BIBECHANA Vol. 20, No. 2, August 2023, 190–199 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Analysis of phytochemical, antioxidant, and α-amylase inhibition capacity of methanol extract of six plants from Kaski, Gulmi and Rupandehi districts of Nepal Bimala Subba1,∗, Namrata Subedi1, Sanju Parajuli2 1Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal 2Botany Department, Central Campus of Technology, Hattisar, Dharan, Nepal ∗Corresponding author. Email: bimalasubba@gmail.com Abstract This study was designed to explore phytochemicals and biological activities of methanolic ex- tract of six traditional medicinal plants ( Clerodendrum trichotomum, Mallotus philipinensis, Dioscorea-bublifera, Rubia cordifolia, Viscum articulatum) of Nepalese origin. Plant extracts were prepared in methanol through cold percolation. Analysis of phytochemical constituent, antioxidant capacities and brine shrimp lethality assay of the test plant samples were carried out using standard methods. The 1,1-diphenyl-2-picryl hydrazyl (DPPH) method was used to study antioxidant activity of different plant extracts. Furthermore, starch-iodine method was used to study the inhibition effect of extract on α-amylase. Phytochemical analysis showed the presence of phytochemicals like alkaloids, flavonoids, phenolic content, glycosides, reduc- ing sugars, etc. in six medicinal plants. Brine shrimp lethality assay suggested the presence of pharmacologically active compounds. Total phenolic content and total flavonoid content of C. trichotomum’s leaves extract were found to be higher with 212.742 mg GAE/g and 112 mg Q/g respectively with strong antioxidant activity. Similarly, the α–amylase inhibition of R. cardifolia’s root extract and C. trichotomum‘s leaves extract was found to be 252.44±0.55 µg/mL and 293.33± 0.81 µg/mL comparative with IC50 value 119.063±0.73 µg/mL of stan- dard acarbose that showed remarkable antidiabetic property among six samples. The results, obtained here suggested that six medicinal plants i.e. C. trichotomum, M. philipinensis, D. deltoidea, D.bublifera, R. cordifolia, and V. articulatum, Nepal origin showed biological ac- tivity by targeting multiple drug targets which justifies their traditional uses. This is the first finding that C. trichotomum‘s leaves can be a promising source for the development of natural antioxidant and antidiabetic agents. Keywords C. trichotomum, M. philipinensis, D. deltoidea, D. bublifera, R. cordifolia, V. articulatum,. Article information Manuscript received: March 25, 2023; Accepted: June 30, 2023 DOI https://doi.org/10.3126/bibechana.v20i2.56226 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 190 http://nepjol.info/index.php/BIBECHANA bimalasubba@gmail.com https://doi.org/10.3126/bibechana.v20i2.56226 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ B. Subba et al./ BIBECHANA 20 (2023) 190-199 191 1 Introduction Many diseases have been cured with plants by re- lating in various formula such as vegetables, fruit, spices, tea, decoction, etc., since ancient time. To- day’s modern society is slowly returning to ancient medicinal system as the number of people who still prefer using traditional folk medicines is increas- ing per year. Plants comprise a wide diversity of compounds, especially secondary metabolites with anticancer, antibacterial, antidiabetics, antitumor, antiviral, analgesic and many other activities to a greater or lesser extent. Notable examples of these secondary metabolites include phenols, flavonoids, alkaloids, terpenoids, saponins, glycosides, tannins etc. [1, 2]. Antioxidants compounds have the ability to pro- tect the body from reactive oxygen species. Reac- tive oxygen species (ROS) become toxic and cause the harmful effects like oxidative stress in absences of proper amount of antioxidants in our body. DPPH scavenging (2, 2-diphenyl-1-picrylhydrazyl) assay is a popular method for screening in-vitro antioxidant of plant extract. DPPH radical in methanol shows strong absorption at 517 nm. On increasing the concentration of extracts decreases the value of maximum absorbance suggest that scavenging of free radical by electron donation [3]. The types of diabetes mellitus according to the American Diabetes Association in 1997 are type 1, type 2, other specific types, and gestational dia- betes. It is characterized by high blood sugar (glu- cose) levels that consequence from defects in in- sulin secretion, or resistant in its action, or both. Normally when there is an elevated level of glu- cose in the bloodstream, the pancreas releases in- sulin which then binds a membrane protein of a cell and results a series of reactions that induce glucose transporters to move into the membrane and facil- itate the movement of glucose into the cell. Type 2 Diabetes [T2D] is characterized by an increase in insulin resistance and decrease beta-cell function and chronic hyperglycemia. Inhibition of α amylase activity is one of the alternative pathways for the control of postprandial hyperglycemia in diabetic patients. Nepal has many plants with medicinal values as it is rich in biodiversity. Some of them are used in traditional medicine that usually has a pharma- cological or biological activity for use in pharma- ceutical drug discovery and drug design and some are still not explored scientifically for medicinal values [3, 4]. The biological activities of the test plants i.e C. trichotomum, M. philipinensis, D. del- toidea, D. bublifera, R. cordifolia, V. articulatum had been reported by many research articles. Al- though these plants are reported for their uses such antioxidant, antidiabetic, antimicrobial, reducing high blood pressure, or improving blood circulation, the same plant grown in Gulmi, Rupandehi, Kaski district of Nepal has not been explored yet. The genetic and geographical variation plays significant role for the dissimilarity of chemical constituents of the same plant. The growing stages of the con- cerned plant at the time of the collection also plays a role for the variation of the chemical constituents of the same plants. Usually synthetic drugs contain a single active compound targeting a specific drug target but plant extracts may contain various active ingredients aiming at multiple drug targets [5]. On the basis of these reports, the present study purposes to calculate the polyphenols and flavonoids in the methanolic extract from different parts of such six medicinal plants i.e. C. trichoto- mum, M. philipinensis, D. deltoidea, D. bublifera, R. cordifolia, V. articulatum to determine their an- tioxidant potential and to evaluate its inhibitory properties on the α-amylase activities. 2 Materials and Methods 2.1 Collection of plant materials and preparation their methanol extract Various plant parts of six medicinal plants were collected from the farmland of Gulmi, Rupandehi, Kaski district of Nepal. The plants were identified with literatures and matched with the voucher spec- imens deposited at National Herbarium and Plant Laboratories, Godavari, Kathmandu. The collected plant parts were cleaned, sliced into small pieces, and shade dried for 10-15 days. Then it was ground into a powder and stored. The leaves extract of six plants was prepared by cold percolation method in methanol solvent. 2.2 Chemicals and reagents The chemicals used in this study were of the com- mercially available analytical grade. The methanol solvent was purchased from Merck, Germany. Sim- ilarly, porcine pancreatic α-amylase, 2,2-diphenyl- 1-picrylhydrazyl (DPPH), and ascorbic acid were purchased from Sigma-Aldrich, USA. 2.3 Qualitative phytochemical analysis Phytochemicals were identified by various color re- actions [6]. In brief, all plant materials were com- pletely extracted by percolation with methanol and subjected to phytochemical screening. The pres- ence of main groups of natural constituents in dif- ferent extractive solutions was analyzed by using different specific reagents. B. Subba et al./ BIBECHANA 20 (2023) 190-199 192 2.4 Brine shrimp lethality assay Brine shrimp lethality assay was carried according to standard protocol [7]. Briefly, sample stock so- lutions were prepared by dissolving 200 mg of each plant extract in 2 mL acetone. The test was con- ducted in 15 mL test tubes and sterilized artificial sea water (final volume 10 mL). From each stock solution 500 µL (equivalent to 1000 ppm), 50 µL (equivalent to 100 ppm) and 5 µL (equivalent to 10 ppm) were transferred to total nine test tubes, three test tubes for each dose level. The control was performed by adding the solvent used to dis- solve the extract (acetone) in the assay. The solvent was evaporated by standing overnight. After com- plete evaporation of solvent, 5 mL of sea water was added to each test tube with gentle shaking to en- sure that the compounds diffused adequately in the aqueous solution. Ten Salina nauplii were counted macroscopically in the stem of a Pasteur pipette against a lighted background and transferred into each sample tube and the solutions were made to 10 mL with artificial sea water. After 24 hours, the numbers of survivor were counted with the help of disposable pipettes. The entire experiment was per- formed in a temperature controlled room at 28 °C under the continuous supply of light by table lamp (60 Watt). The surviving nauplii were counted with the aid of a 3x magnifying glass after 24 hours. The mean mortality at the three dose levels for each ex- tract was determined. No death was observed in the control tubes. The LC50 (Lethal concentration for 50% mortality) values was determined using the probit method, as the measure of toxicity of the extracts [8]. 2.5 Determination of total phenolic con- tent in the plant extracts Total phenolic content in plant extract was calcu- lated by Folin-Ciocalteu Colorimetric method with some modifications [9]. An aliquot of 1 mL of each leaf extract (0.5 -1.0 mg/mL) were mixed with 5 mL Folin–Ciocalteu phenol reagent (10%) and 4 mL of 7% Na2CO3 to get a total volume of 10 mL. The blue colored mixture was shaken well and incubated for 30 minutes at 40 ºC in a water bath. The ab- sorbance of the mixture was measured at 760 nm. The samples were prepared in triplicate for each analysis and the mean value of absorbance was ob- tained. Standard calibration curve for gallic acid in the range of 50–100 g/mL was prepared in the same manner and results were expressed as mg gallic acid equivalent (GAE) per gram of extract. 2.6 Determination of total flavonoid con- tent in the plant extracts Total flavonoid content was determined using the aluminum colorimetric method using quercetin as the standard [10]. An aliquot of 1 mL leaf extract of each concentration range from 0.5-1 mg/ mL in methanol was poured to 15 mL test tube containing 4 mL of double distilled water. At the 0, 5 and 6 minutes’ time interval, 0.3 mL 5% NaNO2, 0.3 ml of 10% AlCl3 and 2 mL of 1 M NaOH was added to the test tube respectively. Immediately volume was maintained 10 mL by adding 2.5 mL double distill water. Absorbance of the obtained pink color mix- ture was determined at 510 nm versus blank con- taining all reagents except quercetin. The samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained. Stan- dard calibration curve for quercetin in the range of 50–100 g/mL was prepared in the same manner. The concentration of flavonoid was expressed as mg quercetin equivalent (QE) per gram of extract. 2.7 Antioxidant assay The antioxidant activity of extract of the test plants and standard (ascorbic acid) was carried by DPPH assay method, following standard protocol [11]. Different concentration of plant extracts (10- 100 µg/mL) and ascorbic acid (10-100 µg/mL) were pre- pared in methanol on the clean and clear test tubes. The sample volume was taken 2 mL. To this sam- ple volume, 2 mL of this 0.2 mM DPPH solution was added. The tube was shaken vigorously for the uniform mixing. These tubes were allowed to stand in dark for half an hour. The control was pre- pared as above without the plant extract or ascorbic acid. Methanol was used to collect the baseline on the spectrophotometer (Thermo Fisher Scientific, Genosystem-10-50. The absorbance was taken on the spectrophotometer at 517 nm. Now the radical scavenging activity was calcu- lated by using the following formula. %Radical scavenging activity = Controlabs − Sampleabs Controlabs × 100% (1) Standard graph was plotted by taking the concen- tration on the X- axis and percentage free radical scavenging activity on the Y-axis. Based on this graph, IC50 value of each sample was calculated and these values of the different species were compared. The species having the lowest IC50 is considered to have the best antioxidant property. 2.8 Alpha-amylase inhibition assay α-amylase inhibition assay was performed following the standard protocol [11]. This protocol is based B. Subba et al./ BIBECHANA 20 (2023) 190-199 193 on that α-amylase converts starch into sugars and plant extracts inhibits the action of α-amylase as acarbose. Substrate was prepared by dissolving 200 mg starch in 25 mL of NaOH (0.4 M) by heating at 100 ºC for 5 minutes and adjusting pH was adjusted to 7.0 after cooling. 400 µL of substrate solution was pre-incubated at 37 ºC for 15 min. Termina- tion of the reaction was carried out by adding 800 µL of HCl (0.1 M). 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 spectrophotometer. Percentage of inhibition was calculated using the formula %Inhibition = ( 1 − Abs2 − Abs1 Abs4 − Abs3 ) × 100 (2) where Abs1 is the absorbance of the incubated mixture containing sample, starch, and α-amylase, Abs2 is the absorbance of the incubated mixture of sample and starch, Abs3 is the absorbance of the incubated mixture of the of starch and α-amylase, Abs4 is the absorbance of the incubated solution containing starch. 3 Results and Discussion 3.1 Plant collections The plants samples on this research work were se- lected and collected from the farmland of Gulmi, Rupandehi, Kaski district of Nepal (Table 1). Table1: List of plants, parts used and therapeutic use. S.N. Scientific name Common name Used part Therapeutic use 1. Clerodendrum-trichotomum Lapche leaves Antioxidant Antidiabetic Antibacterial 2. Mallotus-philipinensis Sindoor leaves Antioxidant Antifilarial 3. Diosocrea- deltoidea Vyakur rhizomes Anti-inflammatory Antiviral Antidiarrhoic 4. Dioscorea-bublifera Gitto rhizomes Antioxidant Anti-inflammatory Antidiabetic 5. Rubia- cordifolia Majitho roots Antidysentric Antioxidant 6. Viscum- articulatum Harchur bark Anticancer Antioxidant Table 2: Phytoconstituents of test plants extract in methanol. Phytochemical CT MP DD DB RC VA Reducing Sugars - - - + - + Polyphenols + + - + + + Alkaloids + + + + + + Glycosides - - + + + + Quinones + - + - + + Saponins + - - + + - Coumarins - - - + - + Flavonoids + + + + + + where, (+) present and (-) absent, CT = C. trichotomum, MP = M. philipinensis, DD = D. deltoidea, DB = D. bublifera, RC = R. cordifolia, and VA = V. articulatum 3.2 Phytochemical screening The results obtained from the phytochemical screening indicating the presence and absence of different types of phytoconstituents are tabulated in table 2. The different extracts of plants depict the pres- ence of various secondary metabolites like alka- loids, glycosides, coumarins, saponins, flavonoids, terpenoides etc. These compound are supposed to be biologically active and act as anticancer, antioxi- dant, antidiabetic etc. [12]. Due to presence of such secondary metabolites V. articulatum be the good source of such biologically active compounds and B. Subba et al./ BIBECHANA 20 (2023) 190-199 194 D. deltoidea and C. trichotomum can be inferred to be potent antioxidant, antidiabetic and antimi- crobial [13–15]. Flavonoids, alkaloids and phenolic compounds are a major source of compounds that act as primary antioxidants or free radical scav- enger. Subsequently, plants containing these phyto- chemicals may be used for the preparation of drug in a systematic way which may be lead to the cure of many ailments in the future [16]. So, many of se- lected medicinal plants can be inferred as the good source of biologically active compounds. 3.3 Brine shrimp bioassay Results obtained from the Brine Shrimp Lethality assay are presented in table 3. Table 3: LC50 Value of Plant samples in Brine shrimp bioassay. Name of Plant Extracts LC50 Value µg/mL C. trichotomum 184.79 M. philipinensis 100 D. deltoidea 193.06 D. bublifera 135.93 R. cordifolia 184.79 V. articulatum 46.39 From the above calculations, sample VA (bark of V. articulatum) had shown the lower LC50 value which indicates that it contains highly active com- pound. The result showed that LC50 of different plant extracts ranged from 46.39 to 193.06 µg/mL. V. articulatum can be inferred to have very strong cytotoxic effect. Likewise, M. philipinensis, D. bublifera, C. trichotomum, R. cardifolia, and D. deltoidea were biologically active with LC50 values 100, 135.93, 184.79, 184.791, and 193.06 µg/mL re- spectively. So, these plants can be suggested to use as therapeutic agents as the toxicity activity on them is nominal. Plant extract resulting in LC50 less than 1 mg/mL were considered toxic to the larvae. This brine shrimp lethality test was also a guide for active antitumor agent [17, 18]. 3.4 Total Phenolic and Flavonoid Con- tents The TPC and TFC were expressed as the GAE/g, and QE/g of extract using a calibration curve of gal- lic acid and quercetin, respectively which is shown in fig. 1 & 3. The total phenolic content of all selected medic- inal plant extracts showed varied data ranging from 54.742±0.025 to 212.742±3.34 mg GAE/g in C. tri- chotomum and D. deltoidea respectively which is shown in figure 2. The total phenol content of rest of plant extract lied between these two extremes. The results showed that the total phenolic con- tent in C. trichotomum (CT) was highest among six selected medicinal plants. Leaf extract of C. tri- chotomum (CT), bark of V. articulatum (VA) also showed relatively high phenolic content. Figure 1: Calibration Curve of Gallic acid. Figure 2: Comparison of TPC in different plant extracts. B. Subba et al./ BIBECHANA 20 (2023) 190-199 195 Figure 3: Calibration Curve of Gallic acid. Figure 4: Comparison of TPC in different plant extracts. From the calculation of total flavonoid content in different plant extract, it was found that sam- ple (CT) C. trichotomum has highest TFC. It was also seen that TFC in (DD) D. deltoidea was high enough. The total flavonoid content of different medicinal plants was found and the result revealed that the TFC varied from 72.39±112 mgQE/g in M. philipinensis to 109.964±1.25 mgQE/g is D. deltoidea. All the remaining plant extract showed the TFC in two extremes as shown in fig 4. The literature revealed that presence of phenols and flavonoids in plants extract had been reported to be associated with antioxidative action in biologi- cal system, acting as scavengers of singlet oxygen and free radicals. Thus, greater TPC and TFC had positive cor- relation with greater antioxidant activities. In this study, the high value TPC (212±2.742 mgQE/g) and TFC (112±2.42 mgQE/g) could be attributed to antioxidant property of C. trichotomum [19]. 3.5 DPPH free radical scavenging activity The free radical scavenging activity of methanol ex- tractives of C. trichotomum, M. philipinensis, D. deltoidea, D. bublifera, R. cordifolia, and V. ar- ticulatum were evaluated by DPPH assay. Table 4 show the value of % DPPH free radical scav- enging activity at different concentrations of ascor- bic acid, C. trichotomum, M. philipinensis, D. del- toidea, D. bublifera, R. cordifolia, and V. articula- tum. The graph of concentration against the corre- sponding percentage radical scavenging activity of different samples was plotted fig 5 and IC50 value was determined. Ascorbic acid was used as the standard in this experiment. Among six different plant extract, methanol extract of leaves of C. tri- chotomum had the IC50 value 40.62±0.16 g/mL and rhizome of D. deltoidea had the IC50 value 41.18±0.81 very close to that of ascorbic acid i.e. 26.64±0.14 g/mL respectively. Remaining plants M. philipinensis’s leaves (60.45 ± 0.99 g/mL), V. articulatum’s bark (63.12±0.85g/mL), D. bublif- era’s rhizome (69.58±0.15 µg/mL), R. cordifolia’s root (89.71±0.13 µg/mL) extract had moderate an- tioxidant activity table 5. Literature revealed that the majority of the an- tioxidant activity is due to the flavones, isoflavones, flavonoids, anthocyanin, coumarins, lignans, cat- echins and isocatechins. The obtained IC50 of methanolic extract of test samples are supported by previously reported value of the same plants from different region with slight variation in value [20, 21]. Compounds like trichotomoside (phenyl- propanoid glycoside), isoacteoside, jionoside D had been isolated and proved to be responsible for the antioxidant activity of C. trichotomum [22–24]. Strong antioxidant activity for acetone and ethanol extract of V. articulatum bark had been reported by Kakpure, 2020 [25]. While weak antioxidant ac- tivity of methanol extract of V. articulatum bark extract had been reported for plant collected from Kathmandu valley by Kumal et al 2020 [26]. B. Subba et al./ BIBECHANA 20 (2023) 190-199 196 Table 4: % inhibition of ascorbic acid, CT, MP, DD, DB, RC, VA at different concentrations Concentration % Inhibition (µ g/mL) Ascorbic acid CT MP DD DB RC VA 20 64.06±0.56 52.5±3.23 35.97±0.85 64.37±0.51 45.23±2.82 1.54±4.04 23.73±2.34 40 75.45±0.05 61.76±2.74 45.27±2.65 65.52±0.08 50.59±2.8 2.97±3.63 55.49±7.74 60 86.79±0.06 69.86±1.52 55.52±0.52 64.85±1.33 51.27±2.86 20.91±2.56 57.99±0.65 80 92.5±0.06 75.40±2.17 60.44±0.99 64.22±3.31 52.44±2.85 54.89±2.01 58.49±19.25 100 94.06±1 78.09±0.64 65.23±0.66 65.55±0.20 52.77±3.73 59.02±3.56 59.62±23.22 Figure 5: A plot of % free radical scavenging of methanol extracts against concentration of plant extract, and ascorbic acid. IC50 values of the plant extracts along with the standard Ascorbic acid is tabulated below in table 5. Table 5: Comparison of IC50 values of different plant extracts with ascorbic acid Sample IC50 (µg/mL) Ascorbic acid 26.64±0.14 C. trichotomum 40.62±0.16 M. philipinensis 60.45±0.99 D. deltoidea 41.18±0.81 D. bublifera 69.58±0.16 R. Cardifolia 89.71±0.13 V. articulatum 63.12±0.91 IC50 values are expressed as mean±SD (n = 3) 3.6 Alpha-amylase inhibition test Alpha-amylase inhibitory activity of plant extracts was determined from quantitative starch-iodine method. Alpha-amylase inhibition % values at dif- ferent concentrations of standard acarbose and six plant extracts are shown in the below table 6. The graph of concentration against the corresponding percentage radical scavenging activity of different samples was plotted fig 6 and IC50 value was deter- mined. The percentage inhibition of different plant extracts versus concentration and antidiabetic ac- tivity in term of IC50 values of different extract are given in table 7. Table 6: % Inhibition at different concentrations of Plant extract. Concentration % Inhibition (µg/mL) Acarbose CT MP DD DB RC VA 40 53.07±1.02 30±0.29 22.89±4.89 13.81±3.06 45.01±3.99 60.96±3.05 37.37±5.33 80 60.73±0.95 41±0.82 30.46±0.39 13.61±1.23 59.01±3.5 63.09±3.09 53.56±0.25 160 67.12±0.95 49±1.13 40.22±0.20 18.77±3.42 57.31±2.4 57.21±2.01 57.12±2.8 320 81.31±0.59 65±5.47 45±2.56 32.08±2.42 62.46±3.2 75.20±1.89 54.92±0.21 640 88.20±0.58 70±0.29 47.24±4.21 38.92±1.23 64.16±4.5 70.52±1.55 56.1±0.69 where CT = C. trichotomum, MP = M. philipinensis, DD = D. deltoidea, DB = D. bublifera,RB =R. Cardifolia, VA = V. articulatum. Mean % Inhibition are expressed as means±SD (n = 3). B. Subba et al./ BIBECHANA 20 (2023) 190-199 197 Figure 6: Inhibition of α-amylase activities by plant extracts. IC50 values of the six plant extracts along with the standard acarbose is tabulated below in table 7. Table 7: Comparison of IC50 values of different plant extracts with standard acarbose Sample IC50(µg/mL) Acarbose 119.063 ± 0.73 Clerodendrum trichotomum 293.33 ± 0.81 Mallotus philipinensis 554.18 ± 0.75 DIoscorea deltoidea 785.15 ± 0.92 Dioscorea bublifera 352.71± 0.59 Rubia cordifolia 252.44 ± 0.55 Viscum articulatum 344.58 ± 0.61 IC50 values are expressed as mean±SD (n = 3) Here, IC50 value of standard acarbose was found to be 119.063 ± 0.73 µg/mL. C. trichoto- mum, M. philipinensis, D. deltoidea, D. bublifera, R. Cardifolia, V. articulatum showed α-amylase inhibitory activity with IC50 value 293.33±0.81 µg/mL, 554.18±0.75 µg/mL, 785.15±0.92 µg/mL, 352.71±0.59 µg/mL, 252.44±0.55 µg/mL, 252.44± 0.55µg/mL respectively in table 7. There was a dose dependent increase in percentage inhibitory activ- ity against α-amylase by all the six plant extracts. The result obtained here are in good correlation with previously reported results [27,28]. Strong an- tioxidant and antimicrobial activity of leaves of C. trichotomum collected from Dhankuta had been re- ported by Subba et al, 2016 [29]. Among six medic- inal plants, leaves of C. trichotomum is found to be good property as antidiabatic, antioxidant, pheno- lic and flavonoid content with low cytotoxic effect. 4 Conclusion The present study revealed that the methanolic ex- tract of the C. trichotomum’s leaves, M. philipine- sis’s leaves, D. deltoidea’s root, D. bublifera’s root, R. cardifolia’s stem, V. artculatum’s bark has no- table antioxidant potential and inhibitory activity on the α-amylase enzyme. These effects would be due to its important phenolic composition, whose quantitative study has revealed the varied pres- ence of polyphenols and flavonoids. These results could justify the use of these plants in traditional medicine for the treatment various health problem including type 2 diabetes and complications. Our study is the first examination of anti-α-amylase ca- pacities of C. trichotomum leaves extract. Study of chemical constituents of these plants using so- phisticated technologies like NMR, HPLC, etc. can provide a way for extensive research that can be used for commercial drug production with lesser or no side effects. Acknowledgement Central Department of Chemistry, Tribhuvan Uni- versity, Kirtipur. References [1] Yizhong Cai, Qiong Luo, Mei Sun, and Harold Corke. Antioxidant activity and phenolic com- B. Subba et al./ BIBECHANA 20 (2023) 190-199 198 pounds of 112 traditional chinese medicinal plants associated with anticancer. Life Sci- ences, 74(17), 2004. [2] G. Miliauskas, PR. Venskutonis, and TA. Beek van. Screening of radical scavenging ac- tivity of some medicinal and aromatic plant ex- tracts. Food Chemistry, 85(2):231–237, 2004. [3] Jayalaxmi Revankar, Divya K., Ankita Sham- nani, and Podili Koteswaraiah. Antioxidant activities of pearl millet (pennisetum glaucum) and little millet (panicum sumatrense) in dif- ferent in vitro models. International Journal of Bioassays, 7(2):5595–5601, 2018. [4] Norman R Farnsworth, Olayiwola Akerele, Au- drey S Bingel, Djaja D Soejarto, and Zhengang Guo. Medicinal plants in therapy. Bulletins of the world health organization, 63(6):965–98, 1985. [5] Nicholas Ekow Thomford, Dimakatso Alice Senthebane, Arielle Rowe, Daniella Munro, Palesa Seele, Alfred Maroyi, and Kevin Dzobo. Natural products for drug discovery in the 21st century: Innovations for novel drug discovery. International Journal of Molecular Sciences, 19(6):1578, 2018. [6] I. Culie. Methodology for analysis of vegetable drugs. Phytochemistry, 63:97–104, 1982. [7] B. N. Meyer, N. R. Ferrigni, J. E. Putnam, L. B. Jacobsen, D. E. Nichols, and J. L. McLaughlin. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica, 45(5):31–34, 1982. [8] D.J. Finney. Probit Analysis. Cambridge Uni- versity Press, Cambridge, 3rd edition, 1971. [9] N Arun, MG Ragunathan, and J Jayanthi. An- tioxidant activity, total phenol, flavonoid, alka- loid, tannin, and saponin contents of leaf ex- tracts of salvinia molesta d. s. mitchell. Asian Journal of Pharmacy and Clinical Research, 9:185–188, 2016. [10] Pallab Kalita, T.K. Barman, Tapas Pal, and Ramen Kalita. Estimation of total flavonoids content (tfc) and antioxidant activities of methanolic whole plant extract of biophytum sensitivum linn. Journal of Drug Delivery and Therapeutics, 3(4):10–15, 2013. [11] R. Kusano, S. Ogawa, Y. Matsuo, Y. Yazaki, and I. Kouno. -amylase and lipase inhibitory activity and structural characterization of aca- cia bark proanthocyanidins. Journal of Natural Products, 74:119–126, 2011. [12] M. A. Bhutkar and S. B. Bhise. In vitro assay of alpha-amylase inhibitory activity of some indigenous plants. International Journal of Chemical Sciences, 10(1):457–462, 2012. [13] P. M. De Sales, P. M. de Souza, L. A. Simeoni, P. O. Magalhães, and D. Silveira. -amylase inhibitors: a review of raw material and iso- lated compounds from plant source. Jour- nal of Pharmacy & Pharmaceutical Sciences, 15(1):141–183, 2012. [14] M. Kumari and S. Jain. Tannins: an antinu- trient with positive effect to manage diabetes. Research Journal of Recent Sciences, 1(12):70– 73, 2012. [15] Mrinmoy Nag, Pulok K. Mukherjee, Rajarshi Biswas, Joydeb Chanda, and Amit Kar. Eval- uation of antimicrobial potential of some in- dian ayurvedic medicinal plants. Pharmacog- nosy Journal, 8(6):525–533, 2016. [16] Gopichand, R. D. Singh, R. L. Meena, V. K. Kaul, and B. Singh. Influence of manure and plant spacing on growth and yield of Dioscorea deltoidea walls: An endangered species. Jour- nal of Medicinal Plants Studies, 1(3):184–190, 2013. [17] Pendli Sreenu, Talari Samatha, Nemali Gandhi, and S. N. Azmeera. Phytochemical analysis of root, stem, and leaf extract of Ru- bia Cardifolia. World Journal of Pharmacy and Pharmaceutical Sciences, 3(10):826–838, 2014. [18] J. Sharma and R. Varma. A review on endan- gered plant of Mallotus philipinensis (lam.). Pharmacologynoline, 3:1256–1265, 2011. [19] M. J. Hossain, J. Khaleda, A. M. A. Chowd- hury, M. Arifuzzaman, and AlForkan. Phyto- chemical screening and evaluation of cytotoxic- ity and thrombolytic properties of Achyranthes aspera leaf extract. Journal of Pharmacy and Biological Sciences, 6(3):30–38, 2013. [20] M. Amir, M. Mujeeb, A. Sayeed, A. Aftab, and M. Aqil. Antioxidant and hepatopro- tective activity of rhizome and callus culture of Dioscorea deltoidea against d-galactosamine induced hepatotoxicity in rats. Planta Medica, 77(5):141, 2011. [21] M. Gangwar, M. K. Gautam, A. K. Sharma, Y. B. Tripathi, R. K. Goel, and G. Nath. An- tioxidant capacity and radical scavenging ef- fect of polyphenol-rich Mallotus philippinen- sis fruit extract on human erythrocytes: an in vitro study. The Scientific World Journal, 2014:1–12, 2014. B. Subba et al./ BIBECHANA 20 (2023) 190-199 199 [22] S. Chae, K. A. Kang, J. S. Kim, J. W. Hyun, and S. S. Kang. Trichotomoside: a new antiox- idative phenylpropanoid glycoside from Clero- dendron trichotomum. Chemistry & Biodiver- sity, 3(1):41–48, 2006. [23] S. Chae, JS. Kim, KA. Kang, HD. Bu, Y. Lee, YR. Seo, JW. Hyun, and SS. Kang. Antioxi- dant activity of isoacteoside from clerodendron trichotomum. Journal of toxicology and envi- ronmental health. Part A, 68(5):389–400, 2005. [24] S. Chae, J. S. Kim, K. A. Kang, H. D. Bu, Y. Lee, J. W. Hyun, and S. S. Kang. An- tioxidant activity of jionoside d from Cleroden- dron trichotomum. Biological & Pharmaceuti- cal Bulletin, 27(10):1504–1508, 2004. [25] Manoj Kakpure. Phytochemical profiling and in vitro antioxidant activity of leaflet mistle- toe Viscum articulatum burm.f. by dpph assay. SSR Institute of International Journal of Life Sciences, 6:2709–2716, 2020. [26] K. Kumal, D. R. Pant, B. Aryal, G. R. Tripathi, and G. P. Joshi. Phytochemi- cal and antioxidant properties of traditionally used mistletoes in nepal. Scientific World, 14(14):83–89, 2021. [27] S. Ghosh, P. More, A. Derle, A. B. Patil, P. Markad, A. Asok, N. Kumbhar, M. L. Shaikh, B. Ramanamurthy, V. S. Shinde, D. D. Dhavale, and B. A. Chopade. Diosgenin from Dioscorea bulbifera: novel hit for treatment of type ii diabetes mellitus with inhibitory activ- ity against -amylase and -glucosidase. PloS One, 9(9):e106039, 2014. [28] B. N. Devi, S. Salma, and V. Lavanya. Phyto- chemical evaluation and in vitro antidiabetic activity of ethanolic extract of Viscum artic- ulatum. International Journal of Pharmaceu- tical Sciences Review and Research, 60(1):99– 104, 2020. [29] B. Subba, C. Srivastav, and R. C. Kandel. Sci- entific validation of medicinal plants used by yakkha community of chanuwa vdc, dhankuta, nepal. SpringerPlus, 5:155, 2016. Introduction Materials and Methods Collection of plant materials and preparation their methanol extract Chemicals and reagents Qualitative phytochemical analysis Brine shrimp lethality assay Determination of total phenolic content in the plant extracts Determination of total flavonoid content in the plant extracts Antioxidant assay Alpha-amylase inhibition assay Results and Discussion Plant collections Phytochemical screening Brine shrimp bioassay Total Phenolic and Flavonoid Contents DPPH free radical scavenging activity Alpha-amylase inhibition test Conclusion