Analysis of morphological characteristics and chemical composition of pomegranate seeds from 19 pomeg- ranate varieties in China

【Objective】Pomegranate (Punica granatum L.) is globally esteemed for its nutritional and health-promoting properties. Traditionally research on pomegranate mainly focuses on its arils (juice) and peel. The seeds, often considered a by-product of fresh consumption and juice processing, have received comparatively less systematic attention despite their significant influence on sensory quality and their potential reservoir of bioactive compounds. Seed hardness, a critical trait affecting consumer acceptance and a common genotypic classification marker (soft, semi-soft, hard), is not fully understood in terms of its underlying morphological and biochemical determinants within the diverse germplasms in China. Furthermore, comprehensive data on the chemical composition, particularly the detailed fatty acid profiles of the pomegranate seeds from major Chinese production regions such as Xinjiang, remains scarce. This study aimed to conduct an integrated morphological and chemical characterization of the seeds of 19 pomegranate cultivars sourced from distinct provinces across China. The primary objectives were: (1) to quantify key morphological parameters, including seed dimensions, weight, and hardness; (2) to elucidate the microstructural basis of hardness using scanning electron microscopy; (3) to systematically analyze the proximate composition (ash, fat, protein, fiber) and bioactive compound profiles (phenolics, flavonoids, proanthocyanidins); (4) to determine, for the first time, the detailed fatty acid composition of seed oils from key Xinjiang cultivars; and (5) to evaluate the antioxidant capacity and establish its relationship with the identified chemical constituents. This work seeks to establish a scientific foundation for the use of pomegranate seeds in nutraceutical, pharmaceutical, and functional food industries.【Methods】The seeds of 19 pomegranate cultivars (Anhui, Henan, Sichuan, Shandong, Shaanxi, Xinjiang, Yunnan) were analyzed. For morphological analysis, the seed length, width, and individual seed weight were measured. The hardness was determined using a sclerometer (FHT-15). The microstructure and thickness of the sclerotesta (hardened seed coat) were examined via Scanning Electron Microscopy (SEM, Hitachi SU1510), with thickness measured on acquired images using ImageJ software. Chemical analyses followed standardized protocols. The proximate composition was determined as follows: crude protein via the Kjeldahl method; crude fat by Soxhlet extraction with petroleum ether; ash content by incineration; acid-insoluble lignin (Klason lignin) by TAPPI T222 om-83; and cellulose content using the anthrone-sulfuric acid method. For detailed lipid profiling, the fatty acid composition was analyzed specifically for six cultivars from Xinjiang. The fatty acid methyl esters (FAMEs) were prepared by saponification and esterification and analyzed by Gas Chromatography-Mass Spectrometry (GC-MS, Thermo Fisher ISQ 7000) using a DB-WAX column for separation, identification, and quantification. The bioactive compounds were extracted and quantified spectrophotometrically: the total soluble (SPC) and insoluble phenolic content (InSPC) determined via the Folin-Ciocalteu method; the total flavonoid content (TFC) using the NaNO2-AlCl3-NaOH assay; and the total proanthocyanidin content (TPAC) by the HCl-butanol method. The antioxidant capacity was evaluated as Trolox Equivalent Antioxidant Capacity (TEAC) using the ABTS+ radical scavenging assay. All measurements were performed with at least three independent replicates. Statistical analysis included one-way ANOVA with Tukey's post-hoc test for mean comparisons and Pearson correlation analysis to examine relationships between variables, conducted using R software (v4.5.0).【Results】Morphological analysis revealed considerable diversity. The seed length ranged from 5.30 to 8.33 mm, width from 2.70 to 4.03 mm, and the lengthto- width ratio indicated shapes from nearly round (e.g., AH1, 1.51) to elongated (e.g., AH2, 3.10). Based on established hardness standards, the varieties were classified into 7 soft-seed (＜3.67 kg· cm-2 ), 3 semi-soft-seed (3.67-4.2 kg · cm- 2 ), and 9 hard-seed types (＞4.2 kg · cm- 2 ). A very strong positive correlation (r = 0.9, P＜0.001) was found between the seed hardness and sclerotesta thickness (0.25- 0.54 mm). The SEM micrographs provided structural evidence, showing that the harder seeds possessed a more ordered, thicker honeycomb structure of sclerotesta cells, whereas the softer seeds exhibited a more disordered structure with thinner cell walls and significantly more perforations. The proximate composition varied widely: the crude fat (11.20%-34.90%), the crude protein (9.93%-18.85%), the cellulose (10.63%-21.88%), and the lignin (14.38%-27.50%). The lignin content showed a weak but significant negative correlation with the hardness (r = -0.6, P＜0.01). The fatty acid profile of the six Xinjiang seed oils was exceptional, dominated by polyunsaturated fatty acids (PUFAs, 83.77%-87.24% ). The punicic acid, a conjugated linolenic acid isomer characteristic of pomegranate seed oil, constituted 77.13% to 79.86% of the total fatty acids. The saturated fatty acids (SFAs, mainly palmitic and stearic acid) ranged from 4.70% to 6.60%, and the monounsaturated fatty acids (MUFAs, solely oleic acid) accounted for 3.37%-5.27%. Regarding bioactives, the soluble phenolic content (SPC) ranged from 0.83 to 1.53 mg · g- 1 , and the insoluble phenolic content (InSPC) from 0.43 to 1.20 mg · g- 1 , with SPC being the predominant fraction (-58% of the total phenolic content, TPC). The total flavonoid content (TFC) and total proanthocyanidin content (TPAC) varied between 0.31-0.50 mg·g-1 and 0.28-0.57 mg·g-1 , respectively. The TEAC values, indicating antioxidant capacity, ranged from 9.75 to 12.88 μmol· g-1 . The statistical analysis revealed strong positive correlations between the TEAC and both TPC (r = 0.96, P＜ 0.001) and TFC (r = 0.96, P＜0.001). Importantly, the correlation between the insoluble phenolics and the TEAC was stronger than that of the soluble phenolics, whereas the TPAC showed only a moderate correlation (r = 0.46, P＜0.01) with antioxidant activity.【Conclusion】This comprehensive study demonstrated that the pomegranate seeds from Chinese cultivars should be valuable source of lipids, proteins, fiber, and potent bioactive compounds, far surpassing their status as mere agricultural by- products. The research revealed that the seed hardness, a primary sensory attribute, was predominantly determined by the thickness and microstructural organization of the sclerotesta, providing a reliable morphological marker for cultivar classification and breeding selection. The exceptionally high and well-characterized punicic acid content in Xinjiang’s pomegranate seed oils underscored their potential as a premium, region-specific source of the unique and health-promoting fatty acid. Furthermore, the significant antioxidant capacity, which is strongly linked to the total phenolic content—and particularly the insoluble-bound fraction—highlighted the importance of considering the entire phenolic matrix, not just soluble extracts, when assessing the health benefits and processing potential of the pomegranate seeds. These findings collectively would establish a solid scientific foundation for the integrated and valueadded utilization of the pomegranate seeds.