Quantification of Quercetin in Solid Oral Polyherbal Formulation Containing Carica papaya L. Leaf Extract by High Performance Thin Layer Chromatography

Introduction

Herbs are the store house of enormous phyto-constituents which vary in structure and pharmacological activity. Herbal plants are useful as starting material for the semi synthetic preparation of other drugs. Herbal medicinal products can be used as dietary supplements or medicines that people take to improve their health and cure various ailments. Herbs are sold as tablets, capsules, powders, teas, extracts, and fresh or dried plants.

Carica papaya (Carica papaya L.) belongs to family Caricaceae, a herb which is reported for various pharmacological activities. Papaya leaves are rich in flavonoids(kaempferol and quercetin), alkaloids (carpaine, pseudocarpaine, dehydrocarpaine I and II), phenolic compounds (ferulic acid, caffeic acid, chlorogenic acid), and cynogenetic compounds. It is a great challenge for herbal industries to convince the modern society regarding efficacy, purity, and quantity of herbal drugs. Analytical parameters and documentation will be a top priority to prove the quality of herbal drugs.¹

There are three kinds of herbal medicines; raw plant material, processed plant material, and medicinal herbal products. Herbal medicinal products can be used as dietary supplements or medicines that people take to improve their health and cure various ailments. Herbs are sold as tablets, capsules, powders, teas, extracts, and fresh or dried plants. Herbals are traditionally considered harmless and increasingly being used by people. Quality evaluation of herbal preparation is a fundamental requirement of industry and other organization and dealing with Ayurveda and herbal products.^2,3

Exclusive use of synthetic compounds has prominent drawbacks such as handling hazards, threat to human environment, etc. For better alternative, plant-based medicine via a formulation and by replacing synthetic additives, preservatives with herbs and spices (garlic, basil, pepper, ginger, rosemary, clove, etc.) which are natural, effective, and non-toxic which have been used as food additives from ancient time as flavouring agent and natural food preservatives, etc. Consuming plant based formulations is safe since they are natural and will be having no or lesser side effects than the prescribed synthetic drugs.⁴

High performance thin layer chromatography (HPTLC) is a sophisticated instrumental technique based on the full capabilities of thin layer chromatography. The advantages of automation, scanning, full optimization, selective detection principle, minimum sample preparation, hyphenation, etc. enable it to be a powerful analytical tool for chromatographic information of complex mixtures of inorganic, organic, and biomolecules.⁵

Carica papaya is a large plant of height upto 20- 30 feet. Consisting of large leaves with entire margins and petioles of length about 1- 3 feet. It is proven that Carica papaya possess antiameobic, anthelmintic, antifertility, diuretic, antifungal, antimicrobial, and many more activities.As such POP-e and “Caripill” are formulations prepared by majorly of leaf extracts which are used prominently in the treatment of peptic ulcers and dengue, respectively. Also,these formulations are used in studies like platelet storage solution, liver healing, synthesis of nano particles, etc.^{6, 7}

Quantification of Carica papaya leaf extract in which the methanolic extract quercetin was less than the limit of detection and in butanolic extract it showed 0.55mg/g. Studies involving whole shoot part of C.papaya aqueous methanolic extract showed 1.2% of the quercetin presence and methanolic extract of Carica papaya leaf showed 3.11mg/g of quercetin presence. In a study extraction with hot water of Carica papaya the flavonoid (quercetin) yield was found to be 0.0% with the taken quantity of the drug for extraction.^{8, 9 , 10}

Materials and Methods

Collection and authentication of plant material

Fresh green matured and young leaves of Carica papaya were collected from the local botanical garden, University of Agricultural sciencesGKVK, Bangalore. Identification and authentication of plant material was done at Regional Ayurveda Research Institute for Metabolic Disorders, Jayanagar (Bangalore). Identification and authentication carried out by Dr. K. Rama Rao, Research Officer, Department of Botany.

Preparation of the leaf and formulation extract

The fresh leaves of Carica papaya were air dried at room temperature for 8 to 14 days, powdered in a blender. The aqueous extract was prepared at the ratio of 1:10 by suspending 25g of powdered leaves in 250mL of distilled water in Soxhlet apparatus. The powdered drug was charged into the thimble of Soxhlet extractor and extracted with water till the siphon tube turned colourless. Then the extract was collected and concentrated using rotavapor.

The herbal formulation Pop-e and Caripill were obtained fromKAPL, Peenya, Bangalore. The tablets were hard and sticky and could not be powdered so the tablets of POP-e and Caripill were individually weighed and dissolved in distilled water. The coating material was easily removed by dissolving in water. After removing the coating material, the tablets were weighed and dissolved in 100mL of distilled water. Further these solutions containing the tablets were concentrated at the temperature of 105^O C.¹¹

Phytochemical evaluation and quantitative estimation

Standard procedures were followed in carrying out phytochemical screening and determining phenolic, flavonoid, and glycoside content of the extract and samples.¹²

Anti-oxidant activity

The antioxidant activity of the aqueous leaf extract of Carica papaya and formulation samples were measured on the basis of scavenging activity of DPPH.One mL of 0.1mM DPPH solution in methanol was mixed with 1mL of plant extract solution of varying concentrations (50, 100, 150, 200, and 250 µg/ml). Corresponding blank samples were prepared and Rutin (50-250 µg/mL) was used as the reference standard. Control was prepared by mixing 1mL methanol and 1mL DPPH solution. All preparations were kept in dark for 30 min. Activity was carried out in triplicate and the decrease in absorbance was measured at 517nm using UV-Vis spectrophotometer. The inhibition % was calculated using the formula where A0 = absorbance of the blank/control; A1 = absorbance of the extract or sample.^13,14

% inhibition of DPPH = (A₀ – A₁) X 100 / A₀

HPTLC

Chromatographic analysis was performed on 20cm x 10cm aluminium plate coated with 0.2mm layer of silica gel 60 F254. Samples were applied to the plate as a band width of 6mm by using CamagLinomat 5 applicator fitted with 100µl syringe. The application rate was kept constant 150nL/sec, space between the bands was14mm. Then TLC plate was kept in twin trough glass chamber with previously saturated mobile phase (20min) for ascending development at room temperature. The chromatogram run was 85mm. Then plate was removed and air dried. Densitometric analysis was performed with the scanning at the absorption maxima using the Camag TLC scanner 3 and was documented.^15,16

Preparation of standard stock solution

Twomg of quercetin was dissolved in methanol to prepare stock solution of 200µg/mL and volume was made to 10mL. Different amount was applied in triplicates using the Camag applicator.

Preparation of sample stock solution

The samples were weighed, added to methanol to prepare a concentration of C.papaya leaf extract, and POP-e of 10mg/mL and Caripill of 5mg/mL and sonicated for 20 min. Then it was filtered through microfilters and clear solution was collected. Samples were then applied on the plates using the Camag applicator.

Results

Collection and authentication of plant material

Fresh green matured and young leaves of papaya plants were collected from the botanical garden, University of Agricultural Sciences, GKVK, Bangalore. The leaves were washed thoroughly 3–10 times in sterile distilled water. Then, they were air-dried under shade at room temperature for about a week. Identification and authentication of plant material was done at Regional Ayurveda Research Institute for Metabolic Disorders, Jayanagar (Bangalore) carried out by Dr. K. Rama Rao, Research Officer, Department of Botany. The certificate bearing the Authentication. No./S.MPU/RARIMD /BNG /2017- 18/408 is deposited at the department of Pharmacognosy, Government College of Pharmacy, Bengaluru for future reference.

Phytochemical evaluation and quantitative estimation

In the phytochemical screening Carica papaya leaf extract exhibited the presence of various phytoconstituents like alkaloids, glycosides, saponins, phenols, and flavonoids. The formulations POP-e and Caripill were also subjected for preliminary phytochemical screening. They also exhibited the presence of alkaloids, glycosides, saponins, phenols, and flavonoids. Hence the Carica papaya leaf used is of same quality as the formulations. The content of phytoconstituents present in both extract and formulation samples are mentioned in the Table 1.

Antioxidant activity

Inhibitory potential of Carica papaya aqueous leaf extract and herbal formulation samples on DPPH in comparison with standard Rutinare graphically presented in the Fig1. The extent of activity at various concentrations (50-250µg/mL) mentioned with solution of 10mg/mL of extract was measured in comparison with the standard Rutin. The inhibitory activity was comparatively less than the standard. Study showed that 250µg/mL concentration with highest inhibitory activity 80.97% of the leaf extract and the comparison is mentioned in Table 2.

HPTLC

Development of suitable mobile phase: Various relevant mobile phases were tried to observe clear spot without any tailing of spot. Finally toluene:ethyl acetate:formic acid in the ratio of (4.5:4:1) (v/v/v)provided a good resolution, sharp and clear spotwith Rf value of 0.57 which corresponds to quercetin. The samples were applied on TLC plate with a concentration of Carica papaya leaf extract 10, POP-e 10, and Caripill 5mg/mL.

Method Validation

Sensitivity: sensitivity of the method was tested with respect to LOD and LOQ of quercetin. The lowest amount of the compound that could be detected was found to be 90.00 ng/spot and the lowest amount that could be quantified was found to be 186.96 ng/spot as mentioned in Table 3.

Specificity

The comparison of the Rf value of the standard quercetin with that of the sample confirmed that the developed method is specific for the study. Identity of the quercetin peak was confirmed by comparing the UV-visible absorption spectrum of the peak from the standard with the sample which were found to be superimposable as presented in Fig2and3. The eluted bands of the standard and samples on the TLC plate are scanned under 254nm and 366nm as shown in Figure 4.

Calibration curve and Linearity: Developed HPTLC method of estimation for quercetin showed a good correlation coefficient (R2 = 0.96269 and 0.95681).The calibration curve was prepared by plotting the concentration of quercetin versus average area of the peak and it was linear in the range of 20 - 120 μg /mL for quercetin. It was defined by the linear equation Y = 53.4 + 0.4961×X with respect to height and Y = 978.7 + 10.79×X with respect to area as presented in Fig 5 & 6.

Precision & Repeatability: Replicate estimation of the quercetin sample preparation provided concurrent results showing that the method is precise.

Robustness : Small detectable deliberate changes were observed in the method in the chromatographic condition (mobile phase composition, elute volume, development distance, temperature, humidity and chamber saturation), sample application (band, spot stability), spot visualization and quantitative evaluation (Plate drying and detection wavelength used).

In comparison of the Carica papaya leaf extract and the formulation samples, total amount of quercetin content present in each samples were calculated per gram of sample based on the analytical data obtained from the analysis and are listed in the Table 4.

Discussion

The present study is first to report the quantification of quercetin in Carica papaya leaf extract and herbal formulations containing papaya extract by HPTLC method. The calibration curve was prepared by plotting the concentration of quercetin versus average area of the peak and it was linear in the range for quercetin. The experiment was performed in triplicate for reproducibility and accuracy was found to be correct. The obtained data was analysed statistically. A good linear relationship with respect to peak area and heightwas observed between the concentration ranges 20-120 μg [Graph 1 showing the peaks with standard and sample]. The regression equation was found to be Y = 53.4 + 0.4961×X with respect to height and Y = 978.7 + 10.79×X with respect to area. The content of quercetin in Carica papaya extract, POP-e, and Caripill were 9.2075, 9.3480, and 9.00mg/gm, respectively. The results were expressed as mg quercetin equivalent per g of the extract and formulations weight.

Antioxidant activity performed by DPPH assay method for aqueous extract of Carica papaya leaf and herbal formulations, all samples exhibited high antioxidant activity and percentage inhibition of Carica papaya extract, POP-e, and Caripill were calculated. The highest percentage inhibition by DPPH exhibited was in Caripill 81.93±0.4371 at 250μg/mL and lowest percentage inhibition was in POP-e 78.766±0.3844 at 250μg/mL.

This study shows that aqueous extract of Carica papaya leaf has greater antioxidant potential and is having anti-cancer, anti-diabetic, prevents CV diseases, etc. due to the presence of quercetin content which is equivalent in comparison with herbal formulations available.

Herbal formulations have a growing demand in the world market. In this study, an attempt has been done to develop chromatographic profile for formulation containing Carica papaya leaf extract. The HPTLC method was developed and validated for the determination of quercetin in formulation containing Carica papaya leaf extract. The aqueous extract of Carica papaya leaf and marketed formulations POP-e and Caripill showed the presence of 0.92, 0.93, and 0.9% w/w of quercetin, respectively. The method was found simple, rapid, accurate, specific and robust for the analysis of quercetin in crude drug and in formulation using the same method.

This study comprehends the optimum potency of the active therapeutic constituent with the conventional method of aqueous extraction. The comparative value quotes the importance of the method and simple technique used in the extraction of significant therapeutic yield. As the study carried out with simple aqueous extraction method, it will provide clear idea on the method and for any other simple innovative methods to come in this aspect for further study. Also mentioning the importance of plant-based formulation in treating ailments.

Conclusion

The HPTLC method was developed and validated for the determination of quercetin in formulation containing Carica papaya leaf extract.The proposed method can be adopted by any laboratory for the quality control of crude drugs and formulations that contain quercetin as active marker compound. Natural remedies are more acceptable with belief that they are safe and have a few side effects than the synthetic ones.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgement

I would like to thank the Principal of Government College of Pharmacy, Bengalurufor providing all the facilities in completion of this project.