UHPLC assessment of embelin in specialized mangrove plant Aegiceras corniculatum (L) Blanco

Abstract

Background

Embelin is one of the biologically charming natural benzoquinones with wide medicament use in therapeutics that is pertained to its unique biochemical structure. Mostly, the plants belonging to Myrsinaceae family especially the fruits of genus Embelia are rich source of embelin. However, the colossal use of these plants as an active ingredient in several drug formulations has led toward their decrease population number increasing the threat status. This major issue requires a well-versed scientific approach to search for suitable alternative substitute that can be used as an active embelin source. On this panorama, Aegiceras corniculatum (L.) Blanco can be used as alternate source of embelin. On this aspect, all the plant parts (root, stem bark, leaf and fruit) are being estimated for embelin content both qualitatively and quantitatively using spectrophotometric and ultra-high-performance liquid chromatographic (UHPLC) methods and also asserted through histochemical analysis. All the aforesaid plant parts are extracted through Soxhlet and water bath methods separately and analyzed for embelin content after isolation through column chromatography and thin layer chromatography.

Results

From results, it was evaluated that all plant parts showed the presence of embelin in a range of 0.17–1.95% dry wt. through UHPLC method, while the highest content was found in fruit followed by root, bark and leaves. The retention time for embelin was found to be 2.7 min.

Conclusions

From the above experimentation, both vegetative and reproductive parts of A. corniculatum have shown the presence of embelin that can be utilized in ample amount so as to reduce the threat status of its primary source.

Background

Aegiceras corniculatum (L) Blanco, commonly known as River/black Mangrove, is a specialized Mangrove plant in the Myrsinaceae family category. Mangroves are intertidal productive forested wetlands, encumbered to the specific tropical and subtropical estuarine zones (Kumar 2000) and constitute a vital component of marine flora having significant ecological and socioeconomic value. A. corniculatum is a small evergreen mangrove tree commonly found along the eastern and western coasts of India (Clarke 1995). It is widely reported to contain several secondary metabolites, namely benzoquinones, tannins, coumarins, flavonoids, polyphenols, saponins, triterpenes, etc. (Bandaranayake 2002), with potent pharmacological activities, viz. antifungal, piscicidal activity (Aseer et al. 2009), anti-inflammatory, antioxidant (Banerjee et al. 2008; Roome et al. 2008), hepato-protective (Roome et al. 2011), antidiabetic (Gurudeeban et al. 2012) and anticancer (Rahman and Khan 2013). It is being scatteredly reported to have embelin (Bandaranayake 2002; Alongi 2009; Rahman and Khan 2013; Thota et al. 2016; Swami et al. 2017), yet no validated study has till now been carried out to explore embelin quantity from this less known but special group of plant. Though structural elucidation through crystallography study was seen (Thota et al. 2016), no report on the UHPLC study is grounded from each and every plant part. Moreover, several studies have been carried out for determination of many unknown compounds through UHPLC-based technique utilization (McCalley 2012) even used as biomarkers for disease (Denoroya et al. 2013). It is an efficient method for rapid identification of compounds. Hence, the identification of embelin through UHPLC method would validate its content in all the plant parts of A. corniculatum. Due to the lack of concrete evidence regarding the presence of embelin in this special group of plants, the present piece of work has been taken forward to validate the presence of embelin in each and every A. corniculatum plant parts and to quantity its quantity.

Methods

Plant materials

Aegiceras corniculatum (L) Blanco (Myrsinaceae) different plant parts (minimum number of preferential specimens) were collected during the month of July from Bhitarkanika mangrove forest areas (procuring permission and approval of local and government administration), Odisha coast, India (20°18′–20°32′N and 86°41′–86°48′E). The species is identified by one of the authors Dr. Uday C. Basak, from the Regional plant Resource Center, India. The species was deposited in the herbarium of Regional plant Resource Center, India, for the purpose of verification (bearing voucher specimen no 4618) and also verified through the reference book “The Flora of Odisha” (Saxena and Brahmam 1995).

Standard preparation

For comparison and validation with the unknown extracted plant samples, standard embelin solution was prepared taking synthetic embelin (Sigma-Aldrich, Germany) in HPLC Grade methanol (1 mg/ml) (Spectrochem, Bangalore, India) and kept at 4 °C temperature for further use. The purity of the synthetic embelin was verified by measuring its λmax and HPLC chromatogram.

Pre-treatment and preparation of sample extract

All shed dried samples were pulverized, and 10 g of each was extracted through Soxhlet apparatus using methanol and chloroform as solvent system (Spectrochem, Bangalore, India) termed as Extraction-1 (Rastogi et al. 2014). Similarly, 5 g of each was extracted through water bath using same methanol and chloroform as solvent system (Spectrochem), termed as Extraction-2 (Ganesan et al. 2010). The extracted samples were crystallized using ice-cold absolute ethanol (Spectrochem, Bangalore, India) (Radhakrishnan et al. 2011; Gupta et al. 2012) for further use.

Embelin isolation (column chromatography method)

For isolation of embelin, the crude extracts were subjected to column chromatography procedure using petroleum ether/benzene as column solvent with a ratio of 2:3 and silica gel (60–120 mesh) as packing material (Gupta et al. 2013). The chromatographically isolated compound was re-crystallized as shiny orange compound with ethanol, and its purity was confirmed through UHPLC method (Rastogi et al. 2014).

Embelin identification and isolation (TLC method)

After column chromatography, eluted samples were further isolated using thin layer chromatography (TLC) method. For the isolation process, TLC was performed on TLC plates (Kukkar et al. 2010). Various mixtures of solvent systems were used to develop the optimal suitable mobile phase for isolation of embelin. Among all, the mobile phase selected for TLC process was a mixture of n-propanol/n-butanol/ammonia (SRL, India) in a ratio of 7:1:2. For viewing the plates, 1% vanillin sulfuric acid (SRL, India) solution was used as chromatogenic reagent. After TLC, the retention factor (Rf) value of the test samples was determined against the standard embelin solution (Vandana and Arora 2010).

Embelin estimation (spectrophotometric method)

Isolated embelin compound from each extract was dissolved in methanol and chloroform; the solution was scanned within the range of 200–800 nm on UV–visible spectrophotometer in a 1-cm quartz cell get the maximum wavelength at 298 nm. Now known amount of sample extracts were measured through UV spectrophotometer at 298 nm wavelength for embelin content (Sudani and Vidyasagar 2012; Belete et al. 2014).

Embelin estimation (UHPLC method)

UHPLC instrumentation

The analysis was done in UHPLC system (Elizabeth and Jashna 2021), Dionex Summit (Dionex Corp., Sunnyvale, CA, USA) equipped with a P680 quaternary low-pressure gradient pump unit, TCC-100 Thermostatted column compartment and DAD UV 340U UV detector. C18 column (4.6 × 150 mm column, 5 μm diameter pore size) was used for the separation process. The instrument was loaded with Chromeleon Chromatography Management System (version 6.7) for data collection and acquisition. Processing was done using HPLC Syringe of 25 µL capacity (Model-Hamilton 1702 RNR, Sigma-Aldrich, Germany).

Mobile phase optimization

Mobile phase optimization was done based on the level of resolution of the analyte that was separated along with the peak properties and applicability of the method involved. For the assessment, methanol (Spectrochem) along with 0.1% TFA (SRL, India) in water with a ratio 65:35 was selected as a suitable solvent system for the separation process (Rastogi et al. 2014).

Chromatographic conditions

Chromatographic separation was performed on a Dionex make UHPLC system (Rastogi et al. 2014). The detailed chromatographic conditions are given in Table 1.

Table 1 Chromatographic condition for assessment of embelin

Method validation

The purposed UHPLC method was validated by defining linearity, peak purity, correlation coefficient, limit of quantification, limit of detection, relative standard deviation, accuracy, peak purity, recovery, sensitivity and precision.

Reproducibility and precision

The reproducibility and precision of embelin were verified by carrying out replicate measurements of the same samples. Ten replicate injections of standard and three replicate injections of extracted samples were used for this purpose. For determining the intra-day accuracy and precision, five replicates of each sample were analyzed thrice on the same day. The inter-day accuracy and precision were assessed by analysis of five replicates of samples on three different days.

Linearity

The calibration curve was developed by the least-square regression for peak area and concentration of the analyte. A linear calibration curve was developed for the concentration range of 0–1000 µg/ml. The relative standard deviation (% RSD) values did not exceed 0.01 for any of the concentrations. After linear regression analysis, the slope (± SD of mean) for the calibration curve of embelin was found to be 15,691 (± 0.17) with a regression coefficient (r2) value of 0.997 (Table 2).

Table 2 Statistical validation of UHPLC method

Limit of detection and limit of quantitation

Limit of quantification and limit of detection were determined from the standard deviation and slope of the calibration curve. Limit of detection (LOD) is the lowest concentration of analyte that can be detected in an injected sample extract under optimized conditions. The limit of quantification (LOQ) is the lowest concentration of the sample that can be quantified with accuracy and precision. These were determined based on the signal-to-noise ratio as per International Council on Harmonisation (ICH) guidelines.

Accuracy and extraction recovery

To check the accuracy of the developed method, a recovery experiment was carried out by standard addition method. A known amount of sample was taken. To each tube known amount of embelin was added. Each sample was analyzed by the developed UHPLC method, and the amount of embelin recovered for each level was calculated. Percent recovery of embelin from all the plant parts of A. corniculatum was 97.89%. Low % RSD (relative standard deviation) values established extraction efficiency robustness of the selected method (Table 3).

Table 3 Accuracy and extraction recovery studies for embelin analysis

Histochemical localization

As the major non-harmful plant part is leaf, the presence of deposition of embelin in the leaf parts is a matter of search and this was achieved by the ammonia solution method (Belete et al. 2014). The deposition of embelin in the mesophyll cells is observed by taking the A. corniculatum matured leaf TS sections. Microphotographs of sections were analyzed using Nikon Eclipse Microscope (Japan, Model 50i) equipped with Nikon Y-IDT. Images were concomitantly viewed and analyzed for pharmacognostic characteristics, and quantitative measurements were taken using Nikon Digital Sight DSL Camera (Model No.-215,492).

Statistical analysis

All the results were validated by using two-way RM ANOVA (repetitive measures) using GraphPad Prism software (Version 6.05) at 99.9% significant level. All the data were expressed as mean ± SD.

Results

Crude Yield

The crude yield of embelin in case of A. corniculatum was found to be in a range of 0.12–2.74% dry wt. (Table 4). The fruit part showed the highest yield of crude embelin followed by the root parts followed by stem bark and then finally by the leaf parts. When the solvent systems were considered, the methanol solvent system was prevailed to be eminent to that of chloroform solvent system. All data were analyzed statistically at 99.9% confidence interval of difference through one-way RM ANOVA along with Holm–Sidak's multiple comparisons test. In the multiple comparison analysis, the row factors, i.e., the plant parts extracted through different processes with various solvent systems, were found to be significant at P value = 0.0243 and column factors, i.e., the crude yield of embelin, were found to be significant at P value = 0.5260.

Table 4 Crude extract yield (% dry wt.) in A. corniculatum

Embelin content (% dry wt.) through spectrophotometric method

In the case of A. corniculatum samples, embelin content in the crude extracts was found to be in a range of 0.23–1.95% dry wt. Among all the selected plant parts, crude embelin content was found to be highest in fruit (1.95% dry wt.), when extracted with Soxhlet using chloroform solvent, while in the case of leaves the least embelin content was found in water bath using methanol solvent extracts (0.23% dry wt.) (Table 5). All data were analyzed statistically at 99.9% confidence interval of difference through two-way RM ANOVA along with Sidak's multiple comparisons test. In the multiple comparison analysis, the row factors, i.e., the plant parts extracted through different processes with various solvent systems, were found to be significant at P value < 0.0001 and column factors, i.e., the crude and purified isolates, were found to be significant at P value = 0.0030.

Table 5 Embelin content (% dry wt.) in extracts and isolates of A. corniculatum through spectrophotometric method

Identification and isolation of pure Embelin

The Rf value of the synthetic embelin standard was 0.35. However, the crude extracted samples during the identification process for the presence of embelin showed Rf in a range of 0.345–0.356 in case of A. corniculatum. Fruit samples, extracted with Soxhlet—chloroform, showed highest Rf value (0.356) and leaf samples, extracted with water bath—methanol, showed least Rf value (0.345). The isolated compound from samples of A. corniculatum showed Rf in a range of 0.342–0357. Fruit samples, extracted with Soxhlet—chloroform, showed highest Rf value (0.357) and leaf samples, extracted with water bath—methanol, showed least Rf value (0.342) (Table 6).

Table 6 Rf values of crude and purified isolates of A. corniculatum

TLC sheets confirming the presence of embelin in the crude extracts and the isolates are given in Fig. 1. All data were analyzed statistically for 99.9% confidence interval through two-way RM ANOVA along with Sidak's multiple comparisons test. In the multiple comparison analysis, both the row factors, i.e., the plant parts extracted through different processes with various solvent systems, and column factors, i.e., the isolates, were found to be significant at P value < 0.0001. The difference in Rf for all the isolates was due to the different types of solvents used along with the time.

Fig. 1

TLC sheets with sample extracts from different plant parts of A. corniculatum

Embelin content (% dry wt.) through UHPLC method

In the case of A. corniculatum samples, embelin content in the crude extracts was found to be in a range of 0.17–1.64% dry wt. Among all the plant parts, embelin content was found to be highest in fruit (1.64% dry wt.), when extracted with Soxhlet method using chloroform as solvent, while in the case of leaves the least embelin content was found in water bath method using methanol solvent extracts (0.17% dry wt.). Embelin content in isolates of A. corniculatum was found to be in a range of 0.028–1.60% dry wt. The highest embelin content was found in fruit (1.60% dry wt.) extracted with chloroform solvent, while in the case of leaves, the least embelin content was found in methanol extracts from water bath method (0.028% dry wt.) (Table 7). The chromatogram peak value of eluted samples is shown in Fig. 2. All data were analyzed statistically at 99.9% confidence interval (CI) of difference through two-way RM ANOVA along with Sidak's multiple comparisons test. In the multiple comparison analysis, the row factors, i.e., the plant parts extracted through different processes with various solvent systems, were found to be significant at P value < 0.0001 and column factors, i.e., crude and purified isolates, were found to be significant at P value = 0.0004.

Table 7 Embelin content (% dry wt.) in extracts and isolates of A. corniculatum through UHPLC method

Fig. 2

Embelin chromatograms of different samples of A. corniculatum

However, the embelin content available in the crude extracts and purified samples are correlated with their corresponding Rf values for embelin and the correlation between its content and Rf value is depicted in Fig. 3.

Fig. 3

Comparative graph between embelin content and its Rf value

Microscopic identification of Embelin

The microscopic figures have revealed the presence of embelin in specialized vacuoles mainly below the epidermal layer (both upper and lower) and in mesophyll cells. After ammonia treatment, the deposition of embelin in specialized vacuoles was visible as brownish red-colored patches (Fig. 4). The deposition of embelin in case of mesophyll cells was found to be more prominent as compared to the deposition in the epidermal layer.

Fig. 4

TS of leaf samples of A. corniculatum showing localization of embelin in a mesophyll cells, b below the epidermal layer (arrowhead), c presence of two-embelin deposition near epidermal layer (arrowhead) (All three in 40× Magnification), d presence of two-embelin deposition (arrowhead) (10× Magnification)

Discussion

Embelin is one of the biologically active benzoquinone derivatives that act as the active principle compound in the fruits of Embelia ribes, a well-known yet very rare and endangered medicinal plant of the Myrsinaceae family (Ferreria and Laddha 2013). Due to the rare, endangered and threatened (RET) status, heavy consumption of fruits, for embelin extraction, led to a disturbance in the production of new plants through the seed germination process. To mitigate the difficulty in regulating and elevating their minuscule population and to meet the market demand of embelin, possible and suitable alternative substitutes are considered for extraction of embelin. To this aspect, allied plants in the same Myrsinaceae family, viz. E. tsjeriam-cottam, Ardisia solanacea, A. japonica, Aegiceras corniculatum and many more, can enlighten the path (Bandaranayake 2002; Podolak and Strzałka 2008; Alongi 2009; Stasiuk and Kozubek 2011; Poojari 2011; Rahman and Khan 2013). However, A. corniculatum act as an interesting substitute, available in Mangrove category that can act as a prominent embelin source and has been scatteredly reported to contain embelin (Bandaranayake 2002; Alongi 2009). The main intention of this research work is to find out suitable alternative substitutes besides the traditionally used E. ribes as a source of embelin to minify the pressure on the later plant, which will lead to its least exploitation. A. corniculatum is the least concerned mangrove flora that could be utilized as an alternative substitute for the yield of embelin.

The crude extracted embelin was yielded in crystallized manner to get orange yellowish needle-like structures. For the yield of crude extracts of embelin, methanol and chloroform solvent systems were used separately. The structural complexity of embelin with the presence of long alkyl chain leads to its solubility in both the solvent systems (Rahman and Khan 2013). In our study, the crude embelin yield was found to be in a range of 0.12–2.74% dry wt., which corroborated to other findings with embelin yield in a range of 0.23–0.28% dry wt. (Swami et al. 2017). Though structural elucidation of embelin in A. corniculatum had been described previously (Thota et al. 2016) along with its quantification in various plant parts (Swami et al. 2017), its detailed quantitative analysis yet has not been done. In the study of Thota et al. (2016), Aegiceras corniculatum leaf and stem parts were taken from Godavari estuary, Andhra Pradesh, and were extracted through subsequent extraction system taking chloroform/methanol (1:1) followed by ethyl acetate. All the column chromatographed and TLC eluted samples were identified through ¹H and ¹³C NMR and mass spectral study. However, in our study all of the Aegiceras corniculatum samples (fruit, leaf, stem bark and root) were collected from Bhitarkanika Mangrove forest, Odisha, followed by the sample extraction through Soxhlet and water bath method taking both methanol and chloroform solvents. All column chromatographed followed by TLC eluted samples is evaluated for embelin content through spectrophotometric and UHPLC method of analysis. The major novelty in the current study in response to the study carried out by Thota et al. 2016 is the estimation of embelin content in all the vegetative and reproductive plant parts of Aegiceras corniculatum (fruit, leaf, stem bark and root) samples. The prime focus in this study was being given only to isolate and estimate embelin content from various plant parts. In the study of Thota et al. (2016), only compound identification was done where as in our study both identification and quantification of each plant part were done for embelin. This is the first attempt made for the estimation of embelin in all the plant parts of A. corniculatum, collected from Odisha, through both spectrophotometric and UHPLC method of analysis. For isolation and separation of compounds, mobile phase optimization was done based on the solubility and polarity index of the desired compounds along with the mobile phase and compound’s affinity with the stationary phase. As all the extracts are being isolated using different solvents, this would definitely have a direct impact during desires compounds separation process (Bele and Khale 2011). Similarly, all the TLC plate’s varying stationary phase thickness and unequal time interval used for TLC plate development also affect the change in Rf values of the result (Elizabeth and Jashna 2021). Several studies have reported embelin content in most of the Embelia tsjeriam-cottam plant to be in range of 0.3–0.35 (Vandana and Arora 2010). Even in case of A. corniculatum, similar kind of Rf value was found that validated our study, of getting Rf values in a range of 0.345–0.356 (Thota et al. 2016). Embelin is widely used in several Ayurvedic and Pharmaceutical industries, which increases the extensive use of the fruit parts, thereby increasing its threat status. Hence, several efforts have been made to localize this bioactive benzoquinone in different plant parts of particularly E. ribes (Nakve et al. 2011; Ferreria and Laddha 2013; Sudhakaran 2015), thereby reducing the difficulties of the extraction procedure. In all their studies, they have localized the presence of embelin in seed and pericarp portion of the fruits. As leaf samples prevailed to contain embelin within it and are the non-harmful source of the concerned plants in terms of yield of embelin, the histochemical localization of embelin within the leaf samples was done. However, anatomical study regarding the localization of embelin in A. corniculatum has yet not been studied so far.

Conclusions

Hence, it could be opined that all plant parts of A. corniculatum do contain embelin and it can act as an alternate substitute source of embelin besides E. ribes and E. tsjeriam-cottam. However, as leaf parts though contain 1/10^th of embelin compared to the fruit parts, it can be used as a suitable alternate nondestructive source of embelin. Leaves are abundantly available, and using leaves for medicinal formulations will also not be a threat to the concerned plant species. Thus, the outcome of this research work will facilitate conservation, domestication and sustainable utilization of potential wild medicinal plant resources.