded to ionic and polar groups whereas free water is
that which is not physically linked to the food matrix and which is freezable
and easily lost by evaporation or drying. Most of the foods are
heterogeneous mixture of substances ,
they may contain varying proportions of the two types. The moisture content of
the sturmeric powder samples analysed ranged between 8.0 to 8.4 %. The moisture
content is within the standard limit of 10.0 prescribed by Agmark.
Total ash and
Acid insoluble ash :
It is observed that results vary from sample to sample. However, levels
of moisture, acid insoluble ash, and starch were consistent in all the samples.
The quality of turmeric powder can also be assessed by considering the total
ash value and acid insoluble ash value. The ash of spices is the inorganic
residue remaining after the organic matter has been burnt away. The ash content
can be regarded as a general measure of the quality or grade of the material
under investigation and often is a useful criterion in identifying the
authenticity of a food. When a high ash figure suggests the presence of an
inorganic adulterant, it becomes necessary to also determine the acid
–insoluble ash. The results of total ash and acid insoluble ash are summarized
in Table 2.It is observed that samples
no. 3 and 1 has the minimum ash levels which indicate that the these samples
are of better quality or grade than the other sample analysed. The samples no 4
and 5 have higher ash value when compared with Agmark standard. High values of
total ash may be due to presence of carbonates, phosphates, silicates in the
sample. The mean acid insoluble ash for
the samples analysed is 0.49 % .The acid insoluble ash is a measure of sandy
material in the spices and the limit
prescribe by Agmark is 1.5 % on dry basis.
Lead and
Chromate:
Many times presence of lead in the samples has been observed. The
presence of lead in turmeric is due to
its occurrence in the roots due to its uptake from the soil and water or due to
external treatment of turmeric for obtaining color and polish to the sample
using lead chromate. The lead content in the present investigation was below
the Agmark limit of 2 ppm. The chromate content was also negative.
Table 1 : Accuracy And Detection
Limits Determination
|
Name of chemical |
λnm |
Test 1 ug/ml |
Test 2 ug/ml |
Test 3 ug/ml |
Test 4 ug/ml |
Test 5 ug/ml |
Test 6 ug/ml |
Test 7ug/ml |
SD |
RSD |
Mean |
LOD ug/ml |
LOQ ug/ml |
|
Curcumin |
425 |
3.2 |
3.4 |
3.3 |
3.5 |
3.4 |
3.3 |
3.2 |
0.12 |
3.6 |
3.34 |
0.40 |
1.20 |
Table 2. Characterization of Turmeric powder as per
Agmark
|
S.no. |
Grade |
Moisture % w/w |
Total ash % w/w |
Acid Insoluble Ash
% w/w |
Starch % w/w |
Lead ppm |
Chromate |
Curcumin % w/w on dry basis |
|
1. |
Turmeric Powder |
8.4 |
7.0 |
0.47 |
52.21 |
< 2.0 |
-ve |
3.34 |
|
2. |
Turmeric Powder |
8.3 |
7.7 |
0.49 |
52.0 |
< 2.0 |
-ve |
3.64 |
|
3. |
Turmeric Powder |
8.4 |
6.5 |
0.47 |
51.7 |
< 2.0 |
-ve |
3.74 |
|
4. |
Turmeric Powder |
8.1 |
8.2 |
0.49 |
51.2 |
< 2.0 |
-ve |
5.06 |
|
5. |
Turmeric Powder |
8.0 |
8.9 |
0.48 |
51.1 |
< 2.0 |
-ve |
9.45 |
|
|
Agmark *Std |
10 |
7.0 |
1.5 |
60.0 |
2.5 |
-ve |
|
*Agmark is a product standardisation logo issued by the Directorate of
Marketing and Inspection (DMI) — a certification body for grading of
agricultural produce.
Curumin:
The most important chemical that is responsible for yellow color in
turmeric is curcumin. The curcumin content in the sample analysed varies from
3.34 to 9.45 %. Higher concentration of curcumin in turmeric samples was found
in samples no.3 and 4 respectively.
Starch:
Starch, a renewable biopolymer, is a versatile agricultural raw
material for industrial purposes because it is inexpensive, relatively easy to
handle, completely biodegradable and widely available in nature from sources
such as cereals, roots, tubers, palms and seeds. Developments in the industrial
sector have increased interest in the identification of new starches with
distinct properties and their potential for processing at large scales.
However, little information is available on practical applicability of
non-conventional sources of starch. Thus, it is necessary that these starches
be studied to obtain and report their structural parameters, information that
is required to gain competitiveness in an international-scale industry.
Turmeric (Curcuma longa) which is
used in industry to obtain food coloring and pharmaceutical products,
may become commercially interesting as starch raw materials. Isolated starch
from turmeric rhizomes after the extraction of its oil contains about 40% (w/w)
starch (8).
Starch accounts for a significant fraction of a large range of crops.
Cereals (e.g., corn, wheat, rice, oat, barley) contain from 60% to 80% of this
carbohydrate, legumes (e.g., chickpea, bean, pea) from 25% to 50%, tubers
(e.g., potato, cassava, cocoyam, arrowroot) from 60% to 90% and some green or
immature fruit (e.g., banana, mango) as much as 70% of the dry weight(9). Starch is frequently isolated and is used in
food industries to impart the desirable functional properties, and modify food
texture and consistency(10). Thus analysis of turmeric samples for
starch would generate information on the unique characteristics of uncommon
starch products and would be helpful for practical applications in food and
non-food processing of end-use products.The starch from turmeric samples was
analyzed and the results are summarized in Table 2. Starch was observed to be a
major component in the turmeric powder with an average level of 51%. Turmeric
is rich in starch. Thus it can be use d as a non-conventional source of starch.
CONCLUSION:
The present investigation reports the validation of analytical method
for curcumin. It also studies the characteristics of active constituents in
turmeric powder available commercially. Such an analysis helps in assessing the
purity of the samples and precision and accuracy of the method through
validation of method. The analysis also brings out the fact that non-
conventional products like turmeric can also be a source for starch.
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Received
on 03.09.2015 Modified on 17.09.2015
Accepted
on 25.09.2015 © AJRC All right
reserved
Asian J. Research Chem. 8(10): October 2015; Page 643-647
DOI: 10.5958/0974-4150.2015.00102.9