Physicochemical and Preliminary Phytochemical
Studies On the Leaves of Crinum latifolium Linn.
Mahima Yadav 1, A. K Meena2, M M Rao2, A. K Mangal2 and Jagbir Chahal1*
1M.M. College of Pharmacy, Mullana,
Ambala, Haryana - 122001 (India)
2National Institute of Ayurvedic
Pharmaceutical Research, CCRAS, Dept. of AYUSH, Patiala -147001, (India).
ABSTRACT:
The present
communication attempts to evaluate the physicochemical and preliminary phytochemical studies on the leaves of Crinum latifolium Linn.
of Amaryllidaceae family. Crinum latifolium Linn. rosette-like herb that arises from an
underground bulb which is used in several traditional medicines to cure various
diseases. It is a stout perennial herb of about 2 m in height. This herb has
been known to
posses Analgesic activity, Anticancer activity and Immuno stimulatory activity. Phytochemical
analysis has recently yielded a vast array of compounds, including more than
150 different alkaloids, tannin, phenolic
compound, flavonoids, terpenoids,
amino acids, steroid saponins and antioxidants. Hippadine, pratorinine, ambelline and lycorine, 2-epilyocorine and 2-epipancrassidine etc.
have been isolated from this plant. As there is no detailed standardization
work reported on fruit, the physicochemical parameters, preliminary phytochemical constants, thin layer chromatography are carried
out. The study revealed specific identities for the particular crude drug which
will be useful in identification and control to adulterations of the raw drug.
KEYWORDS: Extractive values, ash value, TLC, Ayurvedic drug, physicochemical
studies, Crinum latifolium Linn .
INTRODUCTION:
Since origin of
human’s life, plants continue to play a curative and therapeutic role in
preserving human health against disease and decay. The widespread use of herbal
remedies and healthcare preparations, such as those described in ancient texts
like the Vedas and the Bible have been traced to the occurrence of natural
products with medicinal properties.1,2.
The plants of the genus Crinum (Amaryllidaceae)
are used in Asian folk and traditional medicine as rubefacient,
tonic and for treatment of allergic disorders and tumor diseases. These
activities are attributed to the presence of Amaryllidaceae
alkaloids known to possess moderate antitumor and immunostimulating
activities3. The 130
species4 of Crinum
have a pan tropical
distribution with the center of diversity south of the Sahara5. Crinum
species produce tunicate bulbs that, at certain times of the year, are
dormant. But few plants are more striking than this stately Amaryllidaecae6.
Crinum asiaticum (subg. Crinum)
resolved as sister to C. latifolium7.The leaves were investigated at and after flowering, because
significant changes in the alkaloid content of C. latifolium
during different stages of plant growth have been observed. Aqueous extracts of Crinum latifolium L. leaves are used in Vietnamese folk
medicine as an anticancer remedy.
Recently, aqueous extracts from C. latifolium
leaves from Vietnam showed in vitro and in vivo T-lymphocyte
activation 8 and retarded growth of chemically induced tumors
(sarcomas) in rats9. Medicinal
use of C. latifolium
L. is an immunostimulant in disease10.
The chemical composition of leaves
consists of Glucan A and B, phenylalanine, L-leucin, D-L-valin, L-arginin monohydrochloride, latisolin, latisodin, Ambellin, 11-0-acetylambellin, 11-0 acetyl 1,2-β epoxyambellin, crinafolin, crinafolidin, lycorin, epilycorin, epipancrassidin,
9-0-demethylhomolycorin, lycorin-1,0-glucoside, pratorine
(hippadin), pratorinin, pratorimine, pratosin, beladine, latindin, latifin11.
Reports in the
literature suggest widespread use of Crinum species in treating a
variety of ailments. In some cases the Crinum latifolium were used in different countries for the same medicinal purposes like
Earache, Fistula, Rheumatism, Rubefacient , Tubercle , Tumour
and Whitlow12. In most cases these confirm the
therapeutic value of the plants. However where plants have been studied
chemically, a plant potential pharmaceutical value can be assumed, if it is
used widely13. The reason Crinum is used for medicinal
purposes and in a number of countries for similar reason is possibly due to
their alkaloids constituents14.
MATERIALS
AND METHODS:
Plant
material:
The leaves of Crinum latifolium Linn. were collected from the herbal garden
of National Institute of Ayurvedic Pharmaceutical Research (NIAPR), Patiala
(Punjab) during Sep.- Oct. 2010. The voucher specimens were identified and
authenticated by the Taxonomists.
A voucher specimen is kept in the NIAPR Herbarium. The leaves were
dried in shade, pulverized by a mechanical grinder and passed through 40-mesh
sieve to get the fine powder.
Preliminary phytochemical
group test:
The preliminary phytochemical group test of leaf extracts of the plants was
performed by the standard methods15-17.
1)
Test for alkaloids: A small quantity (2 - 5 gm) of the leaf extract of
plant was treated with few drops of dilute hydrochloric acid (HCl) and filtered. The filtrate was treated with Mayer’s
reagent (mercuric chloride and potassium iodide), and the formation of
Yellowish buff colored precipitate indicated positive test for alkaloids.
Small
quantity of leaf extract of plant was treated with few drops of dilute HCl and filtered, and the filtrate was treated with Dragendroff’s reagent (Sodium iodide, basic bismuth
carbonate, glacial acetic acid and ethyl acetate). An orange brown precipitate
indicated the presence of alkaloids.
When small
quantity of leaf extract of plant was treated with few drops of dilute HCL and
filtered, and the filtrate was treated with Wagner’s reagent (Iodine and
potassium iodide) a reddish brown precipitate developed, suggesting the
presence of alkaloids.
Small
quantity of leaf extract of plant was treated with few drops of dilute HCl and filtered, and the filtrate was treated with Hager’s
reagent (aqueous solution of picric acid), a yellowish precipitate demonstrated
the presence of alkaloids.
2) Test
for amino acids: Small quantity of leaf extract of plant was
dissolved in a few ml of distilled water separately and treated with Ninhydrin (tri-ketohydrindene
hydrate) at pH 4 to 8, and the absence of purple coloration suggested the
absence of amino acids.
3) Test
for flavonoids and their glycosides: Small quantity of leaf extract was dissolved in ethanol
and was hydrolyzed with 10% sulphuric acid (H2SO4)
and cooled. Then, the mixture was extracted with diethyl ether and divided into
three portions in three separate test tubes. 1ml of diluted sodium carbonate,
1ml of 0.1M sodium hydroxide and 1ml of diluted ammonia solution were added to
the first, second and third test tubes. The development of yellow color in each
test tube, demonstrated the presence of flavonoid.
4) Test
for steroids and triterpenoids: Libermann-Burchard reaction: The extract(10 mg) of plants was
dissolved in chloroform (1 ml); and then 1 ml of acetic anhydride was added to
the mixture followed by 2 ml of concentrated H2SO4. A
reddish violet ring at the junction of the two layers confirmed the presence of
triterpenoids and steroids.
Salkowski
test: When concentrated H2SO4
was added to chloroform solution (1 ml) of the extract a reddish-blue color was
produced in the chloroform layer and green fluorescence in acid layer,
suggesting the presence of steroids.
5) Test
for reducing sugar: Small quantity (2 - 5 mg) of extract of
plant was dissolved in minimum amount of distilled water and filtered. Equal
volume of Benedict’s reagent was mixed with the filtrate in a test tube and
heated for few minutes, a brick red precipitate confirmed the presence of
reducing sugars.
Small
quantity of extract (2-5 mg) of plants was dissolved in minimum amount of
distilled water and filtered, and to the filtrate equal volume Fehling’s
solutions in a test tube heated for few minutes, leading to the development of
brick-red color indicated the presence of reducing sugars.
6) Test
for gums: Small quantity of extract of plant was
dissolved in minimum amount of distilled water and filtered. The filtrate was
treated with equal volume of concentrated H2SO4, and then
treated with 15% alcoholic solution of α-naphthol
(Molish”s reagent). The formation of red-violet ring
at the junction of sulphuric acid layer and extract
indicated the positive test for gums (Molish’s test).
7) Test
for tannins: Small quantity of extract of plant was
dissolved in minimum amount of distilled water and filtered, and the filtrate
when treated with10% aqueous potassium dichromate solution a yellowish brown
precipitate demonstrated the presence of tannins.
When the
above filtrate was allowed to react with 10% aqueous lead acetate solution, a
yellow color precipitate formation indicated the positive test for tannins.
Again when
the above filtrate of extract are allowed to react with 1 ml of 5% ferric
chloride solution, formation of greenish black coloration demonstrated the
presence of tannins.
8)
Tests for saponins: Small quantity of extract of plant was dissolved in
minimum amount of distilled water and shaken in a graduated cylinder for 15
min. Formation of stable foam suggested the presence of saponins.
RESULTS
AND DISCUSSIONS:
The leaves of Crinum
latifolium Linn
was collected and analyzed the various standardization parameters.
Preliminary phytochemical results showed the presence
or absence of certain phytochemicals in the drug. The
tests performed using various extracts. Phytochemical test revealed the presence or absence of
Alkaloid, glycoside, saponins, flavonoids,
polysaccharides, Steroid, Tannin and results are given in Table1.
Table
1. Preliminary phytochemical tests for different
solvent extract of leaves
of Crinum latifolium Linn.
|
S. No. |
Natural
products |
Test
performed |
Result |
|
1.
|
Alkaloid |
Dragendorff’s test |
+ve |
|
2.
|
Amino acids |
Ninhydrin |
+ve |
|
3.
|
Flavonoids |
Shinoda test |
+ve |
|
4.
|
Steroid/ phytosterols |
Liebermann-Burchard
reagent/ Salkowski test |
+ve |
|
5.
|
Reducing Sugar/ carbohydrates |
Fehling’s solutions / Benedict’s reagent |
+ve |
|
6.
|
Gums |
Molish’s test |
+ve |
|
7.
|
Tannin |
Lead acetate |
+ve |
|
8.
|
Saponin |
Foam test |
+ve |
Physico-chemical parameters of the Crinum latifolium Linn. are tabulated in Table 2. The pH value
of Crinum latifolium Linn is acidic ( 6.06 ). Deterioration time of the plant
material depends upon the amount of water present in plant material. If the
water content is high, the plant can be easily deteriorated due to fungus. The
loss on drying at 105°C was found to be 14.62 %. Total ash
value of plant material indicated the amount of minerals and earthy materials
attached to the plant material. Analytical results showed total ash value content was 11.40 %. The negligible amount of
acid-insoluble siliceous matter present in the plant was 0.48%. The
water-soluble extractive value was indicating the presence of sugar, acids and
inorganic compounds (30.77%). The alcohol soluble extractive values indicated
the presence of polar constituents like phenols, alkaloids, steroids, glycosides,
flavonoids the results given in Table 2.
Table 2. Physico-chemical
parameters of leaves Crinum latifolium Linn
|
S. No. |
Parameters |
Results |
|
1. |
Description |
Dark
green |
|
2. |
Foreign
matter |
<
1.0 % |
|
3. |
pH |
6.06 |
|
4. |
Loss
on drying at 105 0C |
14.62
% w/w |
|
5. |
Fat
content |
0.354
ml |
|
6. |
Water-soluble
extractive |
30.77
% w/w |
|
7. |
Alcohol-soluble
extractive |
11.19
%w/w |
|
8. |
Petroleum
ether - soluble extractive |
1.2
%w/w |
|
9. |
Chloroform
- soluble extractive |
4.3
%w/w |
|
10. |
Total ash |
11.40
% w/w |
|
11. |
Acid-insoluble
ash |
0.48
% w/w |
|
12. |
Sulphated
insoluble ash |
15.8
% w/w |
|
13. |
Water soluble
ash |
5.9
% w/w |
TLC Methodology:
4g of the sample
was soaked in 40 ml of chloroform with occasional shaking for 18 hrs, boiled
for 10 minutes and filtered. The filtrate was concentrated. The solution was
applied on (E.Merck) Aluminium
plate pre-coated with Silica gel 60 F254 of 0.2 mm thickness using Linomat IV applicator. The plate was developed in Toluene:
Ethyl acetate (8:2v/v). After air drying the plate was visualized in UV 254 and
366 nm. The plate was then dipped in Vanillin -Sulphuric
acid and heated in air oven at 105°C till the spots appeared. The result of TLC
Profile (Rf) of Sudarshan
(Crinum latifolium Linn.) given in the Table 3. And TLC of Sudarshan (Crinum latifolium
Linn.) has been given in Figure 1.
Table 3. TLC profile of Crinum
latifolium Linn
|
S. No |
visible light |
254 nm |
366 nm |
After Derivatisation with
Iodine in visible light |
After Derivatisation with
Vanillin sulphuric acid in visible light |
|||||
|
Colour |
Rf. |
Colour |
Rf. |
Colour |
Rf. |
Colour |
Rf. |
Colour |
Rf. |
|
|
1. |
Green |
0.05 |
Light brown |
0.16 |
Grey |
0.03 |
Yellow |
0.36 |
Grey |
0.41 |
|
2. |
Light orange |
0.18 |
Dark brown |
0.20 |
Black |
0.13 |
Yellow |
0.49 |
Grey |
0.47 |
|
3. |
Orange |
0.22 |
Grey |
0.45 |
Blue |
0.16 |
Light yellow |
0.50 |
Purple |
0.55 |
|
4. |
Yellow |
0.55 |
Fluorescent pink |
0.60 |
Pink |
0.43 |
Fluorescent green |
0.56 |
Blue |
0.64 |
|
5. |
Light green |
0.67 |
Brown |
0.65 |
Purple |
0.57 |
Grey |
0.63 |
Purple |
0.71 |
|
6. |
Green |
0.72 |
Pink |
0.70 |
Pink |
0.62 |
Light green |
0.72 |
Green |
0.77 |
|
7. |
Fluorescent green |
0.78 |
Black |
0.76 |
Blue |
0.68 |
Green |
0.78 |
Blue |
0.99 |
|
8. |
Dark green |
0.84 |
Black |
0.98 |
Pink |
0.75 |
Yellow |
0.97 |
- |
- |
|
9. |
- |
- |
|
- |
Black |
0.98 |
- |
- |
- |
- |
Figure 1. TLC profile of Crinum latifolium Linn. (Toluene: Ethyl acetate:: 8 : 2v/v)
CONCLUSION:
Preliminary phyto-chemical
as well as various aspects of the sample were studied and described along with physico-chemical parameters and TLC studies in
authentication, adulteration for quality control of raw drugs. The plant of Crinum
latifolium Linn. exhibits a set of diagnostic
characters, which will help to identify the drug in dried condition.
It has been concluded from this study that estimation
of physico-chemical parameters, heavy metals and
pesticides residue is highly essential for raw drugs or plant parts used for
the preparation of single and compound formulation drugs. The periodic
assessment is essential for quality assurance and safer use of herbal drugs.
ACKNOWLEDGEMENT:
The authors are
very grateful to Director General, CCRAS, New Delhi and Principal, M.M. College of Pharmacy, Mullana, Ambala, Haryana for
providing encouragement and facilities for carrying out this work.
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Received on 27.01.2011
Accepted on 28.02.2011
© A&V Publication all right reserved
Research Journal of Pharmacognosy and
Phytochemistry. 3(3): May- June 2011, 120-123