Phytochemical screening, antibacterial, antioxidant, and anti-diabetic properties of Persicaria chinensis leaves

Introduction

Most countries employ conventional drug to cure numerous diseases, with over 80% using a wide range of plant components like bark, leaves, seeds, roots, flowers and fruits (WHO, 2022) [32] . About 70% of the rural citizens in India depend on herbal remedies to meet their medical care (Rahman et al., 2011; Pandey et al., 2013) [27,22] . Phytochemicals are bioactive molecules derived from plants that have a number of health advantages. These phytoconstituents include dietary fibres, phytosterols, isoprenoids, carotenoids, and polyphenols, which have an impact on immunity, regulate genes, and help lower the risk of getting infectious and non-infectious diseases (Leitzmann, 2016; Kumar et al., 2023) [19,16] . Globally, bacterial infections are the main cause of illness and death, and the emergence of antibiotic-resistant bacteria is currently a major global concern. Plants are a viable option for treating infectious disorders because they have bioactive chemicals with antibacterial qualities. These compounds, which include flavonoids, essential oils, and phenolic compounds, support plant extracts' antibacterial properties (Álvarez-Martínez et al., 2021) [9] . A wide variety of natural sources with antibacterial properties have been found through prior studies (Chassagne et al., 2021) [6] . Antioxidants are substances that prevent the production of free radicals during oxidation. There has been report of antioxidant properties in plants including lignans, coumarin, flavonoids, flavones, isoflavones, catechins, anthocyanin and isocatechins (Dinesh et al., 2011) [8] . Oxidative stress can be averted in cells by the action of enzyme systems like superoxide dismutase, and antioxidants like glutathione, mycothiol, and bacillithiol.

Diabetes mellitus (DM) is a life systemic disease that can alter the metabolism of carbohydrate, fat, and proteins characterized by hyperglycemia, impacting with a frequency of 6.1%. Diabetes is currently one of the top 10 causes of death and disability worldwide. By 2050, there will likely be 1.3 billion cases of diabetes worldwide (Ong, 2023) [12] . The exploration for low-risk and more adequate hypoglycemic agents has increased since the modern anti-diabetic drugs have numerous adverse effects (Campbell, 2007) [5] . Two essential enzymes for the breakdown of carbohydrates are αamylase and α-glucosidase. Reducing the likelihood of developing type 2 diabetes and managing postprandial hyperglycemia can be achieved by inhibiting them. There is a great deal of promise in using medicinal plants to treat DM. To effectively utilize the medicinal benefits of these natural resources, more research is necessary (Kooti et al., 2016; Jacob and Kannan, 2019) [15,4] . The Nagaland state of India has one of the most ethnic diversities, dependent on nature and natural products that intensifies the use and traditional practices of ethnobotanical values within the tribal populations yet, the knowledge on the principal and utilization of these plants persists unexplored. Persicaria chinensis belongs of the Polygonaceae family and it is among the most traditionally utilized plants in various folk medicines in Wokha, Nagaland, India. As a traditional natural plant pharmaceutical, the whole plant is used to eliminate toxins, reduce fever, to treat dysentery, eczema, corneal nebula, inflammatory skin disease, ulcer, allergies, insect sting, and snakebites. The rhizomes of the plant are utilized to remove heat, aid in detoxifying, and reduce edema (Anren, 1998) [1] . Additionally, the whole plant can be eaten as vegetable. Thus, screening of the in vitro techniques could provide the needed preliminary studies obligatory to elect crude extract of this plant with conclusively beneficial properties for further chemical and pharmacological analysis (Mathekaga and Meyer, 1998) [20] . In accountability of the importance of this plant, the present work was carried out.

Materials and Methods

Plant material

Collection of Fresh and healthy leaves of P. chinensis was done at Wokha, Nagaland, India. It was further subjected for identification and authentication at Siddha Central Research Institute (SCRI), Chennai, India. The individual herbarium voucher specimen with the code P20122305C was deposited in Department of Pharmacognosy, SCRI, India. The collected leaves were washed, shade-dried, ground into coarse granulate, and stored at room temperature for further experimental analyses.

Extracts preparation

In accordance with the polarities, 1 kg of the coarse powdered leaves was extracted with 3 L of hexane, followed by ethyl acetate and methanol. The solvent from the filtrate was evaporated at room temperature after being filtered using Whatman No. 1 filter paper. The yield of the extracts was recorded and stored for further experiments at 4 °C.

Phytochemical assay

The extracts were subjected to detect the presence of varied phytoconstituents using standard protocols (Roghini and Vijayalakshmi, 2018) [28] .

Total tannins content (TTC) determination

The TTC was estimated according to the method of Peri and Pompei (1971) [24] . The absorbance of the sample was determined at 725 nm, and TTC were given as mg GAE/g of dry extract.

Determination of Total phenol content (TPC)

According to the method of Lefahal et al. (2018) [8] , the TPC was calculated. The absorbance of the sample was measured at 765 nm and TPC was reported as mg GAE/g of dry extract.

Determination of Total flavonoid content (TFC)

The TFC was carried out according to the technique of Lefahal et al., (2018) [8] . The absorbance of the sample was measured at 510 nm and calculated as mg QE/g of dry extract.

Antibacterial activity

With using the method of the agar well diffusion assay, the antibacterial activities of extracts were assessed against gram-positive (Bacillus subtilis, Enterococcus faecalis, Staphylococcus saprophyticus, and Staphylococcus aureus) and gram-negative (Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumonia, Shigella flexneri, and Salmonella typhi) bacterial cultures. On sterile Mueller Hinton agar medium plates, test cultures that had grown overnight were swabbed, and wells were made to pour the extracts (100 µL; 50-100 µg/mL), and incubated at 37 °C for 24 h. The antibacterial activity of the extracts was revealed by measuring the inhibition zone (in mm).

In vitro antioxidant activity DPPH (2,2-diphenyl-1-picrylhydrazyl) assay

After combining 2.5 mL of 0.1 mM DPPH solution with several concentrations of extracts (5-320 μg/mL), the mixture was incubated for 30 minutes at room temperature in a dark space. At 517 nm, the mixture's optical density, or absorbance, was measured. The standard utilized was ascorbic acid (Perumal and Saravanabhavan, 2018) [25] . The DPPH inhibition (%) was calculated as mentioned below:

ABTS (2,2-axino-bis (3-rthylbenzothiazoline-6-sulphonic acid) assay

Extracts ranging in concentration from 5 to 320 μg/mL were combined with a solution of ABTS (7 mM) and K2S2O8 (140 mM). The mixture was measured at 680 nm after 30 minutes of incubation at 37 °C. The standard utilized was ascorbic acid (Perumal and Saravanabhavan, 2018) [25] . The ABTS inhibition (%) was calculated as mentioned below:

Hydrogen peroxide (H2O2) assay Different concentrations (5-320 μg/mL) of the extracts were added into H 2 O 2 solution (2mM). Following a 10-min incubation time, at 230 nm, the OD was determined with a blank solution. The standard utilized was ascorbic acid (Ruch et al., 1989) [29] . The H 2 O 2 inhibition (%) was calculated as mentioned below:

Nitric oxide (NO) assay Different concentrations (5-320 μg/mL) of the extracts were added into 10 mM solution of sodium nitroprusside. The mixture was incubated at room temperature for about 2h. It was added 0.5 mL of Griess reagent after the incubation period, and the OD was measured at 546 nm (Sreejayan and Rao, 1997) [30] . The NO inhibition (%) was calculated as mentioned below:

Hydroxyl radical scavenging assay (HRSA)

The extracts were diluted with 0.5 mL of 0.018% v/v EDTA solution, 1 mL of 0.85% v/v DMSO, and 0.5 mL of ascorbic acid (0.22%) at varying doses (5-320 μg/mL). The mixture was then incubated for 15 minutes at 80-90 °C in a water bath. After the incubation period, 1 mL of low-temperature pre-cooled TCA at a concentration of 17.5% (w/v) was added to terminate the reaction. Once added, 3 mL of Nash reagent was allowed to sit at room temperature for 15 minutes. Using a reagent blank as a reference, the color intensity was measured at 412 nm (Klein et al., 1991). To determine hydroxyl radical scavenging, the following formula was used:

In vitro antidiabetic assay α-amylase inhibition activity

With some slight modifications, the α-amylase inhibition assay was carried out using Unuofin et al. (2018) [31] methodology. After adding 100 μL of α-amylase (0.1 mg/mL) solution, various doses (10-320 μg/mL) of the crude extracts were incubated for 15 minutes at room temperature. It was then further filled with 100 μL of starch solution and allowed to sit at room temperature for one hour. It was then mixed with 100 μL of iodine reagent and 10 μL of 1M HCl. The standard was acarbose. The mixture was measured at 565 nm and α-amylase inhibition (%) was calculated as mentioned below:

α-glucosidase assay

The α-glucosidase inhibition assay was conducted adopting Apostolidis et al. (2007) [2] methodology. The 100 mM phosphate buffer (pH 6.8) was used to prepare the pNPG substrate solution. For about 10 minutes, 200 μL of αglucosidase solution was combined with extracts at varying concentrations (10-320 μg/mL). Subsequently, 400 μL of the prepared pNPG solution was added, and it was left to incubate for a further 20 minutes at room temperature. 1 mL of 0.1 M Na2CO3 solution was added, and the release of 4nitro phenol-a yellow-colored reaction mixture resulting from the degradation of pNPG-was measured by reading the optical density at 405 nm. Voglibose was employed as positive control. To calculate α-glucosidase inhibition, use this formula:

Statistical analysis

All the experimental data in this study was carried out by mean±standard deviation (mean ± SD).

Results and Discussion

Phytochemical screening

Phytochemicals have drawn a lot of interest since they can expand and shield sophisticated restorative pharmacophores (Patra et al., 2020) [23] . Therefore, for the identification of the chemical status of active substances of the test plant, phytochemical screening was performed with particular attention to their antioxidant and anti-diabetic activity. Various species of Persicaria have been widely used as a traditional ethnomedicinal system of therapeutics in many countries. From the qualitative findings, it was perceived that ethyl acetate extract of P. chinensis leaves substantiated the presence of tannins, alkaloids, terpenoids, phenol, flavonoids, carbohydrate, glycoside, and protein; whereas saponins was completely absent. In the case of hexane and methanol extracts, saponins were found completely absent (Table 1). Different phytochemicals of various extracts contain several compounds, including triterpenoids, saponins, gums and mucilage, and flavones and flavanones (Lai et al., 2012) +. The total content of tannins, phenols and flavonoids is tabulated (as shown in Table 2). Among three extracts tested, total content of tannins, phenolic, and flavonoids were found higher in ethyl acetate extract which showed maximum TTC, TPC, and TFC of 31.27±0.68 mg GAE/g of extract, 31.41±1.56 mg GAE/g dry extract, followed by 36.37±1.1 mg QE/g dry extract respectively. Methanol extract depicted TTC, TPC, and TFC of 23.02±0.23 mg GAE/g dry extract, 26.56±0.42 mg GAE/g dry extract, and 27.33±1.08 QE/g dry extract, respectively; while hexane extract revealed TTC, TPC, and TFC of 17.63±1.12 GAE/g of extract, 23.47±0.56 GAE/g dry extract, followed by 21.20±0.65 QE/g dry extract respectively.

Antibacterial assay

Plethora of studies has reported the different medicinal plant extracts of the antibacterial potential in the past, and our present investigation supported the earlier reports by Astal et al. (2005) [3] and Khan et al. (2007) [13] . Our results illustrated the effectiveness of leaves extracts from P. chinensis in inhibiting all the test bacterial organisms, except for the hexane and ethyl acetate extract against S. flexneri at 50 μg/mL, and methanol extract at the same concentration, which showed no activity against S. typhi. At a concentration of 100 μg/mL, ethyl acetate extract illustrated a maximum antibacterial activity of 26.66±2.08 mm against S. aureus. The present study revealed that ethyl acetate extract exhibited a strong bactericidal effect in comparison to hexane and methanol extracts (Table 3A-C). In vitro antioxidant assay DPPH test is frequently used to assess the antioxidative potency of natural products (Oyaizu, 1986; Prior et al., 2005) [21,26] . In our present study, the P. chinensis solvent extracts of hexane, ethyl acetate and methanol were assessed for their capacity to scavenge radicals using the DPPH radical scavenging test (Fig. 1A), and it was observed that ethyl acetate extract exhibited a substantial dose-dependent scavenging of DPPH, in contrast to hexane and methanol extracts. Furthermore, the IC 50 values of the extracts and the control ascorbic acid were calculated as 35.26±0.15 μg/mL (hexane), 26.88±0.04 μg/mL (ethyl acetate), 57.15±0.34 μg/mL (methanol), and 3.54±0.40 μg/mL (ascorbic acid) respectively. The findings of the ABTS assay revealed that ethyl acetate extract demonstrated the most potent antioxidant properties at higher concentrations (Fig. 1B). The IC 50 values of the extracts and the control ascorbic acid were estimated as 157.39±0.04 μg/mL (hexane), 106.42±0.56 μg/mL (ethyl acetate0, 144.12±0.33 μg/mL (methanol), and 3.54±0.40 μg/mL (ascorbic acid) respectively. The results for HRSA are shown in Fig. 1C. The IC 50 values for hexane, ethyl acetate, methanol extracts, and ascorbic acid were estimated as 226±0.51, 137.04±0.33, 173.10±0.61, and 3.54±0.40 µg/mL, respectively. Hydroxyl radical scavenging results depicted IC 50 values as 192.59±0.36 μg/mL (hexane), 117.86±0.49 μg/mL (ethyl acetate), 145.78±0.45 μg/mL (methanol), and 3.54±0.40 µg/mL (ascorbic acid), respectively (Fig. 1D). On the other hand, in terms of nitric oxide radical scavenging, the IC 50 values for hexane, ethyl acetate, methanol extracts, and ascorbic acid were estimated as 80.43±0.78, 17.04±0.49, 26.58±0.64, and 3.54±0.40 µg/mL, respectively (Fig. 1E). The amount of phenolic hydroxyl groups has a considerable impact on the free radical scavenging activity. The substantial abundance of phenolic compounds in the extract of plants has attracted considerable interest owing to their powerful antioxidant capabilities (Isik, 2020) [11] . In vitro antidiabetic assay Inhibition of α-amylase and α-glucosidase can be a major strategy for managing postprandial blood glucose levels in patients with diabetic type 2. This is because a mixed carbohydrate diet can greatly lower the subsequent increase in blood sugar levels due to the enzymes that regulate the digestion of carbohydrate (Gong et al., 2020) [10] . The present study demonstrated that the IC 50 value of hexane, ethyl acetate and methanol extracts were calculated as 8.17±0.73, 4.05±0.30, and 6.52±1.06 μg/mL, respectively.

Acarbose demonstrated potential inhibitory with an IC 50 value of 2.94±0.03 μg/mL (Fig. 2A). α-glucosidase inhibitor delays the eupepsia of carbohydrate and decelerates the adsorption. α-glucosidase inhibiting prospect of all the solvent leaves extracts were ascertained and the outcomes are illustrated in Fig. 2B. Previous studies had shown that miglitol and acarbose inhibit α-glucosidase competitively, reducing the absorption of starch and disaccharides (Davis and Granner, 2001) [7] . However, in the present study, Voglibose is employed as a standard drug for α-glucosidase inhibitor. In this context, all the extracts revealed prominent α-glucosidase inhibitory activity according to a dosage. However, ethyl acetate extract of P. chinensis displayed potent inhibitory activity with an IC 50 value of 6.81±1.11 μg/mL. The IC 50 value of the control Voglibose was observed as 4.05±0.05 μg/mL.

Conclusion

In summary, P. chinensis leaves extracts indicated the existence of varied phytochemicals. Among these three extracts, ethyl acetate extract depicted maximum TTC, TPC, and TFC of 31.27±0.68 mg GAE/g of extract, 31.41±1.56 mg GAE/g dry extract, followed by 36.37±1.00 mg QE/g dry extract respectively. Ethyl acetate extract displayed a pronounced antibacterial effect against tested pathogens in comparison to hexane and methanol extracts. In addition, ethyl acetate extract showed promising antioxidant activities by scavenging free radicals significantly reduced IC 50 values in a dose-dependent manner. Likewise, ethyl acetate extract depicted satisfactory anti-diabetic activities by inhibiting αamylase and α-glucosidase in a dose dependent manner.

This study will take further research to identify and isolate active bioactive compounds from ethyl acetate extract of P. chinensis leaves and suggest its potent traits for extensive pharmacological applications in future.

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