1. Presented by: S.H. Ansari
Development and validation of HPTLC method for simultaneous estimation of flavonoids and phenolics in Carica papaya leaf juice
Presented by: S.H. Ansari
Department of Pharmacognosy and Phytochemistry
School of Pharmaceutical Education and Research
Jamia Hamdard, New Delhi, INDIA

2. INTRODUCTION
The Carica papaya Linn. (papaya) fruit is consumed as juices, jams, and crystallized dry fruit.
Beneficial effect as an anti-inflammatory, wound healing, anti-tumour, immune-modulatory and antioxidant.
Leaves contain phenolics (caffeic acid, trans ferulic acid, para coumaric acid, vanillic acid) and flavonoids (myricetin, kaempferol, quercetin) etc.
Leaf juice and aqueous extract are commonly used in dengue fever related thrombocytopenia.

3. PHYTOCONSTITUENTS PRESENT
Figure 1: Chemical structure of some phenolic acids and flavonoids

4. RATIONALE OF THE STUDY
Quantification of phenolic acids and flavonoids using HPTLC such as caffeic acid, kaempferol, ferulic acid were detected by visualizing reagents or on cyanopropyl/cellulose derivatized plates.
Our method was simple, simultaneous developed on silica without derivatizing agents which increases sensitivity, repeatability and makes the method more economic.
No reports for quantification of myricetin, caffeic acid, trans-ferulic acid, kaempferol in combination using HPTLC so far.

5. AIMS AND OBJECTIVE Aim: Objective:
To validate and quantify two flavonoids and two phenolics in Carica papaya Linn. leaf juice.
Objective:
Collection and authentication of C. papaya Linn. leaf.
Preparation of leaf juice by squeezing and its freeze dried powder.
Development of polarity based fractions.
Quality control analysis of active fractions and mother juice by HPTLC method.
Quantification and validation of flavonoids and phenolics in mother juice.

6. PREPARATION OF STANDARD SOLUTIONS AND APPLICATION ON HPLTC
QUANTITATIVE ESTIMATION OF MYRICETIN, CAFFEIC ACID, TRANS FERULIC ACID AND KAEMPFEROL USING VALIDATED HPLTC METHOD
PREPARATION OF STANDARD SOLUTIONS AND APPLICATION ON HPLTC
Stock solutions (1000 µg/mL) of myricetin, caffeic acid, trans-ferulic acid and kaempferol were prepared in HPLC grade methanol. By mixing stock solutions, the concentration was found to be 250 µg/mL of each. The mixed stock solution was applied in triplicate in different volumes (0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 4.0, 5.0 µL) on HPLTC plate for calibration plot. Toluene: ethyl acetate: formic acid (50:40:10, v/v/v) was used as solvent system for simultaneous elution of myricetin, caffeic acid, trans-ferulic acid and kaempferol.
Developed chromatograms were scanned at 320 nm for myricetin, caffeic acid, trans-ferulic acid and kaempferol. The data of peak area versus drug concentration was treated by linear least square regressions and concentration range showing best regressions were considered for linearity. The concentrations of unknown samples were determined.

7. VALIDATION PARAMETERS
Validation of developed HPLTC method was followed ICH guidelines for calibration, linearity, precision, accuracy, robustness, specificity, LOD and LOQ, similar to Ahmad et al., 2013; Tiwari et al., 2012; 2015.
Precision: The exactness of the proposed method was obtained by repeatability and intermediate precision. Between day and intra-day, the precision was carried out at three concentration level (50, 500, 1000 ng/spot). The inter-analyst precision was carried out by repeating the same procedure using different system and by different analyst, respectively. The repeatability and intermediate precisions were determined and reported in terms of %RSD.
Robustness: Was a measure of its capacity to persist ineffective by small, but deliberate, differences in the method parameters. Robustness was carried out by introducing small changes in the compositions of mobile phase (toluene: ethyl acetate: formic acid; 5:4:1.1; 5:4:0.9; 5:5:1) and detection wavelength (318 and 322 nm). The effect on the results was studied as %RSD.

8. VALIDATION PARAMETER continued…..
Specificity: Was ascertained by analyzing standard drug sample. The detection of spot for mixed standard in mother extract and its fractions was confirmed by comparing Rf and spectra with that of standards. The peak purity was evaluated by looking at spectra at three different levels i.e. peak start, peak apex and peak end positions of the spot.
Limit of detection (LOD) and limit of quantification (LOQ): LOD is defined as the lowest concentration of analyte in a sample that can be detected and LOQ is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. The LOD was expressed as: LOD = 3.3 σ/slope, whereas LOQ was expressed as LOQ = 10 σ/slope of calibration curve.
Accuracy and recovery: Accuracy was expressed as percent recovery by the assay of added known amounts of analyte. Pre-analyzed samples were spiked with standard at four different concentration levels i.e. 0, 50, 100 and 150% and the mixtures were re-analyzed .

9. RESULTS AND DISCUSSION
Papaya leaf juice yielded 1.44% w/v of freeze dried powder whereas DCM and n-butanol fraction yielded 0.50% and 15.11% w/w, respectively.
HPLTC fingerprinting of mother extract and different fractions were developed on silica gel showed 6 common compounds in all whereas 3 new compounds have been detected at Rf 0.71, 0.80 and 0.92.
Plate was scanned at 320 nm, produced exceptionally well characterized peaks of myricetin, caffeic acid, trans ferulic acid and kaempferol at Rf value 0.39 ± 0.01, 0.44 ± 0.02, 0.50 ± 0.01, 0.55 ± 0.01, respectively.
Developed method was found linear in concentration range ( ng) with good regression coefficient (0.99 ± 0.001).
%RSD of precision and intermediate precision were found in the range of
LOD of markers were in the range of ng/spot, indicating good sensitivity of methods for simultaneous quantification of compounds.
Accuracy for pre-analyzed samples were found in the range of %.

10. DEVELOPED HPLTC PLATE
Figure 2: Developed HPTLC plate showing spots of myricetin, trans ferulic acid, caffeic acid, kaempferol at different concentration in standards and in samples (1: mother extract; 2: DCM fraction; 3: n butanol) at 254 nm

11. HPTLC CHROMATOGRAM
Figure 3: HPTLC chromatogram of standard myricetin, trans ferulic acid, caffeic acid,
kaempferol (A), mother extract (B), DCM fraction (C), n butanol fraction (D) at 320 nm

12. LINEARITY AND VALIDATION DATA
Table 1: Estimation of standards in mother extracts and different fraction of papaya juice
Samples
Content (mg/g)
Myricetin
Caffeic acid
Trans ferulic acid
Kaempferol
Mother extract
± 5.99
± 6.27
± 2.97
± 2.48
DCM fraction
± 4.87
± 5.92
± 6.34
5840 ± 4.36
n Butanol fraction
± 3.47
± 4.68
± 4.13
± 2.19

13. LINEARITY AND VALIDATION DATA
Table 2: Linearity and validation data of chromatographic HPTLC method for mixed standards (n = 3)
Parameters
Biomarkers
Myricetin
Kaempferol
Trans-ferulic acid
Caffeic acid
Linearity range (ng/spot)
Regression equation
Y= x ± (-283.5)
Y= 15.82x ± ( )
Y= x ± 1359
Y= 15.26x ± 2465
Regression coefficient ± SD
0.997 ± 0.001
0.998 ± 0.001
0.993 ± 0.002
Slope ± SD
17.96 ± 0.015
15.82 ± 0.005
11.56 ± 0.015
15.25 ± 0.025
Intercept ± SD
± 0.100
± 0.057
± 0.577
2465 ± 0.577
LOD (ng/spot)
04.502
03.508
05.129
18.767
LOQ (ng/spot)
13.643
10.632
15.544
56.872
Precision (%RSD range)
Inter-day
Intra-day
Inter-laboratory
Accuracy (% drug recovered)

14. PRECISION DATA
Figure 4a (i): Robustness of the HPTLC method for estimation of mixed standards by changing detecting wavelengths (n=3)

15. Precision data
Figure 4a (ii): Robustness of the HPTLC method for estimation of mixed standards by changing detecting wavelengths (n=3) showing Levy Jenning's Plot of %RSD

16. Precision data
Figure 4b (i): Robustness of the HPTLC method for estimation of mixed standards by changing detecting of mobile phase composition (n=3)

17. Precision data
Figure 4b (ii): Robustness of the HPTLC method for estimation of mixed standards by changing detecting of mobile phase composition (n=3) showing Levy jenning's plot of %RSD

18. SUMMARY
Myricetin, caffeic acid, trans ferulic acid and kaempferol in mother extract was confirmed by matching Rf and spectra spot with standard.
LOD of markers were found in range indicated good sensitivity.
Accuracy showed good recovery of all markers used in the range of %.
Peak purity tests of myricetin, trans-ferulic acid, caffeic acid and kaempferol were conducted by comparing UV spectra of standards with sample tracks.
Contents ( mg/g) of all the active marker ingredients were recorded in leaf juice and compared to fractions ( mg/g).
In the present study we could observe the maximum contents of myricetin, trans-ferulic acid, caffeic acid and kaempferol in leaves.

19. CONCLUSION
A stability indicating HPLTC densitometry method was established for quantitative analysis of myricetin, trans ferulic acid, caffeic acid and kaempferol in mother extract and different fractions.
Method was validated according to ICH guidelines for recovery, specificity, linearity, LOD, LOQ and robustness.
Method is simple, rapid, reproducible, accurate, and selective alternative to HPLC for the separation of the myricetin, trans ferulic acid, caffeic acid and kaempferol in Carica papaya.
Method is less consuming and more cost effective than other methods and can be used to separate the drug from its degradation products and from excipients used in preparation of dosage form.
Advantage of TLC is the high sample throughput with results from the small amount of sample preparation required and the simultaneous quantification of several samples.

20. REFERENCES
B.V. Owoyele, O.M. Adebukola, A.A. Funmilayo, and A.O. Soladoye, Inflammopharmacol, 16 (2008) 168–173. 
D.S. Seigler, G.F. Pauli, A. Nahrstedt, R. Leen, Phytochem, 60 (2002)
H.J.Chen, B.S. Inbaraj, B.H.Chen, Int J Mol Sci, 13 (2012)  
N. Otsuki, N.H. Dang, E. Kumagai, A. Kondo, S. Iwata, and C. Morimoto, J Ethnopharmacol, 127 (2010) 760–767. 
N.A. Imaga, G.O. Gbenle, V.I. Okochi et al., Scientific Res Essays, 5 (2010)
D.C. Bhatt, K.D. Mitaliya, N.A. Pandya, U.S. Baxi, Adv Plant Sci, 14 (2001)
S. Ahmad, M. Singh, Y.K. Thajudeen, K. Kamal, R. Parveen, J Planar Chromatogr, 24 (2011)
M.N. Mallick, M. Singh, R. Parveen, W. Khan, S. Ahmad, M.Z. Najm, S.A. Husain, BioMed Res International 2015(In press)
R. Parveen, F.J. Ahmad, Z. Iqbal, M. Singh, Y. T. Kamal, S. Ahmad, Acta Chromatographica, 26 (2014) 391–400.
A. Ahmad, S. Ahmad, M. Rahman, T.E. Tajuddin, R. Verma, M. Afzal, P.S. Mehra, J Planar Chromatogr 28(2015)
S. Ahmad, M. Singh, Y.T. Kamal, M.A. Khan, R. Parveen, Indian J Pharm Edu Res 47(2013)