HPLC study of phenolic compounds in Mirabilis jalapa raw material

Mirabilis jalapa is a popular decorative plant valued for its beautiful multicolored flowers. Folk medicine in various countries applies Mirabilis jalapa as anti-microbial, anti-inflammatory, diuretic, spasmolytic drug. Chemical composition of different types of Mirabilis jalapa has not yet been adequately studied which is an obstacle for its application in medicine. The qualitative composition and quantitative content of phenolic compounds were studied by the HPLC method. The performed experiment revealed presence of hydroxycinnamic acids, flavonoids, isoflavonoids and coumarins in tested herb. The content of phenolic compounds was the highest in Mirabilis jalapa flowers, counting as much as 2977.41 ± 59.55 μg/mg. Total content of phenolic compounds in Mirabilis jalapa herb was 304.25 ± 6.08 μg/mg, in fruits – 67.92 ± 1.36 μg/mg, and in roots – 12.44 ± 0.25 μg/mg. Quantitatively neochlorogenic acid dominated in flowers, chlorogenic acid in fruits, whereas Mirabilis jalapa herb mostly contained rutin and hyperoside. The obtained results will be useful in the development of quality control methods for Mirabilis jalapa herb and manufacture of drug preparations on its basis.


Introduction
Mirabilis jalapa Linn. (clavillia or four-o' clocks) belongs to Nyctaginaceae family. This decorative plant is cultivated all over the world. Mirabilis jalapa flowers bear pink, yellow, red, orange, white colors or several colors simultaneously (Akanji et al. 2016).
Mirabilis jalapa is also known for its healing properties. Folk medicine in various countries applies it to treat dysentery, diarrhea, inflammations, muscular pain, diabetes, urogenital disorders (Ramesh and Mahalakshmi 2014).
Nigerian scientists established anti-malaria activity of Mirabilis jalapa leaves extract (Akanji et al. 2016), whereas scientists from Mexico found spasmolytic effect of Mirabilis jalapa flowers extract (Aoki et al. 2008).
The object of the present work was the study of phenolic compounds in Mirabilis jalapa herb, flowers, fruits and roots.

Sample preparation
Plant. The powdered materials of plant (0.3 g) were weighed into a volumetric flask and extracted with methanol (10 mL) in an ultrasonic bath at room temperature (20 ± 2 °C) for 20 min. The solutions were filtered through a membrane filter (0.45 μm) prior to use.
The identification of the chromatographic peaks was achieved by comparing the retention times and spectral characteristics (λ = 200-600 nm)of the eluting peaks with those of the reference compounds (Table 1). The compounds identified were confirmed by spiking the sample with the standard compound and monitoring the changes in the peak shape and spectral characteristics. For quantitative analysis, a calibration curve was obtained by the injection of known concentrations of different standard compounds. The concentrations of phenolic compounds identified in the extracts were within the limits of the calibration curves.
The following validation characteristics of method were evaluated as specificity, reproducibility of results (accuracy), limits of detection and quantitative evaluation, linearity (Tables 2, 3).
The method specificity was confirmed by coincidence between peak retention time of identified phenolic compounds and the corresponding peak retention time in standard samples and divisibility of substance peaks in chromatogram.
The limit of detection (LOD) may be expressed as: DL = 3.3 *σ/S, where σ = the standard deviation of the response, S = the slope of the calibration curve. The limit of quantification (LOQ) may be expressed as DL = 3.3 *σ/S, where σ = the standard deviation of the response, S = the slope of the calibration curve. The determined LOQ was within the range from 14.00 ng/mL to 420.00 ng/mL. The obtained results confirmed method eligibility for quantitative evaluation of phenolic compounds.
The correlation coefficient (R 2 ) was always above 0,999, which confirmed the linearity of quantitative evaluation method.
The total content of phenolic compounds was calculated by summing the identified components.

Results and discussion
The results of experiment enabled the identification of hydroxycinnamic acids, flavonoids, isoflavonoids and coumarins in tested Mirabilis jalapa herb.
The HPLC chromatogram of Mirabilis jalapa flowers is specified in Fig. 1, of roots in Fig. 2, of fruits in Fig. 3 and herb in Fig. 4.
The quantitative content of phenolic compounds as determined by the HPLC method in Mirabilis jalapa raw material is presented in Table 4.
The highest content of phenolic compounds was found in Mirabilis jalapa flowers, as much as 2977.41 µg/mg. Among hydroxycinnamic acids the most abundant was neochlorogenic acid (2060.10 µg/mg). The amount of flavonoids in flowers was 749.80 µg/mg, mostly avicularin (322.30 µg/mg), hyperoside (292.67 µg/mg) and rutin (131.63 µg/mg). Coumarin and ononin content in Table 1. Retention time, detected at wavelength and UV spectrum of identified phenolic compounds.      In Mirabilis jalapa fruits chlorogenic acid dominated (51.73 µg/mg). The total content of identified phenolic compounds in fruits was 67.92 µg/mg.
The lowest content of phenolic compounds among tested parts of herb was in roots -12.44 µg/mg. The amounts of ferulic and caffeic acids in these was almost identical (4.57 µg/mg and 4.40 µg/mg respectively).

Conclusion
Our experiment enabled the identification of flavonoids, isoflavonoids, hydroxycinnamic acids and coumarins in Mirabilis jalapa raw material. The neochlorogenic acid (2060.10 µg/mg) dominated in Mirabilis jalapa flowers. The rutin (110.33 µg/mg) and hyperoside (93.20 µg/mg) prevailed in herb. The chlorogenic acid content in Mirabilis jalapa fruits was 51.73 µg/mg, much more than all other identified phenolic compounds in this part of herb. The least of all phenolic compounds were found in roots.
The obtained results will be useful in the development of quality control methods for Mirabilis jalapa herb and manufacture of drug preparations on its basis.