DNA protection ability, antihyperglycemic potential and in-vivo toxicity assessment of arrowleaf tearthumb (Persicaria sagittata L.), a neglected aquatic plant species of Eastern Asia

Abstract Persicaria sagittata L. (common name arrowleaf tearthumb, American) is an herbaceous edible plant with characteristics sessile leaves mainly found in wetland areas of North America and Eastern Asia. In Eastern Himalayan Region of India, the ethnic communities consumed this plant as vegetables. The present investigation suggests the plant is endowed with bioactive compounds having potential DNA protection ability and antihyperglycemic activity. The DNA nicking assay revealed that the methanolic extract of this plant has the potential to protect plasmid DNA against hydroxyl damage. The α-glucosidase and α-amylase inhibitory assay of this methanolic extract suggest more effectiveness in inhibition of α-amylase than the α-glucosidase. Further, proximate composition, micronutrient, total phenolic and flavonoid content of this underutilised aquatic plant was determined. And lastly the in-vivo cytotoxicity study of Persicaria sagittata L. plant extract suggest that the plant is less toxic to in-vivo system. Graphical Abstract


Introduction
Diabetes mellitus is a metabolic disorder, which is associated with hyperglycemia and can be categorised into two types (Type 1 diabetes mellitus and Type 2 diabetes mellitus) depending on the mode of progression of the disease which can be categorised into two types: Type 1 diabetes mellitus and Type 2 diabetes mellitus. Moreover, hyperglycemic conditions when not checked and sustain for a longer duration, may cause severe long-term complications like cardiovascular disease, diabetic retinopathy, etc. (Pirart 1978;Reinehr 2013). The usage of carbohydrate digestion enzyme inhibitors (a-amylase and a-glucosidase) is one of the effective ways for managing diabetes by reducing postprandial hyperglycemia (Toeller 2010). The identification of such inhibitors of plant origin will have great importance for therapeutic purposes.
Persicaria (Polygonaceae) comprises about 131 species and broadly found in different part of the world. Many Polygonum spp. has been used as traditional medicines for the treatment of diabetes, diarrhoea, cough, urinary inflammation, etc. (Bothon et al. 2013;Uddin et al. 2016). In Polygonum aviculare L. the metabolites such as myricitrin, quercetin, polydatin and isoquercitrin were found to be responsible for its antihyperglycemic properties (Cai et al. 2020). Anthraquinone glucosides having bacterial neuraminidase inhibitors were found in Polygonum cuspidatum (Uddin et al. 2016).
Arrowleaf tearthumb (Persicaria sagittata L. syn. Polygonum sagittatum L.) is found in wetland areas of North America and Eastern Asia. The plant is a 50-70 cm long annual herb with alternate and widely spaced heart or arrowhead-shaped leaves and prickle along the stem. The flowers are white to pink in color and borne in 2-3 clusters. Even though ethnic people of North East Himalayan Region of India consumed the arrowleaf tearthumb as part of the food, the scientific validation for its nutritional and therapeutic properties is still missing, the present study emphasises the bioactivity (DNA protection ability and antihyperglycemic potential), in-vivo cytotoxicity (using Zebrafish embryo), proximate parameters and micronutrient compositions of this plant.

Results and discussion
Persicaria sagittata L. is an herbaceous plant mainly found in wetland habitats. The methanolic extract of P. sagittata L. displayed promising in-vitro DNA protection and anti-diabetic activity.
The fresh plant contained 85.96% of moisture. The proximate analysis revealed 1.19% ash, 0.24% crude fat, 10.87% total carbohydrates and 1.74% crude protein (Table S1). The results of the proximate analysis indicated higher total carbohydrates (10.87%) content. The plant has an energy value of 52.57 kcal per 100 g of fresh weight.
The micronutrient content per gram dry weight of the plant was found to be 46.08 mg Fe, 66.03 mg Mn, 16.94 mg Zn and 10.52 mg Cu (Table S1). Mn, being a part of essential nutrients, plays a critical role in many metabolic processes involved in the development process as a cofactor of metalloenzymes, immune system, nervous system and antioxidant enzyme system etc. (Trumbo et al. 2001). Iron is also one of the essential nutrients, which contributes to diverse metabolic processes, such as oxygen transport, nucleic acid synthesis and electron transport chain (Abbaspour et al. 2014).
The total phenolic and flavonoid content of P. sagittata L. methanolic extract was found to be 123.45 mg GAE and 4.19 mg QE per g of extract weight, respectively (Table S2). The earlier studies on P. cuspidatum reported the presence of health-beneficial bioactive metabolites such as flavonoids, anthraquinones and stilbenes (Shen et al. 2018).
In the in-vitro DNA nicking assay, we achieved a similar inhibition at lower concentration of the methanolic plant extract then trolox, suggesting a promising DNA protection ability against hydroxyl radicals generated by the Fenton reagent. The densitometry analysis of DNA nicking assay gel did not portray any nicked plasmid in the negative control (without Fenton reagent), while 95.7% nicked plasmid was observed in the positive control (with Fenton reagent) (Figure 1). The nicked plasmid percentage by Fenton reagent was reduced to 64.4%, 37.3% and 16.6% when treated with 2.5, 5 and 10 mg/mL of P. sagittata L. extract. On the other hand, nicked of 16.2%, 3.8% and 1.5% were observed in plasmids treated with 25, 50 and 100 mg/mL of Trolox ( Figure 1). This indicates the extract showed more effective than Trolox in protecting the plasmid DNA from hydroxyl free radical (Figure 1).
The earlier scientific investigation of a few Polygonum species suggests the antihyperglycemic potential (Nigam 2013;Faheemuddin et al. 2016;Rodrigues et al. 2017, Shen et al. 2018. Similarly, the in-vitro antihyperglycemic assay of P. sagittata L. methanolic extract indicated promising a-glucosidase and a-amylase enzyme inhibitory activity. In the a-glucosidase and a-amylase inhibitory assay, the IC 50 value of the methanolic plant extract was found to be 45.21 mg/mL and 23.33 mg/mL, respectively; whereas, the IC 50 values of the standard inhibitor acarbose in a-glucosidase and a-amylase inhibitory assay were 132.13 mg/mL and 11.66 mg/mL, respectively (Table S3). The lower IC 50 value of the methanolic plant extract as compared to the IC 50 value of acarbose in inhibiting a-glucosidase inhibitory assay indicates more specificity of the plant extract in inhibiting a-glucosidase than a-amylase enzyme. Even though commercially available inhibitors such as acarbose, voglibose, and miglitol are effective in controlling hyperglycemia, they tend to exhibit side effects like gastrointestinal discomforts including diarrhoea and flatulence (DiNicolantonio et al. 2015). On contrary, many of the plant-derived secondary metabolites have already been identified as good candidates for carbohydrate digestion inhibitors and can be used as an alternative to synthetic ones without adverse side effects (Kapoor 1990).
Further, the in-vivo cytotoxicity assay of P. sagittata L. methanolic plant extract was also performed using 72-hour post-fertilisation (hpf) zebrafish embryo. The LC 50 value of P. sagittata L. was found to be 69.8 ± 3 mg/mL. The study showed the death of embryo and yolk sac edema at very high concentrations of plant extract ( Figure 2B-C). In-vivo cytotoxicity assay indicated some toxicity of this plant at a very high concentration. The LC 50 value (69.8 ± 3 mg/mL) of the methanolic plant extract in the in-vivo cytotoxicity assay was found to be much higher than the IC 50 values for in-vitro a-glucosidase inhibition (45.21 ± 0.15 mg/mL) and a-amylase inhibition (23.33 ± 2.46 mg/mL). Moreover, the LC 50 value (69.8 ± 3 mg/mL) has been found to be 7 times higher than the concentration of the plant extract (10 mg/mL) resulting in only 16.6% nicked plasmid DNA in the DNA nicking assay. The higher LC 50 value than the IC 50 values of other assays performed suggests that it is safe for consumption as evident from less toxicity of the plant in in-vivo system at the concentration required for desired bioactivity. The overall result suggests a strong DNA protection ability of P. sagittata L. against hydroxyl free radicals and antihyperglycemic activity especially by inhibiting a-glucosidase enzyme.

Experimental
The experimental section is provided in supplementary material.

Conclusion
To the best of our knowledge, the present study is the first report on proximate composition, micronutrient contents, total phenolic content, total flavonoid content, invitro DNA protection ability and in-vivo toxicity study of Persicaria sagittata L. This is also the first report on the anti-hyperglycemic activity of the methanolic plant extract. The present study suggests that arrowleaf tearthumb (P. sagittata L.) is a potential candidate for developing nutraceuticals or functional foods and its consumption would contribute to healthy living.