Quantification of Primary Metabolites of Nerium Indicum Mill
Quantification of Primary Metabolites of Nerium Indicum Mill
Quantification of Primary Metabolites of Nerium Indicum Mill
1, 2007, 123-128
123
Vijayvergia R. and Kumar J. (2007) Asian J. Exp. Sci., 21(1), 123-128
macerated in a grinder with 20 ml of ethanol tube was kept on a water-bath (26º – 30º
and left for 12 hrs. and mixtures was C) for 20 min, and the optical density
centrifuged (1200 rpm) for 15 min, the (ODs) of the yellow orange colours thus
supernatants were removed and was developed were taken at 490 nm in a
concentrated on a water-bath. The volume Spectrophotometer after having set it for
of these aqueous concentrates were raised 100% transmission against the blank. Four
to 50 ml with distilled water (Ext. A) and replicates of each sample were run and
processed further by following the method there mean values were calculated. A
of Loomis and Shull (1937) for soluble regression was computed between its
sugars. However, the residual pellet known concentrations and their respective
obtained by centrifugation was used for the ODs. which followed the Beer’s Law. The
estimation of starch. concentration (mg/gdw) of the total soluble
(B) Starch : The above residue of sugars was directly worked out from the
each test sample was suspended in a regression curve of the standard glucose.
mixture of 5 ml of 52% perchloric acid Four replicates of each experimental
solution and 6.5 ml of distilled water, sample were taken and their mean values
shaken vigorously (5 min) and centrifuged recorded. The sugar content in terms of
(2500 rpm). This step was repeated three glucose equivalent and the use of
times and the supernatants of each sample conversion factor (0.9 to convert the values
was pooled and the volume was raised to of glucose to starch) was made in each
100 ml with distilled water (Ext B). Out case.
of this (Ext. B), 1 ml aliquot was taken Extraction of Proteins : A 60 mg of
separately to estimate starch quantitatively the dried test sample was macerated
(McCready et al., 1950). (Osbrone, 1962) in 10 ml of cold TCA
Quantification of carbohydrates : (10%) for 30 min kept at low temperature
Aliquot (1 ml) of each of the test sample 4º C for 24 hr and then centrifuged. Each
from Ext. A and B were used to quantifying of the supernatants was discarded and the
the total levels of carbohydrates using resultant pellet was re-suspended in 5%
phenols-sulphuric acid method (Dubois et TCA (10 ml) and heated on a water bath
al., 1951). A regression curve for standard at 80º C for 30 min. Each of these samples
sugar (glucose) was also prepared. was cooled, re-centrifuged and each time
the supernatant discarded. Later the pellet
A stock solution of glucose (100 mg/ was washed with distilled water,
ml) was prepared in distilled water, out of centrifuged and each of the residues was
which 0.1 to 0.9 ml was transferred to test dissolved in 1N NaOH (10 ml) and left
tube and the volume was raised to 1 ml overnight at room temperature.
with distilled water. To each of these, 1 ml
of 5% aqueous phenol was added rapidly Quantification of Proteins : In each
having kept in an ice chest and shaken of 1 ml extract, total protein content were
gently. Later 5 ml of Conc. H2SO4 was estimated using the protocol of Lowry et
rapidly added by agitating gently during al., 1951. A stock solution (1mg/ml) of
the addition of the acid subsequently, the bovine serum albumin (Sigma Chemicals)
was prepared in 1 N NaOH, from which
124
Primary Metabolites of Nerium indicum Mill
0.1 to 0.9 ml of the solution was dispensed Extraction of Phenols : Each of 200
separately in a test tube. After this, the mg dried and milled test samples was
volume of each was raised to 1 ml by homogenized in 80% ethanol (10 ml) for
adding distilled water. To each test sample, 2 hrs and left over night at room
5ml of freshly prepared alkaline solution temperature. It was centrifuged, the
(prepared by mixing 50 ml of 2% Na2CO3 supernatants were collected individually
in 0.1 N NaOH and 1 ml of 0.5%CuSO4. and the volume of each was raised to 40
5H2O in 1% sodium potassium tartrate) ml with 80% ethanol.
was added at room temperature and left Quantification of Phenols : To
undisturbed for a period of 10 min. estimate total phenols in each of the test
Subsequently, to each of these mixture sample, the protocol of Bray and Thorpe
tubes 0.5 ml of Folin-Ciocaltcau reagent (1954) was followed, wherein a standard
(CSIR centre for Bio-chemicals, Delhi: curve of caffeic acid (a phenol) was
diluted with equal volume of distilled prepared
water just before use) was rapidly added
and after half an hr, the OD of each was A stock solution (40 mg/ml) of caffeic
measured at 750 nm using a acid was prepared in 80% ethanol, from
spectrophotometer against the blank. Three which 0.1 to 0.9 ml was transferred into
replicates of each concentration were taken test-tubes separately and the volume in
and there mean values were used to each case was raised to 1 ml with 80%
compute a regression curve. The total ethanol. To each of these tubes, 1 ml of
protein contents in each sample was Folin–Ciocalteau reagent (prepared by
calculated by referring the ODs of test diluting the reagent with distilled water in
sample with the standard curve of BSA. 1:2 ratio just before use) accompanied by
Three replicates were examined in each 2 ml of 20% Na2CO3 solution was added
case and their mean values were recorded. and the mixture was shaken vigorously.
Each of these were boiled on a water bath
Extraction of Lipids : One g of each (1 min), cooled and diluted to 25 ml with
of the dried and milled test sample was distilled water. The OD was taken at 750
macerated with 10 ml distilled water nm using a spectrophotometer against a
(Jayaraman, 1981). To this, 30 ml of blank. Three such replicates were taken for
chloroform-methanol (2 : 1, v/v) was each concentration and the average OD
added and mixed thoroughly. Each mixture was plotted against the respective
was left overnight at room temperature, 20 concentration to compute a regression
ml of chloroform and the equal volume of curve.
distilled water was added and centrifuged.
Out of the three layers, a clear lower layer Each test sample was processed in this
of chloroform containing all lipids was similar manner, ODs were measured and
collected in pre-weighted beaker, the the total level of phenols was calculated
solvent evaporated completely and from the mean values (with reference to
weighed, which was taken as the weight caffeic acid) by referring the OD of the test
of total lipids/g of the dried tissue sample. sample with the regression curve of the
standard.
125
Vijayvergia R. and Kumar J. (2007) Asian J. Exp. Sci., 21(1), 123-128
126
Primary Metabolites of Nerium indicum Mill
127
Vijayvergia R. and Kumar J. (2007) Asian J. Exp. Sci., 21(1), 123-128
128