1 Introduction

Northeast India is a biodiversity hotspot region with varieties of natural resources having use in traditional practices for the treatment of different diseases and disorders. The local people of these regions utilized these resources, especially some plants, to prepare traditional herbal remedies [1]. People from rural areas are treated with these traditional remedies by local practitioners. Citrus maxima (Burm.) Merr. or Pomelo (Assamese: Robab Tenga) is such a wild plant that is reported to have many significant biological activities like antioxidant [2], antibacterial [2], antidiabetic [3], hepatoprotective [4], anticancer [5], antidepressant [6] etc. During field survey, it was found that the plant’s root part is being used to prepare an antimalarial herbal remedy. Along with root, the unripe fruit of the plant is also used in the treatment of fever associated with malaria. Later, in extensive literature review, the utilization of the different parts of the plant C. maxima in the treatment of malaria was reported [7]. In one of our previous studies, we found that the fruit part is rich in flavonoid compounds. Among the flavonoids, luteolin showed moderate activity against the chloroquine sensitive (3D7) and chloroquine resistance (RKL-9) strains of Plasmodium falciparum [8]. In another study, the hydroalcoholic root extract (CM-HA) of the plant was found to be potent (< 5 µg/ml) against both the 3D7 and RKL-9 strains of P. falciparum [9]. Later during thin layer chromatography analysis, the presence of luteolin in the CM-HA extract was detected. Hence, this study developed a method for quantitative estimation of luteolin in the CM-HA extract using high performance thin layer chromatography (HPTLC) technique. The HPTLC densitometric technique is a very simple and fast method for the quantitative estimation of marker compounds in plant-based samples [10, 11]. The developed method was validated according to International Council on Harmonization (ICH) guidelines; hence it can be utilized for quality control related analysis of the raw materials or any phytopharmaceuticals developed from the extract [12].

2 Methods

2.1 Instrumentation and Reagents

The analysis was conducted using TLC aluminium plates (20 cm × 10 cm and 10 cm × 10 cm) precoated with silica gel 60 F254 (Merck) (0.2 mm thickness). Using a 100 µl syringe (Hamilton), spotting was done with the CAMAG Linomat V semiautomatic Sample Spotter (CAMAG Muttenz, Switzerland). Based on the size of the aluminium plates, the different CAMAG glass twin trough chambers (20 cm × 10 cm and 10 cm × 10 cm) were used for development. Using visionCATS 2.5 software, a CAMAG TLC Scanner IV carried out the densitometric study. After development, the plates were examined in a CAMAG UV cabinet (254 and 366 nm).

The analytical-grade solvents from Merck included ethanol, methanol, toluene, ethyl acetate, and formic acid (Mumbai, India). Merck supplied the analytical-grade aluminium TLC plates that were coated with Silica Gel 60 F254 (Mumbai, India). We bought luteolin (97%), an analytical-grade marker compound, from Alfa aesar (Thermo Fisher), India. Quercetin (≥ 95%) and Gallic acid (≥ 97%) were purchased from Sigma-Aldrich, India.

2.2 Plant Material

In February 2021, the root of C. maxima was collected from the wild sources in the Longpotia region of Charaideo District, Assam. The plant was identified and authenticated by the Botanical Survey of India, Shillong, India. The coarsely powdered single batch plant materials were extracted using a hydroalcoholic solvent system (1:1) for 72 h by cold maceration [13]. The solvent was evaporated using a rotary vacuum evaporator (IKA Rotary Evaporator RV 8 V) and finally dried by lyophilization technique (IIC Industrial Corporation). The extract was kept in an airtight container in 4℃ for future use.

2.3 Total Phenolic Content and Total Flavonoid Content

The total phenolic content (TFC) of the extract was determined utilizing the Folin-Ciocalteu reagent with gallic acid as the standard using spectrophotometric method [14]. The absorbance was measured at a wavelength of 765 nm using a UV-Vis spectrophotometer (Shimadzu UV-1800) and content was expressed as a milligrams of gallic acid equivalents (GAE) per gram of dry extract weight.

The total flavonoids content (TFC) of the extract was determined by the aluminium chloride based colorimetric method with quercetin as the standard [15]. The absorbance was measured at a wavelength of 415 nm using a UV-Vis spectrophotometer (Shimadzu UV-1800) and the content was expressed as a milligrams of quercetin (QE) equivalents per gram of dry weight. All samples were analysed in triplicate to ensure accuracy and reliability of the results.

2.4 Preparation of Standard and Test Sample

In a volumetric flask, 10 mg of luteolin were dissolved in 10 ml of methanol to create 1 mg/ml of luteolin stock solution. 200 mg of the CM-HA extract was dissolved in 10 ml of methanol to create the test sample with a 20 mg/ml concentration. Then, for ten minutes, the test and standard samples were sonicated (Ultrasonic cleaner STD Rivotek, India). Before being used in the analysis, the samples were centrifuged (RM-03 Plus, REMI, India) for 15 min at 4000 rpm following sonication [16].

2.5 TLC Fingerprint and Chromatography

2.5.1 Chromatographic Conditions

The experiment was conducted at 25 ± 2 °C and 55 ± 3% relative humidity. The slit size was held constant at 5 mm × 0.2 mm, and 20 mm/s was used for the scanning speed [17]. For chromatography, luteolin and CM-HA extract were added in triplicate on a silica gel 60 F254 coated TLC aluminium plate. Depending on the plate size, either 10 ml or 20 ml of mobile phase were used for each chromatography.

2.5.2 Qualitative Fingerprint Analysis and Optimization of Mobile Phase

To achieve optimal separation with clear resolution, the marker compound and the test extract (CM-HA) were applied onto a TLC plate measuring 10 cm × 10 cm. A suitable mobile phase was employed to facilitate the separation process. The plate was subsequently examined using absorbance mode at 254 nm to visualize the distinct bands formed as a result of separation. Upon identifying the plate with satisfactory separation and resolution, it was scanned at 254 nm to generate a chromatogram.

2.5.3 Quantification of Luteolin using HPTLC Densitometric Technique

An accurate and easy approach was established for quantifying luteolin in the hydroalcoholic extract of C. maxima roots since there was no previous report of luteolin quantification in C. maxima using HPTLC. Toluene, ethyl acetate, and formic acid were used as the mobile phase in a 10:8:1 ratio during the optimization of the TLC process. Following sample application, the plates were saturated with the mobile phase (10 ml or 20 ml) for 20 min before being developed in CAMAG twin trough glass tanks. At room temperature, the plates were developed to a height of 7 cm. In order to acquire the maximum quality of the sample and reference in their natural state, derivatization was skipped after development because dark brown bands had developed on the plates. By scanning the plates at 350 nm in absorbance mode, the CAMAG TLC Scanner IV quantitatively analyzed the chemicals. The identification of luteolin was verified by superimposing the UV spectra of the samples and standards within the same Retention factor (Rf) window.

2.5.4 Calibration Curve of Luteolin

A calibration curve employing a reference luteolin and a concentration range of 400–1400 ng/band was used to ascertain the luteolin content. Standard luteolin (1 mg/ml) was produced as a stock solution in methanol. Using a semiautomatic sample spotter, various stock solution volumes (0.4, 0.6, 0.8, 1.0, 1.2, and 1.4) were spotted on TLC plates to produce concentrations of 400, 600, 800, 1000, 1200, and 1400 ng/band, respectively (band width of 8 mm, distance between tracks 11.8 mm).

2.5.5 Validation of HPTLC Method

According to ICH guidelines, the method developed for luteolin identification and quantification in the CM-HA extract was verified for repeatability, precision, and accuracy [8, 16, 18].

2.5.5.1 Precision

The precision, reproducibility, and accuracy of the developed method were assessed for the analytical method’s validation in accordance with ICH recommendations. The instrument precision was evaluated using replicate luteolin solutions (n = 6). To assess the intraday precision, replicate administrations of newly made luteolin solution at the same concentrations (400–1400 ng/band) were used at six different time points (n = 6). Intermediate precision was ascertained by analyzing repeated (n = 6) applications of luteolin solution at the same doses (400–1400 ng/band) on six different days. To indicate the repeatability of the method, the results were represented as a percentage coefficient of variation (% CV) of the peak area.

2.5.5.2 Repeatability

The reproducibility of the method was confirmed by testing 1000 ng/band of luteolin on a TLC plate (n = 10). The results were given as a percentage CV.

2.5.5.3 Limit of Detection and Limit of Quantification

Several concentrations of the luteolin standard solutions were used, together with methanol as a blank, to evaluate the limits of detection and quantification. The results were calculated using the signal to noise (S/N) ratio. Limits of quantification (LOQ) and detection (LOD) were established at S/N ratios of 10:1 and 3:1, respectively.

2.5.5.4 Selectivity

The selectivity of the procedure was determined by examining standard luteolin and CM-HA extract. By comparing the Rf value and the spectra of the band of the standard with those of the sample, it was possible to identify the band for luteolin in the sample. By examining the spectra at three different levels, namely the band’s peak start, peak middle, and peak end positions, it was possible to determine the peak purity of luteolin.

2.5.5.5 Robustness

Within the tolerance of 10%, the mobile-phase volume, mobile-phase composition, and mobile-phase saturation time were all calculated. The % CV between the data for each variable condition was computed to get the results.

2.5.5.6 Accuracy

To check the accuracy of the experiment using the conventional addition approach, the recovery experiment was conducted at three levels: 50%, 100%, and 150% addition of luteolin. By spiking, luteolin was added at a rate of about 500 ng/band. Calculations were made to determine the percentage recovery values and average percentage recovery values for luteolin.

2.5.5.7 System Suitability

The system suitability tests were conducted to confirm the experiment’s repeatability and resolution. Using the same chromatographic parameters, a freshly made standard solution of luteolin was applied at a concentration of 1000 ng/band (n = 6), and the densitograms were then obtained by scanning.

3 Results

3.1 Total Phenolic and Flavonoid Contents

The yield of the extract (CM-HA) was found to be 8.305% w/w in comparison to the raw plant material taken for the extraction process. The different plant secondary metabolites present in the extract were already reported in our previous publication [9]. The Citrus plants contains sufficient amounts of flavonoids which possess significant antimalarial activity [19]. Hence, here we determined the equivalent quantity of phenolic and flavonoid contents which were found to be 68.83 ± 1.83 mg gallic acid equivalent per gm and 40.04 ± 0.97 mg quercetin equivalent per gm of raw extract respectively. The calibration curves of the standards are given in supplementary materials (Fig. S1 and Fig. S2).

3.2 TLC Fingerprint and Chromatography

After developing the plates up to a height of 7 cm, the Rf values and colour of the resolved bands were observed. Under 254 nm in absorbance mode, the bands of luteolin were discovered to be dark brown in colour. It was discovered that the luteolin band’s Rf was 0.532 ± 0.029. The image of a TLC plate showing luteolin and CM-HA extract in absorbance mode at 254 nm is shown in Fig. 1. Luteolin’s Rf value coincided with the Rf value of the band observed in CM-HA extract, which was determined to be in the range of 0.532 ± 0.029.

Fig. 1
figure 1

Qualitative fingerprint analysis of CM-HA in presence of luteolin; (A) TLC plates observed under 254 nm in absorbance mode, (B) Chromatogram of luteolin and (C) Chromatogram of CM-HA extract

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3.3 TLC Densitometric Quantification of Luteolin Using HPTLC

The chosen solvent system provided clear peak resolution. The band at Rf value 0.532 ± 0.029 was identified as luteolin using the chromatogram and UV spectra between 190 and 450 nm (Figs. 2 and 3). The spectra of CM-HA extract’s corresponding band at wavelengths between 190 and 450 nm matched the spectra generated for luteolin, proving that luteolin was present in the sample. For the development of the TLC plates, the twin trough chamber was saturated for 20 min at room temperature. In a CAMAG visualization cabinet, the TLC plates were observed at 254 and 366 nm. As a result of the marker compound’s absorbance property, better visibility was obtained at 254 nm (Fig. 2). The spectra produced in the range of 190–450 nm in absorbance mode revealed that the maximum absorbance of the luteolin was shown at 350 nm (Fig. 3). The presence of luteolin was established by comparing the densitometric chromatogram produced from the CM-HA extract with that produced from the luteolin at 350 nm in absorbance mode (Fig. 4). The Rf value for the peak corresponding to luteolin in the CM-HA extract was the same as for the standard luteolin (Rf 0.532 ± 0.029).

Fig. 2
figure 2

Luteolin in different concentrations and CM-HA in triplicates for quantitative analysis

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Fig. 3
figure 3

Spectra obtained from the band of standard luteolin and CM-HA extract within the 190–450 nm wavelength range

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Fig. 4
figure 4

Chromatogram of luteolin and CM-HA at 350 nm in absorbance mode

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The equation for the linear regression line was y = 1.694 × 10− 7x + 1.946 × 10− 1. The standard curve’s linear regression coefficient (R) was determined to be 0.99032. The regression data showed a solid linear association for the 400–1400 ng/band concentration range. High correlation coefficient values confirmed the system’s adherence to Beer’s law and the linearity of calibration graphs. In the supplementary data, the calibration curve for standard luteolin is illustrated (Fig. S3). As indicated in Tables (1, 2 and 3), the TLC densitometric approach was validated in terms of precision, accuracy and repeatability. The percentage of luteolin recovered for the experiment was between 94.78 and 96.88%. The accuracy and repeatability of the approach were examined by measuring the peak area at six distinct concentration ranges. The results revealed low values of % CV (< 2%) for intraday (1.15–1.60%) and interday (0.37–1.51%) variation for various concentrations of luteolin (Table 3). These findings revealed that the approach had outstanding precision and reproducibility. For luteolin, the LOD value was determined to be 30 ng/band and the LOQ value to be 80 ng/band. The recovery investigations confirmed the study’s accuracy because the percentage recovery (90.74–96.47%) was discovered to be within allowable bounds. The low percentage CV (1.18%) demonstrated the robustness of the experiment and the reproducibility of the approach, demonstrating that luteolin remained stable throughout the analysis process. Based on this TLC densitometric analysis, the content of luteolin in the hydroalcoholic extract of the roots of C. maxima was found to be 7.203 ± 0.557 mg per 1 g of extract.

Table 1 Method development parameters for the quantification of luteolin in the CM-HA extract of Citrus maxima (Burm.) Merr
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Table 2 Recovery studies of luteolin at 50%, 100% and 150% spiking by TLC Densitometric Method
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Table 3 Results of Interday and Intraday Precision of the Developed HPTLC Method (n = 6)
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4 Discussion

The citrus plants are very much rich in phenolic and flavonoid contents specially the fruits. In this study we found the presence of both these two categories of compounds also in the root of C. maxima in significant quantities. Then we found the presence of luteolin in the qualitative TLC analysis of the extract. Luteolin is a moderately active antimalarial compound found in different plants. Luteolin is present in the fruit part of Citrus plants but in this study, we identified the presence of this compound in the root part of C. maxima plant [20]. Hence to quantify this compound in the hydroalcoholic extract of the root part, a simple and cost effective method was developed. The devised HPTLC method for luteolin determination was found to be exact, accurate, and selective. The optimized mobile phase gave good separation and resolution for luteolin in both the standard and the test extract. Fig. S4 displays the resolution of the separated luteolin peak in the test sample and the peak of the reference luteolin. The chromatogram of the hydroalcoholic extract with this mobile phase developed through densitometric HPTLC analysis can be employed as a standardized fingerprint for the quality assessment of the plant material. High correlation coefficient value (R = 0.99032) of the six-point calibration curve obtained from various concentrations indicated that the devised method had strong linearity and peak uniformity. The LOD and LOQ values showed that the new approach produced low noise levels. Consequently, within the specified experimental circumstances, this study establishes a novel, validated approach for quantification of luteolin and demonstrates that it can produce accurate results.

5 Conclusion

The root of the plant C. maxima is found to be rich in phenolic and flavonoid contents and we identified and quantified the presence of an antimalarial flavonoid, luteolin in the extract. The investigation showed that the suggested approach is selective for quantifying luteolin in herbal medication formulations and reproducible. The amount of luteolin determined by the verified approach is substantial for isolation as a pure chemical. This technique can be utilized proactively for quality control of raw pharmaceuticals containing luteolin in process and completed goods because it separates luteolin from other elements with good band resolution.