Flower- and Grass-like Self-Assemblies of an Oleanane-Type Triterpenoid Erythrodiol: Application in the Removal of Toxic Dye from Water.

Erythrodiol (3β-olean-12-ene-3, 28-diol) (C30H50O2) 1 is a nanosized oleanane-type fused 6-6-6-6-6 pentacyclic triterpeneoid extractable from the dried leaves of olive (Olea europia). One step reduction of oleanolic acid extracted from Lantana camara also yields the same compound. The triterpenoid has one secondary -OH group attached at C3 of the "A" ring and one primary -OH group at C28 present at the junction of the "D" and "E" rings. Here, we report the spontaneous self-assembly of erythrodiol in different neat organic liquids and aqueous-organic liquid mixtures. The nanosized dihydroxy triterpenoid having an oleanane-type lipophilic rigid skeleton self-assembled in liquids, yielding nanosized fibrils, microsized flowers, and grass-like architectures via formation of densely assembled fibrils and petals or 2D sheets. The microstructures of the self-assemblies have been characterized by different techniques like optical microscopy, electron microscopy, atomic force microscopy, FTIR, and wide angle X-ray diffraction studies. The porous self-assemblies having a large surface area obtained from 1 were capable of adsorbing toxic fluorophores like rhodamine-B, rhodamine-6G, methylene blue, and crystal violet (CV). Moreover, removal of the aforementioned toxic pigments has also been demonstrated from their aqueous solutions by using UV-visible spectrophotometry and epifluorescence microscopy.

Introduction

Terpenoids, the largest class of natural products containing a multiple of C5 units, have drawn significant research interests in recent years due to their diversified structural features, interesting self-assembly properties, and the applications of the resulting self-assemblies in advanced functional materials and biological research.[1,2] The mono-terpenoids (C10) to higher terpenoids (C15–C40), all having nanometric lengths and properly positioned functional groups, offer innumerable opportunities for their utilization as molecular functional nanos (MFNs).[1] The inherently renewable nature of the plant-based terpenoids have drawn significant research interest in recent years for the development of sustainable society. This large and structurally diverse group of natural products of nanometric dimensions and properly positioned functional groups have made them useful as an interesting class of amphiphiles for the study of their self-assembly properties. Though a significant research advancement has taken place on functional metal nanoparticles (FMNPs) and the self-assembly of different class of compounds such as peptides, sugars, steroids, fatty acids, and lipids, only a few examples on the self-assembly of terpenoids have been reported till date.[3−16]

Previously, we have reported the self-assembly property and applications of several triterpenoids like arjunolic,[17,18] oleanolic,[19] glycyrrhetinic,[20] ursolic,[21] maslinic,[22] corosolic,[23] and betulinic acids;[24] di-hydroxy lupane-type triterpenoid betulin;[25] and C2-symmetric dihydroxy seco-triterpenoid α-onocerin.[26] Self-assembly of a macrocyclic diterpenoid crotocembraneic acid has also been demonstrated in aqueous liquids.[27] These MFNs spontaneously self-assembled as natural amphiphiles without any functional group transformations. Different morphologies such as vesicles, tubes, flowers, and fibrillar networks were obtained by self-assembly of different terpenoids, indicating structure property relationships though predictability of the morphology of a given terpenoid in a medium is in its infancy.

Erythrodiol 1 is a dihydroxy triterpenoid extractable from olive (Olea europia). In plants, erythrodiol is proposed to be the precursor of oleanolic acid. It has been shown to possess a wide range of biological activities such as anticancer activity on different cell lines,[28−30] antitumor activity, vasorelaxant, and cardio-protective activities.[31−33] It is also found to be effective in skin chronic inflammation.[34] However, to our knowledge, the self-assembly property of this compound in liquids has not yet been reported.

In this study, we have investigated the self-assembly property of this triterpenoid erythrodiol 1 in different organic liquids and aqueous-organic liquid mixtures. Nanosized fibrils, microsized flowers, and grass-like architectures were obtained via formation of densely assembled fibrils and petals or 2D sheets. The morphology of the self-assemblies has been analyzed by SEM, AFM, HRTEM, and wide-angle X-ray diffraction studies. The porous self-assemblies having high surface area were utilized as adsorbents of toxic dyes like crystal violet (CV), methylene blue (MB), rhodamine-B (rho-B), and rhodamine-6G from their aqueous solution, as demonstrated by epifluorescence microscopy as well as UV–vis spectrophotometry.

Results and Discussion

Isolation of Erythrodiol and Its Structural Characteristics

Oleanolic acid extracted from the root bark of Lantana camara yielded erythrodiol 1 on reduction with LiAlH4 as a white crystalline solid (Scheme ).[7,35] Solvent extraction of the leaves of olive also yielded the same (Scheme ).[36],[37] Erythrodiol 1 has a fused 6-6-6-6-6 pentacyclic triterpenoid moiety with two polar “–OH” groups: one attached at the C3 position of the “A” ring and the other −OH is attached at C28 present at the junction of cis-fused “D” and “E” rings. Computations carried out by DFT calculation and by molecular mechanics calculation using Allinger’s MMX algorithm revealed the molecular length of 1.57 nm (Figure S1 and S2). The lipophilic 6-6-6-6-6 fused pentacyclic backbone and the presence of polar functional groups at the two ends of the nanosized triterpenoid backbone makes it a unique amphiphile for studying its self-assembly properties in different liquids (Table ).

Scheme 1

Schematic Presentation of Isolation and Synthesis of Erythrodiol 1 and Its Self-Assembly Property Yielding Flowers, Fibers, and Grass-like Architectures

Table 1

Self-Assembly Studies of Erythrodiol 1

entry	medium	conc.(mM)	statea
1	o-xylene	56.5	CS
2	m-xylene	45.2	CS
3	p-xylene	45.2	CS
4	mesitylene	90.4	CS
5	chlorobenzene	45.2	CS
6	o-dichlorobenzene	56.5	CS
7	2-propanol	45.2	CS
8	EtOH	22.6	S
9	DMSO	45.2	S
10	EtOH-H2O (1:1)	45.2	CS
11	DMSO-H2O (1:1)	22.6	CS
12	H2O	22.6	I

S = soluble, I = insoluble, CS = colloidal suspension.

Self-Assembly Studies

Compound 1 was only sparingly soluble in most of the common organic liquids and remained insoluble in water. Self-assembly properties of 1 were studied in different types of neat organic liquids and alcohol–water mixture. For this purpose, a weighed amount of 1 (usually 1–5 mg) was dissolved in neat liquids under hot conditions (40–50 °C) with magnetic stirring. For studying the self-assembly in neat liquids, the hot solution was allowed to cool at room temperature and observed visually after 4–8 h. In o-xylene, m-xylene, p-xylene, mesitylene, chlorobenzene, o-dichlorobenzene, and 2-propanol it remained as a colloidal suspension of 1 in the liquids. For study of the self-assembly of 1 in the ethanol–water mixture, the hot solution of 1 in ethanol was mixed with an increasing amount of water till cloudiness appeared, then it was re-dissolved by heating, and then the clear solution was cooled at room temperature. A colloidal suspension of 1 was also obtained in ethanol–water mixture. Similarly, a colloidal suspension was also obtained in DMSO-water.

Morphology of the Self-Assemblies

Morphologies of the self-assemblies of 1 were studied by optical microscopy (OPM), atomic force microscopy (AFM), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), FTIR, and X-ray diffraction studies.

Scanning Electron Microscopy

SEM analysis of the dried self-assemblies of 1 prepared from its colloidal suspension in o-xylene (2% w/v, 45.20 mM) showed marigold-type flowers with a diameter of 15–20 μm (Figure ). Detailed investigations of the dried self-assemblies prepared from o-xylene by SEM at a higher magnification indicated that the flowers were composed of densely packed petals (2D sheet) with an average thickness of 50–200 nm (Figure d,h and Figure S6c,e,f). SEM carried out with the dried self-assemblies of 1 prepared from the colloidal suspensions in m-xylene (2% w/v, 45.2 mM), p-xylene (2% w/v, 45.2 mM), and mesitylene (4% w/v, 90.40 mM) revealed cauliflower-like flowers having an average diameter of 15–20 μm (Figure and Figures S7 and S8).

Figure 1

SEM images of dried self-assembled 1 in o-xylene at different concentrations: (a–d) in 2% w/v (45.2 mM); (e–h) in 2.5% w/v (56.50 mM).

Figure 2

Electron microscopy images of dried self-assemblies of 1 (a–c) in m-xylene (2% w/v, 45.2 mM) and (d–f) in mesitylene ( 4.0% w/v, 90.4 mM).

Though flower-shaped microstructures having a micrometer diameter were obtained from the dried self-assemblies of 1 in four liquids, o-, m-, and p-xylenes, and mesitylene, their shapes and size were not identical. The petals and the arrangement of the petals in the microstructures were also different. This difference might be due to a very fine balance between the polarity of the medium, structure of the solvent molecules, and solvent–solute and solute–solute interactions.

SEM analyses were also carried out with the dried self-assemblies of 1 prepared from chlorobenzene (2% w/v, 45.20 mM) and o-dichlorobenzene (1.5% w/v, 33.90 mM). Investigations of the self-assembled microstructures prepared from dried self-assemblies of 1 in chlorobenzene (2% w/v, 45.20 mM) and o-dichlorobenzene (2% w/v, 45.20 mM) revealed hierarchical self-assembly of molecules yielding a densely packed fibrillar network and grass-like morphology (Figure ). In chlorobenzene, the fibers were of 22–45 nm in diameter and nano- to micrometer in lengths, whereas the grass leaves observed in o-dichlorobenzene (2% w/v, 45.20 mM) were of several micrometer lengths formed from nanometer diameter fibers (Figure and Figure S9b).

Figure 3

FESEM micrographs of dried self-assemblies of 1: (a) porous nanofibers and (b, c) grass-like architecture in o-dichlorobenzene (2% w/v); (d–f) nanofibers in chlorobenzene (1.5% w/v).

Flower-like self-assembled nanostructures from inorganic metal oxides and inorganic hybrid nanoparticles such as SnO2,[38] TiO2,[39] ZnO,[40] Fe2O3,[41] and Ni(OH)2[42] have been reported in the literature, but such flower- and grass-like self-assembled microstructures resembling naturally occurring marigold (Calendula officinalis), cauliflower, and grass from nanosized organic molecules are rare.

Transmission Electron Microscopy

To further elucidate the self-assembled microstructures observed by electron microscopy, we carried out HRTEM studies.[43] TEM images obtained from very dilute colloidal suspension of 1 prepared from mesitylene (0.8% w/v, 18.08 mM) in dried conditions clearly indicated microsized flowers (Figure and Figure S11c) composed of fibrillar networks and sheets. A TEM micrograph obtained from o-xylene (0.8% v/v, 18.1 mM) also indicated the flower-like objects and gel-like morphology (Figure e and Figure S11a,b). Flower-like morphology observed by HRTEM supported the observation by SEM analyses discussed earlier.

Figure 4

(a–d) HRTEM image of 1 in mesitylene (1% w/v, 22.6 mM) and (e,f) HRTEM image of 1 in o-xylene ( 0.8% w/v, 18.08 mM) in a dried state.

AFM Studies

Atomic force microscopy was carried out with the dried self-assemblies of 1 prepared from a dilute colloid of 1 in neat organic liquid such as o- and p-xylenes (0.8% w/v, 18.1 mM), mesitylene (1% w/v, 22.6 mM), and o-dichlorobenzene. Flowers, petals, fibrillar networks, and spherical objects were observed (Figure , Figure S12) in the dried self-assemblies prepared from all the liquids supporting our earlier observation from electron microscopy (Figure –4).

Figure 5

AFM images of 1 (a,d) in p-xylene (1.0% w/v) and (b,c) in mesitylene (0.8% w/v, 18.08 mM) in a dried state.

Optical Microscopy

Optical microscopy (OM) was carried out to investigate the morphology of the self-assemblies of 1 in various neat organic liquids like o-xylene, m-xylene, p-xylene, and mesitylene (2% w/v, 45.20 mM) in their native state. Erythrodiol 1 self-assembled in o-xylene and m-xylene (2% w/v, 45.20 mM) forming microsized spherical objects and flower-like self-assemblies (Figure and Figure S13). Identical images were also observed in p-xylene and mesitylene. The nanosized spherical objects were not observed by OM due to the size limitations in optical microscopy.

Figure 6

OM images of 1 (a) in o-xylene (2% w/v, 45.2 mM) and (b) in mesitylene (2% w/v, 45.2 mM) in a native state.

FTIR Studies

Self-assembly of the bola-type amphiphile having polar H-bonding groups at the two extreme ends may have been driven by the intermolecular H-bonding along with the dispersive interactions from the large lipophilic rigid terpenoid backbone.[44−47] To investigate the role of H-bonding in the self-assembly, FTIR spectra were recorded taking the neat powder and the dried self-assemblies prepared from o-, m-, and p-xylenes (2% w/v, 45.2 mM), o-dichlorobenzene (2% w/v, 45.2 mM), and mesitylene (4% w/v, 90.4 mM) and their stretching frequencies were compared. The −OH stretching frequency of the neat powder of 1 (3354 cm–1) shifted to 3345, 3346, and 3348 cm–1 in the dried samples prepared from o-, m-, and p-xylenes. The lowering of −OH stretching frequency observed in the dried self-assemblies compared to neat powder indicated stronger intermolecular H-bonding interactions in the self-assemblies (Figure S15) supporting the role of H-bonding during self-assembly.

X-Ray Diffraction Studies

Powder X-ray diffraction studies were carried out to know more detail about the patterns of the self-assemblies. Wide-angle powder XRD studies of neat powder and dried self-assemblies prepared from o-xylene (2.5% w/v, 56.5 mM), m-xylene (2%w/v, 45.2 mM), and p-xylene (2%w/v, 45.2 mM) in the range of 2θ = 5–40° were carried out and their diffraction patterns were compared with neat powder of 1. The wide angle X-ray diffraction peaks observed in the dried self-assemblies of 1 prepared from o- and m-xylene were at d = 1.57, 0.78, 0.52 nm (Table S1), which were in the ratio of 1:1/2:1/3 along with additional peaks. The optimized molecular length of erythrodiol 1 being 1.57 nm, a lamellar pattern of self-assembly of the molecules could be considered in o-xylene and m-xylene. The diffraction pattern of the neat powder also had identical peaks along with additional peaks, though the peaks from the self-assemblies were sharper compared to that in the neat powder indicating comparatively more order assembly of molecules in the self-assemblies (Figure S16 and Table S1). Almost identical d values (similar type of diffraction patterns) found in all the xerogel samples studied were reflecting the presence of identical morphologies at the nanoscale, giving rise to distinct nano- to microsized self-assemblies observed from different liquids.

Proposed Model for the Self-Assembly of 1

A model for the self-assembly of 1, yielding flowers, grass, and a fibrillar network has been proposed as shown in Figure . Erythrodiol 1 having a β-amyrin type skeleton can be schematically represented as an unsymmetrical bola-type amphiphile M-spacer-P (Figure ). It can self-assemble forming a linear 1D chain (Figure A,B) via intermolecular H-bonding. The fibrillar network yields the petals and long-leaf like architectures, leading to the formation of flower- and grass-like morphologies, as evident from electron microscopy.

Figure 7

Possible molecular packing of the self-assembly of 1 yielding nanofiber, flower, and grass via H-bonding and non-covalent interactions.

Adsorption of Fluorophores

Optical and electron microscopies, AFM, and HRTEM analysis of the dried assemblies of 1 prepared from various neat organic liquids confirmed the porous nature of the microsized flower- and grass-like self-assemblies in all the cases. These observations encouraged us to study the adsorption capability of the self-assembled microstructures of 1. Initial adsorption studies of the self-assemblies were carried out with toxic dyes like rhodamine-B (rho-B) and methylene blue (MB). For this study, 1 (2 mg) was dissolved in isopropanol (1% w/v, 200 μL) and was mixed with aqueous rho-B (0.25 mM, 0.03 mL) and cloudiness appeared. Then, the mixture was heated followed by magnetic stirring and allowed to cool at 15 °C for 6 h. Then, an aliquot of 10 μL was taken from the colloidal suspension for studying epifluorescence microscopy. Bright green and red fluorescence observed from the surface of the microsized self-assemblies under fluorescence light indicated that both rhodamine-B and MB were adsorbed on the surface of the porous self-assemblies of 1 (Figure a–f and Figure S14).

Figure 8

Epifluorescence micrograph of (a–c) adsorbed rho-B and (d–f) adsorbed methylene blue (MB) on the surface of the self-assemblies of 1.

Dye Removal Study by UV Spectrophotometry

Toxic dye removal from waste water is of great concern for environmental reasons during the last few decades.[48−51] Adsorption of fluorophores in the self-assemblies as observed by epifluorescence microscopy inspired us to investigate whether the porous self-assemblies of the flower and fibrillar network could be capable of removing the toxic dyes like rhodamine-B, methylene blue (MB), crystal violet (CV), and rhodamine-6G from their respective aqueous solution. For these studies, aqueous solutions of dyes were prepared by dissolving a weighed amount of dye in distilled water. For the preparation of aqueous solution of cationic dye of CV, a weighed amount (4 mg) of CV was taken in a clean dried vial and dissolved in 2 mL distilled water and from this solution, 10 μL was taken and was further diluted to 2 mL by adding distilled water to prepare aqueous CV solution (10 mg/L). By following the similar procedure, aqueous solutions of rho-B, MB, and rhodamine-6G were prepared having the concentration of 10 mg/L. Colloidal self-assemblies of 1 were prepared by dissolving 5 mg of 1 in 0.5 mL dry distilled o-dichlorobenzene (22.50 mM) in four clean vials. Then, 2 mL aqueous solution of all the dyes prepared were placed carefully on the organic colloidal suspension of 1 (22.50 mM) and absorbance were measured taking the aqueous aliquot of 2 mL from the upper layer very carefully at different time intervals with UV–visible spectrophotometry. It was evident from the results of UV–visible spectrophotometry that with increasing time, absorbance was decreased for all the cationic dye solutions. For MB and CV, absorbance decreased within 45 min–1 h and for rhodamine-B and rhodamine-6G, absorbance decreased within 1–1.5 h. Control partition experiments carried out with all the above dyes did not show significant removal even after 4–6 h (Figure S17). All these observations support the absorbing efficiency of the porous self-assembled microstructures (Figure ). These investigations also lead the way for the removal of toxic carcinogenic dyes from water paving the way for sustainable removal of dyes from contaminated water based upon renewable terpenoids.

Figure 9

UV–visible spectroscopy demonstrating the ability of self-assemblies of erythrodiol in o-dichlorobenzene (22.50 mM) for the removal of dye from their aqueous solutions: (a) crystal violet (0.02 mM) removal, (b) methylene blue (0.03 mM) removal, (c) rhodamine-6G (0.02 mM) removal, and (d) rhodamine-B (0.02 mM) removal. Plots of absorbance versus time for different dyes: I, II, III, and IV for CV, MB, rho-6G, rho-B, respectively. Inset: structures of dye molecules and photograph of vials containing dyes in contact with colloidal self-assemblies of 1 before and after removal of corresponding dyes.

Conclusions

In conclusion, spontaneous formation of flower- and grass-like porous self-assemblies of oleanane-type triterpenoid erythrodiol has been reported. According to our knowledge, this is the first report of the formation of flower- and grass-like microstructures by naturally occurring 6-6-6-6-6 fused pentacyclic dihydroxy oleanane-type triterpenoid. Detailed morphological characterization of the self-assemblies has been carried out by AFM, optical microscopy, SEM, FESEM, HRTEM, and wide-angle X-ray diffraction studies. Utilization of the porous self-assemblies has been demonstrated for the removal of toxic dyes such as rho-B, CV, and MB from their aqueous solution. The studies described here demonstrate the generation of advanced materials based on natural terpenoids for applications in material science and medicine.

Experimental Section

Dye Removal Study

The colloidal self-assemblies of 1 prepared from o-dichlorobenzene were used for the study of its dye removal ability. Compound 1 (5 mg) was taken in a vial and dissolved in distilled o-dichlorobenzene (0.5 mL) under hot conditions, and the resulting solution was allowed to cool at 15 °C temperature for 6 h. For the preparation of aqueous solution of cationic dye CV, a weighed amount (4 mg) of CV was taken in a clean dried vial and dissolved in 2 mL distilled water and from this solution, 10 μL was taken and was further diluted to 2 mL by adding distilled water to prepare aqueous CV solution (10 mg/L, 0.02 mM). By following the similar procedure, aqueous solution of rho-B (0.02 mM), MB (0.03 mM), and rhodamine-6G (0.02 mM) were prepared having the concentration of 10 mg/L for all the prepared aqueous solutions. Colloidal self-assemblies of 1 were prepared by dissolving 5 mg of 1 in 500 μL dri-distilled o-dichlorobenzene (22.50 mM) in four clean vials. Then, 2 mL aquous solution of all the dyes prepared were placed carefully on the organic colloidal suspension of 1 (22.50 mM) and absorbance were measured taking the aqueous aliquot of 2 mL from the upper layer very carefully in a quartz cuvette (2 mm path length), and the absorption was measured at λmax = 586 nm (CV), λmax = 660 nm MB), λmax = 550 nm (rho-B), and λmax = 527 nm (rho-6G) after different time intervals.