SEPARATION AND IDENTIFICATION OF CAROTENOIDS IN FLOWERS

OF CHELIDONIUM MAJUS
Introduction
Today more than 600 different naturally occurring carotenoids are
known to exist in nature. Because of the large number of potential
geometrical (E/Z or trans/cis) isomers of carotenoids it is important to
investigate the most appropriate isolation, separation and identification
procedure of these materials.
Chemically carotenoids are isoprenoid compounds which are
important dietary nutrients with antioxidant potential. Different
carotenoids are derived from the same basic C40 isoprenoid skeleton by
modifications such as cyclization, substitution, elimination, addition and
rearrangement. Because of the large number of double bonds, there are
extensive possibilities for geometrical (E/Z or trans/cis) isomerism of
carotenoids.
The first indication of a (Z)-isomer structure is usually given by the
UVVis-spectrum, whose fine structure is reduced in (Z)-isomers
compared to that of (E)-isomers. The kmax values of (Z)-isomers show a
47 nm hypsochromic shift for each double bond compared to the kmaxvalues of (all-E)- substances. Peripheral (Z)-isomers have a low cispeak, and central (15Z)- and next-to-central (13Z)- and (130 Z)-isomers
have a high cis-peak].
There are many natural carotenoids in the flora of the world,
particularly, in medicinal plants. For pharmacological experiments it is
important to screen, analyse and identify these substances. The majority
of previous studies have been concerned with -carotene, but in recent
years, other carotenoids found in the human diet have also begun to
receive attention . Carotenoids may give protection against some lifethreatening conditions, for example, cancer and heart disease. The tea
made from Solidago canadensis flowers is used in the treatment of
diarrhoea, body pains, fevers and snakebites. The main secondary
metabolites of the plant are flavonoids (e.g. quercetin, used in
haemorrhagic nephritis) and antiinflammatory phenolic glycosides,
saponins with antifungal activity, essential oil, tannins and fructan [15].
Chelidonium majus L. (greater celandine) is a member of the family
Papaveraceae. The flowers comprise four yellow petals and two sepals.
The whole plant is toxic as it contains a range of isoquinoline alkaloids,
but there are numerous therapeutic uses when applied at the correct
dosage. The effect of the fresh herb is of a mild analgesic, cholagogic,
antimicrobial and central nervous system sedative. Externally, the
otherwise purgative, caustic, orange sap is an ancient remedy for warts,

corns and ringworm . The current research project aimed at the

carotenoid analysis of total extracts of the inflorescences of Canadian
goldenrod (Solidago canadensis L.), and the flower of greater celandine
(Chelidonium majus L.)
Extraction & isolation of carotenoids was carried out under
nitrogen in darkness and at low temperatures (22 C) using methanol for
dehydration, to avoid pigment decomposition.
The fresh flowers and inflorescences (83.91 g fresh weight for S.
canadensis and 1.28 g for C. majus) were extracted three times with
MeOH and once with Et2O.
The methanolic extracts were combined and transferred into the
mixture of toluene and hexane (1:1) in a separation funnel.
After evaporation of the latter solution the residue was dissolved in
Et2O.
The ethereal solutions were combined and this total extract was
saponified in heterogeneous phase (30% KOH/MeOH) overnight.
The saponified ethereal extract was partitioned between MeOH :
H2O (9:1) and hexane. The partition resulted in a hypophasic and an
epiphasic fraction (hypophasic and epiphasic carotenoids), which were
analyzed separately by CLC and LC.
Experimental Conditions of LC Separations
The carotenoid content was determined using the chromatographic
method suitable for different plant materials and the multi-component
plant extracts. Reverse phase LC analysis was performed on a 250 9 4.6
mm endcapped LiChrospher 100 RP-18 column (Merck, Darmstadt,
Germany), sphere diameter 5 lm. The injection volume was 20 lL and the
column temperature 20 C.
The separation was performed by gradient elution at a flow rate of
1.25 mL min-1 and peak identification was at 450 nm. During the analysis
three different mobile phases were used, namely 12% (v/v) water in
methanol (A), methanol (B) and 50% (v/v) acetone in methanol (C).
The gradient program was the following: 100% A eluent initially for 2
min, increased to 20% B in 8 min, 50% B in 8 min, 100% B in 9 min, 100%
C in 7 min, returned to 100% B in 9 min and finally 100% A eluent in 2
min (linear steps). Experimental Conditions of CLC Separations In the
experiments columns with a length of 30 cm and inner diameter of 6, 5, 4
and 3 cm were used. The adsorbent CaCO3 was pharmacopeal quality
(Ph. Hg. VI., Biogal, Debrecen, Hungary). Toluene-hexane and acetonehexane mixtures with different compositions were used as eluents [18].
Qualitative Analysis and Identification of Carotenoids

The total carotenoid content of the plant samples was determined by

UVVis methods. The determination of the carotenoid composition of the
different fractions (total extracts, hypo- and epiphasic fractions) was
carried out by LC. The peak area was used to determine the percentage
of individual compounds in the extracts [19]. Carotenoids were identified
on the basis of their UVVis spectroscopic properties in different solvents,
chemical reactions [(E/Z)-isomerization (thermal isomerization and iodinecatalized photoisomerization), 5,6-epoxide ? 5,8-epoxide (furanoid oxide)
rearrangement, NaBH4-reduction], by co-chromatography with authentic
reference samples (purity: >95%; LC) and by LC retention times

EXTRACTION AND STANDARDIZATION OF WITHANIA /

ASHWAGANDHA

The process involves

1. Sorting and sieving of roots, to remove dust & contaminants
2. Pulverization of roots
3. Cold extraction and filtration
4. Hot extraction and filtration
5. Concentration of extract
6. Drying of extract
BRIEF DESCRIPTION OF FIGURE:
FIG 1 : Flow chart for Extraction of Ashwagandha.

DESCRIPTION OF THE PROCESS

The plant material i.e. Ashwagandha {Withania somnifera) roots are
collected from Local market
The extract is standardized to contain Withanoside IV and Withanoside
V within the range of not less than 0.8 % and 0.4 % respectively.

The process describes extraction of Withanoside IV and Withanoside V

from Ashwagandha {Withania somnifera) roots comprises following
steps^
a) Sorting and sieving of roots to remove dust and contaminants;
b) pulverizing of roots to obtain pulverized powder;
c) cold extraction of pulverized powder of step (b) with polar solvent
followed by centrifuging to obtain the residue;
d) subjecting to hot extraction of residue obtained in step (c) by adding it
to polar solvent followed by refluxing and centrifuging;
e) collecting the filtrates of step (c) and step (d) after centrifugation,
followed by distillation to obtain an extract;
f) adding and mixing methyl paraben IP, Propyl paraben IP, dicalcium
phosphate anhydrous IP and Colloidal silicone Dioxide IP to the extract of
step (e) to obtain pasty mass;
g) transferring the pasty mass of step (f) to evaporator, followed by
stirring and heating to obtain granular powder of 30 to 60 mesh.
Alternatively following method can be followed
Step 1: Sorting and sieving of roots to remove dust and
contaminants
Ashwagandha roots are cut into small pieces and spread on SS sieve of
size 10 mesh to 30 mesh. The contaminants, infected roots or adhered
dust etc. from the roots are removed by sieving or simply by hand
picking. After sorting and sieving, roots are filled in clean bag for
Pulverization.
Step 2: Pulverization of roots
Suitable commercial pulverization machine with 30 to 60 mesh sieve is
used for pulverization & plastic bag is used for collecting the pulverized
material.
Step 3: Cold extraction and filtration
Mixing and stirring fixed quantity of pulverized Ashwagandha powder of
Step 2 with sufficient quantity of polar solvent in first vessel. Heating the
mixture from Room Temperature to below boiling point and maintain there
for a period of 8 to 15 hours with continuous stirring. Cool the mixture at
room temperature followed by centrifugation (I) to obtained residue. The
filtrate of centrifuge (I) is taken in a second vessel for concentration. The
residue is charged to first vessel for hot extraction.
Step 4: Hot extraction and filtration
The residue obtained from Step 3 is added to the first vessel containing
enough of same polar solvent (30% recovered polar solvent and 70%
fresh polar solvent, wherein the recovered polar solvent is from
Ashwagandha of previous batch), followed by heating and refluxing at
from 60C to 100C over a period of 1 to 4 hours. The mixture is cooled to
Room Temperature and discharge the mass in SS tank followed by
centrifugation.
After centrifugation, the filtrate is added to the second vessel of Step 3.
Step 5: Concentration of extract

The filtrates of Step 3 and Step 4 are collected in second vessel and
heated slowly and further raising the temperature of mixture to distill out
polar solvent, followed by cooling to Room Temperature Discharge pasty
mass in previously weighed SS storage tank. The test sample is sent for
quality control and to the extract obtained is added methyl paraben IP,
Propyl paraben IP and mixed well followed by addition of Dicalcium
phosphate anhydrous IP and Colloidal silicon dioxide IP in the quantity
based on analysis of test sample and total quantity of the extract, and
further mixing well to get the pasty mass.
Step 6: Drying of extract
Transferr the pasty mass of Step 5 to evaporator, followed by stirring and
heating from Room Temperature to 100C, till it is converted into granular
powder. Cool to Room Temperature ,Pass the granular powder from
pulvarizer or multimill, to get the final dry powder of 30 to 60 mesh.
The polar solvent used in the above mentioned process is
selected from lower alcohols such as methanol, ethanol, acetone,
isopropyl alcohol etc., ketones such as acetone, methyl ethyl
ketone, diethyl ketone, ethyl butyl ketone etc., ethers such as
dimethyl ether, diethyl ether, tetrahydrofuran (THF) etc. The
polar solvent used in the abovementioned process is preferably
methanol.
The desired percentages of Withanoside IV and Withanoside V extracted
from Ashwagandha roots are NLT 0.8 % and NLT 0.4 %
respectively.Accordingly, the pharmaceutically acceptable excipients are
selected from the group comprising diluents, binders, wetting agents,
disintegrants and lubricants.Suitable excipients such as Lactose, Starch,
Sodium starch glycolate, Talcum, Magnesium stearate etc. can be used.
The amounts of active constituents such as Withanoside IV and
Withanoside V are analyzed by using HPLC method. The presence of
residual polar solvent, in final dried extract is analyzed by using GC
method. The specifications of residual solvents are shown in Table 2.
In another embodiment of the present invention, trial batches using
various polar solvents were analyzed for the presence of Withanoside IV
and Withanoside V. The results are shown in Table 3.
Table 1 : Results of HPLC Analysis:

Table 3:

Extraction and recovery of rutin

This invention relates to a method for the extraction and recovery of

rutin, comprising extracting rutin-bearing plant material with a distillable
polar solvent, adding a relatively highboiling hydrocarbon to the extract,
distilling

off the polar

solvent from the mixture and separating

precipitated rutin from the distillation residue.

Rutin is a glucoside found in many species of plants; it has been known
for about a century and its formula has been known for about fifty years.
Chemically it is quercitin-p--l--rhamnoside-6-d-glucose.
It has vitamin-P activity, counteracting fragility of the capillaries due to
pathologic conditions or to the action of other drugs. It has been
suggested for therapeutic use in such conditions as diabetes, retinal
hemorrhage, apoplexy, phlebitis and the like and counteracts the adverse
effect on the capillaries of salicylates, arsenicals and thiocyanates.
Because of the growing therapeutic interest in rutin and since its largescale synthesis is not practical, methods of recovery from natural sources
are important.
Rutin is found in at least 38 species of plants including tobacco, but its
best source in this county is buckwheat, Rutin is present chiefly in the
young green tissues of the plant. These are preferably dried soon after
harvesting to secure a maximum yield.
Previous to the present invention an extensively used method of
recovering rutin from buckwheat was the following:
a. Green fresh buckwheat or flash-dried buckwheat is macerated with
ethanol, which may be denatured with methanol or benzene.
b. The extract is withdrawn and the extraction repeated with a fresh
batch of ethanol. c. The extract is withdrawn and the solvent distilled off
from the combined extracts at atmospheric pressure.
d. The residue is extracted with benzene and the extract discarded.

e. The residue is extracted with hot water and the extract allowed to cool.
Crude rutin crystallizes out and is filtered off.
f. The rutin is recrystallized from water.
Since rutin is readily oxidized, rapid extraction is preferable to slow
percolation. The temperature-solubility coefficient of rutin in water is high,
so that water is a favorable crystallization medium. Approximately 5.5 g.
rutin is soluble in a liter of hot water, but only 135 mg. in a liter of cold
water.
Modification : after extraction with alcohol (steps a and b above) and
before distillation (step c), I add a volatile hydrocarbon fraction having a
higher boiling point than the alcohol-e. g. a refined mineral spirit distilling
substantially in the range 160-190 C.;
then distill the mixture until alcohol and water are driven over--e. g. until
the distilling liquid reaches a temperature of 110 0 C. The rutin, being
insoluble in hydrocarbons, forms a viscous residue, from which the
remaining hydrocarbon can be decanted; the decanted hydrocarbon
contains dissolved in it plant resins, chlorophyll and other dark colored
impurities extracted by the alcohol from the buckwheat. Because of the
removal of these impurities one recrystallization of the rutin residue from
water suffices.
This process has, among others, two substantial advantages over the
prior process described above:
(1) It provides for ready removal from the original extract of alcohol and
of any water present without the danger of deleterious local overheating,
since the hydrocarbon left in the still at the end of the distillation provides
a liquid "cushion."
(2) Distillation in the presence of the hydrocarbon fraction simultaneously
effects removal of the extractant and a clean-cut separation of dark
colored oil-soluble impurities from the crude rutin.