INTRODUCTION:

Natural products continue to play a pivotal role in pharmaceutical sciences, particularly as sources of bioactive compounds with therapeutic and preventive potential¹. Among these, plant-derived phenolic compounds and flavonoids have attracted considerable attention due to their well-documented antioxidant, anti-inflammatory, cardioprotective, neuroprotective, and chemo preventive properties^2,3.

Oxidative stress, resulting from an imbalance between the generation of reactive oxygen species (ROS) and endogenous antioxidant defences, is implicated in the pathogenesis of numerous chronic disorders including cardiovascular diseases, metabolic syndromes, neurodegenerative conditions, cancer, and aging-related complications^4,5. Consequently, the identification, characterization, and standardization of natural antioxidants remain a major focus in pharmaceutical and phytochemical research⁶.

Phenolic acids, flavonoids, tannins, stilbenes, and related polyphenols contribute significantly to the antioxidant capacity of medicinal plants and functional foods⁷. Their biological activity is strongly governed by structural attributes such as hydroxylation patterns, degree of conjugation, stereochemistry, and glycosylation⁸. Subtle structural differences often result in marked variations in antioxidant mechanisms, including free-radical scavenging, metal ion chelation, inhibition of lipid peroxidation, and modulation of redox-sensitive signalling pathways. Therefore, accurate structural elucidation and reliable quantification of these phytochemicals are essential not only for understanding structure–activity relationships⁹ but also for ensuring quality control, reproducibility, and regulatory acceptance of herbal medicines and plant-based formulations¹⁰.

Conventional analytical techniques such as spectrophotometric antioxidant assays and chromatographic methods are widely used for evaluating plant antioxidants¹¹. While these approaches are valuable, they often provide indirect or incomplete information. Spectrophotometric assays estimate total antioxidant capacity but do not identify individual active constituents, whereas chromatographic techniques typically require reference standards and extensive sample preparation¹². In this context, nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful, non-destructive analytical tool capable of delivering comprehensive qualitative and quantitative insights into complex phytochemical matrices¹³.

NMR spectroscopy offers unique advantages for pharmaceutical and phytochemical investigations. One-dimensional (^1H and ^13C) and two-dimensional (COSY, HSQC, HMBC, NOESY) NMR techniques enable unambiguous structural elucidation of phenolic and flavonoid compounds, including information on molecular connectivity and substitution patterns^14,15.

Plant Material and Authentication:

Leaves and fruits of Ziziphus nummularia (Burm. f.) Wight & Arn. used in this study were obtained from cultivated plants at Mohanlal Sukhadia University, Udaipur, Rajasthan, India. As the material was derived from cultivated sources, no specific collection permits were required. All procedures complied with institutional and national guidelines for the ethical use of plant materials in research^16,17.

Fresh leaves and fruits were collected from the botanical garden of Government Science College, Udaipur (approximate coordinates: 24.5854°N, 73.7125°E) and processed immediately for extraction and analysis.

Proton NMR Analysis:

¹H Nuclear Magnetic Resonance (NMR) spectroscopy was employed for the identification of phytoconstituents present in the leaf and fruit extracts of Z. nummularia. NMR spectroscopy is based on the principle that nuclei with a non‑zero spin absorb and re‑emit radiofrequency energy when placed in an external magnetic field; this interaction produces characteristic spectral signals that reflect the molecular environment of hydrogen atoms in the sample^18,19.

Purified samples were dissolved in suitable deuterated solvents, including deuterated chloroform (CDCl₃), deuterium oxide (D₂O), carbon tetrachloride (CCl₄), or deuterated dimethyl sulfoxide (DMSO), and transferred into NMR tubes for analysis. Tetramethylsilane (TMS) was used as an internal reference standard. Chemical shifts (δ) were reported in parts per million (ppm) and calculated using the equation:

where Δν represents the difference in resonance frequency between the sample and the reference (TMS) in Hertz, and ν₀ is the operating frequency of the spectrometer^18,20.

All ¹H NMR spectra were recorded at the NMR facility, Punjab University, Chandigarh, India.

RESULTS AND DISCUSSION:

¹H NMR Analysis of Extracts

The proton NMR spectral data of leaf and fruit extracts of Ziziphus nummularia, prepared by both maceration and Soxhlet extraction methods, are summarized in Tables 1.1 and 1.2. The tables present chemical shift ranges (in ppm), relative peak areas (%), and the corresponding functional groups identified based on literature assignments^21,22,23.

In the leaf extracts obtained by maceration, prominent signals in the range of 3.310–3.127ppm were observed, which are characteristic of phenolic protons ²⁴. Similarly, in the Soxhlet‑extracted leaf sample, peaks in the range of 0.994–0.901ppm, consistent with phenolic groups, were detected. In the fruit extract prepared by the Soxhlet method, phenolic signals were also identified in the range of 3.725–3.556ppm.

Functional group assignments from the ¹H NMR spectra indicate that the leaf extracts of Z. nummularia contain a mixture of phytochemicals including phenolic compounds, carbonyl derivatives, alkenes, and carboxylic acids (Table 1.1). Comparable profiles were observed in the fruit extracts, suggesting that both leaves and fruits share several metabolite classes, although the relative intensities of specific signals varied between extraction methods (Table 1.2).

The detection of phenolic protons in multiple spectral regions aligns with previous reports on medicinal plants, where such groups contribute to antioxidant activity and other bioactivities^26,27. The presence of carboxylic acids and carbonyl functionalities supports earlier phytochemical characterizations of the genus Ziziphus, which has been associated with various therapeutic effects²⁷.

Taken together, the ¹H NMR results demonstrate that both maceration and Soxhlet extraction successfully enriched the extracts with diverse bioactive constituents, and that ¹H NMR spectroscopy provides rapid qualitative insights into the metabolic composition of Z. nummularia extracts.

Table: 1.1 NMR analysis of leaves extract by maceration:

S. No.	Peak range	Functional groups	% area
1	3.310 – 3.271	Alcohols, phenols ethers, or esters	0.89
2	3.261 – 3.2440	Alcohols, phenols ethers, or esters	1.65
3	3.169 – 3.127	Alcohols, phenols ethers, or esters	3.64
4	2.8852 – 1.234	Aromatics, carbonyls, (ketones, esters, aldehydes, acids, amides) • alkenes	0.58
Fruits extract spectra maceration
5	4.90 – 4.89	Alkene sp2 hybridized C-H’s	1.65

Table: 1.2 NMR analysis of leaves and fruits extract by soxhlation:

S. No	Peak range	Functional groups	% area
1	1.760 – 1.721	Carbonyls, (ketones, esters, aldehydes, acids, amides) • alkenes, or • aromatics	0.63 – 1
2	1.410 – 1.299	Carbonyls, (ketones, esters, aldehydes, acids, amides) • alkenes, or • aromatics	2.82 – 2.92
3	1.169 – 1.113	Carbonyls, (ketones, esters, aldehydes, acids, amides) • alkenes, or • aromatic	0.85 – 1.84
4	0.994 – 0.901	Alcohols, phenols • carboxylic acids	3.08
Fruits extract by soxhlet method
1	3.725 – 3.670	Alcohols, phenols • ethers, or • esters	3.58
2	3.602 – 3.5561	Alcohols, phenols • ethers, or • esters	3.82
3	3.196 – 3.066	Alcohols, • ethers, or • esters	1.99
4	2.95 – 2.67	Alcohols, • ethers, or • esters	0.49 – 0.83

Figure 2.1 Chromatogram of NMR spectra of leaves extract using maceration method

Figure 2.2 Chromatogram of NMR spectra of fruits extract using maceration method

Figure 2.3 Chromatogram of NMR spectra of leaves extract using soxhlet method

Figure 2.4 Chromatogram of NMR spectra of fruits extract using Soxhlet method

CONCLUSION:

Ziziphus nummularia is a medicinally important plant traditionally used for the management of metabolic, inflammatory, and oxidative stress–related disorders²⁸. In the present study, comparative phytochemical profiling of leaf and fruit extracts was conducted using proton nuclear magnetic resonance (¹H NMR) spectroscopy following maceration and Soxhlet extraction.

The ¹H NMR spectra revealed the presence of several major functional groups, including phenolic compounds, carbonyl derivatives, alkenes, and carboxylic acids, in both leaf and fruit extracts. Signals attributable to phenolic protons were comparatively abundant, suggesting a high content of phenolic metabolites, which are often correlated with antioxidant potential^25,29.

These findings demonstrate that ¹H NMR spectroscopy is a rapid, non‑destructive, and reliable analytical tool for profiling major metabolite classes in complex plant extracts. The observed phytochemical diversity supports the traditional therapeutic uses of Z. nummularia and provides a foundation for further targeted pharmacological and bioactivity studies.

DATA AVAILABILITY STATEMENT:

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

COMPETING INTERESTS:

The author declares that there are no competing interests

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Received on 28.01.2026 Revised on 02.03.2026

Accepted on 26.03.2026 Published on 08.07.2026

Available online from July 13, 2026

Res. J. Pharmacognosy and Phytochem. 2026; 18(3):249-252.

DOI: 10.52711/0975-4385.2026.00036

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