LC-HRMS and HPLC Optimization of Sulforaphane Extraction from Broccoli Seed Hydrolysate for Anti-Obesity Research

Current pharmacotherapies combating obesity (which remains a major global health challenge) exhibit limitations in long-term efficacy, safety, and cost. While broccoli (Brassica oleracea L. var. italica) seeds are a rich source of sulforaphane (SFN), a compound which has demonstrated anti-obesity effects, the bioavailability of SFN from those seeds is limited due to suboptimal conversion conditions and competing nitrile-formation pathways. A joint study conducted by researchers at Kyung Hee University, Kookmin University, and Milaebioresources Co., Ltd (all in Seoul, Republic of Korea), aimed to standardize a high-yield, SFN-rich broccoli seed hydrolysate (BSH), to evaluate its anti-obesity efficacy in mice fed a high-fat diet (HFD), and to elucidate its mechanism via molecular docking and molecular dynamics (MD). SFN hydrolysis was optimized by Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and High-performance liquid chromatography (HPLC) across pH, temperature, time, and L-ascorbic acid. A paper based on this study was published in Phytomedicine.¹

Defined by the World Health Organization (WHO) as an abnormal or excessive fat accumulation that presents a risk to health—commonly classified as a body mass index (BMI) ≥ 30 kg/m²—obesity greatly increases the potential of major chronic diseases, such as type 2 diabetes, cardiovascular disease, and several cancers.^2,3 The global prevalence of the disease has increased over recent decades, with more than 890 million adults (about 13 % of the world’s adult population) currently living with obesity; thenumber is expected to reach around 1.02 billion (18 % of adults) by 2030.⁴ Once primarily attributed to overeating and physical inactivity, obesity is now recognized as a possible result of interactions between genetic predisposition and neuroendocrine dysregulation.⁵

Broccoli is widely recognized for its health benefits, stemming from the vegetable’s diverse bioactive compounds, including glucosinolates, isothiocyanates, flavonoids, and vitamins.⁶ Broccoli seeds have been reported to contain higher levels of glucoraphanin (GRA)—a glucosinolate precursor of the potent bioactive compound sulforaphane (SFN)—compared to mature plants or sprouts.⁷ SFN displays a range of pharmacological properties, such as antioxidant, anti-inflammatory, and metabolic regulatory effects.^8,9

In this study, 5-hydroxytryptamine receptor 2A (5-HT2A) binding was probed by network pharmacology, molecular docking, and 100 ns MD. HFD-fed male C57BL/6 mice (n = 6 per group) received oral broccoli seed water extract (BWE), BSH, or SFN for 8 weeks. Anti-obesity efficacy was then assessed by body/tissue weights, micro-computed tomography (Micro-CT), histology, and serum lipid profiling, and 5-HT2A, adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and other markers in epididymal white adipose tissue (eWAT) and liver were assessed by quantitative real-time PCR (qRT-PCR) and Western blot.¹

According to the researchers,production of SFN from broccoli seeds was maximized under two hydrolysis regimes: short-term (pH 4 at 35°C for 2 h) and long-term (pH 5 at 25°C for 24 h). In silico analyses predicted stable binding of the sulforaphane-glutathione conjugate (SFN-GSH) to 5-HT2A. In HFD-fed mice, BSH (40-800 mg/kg, p.o.) dose-dependently attenuated HFD-induced body weight gain (800 mg/kg vs HFD, p < 0.001). BWE 400 mg/kg and BSH 40 mg/kg showed broadly comparable effects on body weight. Additionally, gene and protein analyses in eWAT and liver showed 5-HT2A (Htr2a) suppression, restored AMPK phosphorylation, and downregulation of lipogenic regulators. Co-administration of the AMPK inhibitor Compound C (5 mg/kg, i.p.) with BSH, however, abolished these BSH-induced effects. Oral administration of a sulforaphane standard (1-10 mg/kg) produced similar effects to BSH (10 mg/kg vs HFD, p < 0.01), suggesting that the anti-obesity effect of BSH is mediated primarily by SFN.¹

“In this study,” write the authors of the paper,1 “SFN-rich BSH ameliorated obesity in HFD-fed mice via a peripheral 5-HT2A suppression-AMPK activation axis.” In the opinion of the research team, however, additional mechanistic clarification will require additional studies which much define the role of 5-HT2A in adipocytes more precisely.¹

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References

1. Jeon, Y. J.; Park, J; Park, K. T. et al. Sulforaphane-Rich Aqueous Broccoli Seed Extract Suppresses Diet-Induced Obesity via 5-HT2A/AMPK Signaling in Mice. Phytomedicine 2026, 153, 157935. DOI: 10.1016/j.phymed.2026.157935
2. Kinlen, D.; Cody, D.; O’Shea, D. Complications of Obesity. QJM2018, 111 (7), 437-443. DOI: 10.1093/qjmed/hcx152
3. Shafiee, A.; Nakhaee, Z.; Bahri, R. A. et al. Global Prevalence of Obesity and Overweight Among Medical Students: A Systematic Review and Meta-Analysis, BMC Public Health2024, 24 (1), 1673. DOI: 10.1186/s12889-024-19184-4
4. Lingvay, I.; Cohen, R. V.; le Roux, C. W. et al. Obesity in Adults. Lancet 2024, 404 (10456), 972-987.DOI: 10.1016/S0140-6736(24)01210-8
5. Masood, B.; Moorthy, M. Causes of Obesity: A Review. Clin. Med. (Lond)2023, 23 (4), 284-291. DOI: 10.7861/clinmed.2023-0168
6. Andrés, C. M. C.; Pérez de la Lastra, J. M.; Munguira, E. B. et al. The Multifaceted Health Benefits of Broccoli—A Review of Glucosinolates, Phenolics and Antimicrobial Peptides. Molecules 2025, 30 (11), 2262. DOI: 10.3390/molecules30112262
7. West, L. G.; Meyer, K. A.; Balch, B. A. et al. Glucoraphanin and 4-hydroxyglucobrassicin Contents in Seeds of 59 Cultivars of Broccoli, Raab, Kohlrabi, Radish, Cauliflower, Brussels Sprouts, Kale, and Cabbage. J. Agric. Food Chem.2004, 52 (4), 916-926. DOI: 10.1021/jf0307189
8. Cascajosa-Lira, A.; Prieto, A. I.; Pichardo, S. et al. Protective Effects of Sulforaphane Against Toxic Substances and Contaminants: A Systematic Review. Phytomedicine 2024, 130, 155731. DOI:10.1016/j.phymed.2024.155731
9. Mangla, B.; Javed, S.; Sultan, M. H. et al. Sulforaphane: A Review of its Therapeutic Potentials, Advances in its Nanodelivery, Recent Patents, and Clinical Trials. PhytOther Res.2021, 35 (10), 5440-5458. DOI: 10.1002/ptr.7176