Preliminary
Phytochemistry and HPTLC Fingerprint profile of leaf extract of Latakaranj: A
Pharmaceutically significant plant
Kashyap Trivedi1, Vasu Patel2,
Aayush Parekh3, Mansi Patel4, Jinal Tandel5*,
Heta Kachhiya6
Indukaka Ipcowala College of Pharmacy, The Charutar
Vidya Mandal University,
Beyond GIDC, P.B. No. 53, Vitthal Udyognagar - 388121,
Gujarat, India.
*Corresponding Author E-mail: kash74053@gmail.com, vppatel5695@gmail.com,
parekhaayu2414@gmail.com, mansipatel0714@gmail.com,
jinal.tandel@cvmu.edu.in
ABSTRACT:
The prickly shrub Caesalpinia bonducella,
also referred to as Latakaranj, is a plant part of the Caesalpiniaceae family
and is used worldwide to treat a variety of conditions, including hypotensive,
antihypertensive, anxiolytic, anticonvulsant, anti-inflammatory, analgesic, and
antipyretic effects. The current study focuses on the fingerprinting profile of
C. bonduc leaves using High Performance Thin Layer Chromatography and
phytochemical screening. To identify different chemical elements such as
alkaloids, glycosides, flavonoids, tannin and phenolic compounds,
carbohydrates, fixed oil, steroid and terpenoids, proteins, amino acids, etc.,
a comparative phytochemical screening was conducted after hydro-methanolic
extract was obtained. The mobile phase used for the HPTLC fingerprinting was
n-hexane: Ethyl acetate (6:4% v/v). Several peaks with Rf values between 0.15
and 0.86 were found.
KEYWORDS: Caesalpinia bonduc, Phytochemical screening, Extract, Fingerprinting,
High Performance Thin Layer Chromatography.
INTRODUCTION:
Medicinal plants have become an important part of the global health
system for both humans and animals, not only in cases of illness but also as a
potential source of therapeutic help for preserving good health. It is
necessary to discover which components of the medicinal herb are responsible
for its therapeutic properties.
Globally, there has been a recent increase in interest
in plant study, and a substantial amount of data has accumulated to demonstrate
the enormous potential of medicinal plants employed in distinct traditional
systems. These days, there is a growing need for more medications derived from
plants and a resurgence of interest in conventional medicine.
The present common perception that "green
medicine" is safer and more trustworthy than expensive synthetic
drugs—many of which have unfavorable side effects—is the primary cause of this
resurgence of interest in plant-derived medications. The biological activity of
herbal medicines, medicinal plants, and their extracts and isolated substances
have been shown to be diverse. These have been and still are utilized as
dietary supplements or folk medicine for a variety of illnesses.1-2
C. bonduc is a prickly shrub that is extensively distributed
around the world and belongs to of the Fabaceae/Caesalpiniaceae family. It is
found worldwide in subtropical and tropical areas. The plant is particularly
useful as a medicinal herb that is used in traditional medicine since all
portions of it have therapeutic qualities. The plant has been reported to
possess anxiolytic, antinociceptive, antidiarrhoeal, antidiabetic, adaptogenic,
anthelmintic, antiestrogenic, anti- inflammatory, antimalarial, antimicrobial,
antifungal, antispasmodic, antioxidant, antiproliferative, antipsoriatic,
antitumor, larvacidal, muscle contractile, hepatoprotective, anticonvulsant and
antifilarial activities. Furthermore, having been proven for possessing a wide
range of activities, the plant has also been shown to include triterpenoids,
alkaloids, flavonoids, glycosides, saponins, and tannins. Presence of these
constituents make the plant pharmaceutically important as further study can be
done by isolating the constituents and prepare formulation from them for the
particular disease. The research being conducted focuses on the leaves'
phytochemical and pharmacogenomics features as well as the C. bonduc
HPTLC fingerprinting profile.3-6
Morphology of leaves:
The bipinnate leaves have firm thrones on their rachis
and measure 30 to 60 mm in length. At the base of every leaf is a pair of
shortened pinnae. Seven pairs of pinnae and three to eight pairs of leaflets
with one or two tiny prickles on the lower surfaces separate them.7
as depicted in figure 1.
Figure 1: Latakarnja plant
MATERIALS AND METHODS:
Plant Material: Branch of Caesalpiniaceae bonduc were obtained from
the Navsari district, and Dr. Veena Patel of the Department of Pharmacognocy at
thendukaka Ipcowala College of Pharmacy, CVMU, verified the authenticity of the
herbarian specimen. For this study, fresh Caesalpiniaceae bonduc leaves were
collected from the Navsari district. The latakaranj plant's fresh leaves have
been collected and dried in the shade. The leaves were ground and crushed after
drying, and the resulting powder was sieved. Following that, the fine powder
gets collected and preserved in an airtight container. Flow chart for the same
given below,
Fresh leaves of plant → Dry over shadow → Crushed
and Grind → Pass from sieve → Powder collected → Store Air
tight container
Extraction of the Leaf Powder:
Accurately weighed 10g powder of C. bonduc,
were subjected to successive solvent extraction using Soxhlet‟s
extraction apparatus and extracted subsequently with, hexane, ether, toluene,
benzene, chloroform, ethyl acetate, methanol and water, in order of increasing
polarity. The extract obtained was subjected to vacuum evaporator and dried.
These extracts were further subjected to calculation of extractive value and
phytochemical screening to detect the presence of various phytoconstituents
e.g. alkaloids, flavonoids, saponins, carbohydrates, sterols, terpenoids,
glycosides, coumarins, tannins and phenolic compounds.8
Preliminary phytochemical evaluation:9
1. Tests for Carbohydrates:
· Molisch’s test (General test): to 2-3ml aqueous extract, add few drops of
alpha-naphthol solution in alcohol, shake and add conc. H2SO4
from side of the test tube. Violet ring is formed at the junction of two
liquids.
2. Test for Protein:
· Biuret test (General test): to 3ml T.S. add 4% NaOH and few drops of
1% CuSO4 solution. Violet or pink color appears.
· Million’s test (for protein): mix 3ml T.S. with 5ml Million’s reagent.
White ppt. warm ppt turns brick red or the ppt dissolves giving red colored
solution.
3. Test for Amino acid:
· Ninhydrin test: Heat 3 ml T.S. and 3 drops 5% Ninhydrin solution in
boiling water bath for 10 min. Purple or bluish colour appears.
4. Test for Steroids:
· Salkowski reaction: to 2ml of extract, add 2ml chloroform and
2ml conc. H2SO4. Shake well. Chloroform layer appears red
and acid layer shows greenish yellow fluorescence.
· Liebermann-Burchard reaction: mix 2ml extract with chloroform. Add 1-2ml
acetic anhydride and 2 drop conc. H2SO4 from the side of
test tube. First red, then blue and finally green color appears.
5. Tests for Alkaloids:
Evaporate the aqueous, alcoholic and
chloroform extracts separately. To residue, add dilute HCl. Shake well and
filter. With filtrate, perform following tests:
· Dragendorff’s test: To 2-3ml filtrate, add few drops
Dragendroff’s reagent. Orange brown ppt. is formed
· Mayer’s test: 2-3ml filtrate with few drops Mayer’s reagent gives
ppt.
6. Test for Glycosides:
· Legal’s test: to aqueous or alcoholic extract, add 1ml pyridine and
1ml sodium nitroprusside. Pink or red color appears
· Keller-Killiani test: to 2ml extract, add glacial acetic acid,
one drop 5% FeCl 3 and conc. H2SO4. Reddish
brown color appears at junction of the two liquid layers and upper layer
appears bluish green.
· Borntrager’s test: To 3ml extract, add dil H2SO4
Boil and filter. To cold filtrate add equal volume benzene or chloroform.
shake well. Separate the organic solvent. Add ammonia. Ammoniacal layer turns
pink or red.
7. Test for Tannins and Phenolic Compound:
· Lead acetate solution: white ppt with extract and lead acetate
solution.
8. Test for flavonoids:
· Shinoda test: To the extracts, a small piece of magnesium ribbon
and 3 to 4 drops of concentrated sulphuric acid was added. Pink color indicates
the presence of flavonoids in the extract.
· Lead acetate test: Extracts were mixed with 10 % lead acetate
solution. Formation of yellow-coloured precipitates indicates presence of
flavonoids.
9. Tests for saponins:
· Froth test: 1ml of extracts was vigorously shaken with 5ml of
distilled water in a test tube for 30 seconds and was left undisturbed for 20
min. Presence of persistent froth indicates the presence of saponins in the
extracts.
10. Tests for Coumarins:
· Ammonia test: A drop of ammonia was taken on filter paper and to
this a drop of extract of plant material was added and observed for
fluorescence. Green fluorescence indicated the presence of coumarins in the
extract.
HPTLC method development:10,12-15
Chemical and Reagents: Toluene HPLC Grade from S D Fine - Chem Ltd., Mumbai
India, Ethyl acetate from Suvidhinath Laboratories, Gujarat India, Formic acid
from Suvidhinath Laboratories, Gujarat India, Methanol HPLC Grade, N-Hexane,
ether, benzene, chloroform from– SRL Pvt. Ltd., Mumbai India.
Instrument:
Sample application has been carried out with help of
Hamilton syringe 100μl syringe which give 6mm bands width using Camag
Linomat 5 (Switzerland) sample applicator on pre-coated silica gel aluminium
plate 60 F254, (10cm x 10cm with 0.2mm thickness, E. Merck,
Germany). Camag TLC scanner 4 is used for the densitometry scanning of the
developed chromatogram. To ensure accuracy, all extracts were weighed on a
Mettler Toledo electronic balance (ME 204, Mettler Toledo group, Mumbai,
India).
Preparation of Stock Solution:
Stock solution was prepared by weighing 10mg of extract
and then transferred to 10ml volumetric flask and volume has been made up to
the mark with methanol to produce concentration of 1000µg/ml of extract. From
above volumetric flask, 1ml extract was pipette out in 10 ml volumetric flask
and then volume was made up to the mark with methanol to produce final
concentration of 100µg/ml extract. 1µl volume was applied on pre-coated silica
gel G60-F254aluminum sheet (E. marc, Germany) 10 x 10cm, Thickness
layer 0.2mm stationary phase
Chromatographic Condition
Sample application- Different extract of C. bonduc was applied on the HPTLC plates in the form of narrow
bands of 6mm length, 10mm from the bottom and left edge, and with 8 mm space
between two bands. Samples application has been carried out under a continuous
drying stream of nitrogen gas.
Mobile phase development:
Plates were developed using a
mobile phase consisting of n-hexane:
ethyl acetate (6:4v/v) Linear
ascending development was carried out in a twin-trough glass chamber
equilibrated through chamber saturation with the mobile phase vapours for 30
min at 250C ± 20C. 10 ml of the mobile phase (5 ml in the
trough containing the plate and 5 ml in the other trough) was used for each
development and was allowed to migrate a distance of 80 mm. Once development
was over, the HPTLC plates were dried completely.
Densitometry analysis:
Developed plate was observed under 254 nm
and 366 nm light. Further derivatization was carried out with Anisaldehyde in H2SO4
solution. Densitometric scanning was performed in the absorbance mode under
control by winCATS planar chromatography software. The source of radiation was
the deuterium lamp, and bands were scanned at λmax 580 nm. The slit
dimensions were 5 mm length and 0.45 mm width, with a scanning rate of 20 mm/s.
RESULT:
Macroscopic review of plant:
100gm Collected leaves powder of Caesalpinia bonduc
were weighed after sieving process and final weight of leaves powder was found
to be 37.5 gm and it was green in colour as shown in figure-2. Table 1 describe
the macroscopic evaluation of plant.
Figure 2: macroscopic
evaluation of plant and powder.
Table 1: Macroscopic
character of Leaves and leaf powder of C. bonduc
|
Leaves
|
Leave Powder
|
|
Leaf type- Bipinnate, ovate shaped
|
Colour- Green
|
|
Leaf arrangement- Alternate
|
Odour- Strong and pungent
|
|
Leaf colour- Green
|
Taste- Bitter
|
|
Surface- Glossy
|
Taxture- smooth and powdery
|
Extractive value:
Successive solvent extraction method was applied to
prepare various solvent extract of plant. Extractive value of the study was
shown in table 2.
Phytochemical screening:
Phytochemical analysis on methanolic leaf extract
showed presence of carbohydrates, steroids, anthraquinone glycosides, coumarin,
flavonoids, alkaloids, tannins and phenolic compound. Phytochemical screening
is shown in Table 3.
Table 2: of extractive value of different solvent
extract of C. bonduc
|
Sr. No.
|
Extract
|
Colour and
consistancy
|
Average
extractive value ± SD (%w/w)
|
|
1
|
Ether
|
Brown, solid
|
6.5 ± 0.51
|
|
2
|
Toluene
|
Dark green,
sticky
|
0.2 ± 0.02
|
|
3
|
Benzene
|
Yellowish green,
sticky
|
7.3 ± 0.46
|
|
4
|
Chloroform
|
Reddish Brown,
sticky solid
|
1.3 ± 0.15
|
|
5
|
Ethyl acetate
|
Yellowish brown,
sticky
|
7.8 ± 0.35
|
|
6
|
methanol
|
Green, sticky
|
8.2 ± 0.6
|
|
7
|
water
|
Brown, dry solid
|
9.0 ± 0.5
|
Table 3: Phytochemical test on methanolic
leaf extract
|
Sr. No.
|
Phyto-constituents
|
Chemical Tests
|
Remark
|
|
1.
|
Carbohydrate
|
Molisch’s test
|
-
|
|
2.
|
Protein
|
Biuret test
|
+
|
|
3.
|
Amino acids
|
Ninhydrin test
|
+
|
|
4.
|
Triterpenoid
|
Salkowski test
|
+
|
|
5.
|
Steroids
|
Liebermann- Burchard test
|
+
|
|
6.
|
Cardiac glycoside
|
Legal’s test
|
+
|
|
7.
|
Anthraquinone Glycosides
|
Borntrager’s test
|
+
|
|
Modified Borntrager’s test
|
+
|
|
8.
|
Saponin Glycosides
|
Foam test
|
-
|
|
9.
|
Coumarin Glycosides
|
Fluorescence test
|
+
|
|
10.
|
Flavonoids
|
Shinoda test
|
+
|
|
11.
|
Tannins and Phenolic compounds
|
5% FeCl3
|
+
|
|
Lead acetate solution
|
+
|
|
Dil. KMnO4
|
+
|
|
12.
|
Alkaloids
|
Dragendorff’s test
|
+
|
|
Wagner’s test
|
+
|
High performance thin layer chromatography:
For comprehensive plant and phytoconstituent
identification, HPTLC fingerprint analysis can be applied as a diagnostic
approach. The results of phytochemical screening were confirmed by HPTLC
fingerprint analyses, which demonstrated the presence of different colored
bands at different wavelengths with particular solvent systems, indicating the
presence of particular phyto-compounds. In the present study C. bonduc showed
best results in n-hexane: Ethyl Acetate (6:4 v/v) solvent system for all the
extracts, after scanning and visualizing the plates in absorbance mode at 254
nm, 366 nm and visible light (580 nm after spraying with Anisaldehyde Sulphuric
acid reagent). The HPTLC images shown in Figure. 3 indicate that all sample
constituents were clearly separated without any tailing and diffuseness.
Different colour band at different Rf value
was compared with standard as shown in figure 4 and concluded the presence of
bitter compound, flavonoids, steroid, triterpenoid, alkaloid etc.
HPTLC finger print analysis showed
prominent peaks in water, hexane, chloroform and methanol leaf extract.
The HPTLC profile of leaf showed the
presence of ten to twelve spots at different wavelength in all four extracts
where as in remaining extract of toluene, benzene, ethyl acetate, ether band
was not moving from the initial spot. 3D overlay of all extract shown in figure
5. Densitogram of all extracts are shown in figure 6
Figure 5: 3D Overlay chromatogram of C.bonduc
leaves extract in different solvent
Track 1 of water extract
Track 2 of methanol extract
Track 3 of chloroform extract
Track 4 of ether extract
Track 5 of toluene
Track 6 of ethyl acetate
Figure 6: Densitogram of all 6 solvent extract of C.
bonduc leaf
DISCUSSION:
Phytochemical screening gives idea about presence of
various constituents in different solvent extract of C. bonduc. Which
can further be estimated for pharmacological importance. The HPTLC study's
findings demonstrated the large number of identical spots, which stand for
related chemicals. Different constituents responsible for the different
pharmacological condition. So for different disease further research can be
done to identify the constitute for various disease. Therefore, the existence
of comparable elements in leaf extracts of methanol, chloroform, ether, and
ethyl acetate is indicated. The current study offers enough details regarding
the phytoconstituents found in C. bonduc leaf extracts, as well as the
identification, standardization, and quality assurance of this therapeutic
plant. With additional sophisticated testing and purification techniques, as
well as in vivo investigations on animal models, these theories can be convincingly
demonstrated with a wealth of data. Future research can be done to further
identify and quantify each.
CONCLUSION:
In conclusion, the widespread therapeutic use of C.
bonduc leaves can be attributed to their rich composition of bioactive
compounds such as proteins, cardiac glycosides, triterpenoids, steroids, and
alkaloids. The consistent presence of these constituents across various solvent
extracts, as evidenced by distinct color bands in fingerprinting analysis,
underscores their pharmacological potential. Further research into the specific
mechanisms of action and clinical efficacy of these compounds is warranted to
fully harness the therapeutic benefits of C. bonduc in modern medicine.
CONFLICT OF INTEREST:
The authors have no conflicts of interest
regarding this investigation.
ACKNOWLEDGMENTS:
The authors would like to thank Indukaka Ipcowala
College of Pharmacy for providing necessary requirement to carry out this
research study.
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