Antioxidant and Antibacterial Activities Evaluation, Phytochemical Characterisation of Rhizome from Angiopteris helferiana and Barks from Saurauia fasciculata in Nepal

Ethnomedicinally, more than 2000 plants were found to be used in Nepal. Among them, the red colored rhizome of Angiopteris helferiana and the bark of Saurauia fasciculata have been used widely to treat muscle fatigue, bone pain, fever, postpartum hemorrhage, and thirst by healers in Kaski and Tanahun districts, Nepal. However, scientific evidence towards their traditional uses is lacking till December, 2023. Therefore, we report the phytochemicals, total phenolic content (TPC), total flavonoid content (TFC), total carbohydrate content (TCC), antioxidant and antibacterial activities of A. helferiana and S. fasciculata extracts. Phytochemical analysis indicated that A. helferiana and S. fasciculata extracts were potential sources of chemicals such as phenols, flavonoids, tannins, terpenoids, saponins, and carbohydrates. The TPC, TFC, and TCC of extracts were determined by using an ultraviolet visible spectrophotometer. Among the extracts tested, A. helferiana extracts showed the highest phenolic and carbohydrate contents of 208.33 ± 12.96 mg of gallic acid equivalent/g and 564.16 ± 2.92 mg of D-glucose equivalent/g of dry extract, respectively. Similarly, S. fasciculata revealed the highest flavonoid content of 30.35 ± 0.1 mg quercetin equivalent/g of dry extract. The extract of A. helferiana and S. fasciculata exhibited potent antioxidant activity by scavenging 2,2-diphenyl-1-picrylhydrazyl radicals with an IC50 of 25.9 µg/ml and 31.07 µg/ml, respectively. The antibacterial activity of the A. helferiana and S. fasciculata extract against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was determined using an agar-well diffusion protocol that revealed the potential antibacterial activity of A. helferiana against E. coli. The present study will help validate the traditional uses of A. helferiana rhizomes and S. fasciculata barks as a healing medicine and inspire the researcher towards further research, development, and formulation.


Introduction
Nepal's diverse altitude, topography, and climate contribute to its signifcant foral biodiversity with 6500 fowering plants and fern species [1].Te people of Nepal have a deep respect and belief in medicinal plants.Approximately 2000 medicinal and aromatic plants are traditionally used for their healing potential [2].Among them, Angiopteris helferiana and Saurauia fasciculata are well-recognized medicinal plants.
Angiopteris helferiana C. Presl.belongs to the Marattiaceae family and is characterised as a gigantic feshy fern with a massive terrestrial rhizome.It is distributed at an altitude of 900-1400 meters in moist forests throughout Southeast Asia, mostly in Nepal, India, China, and Sri Lanka [3].Rhizome of A. helferiana is traditionally prescribed to cure muscle and bone pain/fatigue in Nepal [3], scabies in India [4], and dysentery, infection, scabies, and muscle pain in Bangladesh [5].
To the knowledge of ethnomedicinal healers from the Kaski and Tanahun districts of Nepal, Gai-Khurey (A. helferiana) exists in two local cultivars which are locally recognized by its rhizome color as Rato Gai-Khurey (red colored rhizome) and believed to be more ethnomedicinally potent in muscle and bone pain/fatigue than Seto Gai-Khurey (light yellow colored rhizome).Previous studies conducted on A. helferiana (light yellow rhizome) have reported antioxidant, anti-infammatory, antiobesity, and antidiabetic activities.Furthermore, few lactones such as (−)-epi-osmundalactone and angiopteroside were isolated from the same cultivars [4].However, A. helferiana with red color rhizome remains unexplored until December 2023.
Saurauia fasciculata Wall.(Syn Ternstroemia fasciculata), a member of the Actinidiaceae family, is a 3-6 m tree with 10-25 by 3-8 cm elliptic-oblong to ellipticlanceolate leaves, 5-9 cm long inforescence crowded behind the tip of branchlets, and 6.5-9 by 5-5.5 mm fower [6] (Figure 1).In Nepal, S. fasciculata is locally recognized as Goban and its bark juice is given orally in fever, postpartum hemorrhage, and thirst [7].Previous studies carried out on various other species of the genus Saurauia revealed several biological activities such as antioxidant, antidiabetic, analgesic, wound healing, and antihyperlipidemic [8].However, no studies have been carried out on the bark of S. fasciculata up to December, 2023.
Te present study therefore, aims to analyze phytochemicals and evaluate antioxidant and antibacterial activity of extracts from the red-colored rhizome of A. helferiana and the bark of S. fasciculata.

Plant Collection and Authentication.
Te fresh fronds and rhizome of A. helferiana were collected in May 2023 from the Vyas municipality, Tanahun district, Nepal, and the bark of S. fasciculata was collected in May 2023 from Hemja, Kaski district, Nepal.Te authenticity of the plant specimen (31/02/2080/Herbarium specimen Nos 2 and 5) was confrmed at the National Herbarium and Plant Laboratories, Godawari-3, Lalitpur, Nepal, by referring to the deposited plant specimen.

Extraction Procedures. Te fresh rhizomes of
A. helferiana and the barks of S. fasciculata were cleaned with tap water, reduced in size with a plant cutter, and then shade dried for one week to eliminate moisture.Using the methodology of [9,10], extraction was carried out.

Preparation of Extracts for Qualitative Phytochemical
Analysis.Te red A. helferiana rhizomes (30 g) and the barks of S. fasciculata (30 g) were separately subjected to extraction using 70% methanol as a solvent in a ratio of 1 : 10 w/v at room temperature (24 °C) with frequent agitation for 24 hours.Te liquid crude extract was fltered using a cotton plug and used for qualitative phytochemical analysis.

Preparation of Extracts to Estimate
Extractive Yield, TLC Profling, Quantitative Phytochemical Analysis, Antioxidant Activity, and Antibacterial Activity.Te A. helferiana rhizomes (100 g) and the barks of S. fasciculata (150 g) were separately subjected to successive extraction using 70% methanol as solvent in the ratio of 1 : 10 w/v at 55 °C for 2 hours followed by room temperature (24 °C) for 22 hours and fnally 70% methanol at room temperature (24 °C) for an additional 24 hours.Te crude liquid extracts were fltered using a thick cotton plug and combined to evaporate under reduced pressure at 55 °C using a rotary evaporator (Biobase RE-2000B, Germany).Te concentrated extracts obtained after evaporation were poured in a beaker, followed by complete drying in vacuum desiccators.Te dried extracts were weighed and stored at 4 °C in refrigerator until use.Te extractive yield was calculated using the formula: % extractive yield � weight of dry extract initial weight of dry sample   × 100.(1) 2.3.Qualitative Phytochemical Analysis.Phytochemicals such as alkaloids, phenols, favonoids, tannins, carbohydrates, saponins, terpenoids, anthraquinone, steroids, and proteins were analysed in the rhizome of A. helferiana and the bark of S. fasciculata according to the methodology of [11,12].All the reagents used for analysis were prepared in distilled water.

Test for Phenols (FeCl 3 Test).
A 2% of FeCl 3 solution was prepared, and 2 ml of it was treated with 2 ml of crude liquid extract.Blue, green, or black suggested the presence of phenolic compounds.

Test for Flavonoids
(1) Alkaline Reagent Test.Plant extracts (2 ml) were treated with 2 ml of 2% NaOH, followed by the addition of a few  Te Scientifc World Journal 3 drops of aqueous HCL.Appearance of an intense yellow with NaOH, which changes to colorless upon addition of HCL, indicated the presence of favonoids.
(2) Shinoda Test.A fragment of magnesium ribbon reacted with 2 ml of extract solution and a few drops of HCL.Appearance of red or pink after a few minutes suggested favonoids.
(3) Lead Acetate Test.Crude liquid extract (2 ml) was mixed with a few drops of lead acetate solution; a yellow precipitate indicated favonoids.

Test for Tannins
(1) FeCl 3 Tests.A 2% of FeCl 3 solution was prepared, and 2 ml of it was treated with 2 ml of crude liquid extract.Blue, green or black suggested the presence of tannins.
(2) Lead Acetate Test.Crude liquid extract (2 ml) was mixed with a few drops of 10% lead acetate solution; a white-yellow precipitate indicated tannins.
(3) Lime Water Test.Lime water was prepared, and 2 ml of it was mixed with 2 ml of crude extract; precipitate formation suggested the presence of tannins.
(4) Gelatin Tests.Gelatin (0.5%) was prepared in 10% of NaCl, and 2 ml of it was treated with an equal volume of test extract; turbidity indicated the presence of tannins.

Test for Saponin (Foam Test).
Test extract (2 ml) was shaken with 5 ml of distilled water for a few minutes in a closed test tube; the formation of stable foam confrmed the presence of saponin.

Test for Alkaloids
(1) Mayer's Reagent Test. 2 ml of reagent was treated with 2 ml of liquid crude extract; a white-yellow precipitate suggests alkaloids.
(2) Wagner's Reagent Test.Crude extract (2 ml) was treated with a few drops of reagent; a brown precipitate indicated the presence of alkaloids.
(3) Hager's Reagent Tests.A few ml of extract solution was mixed with 1 ml of reagents.A creamy white precipitate indicated alkaloids.

Test for Terpenoids and Steroids (Salkowski Test).
2 ml of concentrated H 2 SO 4 was added to the mixture containing an equal volume of extract solution and CHCl 3 .
After gently shaking, the appearance of a reddish-brown color in the layer of CHCl 3 and green fuorescence in the layer of H 2 SO 4 layer confrmed the presence of steroids or triterpenoids.

Test for Carbohydrates
(1) Fehling's test.A mixture of test extract (2 ml) and Fehling's solution (2 ml) was heated for 10 min over a hot water bath; a precipitate with a brick red color confrmed the presence of reducing sugar.
(2) Benedicts test.A mixture containing test extract (2 ml) and Benedict's solution (2 ml) was heated for 2 min over a hot water bath; a precipitate with reddish-brown color indicated the presence of carbohydrates.

Test for Protein (Ninhydrin Test
).A 0.2% of ninhydrin solution was prepared, and 1 ml of it was treated with an equal volume of extract solution and boiled for 5 min; a purple color suggested the presence of amino acids or proteins.

Anthraquinone Test (Borntrager's Tests
). 2 ml of extract was mixed with 2 ml of 10% NaOH solution.A red color developed.Afterwards, a small volume of 30% H 2 O 2 solution was added and the mixture was heated at 60 °C.HCl solution was then added, and the red color disappeared; fnally, NaOH solution was added and the development of a red color indicated the presence of anthraquinones.

Estimation of the Total Phenolic Content (TPC).
Te TPC was estimated spectrophotometrically by implementing the Folin-Ciocalteu's reagent method adopted previously [13], with slight modifcations.Briefy, 100 μl of extract (1000 µg/ml in distilled water), 6 ml of distilled water, and 0.5 ml of Folin-Ciocalteu phenol reagent (2 N) were homogenised in a test tube for 10 seconds using a Vortex mixer.Subsequently, after 5 minutes, 1.5 ml of sodium carbonate (7.5%) and 1.9 ml of distilled water were added, homogenised, and incubated for 2 hours in the dark.For the calibration curve, gallic acid (31.25 µg/ml-500 µg/ml) was considered a reference phenolic compound and treated similarly to the extract.At 750 nm, a single beam UV-VIS spectrophotometer (Agilent Cary 60, Malaysia) was used to read the absorbance of the solution.In the blank solution, extract (100 μl) was replaced with water (100 μl) and treated identically to extract.Te mean absorbance of triplicates per sample was used, and TPC was expressed as milligrams of gallic acid equivalents (mg of GAE)/g of extract.

Estimation of the Total Flavonoid Content (TFC).
Te TFC was estimated by implementing the AlCl 3 method adopted by [11], with slight modifcations.Briefy, 2 ml of AlCl 3 (2% in distilled water) and 2 ml of extract (100 µg/ml of water) were mixed together and incubated at room temperature for 10 minutes.For the calibration curve, quercetin (1.25-100 µg/ml) was considered as a reference favonoid and treated in a similar way to the extract.At 415 nm, a single beam UV-VIS spectrophotometer was used to read the 4 Te Scientifc World Journal absorbance of the solution against a consisting of extract with distilled water.Te mean absorbance of triplicates per sample was used to express TFC as milligrams of quercetin equivalent (mg of QE)/g of extract.

Estimation of the Total Carbohydrate Content (TCC).
Te TCC was estimated spectrophotometrically as described by [11], with slight modifcation.Briefy, 1 ml of plant extract (250 µg/ml), 0.5 ml of phenol aqueous solution (5%), and 2.5 ml of concentrated H 2 SO 4 were mixed in a test tube and incubated for 30 min.A calibration curve was produced based on D-glucose (12.5-200 µg/ml) as a standard.At 490 nm, absorbance was measured against a blank consisting of distilled water instead of extract.Te mean of three readings per sample was used, and TCC was expressed in milligrams of D-glucose equivalents (GE)/g of dry extract.

TLC Profling.
Adopting the methodology of [14], TLC profling of extracts from the rhizome of A. helferiana and the bark of S. fasciculata was carried out.Te particle-free extract solution in methanol was used for the TLC profling.
Te solution band was applied to the silica gel F 254 plates using a microcapillary tube.Initially, the extract-loaded plates were exposed to a hot air blow and then developed in a saturated glass beaker containing solvents (chloroform, methanol, and water) in a 6 : 4 : 1 proportion.Te developed plates were again faced with blow to air the solvent.Subsequently, it was visualized in a UV chamber at (1) 254 nm, (2) 365 nm, (3) 10% v/v FeCl 3 spray/dry, (4) 10% v/v H 2 SO 4 spray/heat, and (5) dipped in 500 µM DPPH solution in methanol.
2.6.DPPH Scavenging Assay.Spectrophotometrically, extracts of A. helferiana and S. fasciculata were assessed for their potency in scavenging free radicals from DPPH by implementing the method outlined by [11,15].In brief, test extracts (1.5 ml) were treated with 1.5 ml of fresh DPPH methanolic solution (100 µM) in a microtitter plate.Te plate was gently shaken for 10 seconds to mix the content.Subsequently, after 30 min of dark incubation at room temperature, at 517 nm, the absorbance of DPPH • (oxidised form) in the mixture was measured against the DPPH control (containing 1.5 ml of distilled water instead of extract) and blank (distilled water).Ascorbic acid (0.6125 µg/ml-5 µg/ml) was considered as a reference antioxidant.Te mean absorbance of triplicates per sample was utilized to calculate percentage DPPH • scavenging.

% DPPH radical scavenging �
where the A 0 arrow is the absorbance of the DPPH • control and A 1 arrow is the absorbance of the test sample or the reference sample.Te antioxidant potency of each extract sample and positive control (ascorbic acid) was expressed as an IC 50 value (mean ± standard deviation), i.e., the concentration in µg/ml that scavenged DPPH • absorbance by 50%.Te linear graph that plots the percentage of DPPH • scavenging against extract concentration and ascorbic acid was utilized to calculate IC 50 .

Antibacterial Activity.
Clinical isolates of S. aureus, E. coli, and P. aeruginosa were used to evaluate the antibacterial activity of extracts adopting the well difusion method [16] with a slight change.Bacterial colonies were transferred to a sterile test tube containing normal saline, and a 0.5 McFarland was prepared by maintaining an optical density (OD) of 0.1 at 620 nm.Te solution of Muller-Hinton agar was prepared using distilled water.After autoclaving, the agar was aseptically poured into the sterile Petri plate.After solidifcation of the culture medium, the entire surface of each MHA plate was swabbed with 0.5 McFarland standard bacterial inoculums.In each petri plate, fve wells were created using 6 mm sterile tips.20 μl of molten MHA was dropped to seal the base of the wells.100 µl of extract was added at doses of 25, 50, and 100 mg/ml in sterile normal saline to the corresponding wells, and plates were incubated at 37 °C.As a negative control, normal saline was used, and as a positive control, standard antibiotic meropenem (MP-10 μg) were used.After 48 hours, the clear zone of inhibition around the wells was measured in millimeters (mm).

Statistical Analysis.
Statistical analysis was performed using Microsoft Excel 2019 software.Each experiment was carried out in triplicate, and the data were presented as mean ± standard deviation.Te TPC, TFC, TCC, and antioxidant activity (IC 50 ) were determined by linear regression analysis.1, revealed the presence of numerous bioactive chemical constituents such as phenols, tannins, favonoids, saponins, terpenoids, proteins, and carbohydrates.Alkaloids and anthraquinones were not detected.

Extraction
Yield.Te highest yield (22.81% w/w) was obtained from the rhizomes of A. helferiana, while the lowest yield (5.33% w/w) was obtained from the barks of S. fasciculata (Table 2).

TLC Profling. Te TLC profles of extracts of
A. helferiana and S. fasciculata are shown in Figure 2.

Antioxidant Activities.
Te antioxidant abilities of the extracts of A. helferiana, S. fasciculata extracts, and standard ascorbic acid against DPPH free radicals are shown in Table 3 and Figure 3. Te extract of A. helferiana possesses strong DPPH free radical scavenging activity with an IC 50 of 25.09 µg/ml, whereas S. fasciculata and standard ascorbic acid possess an IC 50 value of 31.07 µg/ml and 2.79 µg/ml, respectively.4 summarizes the antibacterial activity of the extract of A. helferiana and S. fasciculata against S. aureus, P. aeruginosa, and E. coli.Among the extracts tested against the selected bacteria, only A. helferiana rhizome extract exhibited potent antibacterial activity against E. coli with a zone of inhibition ranging from 15 to 20 mm.

Discussion
Plants have been used for their remedial properties since ancient times, and their secondary metabolites have long inspired researchers in the quest for novel chemical compounds with bioactive properties such as antioxidant, antiviral, anticancer, antibacterial, and anti-infammatory activities [17][18][19].Approximately 2000 plants are reported to be used for medicinal purposes in Nepal [1,2].Based on these facts, A. helferiana and S. fasciculata used in this study were selected in compliance with their ethnomedicinal uses in Nepal [3,7].
70% MeOH was used for extraction due to its ability to partition a wide range of polarities and soluble phenolic compounds such as phenolic glycosides, favonoids, phenolic acids, lignans, xanthones, coumarins, iridoids, chalcones, and terpenoids [9,10,14,20].Te high yield of the A. helferiana rhizome extract could be due to the high content of polar and 70% methanol-soluble components (polyphenols, favones, and carbohydrates) in the A. helferiana rhizome.As a result, 22% of the extraction yield is currently obtained from the red rhizome of A. helferiana, which is higher than the yield obtained from the local cultivar of A. helferiana with the yellow rhizome [3,4].6 Te Scientifc World Journal Phytochemical analysis indicated that extracts of rhizome of A. helferiana and the bark of S. fasciculata were potential sources of bioactive chemicals (Table 1).Te current study on 70% MeOH extracts from the A. helferiana rhizome revealed higher TPC and TFC than those reported by previous studies carried out in the MeOH extract of  Te Scientifc World Journal helferiana rhizome [3].Since 70% MeOH is more polar than MeOH [21], the higher TPC and TFC in 70% MeOH extract could be due to the partition of highly polar and water soluble compounds such as saponins, glycosides, and tannins [3,21].However, this study was the frst to analyze the presence of phytochemicals and quantify (phenols, favonoids, and carbohydrates) in the bark of S. fasciculata extracts.
Te presence of several compounds in A. helferiana and S. fasciculata was confrmed by separation of several bands in chromatogram on visualized under UV at 254 and 365 nm (Figure 2).After 10% sulfuric acid spray/heat, yellow, black, and reddish-brown on the chromatogram of A. helferiana suggested the presence of favonoids, carbohydrates, and terpenoids (including saponins), respectively [14].Ferric chloride (FeCl 3 ) is commonly used as a spray reagent to detect the phenolic compounds [14].Te chromatogram of S. fasciculata on FeCl 3 spray showed spots at the bottom suggesting the presence of phenolic compounds.On dip in DPPH, a yellow band on the violet or purple background of the chromatogram revealed the presence of antioxidants in A. helferiana and S. fasciculata [14,22,23].
Plant-derived favonoids and phenolic compounds have raised therapeutic interest due to their various medicinal properties, such as antioxidant, antiaging, and antiinfammatory [15,24].Since the presence of favonoids, phenolic compounds, and antioxidants in the extract of A. helferiana rhizomes and S. fasciculata barks was confrmed by present phytochemical analysis, the TPC, TFC, and TLC profling.It seems necessary to estimate the antioxidant potency of the extracts.Terefore, we performed a DPPH radical scavenging assay to evaluate the antioxidant potency of the extracts.
Oxidative damage caused by free radicals is the pathophysiological basis for a wide array of disease conditions such as diabetes, hypertension, neurodegenerative disorders,  8 Te Scientifc World Journal coronary heart disease, and hepatotoxicity [11].dants scavenge DPPH radicals by hydrogen atom transfer (HAT) or single electron transfer (SET), which ultimately leads to a change in color from purple to yellow after reduction [25,26].In this study, the 70% MeOH extract from red rhizomes of A. helferiana possessed dose-dependent free radical scavenging activity (Figure 3) with IC 50 values of 25.9 µg/ml, which is more potent than the yellow rhizomes of A. helferiana reported by [3].However, the bark of S. fasciculata was studied for the frst time and revealed potent antioxidant activity with an IC 50 of 31.07 µg/ml.Te potent antioxidant activity could be due to phenolic, favonoid, and terpenoid compounds [15,24,26,27].Globally, the emergence and spread of antibiotic resistance, as well as the evolution of new bacterial strains, has become a great concern to the human health [18,28,29].Terefore, efective treatment of infectious diseases entails that the research and development of novel drugs from potent medicinal plants is necessary.Te extract of A. helferiana exhibited the dose-dependent antibacterial activities against E. coli, similar to that of standard meropenem.Previously, Lamichhane et al. reported the angiopteroside from the yellow cultivar of A. helferiana [4].Likewise, the authors in [30,31] revealed signifcant antibacterial activity of angiopteroside against E. coli.Terefore, the potent antibacterial activity of the red cultivar of A. helferiana might be due to the angiopteroside.Additionally, the antibacterial activity might be contributed by phytoconstituents such as saponins [32], phenolic compounds [33], favonoids [19], and terpenoids [34].Several research studies have suggested that the phytoconstituents such as terpenoids, phenolic compounds, and favonoids bind via hydrogen bonding with proteins and enzymes of bacterial cells, causing the disruption of cell membranes [35], inhibition of cell wall synthesis, and inhibition of enzymes necessary for amino acid synthesis [34], which leads to bacterial death.Interestingly, standard meropenem was found to be resistant with minimal zone of inhibition against P. aeruginosa.Similarly, both extracts were also found to be resistant against S. aureus and P. aeruginosa.Tis could be due to upregulation of hydrolytic beta lactamase, efux pump, and porin mutations and downregulation of penicillin-binding proteins and infux passage of antibacterial agents into the bacterial cells [35][36][37].Provided it acquires further isolation and experimental validation for antibacterial candidate development.

Conclusions
Te current study confrmed the potent antioxidant activity of red rhizomes of A. helferiana and the barks of S. fasciculata, including the phytoconstituents such as such as phenols, favonoids, and terpenoids.Additionally, A. helferiana rhizomes exhibited antibacterial activity against E. coli.Terefore, these plant extracts may serve as a source for novel lead structures in the development of antioxidant and antibacterial drugs to protect humans from several diseases related to oxidative stress and bacterial infections.

Figure 3 :
Figure 3: Photograph of the DPPH radical scavenging activity of A. helferiana, S. fasciculata, and ascorbic acid.

Table 1 :
Qualitative phytochemical analysis of A. helferiana and S. fasciculata extracts.

Table 2 :
Extraction yield and total phenolic, favonoid, and carbohydrate content of extracts of A. helferiana and S. fasciculata.
Extraction yield was expressed as % yield � (weight of dry extract/initial weight of dry sample) × 100.TPC: total phenolic content.TFC: total favonoid content.TCC: total carbohydrate content.GAE: gallic acid equivalent.QE: quercetin equivalent.GE: D-glucose equivalent.Te TPC, TFC, and TCC values are the mean ± standard deviation of triplicate experiments.
* Used as a standard antioxidant.Percent scavenging values are expressed as mean ± standard deviation.

Table 4 :
Antibacterial activity of extracts of A. helferiana and S. fasciculata using the agar-well difusion method.