RP-HPLC-DAD determination and quanti ication of Quercetin and Luteolin in plant extracts of Merremia aegyptia and Merremia dissecta

Medicinal plants produce various useful metabolites such as alkaloids, lavonoids, tannins etc those are widely used for the preparation of various pharmaceutical products, or as food additives. A simple and precise reverse phase high performance liquid chromatographic (RP-HPLC) seperation method has been developed for determination and quanti ication of the lavonoids, quercetin and luteolin simultaneously, from methanolic extracts of Merremia aegyptia and Merremia dissecta after optimization of extracting solvent and chromatographic conditions through HPLC coupled to a Diode Array Detector(DAD). HPLC analysis estimated contents of the quercetin to be 20 ng/μl inM. aegyptia stem and 13.2 ng/ μl inM. dissecta callus whereas luteolin was found to be 0.4 mg/ml in M. dissecta callus. From the previous published literature, it appears that this is the irst report of quanti ication of these lavonoid compounds as they have not been reported earlier in both the species under study.


INTRODUCTION
High Performance Liquid Chromatography is an advanced form of liquid chromatography in which small particle columns are used through which the mobile phase is lown at high pressure for better isolation, separation, identi ication and quanti ication of component of interest from a mixture. It is a highly automated technique that generates reports on its own using autosamplers and data systems thus, extending detection limits to nanogram, pictogram and even below levels. It was once gravity-driven process but now automation of this technique has lead to divergent methods of chromatography.
The methods of determination and quanti ication of plant biochemicals that are used on regular basis include Ultra Violet-Visible spectrophotometry, HPLC, High Performance Thin Layer Chromatography and Mass Spectrometry. High performance liquid chromatography coupled with diode array detector (HPLC-DAD) has been reported to quantify isolated compounds present in the polar soluble fraction of plant samples. Many compounds from various plants have been isolated by advanced HPLC methods from Glycyrrhiza glabra (Khalaf et al., 2010) and Cayratia trifolia (Gour et al., 2013), Tridax procumbens (Sanghavi et al., 2014), Citrus macroptera (Nongalleima et al., 2017) and Matricaria chamomilla (Dong et al., 2017).
Its anticancer property includes inducing apoptosis, inhibiting cell proliferation, metastasis, angiogenesis and also have antiplatelet and vasodilatatory activities.
Luteolin is also used as antioxidant inhibiting Reactive Oxygen Species(ROS) induced damage of lipids, DNA, and protein (Brown and Rice-Evans, 1998) and have showed in vitro activities like anti-in lammatory activity (Kumazawa et al., 2006), a phosphodiesterase inhibitor (Yu et al., 2004) and an interleukin 6 inhibitor (Xagorari et al., 2001) . In vitro and in vivo experiments also suggest luteolin may inhibit the development of various types of cancer such as skin cancer (Han et al., 2002).
All of these activities suggest that quercetin and luteolin are compounds with potential clinical application. Determination of contents of quercetin and luteolin in Merremia aegyptia and Merremia dissecta have not been reported so far. This work was therefore designed to develop an RP-HPLC-DAD system to quantify quercetin and luteolin from methanolic extractive solutions of Merremia aegyptia and Merremia dissecta Figure 1.

Chemicals and reagents
Methanol (Chromatographic grade, Merck), phosphoric acid (Analytical grade, Merck ), acetonitrile (Chromatographic grade,Merck ) and acetic acid (Analytical grade, Merck) were used for the mobile phase preparation. Quercetin (Sigma, St Louis), Luteolin (Sigma, St.Louis) were used as external standards. HPLC grade MilliQ water was obtained by Millipore water puri ication system.

Plant Material
The whole plants of Merremia aegyptia and Merremia dissecta were collected from Jaipur. The specimens were identi ied by Herbarium, Department of Botany,University of Rajasthan, Jaipur (RUBL211617 and RUBL211618). The plants collected were washed with water and dried in the shady area for several days.

Standard stock solution and Sample preparation
Standard stock solutions of luteolin, quercetin were prepared by dissolving them in methanol, at concentration of 1.0 mg/mL. All standard solutions were iltered through 0.45 µm syringe ilter. The purity was checked through HPLC analysis monitoring individual compounds absorption maximum.
All the plant samples viz., Leaf, Stem, Seed and Callus each from M. aegyptia and M. dissecta were weighed (5gm) and powdered in a mechanical grinder to ine powder and soxhlet extracted in 80 percent methanol for 24 hrs at 40 • C. The methanol phases were iltered the next day and evaporated in a vacuum to obtain extracts. The dried extracts were dissolved in methanol and diluted. All sample solutions were also iltered through 0.45 µm syringe ilter (Chen and Xiao, 2005).

Instrumentation
5µl of each samples were injected with a HP Agilent 1200 in inity series quartenery pump autosampler with degasser and diode array detector(DAD), data analysis was performed with Agilent Chemstation software.

Determination and quantitative analysis
Determination and quantitative analysis of Quercetin and Luteolin in extracts were done through RP-HPLC following the protocol given by Chen and Xiao (2005) Flavonoids were analyzed on a RP-C 18 column((ZORBAX SB-C 18 ,4.6*250mm, 5µM).; using a mobile phase, consisting of methanolacetonitrile-acetic acid-phosphoric acid-H 2 O (200:100:10:10:200, V/V); under the following conditions: detecting wavelength, 350 nm; low rate, 1.0 ml/min; the sensitivity, 0.05 absorbance units full scale (AUFS) and the volume of injecting sample, 5.0 µl. The HPLC system was operated at ambient temperature (28±1 • C). The method showed linearity for quercetin and luteolin in the range 2.1-20.6, 2.0-24.1 g/ml respectively, and Analytes in each sample were identi ied by comparing the retention time and UV-Vis spectra at 260nm, reference 360nm with those of authentic compounds. Peaks were scanned between 190-400 nm for identi ication purpose and peak purity was checked to exclude any contribution from interfering peaks.

RESULTS AND DISCUSSION
HPLC is a versatile technique widely used for analysis of pharmaceuticals biomolecules, polymers and many organic and inorganic compounds. Chlorogenic acid and hippuric acid have been isolated and quanti ied from Merremia emarginata (Angappan et al., 2018) and comparative analysis of Merremia tridentata and Paederia foetida have been also conducted to analyse the similarity between the active component found in both the plants used for treating vatarogas (Rajashekhara et al., 2011) using HPLC methods.
The retention time and peak area observed for standard quercetin was 6.208, 3143.062 (Figure 2) whereas for the stem extract of M. aegyptia (RT The amount of luteolin in plant samples was analysed by comparing standard curve with known amount of luteolin. The amount of luteolin was found maximum in the M. dissecta callus extract (400.3 ng/µl or .4 mg/ml) (Figure 4).

CONCLUSIONS
The RP-HPLC method mentioned in this paper can be used for simultaneous determination of luteolin and quercetin in the extracts of M. aegyptia and Merremia dissecta plant samples.This method showed good sensitivity, precision, resolution and reproducibility. Better resolution among the two compounds with the analysis time (25 min) was reported and hence can play a reference role in the determination of polyphenolic compounds from other medicinal plants or pharmaceutical preparations as well.

ACKNOWLEDGEMENT
The corresponding author is highly thankful to UGC for the UGC-JRF grant and Centre for converging technologies, University of Rajasthan for the HPLC facility where the work was conducted.

Con lict of Interest
The authors declare that they have no con lict of interests.

Funding Support
None.