Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y
ISSN: 0975-766X Available Online through Research Article www.ijptonline.com DEVELOPMENT OF RP-HPLC METHOD FOR THE QUANTIFICATION OF KARANJIN IN THE SEED EXTRACTS OF PONGAMIA GLABRA Vejendla.Ravikumar*, Md.SirajuddinKhan 1, M.Manoj kumar 2 1Departmant of Pharmacy, Sri Indu Institute of Pharmacy, Hyderabad. 2Departmant of Pharmacy, Sir C.R.Reddy College of Pharmaceutical Sciences, Eluru. Email: [email protected]
Received on 19-12-2010 Accepted on 01-01-2011
ABSTRACT:
The main aim of the work, done by the author, is to estimate the active ingredient Karanjin, a cyclic furano- flavonoid, present in the different parts of seed extracts of karanja ( Pongamia glabra ) mentioned the structure in
Fig.1. and to carry out the Quantitative Analysis of the Karanjin. Reporting the % of Karanjin present in the different solvent extracts namely: ethyl acetate, methanol & hexane of dry seed, kernel, shell and fresh seed & kernal of Pongamia glabra. By taking 200g. of each part found that in dry kernel maximum amount of 36.92g. is extracted by using methanol solvent, which was quantified by using HPLC was reported as 18.46% which is maximum when compared with other parts or by using other solvents.
O O
OCH3 O Fig1. Structure of Karanjin INTRODUCTION 1:
The author thought to discuss some of the important features of flavonoid compounds as the present phytochmical work carried out was mainly concern with the flavonoid compounds.
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1433 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y Flavonoids are yellow pigments, which occur in plant kingdom either in the free state or as glycosides or associated with tannins. These are also known as the anthoxanthins. Chemically, the flavones are hydroxylated derivative of flavone (2-phenyl-4-chromone), which are partially alkylated. In most of the flavones, positions 5 and 7 are hydroxylated and also one or more of positions 3, 4, 5 are also hydroxylated. Further, positions 3 1 and
51 are often methylated whereas positions 5, 7 and 4 1 are usually unmethylated.
Figure: 2. Basic structure of flavone, flavonol, and flavanone (flavonoids). Quercetin is a flavonol with OH
(hydroxy) group at 3, 5, 7, 3 1, 4 1 positions. Kaempferol is a flavonol with OH group at 3, 5, 7, 4 1 positions.
Quercetagetin is a flavonol with OH group at 3, 5, 6, 7, 3 1, 4 1 position.
Flavones show two absorption bands, one at 330-350 nm and other at 250-270 nm. Thus, it becomes possible to distinguish flavones from the anthocyanins on the basis of absorption bands and also by colour reactions.
PROPERTIES OF FLAVONOIDS: 1
• Many flavones are yellow solids
• Mostly flavones are soluble in water, ethanol, dilute acids and alkalis.
• Flavones are precipitated by lead salt.
• With ferric chloride, flavones give either a dull green or a red brown colour.
• Shinoda test: Alcoholic solution of flavones in the presence of magnesium ribbon and concentrated
hydrochloride gives magenta colour.
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1434 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y PHARMACOLOGICAL ACTIVITIES OF FLAVONOIDS:
Flavonoids are phenolic compounds with nuclei arranged in a C6 - C3 - C6 configuration. They have several beneficial properties for plants. The co-evolution of plants and insects has resulted in different plant defense mechanisms, one of which is the accumulation of phytochemicals, which may affect feeding, or growth of insects. 1 For example, the resistance of soya bean, Glycine max L., to the cabbage looper, Trichoplusia ni, appears to be due to the presence of the leaf flavonoids, daidzein, glyceolin, sojagol and coumestrol.2 The generalist feeder, Spodoptera frugiperda, avoids plants known to contain flavone. 3 Similarly, the C-glycosyl flavone (maysin) in maize silk has antibiotic activities against corn earworm larvae, Helicoverpa zea (Boddie). 4
Flavonoids also function to shield plants from UV radiation, act as signaling molecules in plant bacterium symbioses, and are the primary pigments that attract pollinators and seed dispersers 5 although flavonoids are widely distributed among the flowering plants, particular classes of flavonoids have distinct functions in different plant groups. For example, flavonols are essential for male fertility in maize and petunia 6, but they do not appear to have a similar function in Arabidopsis. 7
Isoflavonoids are the major phytoalexins in legumes 8, whereas 3- deoxyanthocyanidins fulfill similar functions in Sorghum bicolor and other grasses 9 multiple mechanisms have been proposed to explain the diversity of phytochemicals between different plants. 10,11
From a pharmaceutical perspective, flavonoids possess a remarkable spectrum of biochemical and pharmacological activities. Different flavonoids exhibit antioxidative, antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic, anti-thrombotic, and vasodilatory properties. 12,13 Catechin and catechin derivatives, oligomeric proanthocyanidins, quercetin and quercetin chalcone, Ginkgo flavone glycosides, silymarin, and others are utilized in the prevention and treatment protocols for cardiovascular disease, cancer, asthma, periodontal disease, liver disease, cataracts and macular degeneration. 14-18 Kuntz et al 19 reported that dietary polyphenols could have a significant role in the prevention of colon cancer by blocking hyper- proliferation of the epithelium and by promoting apoptosis.
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1435 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y BOTANICAL CHARACTERISTICS AND SIGNIFICANCE OF P. GLABRA:
TAXONOMICAL CLASSIFICATION: [20]
Kingdom : plantae (plants)
Sub kingdom : tracheobionta (vascular plants)
Super division : spermatophyte (seed plants)
Division : magnoliophyta (flowering plants)
Class : liliopsida (monocotyledons)
Sub class : arecidae
Order : arecales
Family : leguminacea
Genus : Pongamia
Species : glabra / pinnata
VERNACULAR NAMES: [21]
Telugu : Gaanuga, Pungu
Tamil : Ponga, Pongam.
Oriya : Koranja.
Kanada : Honge.
Assam : Karchaw.
USES OF PLANT PONGAMIA: [22]
Traditional Uses:
The wood of the plant is used in building material, ploughs, combs, yokes, oil-mills and solid wheel of a
cart and as fuel.
The oil extract from the seeds is used in lamps by the weaker section of the society and also in soap
making.
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1436 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y Seeds are used as fish poison.
Leaves are used as fodder, as manure for rice and sugarcane fields.
Pharmacological Uses:
The fresh bark of P.pinnata is used internally to cure bleeding piles.
The root and the bark are bitter, anthelmintic and used in vaginal and skin diseases.
A poultice of the leaves is applied on ulcers infected with worms.
Seeds are anthelmintic, bitter, acrid and carminative they are useful in inflammations, pectoral diseases,
chronic fever, hemorrhoids and anemia.
The oil is styptic, anthelmintic, good in leprosy, piles, ulcers, chronic fever and pain in liver.
A decoction of dried flowers is given for diabetes.
The juice of the roots is used for cleaning teeth, strengthening gums, against gonorrhea and cleaning foul
ulcers.
The juice of the plant is used for treating diarrhea, cough, leprosy and gonorrhea.
Aqueous extract of stem bark exhibits significant CNS sedative and antipyretic activities.
MATERIALS:
All chemicals were analytical grade: Methanol from Qualigens fine chemicals (Mumbai, India), Millipore-
Water used for the preparation of mobile phase solutions, was obtained from All Quartz Double Distilled, Bhanu
Scientific Instruments Company Pvt. Ltd. (Bangalore, India). All the above solutions were de-gassed in an ultrasonic bath (Sonicator), for 30min.
Stock solution of karanjin for generating standard curves were prepared by dissolving 1.2mg of compound in 20ml s.v.f with methanol to yield concentration of 6000mg/ml. Working standard solution of karanjin obtained by diluting 1, 0.8, 0.6, 0.4, 0.2, 0.1ml in 10ml standard volumetric flask so as to get 60, 48, 37, 24, 12, 6µg/ml concentration of karanjin respectively to get a calibration curve
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1437 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y Chromatography was performed on HPLC equipment consisting of LC-10AS pumps, SPD – 10A UV-
Visible detector, and an injector equipped with a 20ul sample loop (Rheodyne, USA). Analytical separation was on column packed with 3µm Waters LC - 18 stationary phase (Waters, USA). The dimension of the separation column was 150 x 4.6mm i.d. with 3um particle size. Data and chromatograms were collected using C-7RA chromatopac software system (Shimadzu, Japan). Dissolution of the compound was enhanced by sonication on
Bandlin-Sonerex (Bandelin, Berlin). UV spectra of karanjin for selecting the working wavelength of detection were recorded on Cintra 5 UV-Visible spectrophotometer (GBC Scientific equipments, Australia).
METHADOLOGY:
A collected dried seed were crushed into powder and was taken into column weighed amount of powder as 200g.
Kept socking for about 12hours with the solvent Hexane so as to remove fat solubles, then after run with
Methanol and collect the methanol extract and kept for concentration through rotovapour so as to get the methanol extract weight 22.36g . This methanol extract was treated with hexane to get Hexane solubles weighted
4.91g . remained Hexane insolubles was treated with Ethyl acetate to get Ethyl acetate solubles, EtOAc.50ml +
H2O 5ml was added and kept for steering on mechanical stirrer separate organic layer through separating funnel collected Ethylacetate solubles, weighed 1.85g . which was analysed for the %purity by HPLC instrument. In the same way kernels and shell were treated to get the ethylacetate solubles weighed 4.46g . & 0.59g . respectively to find out %purity for the respective seed parts and reported the highest %yield of active constituent Karanjin.
FRACTIONISATION OF METHANOLIC EXTRACT:
Separation of hexane solubles from methanolic extract:
Hexane solubles were separated from methanolic extract by means of mechanical stirring. In this, the methanolic extract was taken in hexane solvent and applied mechanical stirring. The traces of hexane solubles are goes in to hexane solvent. This process was repeated by continuous replacement of saturated hexane with that off fresh hexane solvent up to clear hexane solvent obtained. The hexane solubles were concentrated under vacuum, by using Rota vapor, dried completely and weighed.
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1438 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y Separation of ethyl acetate solubles from methanolic extract:
Ethyl acetate solubles were separated from methanolic extract by means of mechanical stirring. In this, the methanolic extract was taken in ethyl acetate: water mixture (90:10) and applied mechanical stirring. The ethyl acetate solubles goes in to ethyl acetate portion and remained polar solubles goes in to water portion in the mixture. The ethyl acetate portion was separated from the water portion by using separating funnel. This process was repeated by continuous replacement of saturated ethyl acetate with that off fresh ethyl acetate solvent up to clear ethyl acetate solvent obtained. The ethyl acetate solubles were concentrated under vacuum, by using Rota vapor, dried completely and weighed.
This was done for the different seed taken those are seed (dry), Kernal (dry), shell (dry), seed (fresh) & kernel (fresh). The weights taken and the concentrated methanol, hexane and ethyl acetate solubles obtained dry weights are given in below table no 1.
Table 1: Amount of Ethyl acetate solubles with different parts of P.glabra seed S.No. Parts of P.glabra Methanol Hexane solubles Ethylacetate solubles seed taken 200g extract (g) weight (g) weight (g) 1. Seed (dry) 23.36 4.91 1.85 2. Kernel (dry) 36.92 8.63 4.46 3. Shell (dry) 5.37 0.87 0.59 4. Seed (fresh) 189 2.79 0.98 5. Kernel (fresh) 22.31 2.71 5.24
IDENTIFICATION OF FLAVONOIDS IN THE SEED EXTRACT OF PONGAMIA GLABRA :
Chemical tests:
a) Alcoholic solution of root extract reacts with freshly prepared ferric chloride Solution and given blackfish
green colour.
b) Alcoholic solution of root extract reacts with 10% lead acetate solution and given yellow precipitate.
c) Shinoda test: Alcoholic solution of root extract in the presence of magnesium ribbon and concentrated
hydrochloric acid given magenta colour.
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1439 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y d) Alcoholic solution of root extract in the presence of galcial acetic acid and concentrated sulfuric acid
given green colour.
e) Alcoholic solution of root extract reacts with few drops of aluminum solution and given yellow colour.
PAST WORK RELATED TO PONGAMIA GLABRA
1. Quantification of Karanjin, tannin and trypsin inhibitors in raw and detoxified expeller and solvent
extracted Karanj ( Pongamia glabra ) cake [23]
2. Quantification of karanjin using high performance liquid chromatography in raw and detoxified karanjin
(Pongamia glabra vent) seed cake [24]
3. Determination of pongamol and karanjin in karanja oil by RP-HPLC [25]
EXPERIMENTAL PART:
Chromatographic Conditions:
The mobile phase consisting of methanol: water (85:15 %v/v). Prior to use, the mobile phase was de-gassed by sonication. Between the samples the injection needle was washed with methanol. The mobile phase was pumped through the system at a flow rate of 0.5ml/min. All experiments were carried out at ambient temperature of 23 oC.
The UV wave length was set at 305nm. The retention time of karanjin was 4.561min. The optimized chromatographic conditions of karanjin were shown in table 2.
Table 2: Optimized chromatographic conditions of karanjin
S.No. PARAMETERS CONDITIONS
1. Mobile phase Methanol: Water (85:15%v/v) 2. Stationary phase Waters LC-18 (150 x 4.6 i.d, 3 µm.) 3. Flow rate (ml/min.) 0.5 4. Run time (min.) 15 5. Column temperature ( oC) 23
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1440 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y 6. Volume of injection (µl) 20 7. Detection wavelength (nm) 305 8. Retention time of karanjin (min) 4.573
RESULTS: METHOD VALIDATION: 1. SPECIFICITY:
To justify the purity of the compound FT-IR and 1H-NMR Spectrums were also attached in below for FT-
IR spectrum fig 5 and for 1H-NMR spectrum fig 6 were illustrated.
The HPLC chromatograms recorded for the crude extracts i.e., other peaks along with karanjin with in a retention time range of 15min. and the standard pure karanjin fig.7 was compared the retention time for pure and crude extracts from different parts of seed such as from seed (d), kernel (d), and for shell (d) in and for different solvents used .The compounds are well separated from each other. Thus, the HPLC method presented in this study is selective for karanjin quantification.
2. LINEARITY:
The regression analysis of standard concentrations of karanjin using weight regression analysis (weight +
1 / (concentration) 2), the calibration curve was linear in the range as illustrated in fig 9. The mean ± standard deviation (SD) for the slope, intercepts and correlation coefficient of standard curves (n=5) were calculated. The regression data was shown in table 3
Table3: Linearity of calibration curves for karanjin. 1. 60 14112 2. 48 10864 3. 37 9335 4. 24 5991 5. 12 2732 6. 06 1405
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1441 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y 3. RECOVERY:
Percent recoveries of compounds from spiked blank were found and are represented as mean ± standard deviation. For karanjin, at concentrations 6.3, 50, 100 & 200ng/ml. For pure karanjin, at a concentration of 25 ng/ml, the recovery was 95.10. The data of recovery studies of crude extracts shown in Table 4.
Table 4: Recovery studies of crude extract of karanjin. Nominal Measured concentration concentration (ng/ml) ± Standard Recovery (ng/ml) Deviation 6.3 06.10 ± 0.05 96.82 50 50.10 ± 0.52 100.02 100 99.92 ± 3.01 99.92 200 200.1 ± 3.80 100.05
4. PRECISION & ACCURACY:
Intra – assay precision of the method was shown in Table 5. This was estimated by assaying the quality control samples (6.3, 50, 100 & 200 ng/ml) five times in the same analytical run. The precision was less than 3.0 and the % relative error less than -3.0.
Inter – assay precision of the method was shown in table 6. This was estimated by assaying the quality control samples (6.3, 50, 100 & 200 ng/ml) for replicate samples (n=5). The precision was less than 3.2 at all levels meeting the acceptance criteria of ± 20% at LOQ and ± 15% at other levels.
Table 5: Precision (C.V.) and accuracy (relative error) of intra-day assay measurements of karanjin at UV detection 305 nm. S.No. Nominal Measured concentration % C.V. concentration (ng/ml) (ng/ml) ± S.D. 1. 6.3 06.10± 0.05 0.893 2. 50 50.10 ± 0.52 0.998 3. 100 99.92 ± 3.01 3.009 4. 200 200.1 ± 3.80 0.184
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1442 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y
Table 6: Precision (C.V.) and accuracy (relative error) of inter – day assay measurements of karanjin at UV detection 305 nm.
S.No. Nominal Measured concentration concentration (ng/ml) (ng/ml) ± S.D. % C.V. 1. 6.3 6.01 ± 0.057 0.945 2. 50 50.02 ± 0.59 1.102 3. 100 98.95 ± 3.19 3.210 4. 200 200.01 ± 3.98 1.981
5. LIMIT OF QUANTIFICATION & LIMIT OF DETECTION (LOQ& LOD)
It was found that below 5ng/ml, the back calculation values failed to meet the acceptance criteria. Hence
5ng/ml levels were five times injected. It was found that RSD was 2.75%. Accuracy, defined as the deviation between the true values expressed, as a percentage was 3.9% at this concentration (5ng/ml). So 5ng/ml was established as LOQ and 3ng/ml was established as LOD.
6. SYSTEM SUITABILITY
for system suitability, five replicates of standard samples were injected and studied the parameters like theoretical plate number (N), tailing factor (T), resolution (R), relative retention time ( α), high efficiency theoretical plates (HETP), capacity factor (k´), plate per meter and peak symmetry of samples. These values are mentioned in below table 7.
Table-7: System suitability parameters of karanjin.
S.No. Parameters Values 1. Theoretical plates (N) 2291 2. Tailing factor (T) 0.98 3. Resolution ( R) 2.10 4. Relative retention time ( α) 4.561 5. HETP 0.074
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1443 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y 6. Capacity factor (K´) 1.80 7. Plates per meter 15163 8. Peak symmetry 1.2
7. ROBUSTNESS
As per the results of the percentage recoveries of karanjin was good under most conditions and did not show any significant change when the critical parameters were modified. The components were well separated under all the conditions carried out considering the modifications in the system suitability parameters and the specificity of the method, as well as carrying the experiment at room temperature may conclude that the method conditions are robust.
8. PURITY STUDIES OF KARANJIN FROM PLANT CRUDE EXTRACTS
The purity study for different crude extracts was calculated according to the formulae given. The %purity studies were given in the below table 8 for different parts of seed extracts and for different solvents given.
Table 8: Karanjin content in different parts of seed extract.
S.No. Part of seed from MeOH extract EtOAc solubles of % of P.glabra (grams) MeOH extract (g) karanjin
1. Seed (d) 11.20% 0.92% 38.35
2. Kernel (d) 18.46% 2.18% 53.52
3. Shell (d) 2.68% 0.32% 12.35
4. Seed (f) 11.54% 1.99% 38.25
5. Kernel (f) 10.93% 2.55% 14.26
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1444 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y Here the graphs of the developed methods are given below:
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1445 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y DISSICUSION:
The Chromatographic method was optimized by changing various parameters, such as pH of the mobile phase, the separation of peaks are dependent on the percentage of methanol. Methanol was used instead of acetonitril to shorten tailing of karanjin. In case of acetonitril, the karanjin show more tailing, even though the retention time is less than the methanol.
Under the presently prescribed conditions, the recovery of karanjin was found to be 95.102%, a very low concentration.
Till now there is no single method was developed for the determination of karanjin with out buffer. So this method is useful for determining the %purity of karanjin in crude extracts.
The observation of C.V. less than 4.0 for both inter-day and intra-day measurements also indicates the high degree of precision.
In the present method, we have established a linearity range of 6 – 60 µg/ml; this linearity range covers all the strengths of karanjin. Hence this method can be applied for quantifying the low levels of karanjin in extracts and other pharmacokinetics studies if necessary.
CONCLUTION
The aim of the present study was to develop simple, fast and sensitive HPLC methods for the estimation of Karanjin from P. glabra few analytical methods are reported in literature, but those methods have some drawbacks like gradient elution, long run time, less resolution and lack of sensitivity like precision and accuracy.
Further more for karanjin there was no proper documentation and validation, which are crucial for analytical method here, it was mentioned clearly.
The new reverse-phase high performance liquid chromatographic methods were developed and validated for the determination of Karanjin in crude extreact & method was developed.
The method described herein is simple validated assay procedures that can readily be used in any laboratory for the quantitative determination of crude extracts. Analytical figures of merit demonstrated during
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1446 Vejendla.Ravikumar * et al. /International Journal Of Pharmacy&Technolog y the method validation protocol compare well with those of known methods for the determination of crude extracts from different parts of seed. The assay procedure was simple with satisfactory precision and accuracy in terms of relative error. We believe that this method fulfills requirements for determining the crude karanjin and for %purity studies from different extracts of the plant parts.
Practically, low cost of single analysis is central features of routine laboratory and the herein described
RP-HPLC method, because it uses low cost of solvents and is easily affordable by clinical laboratories equipped with standard high performance liquid chromatographic systems.
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Corresponding Author: Vejendla.Ravikumar*, Departmant of Pharmacy, Sri Indu Institute of Pharmacy, Hyderabad. Email: [email protected]
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