Abstract

   Blackberries have gained considerable attention due to their high
   antioxidant content and potential health benefits. This study compared
   the metabolite profiles of six blackberry cultivars and investigated
   their biological activities. The metabolites extracted from
   blackberries were analyzed using metabolomics, and their biological
   activities and mechanisms were confirmed using in vitro models and
   network pharmacology. Among the cultivars examined, “Kiowa” ripe
   berries exhibited the highest antioxidant and anti-inflammatory
   activities. These effects were primarily attributed to the accumulation
   of flavonoids (quercitrin and luteolin) and anthocyanin (cyanidin
   3-O-glucoside) in the phenylpropanoid pathway. Furthermore, our
   research identified 13 blackberry metabolites that interacted with 31
   genes, including AKT1, CASP3, JUN, MAPK8, NOS3, NQO1, and HMOX1 which
   play roles in reducing oxidative stress, protecting cells from damage,
   and suppressing inflammation. These findings suggest that blackberry
   metabolites, such as quercitrin, luteolin, and cyanidin 3-O-glucoside,
   may exert therapeutic effects by modulating specific genes and pathways
   associated with antioxidant and anti-inflammatory responses. This
   research is promising not only for plant breeders but also for those
   interested in harnessing the health-promoting properties of
   blackberries.

   Keywords: blackberry, metabolomics, antioxidant, anti-inflammation,
   network pharmacology

1. Introduction

   Scientific investigations have unequivocally established the manifold
   health benefits of incorporating fruits and vegetables into human diet
   [[30]1]. Among the numerous bioactive compounds found in these
   nutritional powerhouses, flavonoids, a type of polyphenolic compounds,
   have gained prominence due to their roles in conferring antioxidant,
   anti-inflammatory, and anticancer properties [[31]2]. The inclusion of
   fruits and vegetables in the daily dietary practices has garnered
   strong recommendations from a plethora of studies. Notably, berries,
   which ranked among the top 50 antioxidant-rich foods, are particularly
   encouraged [[32]3].

   Blackberries, a delectable fruit produced by plant species within the
   genus Rubus in the family Rosaceae [[33]4], have attracted attention
   due to their substantial nutritional values. The commercial production
   of blackberries is estimated to be approximately 154,578 tons annually
   in North America, Europe, Asia, South America, Oceania, Central
   America, and Africa [[34]5]. Blackberry cultivation has rapidly
   increased because of the introduction of new and improved cultivars and
   consumers’ interest in its high nutritional and medicinal value
   [[35]6,[36]7]. Blackberry is a rich source of phenolic compounds such
   as anthocyanins, flavan 3-ols, and phenolic acids [[37]4]. In
   particular, cyanidin 3-O-glucoside is consistently the predominant
   anthocyanin, which is responsible for the dark color of blackberries
   and has been reported to exert prominent bioactivity, including
   anti-inflammatory, antioxidant, anti-cancer, and anti-neurodegenerative
   activities [[38]8,[39]9]. A previous study reported that the
   antioxidant capacity of blackberries was higher than those of
   blueberries, raspberries, and cherries [[40]10]. Moreover, blackberries
   have a higher inhibitory activity against free radicals than
   cranberries and strawberries [[41]11]. The demand for high-quality
   blackberries has led to the development of numerous cultivars with
   varying levels of bioactive compounds and antioxidant capacities.
   Considering the rapid expansion of applications using various
   blackberry cultivars and the importance of their efficient use, it is
   essential to understand their nutritional profiles. Comparing different
   blackberry cultivars on the basis of their metabolite profiles and
   biological activities can help consumers, food processors, and health
   professionals in selecting the most appropriate cultivars for their
   specific needs. In addition, breeders can focus on selecting cultivars
   with the desired traits and genetic markers, thereby accelerating the
   development of blackberry varieties that satisfy both consumer
   preferences and industry demands.