New insights into the chemical profiling, cytotoxicity and bioactivity of four Bunion species

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ScienceDirect

Food Research International

journal homepage:

www.elsevier.com/locate/foodres

New insights into the chemical profiling, cytotoxicity and bioactivity of four

Bunium species

Gokhan Zengin

a,⁎

_{, Mehmet Yavuz Paksoy}

b

_{, Muhammad Zakariyyah Aumeeruddy}

c

_,

Jasmina Glamocilja

d

_{, Marina Sokovic}

d

_{, Alina Diuzheva}

e

_{, József Jekő}

f

_{, Zoltán Cziáky}

f

_,

Maria João Rodrigues

g

_{, Luisa Custodio}

g

_{, Mohamad Fawzi Mahomoodally}

c

a_{Department of Biology, Science Faculty, Selcuk University Campus, Konya, Turkey}

b_{Department of Environmental Engineering, Faculty of Engineering, University of Munzur, Tunceli, Turkey} c_{Department of Health Sciences, Faculty of Science, University of Mauritius, 230 Réduit, Mauritius} d_{Institute for Biological Research “Siniša Stanković” University of Belgrade, Belgrade, Serbia} e_{Department of Analytical Chemistry, Pavol Jozef Šafárik University in Košice, Košice, Slovakia}

f_{Agricultural and Molecular Research and Service Institute, University of Nyíregyháza, Nyíregyháza, Hungary}

g_{Faculty of Sciences and Technology, Centre of Marine Sciences, University of Algarve, Ed. 7, Campus of Gambelas, 8005-139 Faro, Portugal}

A R T I C L E I N F O Keywords: Bunium phytoconstituents cytotoxicity antioxidant enzyme inhibitors A B S T R A C T

Bunium species have been reported to be used both as food and in traditional medicines. The scientific com-munity has attempted to probe into the pharmacological and chemical profiles of this genus. Nonetheless, many species have not been investigated fully to date. In this study, we determined the phenolic components, anti-microbial, antioxidant, and enzyme inhibitory activities of aerial parts of four Bunium species (B. sayai, B. pin-natifolium, B. brachyactis and B. macrocarpum). Results showed that B. microcarpum and B. pinnatifolium were strong antioxidants as evidenced in the DPPH, ABTS, CUPRAC, and FRAP assays. B. brachyactis was the most effective metal chelator, and displayed high enzyme inhibition against cholinesterase, tyrosinase, amylase, glucosidase, and lipase. The four species showed varied antimicrobial activity against each microorganism. Overall, they showed high activity against P. mirabilis and E. coli (MIC and MBC < 1 mg mL−1_{). B. brachyactis}

was more effective against Aspergillus versicolor compared to the standard drug ketoconazole. B. brachyactis was also more effective than both ketoconazole and bifonazole against Trichoderma viride. B. sayai was more effective than ketoconazole in inhibiting A. fumigatus. B. sayai was most non-toxic to HEK 293 (cellular viability = 117%) and HepG2 (cellular viability = 104%). The highest level of TPC was observed in B. pinnatifolium (35.94 mg GAE g−1_{) while B. microcarpum possessed the highest TFC (39.21 mg RE g}−1_{). Seventy four compounds were detected}

in B. microcarpum, 70 in B. brachyactis, 66 in B. sayai, and 51 in B. pinnatifolium. Quinic acid, chlorogenic acid, pantothenic acid, esculin, isoquercitrin, rutin, apigenin, and scopoletin were present in all the four species. This study showed that the four Bunium species are good sources of biologically active compounds with pharma-ceutical and nutrapharma-ceutical potential.

1. Introduction

Since ancient times, nature has been the prime rescue for diseases.

Although there was not enough information on the etiology of

life-threatening diseases, treatments were based on trials and experience

using natural resources such as herbs and animal products (

Awortwe,

Bruckmueller, & Cascorbi, 2019

). The healing properties of medicinal

plants were identified, noted, and passed on to the successive

genera-tions via oral communication and also written documents and preserved

monuments. This interest in medicinal plants has brought about today's

modern fashion of their processing and usage (

Mollica et al., 2017

;

Petrovska, 2012

). In fact, there has been a tremendous surge in the

https://doi.org/10.1016/j.foodres.2019.05.013

Received 1 January 2019; Received in revised form 5 May 2019; Accepted 8 May 2019

Abbreviations: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid; ACAE, acarbose equivalent; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; CUPRAC, cupric reducing antioxidant capacity; DPPH, 1,1-diphenyl-2-picrylhydrazyl; EDTAE, EDTA equivalent; FRAP, ferric reducing antioxidant power; GAE, gallic acid equivalent; GALAE, galatamine equivalent; HEK, human embryonic kidney; KAE, kojic acid equivalent; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; RE, rutin equivalent; TE, trolox equivalent; TPC, Total phenolic content; TFC, Total flavonoid content

⁎_{Corresponding author.}

E-mail address:gokhanzengin@selcuk.edu.tr(G. Zengin).

Available online 10 May 2019

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public’s acceptance and interest in natural therapies in recent years,

especially herbal remedies, which are available not only in drug stores,

but also in food stores and supermarkets (

Ekor, 2014

).

The genus Bunium is close to Carum. The seeds and essential oils of

members of these two genera have been used as food and for

ther-apeutic purposes all over the world since ancient times (

Bousetla,

Zellagui, Derouiche, & Rhouati, 2015

). The genus Bunium (Family:

Apiaceae), consists of 50 accepted species (

http://www.theplantlist.

org

). Plants from this genus are distributed in Asia, Europe, and North

Africa (

Çelik & Bağci, 2017

). Interestingly, Turkey has the highest

number of species density of Apiaceae family (about 486 species), out

of which 8 species from the genus Bunium are endemic to Turkey, with

an endemism ratio of 40% (

Çelik & Bağci, 2017

).

Several Bunium species are consumed as traditional food in

Mediterranean regions including Turkey. For instance, B. persicum

(Boiss.) B. Fedtsch. also known as black cumin, is an important

aro-matic food plant whose seeds are consumed widely as a condiment and

has economic value (

Demirci & Ozkan, 2014

). The seeds are regarded as

stimulants, carminatives and also useful for the treatment of diarrhea

and dyspepsia (

Azizi, Davareenejad, Bos, Woerdenbag, & Kayser,

2009

). Another species, B. paucifolium var. brevipes, is used for urinary

inflammations (

Khatun, Parlak, Polat, & Cakilcioglu, 2012

) and the

tuber and whole plant are eaten after the bark is peeled (

Çakır, 2017

).

Moreover, several parts of Bunium macrocarpum (Boiss.) Freyn Bunium

paucifolium DC. and B. mauritanicum L. are taken raw orally as food and

used to make food products such as bread (

Doğan, Bulut, Tuzlacı, &

Şenkardeş, 2014

). In addition, they also claimed to manage allergy,

bronchitis, and cough (

Benarba et al., 2015

). In Morocco, a herbal

mixture known as Msahan contains 13 medicinal plants including B.

bulbocastanum, and is used for general health improvement, and for

gynecological and musculoskeletal problems (

Teixidor-Toneu, Martin,

Ouhammou, Puri, & Hawkins, 2016

).

Among the Bunium species, most scientific studies have probed into

the biological potential of B. persicum. Pharmacological studies have

proved its antibacterial, antifungal (

Bhat, Gani, & Zargar, 2017

),

anti-oxidant (

Sharafati Chaleshtori, Saholi, & Sharafati Chaleshtori, 2018

),

antinociceptive and anti-inflammatory activities (

Hajhashemi, Sajjadi,

& Zomorodkia, 2011

). As for other species, two coumarins, scopoletin

and scoparone, were identified in the roots of Algerian B. incrassatum

(

Bousetla et al., 2015

). Two prenylated isocoumarins were detected in

the roots and fruits of B. paucifolium var. paucifolium (

Appendino, Ozen,

& Jakupovic, 1994

). Ten main components isolated and identified in

the seeds of B. cylindricurn were dillapiole, myristicin, 1(-)-bornyl

acetate, α-, β-, γ-elemene, β-selinene, 7(11)-selinen-4-ol (juniper

cam-phor), elemol, and 4-methyl-4-hydroxy-penten-2-oic acid (

Agarwal,

Vashist, & Atal, 1974

).

Nonetheless, many species of this genus have not been investigated

to date. Given the pharmacological potential of Bunium species, the

present study aimed to shed light on the biological and chemical profile

of four commonly consumed species in Turkey. To determine the

bio-logical properties, namely antimicrobial, antioxidant, and enzyme

in-hibitory activities of four Bunium species (B. sayai, B. pinnatifolium, B.

brachyactis, and B. microcarpum). The in vitro cytotoxicity of the extracts

were also evaluated against three mammalian cell lines. The

char-acterization of the phenolic composition of the extracts, which are

known bioactive compounds, was undertaken using HPLC-MS/MS, in

an endeavour to support any biological activities observed.

2. Materials and Methods

2.1. Plant material and preparation of extracts

Bunium species (Bunium brachyactis (Post) Wolff; Bunium

pinnatifo-lium Kljuykov; Bunium sayai Yıld and Bunium microcarpum subsp.

mi-crocarpum (Boiss.) Freyn) were collected in different regions of Turkey

in season 2018 (June). The taxonomical classification was performed by

the botanist Dr. Mehmet Yavuz Paksoy (Munzur University, Tunceli).

The location details are given in

Table 1

. The aerial parts (including

flowers) were divided and dried for 10 days at room temperature (RT)

(25 °C, in shade). Then, these samples were powdered with a laboratory

mill.

To prepare methanol extracts, dried samples were stirred overnight

(24 h, 250 rpm in a shaker (Lab Companion SI-300 Benchtop Shaker) at

RT (5 g in 100 mL solvent). After filtration, the extracts were

con-centrated using a rotary evaporator under vacuum at 40 °C. The extracts

were completely dried at 40 °C in the oven (Memmert- UNB 400) and

they were stored at 4 °C until further analysis.

2.2. Chemicals and reagents

All reagents and standards were of analytical grade. Most of

che-micals were supplied by Sigma-Aldrich (Germany), including DPPH,

ABTS, cupric chloride, ferric chloride, ferrous sulphate, trolox, EDTA,

neucuproine, 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), ammonium

acetate, sulphuric acid (96%), gallic acid, rutin, aluminum chloride,

Folin-Ciocalteau reagent, DTNB (5,5-dithiobis (2-nitrobenzoic) acid),

and AChE (acetylcholinesterase (Electric ell

acetylcholinesterase,Type-VI-S, EC 3.1.1.7)), BChE (butyrylcholinesterase (horse serum

ylcholinesterase, EC 3.1.1.8)), acetylthiocholine iodide (ATCI),

butyr-ylthiocholine chloride (BTCl), α-amylase (ex-porcine pancreas, EC

3.2.1.1), acarbose, α-glucosidase solution (from Saccharomyces

cerevi-siae, EC 3.2.1.20), tyrosinase (from mushroom, EC1.14.18.1),

l-2,3-di-hydroxyphenylalanine (L-DOPA), kojic acid, orlistat, pancreatic lipase

(type I, from porcine pancreas, EC. 3.1.1.3), streptomycin, ampicillin,

ketoconazole and bifonazole, MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium). Methanol (HPLC grade) was purchased from

Merck (Germany).

2.3. Profile of bioactive compounds and HPLC-MS/MS analysis

With reference to our previous studies (

Uysal et al., 2017

), the total

amount of phenolics (TPC) (by standard Folin-Ciocalteu method) and

flavonoids (TFC) (by AlCl

3

method) were determined. Standard

com-pounds (gallic acid (mg GAE g

−1

_{) for TPC and rutin (mg RE g}

−1

_{) for}

TFC, respectively) were used to express the obtained results.

All HPLC-MS/MS experiments were carried out on a Dionex HPLC

Table 1

Locations, extraction yields, total phenolic and flavonoids contents of the Bunium species.

Samples Locations Yields (%) Total phenolic content

(mg GAE g−1_extract) Total flavonoid content_{(mg RE g}−1_extract)

B. sayai Hakkari; Depin, Sümbül River around, 1300 m. 5.79 27.52 ± 0.36c⁎ _{13.95 ± 0.15}d

B. pinnatifolium İzmir; Kemalpaşa, Nif Mounth, 650 m 6.21 35.94 ± 1.89a _{38.33 ± 0.22}b

B. brachyactis Kahramanmaraş; Ahir Mounth, Çallıbalma aound, 1800 m 2.31 28.18 ± 0.37c _{23.60 ± 0.16}c

B. microcarpum Antalya, Elmalı-Avlan, 1000 m 8.62 32.87 ± 0.18b _{39.21 ± 0.06}a

⁎ _{Values expressed are means ± S.D. of three parallel measurements. GAE: Gallic acid equivalent; RE: Rutin equivalent. Different letters indicate the differences in}

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Ultimate 3000RS UHPLC instrument. A chromatographic method was

developed using a Thermo Accucore C18 (100 mm × 2.1, mm i. d., 2.6

μm) column (

Zengin et al., 2018

). All analytical details were given in

supplementary material.

2.4. Assays for enzyme inhibition and antioxidant capacity

Lipase, tyrosinase, α-amylase, α-glucosidase and cholinesterases

were selected as target enzymes and the procedures of these assays are

described in our earlier work (

Grochowski et al., 2017

;

Uysal et al.,

2017

). Standard inhibitors (acarbose (for α-amylase and

α-glucosi-dase), galantamine (for AChE and BChE), kojic acid (for tyrosinase) and

orlistat (for lipase)) were used to express the enzyme inhibitor

prop-erties.

The antioxidant capacity of the extracts was spectrophotometrically

screened by different experiments, namely the ferrozine assay (for

chelating abilities), phosphomolybdenum, reduction potentials (by

FRAP and CUPRAC assays) and radical scavenging (using DPPH and

ABTS radicals). Standard compounds (Trolox equivalent –TE g

−

_and

ethylenediaminetetraacetic acid equivalent – EDTAE g

−1

_{) were used to}

express the antioxidant properties. Trolox was used as a standard in

phosphomolybdenum, radical scavenging and reduction abilities, while

EDTA was a reference in metal chelating activity. The procedures of

assays are reported in our earlier work (

Uysal et al., 2017

).

2.5. Antibacterial and antifungal activities

Antimicrobial and antifungal activities were determined using the

microdilution method as described previously (

Zengin et al., 2017

). The

antibacterial activity of the studied extracts was evaluated using several

bacterial strains. Escherichia coli (ATCC 35210), Pseudomonas aeruginosa

(ATCC 27853) and Salmonella Typhimurium (ATCC 13311), were used

as Gram-negative bacteria. For Gram-positive bacteria, Proteous

mir-abilis (human isolate), Enterobacter cloacae (ATCC 35030), Bacillus

cereus (clinical isolate), Micrococcus flavus (ATCC 10240), and

Staphy-lococcus aureus (ATCC 6538) were used. The results were expressed as

minimum inhibitory (MICs) and minimum bactericidal concentrations

(MBCs). Streptomycin and ampicillin were used as standards in the

antibacterial assay.

The antifungal activity of the extracts was evaluated using different

fungal species, including Aspergillus versicolor (ATCC 11730), A.

fumi-gatus (plant isolate), A. terreus (soil isolate), A. niger (ATCC 6275),

Penicillium ochrochloron (ATCC 9112), P. funiculosum (ATCC 36839), P.

verrucosum (food isolate) and Trichoderma viride (IAM 5061). Antifungal

results were expressed by MICs and minimum fungicidal concentrations

(MFCs). Ketoconazole and bifonazole were used as standards in the

antifungal assay.

2.6. Cell culture

The murine RAW 264.7 macrophages, the human embryonic kidney

(HEK) 293, and human hepatocellular carcinoma (HepG2) cell lines

were respectively provided by the Faculty of Pharmacy and Centre for

Neurosciences and Cell Biology (University of Coimbra, Portugal), the

Functional Biochemistry and Proteomics, and the Marine Molecular

Bioengineering groups (Centre of Marine Sciences, Portugal). RAW cells

were maintained in RPMI 1640 culture media, while HEK and HepG2

cells were cultured in DMEM culture media, both supplemented with

10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine (2

mM), and 1% penicillin (50 U mL

−1

_{)/streptomycin (50 μg mL}

−1

_{), and}

were kept at 37°C in moistened atmosphere with 5% CO

2

.

2.7. Determination of the cytotoxicity of the extracts

Cells at 80% confluency were seeded in 96-well microplates at a

density of 1 × 10

4

_{cells well}

−1

_{(RAW 264.7) and 5 × 10}

3

_{cells well}

−1

(HEK 293 and HepG2) and left to adhere for 24h. Then, the extracts

were applied at the concentration of 100 μg mL

−1

_{for 72h, and control}

cells were treated with DMSO at maximum concentration used in the

extracts (0.2%). Cellular viability was determined by the MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric

assay, using a microplate reader (Biotek Synergy 4), as described

pre-viously (

Rodrigues et al., 2014

). Results were expressed as cellular

viability (%).

2.8. Data evaluation

The obtained results were expressed as mean ± standard deviation

(SD) and statistically evaluated by one-way ANOVA (by Tukey test,

p < 0.05), to indicate differences among the tested extracts. To further

statistical evaluation, Pearson correlation, heat map and Principal

component (PCA) analysis were carried out to observe variabilities of

the tested extracts. The statistical procedures were performed by R

software v. 3.5.1.

3. Results and discussion

3.1. Chemical composition

The quest for phytochemical plays a major role in the search for

novel chemical entities with potential as leads for drug discovery (

Gu

et al., 2014

). Plants contain several constituents which are generally

believed to act together synergistically (

Ekor, 2014

).

In order to evaluate the total bioactive compounds of the four

Bunium species, TPC and TFC were determined (Table 1

). TPC ranged

between 27.52 and 35.94 mg GAE g

−1

_{. The highest level was observed}

in B. pinnatifolium (35.94 mg GAE g

−1

_{) followed by B. microcarpum}

(32.87 mg GAE g

−1

_{), while the lowest amount was displayed by B.}

sayai (27.52 mg GAE g

−1

_{). The level of TFC was also quite similar to}

TPC, ranging from 13.95 to 39.21 mg RE g

−1

_{. The species B.}

micro-carpum exhibited the highest TFC (39.21 mg RE g

−1

_{). B. pinnatifolium}

also had a quite similar amount (38.33 mg RE g

−1

_{) while the two other}

plants revealed lower levels, with the least amount in B. sayai (13.95

mg RE g

−1

_).

With regards to the individual phytocompounds, 74 compounds

were detected in B. microcarpum, 70 in B. brachyactis, 66 in B. sayai, and

51 in B. pinnatifolium (

Tables 2–5

). The chromatograms for the extracts

are reported in Supplementary materials (Figs. S1–S4). Quinic acid,

chlorogenic acid, pantothenic acid, esculin, isoquercitrin, rutin,

api-genin, and scopoletin were present in all species. Vitexin, cosmosiin,

diosmin, luteolin, angelicin, salcolin B, and vicenin-2 were present in B.

brachyactis, B. microcarpum, and B. sayai. Naringenin was identified in

B. brachyactis, B. microcarpum, and B. pinnatifolium. In addition,

kaempferol was detected in B. microcarpum, B. pinnatifolium, and B.

sayai. Afzelin was detected in B. brachyactis, B. pinnatifolium, and B.

sayai, while orientin was present only in B. brachyactis and B.

micro-carpum. Several studies have been performed on chemical composition

of essential oils from the members of the Bunium genus (

Sharafati

Chaleshtori et al., 2018

;

Talebi, Moghaddam, & Pirbalouti, 2018

), but

there is still insufficient data for phytochemical characterization of their

extracts. At this point the present work could provide valuable

in-formation for the genus.

3.2. Antimicrobial activity

Due to the emergence of infectious diseases and the rise of microbial

resistance, new strategies are being explored to tackle this issue.

Because of the fact that plants have been used for centuries to treat

infections, they are considered as an important source of new

anti-microbial agents (

Bereksi, Hassaïne, Bekhechi, & Abdelouahid, 2018

).

With regards to the antibacterial activity of the four tested Bunium

species (see

Table 6

), the plants showed varied activity depending on

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Table 2

Chemical composition of B. brachyactis.

No. Compounds Formula Rt, min [M + H]+ _[M–H]− _{Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5}

1 Quinic acid C7H12O6 1.23 191.0556 173.0448 171.0289 127.0389 111.0438 85.0280

2 Pantothenic acid C9H17NO5 5.42 220.1185 202.1079 184.0974 174.1131 116.0347 90.0556

3 Neochlorogenic acid (5-O-Caffeoylquinic acid) C16H18O9 8.95 355.1029 163.0393 145.0289 135.0445 117.0340 89.0391

4 Esculin (Esculetin-6-O-glucoside) C15H16O9 12.28 341.0873 179.0344 151.0393 133.0289 123.0446

5 Coumaroylquinic acid isomer 1 C16H18O8 12.59 337.0924 191.0553 173.0444 163.0391 119.0489 93.0328

6 Esculetin (6.7-Dihydroxycoumarin) C9H6O4 14.10 179.0344 151.0393 133.0288 123.0445 105.0341 89.0391

7a _{Chlorogenic acid (3-O-Caffeoylquinic acid)} _C16H18O9 _14.29 _355.1029 _163.0393 _145.0287 _135.0445 _117.0340 _89.0392

8 Chryptochlorogenic acid (4-O-Caffeoylquinic acid) C16H18O9 15.62 355.1029 163.0393 145.0287 135.0445 117.0340 89.0391

9 Syringic acid C9H10O5 15.93 197.0450 182.0215 166.9976 153.0547 138.0311 123.0074

10 Dihydroxy-methoxycoumarin-O-hexoside C16H18O10 15.99 369.0822 207.0294 206.0216 192.0058 190.9980 163.0025

11 Ethyl syringate C11H14O5 16.82 227.0920 181.0500 155.0706 140.0472 123.0443 95.0496

13 Naringenin-6.8-di-C-glucoside C27H32O15 16.99 595.1663 505.1402 475.1247 415.1037 385.0941 355.0830

14 Feruloylhexose C16H20O9 17.04 355.1029 235.0608 193.0501 175.0392 149.0598

16 Sinapoylhexose C17H22O10 17.95 385.1135 267.0725 249.0612 223.0609 208.0374 164.0469

17 Feruloylquinic acid C17H20O9 18.01 367.1029 193.0500 191.0555 173.0447 134.0362 93.0331

18 Scopoletin (7-Hydroxy-6-methoxycoumarin) C10H8O4 18.61 193.0501 178.0264 165.0549 149.0595 137.0602 133.0288

19 Vicenin-2 (Apigenin-6.8-di-C-glucoside) C27H30O15 19.04 593.1507 575.1415 503.1197 473.1094 383.0780 353.0673

20 Indole-4-carbaldehyde C9H7NO 19.08 146.0606 118.0656 117.0580 91.0549

22 Apigenin-C-hexoside-C-pentoside isomer 1 C26H28O14 19.74 563.1401 473.1088 443.1016 413.0864 383.0781 353.0673

23 N-(2-Phenylethyl)acetamide C10H13NO 20.16 164.1075 122.0969 105.0705 103.0549 90.9484 79.0550

24 Trimethoxyphenylacetyl hexose C17H24O10 20.26 387.1291 225.0766 210.0530 195.0294 71.0124 59.0125

26 Coumaroylshikimate C16H16O7 20.44 319.0818 163.0391 155.0338 119.0489

27 Orientin (Lutexin. Luteolin-8-C-glucoside) C21H20O11 20.45 449.1084 431.0984 413.0879 353.0662 329.0663 299.0556

28 Tetrahydroxyflavanone-O-(deoxyhexosyl)hexoside C27H32O15 20.77 595.1663 287.0567 175.0024 151.0027 135.0442 107.0126

30 Isoorientin (Homoorientin. Luteolin-6-C-glucoside) C21H20O11 20.80 449.1084 431.0983 413.0877 353.0662 329.0662 299.0556

31 Apigenin-O-(acetyl)hexoside C23H22O11 20.81 473.1084 269.0466 268.0380 151.0018

32 4-Acetamidobenzoic acid C9H9NO3 21.05 178.0504 134.0600 92.0491

33a _{Vitexin (Orientoside. Apigenin-8-C-glucoside)} _{C21H20O10 21.44} _433.1135 _415.1032 _397.0928 _337.0710 _313.0712 _283.0606

34 Luteolin-O-glucuronide C21H18O12 22.31 461.0720 285.0410 217.0506 199.0391 151.0026 133.0280

35 Isovitexin (Avroside. Apigenin-6-C-glucoside) C21H20O10 22.34 433.1135 415.1031 397.0926 337.0712 313.0712 283.0606

36 Di-O-caffeoylquinic acid isomer 1 C25H24O12 22.39 515.1190 353.0883 191.0555 179.0341 173.0447 135.0440

37 Luteolin-7-O-glucoside (Cynaroside) C21H20O11 22.43 447.0927 327.0515 285.0411 284.0332 256.0375 151.0027

38 Tetrahydroxyflavone-O-deoxyhexoside C21H20O10 22.46 431.0978 285.0411 284.0332 255.0301 227.0347

39 Methoxy-trihydroxyflavone-C-hexoside C22H22O11 22.71 463.1240 445.1139 427.1034 343.0819 313.0713 287.0557

40a _{Isoquercitrin (Hirsutrin. Quercetin-3-O-glucoside)} _{C21H20O12 22.96} _{463.0877 301.0360} _300.0279 _271.0252 _255.0302 _151.0026

41a _{Rutin (Quercetin-3-O-rutinoside)} _{C27H30O16 23.06} _611.1612 _465.1011 _303.0504 _129.0551 _85.0291 _71.0499

42 Apigenin-O-(deoxyhexosyl)hexoside C27H30O14 23.97 579.1714 433.1138 271.0605

43 Apigenin-O-glucuronide C21H18O11 24.02 445.0771 269.0459 175.0238 113.0231

44a _{Cosmosiin (Apigetrin. Apigenin-7-O-glucoside)} _{C21H20O10 24.04} _433.1135 _271.0605 _153.0184 _145.0280 _119.0495

46 Methoxy-trihydroxyflavone-O-glucuronide C22H20O12 24.29 477.1033 301.0712 286.0479 258.0525

47 Methoxy-trihydroxyflavone-O-hexoside C22H22O11 24.31 461.1084 446.0864 299.0574 298.0487 283.0253 255.0300

48a _{Diosmin (Diosmetin-7-O-rutinoside)} _{C28H32O15 24.59} _609.1820 _463.1247 _301.0712 _286.0477 _129.0552 _85.0291

49 Psoralen C11H6O3 24.79 187.0395 159.0446 143.0495 131.0496 115.0547

50 Tetrahydroxyflavone-O-(deoxyhexosyl)hexoside C27H30O15 24.90 593.1507 285.0410 284.0331 255.0300 227.0347 151.0023

51 Isorhamnetin-O-hexoside C22H22O12 24.98 477.1033 315.0511 314.0441 299.0195 285.0409 271.0258

52 Isorhamnetin-O-(deoxyhexosyl)hexoside C28H32O16 25.25 623.1612 315.0517 314.0439 300.0281 299.0202 271.0253

53 Bergapten (5-Methoxypsoralen) C12H8O4 25.32 217.0501 202.0266 174.0314 146.0362 131.0496 118.0415

54 Psoralen isomer C11H6O3 25.65 187.0395 159.0445 143.0496 131.0497 115.0547

55 Afzelin (Kaempferol-3-O-rhamnoside) C21H20O10 26.46 431.0978 285.0411 284.0332 255.0301 227.0347

56 Dillapiole C12H14O4 27.05 223.0970 208.0734 195.0657 180.0421 165.0913 133.0652

57a _Naringenin _C15H12O5 _27.25 _{271.0607 177.0187} _165.0188 _151.0027 _119.0489 _107.0125

58a _{Luteolin (3'.4'.5.7-Tetrahydroxyflavone)} _C15H10O6 _27.89 _{285.0399 217.0502} _199.0395 _175.0392 _151.0026 _133.0283

59a _{Apigenin (4'.5.7-Trihydroxyflavone)} _C15H10O5 _29.70 _{269.0450 225.0554} _201.0551 _151.0025 _149.0234 _107.0125

60 Dimethoxy-trihydroxy(iso)flavone C17H14O7 29.90 329.0661 314.0438 299.0202 271.0254 61 Salcolin A (Tricin-4'-O-(erythro-β-guaiacylglyceryl) ether) C27H26O11 29.93 525.1397 477.1179 329.0670 314.0442 299.0203 195.0657 62 Methoxy-trihydroxy(iso)flavone C16H12O6 30.01 299.0556 284.0331 256.0379 227.0341 151.0025 107.0124 63 Angelicin C11H6O3 30.51 187.0395 159.0443 143.0496 131.0497 115.0546 64 Salcolin B (Tricin-4'-O-(threo-β-guaiacylglyceryl) ether) C27H26O11 30.73 525.1397 329.0671 314.0439 299.0201 195.0659 165.0547 65 Tetrahydroxyflavone-O-(cinnamoyl)hexoside C30H26O12 32.20 577.1346 285.0410 284.0331 255.0300 227.0346 66 Imperatorin C16H14O4 32.99 271.0970 203.0344 175.0394 147.0444 131.0495 69.0707 67 Dihydroxy-dimethoxy(iso)flavone C17H14O6 34.85 313.0712 298.0488 283.0252 255.0300 68 Dihydroxy-trimethoxy(iso)flavone C18H16O7 34.95 343.0818 328.0592 313.0361 298.0121 69 Isopropyl-methoxy-furocoumarin C15H14O4 34.97 259.0970 244.0736 229.0499 215.0342 185.0613 171.0805 70 Selinidin C19H20O5 35.84 329.1389 229.0864 201.0915 187.0395 175.0394 159.0445 a _{Confirmed by standard.}

(5)

Table 3

Chemical composition of B. microcarpum.

No. Compounds Formula Rt. min [M + H]+ _[M–H]− _{Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5}

1 Quinic acid C7H12O6 1.23 191.0556 173.0447 171.0292 127.0389 111.0437 85.0280

2 Salvianic acid A (Danshensu. 3-(3.4-Dihydroxy)

phenyllactic acid) C9H10O5 4.55 197.0450 179.0343 135.0440 123.0439 72.9916

5 Kynurenic acid C10H7NO3 12.89 190.0504 162.0553 144.0441 116.0498 89.0394

13 4-Hydroxymellein C10H10O4 17.04 195.0657 177.0550 167.0706 149.0600 103.0552

14 Quercetin-di-O-hexoside C27H30O17 17.23 625.1405 463.0894 462.0822 301.0358 299.0203 271.0247

18 Caffeoylshikimic acid C16H16O8 18.04 335.0767 179.0342 161.0234 135.0440

23 Methyl O-caffeoylquinate C17H20O9 19.62 367.1029 191.0557 179.0342 161.0234 135.0440 85.0280

24 Luteolin-C-hexoside-C-pentoside isomer 1 C26H28O15 19.67 579.1350 489.1043 459.0943 429.0846 399.0727 369.0620

25 Trimethoxyphenylacetyl hexose C17H24O10 20.24 387.1291 225.0765 210.0529 195.0293 71.0117 59.0125

28 Orientin (Lutexin. Luteolin-8-C-glucoside) C21H20O11 20.46 449.1084 431.0983 413.0875 353.0661 329.0661 299.0555

29 Tetrahydroxyflavanone-O-(deoxyhexosyl)hexoside C27H32O15 20.75 595.1663 287.0567 175.0025 151.0026 135.0440 107.0126

31 Isoorientin (Homoorientin. Luteolin-6-C-glucoside) C21H20O11 20.80 449.1084 431.0982 413.0873 353.0662 329.0662 299.0555

32 Apigenin-O-(acetyl)hexoside C23H22O11 20.82 473.1084 269.0466 268.0458 151.0026

33 Luteolin-C-hexoside-C-pentoside isomer 2 C26H28O15 20.99 579.1350 489.1072 459.0942 429.0837 399.0739 369.0613

35 Quercetin-O-(pentosyl-deoxyhexosyl)hexoside C32H38O20 21.63 741.1878 301.0359 300.0281 271.0263 255.0295

36 Methyl O-(p-coumaroyl)quinate C17H20O8 21.69 351.1080 163.0389 145.0284 119.0489

37 Quercetin-O-(hexosyl)hexoside C27H30O17 21.87 625.1405 301.0356 300.0281 271.0256 255.0302

38 Methyl O-feruloylquinate C18H22O9 22.15 381.1186 193.0501 175.0390 160.0155 149.0596 134.0362

42 Luteolin-7-O-glucoside (Cynaroside) C21H20O11 22.43 447.0927 327.0527 285.0409 284.0330 256.0379 151.0026

43a _{Narirutin (Naringenin-7-O-rutinoside)} _{C27H32O14 22.49} _581.1870 _435.1290 _315.0866 _273.0762 _153.0185 _85.0291

44 Methoxy-trihydroxyflavone-C-hexoside C22H22O11 22.75 463.1240 445.1143 427.1040 343.0817 313.0710 287.0559

47 Quercetin-O-(malonyl)hexoside C24H22O15 23.71 549.0881 505.0996 463.0859 301.0359 300.0279 271.0252

49 Apigenin-O-(crotonoyl)hexoside C25H24O11 23.99 499.1240 431.0988 269.0459 268.0380 93.0332

54 Quercetin-O-(acetyl)hexoside C23H22O13 24.51 505.0982 463.0869 301.0356 300.0279 271.0251 255.0301

55 Hydroxy-methoxy(iso)flavone-O-hexoside C22H22O9 24.55 431.1342 269.0814 254.0580 237.0550 213.0914

58 Isorhamnetin-O-hexoside C22H22O12 24.98 477.1033 315.0520 314.0438 299.0201 285.0409 271.0252

60 Bergapten (5-Methoxypsoralen) C12H8O4 25.32 217.0501 202.0265 174.0314 146.0363 131.0494 118.0418

62a _Naringenin _C15H12O5 _27.25 _{271.0607 177.0188} _165.0188 _151.0026 _119.0489 _107.0125

64a _{Kaempferol (3.4'.5.7-Tetrahydroxyflavone)} _C15H10O6 _29.35 _287.0556 _258.0527 _213.0550 _165.0184 _153.0183 _121.0286

66 Dimethoxy-trihydroxy(iso)flavone C17H14O7 29.89 329.0661 314.0437 299.0201 271.0252 67 Salcolin A (Tricin-4'-O-(erythro-β-guaiacylglyceryl) ether) C27H26O11 29.94 525.1397 477.1179 329.0678 314.0433 299.0209 195.0649 68 Methoxy-trihydroxy(iso)flavone C16H12O6 30.02 299.0556 284.0331 256.0373 227.0340 151.0023 107.0125 69 Angelicin C11H6O3 30.51 187.0395 159.0442 143.0494 131.0497 115.0548 70 Salcolin B (Tricin-4'-O-(threo-β-guaiacylglyceryl) ether) C27H26O11 30.73 525.1397 329.0674 314.0446 299.0209 195.0651 165.0547 71 Dihydroxy-trimethoxy(iso)flavone C18H16O7 31.29 343.0818 328.0594 313.0361 298.0127 285.0417 270.0170

(6)

each tested bacteria. Overall, the four species showed high activity

against P. myrabilis and E. coli with MIC and MBC values of less than 1

mg mL

−1

_{. The four species were equally effective against S. aureus}

(MIC = 1.13 mg mL

−1

_{; MBC = 1.50 mg mL}

−1

_{) and E. coli (MIC = 0.56}

mg mL

−1

_{; MBC = 0.75 mg mL}

−1

_{). Besides, B. cereus (MIC = 0.28 mg}

mL

−1

_{; MBC = 0.37 mg mL}

−1

_{), P. aeruginosa (MIC = 0.18 mg mL}

−1

_;

MBC = 0.37 mg mL

−1

_{), and S. Typhimurium (MIC = 1.50 mg mL}

−1

_;

MBC = 3.00 mg mL

−1

_{) were most susceptible to B. brachyactis. In fact,}

B. brachyactis was more effective than the standard, ampicillin

(MIC = 0.30 mg mL

−1

_{; MBC = 0.50 mg mL}

−1

_{) against P. aeruginosa}

and was slightly less effective compared to streptomycin (MIC = 0.10

mg/ml; MBC = 0.20 mg mL

−1

_{). On the other hand, P. myrabilis was}

most susceptible to B. microcarpum with a MIC value of 0.14 mg mL

−1

and MBC value of 0.18 mg mL

−1

_{, which was comparable to}

strepto-mycin (MIC = 0.10 mg mL

−1

_{; MBC = 0.20 mg mL}

−1

_{). In addition,}

among the four species, B. pinnatifolium exhibited the highest

Table 3 (continued)

No. Compounds Formula Rt. min [M + H]+ _[M–H]− _{Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5}

72 Tetrahydroxyflavone-O-(cinnamoyl)hexoside C30H26O12 32.13 577.1346 285.0409 284.0331 255.0300 227.0347

73 Imperatorin C16H14O4 32.98 271.0970 203.0344 175.0394 147.0442 131.0497 69.0707

74 Dihydroxy-dimethoxy(iso)flavone C17H14O6 34.85 313.0712 298.0488 283.0253 255.0300

a _{Confirmed by standard.}

Table 4

Chemical composition of B. pinnatifolium.

No. Compound Formula Rt. min [M + H]+ _[M–H]− _{Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5}

1 Quinic acid C7H12O6 1.20 191.0556 173.0445 171.0288 127.0388 111.0439 85.0280

5 Esculin isomer C15H16O9 13.67 341.0873 179.0344 151.0393 133.0287 123.0447

8 Syringic acid C9H10O5 15.92 197.0450 182.0214 166.9976 153.0550 138.0313 123.0075

14 Caffeoylshikimic acid isomer 1 C16H16O8 17.21 335.0767 179.0343 161.0233 135.0441

18 Caffeoylshikimic acid isomer 2 C16H16O8 18.05 335.0767 179.0342 161.0234 135.0441

22 Ferulic acid C10H10O4 19.38 193.0501 178.0264 149.0597 137.0233 134.0362 121.0281

25 Methyl O-(p-coumaroyl)quinate C17H20O8 21.70 351.1080 163.0391 145.0287 119.0489

26 Quercetin-O-(hexosyl)hexoside C27H30O17 21.71 625.1405 301.0358 300.0280 271.0255 255.0294

27 Dihydrokaempferol (Aromadendrin. Katuranin) C15H12O6 21.97 287.0556 259.0617 243.0650 201.0554 177.0549 125.0231

30 Hyperoside (Hyperin. Quercetin-3-O-galactoside) C21H20O12 22.73 463.0877 301.0361 300.0280 271.0253 255.0300 151.0026

33 Quercetin-O-pentoside isomer 1 C20H18O11 23.30 433.0771 301.0370 300.0279 271.0252 255.0291

36 Astragalin (Kaempferol-3-O-glucoside) C21H20O11 24.72 447.0927 327.0523 285.0410 284.0332 255.0300 227.0347

38 Isorhamnetin-O-glucuronide C22H20O13 25.19 491.0826 315.0519 300.0281 271.0257 255.0302

40 Kaempferol-O-(malonyl)glucoside C24H22O14 25.64 533.0931 489.1053 285.0411 284.0332 255.0299 227.0355

42a _Quercetin _C15H10O7 _27.01 _303.0505 _285.0395 _257.0447 _229.0500 _153.0184 _137.0237

43a _Naringenin _C15H12O5 _27.23 _{271.0607 177.0181} _165.0188 _151.0028 _119.0490 _107.0125

46a _Isorhamnetin (3'-Methoxy-3.4'.5.7-tetrahydroxyflavone) C16H12O7 29.84 315.0505 300.0280 283.0250 271.0250 164.0104 151.0027 47 Dimethoxy-trihydroxy(iso)flavone C17H14O7 29.88 329.0661 314.0442 299.0205 271.0252 48 Methoxy-trihydroxy(iso)flavone C16H12O6 29.92 299.0556 284.0331 256.0374 227.0340 151.0023 49 Tetrahydroxyflavone-O-(cinnamoyl)hexoside C30H26O12 32.12 577.1346 285.0410 284.0332 255.0300 227.0347 50 Dimethylallyl-methoxycoumarin C15H16O3 34.96 245.1178 189.0551 161.0600 159.0444 131.0496 103.0550 51 Isoimperatorin C16H14O4 35.72 271.0970 203.0343 175.0395 147.0445 131.0495 69.0707 a _{Confirmed by standard.}

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Table 5

Chemical composition of B. sayaii.

No. Compound Formula Rt. min [M + H]+ _[M–H]− _{Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5}

1 Quinic acid C7H12O6 1.20 191.0556 173.0446 171.0290 127.0390 111.0439 85.0281

8 Feruloylhexose isomer 1 C16H20O9 16.70 355.1029 235.0609 193.0501 175.0396 149.0598

12 Feruloylhexose isomer 2 C16H20O9 17.02 355.1029 235.0611 193.0500 175.0396 149.0598

16 Caffeoylshikimic acid C16H16O8 18.04 335.0767 179.0344 161.0235 135.0442

21 Ferulic acid C10H10O4 19.36 193.0501 178.0264 149.0598 137.0234 134.0363 121.0283

22 Quercetin-O-(hexosyl)hexoside isomer 1 C27H30O17 20.02 625.1405 301.0360 300.0285 271.0252 255.0306

29 Quercetin-O-(hexosyl)hexoside isomer 2 C27H30O17 21.87 625.1405 301.0360 300.0283 271.0250 255.0298

34 Tetrahydroxyflavone-O-(deoxyhexosyl)hexoside

isomer 1 C27H30O15 22.48 593.1507 285.0412 284.0334 199.0396 151.0028

35 Hyperoside (Hyperin. Quercetin-3-O-galactoside) C21H20O12 22.71 463.0877 301.0361 300.0282 271.0254 255.0302 151.0027

40 Quercetin-O-(malonyl)hexoside C24H22O15 23.70 549.0881 505.0999 463.0884 301.0360 300.0281 271.0254

isomer 2 C27H30O15 24.23 593.1507 285.0413 284.0333 255.0303 227.0349 151.0025

47a _{Quercitrin (Quercetin-3-O-rhamnoside)} _{C21H20O11 24.55} _449.1084 _303.0505 _229.0501 _129.0552 _85.0291 _71.0499

49 N-trans-Feruloyltyramine C18H19NO4 24.73 314.1392 177.0550 145.0287 121.0653

50 Isorhamnetin-O-hexoside isomer 1 C22H22O12 24.76 477.1033 315.0519 314.0440 299.0206 285.0414 271.0255

isomer 3 C27H30O15 24.86 593.1507 285.0412 284.0333 255.0302 227.0349 151.0028

56 Methoxy-tetrahydroxy(iso)flavone-O-deoxyhexoside C22H22O11 26.60 461.1084 315.0518 314.0440 300.0284 299.0205 271.0255

57a _Quercetin _C15H10O7 _27.07 _303.0505 _285.0404 _257.0449 _229.0494 _153.0190 _137.0235

61 Dimethoxy-trihydroxy(iso)flavone C17H14O7 29.86 329.0661 314.0442 299.0203 271.0255 62 Salcolin A (Tricin-4'-O-(erythro-β-guaiacylglyceryl) ether) C27H26O11 29.90 525.1397 477.1179 329.0678 314.0433 299.0209 195.0649 63 Methoxy-trihydroxy(iso)flavones C16H12O6 29.98 299.0556 284.0332 256.0378 227.0348 151.0017 107.0116 64 Angelicin C11H6O3 30.54 187.0395 159.0445 143.0496 131.0497 115.0545 65 Salcolin B (Tricin-4'-O-(threo-β-guaiacylglyceryl) ether) C27H26O11 30.70 525.1397 329.0674 314.0443 299.0208 195.0656 165.0550 66 Tetrahydroxyflavone-O-(cinnamoyl)hexoside C30H26O12 32.13 577.1346 285.0412 284.0335 255.0303 227.0349 a _{Confirmed by standard.}

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antibacterial effect against M. flavus and E. cloacae (MIC = 0.56 mg

mL

−1

_{; MBC = 0.75 mg mL}

−1

_).

As for the antifungal activity of the extracts, B. brachyactis showed

the highest effect against A. versicolor (MIC = 0.18 mg mL

−1

_;

MFC = 0.37 mg mL

−1

_{), T. viride (MIC = 0.02 mg mL}

−1

_{; MFC = 0.03}

mg mL

−1

_{), and P. funiculosum (MIC = 0.18 mg mL}

−1

_{; MFC = 0.37 mg}

mL

−1

_{). B. brachyactis was more effective against A. versicolor compared}

to the standard drug ketoconazole (MIC = 0.20 mg mL

−1

_{; MFC = 0.50}

mg mL

−1

_{), and was also more effective than both ketoconazole and}

bifonazole against T. viride. On the contrary, A. fumigatus was most

susceptible to B. sayai (MIC = 0.14 mg mL

−1

_{; MFC = 0.28 mg mL}

−1

₎

while P. ochrochloron was most susceptible to B. pinnatifolium

(MIC = 0.275 mg mL

−1

_{; MFC = 0.37 mg mL}

−1

_{). Moreover, B. sayai}

was more effective than ketoconazole (MIC = 0.20 mg mL

−1

_;

MFC = 0.50 mg mL

−1

_{) in inhibiting A. fumigatus and was slightly less}

effective but comparable to bifonazole (MIC = 0.15 mg mL

−1

_;

MFC = 0.20 mg mL

−1

_{). On the other hand, B. sayai and B. pinnatifolium}

were equally effective against A. terreus (MIC = 0.28 mg mL

−1

_;

MFC = 0.56 mg mL

−1

_{) and P. verrucosum (MIC = 0.28 mg mL}

−1

_;

MFC = 0.37 mg mL

−1

_{) while B. pinnatifolium and B. brachyactis showed}

similar activity against A. niger (MIC = 0.56 mg mL

−1

_{; MFC = 0.75 mg}

mL

−1

_).

A number of compounds identified in the Bunium species were

previously found to possess antimicrobial properties. For instance,

apigenin was found to inhibit several bacteria tested in our study,

in-cluding P. aeruginosa, S. Typhimurium, and P. mirabilis (

Nayaka,

Londonkar, Umesh, & Tukappa, 2014

). Chlorogenic acid showed

anti-bacterial effects by significantly increasing the outer and plasma

membrane permeability, resulting in the loss of the barrier function,

and induced slight leakage of nucleotide, which led to cell death (

Lou,

Wang, Zhu, Ma, & Wang, 2011

). Esculin and rutin were also found to

inhibit S. aureus (

Amin, Khurram, Khattak, & Khan, 2015

;

Kostova,

Nikolov, & Chipilska, 1993

). Vitexin also modulated S. aureus cell

surface hydrophobicity and reduced the intracellular adhesion of

planktonic cells to form biofilm (

Das et al., 2018

). Also, luteolin

in-hibited the activity of DNA topoisomerase I and II in S. aureus, which

resulted in some decrease in the nucleic acid and protein synthesis

(

Wang & Xie, 2010

).

Table 6

Antibacterial activity (mg mL−1_{) of the tested extracts}⁎_. Antibacterial properties

Bunium species S. aureus B. cereus P. mirabilis M. flavus P. aeruginosa E. coli S. Typhimurium E. cloacae B. sayai MIC 1.13 ± 0.26b _{0.56 ± 0.10}c _{0.56 ± 0.10}d _{1.13 ± 0.22}d _{0.28 ± 0.02}b _{0.56 ± 0.10}b _{2.25 ± 0.20}bc _{1.13 ± 0.10}c MBC 1.50 ± 0.20b _{0.75 ± 0.03}d _{0.75 ± 0.20}d _{1.50 ± 0.20}c _{0.37 ± 0.03}b _{0.75 ± 0.01}b _{3.00 ± 0.30}b _{1.50 ± 0.90}c B. pinnatifolium MIC 1.13 ± .0.10b _{0.37 ± 0.02}b _{0.28 ± 0.10}c _{0.56 ± 0.10}c _{0.28 ± 0.03}b _{0.56 ± 0.03}b _{2.15 ± 0.40}bc _{0.56 ± 0.20}b MBC 1.50 ± 0.25b _{0.75 ± 0.04}d _{0.37 ± 0.03}c _{0.75 ± 0.10}b _{0.37 ± 0.04}b _{0.75 ± 0.04}b _{3.00 ± 0.50}b _{0.75 ± 0.03}b B. brachyactis MIC 1.13 ± 0.10b _{0.28 ± 0.03}b _{0.56 ± 0.040}d _{0.75 ± 0.10}c _{0.18 ± 0.01}ab _{0.56 ± 0.05}b _{1.50 ± 0.60}b _{1.13 ± 0.20}c MBC 1.50 ± 0.30b _{0.37 ± 0.02}c _{0.75 ± 0.050}d _{1.50 ± 0.20}c _{0.37 ± 0.02}b _{0.75 ± 0.06}b _{3.00 ± 0.80}b _{1.50 ± 0.10}c B. microcarpum MIC 1.13 ± 0.10b _{1.13 ± 0.10}d _{0.14 ± 0.003}b _{1.13 ± 0.25}d _{0.28 ± 0.03}b _{0.56 ± 0.02}b _{3.00 ± 0.90}c _{1.13 ± 0.20}c MBC 1.50 ± 0.28b _{1.50 ± 0.20}e _{0.18 ± 0.02}b _{1.50 ± 0.26}c _{0.37 ± 0.04}b _{0.75 ± 0.03}b _{4.50 ± 1.20}c _{1.50 ± 0.10}c Streptomycin MIC 0.10 ± 0.10a _{0.03 ± 0.002}a _{0.10 ± 0.03}b _{0.05 ± 0.003}a _{0.10 ± 0.02}a _{0.10 ± 0.02}a _{0.10 ± 0.03}a _{0.10 ± 0.03}a MBC 0.20 ± 0.18a _{0.05 ± 0.003}a _{0.20 ± 0.02}b _{0.10 ± 0.02}a _{0.20 ± 0.02}a _{0.20 ± 0.03}a _{0.20 ± 0.03}a _{0.20 ± 0.03}a Ampicillin MIC 0.10 ± 0.012a _{0.10 ± 0.020}d _{0.01 ± 0.002}a _{0.10 ± 0.03}b _{0.30 ± 0.03}b _{0.15 ± 0.01}a _{0.10 ± 0.02}a _{0.15 ± 0.002}a MBC 0.15 ± 0.10a _{0.15 ± 0.020}b _{0.01 ± 0.001}a _{0.15 ± 0.02}a _{0.50 ± 0.04}c _{0.20 ± 0.03}a _{0.20 ± 0.02}a _{0.20 ± 0.03}a Antifungal abilities

Bunium species A. fumigatus A. versicolor A. terreus A. niger T. viride P. ochrochloron P. funiculosum P. veruccosum B. sayai MIC 0.14 ± 0.02a _{0.75 ± 0.03}d _{0.28 ± 0.02}b _{1.13 ± 0.25}d _{0.09 ± 0.002}b _{0.56 ± 0.03}b _{0.28 ± 0.02}b _{0.28 ± 0.02}ab MFC 0.28 ± 0.01a _{1.50 ± 0.20}e _{0.56 ± 0.03}b _{1.50 ± 0.28}d _{0.18 ± 0.02}b _{0.75 ± 0.03}b _{0.37 ± 0.01}ab _{0.37 ± 0.02}b B. pinnatifolium MIC 0.50 ± 0.03c _{0.56 ± 0.02}c _{0.28 ± 0.01}b _{0.56 ± 0.02}b _{0.28 ± 0.03}d _{0.28 ± 0.01}a _{0.28 ± 0.02}b _{0.28 ± 0.03}ab MFC 1.13 ± 0.10c _{0.75 ± 0.03}d _{0.56 ± 0.02}b _{0.75 ± 0.03}c _{0.37 ± 0.04}c _{0.37 ± 0.02}ab _{0.37 ± 0.03}ab _{0.37 ± 0.03}b B. brachyactis MIC 0.42 ± 0.02c _{0.18 ± 0.02}b _{0.56 ± 0.03}c _{0.56 ± 0.02}b _{0.02 ± 0.002}a _{0.37 ± 0.010}ab _{0.18 ± 0.01}a _{0.37 ± 0.03}b MFC 0.56 ± 0.03b _0.380.03b _{1.25 ± 0.20}c _{0.75 ± 0.03}c _{0.03 ± 0.003}a _{0.75 ± 0.02}b _{0.37 ± 0.02}ab _{0.75 ± 0.04}c B. microcarpum MIC 0.56 ± 0.02c _{1.50 ± 0.20}e _{0.56 ± 0.03}c _{0.75 ± 0.02}c _{0.28 ± 0.02}d _{0.56 ± 0.03}b _{0.56 ± 0.03}c _{0.56 ± 0.02}c MFC 1.13 ± 0.06c _{3.00 ± 0.10}f _{1.13 ± 0.30}c _{1.50 ± 0.20}d _{0.37 ± 0.03}c _{0.75 ± 0.04}b _{0.75 ± 0.04}c _{0.75 ± 0.04}c Ketoconazole MIC 0.20 ± 0.02b _{0.20 ± 0.02}b _{0.15 ± 0.02}ab _{0.20 ± 0.02}a _{1.00 ± 0.10}e _{1.00 ± 0.30}c _{0.20 ± 0.01}ab _{0.20 ± 0.02}a MFC 0.50 ± 0.05b _{0.50 ± 0.03}c _{0.20 ± 0.01}a _{0.50 ± 0.03}b _{1.50 ± 0.20}d _{1.50 ± 0.20}c _{0.50 ± 0.02}b _{0.30 ± 0.03}b Bifonazole MIC 0.15 ± 0.02a _{0.10 ± 0.01}a _{0.10 ± 0.01}a _{0.15 ± 0.02}a _{0.15 ± 0.02}c _{0.20 ± 0.03}a _{0.20 ± 0.01}ab _{0.10 ± 0.01}a MFC 0.20 ± 0.03a _{0.20 ± 0.03}a _{0.15 ± 0.01}a _{0.20 ± 0.02}a _{0.20 ± 0.01}b _{0.25 ± 0.02}a _{0.25 ± 0.02}a _{0.20 ± 0.02}a

⁎ _{Different letters indicate the differences in the Bunium species for each bacterial and fungal strains (p < 0.05).}

Table 7

Antioxidant properties of the Bunium species⁎_.

Samples Phosphomolybdenum (mmol TE g−1_extract) DPPH (mg TE g −1 extract) ABTS (mg TE g −1 extract) CUPRAC (mg TE g −1 extract) FRAP (mg TE g −1

extract) Metal chelating activity(mg EDTAE g−1_extract) B. sayai 1.24 ± 0.16b _{41.15 ± 1.27}c _{68.66 ± 1.09}c _{118.53 ± 2.39}c _{89.05 ± 3.10}c _{40.70 ± 0.60}b B. pinnatifolium 1.53 ± 0.08a _{51.89 ± 2.75}b _{96.66 ± 8.99}a _{155.47 ± 7.60}a _{128.23 ± 5.40}a _{18.29 ± 0.63}c B. brachyactis 1.51 ± 0.17a _{49.79 ± 1.11}b _{84.87 ± 1.64}b _{135.28 ± 1.17}b _{108.64 ± 2.88}b _{52.61 ± 0.49}a B. microcarpum 1.13 ± 0.05b _{69.66 ± 1.52}a _{100.33 ± 3.18}a _{160.64 ± 4.37}a _{121.86 ± 8.34}a _{15.66 ± 0.28}d

⁎ _{Values expressed are means ± S.D. of three parallel measurements. TE: Trolox equivalent; EDTAE: EDTA equivalent. Different letters indicate the differences in}

(9)

3.3. Antioxidant activity

Six assays were used to assess the antioxidant capacity of the tested

Bunium species (Table 7

), namely DPPH and ABTS scavenging, cupric

and ferric reducing, phosphomolybdenum (all expressed in mg TE

g

−1

_{extract), and metal chelating assay (expressed in mg EDTAE/g}

ex-tract). This approach is useful given the variations among the wide

range of antioxidant assays currently used, which do not provide

pre-cise and accurate information on the complete antioxidant power of a

plant sample (

Mahomoodally et al., 2018

).

The most effective DPPH and ABTS scavenger was B. microcarpum

(69.66 and 100.33 mg TE g

−1

_{), respectively) followed by B.}

pinnatifo-lium (51.89 and 96.66 mg TE g

−1

_{, respectively), while the least}

effec-tive scavenger was B. sayai. Similarly, B. microcarpum showed the

strongest cupric reducing activity (160.64 mg TE g

−1

_{) while B.}

pinna-tifolium exhibited the highest ferric reducing effect (128.23 mg TE g

−1

_).

Again, the weakest cupric and ferric reducer was displayed by B. sayai.

On the other hand, the least effective antioxidant in the

phosphomo-lybdenum and metal chelating assays was B. microcarpum while the

strongest antioxidant among the tested Bunium species in the respective

assays were B. pinnatifolium (1.53 mmol TE g

−1

_{) and B. brachyactis}

(52.61 mg EDTAE g

−1

_{), respectively.}

The high antioxidant activity of B. pinnatifolium and B. microcarpum

in most assays (with the exception of metal chelating assay) could be

explained by three reasons; first by the high phenolic and flavonoids

content since a number of previous studies have observed that herbal

extracts possessing high TPC and TFC showed high antioxidant effects

(

Sut et al., 2019

;

Zengin et al., 2019

;

Zengin et al., 2019

). This fact was

also confirmed correlation analysis and the results are given in

Fig. 1

(A). Strong correlation was observed between total bioactive

compounds and antioxidant properties, especially reducing power

as-says. Secondly, it could be that the compounds present in all the four

species are present in higher amount in these two species, but this

as-sumption need to be further investigated using other analytical

tech-niques to measure the amount of these phenolic compounds in the

extracts. Several compounds present in both species, such as

chloro-genic acid, pantothenic acid, esculin, isoquercitrin, rutin, apigenin,

scopoletin, naringenin, and kaempferol, were previously found to

pos-sess antioxidant properties (

Biljali et al., 2012

;

Cavia-Saiz et al., 2010

;

Li, Jiang, Wang, Liu, & Chen, 2016

;

Malik et al., 2011

;

Romanova,

Vachalkova, Cipak, Ovesna, & Rauko, 2001

;

Vellosa et al., 2011

;

Xu,

Hu, & Liu, 2012

;

Yang, Guo, & Yuan, 2008

).

3.4. Enzyme inhibitory activity

The inhibitory properties of the four Bunium species were studied

against six enzymes, namely AChE, BChE, tyrosinase, amylase,

gluco-sidase, and lipase (

Table 8

). The development of innovative natural

cholinesterase inhibitors are presently gaining much interest for the

symptomatic treatment of Alzheimer’s disease. On the other hand, the

inhibition of α-glucosidase and α-amylase (two main enzymes involved

in starch hydrolysis to glucose) has also become an important strategy

to tackle diabetes mellitus (type 2), while tyrosinase inhibition is

ne-cessary to prevent the misbalance in melanin formation, associated

with a variety of skin diseases such as melasma and hyperpigmentation

(

Mahomoodally et al., 2018

). In addition, inhibition of pancreatic lipase

and the associated reduction of lipid absorption is an attractive

ap-proach to manage obesity and its associated diseases such as diabetes

mellitus and coronary heart diseases (

Buchholz & Melzig, 2015

).

It was observed that B. sayai and B. brachyactis were almost equally

effective in inhibiting AChE (3.53 and 3.42 mg GALAE g

−1

_,

respec-tively). The most effective BChE inhibitor was B. brachyactis (3.68 mg

GALAE g

−1

_{). In contrast, B. microcarpum displayed the lowest}

in-hibitory effect against cholinesterases. In addition, the four species

Fig. 1. Statistical evaluations (A: Correlation coefficients between total bioactive compounds and biological activities (Pearson Correlation Coefficient (R). p < 0.05); B: Heatmap of extracts in according to biological activities (n = 4); C: Distribution of the tested Bunium species on the correlation circle based on PCA; PPBD: Phosphomolybdenum; MCA: Metal chelating assay; TYR: Tyrosinase inhibition assay; TPC: Total phenolics content; TFC: Total flavonoids content).