A Pharmacognostical Study on
Albizzia lebbeck bark
Shah U.D.1*, Shah M.B.2
and Saluja A.K.3
1Dept. of
Pharmacognosy, APMC College of Pharmaceutical Education
Research, Himatnagar
2Dept. of
Pharmacognosy, L. M. College of Pharmacy, Ahmedabad
3Dept. of Pharmacognosy,
A. R. College Pharmacy, Vallabh Vidyanagar
ABSTRACT:
Fresh stembark of Albizzia lebbeck was studied for macro and microscopical characters. Betulinic
acid was isolated and identified by M. P., co- chromatography with reference
standard and IR spectral characteristics. HPTLC method was developed for
quantification of betulinic acid using precoated silica gel plates as a stationary phase, Toluene:
Acetone: Acetic acid (100:1:0.1) as a mobile phase
and anisaldehyde sulphuric
acid as a spray reagent. The bark is rough as longitudinal fissures and
transverse cracks are present. TS of bark shows rhytidoma
as a major portion followed by stone cell bands and secondary phloem with
groups of phloem fibres, with starch and prisms of
calcium oxalate being found in all the tissues. Saponins,
tannins, triterpenoids and flavanoids
were found be the major components of the bark. HPTLC method was developed for
quantification of betulinic acid by scanning the
plates at 523nm. The quality parameters and HPTLC method developed would serve
as useful tools in standardization of Albizzia lebbeck.
KEYWORDS: Albizzia lebbeck, HPTLC, quality parameters, betulinic acid
INTRODUCTION
Albizzia lebbeck L. (Benth.) (Fam.: Mimosaceae)
commonly known as “Sirisha” in Gujarat and as woman
tongue tree in western countries is found to be growing in tropical and
subtropical regions throughout the world1,2. Ethnomedically
the bark of the plant is reported to be useful in leucoderma,
bronchitis, piles3,4; Literature survey revealed presence of
alkaloids, saponins, triterpene
acids like betulinic acid and its glycosides, procyanidines and tannins in the bark5-7. The
bark is reported to possess anti-inflammatory8, antianaphylactic9,
mast cell stabilizing9, antihistaminic10, antitumor11,
antidiarrhoeal12, antibacterial13, anticonvulsant14,
immunomodulator15, antifretility16 activity and nootropic17
effect too. Clinical studies of herbal and Ayurvedic preparations containing Albizzia lebbeck as a
key ingredient showed that it is effective in allergic conjunctivitis18
and for anti asthmatic activity19,20. The
present study deals with development of pharmacognostic
standards for Albizzia lebbeck and
in this communication we report new HPTLC method for estimation of betulinic acid, one of the major triterpenoid
acid present in this plant.
MATERIAL AND METHODS:
Plant material:
The
fresh barks of Albizzia lebbeck were collected in March 2005 from Vallabh Vidyanagar campus and
authenticated by a taxonomist at the Department of Bioscience, Sardar Patel University, Vallabh Vidyanagar. A
voucher specimen (No. argh 5) was deposited in Department
of Pharmacognosy, A. R. College of Pharmacy, Vallabh Vidyanagar. The
material was cleaned, dried at room temperature, powdered to 60 # and stored in
airtight container.
Pharmacognostical studies:
Fresh bark and its powder were studied for macro and microscopical characters. Ash values and extractive values
were determined according to the method of Indian Herbal Pharmacopoeia21,
while heavy metal detection was carried out by Inductive Coupled Plasma
Spectrometer.
Phytochemical studies:
Phytochemical
screening was performed with the use of alcoholic extract22.
Isolation and identification of phytomarker as Betulinic acid:
Betulinic acid
was isolated from bark of Albizzia lebbeck
and its identity was confirmed by M. P., overlay IR spectra of isolated and
standard betulinic acid (Aldrich) and by Co-chromatography with
standard betulinic acid (Aldrich) using mobile phase, Toluene:
Acetone: Acetic acid (100:1:0.1), precoated silica
gel G F254 plate as a stationary phase and anisaldehyde
sulphuric acid as a derivatizing
agent.
Estimation of betulinic acid
by HPTLC Method:
1
mg/ml of standard stock solution of betulinic acid was prepared in methanol and was used for
preparation of calibration curve.
Sample preparation:
1 g
of the dried bark of Albizzia lebbeck was exhaustively extracted by subjecting
to reflux for 30 minutes using 10 ml petrol ether (60-800) for three
times. The combined petrol ether (60-800) extract was concentrated
to solid mass (0.5% w/w). 1 mg/ml solution was prepared in petrol ether (60-800).
Instruments:
A Camag TLC system comprising
of a Linomat V sample applicator (Camag,
Switzerland), TLC scanner III controlled by CATS software (version 4.06). Camag 100 µl HPTLC Syringe, Camag twin trough chambers (20X10 cm2) and
automatic TLC sampler III were used for sample application and quantitative
estimation.
Reagent and other materials:
Standard
betulinic acid (Aldrich), toluene, acetone, acetic acid,
distilled water, methanol (HPTLC grade) and silica gel 60F254 precoated HPTLC plate (E-Merck).
Chromatographic conditions:
Ø Stationary phase: Precoated
TLC plate of silica gel 60F254
Ø Mobile Phase: Toluene: Acetone: Acetic acid
(100:1:0.1v/v)
Ø Spotting volume: 800 ng/spot
Ø Wavelength: 523
nm.
Linearity and calibration:
A
calibration curve was derived by plotting peak area (Y axis) versus
concentration (X axis). Linear dynamic response of concentration ranging 800 –
1600 ng/spot of betulinic acid
was observed. The correlation coefficient, slope intercepts and regression
equation was also calculated to provide mathematical estimate of degree of
linearity. The peak area values of standard and sample were used to calculate
the amount of betulinic acid present in extract. The rate of flow or
retention factor (Rf
values) was also determined.
Method Validation:
To
ensure the reliability of method recovery studies were carried out by mixing a
known quantity of standard drug with pre-analysed
sample and contents were re-analysed by proposed
method. The method was validated in terms of linearity, interday,
intraday, precision, repeatability, accuracy, specificity and limit of
detection.
RESULTS AND
DISCUSSION:
Bark measures
1.0 to 2.0 cm in thickness with varying length up to 15- 20cm and is rough due
to presence of longitudinal fissures and transverse cracks. Rhytidoma
forms a major part of the bark and at places gets peeled off in flakes exposing
buff colored inner surface. It shows laminated fracture in outer region and
fibrous in inner region. It is brownish buff externally, reddish brown.
Internally Odor sternutatory and taste astringent.
Microscopic study (Fig. 1):
Bark showed outgrowth which is 15-20
layers of parenchyma. Rhytidoma found inner to the
outgrowth is made up of alternate bands of cork cells and dead phloem. Rhytidoma is followed by secondary phloem consisting of
phloem parenchyma, phloem fibers and medullary rays.
Phloem fibers are lignified in groups of 20-22, associated with idioblasts containing crystals of calcium oxalate while medullary rays are tri-tetraseriate.
Brownish coloring matter, prisms of calcium oxalate crystal and starch grains
are present throughout the section.
Table 1.
Physicochemical parameters
|
Particulars |
Albizzia lebbeck |
|
Total
ash |
06.40
%w/w |
|
Water
soluble ash |
00.60
%w/w |
|
Acid
insoluble ash |
03.55
%w/w |
|
Water
soluble extractive value |
20.88
%w/w |
|
Alcohol
soluble extractive values |
12.80
%w/w |
Table 2. Estimation
of betulinic acid in bark of Albizzia lebbeck
Sample
|
Mean peak area ±
S.D (n=5)
|
Average Amount of betulinic acid (ng/spot)
|
Average % w/w of betulinic acid
± S.D
|
% C.V.
|
A.lebbeck
|
1842.5±7.79
|
10.92
|
0.02±0.067
|
0.42
|
The
identifying features of powdered bark include fungal spores, cork in surface
view, stone cells, sclereids, brownish matter, phloem
fibers with prisms, and tangentially and radially cut
medullary rays.
Data
of Physico-chemical parameters visually ash and
extractive values are given in Table 1. Water-soluble ash value was found to be
more than acid insoluble ash value. The plant showed higher alcohol soluble
components than water-soluble components. Moisture content of plant was found
to be 40% w/w. Heavy metal analysis of bark showed presence of cadmium, lead
and arsenic below respective detection limits.
Qualitative
phytochemical examination revealed the presence of flavonoids, saponins,
carbohydrates, tannins, phenolics, phytosterols and triterpenoids.
Isolation and identification:
The identity of isolated betulinic acid
was confirmed by comparing its M. P. (2940 - 2950 C),
overly IR spectra and co-chromatography with standard betulinic
acid. (fig. 2and 3a)
Estimation of
betulinic acid by HPTLC analysis:
The sharp peaks of betulinic acid,
Rf 0.4
(standard and sample) were obtained when the plate was scanned at 523 nm (fig.
3). The amount of betulinic acid present in the sample was estimated by using
calibration curve. The correlation co-efficient was found to be 0.9937 and
relative standard deviation ranged from 0.38 – 0.82 (Table 3, fig. 3). The
amount of betulinic acid present in plant material was found to be
0.02% w/w on dry basis (Table 2). The validation parameters are given in table
3.
Table 3. Summary of
validation parameters
|
Sr.
No. |
Parameters |
Results |
|
1 |
Linearity |
0.9937 |
|
2 |
Precision
(%C.V.) Repetability of Measurement Repetability of Application Interday Intraday |
0.382-0.82 0.42 0.422-0.81 0.61-0.85 |
|
3 |
Range |
800-1600ng/spot |
|
4 |
Limit
of Detection |
1.66
ng/spot |
|
5 |
Limit
of Quantitation |
5.54
ng/spot |
|
6 |
Accuracy |
99.88-100.20% |
|
7 |
Specificity |
Specific |
The
proposed HPTLC method was precise, accurate and selective. The method was
rapid, sensitive, reproducible and economical. It does not suffer any positive
or negative interference due to other common components present in extract and
can be used for routine quality control analysis and quantitative determination
of betulinic acid in the bark.
CONCLUSION:
The
data generated by qualitative and quantitative evaluations are valuable for
documenting a monograph on Albizzia lebbeck
bark.
ACKNOWLEGEMENT:
The
authors are thankful to A. R. College of Pharmacy for providing basic facilities,
AICTE New Delhi for awarding RPS project and financial assistance and SICART
for providing HPTLC instrument facility.
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Received
on 23.03.2010
Accepted on 30.04.2010
© A&V Publication all right reserved
Research Journal of Pharmacognosy and Phytochemistry.
2(3): May-June 2010, 241-245