**1. Introduction**

Many countries have been trying to eradicate knotweed due to its invasiveness, although it has many practical uses, including improving the health of animals through feed supplementation. This report suggests that it could be used as a feed supplement for horses, thereby improving their fitness. This study was part of a larger project in which knotweed had been administered to several animal species, the health of which improved.

Japanese knotweed (*Reynoutria japonica*, syn. *Polygonum cuspidatum*), including its hybrid (*R.* × *bohemica*), has been thoroughly studied from a range of perspectives, both as an "enemy" of native flora due to its invasiveness and as a plant with many beneficial uses, including positive health effects [1–4]. As a source of resveratrol and other substances with anti-ageing and other positive effects on both animals and humans, the entire knotweed plant can also be expected to have positive effects on the health of horses. Resveratrol has already been used in horses with positive results. Resveratrol is, however, extracted mainly from roots and rhizomes; the aboveground portions of knotweed, which contain equally powerful antioxidants, deserve more thorough investigation [5]. Neochlorogenic acid was identified as an efficient antioxidant in the aboveground knotweed biomass [6]. High amounts of carotenoids in knotweed leaves are comparable to those in spinach [7]. In addition, knotweed plants produce a significant number of bioactive constituents, namely, phenolic substances derived from resveratrol, such as piceid, piceatannol, astringin and emodin, which are also found in aboveground biomass albeit in smaller quantities than in rhizomes and roots.

**Citation:** Kováˇrová, M.; Madˇera, P.; Frantík, T.; Novák, J.; Vencl, Š. Effects of Knotweed-Enriched Feed on the Blood Characteristics and Fitness of Horses. *Agriculture* **2022**, *12*, 109. https://doi.org/10.3390/ agriculture12010109

Academic Editors: Lubomira Gresakova and Emilio Sabia

Received: 28 November 2021 Accepted: 10 January 2022 Published: 13 January 2022

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Among the bioactive components of knotweed, resveratrol and, more recently, piceid (also called polydatin) have been thoroughly studied. Resveratrol was found to modulate many different pathways, as it binds to numerous cell signalling molecules, modulates cell regulatory genes, activates transcription factors, suppresses pro-inflammatory genes' expression and inflammatory biomarkers, induces antioxidant enzymes and inhibits protein kinases and the expression of angiogenic and metastatic gene products. It thus has strong potential as a treatment for inflammatory, cardiovascular, pulmonary and age-related diseases such as cancer, diabetes and even Alzheimer's disease. These chronic illnesses and neurological and autoimmune diseases are accompanied by the dysregulation of multiple cell regulating pathways and are connected with inflammation. Resveratrol targets sirtuin, adenosine monophosphate kinase, nuclear factor-κB, inflammatory cytokines, anti-oxidant enzymes along with cellular processes such as gluconeogenesis, lipid metabolism, mitochondrial biogenesis, angiogenesis and apoptosis. Immunity is regulated by its interfering with immune cell regulation, pro-inflammatory cytokines' synthesis and gene expression [8]. Similarly, piceid regulates lipid metabolism, helps treat cardiovascular diseases [9] and diabetic cardiomyopathy via its anti-inflammatory and anti-oxidative effects [10], preserves mitochondrial function in the central nervous system, offers a therapeutic option for Spinal cord ischemia/reperfusion injury [11], decreases the levels of reactive oxygen species in neurons from the ischemic cortex, ameliorates oxidative stress and mitochondria-dependent apoptosis [12], fights neurodegenerative diseases including Alzheimer's, Parkinson's, cognition/memory dysfunction, brain/spinal cord injuries, ischemic stroke and miscellaneous neuronal dysfunctionalities [13], promotes a radiosensitising effect on osteosarcoma cancer cells, reduces clonogenic survival of tumor cells and induces osteogenic differentiation, alone and in the presence of ionising therapy [14]. Emodin has recently garnered increased interest, with refs. [15,16] reporting its antiviral and anti-inflammatory activities against SARS-CoV-2, for the reason that it is one of the components of the Chinese medicine LQF, the Lianhua-Qingwen formula based on 11 herbs with 61 compounds that is used for the prevention and treatment of viral diseases, including coronavirus disease 2019 (COVID-19).

Three resveratrol analogue glucosides, namely, piceid, piceatannol glucoside and resveratroloside, were found to have antibacterial effects [11], and their bioactivity was comparable to that of resveratrol, which is released in the gu<sup>t</sup> due to glucoside hydrolysis. Piceid exhibited more powerful effects than resveratrol against hepatitis B virus [17,18]. Piceatannol had even more efficient anti-inflammatory, immunomodulatory, anti-proliferative, anti-leishmanial, and anti-leukaemic activities than resveratrol. Resveratroloside competitively inhibited α-glucosidase, thus alleviating postprandial hyperglycemia in diabetic mice [19]. Crude extract from knotweed roots and rhizomes inhibited such troublesome bacteria as *Staphylococus aureus*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, *Bacillus cereus*, *Escherichia coli*, *Listeria monocytogenes* and *Salmonella anatum* [20,21].

It might therefore be expected that there are even greater health effects of knotweed plants than there are of their individual components.

In horses, resveratrol has already been used with positive results. Inflammation is accompanied by increased enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO), resulting in the production of the highly oxidative acid HOCl [22,23]. Resveratrol was shown [24] to substantially decrease HOCl production and mitigate inflammation in horses. Inhibitory effects of resveratrol on equine neutrophil myeloperoxidase were described [25,26]. It was found that four weeks of supplementation with 1 g/day resveratrol in old horses decreased the inflammation-induced production of cytokinin both in vitro and in vivo, indicating that resveratrol has substantial potential for the treatment of acute and chronic inflammatory horse diseases [27]. Resveratrol was also reported to reduce the gene expression of inflammatory mediators, thus allowing even old horses to move freely in training and competitions [28,29]. Resveratrol has been administered to performance horses in a number of food supplements, such as Equithrive Joint® and Resverasyn®, with the aims of reducing the effects of laminitis and slowing ageing.

Metabolic efficiency in animals decreases with age, leading to increased levels of creatine kinase and glucose. A decrease in both creatine kinase and glucose levels in horses receiving resveratrol and hyaluronic acid (EquithriveJoint®) was found [30]. EquithriveJoint® was administered to old, lame horses and a decreased serum malondialdehyde (MDA) concentration and modulated serum levels of glutathionperoxidase (GPx), catalase and superoxiddismutase (SOD) were found, indicating protective effects of EquithriveJoint® against oxidative stress and ageing [31]. A beneficial effect of EquithriveJoint® on the performance of horses with hindlimb lameness treated with triamcinolone was also demonstrated [32]. Supplementation with these plant-derived phenolics in old horses was suggested [33] to lower the doses of nonsteroidal anti-inflammatory drugs and thus reduce their side effects. Since laminitis and other diseases impair older horses, the effects of resveratrol on the health of young horses are unclear.

Based on the above evidence, there is good reason to assume that knotweed would help treat horse health issues in a similar way to resveratrol. Although the diseases occur mainly in older horses, the aim of this project was to find out whether knotweed can also improve the fitness of young horses. Reports on the health effects of supplementation with knotweed herbs in animals are scarce; a drug containing knotweed (Praziver®) helped cure equine helminthiasis [34].

Knotweed is not only one of the best sources of resveratrol, its derivatives, carotenoids [7] and neochlorogenic acid [6], but it also contains other substances, the effects of which are still not well known. A conservative approach to the use of knotweed as a dietary supplement in animals thus persists, although there have been many reports of various animal species grazing on it without problems. It is also on the list of safe plants for fodder, as it was introduced to Europe for use as feed for domesticated animals. Aboveground parts of knotweed are consumed by humans in some areas, e.g., in Japan and North America [6].

This study thus aimed to fill the knowledge gap regarding the effects of knotweed on the health status of young horses. We performed two consecutive experiments, during which the dry aboveground knotweed biomass was administered as a dietary supplement to a large number of young stallions.

#### **2. Materials and Methods**

#### *2.1. Horses and Husbandry*

Most of the horses involved in the two experiments belonged to the warmblood breed known as the Czech Warmblood, while the minority of coldblood horses belonged to two breeds, the Czech-Moravian Belgian Horse, a breed constituted from the Czech Coldblood and Moravian Coldblood, and the Silesian Norik. These horses were kept at the Regional Stud Farm Tlumaˇcov, Czech Republic, which provides breeding services, rears foals and young stallions and performs early testing and training of stallions. Most of the stallions are sold at the age of 2–3 years, and only the selected ones are further kept for breeding and training. In October 2021, i.e., 2 years after the experiments, those warmblood horses that had been involved in the experiments and later sold to the horse keepers in the Czech Republic who go<sup>t</sup> them involved in sport activities, were tracked in the records of the Czech Equestrian Federation: https://www.jezdectvi.org/kone (accessed on 25 October 2021).

In winter, the horses were kept in individual boxes on a deep straw bed and fed individually with hay, with a feed supplement and with an experimental mixture, which were always consumed without leftovers. From May to October/November they stayed on pastures at grass only. In boxes, only the 3-year-old stallions were under training, receiving 30 min of daily exercise. All of the horses were healthy and under veterinary control.
