Nepal is home to more than fifty nine culturally rich ethnic and indigenous groups. Many of them have their medical practices handed down orally from generation to generation. One approximation puts that up to 70-80% of the rural population fully depends on medicinal plants for their primary healthcare (Ghimire et al., 2011).Nepal is a country known for abundant natural resources, endowed with enough medicinal plants that have been consumed by local people since time immemorial without knowing its biological potential and chemical composition (Young and Woodside, 2001).So these medicinal plants are needed to be explored significantly in a scientific basis which would provide a new dimension to the pharmaceu-tical or therapeutic field. These medicinal plants play a significant role in the management of several illnesses caused by the radicals formed in the body by different chain reactions. Formation of free radicals in human body causes oxidative damage to the biomolecules which ultimately lead to health disorders such as cancer, diabetes, inflammation, asthma, cardiovascular diseases, neurodegenerative diseases, and premature aging (Parajuli et al., 2013). At present most of the synthetic antioxidants are commercially available but are known to have carcinogenicity and other side effects when taken in vivo. Hence, their use is being restricted nowadays, and there is increasing interest in finding out safer and bioactive natural antioxidants from medicinal plant sour-ces (Sharma et al., 2018). DPPH free radical scavenging assay has been primarily used by various researchers as a quick, easy, reliable, and reproducible parameter in search of in vitro antioxidant activity of a pure natural compound as well as plant extracts. Antioxidant com-pounds offer hydrogen atom to the free radicals resulting in neutral compounds with loss of color (Subedi et al., 2014). The importance of natural phenolic compounds from plant materials show the antioxidant activity and also enhance their redox properties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quencher (Rezaeizadeh et al., 2011). Polyphenol compounds are secondary metabolites found in numerous plant species, and they are reported to have multiple functions to counteract the free radicals and inhibit different types of oxidizing enzymes (Kazeem et al., 2013).

Diabetes mellitus (DM) is a complex disease that is characterized by a gross derangement in carbohydrate, protein, and fat metabolism. It is a progressive metabolic disorder characterized by chronic hyperglycemia that eventually causes damage, dysfunction, and failure of different organs, mainly the eyes, kidneys, nerves, heart, and blood vessels (Alagesan, 2014). Type I diabetes results from the inadequate synthesis of insulin by β-cells of the pancreas, while type II diabetes is characterized primarily by insulin resistance; a condition in which peripheral cells do not usually respond to insulin (Arumugam et al., 2013).

The disease, especially Type II DM, has turned into a global health problem and is the third leading cause of death having a high occurrence and mortality rate. In Nepal, 526,000 cases of diabetes were reported in 2015 (Najafian, 2014). Due to becoming a major health problem, multiple therapies have been developed for type 2 diabetes. One of the best therapeutic targets is α-amylase. Alpha-amylase catalyzes the hydrolysis of glucosidic linkage in starch to oligosaccharides, whereas membrane-bound intestinal glucosidase hydrolyze oligosaccharides to glucose (Sales et al., 2012). Inhibitio­­n of α-amylase therapy is responsible for delaying the absorption of glucose after a meal. Several α-amylase inhibitory compounds have been isolated from medicinal plants that lead to the development of new drugs with high potentiality and lower adverse effect than the existing ones (Kwon et al., 2007). Synthetic drugs are reported to cause various side effects such as abdominal distention, flatulence, and diarrhea owing to the excessive inhibition of pancreatic α-amylase. This results in abdominal bacterial fermentation of undigested carbohydrates in the colon. Hence, at present, there is an increasing interest among food scientists to identify natural sources of α-amylase and α-glucosidase inhibitors for the dietary management of type II diabetes (Piette and Kerr, 2006). It is estimated that more than 800 plant species have hypoglycemic activity, and more than 450 plants have been experimentally tested (Ciulei, 1982). Therefore, it is urgent to identify and explore the antioxidant, total phenolic and flavonoid contents and alpha-amylase inhibitors from the medicinal plants of Nepal.

Thus, the present investigation was undertaken to make a comparative study of antioxidant activity, estimation of total phenolic and flavonoid contents and alpha-amylase inhibitory activity from some medicinal plants, that is, Woodfordia fructicosa, Tectaria coadunate, Abrus precatorius, Prunuscerasoids, Eclipta prostrate, Eclipta prostrate, Poranopsis paniculata, Chenopodium album, Oroxylum indicum, Curcuma caesia, and Butea monosperma used traditionally for the treatment of a number of illnesses including diabetes.

Chemicals and reagents

Chemicals used in this study were methanol (Merck, Germany), porcine pancreatic alpha-amylase, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and ascorbic acid (Sigma, Aldrich, USA). All the needed chemicals used in this research work were of the commercially available analytical grade.

Plant collection and extract preparation

Ten medicinal plants were collected from 12 kilo VDC of GorkhaDistrict of Nepal based on their ethnobotanical uses as shown in Table 1. All collected plants were identified by an expert from the Central Department of Botany, Tribhuvan University, Kirtipur Kathmandu, Nepal. 100 g powder of each plant was extracted by cold percolation method in methanol (200-300 ml) at room temperature for 48 h with constant agitation. The mixture was filtered through clean cotton and filtrate was concentrated at the temperature lower than the boiling point of methanol under reduced pressure by rotary evaporator to get the crude extract. Finally, the concentrated extracts were fridge dried to yield a dry powder.

Phytochemical screening

Phytochemical screening is the method of finding the primary, secondary metabolites present in the plant extracts. The phytochemicals as secondary metabolites were investigated by following the standard protocol put forward by Kim et al. (2003).The analysis of the presence of leading groups of natural constituents present in the different plant extracts was done by the color reaction using different specific reagents.

Total polyphenol content determination

Total phenolic contents (TPC) of all selected plant extracts were determined using Folin-Ciocalteu Reagent (FCR) and gallic acid as a standard compound following the procedure put forward by Zhou et al. (2010). Briefly, the stock solution of all the extracts was prepared by dissolving 10 mg in 1 ml of methanol (10 mg/ml). Serial dilutions were carried out to get the concentration of 0.125, 0.25, 0.5 and 1.0 mg/ml. To these diluted solutions, 10% FCR and 7% Na₂CO₃ were added and incubated for 30 min, followed by measurement of absorbance at 760 nm using the UV-visible spectrophotometer. The calibration curve was constructed using the solution of gallic acid as standard in methanol using the concentration ranging from 10-100 µg/ml. Based on this standard calibration curve of gallic acid, the concentrations of the individual samples were calculated. The results of TPC are measured in milligrams of gallic acid equivalent (mg GAE) per gram of dry extract (Kalita and Barman, 2013). TPC in the plant extracts taken under study was calculated by using regression equation:

y = 0.0023x

R² = 0.9902, obtained from the calibration curve of gallic acid (Piette and Kerr, 2006).

Total flavonoid content determination

Aluminum chloride colorimetric method was adopted with some modification for flavonoid content determination(Process et al., 2013). Serial dilutions of concentration of 0.125, 0.25, 0.5, and 1.0 mg/ml were made from the stock solution of all the extracts. At zero time 0.3 ml 5% NaNO₂ (sodium nitrite) was added. After 5 min, 0.3 ml of 10% AlCl₃ (aluminium chloride) was added and kept to stand

for 6 min. Then 2 ml of 1M NaOH was added to the mixture and shaken well. The absorbance was recorded at 510 nm using UV-visible spectrophotometer. The calibration curve was constructed with the help of standard quercetin solution in methanol with the concentration ranging from the 10-100 µg/ml. Total flavonoid content (TFC) in the plant extract was expressed in terms of the milligram of quercetin equivalent per gram of the dry mass (mg QE/g) (Jamuna et al., 2012). TFC in the plant extracts was calculated by using regression equation y =0.0174x, R² = 0.9974, obtained from the calibration curve.

Antioxidant activity

The ability of different plant extracts to scavenge DPPH free radicals was performed following the standard protocol adopted by Jamuna et al. with some modification (Jamuna et al., 2012). In brief, 2 ml of different extract solution (20, 40, 60, 80, and 100 µg/ml) of each plant samples was mixed with 2 ml of DPPH solution. The mixture was kept to stand for 30 min. Then the absorbance was measured   at    517 nm  using     UV   spectrophotometer.   Radical scavenging activity of each sample was calculated by using the formula;

Radical scavenging (%) = [(A_o-A_s)/A_o] × 100

Where:

A₀ = Absorbance of the control (DPPH solution + methanol),A_s = Absorbance of the test sample

Control was the test solution without sample, and ascorbic acid was used as the positive control. The absorbance was measured at least in triplicate. The inhibition curve was plotted for the triplicate and represented as a percentage of mean inhibition ± standard deviation, and the IC₅₀ values were obtained. The linear regression of the rate of radical scavenging versus concentration was used for the calculation of the concentration of each plant extract required for 50% inhibition of DPPH activity (IC₅₀).

Alpha-amylase inhibition assay

Alpha-amylase inhibition assay was performed using a standard where the undigested starch due to enzyme inhibition was detected at 630 nm (blue, starch-iodine complex) described by Meyer et al.  (1982). The stock solution of all the plant extracts was made by dissolving 10 mg in 10 mL of dimethylsulphoxide (DMSO) (1000 µg/ml). The substrate was prepared by dissolving 200 mg starch in 25 ml of NaOH (0.4 M) by heating at 100°C for 5 min. The pH of the mixture was adjusted to 7.0 after cooling, and the final volume was made up to 100 ml using distilled water. Acarbose was used as Positive control. 400 µl of substrate solution was pre-incubated at 37°C for 5 min with 200 µl of acarbose or plant extract at varying concentrations (40, 80, 160, 320 and 640 µg/ml), followed by 200 µl of 50 µg/ml α-amylase (20mM phosphate buffer with 6.7 mMNaCl, pH 6.9), and incubated at 37°C for 15 min. Termination of the reaction was performed by adding 800 µl of HCl (0.1M). Then, 1000 µl of iodine reagent (2.5 mM) was added, and absorbance was measured at 630 nm. The assay was carried out in triplicates using a spectrophotometer. Percentage of inhibition was calculated using the formula;

% Inhibition = (1-[Abs2-Abs1/Asb4-Abs3]) × 100

Where:

Abs1 is the absorbance of the incubated mixture containing plant sample, alpha-amylase, and starch. Abs 2 is the absorbance of the incubated mixture of sample and starch.Abs3 is the  absorbance  of the incubated mixture of starch and α-amylase.Abs4 is the absorbance of the incubated solution containing starch.

Statistical analysis

Data were recorded as the mean of (±) standard deviation of three determinations of absorbance for each concentration, from which the linear correlation coefficient (R²) value was calculated using MS Office Excel 2007. The linear regression for a straight line is:

y = mx + c;

The concentration of the extract was calculated using the regression equation.

Toxicity

The toxicity test was performed by using brine shrimp assay following the standard protocol put forward by Joseph et al. (2016) 20 mg of the sample to be tested was dissolved in 2 ml methanol. The solution thus prepared was used as stock solutions. The newly hatched brine shrimp nauplii (Artemia selina) was introduced to the solution of crude plant extracts with different concentrations: 1000, 100 and 10 μg/ml. The control was performed by adding the solvent used to dissolve the extracts. The entire test was performed in a temperature controlled room at 20°C, under continuous light. After 24 h, the numbers of survivors were counted with disposable pipettes, and the number of deaths at each dose was recorded. This method evaluates the toxicity of plant extracts towards the nauplii by determining the LC₅₀ (µg/ml). Compounds of LC₅₀ value less than 1000 ppm are considered as pharmaceutically active.

Phytochemical screening

The results of phytochemical screening are shown in Table 2. The result shows that glycosides and terpenoids were present in most of the extract, whereas some extracts showed the presence of reducing sugars and saponins. Flavonoids were present only in W. fructicosa, P. ceraoids,  A. precatorius,  E. prostrate  and   C. album.

Alkaloids were present only in T. coadunate and B. monosperma. The absence of alkaloids in most of the plant extracts may be due to the decomposition of alkaloids while concentrating the extract by the rotatory evaporator. The results presented in the Table 2 are slightly different from the data present in the literature of some plants. The outcome of phytochemical screening shows the presence of different phytochemicals of similar plants is due to variation in altitude, different environ-mental conditions, method and time of sample collection, extraction procedure and also due to lab setup and chemical grades.

Total phenolic content (TPC)

The TPC of different plant extracts (mg gallic acid equivalent per g dry extract) are tabulated in Table 3. The results demonstrate that the total phenolic content was found high in P. cerasoides (805.48±0.024 mg GAE/g extract) and low in B. monosperma (277.39±0.108 mg GAE/g extract) while the rest have moderate values. The total phenolic content was compared with the previous result in which the total phenolic content of M. oleifera flowers were recorded as 19.31 mg/g of the gallic acid equivalent of total phenolic content in the dry extract (Hasan et al., 2013).The present study showed that plants are the good sources of polyphenols as secondary metabolites.

Total flavonoid content (TFC)

The TFC of different plant extracts is shown in Table 3. The antioxidant property of flavonoid depends on their structure, particularly hydroxyl position in the molecule and their ability as an electron donor to a free radical (Sharma et al., 2018).Among the studied plant extracts, there is variation in total flavonoid contents ranging from O. indicum (16.96±0.015 TFC mg QE/g extract) to W. fructicosa (722.76±0.108 TFC mg QE/g extract). Also, the extract of T. coadunate, P. cerasoides, E. prostrate, C. album showed high total flavonoid content while the rest had a significantly low value of total flavonoid content. Previous results showed that total flavonoid content was found to be 96.15 ± 4.26 mg of quercetin equivalent/100 g (aqueous extract), 85.18 ± 4.21 mg of quercetin equivalent/ 100 g (hydro-alcohol extract), 96.01 ± 2.99 mg of quercetin equivalent/ 100 g (ethanol extract) (Das et al., 2007). This study shows plants are rich in flavonoid content. Although, the quantitative determination of flavonoid compounds in plant extracts is influenced by their structural complexity, diversity, nature of analytical assay method, selection of standard, and presence of interfering substances.

DPPH free radical scavenging activity

DPPH free radical scavenging activities of selected medicinal plants are presented in Figure 1. The percentage scavenging effect on the DPPH radical was concomitantly increased with the increase in concentration of the plant extracts from 20-100 µg/ml. The antioxidant potential is in an inverse relation with IC₅₀ value; the lower value of IC₅₀ indicates high antioxidant potential. The IC₅₀ value of the plant extracts along with the standard ascorbic acid is presented in Table 3.Effective results were obtained from methanolic extract of W. fructicosa (24.40±0.383 µg/ml), T. coadunate (15.17±0.180 µg/ml), P. cerasoides (7.54±0.223 µg/ml), E. prostrate (10.78±0.307 µg/ml) and Chenopodium album (11.69±0.362 µg/ml) which showed the strongest DPPH radical scavenging activity as their IC₅₀ value was close to standard ascorbic acid (39.85±0.025 µg/ml). The previous result showed the IC₅₀ value of P. cerasoides was 56.00±0.242 μg/ml that is the value close to IC₅₀ value of ascorbic acid as standard IC₅₀ value 25.55±0.156 μg/ml (Joseph et al., 2016).

Alpha-amylase inhibition assay

Alpha-amylase inhibitory activity of plant extract was determined using the quantitative starch-iodine method. The result of α amylase inhibition activity is shown in Figure 2. The result of α-amylase enzyme inhibition activity in terms of IC₅₀ value is shown in Table 4.  In thepresent study, methanolic extract of different parts of plants was investigated for their potential to inhibit α-amylase activity. The IC₅₀ value of standard acarbose was 27.93±0.07 µg/ml. Among the selected plants, methanol extract of A. precatorius root showed highest α-amylase inhibition activity with an IC₅₀ value of 70.29±0.14 µg/ml. Along with other methanolic plant extract also showed effective results such as T. coadunata rhizome extract (80.89±0.09 µg/ml),O. indicum bark extract (192±0.08 µg/ml), W. fructicosa flower extract (236±0.15 µg/ml), P. cerasoids bark extract (253±0.16 µg/ml). Lowest α-amylase inhibition property was shown by methanolic plant extract of C. album (1168±0.07 µg/ml),methanolic rhizome extract of C. caesia(950±0.20 µg/ml), methanolic leaf extract of P. paniculata (1335±0.04 µg/ml), methanolic bark extract of B. monosperma (1360±0.05 µg/ml).The result showed a dose-dependent increase in percentage inhibitory activity of α- amylase by all the ten plant extracts. Reddy et al.(2010) analyzed the α-amylase enzyme inhibition activity by oral administration of ethanolic extract of A. precatorius. The serum glucose value of 131.16±1.939 mg/dl was found, whereas standard Glibenclamide value was 96.5±1.607 mg/dl (Wink, 2008).

Brine shrimp toxicity

The degree of lethality was found to be directly proportional to the concentration of the extracts where maximum mortalities of the brine shrimp larvae took place at the concentration of 1000 µg/ml, and least mortalities were at10 µg/ml.  Those  having  LC₅₀  values less than 1000 µg/ml are supposed to be pharmacologically active. The extract of E. prostrate was toxic against brine shrimps having LC₅₀ value of 6.3 µg/ml. P. cerasoides ranks as mild toxic plant species with LC₅₀ value 33.88 µg/ml. Besides, the extracts of C. caesia (138.04 µg/ml), P. paniculata(177.82 µg/ml), A. precatorius (489.78 µg/ml) and W. fructicosa (912.01 µg/ml) showed significant lethality.

This study provides some scientific support for their traditional use for the management of several mentioned ailments. Data on biological activities of many medicinal herbs are tremendously increasing. However, it is im-practical to specify the performance of multi-component mixture, as that plant extracts comprise a wide range of phytochemical constituents, to only a single component from that extract (Singh and Hamal, 2013).Since extracts of W. fructicosa and P. cerasoides are the better sources of bioactive chemical constituents that can be attributed through antioxidant, total phenol, and flavonoid content determination, further in vitro and in vivo bioactivity of these extracts needs to be assayed much to identify the typical modes of action of the extracts and isolated pure compounds that can shape potential drug discovery. Here, it is concluded that further bioassay-guided fractionation and isolation approaches will be required on the active plant extract to identify the compound responsible for the promising in-vitro anti-diabetic activity.

The authors have not declared any conflict of interests.

The authors are grateful to the Central Department of Chemistry, Tribhuvan University for laboratory facilities.