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Journal of Traditional and Folk Practices
Volume 04(2), December 2016, 79 - 95.
ISSN 2278 - 5906
Pharmacognostic standardisation and phytochemical analysis of
Tetracera akara (Burm. f.) Merr.
Ragesh R Nair, S R Suja*, V Vilash, A L Aneeshkumar and S Rajasekharan
Division of Ethnomedicine and Ethnopharmacology, Jawaharlal Nehru Tropical Botanic
Garden and Research Institute, Palode, Thiruvananthapuram 695 562, Kerala, India.
*sujasathyu@gmail.com
Received : 29 Oct 2016 Accepted : 22 Dec 2016
Abstract
Tetracera akara (Burm. f.) Merr. (Dellineaceae) is used by the Kani tribe of Kerala, India
for treating various liver ailments. The plant has pharmacologically active compounds
namely Betulin, Betulinic acid, Lupeol and β-Sitosterol possess anti-HIV, anti-diabetic
and anti-inammatory potential. However, there is no data available regarding the
pharmacognostic standardization of this plant. Therefore, the aim of the present study
was to standardize the pharmacognostic features of T. akara root by physicochemical
characterisation, morpho-anatomical studies of various plant parts and detailed
phytochemical studies by HPTLC proling. The pharmacognostic evaluation of T. akara
revealed the presence of characteristic morphological, organoleptic and physicochemical
features of the plant. The anatomical studies helped to distinguish root and stem material
even in crushed or powdered form. Detailed leaf anatomy showed the presence of
uniseriate trichomes with tapered end and paracytic stomata on the lower leaf surface.
Powder analysis of root showed the presence of calcium oxalate and raphide crystals,
xylem bre, vessel and tracheids. Preliminary phytochemical analysis of crude extract
of T. akara revealed the presence of phytoconstituents like avonoids, phenols, tannins,
saponins and terpenoids. HPTLC studies of crude extract and ethanolic fraction revealed
the comparative HPTLC ngerprinting prole of T. akara at 254 and 366nm for the rst
time. The present investigation thus sets a benchmark in authenticating the genuine plant
material by setting diagnostic indices for the identication and preparation of monograph of
T. akara, a new entry in the eld of therapeutics.
Keywords: Tetracera akara, Kani tribe, HPTLC proling.
1. Introduction
Medicinal plants have a long-standing
history in the practices of traditional medicine,
which is based on hundreds of years of belief
and observations (Jeyaprakash et al., 2011).
Traditionally used medicinal plants are now
moving from fringe to mainstream as people
are becoming more aware of therapeutic
interventions of these medicinal plant resources
and their products in maintaining health and
preventing diseases with an eco-friendly touch.
One-fourth population of the world, mainly from
the developing countries depends on traditional
medicines for the treatment of various ailments
(Jena et al., 2011). A key obstacle, which has
hindered the acceptance of the alternative
medicines including tribal medicines in the
developed countries, is the lack of documentation
and stringent quality control which ensures it’s
safety and efcacy. In this scenario, it becomes
the need of the hour to make an effort towards
standardization of traditionally used medicinal
plants and it can be achieved by authentication
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Ragesh et al.
J. Traditional and Folk Practices
Vol. 04(2); 2016
of the correct plant source by pharmacognostic
and phytochemical evaluation (Padashetty et al.,
2008)
Tetracera akara (Burm. f.) Merr. belongs
to the family Dellineaceae, is a woody climber
distributed in India, Sri Lanka, China, Laos,
Cambodia, Thailand, Myanmar and Indo-
Malayan archipelago. In India, it is found
in the Western Ghats region of Kerala and
Tamil Nadu. The medicinal use of Tetracera
akara was rst reported by S. Rajasekharan
and his team in 1987 during Ethnobiological
studies carried out with the help of Mallan kani
residing in the Chonnampara tribal settlement
of Thiruvananthapuram district. According to
Mallan kani, the paste of the fresh root along
with coconut kernal is administered orally in
empty stomach, before sunrise for 3 days to cure
jaundice and liver related disorders (Saradamma
et al., 1987). Leaf decoction of T. akara is used
to treat pulmonary haemorrhages and gargles
for aphthae (Udayan et al., 2009). It is reported
that T. akara contains terpenoids like betulin,
betulinic acid, lupeol and β-Sitosterol which
have a wide range of bioactivities like anti-HIV,
anti-diabetic and anti-inammatory (Lima et
al., 2014). However, no pharmacognostic study
has been carried out on this plant and hence
the objective of the present study is to evaluate
various pharmacognostic properties including
morpho-anatomical, microscopic, phytochemical
and physicochemical characterization of T.
akara, which will provide some useful markers
for identication of crude drug.
Fig.1 A. Habit of Tetracera akara (woody climber), B. twig with Flowers, C. roots of T. akara and D. twig
with fruits.
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2. Materials and Methods
The chemicals used during the study were of
analytical grade. Solvents viz. petroleum ether,
benzene, chloroform, acetone, ethanol (95%),
n-butanol and reagents viz. phloroglucinol,
glycerine, HCl, chloral hydrate and sodium
hydroxide were procured from Sigma-Aldrich,
USA. Compound microscope, glass slides, cover
slips, watch glass and other common glass wares
were the basic apparatus and instruments used
for the study. Microphotographs were taken
using a Zeiss axiostar plus microscope attached
with Cannon power shot A620.
2.1 Collection and authentication of plant
material
T. akara (Burm. f.) Merr. root was collected
from Kottoor (N 08o35’03.8’’, E 77o10’ 54.8’’
and altitude 585m), Thiruvananthapuram district
of Kerala, India and authenticated by the plant
taxonomist of the JNTBGRI, Palode. Voucher
specimens were deposited at the Jawaharlal Nehru
Tropical Botanic Garden and Research Institute
Herbarium (TBGT 86868 dated 08/08/2015).
The roots of the plant collected were washed in
running water, shade dried, powdered and passed
through 40 mesh sieve and stored in an airtight
container for further use.
2.2 Macroscopic and organoleptic
characterization
Morphological studies were carried out by
observing the plant parts with naked eyes or hand
lens if needed. The macroscopic and organoleptic
characters like phyllotaxy, size, shape, venation,
presence or absence of petiole, apex, margin,
base and lamina of the leaves were noted along
with texture, colour, surface, odour and taste of
leaves, stem and roots (Trease and Evans, 2002
and Wallis, 1985).
2.3 Microscopic characterization
2.3.1 Anatomical studies of the Leaf, Stem
and Root
Free hand transverse sections of lamina and
midrib, stem and root were prepared, stained
with Safranin, mounted on glass slides using
glycerine and observed under light microscope
with camera attachment and photomicrographs
were taken (Trease & Evans, 2002).
2.3.2. Quantitative leaf microscopy
2.3.2.1. Stomatal number and stomatal index
For epidermal studies, Shultze’s method of
maceration was used (Subrahmanyam, 1996).
Leaves were rst treated with conc. nitric acid
for 30 min under sunlight. The upper and lower
epidermis of the leaf were peeled separately and
then boiled it with sodium hypochlorite solution.
The peeled epidermis was placed on a slide and
mounted with a drop of glycerin. An average
number of stomata per mm2 of the epidermis
of the leaf (stomatal number) is calculated and
the values for upper and lower epidermis were
determined separately using the equation:
Stomatal index (SI) = S × 100
E+S
Where, S = the number of stomata per unit
area and E = the number of epidermal cells in the
same unit area of leaf.
2.3.2.2. Determination of vein-islet number
and vein-let termination number
The vein-islet number is the average number
of vein-islets per mm2 of a leaf surface midway
between midrib and margin and the average
number of terminated vein-let per mm2 of a leaf
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was taken as vein-let termination (Trease and
Evans, 2002).
2.4. Powder microscopy
Fresh roots were washed under running
water, shade dried, nely powdered and stored
in air tight container. A small quantity of root
powder was placed on slides and mounted in
2-3 drops of chloral hydrate and each slide was
covered with a cover slip and then examined
under a microscope. Different cell components
i.e. cork cells, sieve tubes bers, lignied bers,
cortex cells, calcium oxalate crystals etc. were
noted and photographs were taken using a digital
camera attached to a microscope (Mehlhorn,
2011).
2.5. Fluorescence analysis
The uorescence character of the root powder
(40 mesh) was studied both in daylight and UV
light (254 and 366 nm) after treatment with
different reagents like sodium hydroxide, picric
acid, acetic acid, hydrochloric acid, nitric acid,
iodine, ferric chloride etc. (Kokashi et al., 1958).
The colour changes were noted using Methuen
handbook of colour (Kornerup & Wanscher,
1978).
2.6. Physicochemical analysis
The following physicochemical parameters of
T. akara root powder were determined according
to the WHO guidelines on quality control
methods for medicinal plant materials.
2.6.1. Determination of pH
Dissolved 1 g of the root powder in 100 mL
and 10 g of the leaf powder in 100 mL of distilled
water, ltered and checked the pH of the ltrate
with a standardized glass electrode.
2.6.2. Determination of soluble extractive
Take 5 g of the air dried root powdered in 100
mL of respective solvents (hexane, chloroform,
petroleum ether, ethanol, methanol and water) in
a closed ask, shaked frequently during 6 h and
allowed to stand for 18 h. The ltrate is collected
and evaporated to dryness in a tared at bottom
shallow dish, dried at 105˚C and weighed. The
percentage of soluble extractive is calculated
with reference to the airdried drug.
2.6.3. Loss on Drying (LOD)
About 2-3 g of root powder is accurately
weighed and kept in a hot air oven maintained
at 105˚C for 5 hin a China dish. After cooling in
a desiccator, the loss in weight was recorded and
repeated till constant weight was obtained.
Loss on
drying
(%) LOD
=
Lose in weight
×100
Weight of the drug in g
2.6.4 Swelling Index
Leaf powder (1 g) was taken in a measuring
cylinder (25 mL) and suspended in 25mL
distilled water for 1 h by thorough mixing every
10 minutes. After 3 h, volume in mL occupied by
the plant material including any sticky mucilage
was measured. The experiment was repeated
three times for accuracy and the swelling index
was calculated.
2.6.5. Foaming index
Finely powdered root (1g) was boiled in 100
mL of water for 30 minutes. Then cooled and
ltered into a 100 mL volumetric ask and added
sufcient water to make up the volume. The
prepared decoction was poured into 10 stoppered
test tubes each ranging from 1 mL, 2 mL …….10
mL and the volume of the liquid in each tube was
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Vol. 04(2); 2016
adjusted to10 mL with water. The tubes were
duly stoppered and shaken them in a lengthwise
motion for 15 sec. (two shakes per second) and
allowed to stand for 15 minutes. The foam height
in each tube was measured.
Foaming index = 1000
a
(‘a’ is the volume of the plant decoction for foaming
foam of height 1cm.)
2.6.6 Determination of total ash, acid
insoluble ash and water-soluble ash
About 6 g root powder in a tared silica dish
was ignited. Scattered the powder drug on the
bottom of the dish and incinerated by gradually
increasing the heat not exceeding dull red heat
until free from carbon, then cooled and weighed.
The % w/w of total ash with reference to the air
dried drug was calculated. One part of the total
ash obtained was boiled for 5-10 min with 25
mL of diluted hydrochloric acid, collected the
insoluble matter in a Gooch crucible, washed
with hot water, ignited and weighed. Percentage
of acid insoluble ash is calculated with reference
to the airdried drug. The % w/w of acid insoluble
ash with reference to the air dried drug was
calculated. Another part of the total ash was
boiled with 25 mL of water for 5-10 min. The
insoluble matter was collected in a Gooch
crucible, washed with hot water and ignited
in a crucible for 15 min at a temperature not
exceeding 450°C. The weight of insoluble matter
was substracted from the weight of the ash. The
difference in weight represents the water-soluble
ash. Percentage of water soluble ash is calculated
with reference to the airdried drug.
2.7. Preparation of serial fractions
T. akara root powder was rst extracted with
ethanol to obtain crude ethanolic extract (TA
CRD). Fresh root powder was then used for serial
extraction with hexane using Soxhlet apparatus,
the powder was then dried and again extracted
with chloroform and nally with ethanol to get;
1. Hexane fraction (TA HEX), 2. Chloroform
fraction (TA CHL) and 3. Ethanolic fraction (TA
ETH). Yield in g/100g root powder of TA CRD,
TA HEX, TA CHL and TA ETH of T. akara root
powder was calculated. Consistency, colour and
odour were also evaluated.
2.8. Phytochemical analysis
2.8.1. Preliminary phytochemical screening
Preliminary phytochemical analysis was
carried out in crude, hexane, chloroform and
ethanol fractions of T. akara using standard
procedures (Kokate et al., 2009).
2.8.2. High Performance Thin Layer
Chromatography (HPTLC) Proling of T.
akara
The standardization of a crude drug is an
integral part of establishing its correct identity.
The results of this investigation could therefore,
serve as a basis for proper identication of the
plant. Chromatographic ngerprint prole of
hexane, chloroform, ethanol and crude extract
were studied by HPTLC (CAMAG). 5 mg/
ml concentration of extracts were prepared in
respective solvents of chromatographic grade
and then ltered by Whatman’s lter paper No.
1. Prepared samples of different extracts were
applied on TLC aluminum sheets silica gel 60
F 254 (Merck), 07 μl each with band length of
5 mm using Linomat 5 sample applicator set
at a speed of 150 mL/sec. A number of solvent
systems were tried for each extract for better
resolution and maximum number of spots, but
the satisfactory resolution was obtained in the
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Table: 1 Macroscopic characters of Tetracera akara
Organoleptic characters
Leaf Stem Root
Surface Rough Scabrid Smooth
Colour
Upper Dark green
Dark brown Light brown
Lower Light green
Odour No characteristic odour No characteristic odour No characteristic odour
Taste No characteristic taste Slightly bitter Bitter
solvent system Benzene: Ethyl Acetate: Acetic
Acid in the ratio 3.6 : 1.2 : 0.2. The chromatograms
were developed in twin trough glass chamber
saturated with solvent Benzene: Ethyl Acetate:
Acetic Acid in the ratio 3.6 : 1.2 : 0.2 for 20
minutes up to the distance of 80 mm. The
airdried plates were viewed under UV and day
light. Spots were visible without derivatization
at 254 and 366 nm wavelengths, but best results
were shown when TLC plates were sprayed
with detection reagent (Anisaldehyde-Sulphuric
acid reagent and plate was heated at 110°C for
5 minutes) and then visualized in visible light.
Scanning was performed by CAMAG HPTLC
Densitometer (Scanner 3) in absorbance mode
at both 254 and 366 nm, the extracts were also
scanned at 350-600 nm using Deuterium and
Tungsten lamp with slit dimension 6.0 X 0.45
macro. The Rf values and colour of the resolved
bands were noted.
3. Results
3.1 Macroscopic and organoleptic
characterization
Macroscopic and organoleptic characters of
the fresh leaves, stem and root were noted and
the results were presented in Table 1 and 2 and
the habit of the plant is shown in Fig. 1.
Table: 2 Organoleptic characters of Tetracera akara
Macroscopic Observation
Phyllotaxy Alternate
Type Simple
Leaf Length 17.5 - 20.5 cm
Width 5.00 - 6.5 cm
Shape Elliptic-oblong
Apex Acuminate
Margin Entire to serrate
Venation Reticulate
Base Attenuate
Petiole 5 - 9 mm long.
3.2 Microscopic characterization
3.2.1 Anatomical studies of T. akara
Root - Cross section of root is circular in out
line with outer bark, secondary cortex, phloem,
peripherally placed proto xylem and meta xylem
towards the pith. Parenchymatous medullary rays
are present. Pith is reduced and parenchymatous
(Fig. 2.A and A1).
Stem - Cross section of stem is circular in
outline with outer periderm, secondary growth
with peripherally placed meta xylem and proto
xylem towards the center. Conjoint, open primary
vascular bundles are arranged as broken ring
above the parenchymatous pith (Fig. 2.B and
B1).Young stem is sccabrid with multicellular
trichomes.
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Fig. 3. A. Transverse section of lamina, B. Adaxial epidermal layer, C. Abaxial epidermal layer D. Uniseriate
trichomes, E. Portion enlarged-upper epidermal layer and F. Paracytic stomatal complex. UE-Upper epidermis,
PC-Palisade cells, SP-Spongy parenchyma, LE-Lower epidermis, Stomata (→) EC-Epidermal cells, SP-
Stomatal pore, SC-Subsidary cell and GC-Guard cell.
Fig: 2 A. Cross Section of root and A1. a portion of root enlarged, B. Section of Stem and B1. a portion of
stem enlarged, C. Section through midrib and C1. a portion of midrib enlarged. Cr-Cork, Cc-Cork cambium,
Pd-Phelloderm, Ph-Phloem, Mr-Medullary rays, PrX -Protoxylem, MeX-Metaxylem, Pt-Pith, PVB- Primary
vascular bundles, SeG-Secondary growth, H-Collenchymatous hypodermis, Sc-Sclernchyma, Xy-Xylem, Sd-
Starch deposition and Pc-Parenchymatous pith.
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Leaf - Section of the leaf shows two distinct
regions, midrib and lamina.
Midrib - Single layered epidermis of
rectangular cells with thin layer of cuticle.
Abaxial epidermis with small rectangular cells
and uniseriate trichomes with tapering end.
2-3 layers of collenchymatous hypodermis
followed by 5-6 layers of parenchymatous
cortex. Compactly packed parenchyma with
cluster of raphide crystals are also present in
cortex.1-2 layers of sclerenchyma present below
the endodermis, followed by phloem and xylem.
(Fig. 2.C and C1)
Lamina - Narrow lamina with single layered
epidermis. The adaxial epidermis completely
lacks stomata. Below the upper epidermis,
compactly arranged palisade tissue is present
which is followed by spongy parenchyma.
Abaxial epidermis possess paracystic stomata
(Fig.3).
3.2.2 Quantitative leaf microscopy
Quantitative leaf characteristics were
observed and the results were shown in the
Table: 3
Table: 3 Tetracera akara quantitative leaf microscopy
Parameter Range Mean ± SD
Stomatal number-
Upper epidermis 0 0
Stomatal number-
Lower epidermis 56 - 68 64.56 ± 5.68
Subsidiary cell
length 22 - 27 μm 24.85 ± 4.2 μm
Subsidiary cell
width 3 - 5 μm 4.04 ± 0.26 μm
Stomatal index-
Upper epidermis 0 0
Stomatal index-
Lower epidermis 15.12-21.28 18.67 ± 1.23
Vein islet number 19 - 26 22.8 ± 1.42
Vein let
termination
number
34 - 46 38.42 ± 1.82
Values are expressed as mean ± SD of ten values
Fig. 4 Powder analysis of Tetracera akara shows the presence of A- Calcium oxalate crystals, B- Raphide
crystals C- Xylem bre, D - Xylem vessel, E-xylem trachieds, F- Xylem vessels with pitted thickening and
G- fragments of bark.
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3.3 Powder analysis
Analysis of T. akara root powder revealed the presence of calcium oxalate crystals (A), raphide
crystals (B) xylem bre (C), xylem vessel (D), xylem trachieds (E), Xylem vessels with pitted
thickening (F) and periderm (G) shown in Fig. 4.
3.4. Fluorescence analysis
Chemical tests of T. akara root powder was carried out with different reagents and observed under
UV- 254 nm and UV-366 nm. The results were compared with their respective observations in visible
light and they were represented in Table:4 and Fig. 5.
Table: 4 Observations of Tetracera akara root powder under visible light and UV (254 nm and 366 nm)
Treatment
Observation (Colour developed)
Visible light UV-254nm UV-366nm
Powder alone Orange brown (5A2) Yellowish brown (5E8) Dark brown (4F5)
Powder + 1 M NaOH Brown (7E6) Dark green (27F4) Violet brown (10E4)
Powder + 1 M NaOH + Methanol English red (8D8) Dull green (26E3) Dull green (27E4)
Powder + 1 M NaOH + Water Dark brown (7F7) Dark green (28F7) Violet brown (10E3)
Powder + 1 M HCl Orange white (5A2) Pale green (28A3) Pale yellow (2A3)
Powder + dil HNO3Butter yellow (4A5 Greenish white (27A2)* Wax white (2B3)
Powder + 5% Iodine Reddish brown (8E4) Dark green (28F2) Dark blue (19E6)
Powder + 5% FeCl2Bronze brown (5E5) Dark green (26F5) Blackish blue (20FC)
Powder + dil Ammonia Orange red (8B7) Megro (6F3) Greyish green (29B5)
Powder + Methanol Butter yellow (4A5) Champagne (4B4) Light green (28A5)
Powder + HCl Yellowish brown (5D8) Brass (4C7) Greenish white (27A2)*
Powder + 1M H2SO4Yellowish white (4A2) Greenish white (28A2) Wax white (2B3)
Powder + HNO3Persian orange (6A7) Golden brown (5D7) * Corn (4B5)
Powder + K2Cr2O7Brownish yellow (5C8) Topaz (5C5) Golden blonde (5C4)
Powder + 95% Ethanol Amber yellow (4B6) Corn (4B5) Mustard yellow (3B6)
Powder + Toluene Colourless Yellowish white (4A2) Paster green (28A4) *
Methuen hand book of colour. *presence of uorescence
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Fig. 5. T. akara root powder after reaction with reagents, A - Root powder observed under visible light after
reaction with reagents, B - Root powder observed under UV 254 nm after reaction with reagents and showing
uorescence in 5 and 12, C -Root powder observed under UV 366 nm after reaction of with reagents and
showing uorescence in 10 and 15.
3.5. Physicochemical analysis
Physiochemical parameters of T. akara root powder were evaluated and the observations are
presented in Table. 5.
Table: 5 Physicochemical parameters of T. akara root powder.
pH of Water solution 1% w/v 7.68 ± 0.42
10% w/v 8.24 ± 0.34
Hexane-soluble extractive 0.58 ± 0.08% w/w
Chloroform-soluble extractive 1.08 ± 0.15% w/w
Petroleum ether-soluble extractive 2.78 ± 0.16% w/w
Ethanol-soluble extractive 11.26 ± 0.38% w/w
Water-soluble extractive 5.72 ± 0.35% w/w
Loss on drying (LOD) 10.8 ± 2.52% w/w
Total ash 17.46 ± 1.46% w/w
Acid-insoluble ash 1.36 ± 0.64% w/w
Water-soluble ash 14.82 ± 0.89 % w/w
Swelling index 7mL
Foaming index 250
Values are expressed as mean ± SD of six values
3.6. Extractive percentage and characteristics of extract and fractions of T. akara root
Physical characteristic and percentage yield of TA CRD, TA HEX, TA CHL and TA ETH were
shown in the Table 6.
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Table: 6 Percentage extractive and characteristics of T. akara root extract and fractions.
Name of the
Extract/ Fractions Consistency Colour Odour Yield
(g/100 g powder)
TA HEX Semi solid Greyish white Characteristic 0.58 ± 0.08% w/w
TA CHL Solid Whitish yellow Characteristic 1.08 ± 0.15% w/w
TA ETH Hard solid Dark brown Characteristic 9.04 ± 0.19% w/w
TA CRD Hard solid Dark brown Characteristic 11.26 ± 0.38% w/w
Values are expressed as mean ± SD of three values
3.7. Phytochemical analysis
Preliminary phytochemical analysis of T. akara crude extract and fractions of hexane, chloroform
and ethanol revealed the presence of phytochemicals like avonoids, phenols, steroids and terpenoids
represented in Table. 7.
Table: 7 Preliminary phytochemical analysis of T. akara extracts
Phytochemicals Test conducted Results
TA HEX TA CHL TA ETH TA CRD
Alkaloids
Mayer’s test ----
Dragendorff’s test ----
Carbohydrates
Benedict test +-++
Molisch’s test --+ +
Cardiac glycosides Keller-killani test -+-+
Flavonoids
Alkaline reagent test -- + +
Shinoda test --+ +
Phenols
Lead acetate test --++
Shinoda test --+ +
Proteins Biuret test +-- +
Saponins Foam test --+ +
Steroids
Salkowski test + + + +
Libermann-burchard test + +-+
Terpenoids Solkowiski test + + + +
Tannins
Ferric chloride test --++
Phenazone test --++
+: Present, -: Absent.
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Fig: 6. a- Tracks of T. akara in HPTLC proling at 254 nm, b- Chromatographic plates at 254 nm and visible
light, c & d- Chromatogram of T. akara crude extract and Ethanol fraction at 254 nm.
Fig: 7. e- Tracks of T. akara in HPTLC proling 366 nm, f- Chromatographic plates at 366 nm and sprayed
with anisaldehyde, g & h- Chromatogram of T. akara crude extract and Ethanol fraction at 366 nm.
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3.7.1. TLC standardization and HPTLC proling of T. akara
TLC of various solvent extract were carried out with many ratios of different solvents and the
best eluent mixture (Benzene: Ethyl acetate: Formic acid in the ratio 3.6:1.2:0.5) was further used
for HPTLC proling. HPTLC proling of T. akara crude extract and various fractions at 254 nm and
366 nm is represented in Fig. 6 & 7and the number of peaks and Rf value of phytoconstituent with %
maximum is represented in Table 8.
Table: 8. Shows the number of peaks and % maximum of phytoconstituent with respective Rf value of T. akara
crude extract and fractions at 254 nm and 366 nm.
Extract
254 nm 366 nm
No. of
Peaks
% max with respective
Rf value
No. of
peaks
% max with respective
Rf value
TA CRD 8 18 % at 0.79 10 21 % at .96
TA ETH 10 14 % at 0.46 11 14 % at .96
4. Discussion
Same plant may have different vernacular
names in different regions of the country, which
causes serious problems in identication of
plants (Shinde et al., 2009) and can even lead
to adulteration (Zhang et al., 2012). According
to World Health Organization (WHO), the
macroscopic and microscopic description
of medicinal plant is the rst step towards
establishing its identity and purity (Anonymous.,
1998). The source and quality of raw materials
play an important role in guaranteeing the
quality and stability of herbal preparations
(Calixto, 2000). Thus, in recent years there has
been an emphasis in standardization of tribal
medicinal plants of therapeutic potential by
pharmacognostical studies, as it is more reliable,
accurate and inexpensive. Pharmacognostic
characterization including physiochemical
evaluation is meant for identication,
authentication, detection of adulteration and
compilation of quality control of the raw drug
material. Since the plant, T. akara is used by
the Kani tribes of Kerala in their traditional
medicinal system, it is necessary to standardize it
as the rst step towards scientically validating
it as a potent source of drug against various liver
ailments.
The morphological characters of T. akara
observed can be used to distinguish it from the
closely related species like Tetracera indica.
The young stem is scrabid in nature due to the
presence of large number of uniseriate trichomes
and at maturity the stem become woody.
Leaves are simple, elliptic to oblong in shape,
which are arranged in an alternate manner.
Anatomical features are important in systematics
for identication, placing anomalous groups in
satisfactory positions in classication and for
indicating patterns of relationships that may
have been observed by supercial convergence
in morphological features (Essiett et al., 2012).
The mature roots and stem of T. akara is woody
because of secondary growth and is difcult
to distinguish in powdered or chopped form.
Anatomical studies of the root showed the
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Vol. 04(2); 2016
presence of collateral vascular bundles with
endarch xylem, while stem show the presence
of conjoint vascular bundles with exarch xylem.
Large parenchymatous pith is present in the C.S
of stem while pith is highly reduced in roots.
The anatomical features described above can be
used to identify the correct raw material, the root
with higher phytochemical content. Transverse
section of leaf shows midrib and laminar regions
with single layer of epidermis. Adaxial surface of
the leaf completely lack stomata and paracystic
stomata are found on the abaxial surface with
a stomatal index of 18.67 ± 1.23. Presence of
uniseriate trichrome on the lower side of leaf is
noted.
Powder microscopic analysis showed the
presence of calcium oxalate crystals, raphide
crystals, xylem bre, xylem vessel, xylem
trachieds, xylem vessels with pitted thickening
and fragments of bark. When physicochemical
methods become inadequate, the plant materials
can be distinguished from their adulterants on the
basis of uorescence characterization. T. akara
root powder produced characteristic uorescence
in short UV when treated with dilute and
concentrated HNO3 and in long UV when treated
with HCl and Toluene. Various physiochemical
parameters evaluated in this study can be used
for adulterant resolution or improper handling
of the raw material and compilation of a suitable
monograph for T. akara. Low moisture content
indicates less chances of microbial degradation
of plant drug during storage (Kunle et al., 2012).
The general requirement of moisture content in
herbal drugs is less than 14 % (Anonymous.,
1980). In this study, the loss on drying indicate
the moisture content which is 10.8 ± 2.52 % w/w
and within the accepted range.
Ash values can be used as reliable aid for
identication of the plant materials and detecting
adulteration (Nayak et al., 2010). It gives an idea
of earthy matter or the inorganic composition
and other impurities present along with drug.
Based on the result obtained, the total ash value
obtained was 17.46 ± 1.46 % w/w, acid-insoluble
ash was 1.36 ± 0.64 % w/w and water soluble ash
was 14.82 ± 0.89 % w/w respectively. The acid
insoluble ash was very low which shows that a
very small amount of the inorganic component
is present which is insoluble in acid and this is
of diagnostic importance. The extract values
give an idea about the nature of the chemical
constituents present in the plant and is useful for
the estimation of specic constituents soluble in
that particular solvent. The ethanol and water
soluble extractive yield were higher than that
of hexane, chloroform and petroleum ether
fractions. Thus, ethanol and water are good
choice of solvent extraction of T. akara.
Phytochemical analysis is one of the important
tool for quality assessment of medicinal plant
which includes preliminary phytochemical
analysis, chemo proling and marker compound
analysis using modern analytical techniques.
Comparative preliminary phytochemical
analysis revealed the phytochemical nature of
various solvent extract of T. akara root, of which
the crude extract and ethanolic fraction were rich
in bioactive phytoconstituents like avonoids,
phenols, tannins, saponins and terpenoids.
Flavonoids and phenols possess cardioprotective,
lipid lowering, antiulcer, hepatoprotective,
anti-inammatory, antineoplastic, antibacterial,
antifungal, anti-allergic, antiviral and antioxidant
properties (Gupta., 2010). The tannins possess
antimicrobial, anti-oxidant and antihypertensive
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J. Traditional and Folk Practices
Vol. 04(2); 2016
properties (Salas et al., 2010). Anti-cancerous
effect of saponins has been reported earlier
(Francis et al., 2002). Triterpenoids possess
wound healing, anti-inammatory, antiviral and
hepatoprotective effects (Jäger et al., 2009).
TLC standardization of various solvent
extract of T. akara with different ratios of
solvents showed that maximum separations
of the phytoconstituents were obtained in the
solvent system Benzene: Ethyl acetate: Formic
acid in the ratio 3.6:1.2: 0.5. This solvent system
was further used for HPTLC proling. HPTLC
method has been used as a reliable tool for the
qualitative and quantitative phytochemical
analysis of herbal drugs and formulations (Sajeeth
et al., 2010). HPTLC ngerprint proling of
crude and ethanolic fraction of T. akara were
carried out for the rst time and it will serve as
a reference standard in medicinal plant research.
Maximum number of peaks at 254 nm were
obtained for T. akara ethanolic fraction. At 366
nm, a maximum of 11 peaks were obtained for
T. akara ethanol fractions. This analysis was the
rst of its kind towards understanding the nature
of active principle and detailed phytochemistry
of T. akara. However, isolation of individual
phytochemical constituents from T. akara will
pave way for the development of a novel herbal
drug with least side effects.
5. Conclusion
The present study was undertaken with an
aim of pharmacognostic standardisation and
phytochemical evaluation of T. akara root which
is used by the Kani tribe of Kerala to treat various
liver ailments. These parameters which are being
reported for the rst time in this study will help
in authenticating the genuine plant material. It
will also help in detecting adulterant and setting
some diagnostic indices for the identication and
preparation of monograph of T. akara.
Acknowledgements
The authors would like to thank Department
of Science and Technology, India, INSPIRE
Programme for nancial assistance and Director,
Jawaharlal Nehru Tropical Botanic Garden and
Research Institute, Palode, for the providing
necessary facilities.
Conict of Interest
The authors have no conict of interest.
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