Structural Characterization and In-silico Evaluation of Bioactive compounds from Neisosperma Oppositifolium Leaves as Anti-diabetic and Anti-cancer agents

Introduction

The exploration of natural products from medicinal plants continues to play a vital role in the discovery of novel therapeutic agents¹. The most challenging and prevalent global health issues of the 21^st century are Diabetes mellitus and cancer². As per the reports of  International Diabetes Federation³, over 530 million people worldwide were living with diabetes in 2023, and this figure is expected to rise significantly due to factors such as sedentary lifestyles, obesity, and unhealthy dietary habits⁴. Among them,  nearly 90% of cases accounts for Type 2 or non-insulin dependent diabetes, which is characterized by impaired glucose metabolism due to insulin resistance⁵. According to the World Health Organisation (WHO), there will be around 10 million cancer-related deaths and 20 million new cancer cases worldwide in 2026, making cancer a major source of morbidity and mortality⁶. Both diseases impose substantial burdens on healthcare systems and economies. Interestingly, a growing body of evidence suggests a bidirectional link between diabetes and certain types of cancer, due to shared risk factors like chronic inflammation, hyperinsulinemia, and oxidative stress ^7,8. Despite advances in treatment, limitations such as drug resistance, adverse effects, and high costs necessitate the exploration of safer, plant-derived therapeutic alternatives⁹.

Indole alkaloids are a structurally diverse group of natural metabolites with wide ranging distribution across various biological sources. They exhibit a broad spectrum of pharmacological effects, including antibacterial, antimalarial, anti-inflammatory, antiviral, and anticancer activities¹⁰. Their chemical diversity and multifaceted therapeutic potential have positioned indole alkaloids as important scaffolds in natural product based drug discovery, particularly in the areas of diabetes management and cancer treatment. For example, vindoline¹¹, tetrahydroalstonine¹², and reserpine¹³ display promising antidiabetic effects by improving insulin sensitivity, regulating glucose metabolism, and inhibiting carbohydrate-hydrolyzing enzymes. Compounds such as vincristine¹⁴, vinblastine¹⁵, and evodiamine¹⁶ are either firmly established as chemotherapeutics or exhibit strong anticancer potential by inducing apoptosis, suppressing cell growth, inhibiting angiogenesis, and interfering with microtubule dynamics.

In this context, the investigation of natural bioactive compounds with dual anti-diabetic and anti-cancer activity holds significant potential for the development of novel and more effective therapeutic agents¹⁷. In recent years, the integration of phytochemical analysis with computational drug discovery tools has accelerated the identification of bioactive compounds and their potential biological targets¹⁸.

No prior scientific studies on the biological activities or chemical constituents of selected plant Neisosperma oppositifolium have been documented in the available literature. In light of this research gap, the current study was undertaken to explore the pharmacological potential of its bioactive compounds. This involved a systematic approach of extraction, isolation, analytical characterization, and biological evaluation, with a focus on predicting anti-diabetic and anti-cancer activities through in silico methods^19-21. The study aims to provide a foundational understanding of the plant’s therapeutic potential and support further pharmacological investigations.

Plant Profile

Neisosperma oppositifolium, commonly known as tonga, tokelau, and aikikiru, belongs to Apocynaceae family and is traditionally recognized for its management in diabetes and hypertension. The biological source is the dried leaves, which are rich in indole alkaloids such as bleekerine, ochropposinine, isorespilline, and 3-epirauvanine. The plant typically grows as a small to moderate tree reaching up to 15m in height and exudes latex. It is widely distributed throughout the South Pacific and extends westward into the Indian ocean, thriving in littoral forests, mangrove swamp edges, and limestone-rich environments. The plant typically flowers during summer, followed shortly by fruiting.

Materials and Methods

Instruments and Chemicals

Various instruments and Software tools used  for the study were single-pan electronic weighing balance (Axis LC/GC), UV chamber (Medimeas, Ambala), ATR-FTIR spectrophotometer (Alpha II Bruker), NMR spectrophotometer (Bruker 500 MHz), ESI MS (G3540A-Agilent 8890) and PASS online and AutoDock 4.2 for in silico prediction of biological activity. Several chemicals and solvents viz ethanol, methanol, petroleum ether, hexane, chloroform, toluene, ethyl acetate, and various acids used were of laboratory-grade and all sourced from Molychem, Mumbai.

Plant Collection and authentication

To prepare various solvents extracts, fresh and healthy leaves of Neisosperma oppositifolium (Fig.1) were procured from Dwaraka medicinal garden, located in Gangireddy Palli village, Yerraguntla, Kadapa District, A.P., India. The authentication of this plant was authorized by botanist from Government Degree college, Rajampet, Annamayya District, A.P., India.

Figure 1: Fresh and dried leaves of Neisosperma oppositifolium. Click here to View table

Extraction of bioactive constituents

Freshly collected leaves were thoroughly washed with water to remove dust particles and shade-dried at room temperature for 10 days (Fig. 1). The dried material was ground into a fine powder, and 50 g of this powder was macerated in 250 ml of different solvents of increasing polarities viz., n-hexane, toluene, chloroform, ethyl acetate, ethanol, methanol, and water for 7 days. After maceration, the extracts were filtered and concentrated under reduced pressure using a rotary evaporator. The total weight of crude extracts were 0.542, 0.683, 0.751, 0.824, 0.967, 0.934, and 0.926 g, with the percentage yields of 0.01, 0.013, 0.015, 0.016, 0.019, 0.018, and 0.018%, respectively.

Phytochemical analysis

It was performed on all extracts to identify the presence of major phytoconstituent groups. Tests for alkaloids, glycosides, tannins, and flavonoids were performed using standard protocols.²² These tests helped in selecting the most promising extract for further isolation and characterization.

Chromatographic analysis

Ethanolic leaf extract of N. oppositifolium was analysed through chromatography²³ to isolate and purify bioactive constituents, particularly indole alkaloids. TLC was performed initially for the preliminary separation and optimization of solvent system. Silica gel based TLC plates containing a fluorescent dye were activated at 110°C for 30 minutes and developed using various freshly prepared solvent systems as shown in table 1. Crude extracts and isolated compounds were spotted on the TLC plates, and chromatograms were developed using saturated chambers, visualized under UV light at 254 nm and 365 nm, and Rf values were computed for analysis. Based on the TLC results, the solvent system viz n-hexane: methanol: ethyl acetate: water (3:1:5:1) was given clear and well-resolved bands, making it suitable for bulk separation via column chromatography. A silica gel-packed column (60–120 mesh) was used as the stationary phase, and polarity was gradually increased to optimize elution. Fractions were collected, monitored using TLC, and those showing similar profiles were pooled, concentrated, and subjected to spectroscopic characterization and in silico biological evaluation.

Table 1: Various mobile phase compositions employed for TLC Studies

Analytical characterization by Spectroscopic analysis

The concentrated fractions obtained were labelled as A, B, C, D, E and F. upon phytochemical analysis, fractions D, E, F tested positive for indole alkaloids. Then they were concentrated and subjected to characterization using ATR-FTIR, ¹H and ¹³C-NMR and ESI-MS²⁴ to elucidate their molecular structures. The combined spectral analysis resulted in identification of functional groups, structural and molecular mass of phytochemical constituents present in these fractions.

In-silico Evaluation of Pharmacological activity

The Prediction of Activity Spectra for Substances (PASS) online tool was used to evaluate possible biological activities based on structure activity relationships, with activities having Pa values > 0.5 considered potentially significant²⁵. To further explore their pharmacological potential, molecular docking studies were conducted using AutoDock software (ver. 4.2) to estimate binding affinities against two therapeutically validated and clinically relevant target proteins. 2JJK, Human fructose-1,6-bisphosphatase, a key enzyme in gluconeogenesis and a validated antidiabetic target²⁶, as its inhibition lowers glucose production without causing hypoglycemia or weight gain, and 3KOK, a central signaling mediator in cell growth and survival, and an established target for anticancer therapy²⁷, were chosen as clinically relevant protein targets for in-silico bioactivity evaluation against diabetes and cancer.

The docking scores, expressed as binding energy (ΔG, kcal/mol), reflect the strength of interaction between each compound and the active site of the respective proteins²⁸. Lower (more negative) binding energy values indicate stronger binding affinity, suggesting potential biological relevance. The number of hydrogen bonds and key interacting residues were also analysed to gain insights into the binding mode²⁹.

RESULTS AND DISCUSSION

Phytochemical screening of ethanolic extract (0.967g) of N. oppositifolium leaves given a positive result for indole alkaloids, which were well resolved through the optimization of n-hexane: methanol: ethyl acetate: water (3:1:5:1v/v) as the mobile phase and purified through thin layer and column chromatography respectively. The purified fractions that showed positive for indole alkaloids were coded as compound NO-1, NO-2 and NO-3 and subjected to characterization using advanced spectroscopic techniques.

Analytical Characterization

Characterization of isolated compound was established using ATR-FTIR, ¹H, ¹³C NMR and mass spectrometry and spectral data are presented below

Compound NO-1

Pale yellow liquid; Thalleoquine test- negative, vitali-morin test- negative, Van urk’s test- positive; Rf-0.64 (solvent: n-hexane: methanol: ethyl acetate: water (3:1:5:1v/v); ATR-FTIR cm^-1: 3408.66, N–H str. (Secondary amide); 1712.47, C=O str. (Ester carbonyl); 1642.12, C=O str. (Amide carbonyl); 1260.98, C–O str. (Methoxy group); 1149.47, C–O–C str. (Cyclic ether) (Fig. 2a); ¹H NMR (Acetone d_6, 400 MHz), δH 3.73 (6H, singlet, OCH₃), 3.81 (3H, singlet, OCH₃), 9.25 (1H, singlet, NH) 1.16-1.18 (3H, doublet, CH₃), 6.33 (1H, doublet, Aromatic), 6.83 (1H, doublet, Aromatic), 7.25-7.27 (1H, doublet, Aromatic), 1.64-1.78 (2H, multiplet, CH₂), 1.88-1.95 (1H, multiplet, CH₂), 2.05-2.11 (1H, multiplet, CH), 2.27-2.30 (1H, multiplet, CH), 2.40-2.47 (1H, multiplet, CH₂), 2.91-3.01 (5H, multiplet, CH₂ & CH), 4.76-4.81 (1H, multiplet, CH) (Fig. 3a); ¹³C NMR (101 MHz, Acetone d₆), δC 201.88(1C), 167.82 (1C), 157.32(1C), 150.36 (1C), 145.32 (1C), 141.09 (1C), 132.21(1C), 117.98(1C), 104.70 (1C), 70.73 (1C), 65.54 (1C), 61.26 (1C), 55.23 (1C), 53.07(1C), 46.23(2C), 29.82 (1C), 13.85 (1C) (Fig. 4a); ESI-MS(m/z): 429.20 ([M+H]⁺) for C₂₃H₂₆N₂O₆ (Fig. 5a).

Compound NO-2

Pale yellow liquid; Thalleoquine test- negative, vitali-morin test- negative, Van urk’s test- positive; Rf- 0.64 (solvent: n-hexane: methanol: ethyl acetate: water (3:1:5:1v/v); ATR-FTIR cm^-1: 3363.92, N–H str. (indole); 2941.24, C-H str. (sp³); 1711.78, C=O str. (ester); 1243.98, C–O str. (Methoxy or ester group); 1149.95/1098/1037, C–N or C–O str. (amine or ether), 851.55 / 799 / 755.10, Aromatic C–H bending (out-of-plane) (Fig. 2b); ¹H NMR (Acetone d_6, 400 MHz), δH 3.59 (6H, singlet, OCH₃), 3.74 (3H, singlet, OCH₃), 9.25 (1H, singlet, NH) 1.17-1.18 (3H, doublet, CH₃), 4.60-4.65 (1H, doublet, Aromatic), 6.32-6.38 (1H, doublet, Aromatic), 7.27-7.29 (1H, doublet, Aromatic), 1.88-2.26 (2H, multiplet, CH₂), 2.58-2.68 (1H, multiplet, CH₂), 2.75-2.88 (1H, multiplet, CH), 2.90-2.92 (1H, multiplet, CH), 2.95-3.01 (5H, multiplet, CH₂ & CH), 4.20-4.23 (1H, multiplet, CH) (Fig. 3b); ¹³C NMR (101 MHz, Acetone d₆), δC 162.40 (1C), 149.70 (1C), 149.30 (1C), 143.50 (1C), 136.90 (1C), 134.50 (1C), 127.05 (1C), 122.60 (1C), 108.12 (2C), 94.80 (1C), 70.86 (1C), 59.85 (1C), 56.16 (1C), 52.25 (1C), 39.20 (2C), 38.20 (1C), 31.80 (1C), 21.60 (1C), 18.10 (1C) (Fig. 4b); ESI-MS(m/z): 413.20 ([M+H]⁺) for C₂₃H₂₈N₂O₅ (Fig. 5b).

Compound NO-3

Pale yellow liquid; Thalleoquine test- negative, vitali-morin test- negative, Van urk’s test- positive; Rf- 0.68 (solvent: n-hexane: methanol: ethyl acetate: water (3:1:5:1v/v); ATR-FTIR cm^-1: 3589.45, 3498.13, O–H str. (broad, strong); 2985.82, 2939.20, 2913.16, C-H str. (sp³, aliphatic); 1712.49, C=O str. (strong); 1642.91, C=C or C=N str.; 1412.96, 1364.14, C-H bending (CH₂, CH₃); 1244.66, 1149.43, C–O str. (ether); 1037.73, C–N or C–O str. (amine or ether), 934.54/856.34, Aromatic indole C–H bending (out-of-plane); 755.94, Ring deformation / aromatic substitution band (Fig. 2c); ¹H NMR (Acetone d_6, 400 MHz), δH 3.54 (6H, singlet, OCH₃), 3.60 (3H, singlet, OCH₃), 9.51 (1H, singlet, NH) 1.39-1.45 (3H, doublet, CH₃), 5.48 (1H, doublet, Aromatic), 6.32-6.38 (1H, doublet, Aromatic), 1.91-1.99 (2H, multiplet, CH₂), 2.08-2.13 (1H, multiplet, CH₂), 2.17 (1H, multiplet, CH), 2.23-2.97 (1H, multiplet, CH), 3.34-3.39 (5H, multiplet, CH₂ & CH), 4.14-4.18 (1H, multiplet, CH) (Fig. 3c); ¹³C NMR (101 MHz, Acetone d₆), δC 208.50 (1C), 149.30 (1C), 136.90 (1C), 134.50 (1C), 127.05 (1C), 108.12 (2C), 94.80 (1C), 60.20 (1C), 59.15 (1C), 56.16 (2C), 52.60 (1C), 46.00 (1C), 40.20 (1C), 35.90 (1C), 33.31 (1C), 27.94 (1C) 21.60 (1C) (Fig. 4c); ESI-MS(m/z): 373.21 ([M+H]⁺) for C₂₂H₂₈N₂O₃ (Fig. 5c). Fig. 2. ATR-FTIR spectra of (a) Compound NO-1 (b) Compound NO-2 (c) Compound NO-3

Figure 2: ATR-FTIR spectra of (a) Compound NO-1 (b) Compound NO-2 (c) Compound NO-3 Click here to View Figure

Figure 3: 1H NMR spectra of (a) Compound NO-1 (b) Compound NO-2 (c) Compound NO-3 Click here to View Figure

Figure 4: 13C NMR spectra of (a) Compound NO-1 (b) Compound NO-2 (c) Compound NO-3. Click here to View Figure

Figure 5: Mass spectra of (a) Compound NO-1 (b) Compound NO-2 (c) Compound NO-3. Click here to View Figure

After spectral interpretation, Compound NO-1, NO-2, NO-3 were identified as Methyl 5,6-dimethoxy-1′-methyl-2-oxa-4a’,5′,5a’,7′,8′,10a’-hexahydro-1’H,10’H-spiro[indoline-3,6′-pyrano[3,4-f]indolizine]-3′-carboxylate with a chemical formula of C₂₃H₂₆N₂O₆ (428 g/mol) (Fig. 6a), Methyl 10,11-dimethoxy-4-methyl-4a,5,7,8,13,13b,14,14a-octahydro-4H-indolo[2,3-a]pyrano[3,4-g]quinolizine-2-carboxylate with a chemical formula of C₂₃H₂₈N₂O₅, (412.47 g/mol) (Fig. 6b) and 1-(3-(2-Hydroxyethyl)-9,10-dimethoxy-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]quinolizin-2-yl)ethan-1-one with a chemical formula of C₂₂H₂₈N₂O₃ (372 g/mol) respectively (Fig. 6c).

The comparison of the three compounds with reported analogues in the chemical database, PubChem, indicates that NO-2 is closely related to a compound called Reserpiline (PubChem CID 67228)³⁰ and Isoreserpiline (PubChem CID 161345) ³¹, which share the same class of indolo-pyranoquinolizine alkaloids with similar substitutions and core structure, suggesting a known type of indole alkaloid derivative and NO-3 aligns well with Corynantheidine (PubChem CID 6540753)³², which has an indoloquinolizine core with similar methoxy and hydroxyethyl substitutions, supporting structural validation as related to known indoloquinolizine alkaloids. while NO-1 may represent a novel spiro structure combining indoline and pyranoindolizine moieties.

Figure 6: Predicted structures of (a) Compound NO-1 (b) Compound NO-2 (c) Compound NO-3. Click here to View Figure

In silico biological evaluation

Prediction using PASS online tool

Based on structure activity relationships, The probable biological activity of the isolated compounds (NO-1, NO-2 and NO-3) from ethanolic extract of N. oppositifolium was predicted using the PASS online tool. The three compounds NO-1, NO-2, NO-3 showed anti-diabetic and anti-cancer activities with Pa and Pi values as indicated in Table 2.

Table 2: PASS prediction results of isolated compounds

 Molecular docking analysis

All three isolated compounds demonstrated distinct binding affinities against diabetic (2JJK) and anticancer (3KOK) targets. Each compound’s binding energy provides insight into its relative interaction strength with both target proteins. NO-1 exhibited the strongest antidiabetic effect, while NO-2 displayed superior anticancer binding. Binding energies and interacting with key residues involved in substrate recognition and activity modulation are shown in Table 3 and Fig. 8 & Fig. 9.

Table 3: Comparative docking results

For the diabetic target protein 2JJK, standard inhibitors such as acarbose³³ generally show binding affinities in the range of –6.5 kcal/mol, interacting with key residues involved in substrate recognition and activity modulation, such as GlyA20, ArgA14, and ThrC19. Compound NO-1, with a binding energy of –7.4 kcal/mol and substantial hydrogen bonding with GlyA18, GlyC18, ThrC19, MetC10, and ArgA14, demonstrates competitive or even superior affinity and interaction compared to these reference drugs, highlighting its strong antidiabetic potential.

For the anticancer target protein 3KOK, commonly used reference drug, imatinib³⁴, tend to display binding energies around –6.5 kcal/mol, with hydrogen bonds formed at residues critical for cancer pathway modulation such as LysA54, ValA92, and ArgA51. Compound NO-2, with a binding energy of –6.0 kcal/mol and multiple hydrogen bonds with ValA92, ArgA51, ThrA52, LysA54, and LeuA53, shows reasonable binding and selectivity, although slightly lower than the standard, yet its targeted interactions can still justify its selection for further anticancer studies.

Figure 7: Binding energy (Kcal/mol) of compounds against target proteins. Click here to View Figure

When all three compounds are considered (Fig. 7), NO-1 stands out as the most effective antidiabetic agent based on its highest binding affinity (–7.4 kcal/mol) and extensive hydrogen bonding with key residues. NO-2, while less effective against the diabetic target, demonstrated the strongest anticancer activity (–6.0 kcal/mol) through multiple hydrogen bonds, reinforcing its candidacy for anticancer research. NO-3 displayed weaker interactions in both categories, suggesting limited therapeutic potential compared to NO-1 and NO-2.

Figure 8: Molecular docking results of (a) Compound NO-1 (b) Compound NO-2 (c) Compound NO-3 against fructose-1,6-bisphosphatase (FBPase, PDB ID: 2JJK) protein for anti-diabetic activity. Click here to View Figure

Figure 9: Molecular docking results of (a) Compound NO-1 (b) Compound NO-2 (c) Compound NO-3 against phosphoinositide 3-kinase (PI3K, PDB ID: 3KOK) protein for anti-cancer activity. Click here to View Figure

Conclusion

This study successfully isolated and characterized three indole alkaloids from Neisosperma oppositifolium leaf extract using maceration, Thin layer and column chromatography. Structural elucidation was confirmed by ATR-FTIR, ¹H NMR, ¹³C NMR, and ESI-MS. In-silico evaluation predicted significant anti-diabetic and anti-cancer activities, with compound NO-1and NO-2 showing the highest activities. These findings support further in-vitro and in-vivo studies to validate the therapeutic potential of N. oppositifolium in modern drug development. Despite promising docking and in silico results, this study has key limitations. The stereochemical configurations of the isolated compounds require further confirmation through techniques such as NOE experiments and X-ray crystallography to fully determine their structures and better understand their biological significance. Moreover, the predicted bioactivities remain unverified as this study lacks experimental biological validation. Future research must include comprehensive in vitro and in vivo testing to assess both efficacy and safety. In addition, investigating pharmacokinetics and toxicity is crucial before these compounds can be considered for therapeutic use.

Acknowledgment

The authors would like to thank all contributors and reviewers whose invaluable insights and expertise greatly enhanced the quality of this manuscript.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

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