Validated HPTLC Method for Quantification of Luteolin and Apigenin in Premna mucronata Roxb., Verbenaceae

A simple, rapid, and precise high-performance thin-layer chromatographic method was developed for quantitative estimation of luteolin and apigenin in Premna mucronata Roxb., family Verbenaceae. Separation was performed on silica gel 60 F254 HPTLC plates using toluene : ethyl acetate : formic acid (6 : 4 : 0.3) as mobile phase for elution of markers from extract. The determination was carried out in fluorescence mode using densitometric absorbance-reflection mode at 366 nm for both luteolin and apigenin. The methanolic extract of Premna mucronata was found to contain 10.2 mg/g % luteolin and 0.165 mg/g % of apigenin. The method was validated in terms of linearity, LOD and LOQ, accuracy, precision, and specificity. The calibration curve was found to be linear between 200 and 1000 ng/band for luteolin and 50 and 250 ng/band for apigenin. For luteolin and apigenin, the limit of detection was found to be 42.6 ng/band and 7.97 ng/band while the limit of quantitation was found to be 129.08 ng/band and 24.155 ng/band, respectively. This developed validated method is capable of quantifying and resolving luteolin and apigenin and can be applicable for routine analysis of extract and plant as a whole.


Introduction
Quality control of medicinal plants is highly essential to ensure authenticity, stability, and consistency. Safety and efficacy studies along with the standardization of bioactive extract on the basis of active principle or major compound(s) as a quality assurance parameter shall open up unlimited possibilities for herbal medicine in pharmacotherapeutics [1][2][3]. Standardization can be carried out by obtaining a chemical fingerprint/profile in terms of one or more marker compounds (chemical or biomarker). Use of chromatography for standardization of plant products was introduced by the WHO and is accepted as a strategy for identification and evaluation of the quality of plant medicines [4][5][6]. HPTLC is becoming a routine analytical technique because of its advantages of low operating cost, high sample throughput, simplicity and speed, the need for minimum sample cleanup, reproducibility, accuracy, and reliability [7][8][9][10].
Premna mucronata, family Verbenaceae, commonly known as Arni is a large shrub or small tree of about 20 to 25 feet's height found in Northern India, the Gangetic plains, Uttar Pradesh, Bihar, and Bengal province of India. Besides, it is also found in hill area from Kumaon to Bhutan up to the height of 5000 feet from sea level [11]. The plant possesses antioxidant and hypocholesterolemics activities and is also known for improving digestion, acts as a blood purifier, cardiac stimulant, and expectorant, and is also useful in skin disorders [11][12][13]. It has been reported that the plant Premna mucronata contains flavonoids, luteolin, apigenin, clerodendrin, hispidulin, and pectolinarigenin. The plant 2 Advances in Pharmacological Sciences also contains clerodin, clerosterol, D-mannitol, palmitic acid, ceryl alcohol, and cerotic acid. Within the complex mixture of phytoconstituents in this plant, luteolin and apigenin can be used as analytical markers for determination of its quality. Preliminary studies performed at our laboratory have established the efficacy of the plant Premna mucronata in cardioprotection against experimentally induced myocardial infarction in rats by isoprenaline and coronary artery ligation method [14][15][16][17][18]. Literature survey also has revealed that the flavonoid glycosides possess antioxidant properties and strong scavenging properties for superoxide radicals [19][20][21][22]. Moreover, luteolin, a flavonoid, is also capable of protecting the myocardium against IR injury, partly mediated through downregulation of NO production and its own antioxidant properties [16]. Hence, further work was focused on identification and quantification of flavonoids, luteolin and apigenin, in Premna mucronata. However, to the best of our knowledge, there is no report on thin-layer chromatographic method for quantitative analysis of luteolin and apigenin in methanolic extract prepared from whole plant Premna mucronata. Here, we have developed HPTLC method for quantitative estimation of luteolin and apigenin. The developed method was also validated in terms of system suitability, specificity, linearity, LOD, and LOQ according to ICH guidelines [23].

Preparation of Standard Luteolin Solution.
A stock solution of luteolin was prepared by dissolving 5 mg of accurately weighed luteolin in methanol and making up the volume to 10 mL with methanol. Working solution of luteolin (200 g/mL) was prepared by appropriate dilutions of the stock solution with methanol.

Preparation of Standard Apigenin Solution.
A stock solution of apigenin was prepared by dissolving 5 mg of accurately weighed apigenin in methanol and making up the volume to 10 mL with methanol. The stock solution was further diluted with methanol to give a standard solution of apigenin (50 g/mL). Lamp: mercury.

Chromatographic
The HPTLC analysis was performed in an air conditioned room maintained at 22 ∘ C and 55% humidity using precoated silica gel 60 F 254 aluminium backed plates (10 × 10 cm, 0.2 mm layer thickness, 5-6 m particle size; Merck, Darmstadt, Germany). 6 L and 5 L of the standard solutions of luteolin and apigenin were spotted using a Linomat 5 autosampler fitted with a 100 m Hamilton syringe (CAMAG, Muttenz, Switzerland) and operated with settings of a band length of 8 mm; distance between bands of 5 mm; distance from the plate edge of 10 mm; and distance from the bottom of the plate of 10 mm. The plates were developed to a distance of 80 mm using toluene : ethyl acetate : formic acid (6 : 4 : 0.3, v/v) mobile phase in CAMAG twin trough chamber presaturated with mobile phase. The developed plates were air dried and scanned with a CAMAG TLC Scanner 4 equipped with winCATS planar chromatography manager (version 1.4.6) software that was used for densitometry measurements, spectra recording, and data processing. The absorption/remission measurement mode was used at a scan speed of 20 mm/s. Zones of luteolin and apigenin were scanned from 200 to 400 nm to record their absorption and fluorescence spectra, respectively. Densitogram was recorded in fluorescence mode for both luteolin and apigenin.

Calibration Curve for Standard Luteolin.
The standard solution of luteolin (200 to 1000 ng/band) was applied in triplicate on HPTLC plate. The plate was developed and scanned as per the chromatographic conditions mentioned above. The peak areas were recorded. Calibration curve of luteolin was prepared by plotting peak area versus concentration of luteolin applied.

Calibration
Curve for Standard Apigenin. The standard solution of apigenin (50 to 250 ng/band) was applied in triplicate on HPTLC plate. The plate was developed and scanned as per the chromatographic conditions mentioned above. The peak areas were recorded. Calibration curve of apigenin was prepared by plotting peak area versus concentration of apigenin applied.
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Preparation of Methanolic Extract of Premna mucronata (MEPM).
Accurately weighed 10 gm of powdered drug of whole plant was extracted for 15 min with methanol (4 × 25 mL) under reflux on water bath at 100 ∘ C. The methanolic extract was filtered through Whatman number 1 filter paper. The filtrates were combined, concentrated, and transferred to a 50 mL volumetric flask, and the volume was made up to 50 mL with methanol.

HPTLC Analysis of Plant Extract.
Samples of methanolic extract of Premna mucronata were filtered through 0.45 m filter, and HPTLC was performed under the conditions optimized for the reference compound. The plates after development were dried in air and photographed at 254 nm. The plates were then derivatized by spraying with NP-PEG reagent followed by photographing the plates in visible and fluorescence mode and scanned at 366 nm for both luteolin and apigenin. The amount of luteolin and apigenin in plant extract was quantified using calibration curve plotted with luteolin and apigenin.
The intermediate precision of the method was studied by analyzing aliquots of standard in triplicate at 3 concentration levels for luteolin and apigenin on the same day for intraday precision. The study was also repeated on different days with freshly prepared samples in order to determine interday precision. The results were expressed as relative standard deviation (RSD). The specificity of the method was ascertained by determining the peak purity of the component by overlaying the fluorescence spectra of luteolin and apigenin in the sample extract with the spectra of reference standard luteolin and apigenin at the start, middle, and end positions of the bands. The accuracy of the method was determined by recovery studies at 3 levels in triplicate. For recovery studies, known amount of standard was added.

Results and Discussion
Different proportions of toluene and ethyl acetate were tried as the mobile phase on silica gel HPTLC plates and a ratio of (6 : 4 v/v) gave good resolution. Well resolved symmetric band for luteolin and apigenin in extract was obtained under the optimized conditions using precoated HPTLC plates with 0.2 mm thickness, 5-6 mm particle size, and the mobile phase toluene : ethyl acetate : formic acid (6 : 4 : 0.3). Luteolin and apigenin on derivatization with NP-PEG reagent (natural products polyethylene glycol reagent) appeared orange in visible mode (Figures 1(a) and 1(b)) and gave bright yellow and parrot green fluorescence in fluorescence mode (Figures 2(a) and 2(b)) [24]. Standard luteolin ( 0.45) and standard apigenin ( 0.49) showed single peak in HPTLC chromatogram (Figures 3 and 4). Calibration curve of luteolin and apigenin was prepared by plotting concentration of luteolin versus area of the peak and concentration of apigenin versus area of the peak, respectively (Figures 5 and 6).

Determination of Marker in Extract.
The methanolic extract of Premna mucronata was found to contain 10.2 mg/g % luteolin and 0.165 mg/g % of apigenin. Furthermore, the methanolic extract of Premna mucronata shows peak in the chromatogram at same value as luteolin (0.45) standard and apigenin (0.49) standard (Figure 7).

Linearity.
Luteolin and apigenin showed good correlation coefficient of 0.9942 and 0.9917, respectively, when peak area of the resolved band was plotted against concentration, thus exhibiting good linearity between concentration and peak area. Table 1 summarizes Beer's law limit, linear regression equation, and correlation coefficient for the method.   Tables 2 and 3. 3.4. Accuracy. The proposed method when used for quantitation of marker after spiking with standard afforded recovery of luteolin and apigenin in the range of 96.67%-102.92% and 97.72%-99.29%, respectively, at three concentration levels as shown in Tables 4 and 5.

Limit of Detection and Limit of Quantification.
For luteolin and apigenin, the limit of detection was found to be 42.6 ng/band and 7.97 ng/band while the limit of quantitation  was found to be 129.08 ng/band and 24.155 ng/band, respectively.
3.6. Specificity. The identity of the bands in the sample extracts was confirmed by comparing , absorption spectra, and fluorescence spectra and by overlaying with those of their respective standard using CAMAG TLC Scanner 4.
Advances in Pharmacological Sciences 5     There were no interfering spots by the plant constituents at values of the marker. The absorption spectra of standard marker luteolin ( 0.45) and apigenin ( 0.49) and the corresponding spot present in extract matched exactly, indicating no interference by the other plant constituents (Figures 8 and 9). The purity of the bands due to luteolin and apigenin in the sample extract was confirmed by overlaying the absorption and fluorescence spectra recorded at start, middle, and end position of the band in the sample tracks, respectively (Figures 10 and 11).

Conclusion
The proposed HPTLC method was found to be rapid, simple, and accurate for quantitative estimation of luteolin and apigenin. The recovery of luteolin and apigenin from whole part of Premna mucronata was found to be 96.67%-102.92% and 97.72%-99.29%, respectively, revealing accuracy of the developed method and hence can be applicable for routine 6 Advances in Pharmacological Sciences    analysis of extract and plant as a whole. Moreover, HPTLC profile by co-TLC can be used for fingerprinting thereby providing assurance of standardization and quality. And HPTLC has various advantages of low operating cost, high sample throughput, simplicity, and the need for minimum sample clean-up. In future, as the plant shows good cardioprotective activity, dosage form of the same can be formulated where fingerprinting and quantification of both the chemical markers will be used.