First crystallization of succinic acid from a plant matrix: isolation of the γ-polymorph from Solanum sanctae-catharinae Dunal.

Abstract

Succinic acid is a platform chemical widely used in the pharmaceutical, food, and materials industries, exhibiting notable polymorphic behavior that directly impacts its physicochemical properties. Succinic acid presents three reported crystal polymorphs, of which the γ-form is the least common and typically arises under specific non-equilibrium conditions. This study reports, for the first time, the isolation and crystallization of the γ-polymorph of succinic acid from a plant matrix (Solanum sanctae-catharinae Dunal). The crystalline compound was obtained from the ethyl acetate fraction following column chromatography. Structural characterization by single-crystal X-ray diffraction (SC-XRD) allowed the unambiguous assignment of the crystalline phase as the γ-polymorph through comparison with previously reported crystallographic data. The formation of the γ-polymorph under mild conditions suggests that the chemical complexity of the plant matrix may have influenced the crystallization pathway, possibly acting as a molecular template. This study contributes to expanding the literature of S. sanctae-catharinae and disclose new possibilities for the study of crystals polymorphism in natural matrices.

Keywords:
succinic acid; crystallography; polymorphism; natural products; X-ray diffraction.

INTRODUCTION

Succinic acid (SA) is a platform chemical with diverse industrial applications, playing a crucial role in the food, pharmaceutical, and chemical industries, particularly as a precursor for biodegradable polymers.¹,² Because of its unique functions, SA is considered one of the critical platform chemicals, which are basically building block molecules used as starting materials for producing a wide range of other chemicals, materials, and products.³ This important organic acid, also known as butanedioic acid, is an intermediate of several biochemical pathways such as the tricarboxylic acid (TCA) cycle and is one of the important fermentation products of energy metabolism.⁴,⁵ SA is a colorless crystal soluble in water, ethanol, and acetone.⁶ It is a member of the four-carbon dicarboxylic acid family, pure SA is solid under atmospheric conditions and its solubility is relatively low.⁴

Succinic acid is an organic acid present in the angiosperm’s metabolism due to its role in TCA cycle. Previous phytochemical analyses⁷-¹¹ have detected succinic acid in several plant species, such as Solanum lasiocarpum Dunal, Euphorbia gaillardotii Boiss., Aquilaria agallocha Roxb., Anabasis aretioïdes Coss. & Moq., and Primula veris L. Solanum sanctae-catharinae Dunal, popularly known as “Joá-Manso”, which is a native species of the genus Solanum that can reach heights of 3 to 6 m.¹²,¹³ In Brazil, its natural distribution is concentrated in the South and Southeast regions, particularly within the Atlantic Forest phytogeographic domain. Despite the relative abundance of pharmacological research involving Solanum species, S. sanctae-catharinae remains underexplored. Although, recent studies¹⁴,¹⁵ have reported the presence of alkaloids, coumarins, flavonoids, and triterpenes/steroids in the aerial parts (leaves and stem) of S. sanctae-catharinae.

This novel finding underscores the phytochemical richness of S. sanctae-catharinae and expands the knowledge of SA crystal polymorphs. Therefore, this study reports, for the first time, the extraction, purification, and crystallization of succinic acid from the aerial parts of S. sanctae-catharinae, followed by crystallographic phase identification of the γ-polymorph using single-crystal X-ray diffraction (SC-XRD) through comparison with previously reported structures.

EXPERIMENTAL

Plant material

The aerial parts (leaves and stems) of S. sanctae-catharinae were collected in 2022 from a remnant forest called Capão CIFLOMA, located at the Jardim Botânico campus of the Federal University of Paraná (UFPR) in Curitiba, Paraná (GPS coordinates 25°26’49.4” S, 49°14’21.7” WO). The species identification was performed by MS Alan Lessa and the collected material was compared with the voucher No. EFC11836, registered at the Herbarium Escola de Florestas, CURITIBA (HFC). Access to genetic heritage was authorized and registered by the Brazilian system SisGen (or the National System for the Management of Genetic Heritage and Associated Traditional Knowledge) registered under code A11D443.

Preparation of extract and partitioning

The S. sanctae-catharinae ethanolic crude extract was obtained from 2 kg of powdered dried aerial parts in ethanol using a Soxhlet apparatus for 11 h. The crude extract was concentrated using a rotary evaporator at 70 °C on a low pressure system, and 155 g of aerial parts crude extract were obtained. The crude extract was then used to obtain fractions by liquid/liquid partitioning with solvents of different polarities in the following order: hexane, chloroform, and ethyl acetate (55, 11.9, and 2.7 g, were respectively obtained from each fraction). The partitioning of the crude extract was performed using a modified Soxhlet extractor.¹⁶ After partitioning, fractions were placed in a hot water bath (70 °C) to evaporate as much of the solvent as possible. All solvents (Sigma-Aldrich) used were of analytical grade.

Isolation and characterization of succinic acid

The ethyl acetate fraction (2.7 g) was fractionated by silica gel column chromatography. The ethyl acetate fraction was dissolved in methanol (15 mL) and adsorbed onto silica gel 60 (8 g; 0.063 0.200 mm). After solvent evaporation under airflow and drying at 50 °C overnight, the dry-loaded sample was applied to a silica gel column (20 cm × 1 cm; 50 g). Column elution was performed using a gradient of hexane-methanol solvent system with increasing polarity, from 100% hexane and increasing the methanol content by 5% for each elution (100 mL) until reaching 100% methanol, to afford 210 fractions. Fractions (10 mL) were collected in vials and evaporated at room temperature under a fume hood. A crystalline compound was evidentiated in 5 vials, chronologically labeled as FL23-FL27.

Single crystals were mounted on a Micro-mount/mesh using inert oil and fixed on a Bruker D8 Venture diffractometer equipped with Photon II C7 detector, using Mo-Kα radiation and a graphite monochromator. Intensity data were measured by thin-slice ω and φ-scans.

Data reduction and integration were processed using the APEX4 software package.¹⁷ The structure was refined by full-matrix least-squares methods, on F2’s, in SHELXL.¹⁷ Furthermore the structure was determined by intrinsic phasing routines in the SHELXT program.¹⁸ Absorption corrections were applied using multi-scan method.¹⁷ Refinement of non-hydrogen atoms was carried out using anisotropic thermal parameters. All hydrogen atoms were located from Fourier difference maps and their positions and isotropic thermal parameters allowed to refine freely. Scattering factors for neutral atoms were taken from the literature.¹⁹ Computer programs used in the analysis were run through WinGX²⁰ and figures that refer to the structure were made using the software Mercury^®.²¹ Crystallographic data and structure refinement are summarized in Table 1 and selected molecular dimensions are given in Tables 2 and 3. Data have been deposited at the Cambridge Crystallographic Data Centre (CCDC) with the following CCDC number: 2527326.

Table 1
Crystal data and structure refinement of succinic acid γ-polymorph

Identification code CCDC	2527326
Elemental formula	C4H6O4
Molar mass / (g mol-1)	118.09
Radiation type	Mo-Kα
Temperature / K	299(2)
Crystal system, space group	monoclinic, C2/c
a / Å	5.7134(2)
b /Å	8.5612(3)
c / Å	10.4265(4)
Angle / degree	α = γ = 90 β = 90.8390(10)
Volume / Å3	509.94(3)
Z	4
Density / (g cm-3)	1.538
F(000)	248
Absorption coefficient / mm-1	0.141
Crystal color, shape	colorless prism
θ range / degree	3.91 to 27.4
Reflections collected	19464
Unique data	561 [R(int) = 0.022]
Observed data, [I > 2σ(I)]	507
Number of parameters	49
Goodness-of-fit on F2	1.075
R1 [I > 2σ(I)], wR2 [I > 2σ(I)]	R1 = 0.035, wR2 = 0.079
R1, wR2 (all data)	R1 = 0.041, wR2 = 0.084
Largest diff. peak and hole / (e Å-3)	0.21 and -0.18
Location of the largest difference peak	0.74 Å from C1

Table 2
Molecular dimensions of succinic acid γ-polymorph

Bond length / Å
O(1)-C(1)	1.3060(15)	C(2)-C(2)i	1.514(3)
O(1)-H(1)	0.94(2)	C(2)-H(2)	0.968(17)
O(2)-C(1)	1.2196(15)	C(2)-H(3)	0.995(17)
C(1)-C(2)	1.4985(18)
Angle / degree
C(1)-O(1)-H(1)	111.3(13)	C(1)-C(2)-H(2)	107.8(10)
O(2)-C(1)-O(1)	123.28(12)	C(2)#1-C(2)-H(2)	112.7(10)
O(2)-C(1)-C(2)	123.52(11)	C(1)-C(2)-H(3)	106.2(10)

Table 3
Selected hydrogen bonds of succinic acid γ-polymorph

bD-H...cA	ad(bD-H) / Å	ad(H...cA) / Å	ad(bD...cA) / Å	bDHcA / degree
O(1)-H(1)···O(2)ii	0.94(2)	1.73(2)	2.6685(14)	177(2)
C(2)-H(2)···O(1)iii	0.968(17)	2.642(17)	3.5305(17)	152.8(13)

• Symmetry transformations used to generate equivalent atoms: (ii) -x + 1, -y + ½, -z + 1, (iii) -x, y, -z + 3/2. Estimated standard deviations in parentheses.
• ^a
  Distance;
• ^b
  donor;
• ^c
  acceptor.

RESULTS AND DISCUSSION

The crystalline material were obtained as colorless prismatic crystals (Figure 1), yielding 3.2 mg, which corresponds to 0.118% of the ethyl acetate fraction and 0.00016% of the total extracted from the aerial parts. The crystals were isolated by natural evaporation of the solvent at room temperature.

Figure 1
Crystal formed on the vial inner wall

Structural elucidation by SC-XRD analysis revealed that the γ-polymorph crystallized from S. sanctae-catharinae adopts a monoclinic system, space group C2/c, with Z = 4 (Table 1). In this centrosymmetric space group, the asymmetric unit contains half of a C₄H₆O₄ molecule, with the complete molecule generated by a crystallographic inversion center. This symmetry constraint leads to a folded molecular conformation, which is a distinctive feature of the succinic acid γ-polymorph.

Hydrogen bonding plays a crucial role in stabilizing this supramolecular framework. An examination of the hydrogen-bonding interactions reveals that the crystal structure is primarily stabilized by strong intermolecular O-H···O hydrogen bonds between carboxylic acid groups (Table 2). The interaction O1-H1···O2ⁱ, with a distance of 1.73(2) Å and an angle of 177(2)° (symmetry code: (ii) -x + 1/2, -y + 1/2, -z + 1) links pairs of molecules into carboxylic acid dimers through the crystal lattice (Table 3). Additionally, the weaker C2-H2···O1ⁱⁱⁱ interactions, with a distance of 2.642(17) Å and an angle of 152.8(13)° (symmetry code: (ii) -x, y, -z + 3/2) contribute to the supramolecular stabilization (Tables 2 and 3).

The diffraction data obtained were consistent with the γ-polymorph of succinic acid, matching the crystallographic parameters reported for CCDC entry 1836394. The identification was based on unit cell parameters, space group, and diffraction pattern comparison, considering expected variations due to temperature differences. The SC-XRD analysis revealed that succinic acid crystallized from S. sanctae-catharinae exhibits a monoclinic crystal system with space group C2/c consistent with the γ-polymorph of succinic acid (Figure 2). The low residual electron density values (± 0.21 e Å^-3) and high data completeness (99.6%) of the crystalline material confirm its structural integrity and phase purity.

Figure 2
Oak ridge thermal-ellipsoid plot (ORTEP) representation of γ-succinic acid grow fragment, indicating the atoms numbering scheme. Displacement ellipsoids of non-H atoms are drawn at the 50% probability level, and isotropic displacement parameters of H atoms are represented by spheres of arbitrary radius

The agreement between the experimental diffraction data and the reference structure supports the assignment of the γ-polymorph (R₁ = 0.035, wR₂ = 0.079), indicating high structural accuracy (Table 1). The crystal structure is stabilized by intermolecular hydrogen bonds between carboxyl groups, forming a robust packing motif, as visualized in Figure 3. No diffraction signals corresponding to other polymorphs (α or β), solvates, or hydrates were detected, confirming the high purity of the SA γ-polymorph.

Figure 3
Packing of molecules of γ-succinic acid, viewed along the a-axis, showing the hydrogen-bonding interactions. Displacement ellipsoids of non H atoms are drawn at the 50% probability level, and isotropic displacement parameters of H atoms are represented by spheres of arbitrary radius

A scientific literature survey⁷-¹¹ demonstrates that succinic acid has been identified in several plant species, including Solanum lasiocarpum, Euphorbia gaillardotii, Aquilaria agallocha, Anabasis aretioïdes, and Primula veris. However, in all these reports, succinic acid was detected exclusively by spectrometric methods (liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-diode array detection (HPLC-DAD)), with no previous reports of isolation in crystalline form or structural elucidation via SC-XRD (Table 4).

Table 4
Occurrence of succinic acid in different plant species and whether crystallization was performed

Plant species	Plant organ	Identification method	Reference
S. lasiocarpum	fruits	LC-MSa	7
E. gaillardotii	leaves	GC-MSb	8
A. agallocha	root	GC-MSb	9
A. aretioïdes	root	HPLC-DADc	10
P. veris	leaves, stem	HPLC-DADc	11

• ^a
  Liquid chromatography-mass spectrometry;
• ^b
  gas chromatography-mass spectrometry;
• ^c
  high-performance liquid chromatography-diode array detection.

Polymorphism in succinic acid crystals has been extensively investigated due to its significant impact on physicochemical properties such as solubility, thermal stability, crystallinity, and bioavailability, which are crucial for both material science and pharmaceutical applications.²²-²⁵ The α-form is a high-temperature modification described as triclinic, although it presents a pseudo-monoclinic habit due to systematic twinning.²⁶ In contrast, the β-form, typically obtained at room temperature, is monoclinic and considered the thermodynamically stable form under mild conditions.²²

The third polymorph is the γ-polymorph of succinic acid first reported by Lucaioli et al.²³ in 2018. In that work, the γ-form was described as crystallizing in the monoclinic space group C2/c, with half a molecule in the asymmetric unit and a markedly folded molecular conformation. The crystal structure is stabilized by R²₂(8) carboxylic acid dimer hydrogen-bonding motifs at both ends of the molecule, forming hydrogen-bonded chains.

Notably, although all known polymorphs of succinic acid contain R²₂(8) hydrogen-bonded carboxylic acid dimers, the γ-polymorph is distinguished by the folded conformation of the succinic acid molecule, whereas the αand β-forms adopt near-planar conformations.²³ The γ-polymorph exhibits a distinct hydrogen bonding pattern and different thermal behavior, tending to convert into the β-form upon mild heating or long-term storage.²³ These crystallographic distinctions are not purely academic but directly influence the performance of succinic acid in diverse applications, affecting solubility, stability, and functional behavior.

The present study reports, for the first time, the isolation and crystallization of succinic acid directly from a plant matrix (Solanum sanctae-catharinae Dunal aerial parts). SC-XRD analysis revealed that succinic acid crystallized are consistent with the γ-polymorph of succinic acid reported in the Cambridge Crystallographic Data Centre (CCDC entry: 1836394) described and characterized by a previous crystalographic study.²³

The spontaneous formation of the γ-polymorph under mild conditions observed was not reported yet. We suggest that the heterogeneous chemical environment of the plant extract influences nucleation and polymorph selection. Previous studies²³ have demonstrated that the γ-form typically arises under non-equilibrium conditions such as heterogeneous systems with complex molecular pools. This observation aligns with the findings of Lucaioli et al.²³ in 2018, where the presence of diverse molecular species in heterogeneous crystallization environments promoted the unexpected formation of the γ-polymorph. Such phenomena are well-documented in the field of crystal engineering, stating that a complex matrix with impurities, co-metabolites, or minor components influence nucleation pathways and crystal polymorph selection. This corroborate with the research of da Rocha in 2025¹⁴ which evidentiated the presence of diverse secondary metabolites in the ethyl acetate fraction, considered a pool of complex molecules. The spontaneous formation of the γ-polymorph from the ethyl acetate fraction suggests that the chemical complexity of the plant matrix may have acted as a template or modulator, favoring this succinic acid metastable form.

This unusual finding highlights the successful isolation and crystallographic characterization of the succinic acid γ-polymorph from a plant extract which disclose new possibilities for the study of crystals polymorphism in natural products matrices.

CONCLUSIONS

This study reports, for the first time, the isolation and crystallographic characterization of succinic acid from aerial parts of S. sanctae-catharinae. The crystalline compound, recovered from chromatographic column fractions was identified through SC-XRD, and assigned as the γ-polymorph of succinic acid, based on comparison with previously reported CCDC crystallographic data (entry code: 1836394). Full crystallographic refinement and deposition at the CCDC (entry code: 2527326) were performed to support the identification of the crystal structure.

The spontaneous formation of the γ-polymorph under mild conditions suggests that the complex chemical environment of the plant extract may influence nucleation pathways and polymorph selection. These findings contribute to a broader understanding of succinic acid polymorphism and highlight natural matrices as relevant systems for investigating crystallization phenomena. The present study provides compelling evidence for the isolation of γ-succinic acid from a plant matrix. In addition this work expands the knowledge of succinic acid crystal polymorphism in natural matrices.

SUPPLEMENTARY MATERIAL

Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 IEZ, UK (fax: + 44-(0)1223-336033 or email: [e-mail]), under CCDC number: 2527326 and 1836394.

ACKNOWLEDGMENTS

This study was financed in part by the CAPES - Finance Code 001.

DATA AVAILABILITY STATEMENT

All data are available in the text and supplementary.

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Edited by

• Associate Editor handled this article:
  Fernanda G. Finelli

Publication Dates

• Publication in this collection
  27 Mar 2026
• Date of issue
  2026

History

• Received
  26 Nov 2025
• Accepted
  09 Feb 2026
• Published
  24 Feb 2026