Isolation and Identification of Bioactive Compounds from Leaf and Flower Extracts of Firmiana colorata

Introduction

The use of plants or plant parts to combat illnesses in humans is as ancient as mankind.¹ These practices are considered as ‘Complementary Medicine’ by the World Health Organization (WHO) and are usually affordable, available and accessible.² Earlier, 80% of the drugs were derived from plants, but later in modern times, synthetic drugs became more popular. However, significant and dangerous resistance to synthetic drugs has once again created a demand for exploring novel compounds from plant sources.³ Providing scientific understanding and authentication to this traditional medicine is the need of the hour and of utmost importance as there is a dependency on plant-based drugs even today. The medicinal significance of a plant is often indicated by the presence of a variety of bioactive compounds.

Bioactive compounds have demonstrated several medicinal properties, in both in vitro and in vivo studies.⁴ Most notable class of bioactives in plants belong to the family of phenolic compounds. Other important phytoconstituents include alkaloids, flavonoids, tannins, saponins and glycosides. Isolation and structure elucidation of these compounds from plant extract are important in determining their activity, safety, and efficacy. This also enables the development of suitable methods to obtain these plant bioactive compounds on a large scale.⁵ Many plant compounds have been explored for their bioactivity and are currently trending in pharmaceutical industry. Nevertheless, search is on for more compounds to be used as medicinal or therapeutic agents.

One such plant to be explored is Firmiana colorata (F. colorata), a deciduous tree native to the rich flora of Western Ghats of India. Different parts of F. colorata were found to be used in traditional medicine to treat various ailments and diseases like jaundice, fever, cholera and intestinal dysfunction.^6-8 F. colorata also has other significant uses; cordage made from its bark is used for bullocks in festivals (Pola), leaves serve as fodder for animals and flowers are used for ornamental purposes.⁹

Our previous studies on F. colorata focused on bridging the gap between its phytochemical makeup and pharmacological importance. Our studies on F. colorata revealed the presence of a variety of phytochemicals, minerals, with potent antimicrobial and anti-inflammatory activities.

Further comprehensive work on its unexplored bioactive compounds is needed to authenticate the efficacy of this plant. Keeping in view the medicinal properties of F. colorata, current study aimed at isolating and characterizing the bioactive compounds from extracts of F. colorata using chromatographic and spectroscopic techniques.

Materials and Methods 

Preparation of plant extracts

Leaves and flowers of F. colorata were collected from Indian Institute of Science, Bengaluru, India. The plant was authenticated by Central Ayurveda Research Institute, Ministry of Ayush, Bengaluru, India, bearing voucher specimen number SMPU/CARI/ BNG/202021/1052. Collected samples were cleaned, shade-dried at room temperature and ground into coarse powder. Leaf and flower extracts (hereafter referred to as LE and FE, respectively) of F. colorata were obtained by hot percolation extraction method through Soxhlet apparatus using ethyl acetate and ethanol as successive solvents. A total of four extracts viz., ethyl acetate-LE, ethanol-LE, ethyl acetate-FE and ethanol-FE were obtained. Ethyl acetate and ethanol leaf extracts were pooled and so were the ethyl acetate and ethanol extracts of flowers. Excess solvent from the extracts was removed using a rotary evaporator under vacuum and the dried extracts were subjected to silica gel column chromatography.¹⁰

Column and thin layer chromatography (TLC)

• a. Isolation of compounds: Customized borosil column of 75 mm diameter X 400 mm height were used for isolation of compounds. Two separate columns viz., Column 1 and Column 2 were packed with silica gel and extracts were loaded onto it. Column 1 was loaded with LE while Column 2 was loaded with FE. The extracts were eluted using mobile phase n-hexane: ethyl acetate: methanol (100~0:100~0). The elution rate was maintained at 2 mL/min with a total elution volume of 800 mL from each column. Each elute from Column 1 and Column 2 was spotted onto silica gel F254 plates (25×25 cm; Merck, Germany), developed in chloroform and methanol (85:15 v/v) solvent system, dried, dipped in 3% phosphomolybdic acid and the resulting spots were visualized under UV light. Elutes were precisely monitored and similar fractions were pooled based on TLC profiles. Two major fractions were obtained - one fraction from Column 1 (hereafter referred to as Compound 1) with n-hexane:ethyl acetate (40:60) and the other fraction from Column 2 (hereafter referred to as Compound 2) with ethyl acetate:methanol (85:15). Compound 1 and 2 were submitted to purification.^11,12
• b. Purification of compounds: Two Sephadex LH20 columns viz., Column 3 packed with Compound 1 and Column 4 packed with Compound 2, were engaged in purification of isolated compounds using ethyl acetate: methanol as solvent system. Compound 1 was purified with ethyl acetate: methanol (55:45) through Column 3. Whereas, Compound 2 was purified with ethyl acetate: methanol (90:10) through Column 4. Elutes containing the pure compounds were collected and the solvent was excluded using a rotary evaporator. Subsequently, these two pure compounds were characterized using various analytical techniques viz., Mass spectrometry, Nuclear magnetic resonanace (NMR)-1H & 13C and Fourier Transform Infrared Spectroscopy (FTIR) and identified via literature survey.¹³

Spectroscopic analysis

FT-IR spectroscopy: FT-IR technique was employed to study the functional groups of the isolated pure compounds viz., Compound 1 and Compound 2. A high throughput screening tensor II (Bruker, Germany) with a globular mid-infrared (IR) source and a Deuterated L-Alanine Doped Triglycine Sulfate (DLATGS) detector was utilized for FTIR studies. Background spectra were acquired before obtaining spectra for each compound. Spectra of pure compounds were obtained in transmittance mode under 4000 to 400 cm-1 spectral band. All spectra were adjusted in the environment using multiplicative signal correction (MSC) with OPUS software version 7.5.

Mass spectrometry: The molecular mass analysis of Compound 1 and Compound 2 was done using Acquity system, equipped with an electron spray ionization source, BEH C18 column (50 x 1.0, 1.7μ) and connected to a Xevo G2-XS QTOF mass detector with capillary voltage: 3.0 KV, collision energy: 20 V, ramp collision energy: 30-90 V, source temp: 150ºC, desolvation temp: 450 ºC, cone gas: 50 L/hr, desolvation gas flow: 800 L/hr. Mobile phase-A (0.1% formic acid) and mobile phase-B (acetonitrile) were used in a gradient system with 5 μL sample injection volume. The gradient program started with 98% mobile phase-A and 2% mobile phase-B from 0 to 1 min, followed by 1:1 mobile phase-A and mobile phase-B from 2 to 6 mins. Later 7 to 16 minutes of run time involved 5% mobile phase-A and 95% mobile phase-B gradient. A 17 to 20 mins run time employed 2% mobile phase-A and 98% mobile phase-B gradient. The flow rate was maintained at 0.2 mL/min throughout the run. The column was maintained at 25±2°C laboratory condition set up. The positive ion mode with the spray voltage of 3.5 kV at the source temperature of 80°C was set to obtain the spectra and the mass spectra were recorded under electron impact ionization at the energy of 70 eV. The recorded mass spectra of Compound 1 and Compound 2 were within the range of 100-500 m/z.

NMR spectroscopy

Sample preparation for analysis: Compound 1 was dissolved in CDCl3 and Compound 2 was dissolved in DMSO-d6 solvent. The resultant solutions were vortexed, sonicated and submitted to NMR spectroscopy.

NMR instrumentation and data acquisition: 1H NMR spectra were recorded on a Bruker ADVANCEII 500MHz (11.7 T) spectrometer operating at proton frequency 500.18 MHz using a 5 mm double resonance broad band observe probe head. An acquisition time of 2.5 seconds and a relaxation delay of 12 seconds were used. The sample temperature was set at 298±2K for all the measurements. 1D-1H NMR experiments were carried out with a zg pulse program from the standard Bruker library. The 90° pulse was optimized and found to be p1: 11.7 µs. 13C-NMR spectra were recorded on 400 Mhz JEOL [Delta V5.3.2] instrument using carbon. jxp experiment with 1065 scans, a relaxation delay of 2 sec and an acquisition time of 0.5 seconds.

Qualitative tests: Molisch’s test and Iodine test were performed for both, Compound 1 and 2 after spectral analysis. A distinctive test, which was performed only for Compound 2 anticipating the presence of phenolic hydroxyl group from spectral data. The reaction involved Compound 2 with 1% vanillin and concentrated sulfuric acid and the resultant solution sprayed with FeCl₃.

Results

The present study aimed at isolating the possible bioactive compounds in the leaf and flower extracts of F. colorata. For this purpose, various combinations of solvents including n-hexane, petroleum ether, ethyl acetate, and methanol were worked out. However, combinations of n-hexane:ethyl acetate (40:60) and ethyl acetate:methanol (85:15) were found to be optimum for the isolation of pure compounds from the extracts of F. colorata.

Two pure compounds, one from LE (Compound 1) and the other from FE (Compound 2) were successfully isolated through column chromatography. FTIR analysis discerned the functional group characteristics of both the isolated compounds and their mass was revealed by MS data. The FTIR, NMR, and MS spectral data are provided below deciphering the identity of Compound 1 and Compound 2.

The data of Compound 1 isolated from LE is as follows:

1. Physical properties: White amorphous powder, melting point of 70-71.5o C, soluble in methanol.

2. Qualitative tests: Molisch’s test - negative and Iodine test - positive.

3. Spectral data:

a. FTIR spectra (Figure 1a): Features indicative of a saturated fatty acid, with an alkyl C-H stretch spanning from 2950–2850 cm^-1 and a broad carboxylic acid with an O-H stretch spanning from 3000–2500 cm^-1. Additionally, a broad peak of alkane C-H bonds were seen.

b. ESI-MS spectra (Figure 1b): Quasimolecular signal at m/z: 283.33 [M-H]-

c. 1H-NMR spectra (Figure 2a): δ 2.35 (2H, t, J=7.5Hz), δ 1.63 (2H, m), δ 1.25- δ 1.30 (28H, m), δ 0.84 (3H, t, J=7.0Hz).

d. 13C-NMR spectra (Figure 2b): δ C1 180.2, δ C2 34, δ C3 31.9, δ C4 –C16 (29.6-30), δ C17 22.6, δ C18 14.1.

4. From the above spectral data, the chemical shift assignments for Compound 1 in leaf extract were obtained (Figure 3a & 3b).

Compound 1 from leaf extract was identified as stearic acid after comparing the above spectral data with the data available in the literature.^14-16

The data of Compound 2 isolated from FE is as follows:

1. Physical properties: White amorphous powder, melting point of 211-212.5o C, soluble in methanol.

2. Qualitative tests: Molisch’s and Iodine tests - negative and in a distinctive test, the reaction with 1% vanillin and concentrated sulfuric acid resulted in a bluish-purple color solution and when sprayed with FeCl3 produced a purple hue suggesting the presence of phenolic hydroxyl group.

3. Spectral data:

a. FTIR spectra (Figure 4a): A pronounced stretching band of the phenolic group -OH in the FT-IR spectrum, ranging from 3550 to 3200 cm⁻¹ confirmed the presence of hydrogen bond formation. Aromatic hydrocarbons showcased absorptions in the 1600- 1585 cm⁻¹ and 1500-1400 cm⁻¹ ranges corresponding to carbon-carbon stretching vibrations within the aromatic ring. Bands in the 1250–1000 cm⁻¹ range are indicative of C–H in-plane bending, although they were faint. A broad carboxylic acid O-H stretch was identified in the 3000–2500 cm⁻¹ range.

b. ESI-MS spectrum (Figure 4b): Quasimolecular signals at m/z: 165.06 [M+1].

c. 1H-NMR spectra (Figure 5a): δ 12 (1H, s), δ 10.03 (1H, s), δ 7.46 (2H, d, J=6.1Hz), δ 6.76 (2H, d, J=6.1Hz), δ 6.25 (1H,d,J=15.6Hz).

d. 13C-NMR spectra (Figure 5b): δ167.9, 115.3, 144.2, 130.7, 115.3, 125.3, 159.6.

3. From the above spectral data, the chemical shift assignments for Compound 2 in flower extract were obtained (Figure 6a & 6b).

Compound 2 from flower extract was identified as p-Coumaric acid after aligning the above spectral data with the data available in the literature.^17,18

Discussion

Malvaceae family holds an important place in traditional medicine due to its wide range of medicinal uses, which are attributed to its rich variety of bioactive compounds and essential oils.¹⁹ F. colorata belongs to the Malvaceae family and is no exception. Like Sterculia urens, Sterculia foetida and other members of the Malvaceae family, F. colorata also exhibits potent antimicrobial and anti-inflammatory properties demonstrated in our previous studies. Our studies on F. colorata have also shown the presence of important bioactive compounds and minerals in its leaves, bark Hajira B B et al., RJAHS 2024;4(2):23-29 and flower extracts. The current study was conducted as part of an ongoing exploration of F. colorata.

In this study, isolation of two pure compounds - Compound 1, identified as stearic acid and Compound 2, identified as p-Coumaric acid was achieved, marking a pioneering effort. The complexity of isolating major fractions from F. colorata extracts, which encompass a wide range of phytochemical compounds, is amplified by the sparse available data on its composition. Despite these challenges, stearic acid and p-Coumaric acid were successfully isolated and identified from extracts of F. colorata using column chromatography.

As selection of solvents for column chromatography requires careful consideration of their polarity relative to the compounds being separated, solvents with varying polarities (n-hexane:petroleum ether:ethyl acetate: methanol) were tried to perform individual separations of compounds. However, only two major fractions were obtained. This outcome emphasizes the need to include a broader range of non-polar to polar solvents for effective column chromatographic separation and isolation of compounds from F. colorata extracts.

Isolation of stearic acid with n-hexane: ethyl acetate (40:60) obtained may be attributed to its bifunctional character, i.e, a polar head group and a nonpolar chain that confers solubility in organic solvents, like ethyl acetate in the current study. However, isolation of coumaric acid with ethyl acetate: methanol (85:15) may be ascribed to the protective role of methanol. It is known that methanol can prevent phenolic compounds from being oxidized by enzymes, such as phenoloxidases.

The study emphasizes on the employment of established spectroscopic techniques such as NMR, FTIR, and MS for structural elucidation of the isolated compounds, ensuring repeatability, reproducibility and facilitating future investigations into F. colorata. The obtained spectral data of the isolated compounds, corroborated by existing literature led to the identity of the Compounds 1 & 2 as stearic acid and p-Coumaric acid, respectively.

Many species of the Malvaceae family exhibit the presence of stearic acid in considerable quantities. There are reports on the presence of a significant amount of stearic acid in the cotyledons of Sterculia urens along with other fatty acids viz., linoleic acid, palmitic acid, eicosadienoic acid and eicosatrienoic acid.²⁰ The current study reporting the presence of stearic acid in the leaf extract of F. colorata, agrees with the results of the reported studies.

Several studies have reported that stearic acid has hypocholesterolemic effects, reduces high blood pressure, improves cardiac functions, and reduces risks of atherosclerosis and cancers.²¹ Studies reported by Kelly et al. indicate that dietary consumption of 19 g of stearic acid/day has favorable effects on thrombogenic and atherogenic risk factors in males and recommends that food industries consider fortifying foods with stearic acid instead of palmitic acid and trans fatty acids.²² In this context, leaves of F. colorata may be considered a rich source of stearic acid and may find application in the food, cosmetic and pharmaceutical industries.

Nevertheless, results of phytochemical analysis carried out in our preceding studies revealed the presence of coumarin, a source for coumaric acid, in the ethyl acetate and ethanol extracts of F. colorata. ¹⁰ Isolation of p-Coumaric from FE substantiates these observations. p-Coumaric acid is a phenolic acid and one of the three isomers of hydroxycinnamic acid that displays various biological activities.²³ Reports revealed that p-Coumaric acid minimizes various cancers by reducing the formation of carcinogenic nitrosamines, downregulating Grp78 and activating UPR-mediated apoptosis.^24,25 It is also investigated that it significantly reduces inflammation by decreasing the expression of cytokines COX-2, IL-6, TNF-α and PGE2 and reduces the expression of p-p65 and p-IκBα.²⁶ Furthermore, p-Coumaric acid has been proven to be an effective antioxidant with notable hepatoprotection by inhibiting Mitogen-activated protein kinase (MAPKs) and apoptosis signaling via enhancing Nrf2 signaling.²⁷ Its synergistic effects on modulating glucose metabolism, and amoebostatic activity against Entamoeba histolytica are also known.^28,29 These properties of p-Coumaric acid may be considered as the contributing factors to the antiinflammatory and antimicrobial properties exhibited by the flower extracts of Firmiana colorata demonstrated in our earlier studies. Chemoprotective activity, antimicrobial activity against a broad spectrum of pathogens and antioxidant potential of p-Coumaric acid have attracted the attention of researchers to identify more plants rich in p-Coumaric acid and flower extract of Firmiana colorata may contribute to the objective.

Conclusion

Isolation, identification, and characterization of stearic acid and p-Coumaric acid were achieved successfully from leaf and flower extracts of F. colorata. These compounds may find application as active ingredients in herbal formulations and pharmaceuticals. The presence of stearic acid and p-Coumaric acid in LE and FE of F. colorata justifies its use in traditional medicine.

Conflict of interest

The authors do not have any conflict of interest regarding the publication of this paper.