1Phytochemical, Antibacterial and Antioxidant Activities of Anthurium Hookerii

2leaves Extracts

4Atmira Sariwati1, Inayah Fitri2, Adi Setyo Purnomo3, and Sri Fatmawati3*

61Department of Chemistry, Faculty of Sciences, Technology and Analysis, Institut Ilmu

7Kesehatan Bhakti Wiyata Kediri.

82Department of Biology, Faculty of Sciences, Technology and Analysis, Institut Ilmu

9Kesehatan Bhakti Wiyata Kediri.

103Department of Chemistry, Faculty of Mathematics and Natural Sciences, Institut

11Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya 60111, Indonesia.

12

13

14

15*Corresponding author

16E-mail: fatma@chem.its.ac.id

17Tel / Fax: +62-31-5943353 / +62-31-5928314

1
 1Abstract

2Many plants of the family of Araceae posses significant benefit as medicinal plants.

3Anthurium hookerii is herbaceous genus of the family of Araceae. Anthurium hookerii leaves

4were extracted by using five different solvents (water, methanol, ethyl acetate, n-hexane and

5dichloromethane). The extracts were tested for their phytochemical and bioactivities such as

6antioxidant and antibacterial activities. The presence of tanins and saponin was found in all

7crude extracts. The steroid was only found in dichloromethane extract, whereas flavonoid was

8obtained in methanol and water extracts. Besides, methanol, ethyl acetate, water and n-hexane

9extracts showed triterpenoid contents. Alkaloid presences in ethyl acetate, methanol,

10dichloromethane, and water extracts. The total phenolic content was examined according to the

11Follin Ciocalteu method, and which varied from 9.52 - 62.66 mg GAE/g. The highest total

12phenolic was found in methanol extract. Antioxidant activity was measured based on 1,.1-

13diphenyl-2-picrylhydrazyl (DPPH) method that showed the scavenging activity was found

14with range 7.24 % - 66.1%, which and the methanol extracts showed the highest antioxidant

15with IC50 value of 232.90 µg/mL. Antibacterial activity of leaves extracts of A.nthurium

16hookerii was screened based on disc diffusion method. Water extract was active against Gram-

17negative and Gram-positive bacteria Klebsiella sp.,, Bacillus subtilis, Pripioni agnes and

18Strepticoccus mutans with maximum diameter of inhibition zone 10.30, 14.20, 9.60 and 15.10

1 2
 1mm, respectively. These results indicated that methanol extract of A. hookerii leaves was

2potential for antioxidant, while water extract was potential source as new antibacterial agent.

3Key words: Antioxidant, Antibacterial , Anthurium hookerii, DPPH assay, Disck

4diffusion method
5
61. Introduction

7 For centuries, herbal medicines have been heavily utilized in the development of

8pharmacopoeias providing a major focus in global health care (Graham et alet al.,

92000). The value of medicinal plants is a potential source of bioactive compounds.

10Those are normally accumulated as secondary metabolites in all plant cells but their

11concentration varies according to the plant part, season, climate and particular growth

12phase (Maji et alet al., 2010).

13 Many plants respond to environmental stressors by producing antioxidant such as

14polyphenol. Natural antioxidants either in the form of raw extracts or their chemical

15constituents are effective to prevent the destructive processes caused by oxidative stress.

16The antioxidant absorbs and neutralizes free radicals, quenching singlet and triplet

17oxygen or inducing expression of peroxides and other toxic metabolites (Djeridane et

18alet al., 2006; Kumar et alet al., 2013; Iloki-Assanga et alet al., 2015). Free radical are

19continiously produced in the mitochondrial respiratory chain or due to exposure to

20environmental stress (atmospheric pollutants) and cause oxidative damage to lipids and

1 3
 1nucleic acids (Naznin et alet al., 209; Shui et alet al., 2004; Jebakumar et alet al., 2012),

2and it also can be implicated in many diseases like cancer, atherosclerosis, arthritis

3parkinson’s disease, and neurodegeneration (Makari et alet al., 2008; Zetola et alet al.,

42002; Jebakumar et alet al., 2012).

5 The aAnother benefit of several medicinal plants is an antibacterial agent. Those

6are proven that have ability to inhibit the growth of pathogenic bacteria. Infectious

7diseases are still becoming health problem in development countries. According to

8Geyid et alet al. ( 2005), mMany infectious disease such as chlera, diarrhoea, lung

9disease and cure skin can be treted by medicinal plants.

10 Within the recent years, infectious diseases have increased to a great extent and

11antibiotic resistance makes this condition more complicated to faced and it needs

12several effort to overcome this problem. The use of synthetic antioxidant are being

13restricted because of their toxicity and carcinogenic effects. It is generally accepted that

14medicines derived from plant products are safer than their synthetic counterpart

15(Vongtou et alet al., 2005; Oluyemi et alet al., 2007; Saeed, 2012). Therefore, worldwide

16movements toward finding out chemical constituents from various parts of plant and the

17bioactivity studies of the novel drugs isolated. The family of Araceae has been proven

18as significant antibacterial and antioxidant agents.

19 Anthurium is herbaceous genus of the family of Araceae found through South

1 4
 1America. Some species of Anthurium are used in traditional medicine for the treatment

2of different diseases (Joly et alet al., 1987; Zamora-Martinez et alet al., 1992). The polar

3extract of the leaves of Anthurium versicolor was tested for antioxidant activity by

4DPPH method (Aquino et alet al., 2001). The flowers and leaves of Anthurium

5cerrocampanense have been used as an antiinflammantory agent (Segura et alet al.,

61998). Anthurium acutangulum was used as a remedy for whooping cough (Duke,

71986). Besides, Anthurium wagnerianum had stimulant activity (Di Carlo et alet al.,

81964), while Anthurium adreanum also had potential against Bacillus, Staphylococcus,

9Escherichiu serratia, Pseudomonas, Proteus, Aerobacter, Klockera, Saccharomyces,

10Mycobacterium, Penicillium, Scopuloriopsis, and Fusarium (Donberger et alet al.,

111982). After studying several literatures, as yet there are no data related to the potential

12of A. hookerii as an antioxidant and antibacterial agents.

13 The main objective of this study was to screen different solvent extracts from

14A. hookerii leaves and tested the biological activities. The antioxidant was evaluated by

15DPPH scavenging, while total phenolic content was evaluated by Follin-ciocalteu,

16phytochemical screening and antibacterial activity were evaluated by disk diffusion

17method as well.

18

192. Materials and methods

202.1. Chemicals

21 The chemicals used in this study were dichloromethane, methanol, n-hexane

22ethyl acetate, dimethyl sulfoxide (DMSO), sulfuric acid (H2SO4), hydrochloric acid

23(HCl), Potassium iodide, iodine, glacial acetic acid, ferric chloride, chloroform, and

24media cultures (nutrient agar (NA) and nutrient broth (NB), which purchased from

1 5
 1Merck in high grade. 2,2-diphenyl-1 picrylhydrazyl (DPPH) was purchased from Tokyo

2Chemical Industries (TCI, Tokyo, Japan). Gallic acid was purchased from Wako Pure

3Chemical Industries (Osaka, Japan). Follin-Ciocalteu reagent was obtained from Merck.

4Mueller Hinton Broth agar (NB, Merck, Darmstadt, Germany).

52.2. Bacteria culture conditions

6 Stock cultures of B. subtilis NBRC 3009, P. aeruginosa NBRC 3080 (NITE

7Biological Resourches Center, NBRC; Chiba, Japan), Klebsiella sp., Streptococcus

8mutans, which obtained from the Collection of laboratory of Microbial Chemistry,

9Department of Chemistry, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia

10were maintained on 9-cm diameter nutrient agar (NA, (Merck, Darmstadt, Germany)

11that had been incubated at 37°C. The colony was inoculated into 60 mL of nutrient broth

12(NB, (Merck, Darmstadt, Germany) medium in 100-mL Erlenmeyer flasks. The cultures

13were pre-incubated at 37°C for 20 h with shaker at 180 rpm (Wahyuni, 2016).

142.3 Plant material and preparation of sample

15 Plant material was collected from Ngawi, Indonesia. Fresh plant material A.

16hookerii leaves were washed with distilled water and cut into small pieces, dried

17overnight in an air dryer at 40oC, ground to a particle size of 25 mesh using a grinder.

182.4 Extraction of A. hookerii leaves

19 Dried powder of A. hookerii leaves samples (20 g) were placed in a 500 ml flask,

20mixed with 200 ml of solvents (water, methanol, ethyl acetate, dichloromethane, and n-

21hexane) separately and tightly wrapped with aluminum foil. Extraction was carried out

22using an orbital shaker at 180 rpm for 24 hours, after which the suspensions was filtered

23using Whatman No 1 filter paper. The filtrate was evaporated using rotatory evaporator

1 6
 1maintained at 68oC for n-hexane extract, 100oC for water extract, 77oC for ethyl acetate

2extract, 40oC for dichloromethane extract and 65oC for methanol extract to get dry

3extracts. Solvent free extracts were transferred to extract vials and store at 4 oC for

4further used.

52.4 Preliminary phytochemical screening of A.nthurium jenmanii

6 Phytochemicals screening of A. hookerii for the presence of alkaloids, saponins,

7 tannins, flavonoids, and triterpenoids wereas done by treating leaves on different

8 extracts by using standard procedure.

92.4.1 Test for alkaloids

10 Test for alkaloids was done by Dragendroff’s test : 2 mg of the A. hookerii

11extracts and was added 5 mL of distilled water .was added, 2 M hydrochloric acid (HCl)

12was then added until an acid reaction occurs, which followed .added To this 1 mL of

13Dragendroff’s reagent was added. Formation of orange red precipitate indicates

14indicaated the presence of alkaloids (Joshi et alet al., 2013; Aabdulahi et alet al., 2013;,

15Iqbal et alet al., 2015).

162.4.2 Test for saponins

17 0.5 g of the A. hookerii extracts (0.5 g) was separately shaken with distilled

18water (10 mL) in test tube. The formation of frothing which persist on warming in a

19water bath for 5 min, shows the presence of saponins (Banso and Adeyemo, 2006; Iqbal,

202015).

212.4.3 Test for tannins

22 0.5 g of the A. hookerii extracts (0.5 g) was separately stirred with distilled

23water (10 mL) and then filtered. A few drops of 5% ferric chloride (FeCl 3) were then

24added. Black or blue-green precipitate iswas taken as positive result for presence of

1 7
 1tanins (Banso and Adeyemo, 2006; Iqbal, 2015).

22.4.4 Test for steroids

3 0.5 g of the A. hookerii extracts (0.5 g) was boiled in 10 mL chloroform

4(CHCl3) and filtered, and then added 1 mL of l acetic anhydride and few drops of

5concentrated sulfuric acid (H2SO4) to the filtrate. Green ring indicatesd presence of

6steroid (Samejo, 2013).

72.4.5 Test for flavonoids

8 0.5 mg of the A. hookerii extracts (0.5 mg) was dissolved in 0.5 mL of its

9solvent (methanol), followed added. A a few drops of dilluted sodium hydroxide

10(NaOH) solution. An intense yellow colour appeared in the extract, which became

11colourless upon the addition of a few drops of diluted sulfuric acid (H 2SO4), .This

12showsn the presence of flavonoids (Alabri et alet al., 2014).

132.4.6 Test for triterpenoids

14 A. hookerii extracts (5 mL) Five milliliters of extracts were mixed with 2 ml of

15chloroform and few drops of H2SO4. Blue/green ring precipitate indicatesd the presence

16of triterpenoids (Samejo, et alet al., 2013).

17
182.5. Total phenolic content

19 20 mg of the test A. hookerii extracts (20 mg) extract A. hookerii was dissolved

20in a solution of 5 mL of 3% HCl in 60 % methanol and the resulting mixture (100 µL)

21was added to 2 mL of aqueous sodium carbonate solution. After 3 min, 100 µL Follin- –

22Ciocalteu phenol reagent were added to the mixture. After 30 min standing, the

23absorbance was measured at 750 nm against a blank. Standar calibration curve for gallic

1 8
 1acid with variation concentration 0.5, 1.0, 1.5, 2.0, and 2.5 mM wereas prepared in the

2same manner and result were expressed as mg gallic acid equivalent (GAE) per gram of

3extract (Tsai et alet al.,. 2009).

42.6 Antioxidant activity (DPPH radical scavenging activity)

5 The free radical scavenging capability of each extracts solution on DPPH

6radicals was determined as described previously (Joly et alet al., 1987; Janet et alet al.,

72015). The stock solution was prepared by dissolving 24 mg DPPH with 100 ml

8methanol. The working solution was obtained by diluting DPPH solution with methanol

9to attain an absorbance of about 0,98 ± 0.02 at 517 nm using spectrophotometer. One-

10milliliter aliquot of this solution was mixed with 33 µLl of sample at various

11concentrations (10-500 µg/ml). The reaction mixture was shaken well and incubated in

12the dark for 20 min at room temperature. The control was prepared as above without

13sample extracts. The scavenging activity was estimated based on the percentage of

14DPPH radical scavenger as the following equation :

15Inhibition radical scavenging (%) =

16
17The IC50 values representing the concentration of sample required to scavenge 50% of

18
the DPPH free radical were calculated by implementing

19 vs normalized response model with a variable slope

20(Fitriana, 2016).

1 9
 12.7 Antibacterial activity

2 The antibacterial screening was carried out by agar disck diffusion method with

3determination of diameters of inhibition zones made by A. hookerii extract against

4various bacterial strains (Klebsiella. sp. and P.ropioni agnes, as Ggram- negative

5bacteria,as well as B. subtilis and S.trepticoccus mutans as Ggram- positive bacteria).

6Sterilized Mueller Hinton Broth (MH) agar (MH) was poured into petri plates

7individually, and then inoculated with 100 µLl suspension of tested bacteria. Five-

8millimeter discs of Whatman No 1 filter paper were prepared and immersed in each of 1

9ml extracts solution (10 mg) in DMSO ( Alabri et al, 2014)[32]. Chloramphenicol was

10used as positive control. The plates were incubated at 377 oC for 24 hours for maximal

11bacterial growth. Antibacterial activity was evaluated by measuring diameter (mm) of

12inhibition zones using a zone reader. Commercial antibiotic disc of Chloramphenicol

13(10 µg/disc) was used as positive controls, while . DMSO was used as a negative

14control.

152.7 Statistical analysis

16 Values were the average of triplicate determinations. Student’s t-test was used

17to detect any significant differences between or within groups during substrate

18transformation. Differences between means at a confidence level of 5% (P < 0.05) were

19considered to be statistically significant.

20

213. Results

22 Extraction yield refers to the percentage of extracts, which obtained from dried

23plant sample through a solvent extractions procedure for further isolation and

1 10
 1utilization. Table. 12 showed that among of five solvents, methanol produced the

2highest extraction yields (1.92 g dried extract). However, this yield was not significantly

3different from the water extract (1.65 g dried extract). Both the of methanol and water

4extracts were found to be significantly different (P < 0.05) compared with those

5dichloromethane (0.76 g), ethyl acetate (0.70 g), and n-hexane extracts (0.44 g) dried

6extracts., respectively.

7 Phytochemical screening was shown in Table 21. The presence of sSome

8secondary metabolites such as tanins and saponin were presented in all extracts. The

9presence of steroid was found only in dichloromethane extract. The presence of alkaloid

10was absent only in n-hexane extract. The presence of flavonoids was found in methanol

11and water extracts and while tri terpenoids contents was found in ethyl acetate,

12methanol, n-hexane and water extracts.

13 Total phenolic content of the A. hookerii was measured using Folin-Ciocalteu

14method. Total phenolic compound values were obtained from the calibration curve

15method using Gallic acid (y = 0.3636x – 0.0077; r2 = 0.9988), where yx is the

16absorbance and xy is the concentration of gallic acid solution (µg/mL) expressed mg

17GAE/g. Total phenolic of different solvent of A. hookerii extracts was shown in the

18Table. 2. Methanol crude extract hads the highest total phenolic content (76.56 mg

19GAE/g) among the various solvent of A. hookerii leaves extracts, closely followed by

20water extract (62.66 mg GAE/g), but siugnificant different with ethyl acetete extract

21(26.85 mg GAE/g), cloesely followed by dichloromethane extract (26.85 mg GAE/g)

22and the lowest was obtained by n-hexane extract (9.52 mg GAE/g) respectively.

1 11
 1 Total antioxidant capacity of various solvent extracts of A. hookeri leaves were

2calculated using the standard curve gallic acid (y = 34.80 ln (x) – 109.8; r 2 = 0.970).

3The DPPH scavenging ability of methanol (66.11%) was the highest than those of ethyl

4acetate (26.77%), dichloromethane (18.18%), water (15.22%) and n-hexane (7.24%)

5extracts. The positive control, gallic acid, showed highest DPPH inhibition (97.80%).

6 In this study, 50 percent inhibition was found only in methanol extract, so Tthe

7best antioxidant was obtained in methanol extract determined with IC50 values of DPPH

8radicals scavenging was obtainedof 232.90 μg/mL .

9 Antibacterial activity of A. hookeri leaves extracts against 4 pathogenic bacteria

10was shown in Table 3. Water extract showed the excelent activities against Klebsiella

11sp., and B. subtilis, with which inhibition zone greater than the positive control

12(chloramphenicol). The inhibition zone of water extract was obtained againstagainst

13P.ropioni agnes (9.6 mm) and S.trepticoccus mutans (15.10). Methanol extract showed

14activity against Klebsiella sp. with inhibition zones of 9.10 mm. However,

15dichloromethane, ethyl acetate, and n-hexane extracts showed the weak low antibacteria

16activities against four bacteria, because they could not yet surpassedsurpass the positive

17control.

184. Discussion

19 Yield results of maceration extracts with different solvent produce different

20percentage yield (Salamah et alet al., 2013). Polar solvents extracted significantly

21greater yield than the nonpolar counterpart. The efficiency of methanol as a solvent

22relates to its intermediate polarity, which allows it to solvate low molecular weight

1 12
 1organic compounds possessing proton table functional groups (e.g. COOH, OH)

2(Nguyen et alet al., 2015). The highest extraction yields was obtained by methanol

3solvent, which t. This may be attributable to the higher solubility of proteins and

4carbohydrates in methanol (Zielinski, 2008; Do, 2014).

5 Phytochemical screening of A. hookerii with different solvents extracts of A.

6hookerii showed varied different results, which . The results of identified bioactive

7compound of A. hookerii are presented in Table. 1. The phytochemical screening of all

8variation solvent of extracts A. hookerii leaves showed the presence major of active

9chemical constituents such as saponin and tannins. Tannins and its derivatives are

10phenolic compounds considered to be primary antioxidants or free radical scavenger

11(Alabri et alet al., 2014; Barile et alet al., 2007; Ayoola et alet al., 2008; Varahalarao,

122012; Sekar et alet al., 2012). Saponins are also bioactive constituents that which

13involved in plant defense system because of their antimicrobial activity (Alabri et alet

14al., 2014; Barile et alet al., 2007; Ayoola et alet al., 2008) and as antioxidant and as well

15as anti inflammatory agents (Najafi et alet al., 2010; Samejo et alet al., 2013). However,

16ethyl acetate, dichloromethane, methanol and water extracts also were detected other

17bioactive compounds, alkaloid. Many alkaloids derived from medicinal plants showed

18biological activities like antimicrobial (Iqbal et alet al., 2015; Benbott et alet al., 2012).,

19The presence of flavonoid was obtained in methanol and water extracts. Besides, The steroid

20was only found in dichloromethane extract. Steroids have been reported to have

21antibacterial (Yadav et alet al., 2013; Samejo et alet al., 2013). Methanol, n-hexane, ethyl

22acetate, and water extracts showed triterpenoid contents. Triterpenoid have been reported to

23have antibacterial properties (Najafi et alet al., 2012; Samejo et alet al., 2013). The previous

1 13
 1reported that n-BuOH extract from leaves of A.nthurium versicolor revealed presence of

2phenolic compounds (including flavonoid and phenyl propanoid derivatives) (Aquino,

32001). Aquaeous, chloroform and ethanol extracts from leaves of A.nthurium adreanum

4revealed presence of tannin (Shazhni et alet al., 2016).

5 Among five extracts of A. hookerii, The total phenol content among the five

6crude extracts such as methanol extract showedwas containing the highest amount of

7phenol (76.56 mg GAE/g), followed by water (62.66) > ethyl acetate (26.85 mg GAE/g)

8> dichloromethane (26.08 mg GAE/g) > n-hexane (9.52 mg GAE/g). The recovery of

9phenolic contents in different samples is influenceds by the polarity of extracting

10solvents and solubility of each compound in the solvent use for extraction process

11(Alothman, 2009; Sulaiman, 2011; Iloki-Assana, 2015). Solvent polarity plays key role

12in increasing phenolic solubility (Naczk et alet al., 2006; Medini 2014). These phenolic

13compounds may posses more phenol in the methanol extract. Chemical group of

14compounds in the meathanol extract implied in this total phenolic content. Phenolic,

15which are aromatic metabolites that have one or more acidic phenolic hydroxyl group,

16are futher classified into hydroxycinnamic acids, flavonoids, anthocyanins and tannins

17(Unal, 2014). The tannins contents are with more half of the polyphenolic contents, so

18the capacity reductive of the Fe(III) in Fe(II) is in the order of the tannin contribution on

19the polyphenolics content (Bangao, 2012). This result similar with previous report that

20methanol have been proven as effective solvent for extraction of phenolic compounds

21(Siddhuraju, 2003; Iloki-Assanga, 2015). n-hexane was the lowest total phenolic content

22that may be attributable to the content of more non phenol compounds such as

23carbohydrate and terpene than in order extract. This result was different significant with

24previous work that confirm the highest total phenol content of crude n-BuOH extract of

1 14
 1A. versicolor was obtained 190.6 µg/mg (Aquiono, 2001). The higher total phenol

2content in plant analysed could have relation with plant stress with the possibility of the

3presence of polyamines as stress indicators (Unal, et alet al., 2014).

4 The free radical scavenging activity of the A. hookerii leaves extract were

5tested through DPPH method. The principle of DPPH method is that the antioxidant

6agents react with the DPPH and produce stable free radicals. Accordingly, DPPH

7change its own colour gradually. This phenomenon indicates the sample contain

8antioxidant agent (Hossain et alet al., 2014).

9 The highest DPPH scavenging ability was obtained by methanol extract

10(64.11%) and the positive control, gallic acid, showed higher DPPH inhibition

11(97.80%). The different extracts obtained difference in the secondary metabolite

12constituent as reflected in the result of antioxidant potential (Janet et alet al., 2015). The

13high antioxidant activity of The methanol extract might be caused by was screened on

14the presence tannin, flavonoid, and saponin (Table 1). Antioxidant properties of

15flavonoids are derived from the ability to transfer an electron or to donate hydrogen

16atom to the free radicals compounds and also form complexes with metal (Redha ,

172010). Saponin compounds are radical scavenger by forming hydrogen peroxide as an

18intermediate and can donate hydrogen to DPPH radical compound that terminate radical

19chain reactions (Xiong et alet al., 2010: Nurjanah, 2015). Tannins have the abilitiesy to

20capture free radicals. These compounds are very effective as an electron donor and

21hydrogen atom and chelating metals, because it have a hydroxyl group and conjugated

22double bonds that allow the delocalization of electron (Nurjanah et alet al., 2015). nN-

23hexane extract requires very high concentration to achieve DPPH radical scavenging

24caused the property of the nonpolar solvent containing only non polar compounds only

1 15
 1such as essential oils, fat and wax that does not potential antioxidant (Suratmo, 2009;

2Nurjaah, 2015).

3 The IC50 of a compound is inversely related to its antioxidant capacity, as it

4express the amount of antioxidant required to decrease the DPPH concentration by 50%,

5which is obtain by interpolation from linear regresion analysis. A lower IC 50 indicates a

6higher antioxidant activity of a compound (Liu, 2009; Do, 2014). In this study, 50

7percent inhibition was found only in methanol extract showed , so the best antioxidant

8determined with IC50 values of DPPH radicals scavenging was obtained 232.90 μg/mL.

9These result are consistent with bioactive compounds of phenol detected in the

10methanol extract include flavonoids, tanins and saponins, and it iswhich proved that the

11metahanol extract was the greater total phenolic content. Usually componentGenerally,

12most of that have antioxidant activity compounds are phenolicphenolic compound that

13have hydroxyl group subtituted in the ortho position to the –OH and –OR (Andayani, et

14alet al. 2008; Nurjanah et al, 2015). Methanol extract of A. hookeri is classified as a

15weak antioxidant because IC50 values more than 200 ppm (Molyneux , 2004; Nurjanah

16et al., 2015). This result similar with chloroform extract of Alocasia fornicate leaves

17(248.85μg/mL) and n-hexane of extract Alocosia marcrorbiza leaves (245.17 μg/mL)

18belong to the family Araceae (Mandal et al, 2010). However, This this result was

19different from significant EC50 (142,6 μg/mL) of n-BuOH extract from the leaves of

20A.nthurium versicolor with EC50 (142,6 μg/mL) (Aquino, 2001).

21 In the present study, tThe correlation between the total phenolic contents and

22antioxidant activity A. hookerii was evaluated. There was correlation between total

23phenolic compound and antioxidant activity and as well as the IC50. A significant and

1 16
 1linear relationship existed between the antioxidant activity and phenolic content of

2methanol extract, thus indicating that phenolic compounds are major contributors to

3antioxidant activity (Maizura, 2011), a property derived from their redox abilities,

4which can quench and neutralize the free radical (Florence, 2011; Iloki-Assanga, 2015).

5This result not difference significant with the previous report which evaluated the

6correlation between antioxidant activityactivities and total phenolic of 133 species of

7medicinal plants of Indiana (Surveswaran et alet al., 2007). This wasis carried as by

8Hardiana et alet al. (2012); Nurjanah et alet al. (2015) stated that phenolic compounds

9known to contribute significantly to the antioxidant activities, the greater the content of

10phenolic compounds either with the antioxidant activity.

11 The in vitro antibacterial activity of the five A. hookeri leaves crude extract against

12the employed four bacteria was qualitative assessed by the presence or absence of inhibition

13zones. An extract is active if it induces an inhibition zone superior at 3 mm around the disc

14(Schulz et alet al. 1995; Bangau 2012). Taking account of this consideration, we can say that

15all extract were active on the following species S.treptococcus mutans, B. subtilis, Klebsiella.

16sp., and P. agnes. The maximum inhibition zones wereas 15.10, 14.20, 10.30 and 9.60 mm

17mm in case S.treptococcus mutans, 14.20 mm in case B. subtilis, 10.30 mm in case

18Klebsiella. sp., and 9.60 mm in caseand P. agnes, respectively, which obtained from water

19extract. as compared with the positive control (Chloramphenicol), these strong antibacteria

1 17
 1activity was found in water extract with a broad antimicrobial spectrum against both Gram-

2positive and Gram-negative bacteria. Water extract was the most effective against Klebsiella.

3sp. and B. subtilis, because the inhibition zone was higher more superior than positive control.

4While trying to understand, the reason of this strong inhibition of water extract because plant

5produce secondary metabolites in order to protect themselves from microorganism, such as

6flavonoid, alkaloid, triterpenoid, and saponin that are producing a better oppurtunity for testing

7wide range of microorganis (Mansour, 2010; Tiwari, 2014). Alkaloids have been reported as

8powerful poison and many alkaloids derived from medicinal plants showed biological

9activities like antimicrobial (Iqbal et alet al., 2015; Benbott et alet al., 2012), which had

10bioactivity against Gram-positive bacteria (Omar et alet al., 1992; Iqbal et alet al.,

112015). Flavonoids have the ability to complex with extracellular and& soluble proteins

12and as well as with bacterial cells walls. Liphophilic flavonoids may also disrupt

13bacterial membranes (Clements et alet al., 2002; Selvi et alet al., 2011). Triterpenoids

14are known to weaken the membranous tissue, which results in dissolving cell wall

15organism (Rao et alet al., 2003; Okwu, 2004; Verughese & Tripathi, 2013). The mode of

16action os saponin against bacteria is due to its ability to cause leakage of protein &

17certain enzymes from the cell ( Selvi et alet al., 2011). In this study, weak inhibition can

18be related to the low content of polyphenolic compounds. Also the phenolic compounds in

19particular the tannins are suitable for precipitation during the reactions of oxidation and that

1 18
 1could be factor of toxicity with respect to the micro-organism (Bakasso, 2009; Bangou et alet

2al., 2012). The best antibacteria activity of methanol extract was obtained on the Gram-positive

3bacteria (S.treptococcus mutans), which are very rich in peptiodoglycane (50-80% of the

4lining); consist of several layers of peptidoglycan an having reticulation (Corvec, 2009;

5Bangau, 2012). One of the peptiodoglycanes roles is to ensure the regidity and the solidity

6lining of the bacterial as well as the protection of the cytoplasmic membran against osmotic

7lysis. Methanol extract of Anthurium sp. leaves had not ability against bacteria (Bonjar

8et alet al., 2004), which similar with our observation. Acetone, chloroform and ethanol

9extracts from leaves of A.nthurium adreanum showed inhibition activity against S.

10aureus, E. coli and Bacillus careus (Shazni, 2016).

115. Conclusion

12 Bioactive compounds are normally accumulated as secondary metabolites in all

13plant cells but their concentration varies according solvent extractions. The presence

14phytochemical such as alkaloids, tannins, steroids, saponins, flavonoid and triterpenoid

15showed the antioxidant and antibacterial activities. The methanol extract had the highest

16total phenolic content 76.56 (mg GAE/g), and DPPH inhibition activity approximately

1766.11% with , while the best IC50 (232.90 µg/ml). Besides, water extract had

18antimicrobial activity against S.treptococcus mutans, B. subtilis, Klebsiella. sp., and P.

19agnes. This study provided first report on the phytochemical screening, as well as

20antioxidant and antimicrobial activities of A. hookeriii leaves extracts. This plant

21extracts could serve as potential sources of new antioxidant and antibacterial agents.

1 19
 1

2Acknowledgments

3 This work was supported by a grant from the Research Project “ Penelitian

4Dosen Pemula “ from the Directorate of Research and Community Service, Directorate

5General of Strengthening Research and Development, Ministry of Research,

6Technology and Higher Education, Indonesia.

8Reference

9Andayani, R., Lisawati, Y., Maimunah., 2008. Penentuan aktivitas antioxidant , kadar

10 fenolat total dan likopen pada tomat (Solanumlycopersium L.) . Jurnal Ssains dan

11 Tteknologi Farmasi, 3(1):, pp. 1-9

12

13Alabri, T.H., Al Musalami, A.H., & Hossain, M.A., 2014. Comparative study of

14 phytochemical screening, antioxidant and antimicrobial capacities of fresh and

15 dry leaves crude plant extracts of Daturametel L., Journal of King Saud

16 University-science, 26:, pp. 237-243.

17Alothman, M., Rajeev, A., Karim, A., 2009. Antioxidant capacity and phenolic content

18 of selected tropical fruits from malaysia extracted with different solvents. Food.

19 Chem. 115:. 785-788.

1 20
 1Aquino ,R., Morelli, S., Lauro, M.R., Abdo, S., Saija, A., & Tomaino, A. 2001.,

2 Phenolic Constituents and antioxidant activity of an extract of Anthurium

3 versicolor leaves., J. Nat Prod , 64: , pp. 1019-1023., 2001.

4Ayoola, G.A., Coker, Y.A.B., Adesegu, S.A., Adepoju-Bello, A.AA., Obaweya, K.,

5 Ezennia, E.C., & Atangbayila, T.O., 2008. Phytochemical screening and

6 antioxidant activities of some selected medicinal plant used for malaria therapy in

7 Southwestern Nigeria. , Trop J Pharm Res , 7(3):, pp. 1019-1024.

8Barile, E., Bananomi, G., Antignani, V., Zolfaghari, B., Sajjadi, S., Porto L.M., Scala,

9 F., & Lanzotti, V., 2007. Phytochemical screening and antimicrobial assessment

10 of Abutilon mauritianum, bacopamonifera and daturastramonium.,

11 Phytochemistry, 68:, pp. 596-603.

12Bakasso , S., 2009. Phytochemical studies and biological potentialities of five

13 Indigofera species used in tarditional medicine in Burkina Faso. PhD. Thesis

14 University of Ougadougou:, 135

15Bangou, M.J., Meda, N.T.R, Thiombiano, A.M.E., Kiendrebeogo, M., Zeba, B.,

16 Millogo-Rasolodimby, J., Nacoulma, O.G., Coulidiaty, Y.D., Yougbar-Ziebrou,

17 M., 2012. Antioxidant and Antibacterial activities of five Verbaneceae species

18 from Burkina Faso. Current Research Journal of Biologica Sciences . 4(6):. 665-

19 672

1 21
 1Benbott, A., Yahyia, A., & Belaidi, A. 2012., Assesment of the antibacterial activity of

2 crude alkaloids extracted from seed and roots and plant Peganumharmala L., J

3 Nat Prod Plant Resour, 2:, pp. 568-573., 2012.

4Bonjar, G.H.S., Aghigi , S., & Nik, K., 2004. Antibacterial and antifungal survey in

5 plants used in indigenous herbal-medicine of South east Regions of Iran., Journal

6 of Biological Sciences, 4(3):, pp. 405-412.

7Clements, J.M., Coignard, F., Johnson, I., Chandler, S., Palan, S., Waller, A., Wijkmans,

8 J., and Hunter, M.G., 2002. Antibacterial activities and characterization of novel

9 inhibitors of LpxC. J. Antimicrbial Agents and Chemotherapy. 46: . 1793-1799.

10Di Carlo, F.J., Haynes, L.J., Sliver, N.J., & Phillip, G.E. 1964., Rheticulo endothelial

11 system stimulants of botanical origin., Journal of the Reticulo endothelial

12 Society, 1: , pp. 224,. 1964.

13Djeridane, A., Yousfi, M., Nadjemi, B., Boutassouna, D., Stocker, P., Vidal, N., 2006.

14 Antioxidant activity of some Algerian medicinal plants extracts containing

15 phenolcompounds. Food Chem, 97:, 654-660.

16

17Duke , J.A. 1986., bIsthmian ethnobotanical dictionary,3rd ed, Scientific Publications,.

18 Jodphar:, 1986, 12.

1 22
 1Do , Q.D., Angkawijaya, AEa.e., Tran-Nguyen, P.L., Huynh, L.H., Soetaredjo, F.E.,

2 Ismadji, S., 2014., Effect of extraction solvent on total phenol content, total

3 flavonoid content, and antioxidant activity of Limnophila aromatica., Journal of

4 Food and drug Analysis , 22:,pp. 296-302.

5Donberger , K., & Lich, H. 1982. ,Screening nach antimicrobiell sowie potentiell

6 cancerostatisch wirksamen pflanzeninhaltsstoffen., Pharmazie, 37:, pp. 215-221,

7 1982.

8Fitriana, W.D., Ersam, T., Shimizu, K., and Fatmawati , S., 2016. Antioxidant activity

9 of Moringa oleifera extracta. Indonesia. J. Chem, 16 (3):, pp. 297-301.

10Florence, O., Adeolu, A., Anthony, J., 2011. Comparison of nutritive value, antioxidant

11 and antibacterial activities of Sonchus asper and Sonchus oleraus. Rec Nat prod ,

12 5(11) : ,pp. 1142-1145.

13Geyid, A., Abebe, D., Debella , A., Makonnen, Z., Aberra, F., Teka, F., Kebede, T.,

14 Urga, K., Yersaw, K., Biza, T., Mariam, B.H., Guta, M., 2004. Screening of some

15 medicinal plants of ethiopia for their anti-microbial properties and chemical

16 profiles. Journal of Ethnopharmacology. 97:. 421-427.

17Graham , J.G., Quinn, M.L., Fabricant, D.S, Farnsworth, N.R., 2000. Plant used against

18 Cancer –an extention of the work of Jonathan Hartwell. J. Ethnopharmacol. 7: 3,

19 347-377.

1 23
 1Hardiana, R., Rudiyansyah, Zaharah, T.A., 2012. Antioksidan senyawa golongan fenol

2 dari beberapa jenis tumbuhan familimal vaceae. JKK , 1(1):, pp. 8-13.

3Hossain, M.A., Al-Kalbani, M.S.A., Al-Farisi, S.A.J., Weli, A.M., Al-Riyami, Q., 2014.

4 Comparative study of total phenolics, flavonoids contents and evaluation of

5 antioxidant and antimicrobial activities of different polarities fruits crude extracts

6 of Datura metel L. Asian Pasific Journal of Tropical Disease . 4(5):, 378-383.

7Iqbal, E., Salim, K.A., Linda, B.L., & Lim. 2015., Phytochemical screening, total

8 phenolics and antioxidant activities of bark and leaf extracts of Goniothalamus

9 velutimus (Airy Shaw) from Brunei Darussalam., Journal of King Saud University

10 – Science, 27:, pp. 224-232, 2015.

11Iloki-Assangaa , S. B., Lidianys, M., Lewis-Lujan., Claudia, L., Lara-Espinoza, Armida

12 , A., Gil-Salido, Fernandez-Angulo, D., Jose , L., Rubio-Pino, Haines , D.D.,

13 2015. Solvent effect on phytochemical constituent profiles and antioxidant

14 activities, using four different extraction formulation for analysis of Bucida

15 buceras L. and Phoradendron californicum. BMC Res Notes , 8:, pp. 396.

16Janet, J.R., Puzon ,M., & Rivera, W.L., 2015. Free radical scavenging activity and

17 bioactive secondary metabolites from various extracts of Glinusoppositifolius (L)

18 Aug. DC. (Molluginaceae) roots, stem, and leaves., Asia Pac J Trop Dis, 5(9): ,

19 pp. 711-715.

1 24
 1Jebakumar, A.Z., Nondo, H.S., George, S.K., Manoj, G., 2012. Natural antioxidant and

2 in vitro metods for antioxidant activity. International Journal of Pharmacoloy

3 research , 2(1):. 46-55

4Joly, L.G, Guerra , S., Septimo, R., 1987. Ethnobotanical inventory of medicinal plants

5 used by the Guaymiindians in Western Panama. Part I., J Ethnopharmacol 20:,

6 pp. 145-171.

7Kumar, S., Mishra, A., Pandey, A.K., 2013. Antioxidant mediated protective effect of

8 Parthenium hysterophorush against oxidative damage using in vitro. BMB

9 Complement Altern Med. 13:; 120.

10
11Maji , S., Dandapat, P., Ojha, D., Maity, C., Halder, S.K., Das Mohapatra, P.K., Pathak,

12 T.K., Ppati , B.R., Samanta, A., and Mondal , K.C., 2010. In vitro antimicrobial

13 potentialities of different solvent extracts of Ethnomedicinal plants against

14 clinically isolated human pathogens. J Phytol 2 (4):, 57-64

15Mandal P, Misra TK, and Singh ID. 2010. Antioxidant Activity in the Extract of two

16 edible Aroids. Indian Journal of Pharmaceutical Sciences 72(1): 105-108

17Mansour, S., Seyydnejad, N., Masumeh, D.I., Hossein, M., 2010. Antibacterial activity

18 of hydroalchlolohic extract Callistemon citrinus and Albiza lebbeck. American

19 Journal of Apllied Science, 7(1): .13-16

20Medini, F., Fellah, H., Ksouri, R., Abdelly, C.., 2014. Total phenolic, flavonoid and

1 25
 1 tannin ntents and antioxidant and antimicrobial activitie of organic extracts of

2 shoots of the plant Limonium delicatulum. Journal of Taibah University for

3 Science . 8:. 216-224.

4Maizura , M., Aminah , A., Wan aida, W.M., 2011. Total phenolic content and

5 antioxidant activity of kesum (Polygonum minus), ginger (Zingiber officinale) and

6 tumeric (Curcuma longa) extract. International Food Research Journal. 18:, 526-

7 531

8Makari, H.K., Haraprasad, N, Pathil, H.B., Kumar, R., 2008. In vitro antioxidant

9 activity of the hexane and methanolic extracts of Cordial wallichi and Celatrus

10 paniculata. The Internet J. Aesthetic and Antiaging Medicine, 1:, 1-10

11Molyneux, P., 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH)

12 for estimating antioxidant activity. Journal Science of Technology. 26 (2):. 211-

13 219.

14Najafi, S., Sanadgol, N., Nejad , B.S., Beiragi, M.A., & Sanadgo , E. 2010.

15 ,Phytochemical screening and antibacterial activity of Citrulluscolocynthis (Linn)

16 Sahrad against Staphylococcus aureus., J Med Plants Res, (22):, pp. 2321-2325,

17 2010.

18Naczk , M., Shahidi, F., 2006. Phenolic in cereals, fruits and vegetables occurance,

19 extraction and analysis., J. Pharm. Biomed. Anal. 41:. 1523-1542.

1 26
 1Naznin , A., Hasan, N., 2009. In vitro antioxidant activity of methanolic leaves and

2 flowers extract of Lippia alba. Research Journal of Medicine and Medical

3 agencies, 4(1):,107-110

4Nguyen, VV.T., Bowyer, M.C., Vuong, Q.V., Altena, L.A.V., & Scarlet, J.C.H.. 2015.,

5 Phytochemicals and antioxidant capacity of Xao tam phan (Paramignyatrimera)

6 rot as affected by various solvents and extractions methods)., Industrial Crops and

7 Products, 67:, pp. 192-200, 2015.

8Nurjanah., Agoes, M.J., Hidayat, T., Shylina, A.., 2015. Bioactive compounds and

9 antioxidant activity of Lindur stem bark (Bruguiera gymnorrhiza). International

10 Journal of Plant Science and Ecology. 1(5):. 182-189.

11Omar, S., Chee, C.I., Fasihuddin, A., Ni, J.X., Jaber, H., Huang, J., & Nakatsu, T.,

12 1992. Penanthrene lactams from Goniothalamus velutinus. ,Phytochem, 31: , pp.

13 4395-4397.

14Okwu , D.E., Okwu, M.E., 2004. Chemical composition of Spondias mombin linn. Plant

15 part. J Sustain Agric. Environ , 6(2):. 140-147.

16Oluyemi, K.A., Okwuonu, U.C., Baxter, D.Gg, Oyesuda, T.Oo., 2007. Toxic effect of

17 methanolic extract os Aspilia africana leaf on the estrous cycle and uterine tissues

18 of Wistar rats. Int J Morphol, 25:, 609-614

1 27
 1Rao, C. V., Ohja , A.S.K., Mehrotra, S., Puspangadan , P., 2003. Analgesic,

2 antiinflamatory and Antiulcerogenic activity of unripe fruits Aegle marmelos . of .

3 Acta Pharmacetica Turcica. 45:. 85-91

4Redha , A.., 2010. Falvonoid: Struktur, sifat antioksidatif dan peranannya dan perannya

5 dalam systembiologis. Jurnal Belian . 9(2):. 196-200

6Samejo, M.Q., Sumbul, A., Shah, S., Memon, S.B,., & Chundrigar , S. 2013.

7 ,Phytochemical screening of Tamarixdiocia Roxb.ex Roch., J Pharm Res, 7:, pp.

8 181-183, 2013.

9Saeed, N., Khan, M.R., and Shabbir, M., 2012. Antioxidant activity, total phenolic and

10 total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC

11 Complementary & Alternative Medicine. 12:, 221.

12Schulz, B. J., Sucher, H.J., Aust, K., Krohn, K., Ludwig., 1995. Biological active

13 secondary metabolites of endophetic Pezcula species. Mucol. Res. 99: , 1007-

14 10015.

15Sekar , D., Kolajinathan, K., Saranraj, P., & Gajendiran, K., 2012. Screening of

16 Phyllanthusamarus, Acalyphaindica and Daturametel for it s antimicrobial

17 activity against selected pathogen. , International Journal of Phermaceutical &

18 Biological Archives, 3(5):, pp. 1231-1235.

1 28
 1Surveswaran, S., Cai Y.Z. Cai., H.Corke H., M.Sum M., 2007. Systematic evluation of

2 natural phenolic antioxidant from 133 Indian medicinal plants. Food Chem, 102:,

3 938-953.

4Tamli Selvi, A., Dinesh, M.G., Satyan, R.S., Chandrasekaran, B., Rse, C., 2011. Leaf

5 and Seed of Bixa orellana L. exert anti-microbial activity against bacterial

6 pathogen. Journal of Applied Pharmaceutical Science. 01(09): ,116-120

7Tiwari, U., Jadon, M., Nigam, D., 2014. Evaluation of antioxidant and antibacterial

8 activities of methanolic leaf extract of Calistemon viminalis. International Journal

9 of Pharmaceutical Sciences and Business Management. 2(12):, 01-12

10Salamah. E., Ayuningrat, E., Purwaningsih., 2008. Penapisan awal komponen bioaktif

11 dari Kijing Taiwan (Anadonta woodiana lea) sebagai senyawa antioksidan.

12 Buletin Teknologi Hasil Perikanan. 11(20):, 119-132

13Siddhuraju, PP., Becker, K.., 2003.., Antioxidant properties of various extracts of total

14 phenolic constituent from three different agroclimatic origins of drumstik tree

15 (Moringa oleifera lam) leaves. J .Agric Food Chem; 51:, 2144-2155.

16Sulaiman, S., Sajak, A., Supriatno, K., Seow, E., 2011. Effect of solvent in extracting

17 polyphenols and antioxidant of selected raw vegetables. Food comp anal ;24: ;

18 506-515.

19Suratmo., 2009., Potensi ekstrak daun sirih merah (Piper crocatum) sebagai antioksidan.

1 29
 1 Journal Penelitian. 205(1):. 1-5

2Shazhni, J.R.A., Renu, A., Murugan, M., 2016. Phytochemical screening and Invitro

3 Antimicrobial activity of Ornamental Plant Anthurium andraeanum. Journal of

4 pharmaceutical. Sciences and Rsesearch, Vol 8:, pp. 668-670.

5Shui, G.H., Leong, L.Pp., 2004. Analysis of polyphenolic antioxidant in star fruit using

6 liquid chromatography and mass spectrometry. J Chromatogr , 1022:, 67-75

7Segura, L,., Vila, S., Gupta, M.P., Avella, M.E., Adzet, T., & Canigueral, S., 1998.

8 Anti-inflammatory activity of Anthurium cerrocampanense croat in rats and mice.,

9 J Enthopharmacol, 61:, pp. 243-248

10Tsai, S.Y., Huang, S.JJ., Lo, S.H., Wu , T.P., Lian, P.Y., Mau, J.L. 2009. Flavour

11 components and antioxidant properties of several cultivated mushrooms. Food

12 Chemistry . 113: , 578-584.

13Unal, K., Susanti, D., Taher, M., 2014. Polyphenol content and antioxidant capacity in

14 organically and conventionally grown vegetables. Journal of Coastal Life

15 Medicine. 2(11): . 864-871.

16Varahalarao, V., & Kaladhar, D.S.V.G.K.., 2012. Antimicrobial study of plant extracts

17 of Daturamatel L against some important disease causing pathogen. ,Asia Pac J

18 Trop Dis :, pp. S594-S597.

1 30
 1Vungtou, H.Oo., Abbah, J, Chindo, B.A., Mosugu, O., Salawu, A.O., Kwanashie, H.O.,

2 Gamaniel, K.S.. , 2005. Central inhibitory effects of the methanol extract of

3 Neoroutanenia mitis root in rats and mice. J Pharm Biol , 43:, 113-120

4Wahyuni, S., Suhartono, M.T., Khaeruni, A., Purnomo, A.S., Asranudin, Holilah,

5 Riupassa, P.A., 2016. Purification and Characterization of Thermostable Chitinase

6 from Bacillus SW41 for Chitin Oligomer Production. Asian Journal of Chemistry ,

7 28(12):, pp. 2731-2736.

8Xiong, Y., Yuan, C., Chen, R, Dawson, T.M., Dawson, V.L., 2010. Preparation and

9 biological activity saponin Ophiogon japonicas. African Jornal of Farmacy and

10 Pharmacology. 6(26):. 1964-1970.

11Yadav, R.N.S., & Agarwala, M. 2011. ,Phytochemical analysis of some medicinal

12 plants., J Phyto L, 3(12):, pp.10-14., 2011

13Zamora-Martinez, M.C, & Pola, C.N.P., 1992. Medicinal plants used in some rural

14 populations of Oaxaca, Puebla and Veracruz, Mexico. , J Eethnopharmacol, 35:,

15 pp. 229-257.

16Zetola, M., Lima, T.C.Mm., Sonoglio, D., 2002. CNs activities of liquid and spray-dried

17 extracts from Lippia alba verbenaceae (Brazihian false Melisa). J

18 ethnopharmacology, 82: ,207-215.

1 31
 1Zielinski, H., Kozlowska, H., 2000. Antioxidant activity and total phenolic in selected

2 cereal grains and their different morphological fractions. J Agric Food Chem , 48:

3 , pp. 2008-2016.

5Table 1. Phytochemical constituents of different extract of A. hookeri leaves.

6
Phytochemical Extracts solvent
Ethyl acetate Methanol Dichloromethane n-Hexane Water
Alkaloids + + + - +
Tanins + + + + +
Steroid - - + - -
Saponin + + + + +
Flavonoids - + - - +
Triterpenoids + + - + +
7
8
9
10Table 2. Extractions yield, DPPH radical scavenging inhibitions and IC50 of A. Hookeri
11 leaves extracts. Values are means with standard deviations (n = 3).
12
Solvents Extraction Total phenol (mg DPPH IC50 (µg/mL)
yield (g) GAE/g) Inhibition (%)
Water 1.40 62.66 ± 0.51a 15.22 ± 0.03a -
Methanol 1.64 76.56 ± 0.45 b
66.11 ± 0.02b 232.90
Ethyl acetate 1.02 26.85 ± 0.12c 26.77 ± 0.01c -
Dicloromethane 1.12 26.08 ± 0.14 d
18.18 ± 0.03d -
n-Hexane 0.66 9.52 ± 0.19e 7.24 ± 0.01e -
13(-) Not measured, DPPH Inhibition (%) < 50% . Data are mean ± standard deviation (n = 3). Data
14followed by the same minor letter on each row are significantly different (P < 0.05).
15
16
17Table 3. Antibacterial activity of A. hookeri leaves extracts.
18
Solvents Diameter of inhibition zone (mm) bacteria
Klebsiella. sp B. subtilis Propioni Strepticoccus mutans
agnes
Water 10.30 ± 1.21aA 14.20 ± 0.30aB 9.60 ± 0.50aA 15.10 ± 0.70aB
Methanol 9.10 ± 0.61aA 6.00 ± 0.10bB 6.60 ± 0.60bB 7.50 ± 0.50bC
Ethyl acetate 7.40 ± 0.10bA 6.20 ± 1.61 bA
6.80 ± 0.20 bA
7.10 ± 0.80bA
Dicloromethane 6.40 ± 1.31bA 6.50 ± 0.61 bA
6.70 ± 0.10 bA
6.60 ± 0.20cA
n-Hexane 6.00 ± 0.20bA 6.00 ± 0.40 bB
6.00 ± 0.80 bB
6.00 ± 0.61cB
Chloramfenicol 6.00 ± 0.41bA 9.40 ± 0.30 cB
22.10 ± 0.51 cC
42.00 ± 1.51dD
(positive control)
19Data are mean ± standard deviation (n = 3). Data followed by the same minor letter on each row or by the
20same capital letter on each coloumn are significantly different (P < 0.05).
21

1 32
1
2

3
4
5
6
7
8

1 33