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journal homepage:www.elsevier.com/locate/indcropPhytochemical pro
filing, in vitro biological properties and in silico studies on
Caragana ambigua stocks (Fabaceae): A comprehensive approach
Saima Khan
a, Mamona Nazir
b, Naheed Raiz
a, Muhammad Saleem
a,⁎, Gokhan Zengin
c,⁎,
Gazala Fazal
a, Hammad Saleem
d,e, Mahreen Mukhtar
a, Muhammad Imran Tousif
f,
Rasool Baksh Tareen
g, Hassan H. Abdallah
h,i, Fawzi M. Mahomoodally
jaDepartment of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, 63100, Bahawalpur, Pakistan bDepartment of Chemistry, Government Sadiq College Women University, Bahawalpur, 63100, Bahawalpur, Pakistan cSelcuk University, Science Faculty, Department of Biology, Konya, Turkey
dSchool of Pharmacy, Monash University, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia eInstitute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal Sciences (UVAS), Lahore, 54000, Pakistan fDepartment of Chemistry, Dera Ghazi Khan Campus, University of Education Lahore, 32200, Dera Ghazi Khan, Pakistan gDepartment of Botany, Baluchistan University Quetta, Pakistan
hSchool of Pharmacy, Universiti Sains Malaysia, Penang, Malaysia
iChemistry Department, College of Education, Salahaddin University, Erbil, Iraq jDepartment of Health Sciences, Faculty of Science, University of Mauritius, Mauritius
A R T I C L E I N F O Keywords: Caragana ambigua Phytochemicals Antioxidant Tyrosinase Molecular docking A B S T R A C T
Plant from Caragana genus have interesting potential in folklore medicines and have been explored for various pharmacological activities. The aim of this study was to probe into the chemical and biological effects of dif-ferent extracts of Caragana ambigua Stocks (Fabaceae). Total phenolic and totalflavonoids contents were de-termined using standard spectrophotometric methods, whereas, the secondary metabolites composition was established by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis. Antioxidant potential was estimated via 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylben-zothiazoline-6-sulphonic acid (ABTS) radical scavenging, ferric reducing antioxidant power (FRAP), cupric re-ducing antioxidant capacity (CUPRAC), phosphomolybdenum and metal chelating assays and the enzyme (cholinesterases,α-amylase, α-glucosidase and tyrosinase) inhibition potential were also assessed. Moreover, in silico docking studies were performed to highlight possible interactions between three major secondary meta-bolites and the tested enzymes. The ethyl acetate extract exhibited higher phenolic (85.87 ± 2.96 mg GAE/g) andflavonoid (66.45 ± 0.37 mg RE/g) contents, which, we propose, are responsible for its higher radical scavenging, reducing power, total antioxidant capacity and tyrosinase inhibition. The n-hexane extract showed stronger anti-cholinesterase and anti-diabetic property. Similarly, UHPLC-MS profiling of methanol and ethyl acetate extracts identified the presence of flavonoids, phenolics, alkaloids, and coumarin derivatives. Three of the dominant compounds (isobergaptene, jujubasaponin IV and phellodensin D), were docked against all en-zymes and their affinity were compared. Among these compounds, phellodensin D showed highest affinity to-wards all the studied enzymes. It is therefore, concluded that extracts from C. ambigua showed potent antioxidant and enzyme inhibition potential with potent bioactive molecules which could open new industrial applications.
1. Introduction
Herbs and herbal products have been the basis for treating several ailments since the dawn of mankind. Such non-conventional and al-ternative medicines are still utilized today with new avenues of po-tential industrial applications (Khalkho et al., 2015). Since its inception,
thefield of ethno-pharmacology has been developed from entirely a collection of data on medicinal plants used by a certain community into a more complex, multidisciplinary research area which have engaged modern researchers to revisit these precious timeworn knowledge of ancestral medicines (Alvin et al., 2014). This approach is established on the hypothesis that the biologically active compounds isolated from
https://doi.org/10.1016/j.indcrop.2019.01.044
Received 9 November 2018; Received in revised form 21 January 2019; Accepted 22 January 2019
⁎Corresponding authors.
E-mail addresses:drsaleem_kr@yahoo.com,m.saleem@iub.edu.pk(M. Saleem),gokhanzengin@selcuk.edu.tr(G. Zengin).
Available online 28 January 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
such natural products are presumably safer as compared to those de-rived from plant species with no historical use by human (Melucci et al., 2018). Consequently, bioactive phytochemicals are getting attention to obstacle the worldwide health related problems such as diabetes mel-litus (DM), cardio metabolic, Alzheimer’s diseases (AD) and cancer (Grochowski et al., 2017a).
The species of the genus Caragana belonging to family Fabaceae, have been traditionally used for the treatment of headache, cough, asthma, strain-induced fatigue, nose bleeds, fevers, inflammation, cancer, dizziness, neuralgia, rheumatism, arthritis and hypertension (Khan et al., 2008;Meng et al., 2009). About ten species of this genus have been known for their uses in different conventional medicine systems of Eastern Asia, China, Inner Mongolia and Tibet. In the in-digenous Traditional Chinese Medicine (TCM), this species is used for multi-functional purposes including nourishing the blood and pro-moting bloodflow (College, 1977).
The current pharmacological investigation has reported a wide range of biological and chemical properties of different Caragana spe-cies, however, still some species of this group are un-explored. Caragana ambigua is one of them, which has not yet been investigated scientifi-cally in terms of its chemical and biological effects and only little work has been done for its phytochemical investigation, which confirmed the presence ambiguanol, 3,3′,4′,5,7-pentahydroxyflvane, 5-hydroxy-3′,4′,6,7-tetramethoxyflav- one, E-cinnamic acid, calycosin and β-si-tosterol 3-O-β-D-glucopyranoside (Majida et al., 2011). Another study conducted on dried root part of C. ambigua reported the presence of alkaloids, tannins and saponins (Kayani et al., 2007). Therefore, this study was designed to evaluate the phytochemical composition (total bioactive contents and secondary metabolites constituents) and anti-oxidant (DPPH, ABTS, FRAP, CUPRAC, phospholybdenum and ferous chelating) potential of methanol, ethyl acetate, n-hexane and aqueous extracts of C. ambigua. Moreover, inhibition potential against key en-zymes involved in neurodegenerative diseases, diabetes and skin pro-blems were also determined along with docking studies (in order tofind any possible interaction between the secondary metabolites and ob-served enzyme inhibition activity). Indeed, molecular docking has be-come one of the essential tools for studying the interactions of the in-hibitors at the active site of the receptor. Besides, the calculation of the binding free energy, docking studies also elucidates the best con-formation which helps in developing new inhibitors for the target en-zymes. As far as the literature review concerns, this research could be regarded as the foremost detailed investigation on such phytochemical composition, enzyme inhibition and antioxidant properties of C. am-bigua.
2. Materials and methods 2.1. Plant material and extraction
Whole plant of C. ambigua was collected from Ziarat valley, Baluchistan in May 2014, and was identified by Prof. Dr. Rasool Bakhsh Tareen, Department of Botany, University of Baluchistan, Quetta, Pakistan, where a voucher specimen No. CA-RBT-06 has been deposited in the herbarium. The whole plant material was dried for 15 days under the shade. The dried plant (15 kg) was ground into coarse powder and was extracted (thrice) with methanol (20 L) at room temperature for seven days. The resulted extract was concentrated under vacuum to obtain 105 g of dark brown gummy mass. The extract was further suspended in water (1.0 L) and was fractionated into n-hexane (39 g) and ethyl acetate (22.5 g) soluble parts, whereas, aqueous layer wad dried to 41 g of the residue.
2.2. Total bioactive contents and UHPLC-MS analysis
Folin-Ciocalteu method was used for determining the total phenolic contents (TPC) using gallic acid as a standard and the results were
expressed as gallic acid (mg GAE/g extract). Similarly, totalflavonoid contents (TFC) were estimated using aluminum chloride calorimetric assay and the amount of total flavonoids were expressed as rutin equivalents (mg RE/g extract) (Slinkard and Singleton, 1977;Zengin et al., 2016b).
Secondary metabolites profiling was done by UHPLC-MS. UHPLC of Agilent 1290 Infinity LC system coupled to Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with dual ESI source was used. Agilent Zorbax Eclipse XDB-C18 column with narrow bore 2.1 x 150 mm, 3.5μm (P/N: 930990-902). Temperature of 4 °C and 25 °C was main-tained for auto-sampler and column respectively. 0.1% Formic acid solution in water was used as mobile phase A while 0.1% formic acid solution in acetonitrile was used as B, their flow rate was kept as 0.5 mL/min. 1.0μL of the methanol extract was injected for the time of 25 min and 5 min were used for post-run time. Nitrogen gas withflow rate of 25 and 600 L/hour was used as a source of nebulizing and drying gas respectively and temperature was maintained at 350 °C. The frag-mentation voltage was optimized to 125 V. Analysis was performed with a capillary 1 voltage of 3500 V.
2.3. Antioxidant assays
The antioxidant activities were estimated using radical scavenging (DPPH•and ABTS•+), reducing power (FRAP and CUPRAC), phospho-molybdenum method (total antioxidant capacity), and metal chelating assays as described previously by Grochowski et al. (Grochowski et al., 2017b). The results were reported as trolox equivalents, while ethyle-nediaminetetraacetic acid (EDTA) was used as reference for metal chelating assay.
2.4. Enzyme inhibition assays
The possible enzyme inhibitory potential of all the extracts against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase and α-glucosidase were determined using previously re-ported standard in vitro bio-assays (Grochowski et al., 2017b;Mollica et al., 2017). The cholinesterase inhibition was expressed as galantha-mine equivalents, amylase and glucosidase as acarbose equivalents and kojic acid equivalents for tyrosinase inhibition.
2.5. Docking calculations
The 3D structures of isobergaptene, jujubasaponin IV and phello-densin D were downloaded from zinc database (Irwin et al., 2012). Austin model 1 (AM1) semi-empirical method implemented in Gaussian 09 software (Frisch et al., 2009) was used to optimize the compounds to their minimum energy level. The crystal structures of the enzymes were downloaded from Protein Databank RCSB PDB. Protein data bank (PDB) structure, 4EY6, was used as AChE receptor in which the enzyme was in complex with galantamine at the active site. 1P0P is re-presenting the crystal structure of the BChE in which the enzyme was crystallized with butyrylthiocholine. For the tyrosinase enzyme, 5I38 crystal structure was used for the docking calculations in which the Table 1
Total phenolic andflavonoid content of C. ambigua extracts*.
Extracts Abbreviation Total phenolic content (mgGAE/g extract)
Totalflavonoid content (mgRE/g extract)
Methanol Ca-M 37.37 ± 0.21b 17.36 ± 0.27b
Ethyl acetate Ca-E 85.87 ± 2.96d 66.45 ± 0.37d
n-Hexane Ca-H 32.39 ± 2.15a 15.06 ± 0.16a
Aqueous Ca-A 44.60 ± 0.89c 58.69 ± 0.15c
* Values expressed are means ± S.D. of three parallel measurements; means with different superscript letters in the same column are significantly (p < 0.05) different; GAE: Gallic acid equivalents; RE: Rutin equivalents.
enzyme was crystallized with Kojic acid. Protein data bank code 7TAA and 3W37 were used to represent the α-amylase and α-glucosidase enzymes, both enzymes were crystallized with the acarbose inhibitor. Prior to the docking calculations, water molecules and co-crystallized molecules were removed from the protein structure.
Autodock 4 software (Molinspiration Database) (Irwin and Shoichet, 2005) was used to scan all the possible conformations, cluster the best poses and rank these clusters according to the binding free energy with the target enzyme. During the process of preparing the receptor, Kollman united atom charges was used to neutralize the protein and the protein is immersed in a grid box with a dimensions, 60 × 60 × 60 with 0.375 Å distance between points. In order to scan all the possible conformations, 250 run for each inhibitor using La-marckian genetic algorithm. The structures of the docked conforma-tions with the intra-molecular interacconforma-tions with the active site were visualized using Discovery studio 5.0 visualizer.
2.6. Statistical analysis
All results were the mean of three equivalent experiments and were expressed as average ± SD of value. One-way ANOVA and SPSS v. 17.0 programs were used for result analysis. The value of p < 0.05 was considered as statistically significant.
3. Result and discussion 3.1. Chemical profile of extracts
Phytochemicals are bioactive substances having significant role in maintenance of overall health of human body by repairing and pro-moting cell growth. Phenolic compounds have the ability to reduce the risk of some chronic diseases like hypertension, diabetes, cancer, and cardiovascular problems (Lin et al., 2016;Mollica et al., 2017;Tănase et al., 2018). In the present study, different extracts of C. ambigua were Fig. 1. Total ion chromatograms of methanol and ethyl acetate extracts of C. ambigua.
Table 2
UHPLC-MS secondary metabolites profile of methanol extract of C. ambigua.
S. No RT(min) Base peak(m/ z)
Diff (DB.ppm)
Peak height AUC Proposed compounds Compound class Molecular formula Molecular mass
1 0.626 215.03 10.96 1091409 3957998 Isobergaptene Coumarin C12H8O4 216.03
2 0.668 683.22 −2.2 118383 750095 Citbismine C Alkaloid C37H36N2O11 684.23
3 7.864 885.26 −0.53 213028 1807731 Kaempferol-3-isorhamninoside-7-rhamnoside Flavonoid C39H50O23 886.27
4 7.921 739.20 −0.71 71317 579281 Robinin Flavonoid C33H40O19 740.21
5 0.868 290.08 −0.25 120873 391537 Sarmentosin epoxide Glycoside C11H17NO8 291.09
6 8.719 609.14 −1.83 163999 1095266 Robinetin 3-rutinoside Flavonoid C27H30O16 610.15
7 9.107 579.20 −0.95 99994 696046 (+)-Syringaresinol O-β-D-glucoside Lignin C28H36O13 580.21
8 9.118 623.16 −1.8 300142 2545922 Tricetin 7-methyl ether 3'-glucoside-5'-rhamnoside
Flavonoid C28H32O16 624.17
9 9.992 1133.53 0.06 123692 1068560 Calendasaponin C Terpene C54H86O25 1134.54
10 11.442 299.05 −0.75 92724 709453 Kaempferide Flavonoid C16H12O6 300.06
11 11.451 329.23 −0.99 139562 1367030 5,8,12-trihydroxy-9-octadecenoic acid Fatty acid C18H34O5 330.24
12 11.463 517.17 −0.33 90511 631280 Phellamurin Flavonoid C26H30O11 518.17
13 12.166 941.51 −0.04 291245 4112074 Jujubasaponin IV Saponin C48H78O18 942.51
14 13.114 355.11 −2.29 250807 1863271 Phellodensin D Flavonoid C20H20O6 356.12
15 13.179 353.10 −1.24 139571 1054198 2,3-Dehydrokievitone Flavonoid C20H18O6 354.11
16 14.099 337.10 −1.41 119429 901058 (-)-Glyceollin I Flavonoid C20H18O5 338.11
appraised for their total bioactive contents and the results are shown in Table 1. The ethyl acetate extract showed highest phenolic contents (85.87 ± 2.96 mgGAE/g extract) followed by aqueous (44.60 ± 0.89 mgGAE/g extract), methanol (37.37 ± 0.21 mgGAE/g extract) and n-hexane (32.39 ± 2.15 mgGAE/g extract) extracts. Similarly, the total flavonoid contents were ranged from 66.45 ± 0.37 to 17.36 ± 0.27 mgRE/g extracts. The higher contents offlavonoids were found in ethyl acetate extract(Table 1).Overall, ethyl acetate extract of C. ambigua was rich in phenolic andflavonoid contents, and the results
of total bioactive contents are in agreement with the previous report which also reported highest phenolic (140.0 ± 10.1 mgGAE/g extract) andflavonoid (140.0 ± 10.1 mgRE/g extract) contents in ethyl acetate extract of C. sinica (Tai et al., 2010).
Liquid chromatography mass spectrometry (LC/MS) analysis of methanol and ethyl acetate extracts of C. ambigua was performed. A typical chromatograms of both the extracts with mass spectrometric detection in negative ionization mode exhibited quite complex patterns of peaks as shown inFig. 1. The LC/MS analysis of C. ambigua methanol Table 3
UHPLC-MS secondary metabolites profile of ethyl acetate extract of C. ambigua.
S. No RT(min) Base peak (m/z) Diff (DB.ppm)
Peak height AUC Proposed compounds Compound class Molecular formula Molecular mass
1 9.014 342.13 84673 84673 578292 Sphalleroside A Phenolic C16H22O8 341.12 2 9.7 272.06 399343 399343 5624494 ( ± )-Naringenin Flavonoid C15H12O5 271.06 3 9.815 288.06 89978 89978 746052 2,6,3',4'-Tetrahydroxy-2-benzylcoumaranone Flavonoid C15H12O6 287.05 4 9.936 534.17 150660 150660 1191160 Phellatin Flavonoid C26H30O12 533.16 5 10.005 300.06 413659 413659 3059898 Kaempferide Flavonoid C16H12O6 299.05 6 10.268 270.05 159020 159020 1315646 Demethyltexasin Flavonoid C15H10O5 269.04 7 10.573 284.06 501543 501543 4588296 Texasin Flavonoid C16H12O5 283.06 8 11.225 298.04 86311 86311 592383 8-Methoxycoumestrol Flavonoid C16H10O6 297.04 9 11.268 370.10 699817 699817 6989022 Neouralenol Flavonoid C20H18O7 369.09
10 11.313 516.16 202694 202694 1939688 Vitexin 2”-O-(2”'-methylbutyryl) Flavonoid C26H28O11 515.15
11 11.335 386.10 290376 290376 2984308 Melisimplexin Flavonoid C20H18O8 385.09
12 11.336 270.05 697173 697173 7127388 Demethyltexasin Flavonoid C15H10O5 269.04
13 11.372 284.06 126016 126016 804765 Texasin Flavonoid C16H12O5 283.06
14 11.445 300.06 1136224 1136224 1.2E+07 Kaempferide Flavonoid C16H12O6 299.05
15 11.476 518.18 649168 649168 6253294 Phellamurin Flavonoid C26H30O11 517.17
16 11.628 370.10 170849 170849 3223872 Neouralenol Flavonoid C20H18O7 369.09
17 11.978 386.13 84864 84864 632581 Samidin Coumarin C21H22O7 385.12
18 12.014 330.24 104995 104995 1162332 5,8,12-trihydroxy-9-octadecenoic acid Fatty acid C18H34O5 329.23
19 12.113 370.10 202388 202388 2028171 Neouralenol Flavonoid C20H18O7 369.09
20 12.117 352.09 210748 210748 1953994 Psoralidin oxide Flavonoid C20H16O6 351.08
21 12.177 372.12 171,312 171,312 1949428 7,8,3',4',5'-Pentamethoxyflavone Flavonoid C20H20O7 371.11
22 12.486 370.10 192907 192907 2199607 Neouralenol Flavonoid C20H18O7 369.09
23 13.118 356.12 3375776 3375776 3.6E+07 Phellodensin D Flavonoid C20H20O6 355.11
24 13.164 294.18 99663 99663 1247070 Gingerol Polyphenol C17H26O4 293.17
25 13.985 314.24 101844 101844 1191607 9,10-Epoxy-18-hydroxystearate Fatty acid C18H34O4 313.23
26 14.268 368.12 524153 524153 6224450 Aurmillone Flavonoid C21H20O6 367.120
RT: retention time; AUC: area under curve. Table 4
Antioxidant properties of C. ambigua extracts*.
Plant extracts
Radical scavenging assays Reducing power assays Total antioxidant activity Ferrous ion chelation
DPPH (mgTE/g extract)
ABTS (mgTE/g extract) FRAP (mgTE/g extract) CUPRAC (mgTE/g extract) Phosphomolybdenum (mmolTE/g) Metal chelating (mgEDTAE/g) Ca-M 55.05 ± 2.00b 103.12 ± 6.10b 162.68 ± 1.99b 204.66 ± 9.17b 1.30 ± 0.10a 23.82 ± 1.24b Ca-E 83.32 ± 6.22d 421.94 ± 13.93d 405.26 ± 11.15d 617.89 ± 7.00d 2.70 ± 0.06a 52.56 ± 3.15c Ca-H 12.66 ± 0.19a 60.10 ± 2.35a 85.36 ± 8.82a 161.88 ± 8.71a 1.96 ± 0.12a 74.27 ± 3.71d Ca-A 60.16 ± 3.01c 115.52 ± 1.22c 227.96 ± 3.13c 236.98 ± 2.11c 1.47 ± 0.06a 10.32 ± 0.21a
* Values expressed are means ± S.D. of three parallel measurements; means with different superscript letters in the same column are significantly (p < 0.05) different TE: Trolox equivalent; EDTAE: EDTA equivalent.
Table 5
Enzyme inhibition activities of C. ambigua extracts*.
Plant extracts
AChE
(mgGALAE/g extract) BChE
(mgGALAE/g extract
α-Amylase (mmolACAE/g extract) α-Glucosidase (mmolACAE/g extract) Tyrosinase (mgKAE/g extract)
Ca-M 4. 55 ± 0.08b 2.91 ± 0.41a 0.54 ± 0.02b na 172.08 ± 1.29b
Ca-E 3.18 ± 0.24a 3.57 ± 0.12a 0.75 ± 0.01b 1.67 ± 0.01c 185.80 ± 1.45d
Ca-H 4.81 ± 0.32b 4.95 ± 0.30b 0.81 ± 0.02b 1.68 ± 0.01b 176.01 ± 1.10c
Ca-A na na 0.08 ± 0.01a 0.78 ± 0.10a 38.57 ± 1.83a
* Values expressed are means ± S.D. of three parallel measurements; means with different superscript letters in the same column are significantly (p < 0.05) different; AChE: Acetylcholinesterase; BChE:Butyrylcholinesterase; GALAE: Galatamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent; na: not active.
extract revealed the presence of 16 different secondary metabolites as presented inTable 2. Most of these compounds were offlavonoid, al-kaloid, saponin and terpenoids. The major flavonoids identified wer-ekaempferol3-isorhamninoside-7-rhamnoside, robinin, robinetin-3-ru-tinoside, tricetin7-methyl ether 3′-glucoside-5′-rhamnoside, kaempferide, phellamurin, phellodensin D, 2,3-dehydrokievitone and (-)-glyceollin I. Other important compounds present were isobergaptene (coumarin), jujubasaponin IV (saponin), citbismine C (alkaloid) and calendasaponin C (terpene). Similarly, the ethyl acetate extract showed the presence of 26 different secondary metabolites belonging to flavo-noid, coumarin and phenolic class of compounds (Table 3). The flavo-noids were identified as ( ± )-naringenin, 2,6,3′,4′-tetrahydroxy-2-benzylcoumaranone, phellatin, kaempferide-8-methoxycoumestrol, neouralenol, vitexin2′'-O-(2′”-methylbutyryl) melisimplexin, de-methyltexasin, texasin, phellamurin, psoralidin oxide, 7,8,3′,4′,5′-pen-tamethoxyflavone, phellodensin D and aurmillone. The presence of flavonoids, alkaloids, saponins and terpenoidsin C. ambigua is in agreement with other Caragana species (Mandal et al., 2016). 3.2. Antioxidant assays
Antioxidants are the compounds which are produced either by biological systems or occur naturally in many foods and a balance among oxidants and antioxidants is of significance for regular body functioning (Kedare and Singh, 2011). In the current study, different antioxidant tests (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum and metal chelating assays) were executed in order to obtain a thorough evaluation of the antioxidant potential of extracts from C. ambigua, and the results are presented inTable 4. The ethyl acetate extract shows highest radical scavenging activity (DPPH: 83.32 ± 6.22 and ABTS: 421.94 ± 13.93 mgTE/g extract, respectively), followed by the aqu-eous and methanol extracts.
Reducing abilities of plant extract can be evaluated by CUPRAC and FRAP assays among other in vitro methods. In these assays, Cu+2and Fe+3ions are reduced to Cu+1and Fe+2ions respectively in the pre-sence of antioxidants (Zengin et al., 2016a). The reducing power ca-pacities results follows the same pattern (Table 4) as in case of radical scavenging assay, in which ethyl acetate extract was the most active one (FRAP: 405.26 ± 11.15, CUPRAC: 617.89 ± 7.0 mgTE/g ex-tract).
Additionally, total antioxidant capacity of the extracts was also determined using the phosphomolybdenum assay. The ethyl acetate extract of C. ambigua exhibited highest total antioxidant capacity with a value of 2.70 mmolTE/g extract (Table 4). It was also noted that the
high antioxidant activity of the ethyl acetate extract in the radical scavenging, reducing power and phosphomolybdenum assays, was proportional to its higher total phenolic and flavonoid contents (Table 1). Indeed, a number of earlier studies have reported a strong correlation of total bioactive contents and the above-mentioned anti-oxidant mechanisms (Barbouchi et al., 2018;Gündüz et al., 2015;Mitic et al., 2018). In addition, it is to be noticed that a previous study conducted by Zhi Gang et al (2010) also found the ethyl acetate extract of C. sinica to be most active in DPPH radical scavenging assay (IC50:
45.0μg/mL) (Tai et al., 2010). Another research reported the isolated compound conferin from ethyl acetate extract of C. conferta to possess significant DPPH radical scavenging potential with IC50 value of
19.1 ± 0.11μM (Khan et al., 2010). Similarly, it was earlier described that the ethyl acetate extract of C. sinica to be most active in FRAP reducing power assay (Meng et al., 2009). Moreover, there is a growing number of evidence that toxic and carcinogenic metals are able to in-teract with nuclear proteins and DNA which results in oxidative dete-rioration of biological macromolecules (Valko et al., 2005). The metal chelating activity of C. ambigua is shown inTable 4. In contrast to other antioxidant activities, n-hexane extract (74.27 mg EDTAE/g extract) showed excellent metal chelating activity as compared to ethyl acetate extract (52.56 mg EDTAE/g extract). Methanol and aqueous extracts were least active for metal chelating activity.
3.3. Enzyme inhibition potential
As most of the therapeutic drugs acts by inhibiting a specific en-zyme, thus, enzyme inhibition is one way in which enzyme activity is regulated (Zengin et al., 2018). In this study, the enzyme inhibitory activity of C. ambigua extracts was tested against cholinesterases (AChE and BChE),α-amylase, α-glucosidase and tyrosinase and the results are shown in Table 5. Acetylcholinesterase (AChE) and bu-trylcholinesterase (BChE) are the key enzymes in the breakdown of acetylcholine and butrylcholine and their inhibition is regarded as one of the management strategies against several neurological problems. As the cholinesterases inhibition results in increase in the concentration of acetylcholine in the brain, which in turn cause an increase in commu-nication between the brain nerve cells (Grochowski et al., 2018). All the extracts of C. ambigua (except aqueous extract) exhibited considerable inhibition potentials against both cholinesterases enzymes. The n-hexane extract showed maximum AChE (4.81 ± 0.32 mgGALAE/g) and BChE (4.95 ± 0.30 mgGALAE/g) inhibition followed by ethyl acetate and methanol extracts. Thisfinding may be linked to the pre-sence of non-phenolic inhibitors in the n-hexane extract and our results Table 6
Binding energy (kcal/mol), inhibition constant, interaction sites and distances between residues at the active site of HMG-CoA and chosen ligands.
Compounds docked Enzymes Binding energy / inhibition constant Ki
Interaction site
Isobergaptene AChE −6.72 (11.9 μM) Ala 127 (HB), Tyr 133 (HB), Trp 86, Tyr 337 BChE −6.36 (21.8 μM) Tyr 440 (HB), Trp 82 (HB), Ala 328, Phe 329 α-amylase −6.07 (35.4 μM) Arg (HB), Asp 340, Trp 83, His 80
α-glucosidase −6.68 (12.1 μM) Ile 358, Asp 469, Met 470, Arg 552 (HB), Asp 568
Tyrosinase −7.47 (3.37 μM) Asn 205 (HB), Glu 195, His 204, Met 61, Ala 221, His 208, Met 215, Val 218 Jujubasaponin IV AChE −4.64 (395.3 μM) Thr 75 (HB), Tyr 7 (HB), Asp 74 (HB), Tyr 341 (HB), Ser 293 (HB), His 287, Trp 286
BChE −4.36 (634.6 μM) Asn 68 (HB), Gln 67 (HB), Gln 71 (HB), Ile 69, Asn 83, His 77, His 126, Gln 270 α-amylase −4.03 (1.1 mM) Gly 167 (HB), Asp 206 (HB), Glu 230 (HB), Asp 297, Trp 83, Tyr 75.
α-glucosidase −4.30 (699.4 μM) Thr 478 (HB), Asp 568 (HB), Lys 506 (HB), Arg 552, His 626, Phe 601, Trp 432, Phe 476 Tyrosinase −2.68 (10.8 mM) Asn 199 (HB), Gly 200 (HB), Glu 158 (HB), Pro 201, Met 184, Met 61, Lys 47, Pro 219, Phe 197. Phellodensin D AChE −9.28 (157.8 nM) Tyr 72 (HB), Asp 74 (HB), Gly 122 (HB), Ser 203 (HB), Tyr 133 (HB), Phe 297, Trp 86, Gly 120
BChE −10.41 (23.3 nM) His 438 (HB), Glu 197 (HB), Tyr 128 (HB), Asn 83 (HB), Asp 70 (HB),Trp 82, Thr 120, Ile 69 α-amylase −6.89 (8.9 μM) Asp 206 (HB), Arg 344 (HB), Gln 35 (HB), Asp 340 (HB), Asp 168 (HB), Tyr 75, Trp 83, His 80, Tyr
82
α-glucosidase −8.24 (914.1 nM) Asp 630 (HB), Asp 357 (HB), Asp 232 (HB), Trp 329, Phe 601, Ala 628, Ala 602, Met 470, Trp 432, Asp 569
Tyrosinase −6.37 (21.5 μM) Asn 205 (HB), Gly 196, Phe 197, His 208, Val 218
are supported by the literature, where no correlation between total phenolic and anti-cholinesterase effects (Russo et al., 2015; Samaradivakara et al., 2016) have been reported. Similarly, previous investigations indicated that Caragana species possess AChE inhibition activity. For example,Ramakrishna and Roshchina (2018)reported the isolation ofα-viniferin and kobophenol A from C. chamlague to possess reversible non-competitive AChE inhibition in a dose-dependent manner (IC50: 2.0 and 115.8 μM, respectively) (Ramakrishna and
Roshchina, 2018).
The enzymes α-amylase and α-glucosidase are involved in the breakdown of carbohydrate, thus their inhibition has been an important strategy for the management of diabetes, ultimately lowering post-prandial glucose level. Similarly, the inhibitors of α-glucosidase are
reported to delay the breaking down of carbohydrate in the gut and decrease postprandial blood glucose peak in diabetic patients (Uysal et al., 2018). Among the tested extracts, n-hexane extract was the most effective against α-amylase (0.81 ± 0.02mmolACAE/g) and α-gluco-sidase (1.68 ± 0.01 mmolACAE/g) enzymes (Table 5). Similarly, ethyl acetate extract was also considerably active against both theα-amylase and α-glucosidase enzymes with the values of 0.75 ± 0.01 and 1.67 ± 0.01 mmol ACAE/g, respectively. Whereas, the methanol ex-tract showed moderate inhibition againstα-amylase but was inactive forα-glucosidase. The aqueous extract was least active against both enzymes. The observed anti-diabetic potential of C. ambigua is in ac-cordance with the previous reports asMeng et al. (2009)reported thein vivo activity of C. intermedia to control blood glucose level (Meng et al., Fig. 2. Phellodensin D at the active site of AChE (A), BChE (B), tyrosinase (C),α-amylase (D) and α-glucosidase (E).
2009) and similarly, C. arborescens has been reported to contain fla-vonoids having hypoglycemic properties (Mandal et al., 2015).
Tyrosinase inhibitors are active agents with the capacity of reducing enzymatic reactions such as food browning and hyperpigmentation of human skin (Zengin et al., 2017). Results of tyrosinase inhibition ac-tivity of C. ambigua methanol extract are presented inTable 5. Ethyl acetate extract showed the highest tyrosinase inhibition (185.80 ± 1.45 mgKAE/g), while n-hexane and methanol extracts also showed significant tyrosinase inhibition with the values of 176.01 ± 1.10 and 172.08 ± 1.29 mgKAE/g, respectively. The ob-served higher tyrosinase inhibition of ethyl acetate extract can be due to higher amount offlavonoids in this extract as flavonoids can act both as substrates and inhibitors against tyrosinase by precipitating the tyrosinase enzyme, thus inhibiting enzymatic activity (Pettersen et al., 2004). Similarly, other species of this genus such as C. sinica has already been documented to inhibit tyrosinase in vitro (Jeon et al., 2012). Overall, from the abovefindings, we found that the ethyl acetate ex-tract, which displayed the significant total phenolic and flavonoid contents, was the most effective inhibitor against all the tested en-zymes. This is thefirst comprehensive report on such enzyme inhibition potential of C. ambigua.
3.4. Docking results
Computational molecular docking studies have been extensively used successfully for the theoretical prediction of ligand-target inter-actions and to better interpreted the molecular basis of the biological activity of natural products (Zengin et al., 2017). In silico studies also anticipates further insights into the possible mechanism of action and binding mode of active compounds against metabolic key enzymes (Mocan et al., 2016). In order to get better insight on the inhibition ability of the studied compounds and to correlate the experimental enzyme inhibition results, three representing compounds of ethyl acetate extract (isobergaptene, jujubasaponin IV and phellodensin D) were selected for the docking calculations. The selected compounds were docked againstfive enzymes, AChE, BChE, tyrosinase, α-amylase andα-glucosidase enzymes.
Table 6summarizes the binding affinity of the studied compounds against the five enzymes with estimated inhibition constants and the interactions with the catalytic amino acid residues at the active site. Generally, phellodensin D has shown the best affinity towards the five enzymes followed by isobergaptene and jujubasaponin IV. This se-quence of binding affinity is attributed to the strong interactions be-tween these molecules and the active site of the enzymes. Among the different types of interactions between the inhibitor and the active site, hydrogen bonds represent the strongest non-bonding interaction. The list of interactions contains beside the hydrogen bonds, pi-pi interac-tions, electrostatic interactions and van der Waals. Fig. 2 shows the docked compound, phellodensin D at the active site of thefive studied enzymes and its interactions with the active site in which hydrogen bonding is the dominant interaction.
4. Conclusion
In this study, we tested the different solvent extracts of C. ambigua for their biological and chemical profiling. The n-hexane and ethyl acetate extracts were found to inhibit key enzymes involved in Alzheimer’s disease (acetylcholinesterase and butrylcholinesterase), diabetes (α-amylase and α-glucosidase) and skin problems (tyrosinase). Furthermore, highest total bioactive contents were observed in ethyl acetate extract and the same extract tends to show the highest anti-oxidant potential (except metal chelation). Docking results have sup-ported the corresponding experimentalfindings and highlighted inter-esting results about the interactions with the active site. Phellodensin D has shown the best binding affinity with the five studied enzymes. This study paved the pathway to scientific community regarding the
pharmacological potentials of C. ambigua and provides the rationale for further identification and characterization of its bioactive antioxidants and enzyme inhibitors with potential industrial applications. Our sub-sequent step is to put forth on the chromatographic isolation and characterization of secondary metabolites from the above extracts which may be responsible for their potent actions in various activities. Acknowledgments
Muhammad Saleem thanks to Mrs. Topsy Smalley for providing various research articles published in literature, which were not avail-able to the author, due to limited access in his institute.
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