Phytochemical Analysis, Antioxidant Potential and Antibacterial Activities of Different Anatomical Parts of Hypericum cordifolium Choisy

The genus Hypericum comprises a large number of species. The flower, leaf, stem, and root of the Hypericum species are widely used in traditional medicine in different cultures. Many Hypericum species have been well investigated phytochemically and pharmacologically. However, only a few reports are available on the H. cordifolium native to Nepal. The present study aims to evaluate the phytochemical composition of different extracts, qualitative analysis of methanol extract of the flower and leaf using thin-layer chromatography (TLC), and the antioxidant properties of components by the TLC-DPPH. assay. The phenolic and flavonoid contents were estimated in different extracts of the leaf and stem, and their antioxidant and antibacterial activities were evaluated. In the phytochemical screening, phenolics and flavonoids were present in ethyl acetate, methanol, and 50% aq methanol extracts of both the leaf and stem. In TLC analysis, the methanol extract of flowers showed the presence of 11 compounds and the leaf extract showed the presence of 8 compounds. Both extracts contained chlorogenic acid and mangiferin. Hyperoside and quercetin were present only in the flower extract. In the TLC-DPPH. assay, almost all of the flower extracts and 5 compounds of the leaf extract showed radical scavenging potential. Estimation of phenolics and flavonoids showed that all the leaf extracts showed higher amounts of phenolics and flavonoids than stem extracts. Among leaf extracts, greater amounts of phenolics were detected in 50% aqueous methanol extract (261.25 ± 1.66 GAE/g extract) and greater amounts of flavonoids were detected in methanol extract (232.60 ± 10.52 CE/g extract). Among stem extracts, greater amounts of flavonoids were detected in the methanol extract (155.12 ± 4.30 CE/g extract). In the DPPH radical scavenging assay, the methanol extract of the leaf showed IC50 60.85 ± 2.67 µg/ml and 50% aq. methanol extract of the leaf showed IC50 63.09 ± 2.98 µg/ml. The methanol extract of the stem showed IC50 89.39 ± 3.23 µg/ml, whereas ethyl acetate and 50% aq. methanol extract showed IC50 > 100 µg/ml. In the antibacterial assay, the methanol extract of the leaf showed the inhibition zone of 12-13 mm and the stem extract showed the inhibition zone of 7–11 mm against S. aureus, E. coli, and S. sonnei, whereas both extracts were inactive against S. typhi. The findings of this study support the traditional use of this plant in Nepal for the treatment of diseases associated with bacterial infections. The present study revealed that the underutilized anatomical parts of H. cordifolium could be the source of various bioactive phytochemicals like other Hypericum species.


Introduction
Te genus Hypericum is one of the most important medicinal plants that comprise 484 species widely distributed in tropical, temperate, and high mountains around the world [1].Among them, H. perforatum is one of the well-studied species.In Germany, the fowers, leaves, stems, and roots are widely used in traditional medicine [2].It is available in pharmacies as licensed medicine for the treatment of anxiety and mild to moderate depression.In Britain, products prepared from H. perforatum are consumed as traditional herbal medicines.In the USA, it is used as a dietary supplement and has very high demand [3,4].Because of the high economic value of this plant to the herbal industry, it is cultivated in addition to wild collection [5].Generally, the dry extract of the aerial part of the herb is applied for internal use and the oily extract is used for external application [6].
Te efectiveness of H. perforatum extracts to treat depression has been studied in several clinical trials.Te results were positive in comparison to placebo.It has a similar efect to standard antidepressants [7,8].In addition, it showed anti-infammatory, hepatoprotective, antiviral, antimicrobial, antioxidant, antitumor, and wound healing activities [9][10][11].Te observed activities are due to the presence of a wide range of secondary metabolites such as naphthodianthrones, acyl-phloroglucinols, favonoids, phenolic acids, and xanthones [12,13].It has been reported that not only H. perforatum but other Hypericum species have also a wide range of pharmacological activities [14,15].Terefore, they need to be investigated phytochemically and pharmacologically.
Tere are many Hypericum species available in Nepal.In Kathmandu valley, at an altitude between 900 and 2000 m, H. cordifolium is one of the easily available species.Locally, it is called Areli, Areto, or Ghod Jatra Phool.Te golden yellow fowers appear in March to April.Te locals use the fowers for religious purposes, plant juice for menstrual disorders, root juice for diarrhea, and bark juice for back pain and dislocation of bone.Similarly, fower paste is eaten to cure dysentery, fresh young shoots to relieve throat pain, and root and fower juice to treat fever, pneumonia, diarrhea, dysentery, cough, and cold [16].In H. perforatum, the bioactive secondary metabolites accumulate in the aerial parts, so herbal products are usually prepared from leaves and fowers [2].Tis has led us to work on fower, leaf, and stem extracts of H. cordifolium separately.In our previous work, we have investigated fower extracts for wound healing activities, some phytochemicals such as polyprenylated aromatic acylphloroglucinols were isolated, and phytochemical analysis, antioxidant, antibacterial, and cytotoxic activities were evaluated [17][18][19].Te aim of the present work was to evaluate the phenolic composition of leaf and fower extracts by thin-layer chromatography, TLC, and antioxidant properties of components of the extracts by the TLC-DPPH .assay.Furthermore, the contents of phenolics and favonoids in diferent leaf and stem extracts were quantifed, and their antioxidant and antibacterial activities were determined.Te fndings of this study support the traditional medical knowledge of the local people to some extent and utilization of the underexplored natural resources of Nepal for the formulation of herbal drugs or nutraceuticals for primary care.

Plant Materials and Extraction.
Plant materials were collected in April 2021 from the pine forest of Nagarkot area of Bhaktapur district.It was identifed by Prof. Dr. R. P. Chaudhary, Research Centre for Applied Science and Technology, RECAST, Tribhuvan University.Te voucher sample (#HC-21-MR) was deposited at RECAST.Te separated fowers, leaves, and stems were shade dried separately and crushed to fne powder using a kitchen grinder.Te crushed leaf and stem (50 g each) were extracted successively with hexane (250 ml), dichloromethane (250 ml), ethylacetate (200 ml), and methanol (200 ml) using a Soxhlet extractor.Te remaining residue after extraction with methanol was dried and refuxed with 50% aqueous methanol (100 ml) for 2 hours and then allowed to cool and flter.Te solvents were evaporated separately under reduced pressure using rotavapor (BUCHI R-200, BUCHI V-800).Te concentrated extracts were kept in a freezer at −20 °C for further use.

Chemical Screening.
Te presence of various classes of phytochemicals like polyphenols, favonoids, tannins, terpenoids, alkaloids, saponins, quinones, glycosides, and reducing sugars were tested by using diferent specifc reagents.Te standard protocol of Culie was adopted for the detection of phytochemicals [20].

Optimization of TLC Separation and TLC-DPPH Assay.
Te powdered leaves and fowers of H. cordifolium, each 1 g, were frst extracted with dichloromethane and then with methanol at room temperature.Te extracts were analyzed by thin-layer chromatography (silica gel 60F 254 Merck, 11 × 2 cm plate, run time 30 min), and diferent combinations of mobile phases (no.1-5) were used [19,21].Te chromatograms were developed with diferent mobile phases to optimize the chromatographic conditions.Te developed plates were dried at room temperature, and the bands were visualized under a UV lamp (DESAGA, HP-UVIS) at 254 and 366 nm.Subsequently, the plate was sprayed with the chromogenic agent, FeCl 3 .For the determination of R f and λ max values of each band, preparative TLC separation was carried out on the optimized mobile phase in silica gel 60 F 254 aluminum plate (10 × 5 cm).Te major bands were cut carefully and extracted with methanol.
For TLC-DPPH ., the chromatogram developed in an optimized mobile phase was sprayed with 0.2% methanolic DPPH solution and allowed to dry.Te antioxidant activities of diferent components were observed as yellow spots on a purple background [22].14, v/v/v/v) (5) Ethyl acetate-formic acid-water (67 : 13 : 20, v/v/v) 2.5.Determination of Total Phenolic (TPC) and Total Flavonoid (TFC) Content.Te total phenolic content in different leaf and stem extracts was estimated by using the Folin-Ciocalteu (FC) reagent as previously described [23].Te calibration curve was constructed using gallic acid.Various concentrations of gallic acid solutions were prepared (10,25,50,75, and 100 µg/mL).In a 20 mL test tube, 1 mL gallic acid solution of each concentration was added, and then 5 mL 10% FC reagent and 4 mL 7% sodium carbonate were added.Te blue mixture was shaken well and incubated for 30 minutes at 40 °C in a water bath.Ten, the absorbance was measured at 760 nm against a blank using the SHIMADZU UV-1900I spectrophotometer.Similarly, various concentrations of the extracts (100, 50, 25, and 12.5 µg/ml) were prepared.Following the procedure applied for gallic acid, absorbance for each concentration of the extract was recorded.TPC was calculated using the formula: C � c V/m, where C represents total phenolic content in mg GAE/g dry extract, c indicates the concentration of gallic acid obtained from the calibration curve in mg/mL, V indicates the volume of the extract in ml, and m is the mass of the extract in gram.It is expressed as mg gallic acid equivalents (GAE) per gram dry extract (mg/g).
Te total favonoid content in diferent extracts of the leaf and stem was estimated by using the aluminium chloride reagent [24].Te calibration curve was constructed using (±)-catechin.Various concentrations of catechin solutions (10,25,50,75, and 100 µg/mL) were prepared.In a 20 mL test tube, 1 mL catechin solution of each concentration, 6.4 mL of double distilled water, and 0.3 mL of 5% NaNO 2 solution were added.After 5 minutes, 0.3 mL of 10% AlCl 3 solution was added and waited for 1 minute.Ten, 2 mL of 1 M NaOH was added with shaking.Te absorbance of the pink mixture was determined at 510 nm using a SHI-MADZU UV-1900I spectrophotometer.Similarly, various concentrations of the extracts (100, 50, 25 and 12.5 µg/ml) were prepared.Following the procedure applied to catechin, absorbance for each concentration of the extract was recorded.Te total favonoid content of the extracts was calculated as described in the case of phenolics.

Determination of Antioxidant Activity.
Te antioxidant activity of the extracts and ascorbic acid was determined using the DPPH free radical as described by Brand-Williams with a slight modifcation [25].DPPH solution (0.10 mM) was prepared in methanol.Plant extracts and ascorbic acid solutions of diferent concentrations (20,30,40,50,60,70,80, 90, and 100 µg/ml) were prepared in methanol.To 0.5 ml of ascorbic acid or extract, 2.5 ml of DPPH solution was added with shaking and was kept in the dark for 30 min.Ten, absorbance was measured at 517 nm using the SHI-MADZU UV-1900I spectrophotometer.Control was prepared by adding 0.5 ml methanol instead of ascorbic acid or extract.Te percentage of DPPH radical scavenging activity was calculated using formula (1), where Ac is the absorbance of control and As is the absorbance of solution.IC 50 value was calculated from the plotted graph of radical scavenging percentage against the concentration of ascorbic acid or extracts.

Determination of Antimicrobial Activity.
Te antimicrobial activities of the extracts were evaluated by the agar well difusion method [26] against one Gram-positive bacterium Staphylococcus aureus (ATCC 25923) and three Gram negative bacteria, Salmonella typhi (ATCC 14028), Escherichia coli (ATCC25922) and Shigella sonnei (ATCC25931).Te extracts were prepared at a concentration of 50 mg/ml in 50% DMSO.Ten, 50 μL of the prepared extract was introduced into the agar well of 6 mm diameter seeded with the respective microorganisms.Negative control experiments were performed using an equivalent volume of 50% DMSO, and positive control experiments were performed using a standard antibiotic, neomycin (1 mg/ml).Te plates were kept in the refrigerator at 4 °C for 4 hours, and then they were turned over to incubate overnight at 37 °C in an inverted position.At the end of the incubation period, the clear inhibition zone of bacterial growth was observed around each well in the presence of diferent extracts/ neomycin that was measured.

Statistical Analysis.
For total phenolic, favonoid content, and antioxidant activity determination, absorbance data were recorded as a mean of three determinations for diferent concentrations.Te total content of phenolic, favonoid, and IC 50 values in the DPPH assay were calculated from the regression equation of the calibration curve, Y � mx + c, where Y is the absorbance of the extract, m is the slope from the calibration curve, x is the concentration of extract, and c is the intercept.Te linear correlation coefcient (R 2 ) values were also calculated.All the data were presented as a mean ± SD.Te mean values were compared using one-way ANOVA.In the case where the results were statistically diferent (p < 0.05), the Tukey-Kramer multiple comparison test was performed using Microsoft Excel 2016.[27,28].Terefore, we have adopted the Soxhlet extraction method.In the case of the leaf, the extract yield was in the order, CH 2 Cl 2 > MeOH > 50% aq.MeOH > EtOAc > nhexane.In the case of the stem, the extract yield was in the order of 50% aq.

Results and Discussion
MeOH > MeOH > nhexane > EtOAc > CH 2 Cl 2 .Tis indicated that the yield depends on the chemical composition of the sample as well as the type of solvent used in the extraction process.For example, the leaf contains high amounts of less polar phytochemicals that are readily soluble in dichloromethane than hexane.Similarly, the stem contains large amounts of more polar phytochemicals that are readily soluble in 50% aq.methanol than methanol.

Screening for Phytochemicals.
Plants are sources of bioactive compounds.Tey are used for curing as well as healing of various human diseases.Screening of the plant extracts for phytochemicals provides a great knowledge about the occurrence of specifc classes of phytochemicals that has great application.Terefore, phytochemical screening was conducted.Phytochemical screening of leaf and stem extracts revealed the presence of many classes of phytochemicals such as phenolics, tannins, favonoids, alkaloids, saponins, quinones, and terpenoids.In the hexane extracts of the leaf and stem, most of the tested phytochemicals were absent and only terpenoids and quinones were present.In the dichloromethane extract of the leaf, alkaloids, phenolics, and quinones were present, whereas in the stem extract, only phenolics and terpenoids were detected.Te positive test for phenolics is due to the presence of less polar acylphloroglucinols which are characteristics of Hypericum species [14].
In the ethyl acetate extract of the leaf, phytochemicals like alkaloids, favonoids, phenolics, tannins, and quinones were present, whereas in the stem extract, terpenoids, favonoids, phenolics, and tannins were present.In the case of methanol extracts of the leaf and stem, most phytochemicals such as terpenoids, favonoids, phenolics, glycosides, and tannins were present.In addition, the leaf extract also contains alkaloids, reducing sugars, and saponins.In the case of 50% aq.methanol extracts of the leaf and stem, terpenoids, favonoids, phenolics, tannins, and glycosides were present.Additionally, the leaf extract also contains saponins.Te phytochemical screening of leaf and stem extracts revealed that leaf extracts are richer sources of diverse classes of phytochemicals than stem extracts.Tis is the frst report on the phytochemical screening of leaf and stem extracts.In a previous study, only the ethanol extract of the leaf was investigated [29].Te results of phytochemical screening of leaf and stem extracts of H. cordifolium are presented in Table 2.

TLC Separation and TLC-DPPH Assay.
Te patterns of phenolics of H. cordifolium fower, leaf, and stem extracts were examined by the TLC fngerprint method.Te plant material was extracted frst with dichloromethane at room temperature to remove nonpolar phytochemicals followed by methanol.As the aim of the present study is to detect the distribution of phenolic acids, favonoids, and their glycosides as key marker compounds for chemical profling, the methanol extract was chosen for TLC analysis as described by Scotti et al. [3].In TLC analysis, the stem extract did not show detectable bands, so it was discarded.Tis method is useful for confrming the quality of the product.Te United States and the European Pharmacopoeia adopted this method to control the quality of H. perforatum [30].In TLC analysis, diferent mobile phase compositions were used, but better separation was observed in ethylacetate-methanolwater, 100 : 13.5 : 10 (v/v/v) with good resolution of bands as observed under UV lamp 366 nm.TLC analysis of the fower extract revealed seven bands at 254 nm, eleven bands at 366 nm, and nine bands with the chromogenic agent, FeCl 3 .Te leaf extract revealed fve bands at 254 nm, eight bands at 366 nm, and fve bands with the chromogenic agent, FeCl 3 .Most of these bands were phenolic compounds, including favonoids, their glycosides, and phenolic acids.Te bands were identifed based on the colour observed under the UV lamp at 366 nm, the R f values, and the UV λ max values [19,[31][32][33].Te observed results are presented in Table 3.
Band 1 (R f 0.12) in both extracts corresponds to chlorogenic acid, and band 3 (R f 0.40) corresponds to mangiferin.Band 4 (R f 0.46) and band 11 (R f 0.75) corresponding to hyperoside and quercetin, respectively, are present in the fower extract only.A bright orange band with R f 0.64 was seen in the leaf extract and a very light pink band with the same R f value was seen in the fower extract, which could be hypericin.In our previous work, we have reported the presence of chlorogenic acid, hyperoside, and quercetin in the fower extract [19], and hypericin and rutin were not detected in TLC [19].Te results of the TLC fngerprint analysis indicated that the phytochemical compositions of the fower and leaf extracts were diferent.Tis may lead to diferent biological activities of the two anatomical parts.In the case of H. perforatum, Scotti et al. also observed diferent chemical compositions of fower and leaf extracts in HPTLC [3].According to the United States and European Pharmacopoeia, in the TLC of H. perforatum, the lower part should contain rutin, chlorogenic acid, and hyperoside, the middle part should contain three yellow bands of favonoids, and the upper part should contain hypericin, pseudohypericin, and quercetin [34].TLC fnger print analysis indicated that the quality of H. cordifolium is somewhat similar to that of H. perforatum with some diferences in the quantity of secondary metabolites.So, H. cordifolium can also be used as a herbal medicine like H. perforatum.
Te TLC plate after derivatizing with DPPH solution was analyzed in daylight.Yellowish bands on a purple background were marked.In fower extracts, bands of chlorogenic acid, mangiferin, hyperoside, quercetin, and other unidentifed bands appeared on the TLC plate turned into yellow, whereas in the leaf extract, only a band of chlorogenic acid, three bands of favonoids, and the uppermost band turned into yellow.Te results of the TLC-DPPH assay revealed that the fower extract has more antioxidant phytocomponents than the leaf extract.Te antioxidant activities of chlorogenic acid, mangiferin, hyperoside, and Te Scientifc World Journal quercetin have been well reported [35][36][37][38].Te antioxidant activity of H. cordifolium could be due to the presence of these metabolites.Te chromatograms visualized under 245, 366 nm, derivatized with FeCl 3 and DPPH, are presented in Figures 1(a)-1(d), respectively.A photo of the plant is presented in Figure 2.

Total Phenolic and Flavonoid
Contents.Polyphenols, mainly phenolic acids, favonoids, and tannins are common in plant-derived foods and beverages.Tey showed the organoleptic properties of such foods and beverages [39].As the antioxidant and other biological properties are directly related to the polyphenol content, it is necessary to estimate the total phenolics and favonoids in plant samples.Tere are diferent methods for the determination of total phenolics.But the Folin-Ciocalteu method (F-C) is the most common and reliable method.In this method, in the alkaline medium, phenolic compounds transfer electrons to phosphomolybdic/phosphotungstic acid complexes to form blue complexes, possibly (PMoW 11 O 4− 40 ) that are determined colorimetrically at 760 nm [40,41].Gallic acid is widely used as the standard because it is very common in plants.Tis method gives a general measurement of phenolic content [42].TPC in diferent extracts was calculated from the regression equation of the calibration curve (Y = 0.011x + 0.021; R 2 = 0.994).In the case of leaf extracts, the TPC values are in the order, 50% aq methanol (261.25 mg GAE/g) > methanol (122.71mg GAE/ g) > ethylacetate (102.37 mg GAE/g).In the case of stem extracts, the TPC values are in the order, methanol (26.48 mg GAE/g) > 50% aq.methanol (24.06 mg GAE/g) > ethyl acetate (7.32 mg GAE/g).Tis indicated that the leaf extracts contained relatively higher amounts of phenolic compounds than stem extracts.In our previous work, we have found that the phenolic content of fower extracts was in the order, 50% aq.methanol (228.19 ± 0.639) > ethyl acetate (227.17 ± 0.736) > methanol (199.28 ± 0.576) [19].Tis indicated that fower and leaf extracts are richer sources of phenolic compounds than stem extracts.Shresta et al. reported that the total phenolic content of the methanol extract of the leaf was 36.28 mg GAE/g dry extract which is much lower than our fnding [43].
Te total favonoid content in plant extracts was determined colorimetrically using aluminum chloride.It is used as a complexing agent and form chelates of Al (III)favonoids due to their many oxo and hydroxyl groups.Tis assay was frst developed by Christ and Muller for the determination of favonol derivatives in drugs [44].Later, it was modifed by the addition of NaNO 2 before the addition of AlCl 3 .It serves as a nitrating agent that is selective for aromatic vicinal diols to produce a favonoid-nitroxyl derivative which shows absorption at 510 nm [24].
Te total favonoid content in diferent extracts was calculated from the regression equation of the calibration curve (Y � 0.002x + 0.002; R 2 � 0.999).In the case of leaf extracts, the TFC values are in the order of methanol (232.60 mg CE/g) > 50% aq.methanol (62.63 mg CE/g) > ethyl acetate (51.47 mg Te Scientifc World Journal  Te Scientifc World Journal CE/g).In the case of stem extracts, the TFC values are in the order of methanol (155.10 mg CE/g) > ethyl acetate (34.10 mg CE/g) > 50% aq.methanol (20.62 mg CE/g).In our previous work, we have found that the favonoid content of fower extracts was in the order ethyl acetate (306.41 ± 0.95) > methanol (231.5 ± 0.93) > 50% aq.methanol (227.4 ± 0.99) [19].Tis indicates that fower and leaf extracts are richer sources of favonoid compounds than stem extracts.Shresta et al. reported that the total favonoid content of the methanol extract of the leaf was 5.89 mg CE/g dry extract which is very much lower than our fnding [43].Te low content of phenolics and favonoids could be due to the chemical diversity arising from diferent collection sites and time.Te results are shown in Table 4.

DPPH Free Radical Scavenging Activity.
Plant phenolics have a preventive efect on chronic diseases caused by oxidative stress.It is well known that antioxidants such as vitamin C, vitamin E, carotenoids, organo sulfur compounds, and mostly polyphenols are very common in fruits, vegetables, nuts, and medicinal herbs.Tese antioxidants break radical chain reactions and prevent oxidative stressrelated damage [45,46].Terefore, it is necessary to test the radical scavenging capacity of medicinal herbs.For this purpose, a stable DPPH free radical was used.When a solution of DPPH is mixed with a substance that can donate a hydrogen atom, DPPH is reduced with the loss of its violet colour to pale yellow.Te Scientifc World Journal In the DPPH free radical scavenging assay, the IC 50 values of the leaf extracts were in the order, methanol (60.85 ± 2.67 µg/ml) > 50% aq.methanol (63.09 ± 2.98 µg/ ml) > ethyl acetate (96.43 ± 3.85 µg/ml).In the case of stem extracts, the order of IC 50 value is methanol (89.39 ± 3.23 µg/ml) > 50% aq.methanol and ethyl acetate extracts (>100 µg/ml).In our previous work, we have found that the IC 50 values of fower extracts were in the order, ethyl acetate (18.79 ± 0.98 µg/ml) > methanol (18.98 ± 0.81 µg/ ml) > 50% aq.methanol (27.00 ± 1.03 µg/ml) [19].Tis indicated that the radical scavenging activity is in the order, fower > leaf > stem.Tis could be due to the presence of more phenolics and favonoids in fower and leaf extracts than in stem extracts.Tis was also supported by the results of the TLC-DPPH .analysis of fower and leaf extracts.In the DPPH free radical scavenging assay, the IC 50 values of ethyl acetate (2.63 ± 0.01 µg/ml), methanol (3.63 ± 0.01 µg/ ml), and methanol-water (4.71 ± 0.04 µg/ml) extracts from aerial parts of H. perforatum have been reported by Öztürk et al.Tey found a strong correlation between higher phenolic/favonoid content and antioxidant activities [42].Similarly, Raut et al. reported the IC 50 value (20.68 µg/ml) of the methanol extract from the aerial parts of H. cordifolium in the DPPH free radical scavenging assay [47] which is close to our results for fower extracts as the aerial parts of H. cordifolium mostly contain fowers.Te results are presented in Table 4.
3.6.Antimicrobial Activity.Bacterial infections and antibiotic drug resistance are the major challenges these days.Phytochemicals alone or in combination with antibiotics have exerted potential antibacterial activities against drugsensitive and drug-resistant pathogens via diferent mechanisms of action [48][49][50].Testing of plant extracts for antibacterial activity may help fnd new compounds that may cause synergistic efects with the existing antibiotics.
Flavonoids are known for their antibacterial activities [51,52].As the methanol extracts of the leaf and stem contained higher amounts of favonoids than other extracts (Table 4), they were selected for antibacterial assay.Te extracts were tested against one Gram-positive bacterium, S. aureus, and three Gram-negative bacteria, E. coli, S. typhi, and S. sonnei.It is well known that S. aureus is mainly responsible for skin infection, S. typhi for typhoid fever, while E. coli and S. sonnei for diarrhea and dysentery.Te tested extracts showed only weak activities with the inhibition zones that ranged from 7 to 13 mm against all tested bacteria except for S. typhi.Te antibacterial activities of the methanol extract of the leaf have been reported by Shrestha et al., against S. aureus and E. coli, as well as against some strains of multidrug resistance bacteria such as K. pneumonia, methicillin-resistant S. aureus, P. aeruginosa, and A. baumannii [43].Similarly, Raut et al. reported the antibacterial activities of methanol extracts of aerial parts against S. aureus and E. coli [47].Te antibacterial activities depend on the season of collection, the plant part used, and the extraction methods [53].Shrestha et al., and Raut et al., extracted the plant materials directly with methanol, and the overall antibacterial activity could be due to the acylphloroglucinols, naphthodianthrones, xanthones, phenolic acids, and favonoids present in the extracts.Acylphloroglucinol derivatives have potential antibacterial and cytotoxic activities [54].In our study, we have adopted the polarity-based extraction method where the antibacterial activity of the methanol extract could be due to polar phenolics and favonoids compounds.Tese results of antibacterial activities support, to some extent, the traditional use of this plant to treat diarrhea, dysentery, and fever due to bacterial infections [16].Te results are presented in Table 5.

Conclusions
Te present study highlights the phytochemical analysis and the antioxidant and antibacterial potential of leaf and stem extracts of H. cordifolium, native to Nepal.Qualitative TLC analysis of the methanol extract of the fower and leaf showed that they difer from each other in chemical composition.Te fower extract showed the presence of more phytochemicals than the leaf extract.However, some common marker compounds such as chlorogenic acid and mangiferin were  8 Te Scientifc World Journal present in both extracts.Hyperoside and quercetin were present only in the fower extract.TLC fnger print analysis revealed that the quality of H. cordifolium was somewhat similar to H. perforatum.In the TLC-DPPH .assay, almost all phytochemicals of the fower extract showed radical scavenging activity, while only few phytochemicals in leaf extracts showed radical scavenging activity.Quantitative estimation of phenolics and favonoids indicated that the leaf extracts were the good source of these phytochemicals than the stem extracts.In the antioxidant assay, the leaf extract showed lower IC 50 values than the stem extracts.However, in statistical analysis using one-way ANOVA followed by the Tukey test, no signifcant diference between the IC 50 values of the ethyl acetate extract of the leaf and the methanol extract of the stem was observed.A similar case was observed between the methanol and 50% aq.methanol extracts of the leaf.But there were signifcant diferences in IC 50 values between the extracts and standard ascorbic acid.In the antibacterial assay, the leaf extracts exhibited greater inhibition zones than the stem extracts.However, in statistical analysis, no signifcant difference in the inhibition zone was observed between the leaf and the stem extracts against S. aureus and S. sonnei.In contrast, signifcant diferences were observed in the inhibition zones produced by extracts and the standard antibiotic, neomycin, against S. aureus and S. sonnei, while no diference was observed against E. coli.In conclusion, leaf and stem extracts of H. cordifolium were weak antioxidants and antibacterials in comparison to the respective standards.However, in general, the fnding of our study indicated that in terms of the content of phytochemicals, antioxidant, and antibacterial potencies, the diferent anatomical parts of H. cordifolium are in the order fower > leaf > stem.Terefore, the underutilized natural resources of Nepal can be used for the formulation of dietary supplements, nutraceuticals, or herbal medications.But a toxicological study is also crucial before formulation of herbal drugs.

Table 1 .
At elevated temperature, the viscosity and surface tension of the solvents are decreased.It helps the solvents reach the sample matrices which facilitate the extraction rate 3.1.Extractive Values in Diferent Solvents.Te analysis of herbal drugs generally requires suitable extraction methods.Extraction with solvents of increasing polarities above room temperature, starting from n-hexane, dichloromethane, ethyl acetate, methanol, and 50% aqueous methanol, provided diferent amounts of extracts.Te results are presented Te Scientifc World Journal in

Table 1 :
Percentage yield of leaf and stem extracts.

Table 2 :
Phytochemical screening of leaf and stem extracts.