In vitro biological propensities and chemical profiling of Euphorbia milii Des Moul (Euphorbiaceae): A novel source for bioactive agents

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In vitro biological propensities and chemical profiling of Euphorbia milii Des

Moul (Euphorbiaceae): A novel source for bioactive agents

Hammad Saleem

_{, Gokhan Zengin}

_{, Marcello Locatelli}

_{, Adriano Mollica}

_{, Irshad Ahmad}

_,

Fawzi M. Mahomoodally

_{, Syafiq Asnawi Zainal Abidin}

_{, Nafees Ahemad}

a_{School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway Selangor Darul Ehsan, Malaysia} b_{Institute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal Sciences (UVAS), Lahore, 54000, Pakistan} c_{Department of Biology, Faculty of Science, Selcuk University, Campus/Konya, Turkey}

d_{Department of Pharmacy, University ‘G. d’Annunzio” of Chieti-Pescara, 66100, Chieti, Italy} e_{Department of Pharmacy, The Islamia University of Bahawalpur, 63100, Pakistan} f_{Department of Health Sciences, Faculty of Science, University of Mauritius, Mauritius}

g_{Liquid Chromatography Mass Spectrometery (LCMS) Platform, Monash University, Jalan Lagoon Selatan, 47500 Bandar Sunway Selangor Darul Ehsan, Malaysia} h_{Tropical Medicine and Biology Multidisciplinary Platform, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway Selangor Darul Ehsan, Malaysia}

A R T I C L E I N F O Keywords: Euphorbia milii Phytochemicals Antioxidant Enzyme inhibition Tyrosinase A B S T R A C T

The plants of Euphorbia genus have been extensively studied for their nutritive and therapeutic purposes. The present research is the foremost effort to investigate and compare the biological activities and chemical com-position of dichloromethane (DCM) and methanol (MeOH) solvent extracts of Euphorbia milli Des Moul aerial and root parts. Antioxidant potential was determined using six different (FRAP, CUPRAC, Phosphomolybdenum, DPPH, ABTS and ferrous chelation) methods. The enzyme inhibition effects of the tested extracts were evaluated against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-glucosidase, α-amylase and tyrosinase. Similarly, the amount of total phenolic and flavonoid contents were assessed via spectrophotometric methods and individual secondary metabolites were also determined using UHPLC-MS analysis. Methanolic extracts from both aerial and root parts contained the highest contents for phenolic and flavonoids which tends to correlate with their significant DPPH, ABTS (radical scavenging), FRAP, CUPRAC (reducing power) and α-glucosidase inhibition potentials. While, both the DCM extracts containing the lowest bioactive contents were most active in the phosphomolybdenum assay, cholinesterases and tyrosinase inhibition. The root extracts proved to be a better source of bioactive antioxidant molecules. Additionally, UHPLC-MS profiling of both the methanolic extracts revealed the presence of total 16 secondary metabolites belonging to five major groups (phenolic, flavonoid, coumarin, glycoside and alkaloid). To conclude, our results suggest that E. milii can be considered as a promising lead origin for natural bioactive enzyme inhibitory and antioxidant compounds which could pave the way for industrial applications.

1. Introduction

Medicinal plants and herbal products are gaining much momentum globally due to complexity of pathologies coupled with side effects associated with the use of synthetic drugs. Natural products have been appraised and verified for their various pharmacological activities such as cytotoxic, anti-oxidants, antidiabetic, antimicrobials and anti-in-flammatory (Buchholz and Melzig, 2015;Grochowski et al., 2018). Si-milarly, it has been observed that the higher utilization of natural products is inversely related with the probability of prime health issues

worldwide, including cancer, cardiovascular disorders and type 2 dia-betes (Benzie and Choi, 2014;González-Gallego et al., 2010). To this effect, the discovery of bioactive products from natural products such as medicinal plants has attained much recognition of the scientific com-munity.

The genus Euphorbia belongs to Euphorbiaceae family, is amongst the largest genus of medicinal plants which is distributed throughout tropical countries including China and Pakistan (Rauf et al., 2014). The phytochemical studies conducted on different Euphorbia species had revealed the existence of different secondary metabolites including

https://doi.org/10.1016/j.indcrop.2018.12.062

Received 16 November 2018; Received in revised form 17 December 2018; Accepted 19 December 2018

⁎_{Corresponding authors at: School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway Selangor Darul Ehsan, Malaysia.} ⁎⁎_{Corresponding author.}

E-mail addresses:hammad.saleem@monash.edu(H. Saleem),gokhanzengin@selcuk.edu.tr(G. Zengin),nafees.ahemad@monash.edu(N. Ahemad).

Available online 25 December 2018

0926-6690/ © 2018 Elsevier B.V. All rights reserved.

diterpenes (Hou et al., 2011), euphorbetin, aesculetin, daphnetin, β-sitosterol, kaempferol-3-glucuronide (Zheng et al., 2009), vitexicarpin, artemetin, daucosterol, p-hyfroxybenzoic acid (Jiao et al., 2010), fla-vones, flavonol glucosides (Surveswaran et al., 2007), hydrocarbons, steroids, triterpenes and fatty acids (Ismaila et al., 2017). The plants of

Euphorbia genus have been detailed to have pharmacological properties

such as anticancer, anti-viral and antimicrobial (Sadeghi-Aliabadi et al., 2009;Yang et al., 2005;Zeghad et al., 2016). Latex of some Euphorbia species have been reported as topical treatment in some skin diseases and sexual transmitted disease like gonorrhea and in case of migraines and gastric parasite in several traditional system (Singla and kamla, 1990;Zeghad et al., 2016). Different Euphorbia species have been lo-cally utilized for different ailments including warts, antibacterial and against intestinal parasites (Mwine and Van Damme, 2011). Ad-ditionally, some of the Euphorbia species have also been reported to possesses anti-arthritis, (Ali et al., 2018) inflammatory, oxidant, antitumor, antispasmodic, anticonvulsant, antidiabetic, anti-eczema, antitussive (Pešić et al., 2011) and anti-proliferative properties (Chaabi et al., 2007). E. milii, commonly named as “Christ thrown” or “Christ plant” is amongst the most important medicinal plant of this genus having folklore medicinal importance for treating warts, cancer and hepatitis. This plant has also been reported for antifungal, anti-nociceptive and molluscicidal properties (Opferkuch and Hecker, 1982; Rauf et al., 2014).

Despite the plethora of studies related to the therapeutic uses of E.

milii, data regarding its chemical composition, antioxidant potential

and enzyme inhibition activities related with most common human diseases is limited. Given the background regarding medicinal proper-ties of E. milii, this work was conducted to probe into the enzymatic inhibitory activities and antioxidant potential of methanol and DCM extracts from aerial and roots of E. milii. All the extracts were chemi-cally characterized by determining their total bioactive contents and both methanolic extracts were also assessed for individual secondary metabolite profiling by ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS). As far as the literature review con-cerns, this research can be regarded as the first comprehensive de-scription on the secondary metabolites profiling, antioxidant and en-zyme inhibitory capabilities of E. milii.

2. Materials and methods

2.1. Plant material and extraction

Aerial and root parts of E. milii plant were collected from Bahawalpur, Pakistan. The plant material was identified by the tax-onomist, Dr. H. Waris, Cholistan Institute of Desert Studies (CIDS), The Islamia University of Bahawalpur (IUB), Pakistan. Moreover, a voucher specimen (EM-WP-01-15-119) for future reference was also deposited in the herbarium of Department of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur, Pakistan. The collected plant material was placed under shade for 15 days. The resultant dried plant parts was ground into coarse powder. The extraction of powdered material was done by maceration with DCM and methanol solvents, successively for 72 h. The resultant extracts were concentrated under rotary evaporator.

2.2. Total bioactive contents

The standard Folin-Ciocalteu and aluminum chloride methods as described previously were used to determine the total bioactive con-tents i.e., total phenolic (TPC) and total flavonoid concon-tents (TFC), re-spectively (Slinkard and Singleton, 1977;Zengin et al., 2016). The re-sults of total phenolic contents were expressed as equivalents of gallic acid (mg GAE/g extract) while the amount of total flavonoid contents were recorded as quercetin equivalents (mg QE/g extract).

2.3. UHPLC-MS analysis

Secondary metabolites profiling was done by RP-UHPLC-MS (re-verse phase ultra-high performance liquid chromatography mass spec-trometry) analysis. Agilent 1290 Infinity UHPLC system which is cou-pled to Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with dual ESI source was utilized. The column used was Agilent Zorbax Eclipse XDB-C18 having narrow bore 2.1 × 150 mm, 3.5 μm (P/N: 930990-902). Temperature of 4 °C and 25 °C was maintained for auto-sampler and column respectively. The mobile phase (A) used was 0.1% formic acid solution in water whereas, acetonitrile and 0.1% formic acid solution was the mobile phase (B). The flow rate of mobile phase was kept at 0.5 mL/min. Plant extract solution (1.0 μL in HPLC grade methanol solvent) was Injected for the time of 25 min and 5 min were used for post-run time. Nitrogen gas with flow 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 analysis was performed with a capillary voltage of 3500 V while, the fragmentation voltage was optimized to 125 V.

2.4. Antioxidant assays

The methods as described earlier by Grochowski et al. (Grochowski et al., 2017) were utilized to estimate the radical scavenging (DPPH•

and ABTS•+_{), reducing power (FRAP and CUPRAC), total antioxidant}

capacity (phosphomolybdenum assay) and metal chelating power of the studied extracts. The antioxidant potential of all the assays were re-ported as trolox equivalents (mg TE/g extract), while metal chelating activity was expressed as mg EDTAE/g extract.

2.5. Enzyme inhibition assays

For the enzyme inhibitory activities, the practicable inhibition po-tential of the tested plant extracts against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-amylase, α-glucosidase and tyrosinase were determined employing in vitro standard methods as described previously (Grochowski et al., 2017;Mollica et al., 2017). Galantamine was used as a standard for AChE and BChE and the cholinesterases inhibition activity was measured as mg GALAE/g extract (as milligrams of galantamine equivalents per gram of extract). Similarly, α-amylase and α-glucosidase inhibition were expressed as millimoles of acarbose equivalents per gram of extract (ACAE/g) and tyrosinase inhibition was recorded as milligrams of kojic acid equivalents per gram of extract (KAE/g).

2.6. Statistical analysis

All the assays were executed as three collateral and different ex-periments. The obtained results were expressed as mean value ± standard deviation (SD). SPSS v.17.0 software was used for the analysis of data. One-way analysis of variance (ANOVA) followed by Tukey's test was used to determine differences between the means. A value of p < 0.05 was considered as statistically significance. The PCA (prin-cipal component analysis) and Pearson linear correlation was de-termined in order to identify any correlation between bioactive con-tents and tested biological assays.

3. Results and discussion

3.1. Total bioactive contents

Plant secondary metabolites are now a days gaining much attention because of their pharmacological effects such as antioxidant, anti-in-flammatory or anticancer (Locatelli et al., 2017;Uysal et al., 2018). The studied E. milii plant extracts were assessed for their total phenolic and flavonoid contents using spectrophotometric methods and the obtained

results are depicted inTable 1. For both phenolic and flavonoids, the highest contents were observed from the methanolic extracts, and specifically the root methanol extract showed the highest phenolic contents (30.81 ± 0.95 mg GAE/g), whereas the highest contents in terms of total flavonoids were obtained in aerial methanol extracts (2.76 ± 0.38 mg QE/g). A similar type of results were earlier reported by Basma et al. (2011)who reported the higher values of flavonoid contents in the methanolic extracts from leaves of E. hirta in comparison with stems, flowers and roots from the same plant (Basma et al., 2011). In another study, the total phenolic and flavonoid content in E.

grand-ialata alcoholic extract were reported as 17.61 ± 1.2 μg GAE/ g and

0.495 ± 0.39 μg RE/g fresh weight, respectively (Ismaila et al., 2017). Similarly,Zhang et al., (2017)has demonstrated the varied total phe-nolic and flavonoid content among different parts of E. lathyris. The highest amount of phenolic were observed in testa (290.46 ± 15.09 mg of gallic acid equiv/100 g dry weight (DW). However, the root contained the highest flavonoids i.e., 215.68 ± 3.10 mg of rutin equiv/g DW) (Zhang et al., 2017).

Apart from total bioactive contents, individual secondary metabo-lite compounds of both the methanolic extracts were detected utilizing UHPLC-MS analysis. A negative ion mode was used for the tentative identification of compounds. The typical total ion chromatograms (TICs) of the extracts with their mass spectrometric detection exhibited quite different peaks as shown in Fig. 1. The UHPLC-MS analysis of aerial methanol extract of E. milii identified five different compounds belonging to sesquiterpene (eremopetasitenin A1), phenolic (lusitani-coside), coumarin (fraxetin), alkaloid (megastachine) and glycoside (peruvoside) classes of phytochemicals (Table 2). Similarly, E. milii root methanol extracts identified the 11 different secondary metabolites

belonging to five different groups i.e., phenolic, flavonoid, sesqui-terpene, glycoside and coumarin (Table 3). Flavonoids and phenolics were the major compounds present. The flavonoids identified were dichotosinin, abruquinone B, kaempferol 3-(6′'-acetylglucoside)-7-glu-coside, kaempferide 5-glucoside-7-glucuronide and herbacetin 8-acetate while, dihydroferulic acid 4-sulfate, ellagic acid and li-cochalcone A were the main phenolics present.

3.2. Antioxidant properties

Antioxidants can be defined as the compounds that can either in-hibit or delay the process of oxidation by acting against the particular oxidizing enzymes or either by reacting with the oxidant molecules, thus protecting the damage of critical biomolecules (Abeywickrama et al., 2016). The antioxidant capacity of E. milii aerial and root extracts were evaluated using six different assays including free radical scavenging (ABTS and DPPH), reducing power (CUPRAC and FRAP), phosphomolybdenum and metal chelating and the results are gathered inTable 4. The free radical scavenging assays measures the potential of

E. milii extracts to scavenge the in vitro formed free radicals. In the

present research, the prominent DPPH and ABTS radical scavenging capacity was manifested by root-MeOH extract (58.54 ± 0.66 and 89.14 ± 2.62 mg TE/g extract, respectively), whereas the lowest scavenging values were observed for aerial-DCM extract (7.23 ± 0.43 and 22.71 ± 0.64 mg TE/g extract, respectively) and this trend was similarly observed for total bioactive contents. The reducing power capacity of E. milii extracts also presented similar results as in case of DPPH and ABTS assays and the root-MeOH extract showed highest FRAP and CUPRAC powers with values of 87.73 ± 1.85 and 136.44 ± 6.41 mg TE/g extract, respectively. Overall, phenolic and flavonoid rich methanolic extracts were the most active for radical scavenging and reducing power antioxidant assays. Clearly, the results of these antioxidant assays can be correlated with the higher amounts of total phenolic and flavonoids from the tested plant extracts. The results of these antioxidant assays are in accordance with earlier stu-dies, which also presented a significant association among total bioac-tive contents and antioxidant capacities (Amessis-Ouchemoukh et al., 2017;Giusti et al., 2017;Wang et al., 2017;Zengin et al., 2018b). As shown inFig. 2, Pearson correlation shows, a strong positive relation-ship was observed between TPC (R = 0.86-0.64), TFC (R = 0.63-0.38), and radical scavenging and reducing power antioxidant (DPPH, ABTS, Table 1

Extraction yields (%), total phenolic and flavonoid contents in E. milii extracts. Extracts Yield (%) Total phenolic content

(mg GAE/g) Total flavonoid content(mg QE/g) Aerial-MeOH 17 25.56 ± 0.52 2.76 ± 0.38 Aerial-DCM 14 8.72 ± 0.17 1.35 ± 0.14 Root-MeOH 19 30.81 ± 0.95 2.42 ± 0.07 Root-DCM 11 27.50 ± 0.45 1.27 ± 0.16

Data from three repetitions, with mean ± standard deviation. GAE: gallic acid equivalent; QE: quercetin equivalent.

FRAP and CUPRAC) assays. The strongest correlation of TPC and TFC was observed with CUPRAC assay (R = 0.86 and 0.63, respectively). Previously, methanol extract of E. grandialata has been reported for DPPH scavenging activity (140.6% μg ascorbic acid/g) when compared with standard ascorbic acid (Ismaila et al., 2017). Similarly,Zhang et al (2017)had reported a positive correlation between total phenolics and DPPH free radical scavenging activity in different parts of E. lathyris (Zhang et al., 2017).

The phosphomolybdenum method is appraised a meaningful and simple method to further determine the total antioxidant power of plant samples (Llorent-Martínez et al., 2017). In this assay, both root extracts exhibited higher values as compared to aerial extracts. However, in both cases, the DCM extracts displayed superior values (Table 4). As this antioxidant assay measures the antioxidant capacity of both phe-nolic and non-phephe-nolic compounds, therefore at this point, tocopherol or vitamin C which are non-phenolic antioxidants can be responsible for the observed activity of DCM extracts. These results are in accordance to some previous reports (Albayrak et al., 2010;Llorent-Martínez et al., 2017) which had also presented the higher phosphomolybdenum an-tioxidant potential for DCM solvent. Moreover, a weak correlation be-tween total bioactive contents and total antioxidant capacity was also observed in previous stidies (Nićiforović et al., 2010;Sarikurkcu et al., 2015). Similarly, a negative correlation was also observed among TPC (R = -0.47), TFC (R = -0.99) and phosphomolybdenum assay (Fig. 2). Chelating agents are the compounds which bind with pro-oxidant metals and are effective as secondary antioxidants (Abeywickrama et al., 2016). As shown in Table 4, the current study showed

considerable chelating activity for aerial-DCM followed by aerial-MeOH and root-MeOH extracts (49.92 ± 1.23, 23.51 ± 3.02 and 23.36 ± 0.25 mg EDTAE/g extract, respectively). However, the root-DCM extract was found to be inactive for this activity.

3.3. Enzyme inhibition assays

The search for safe enzyme inhibitors of natural origin to overcome global health problems including neurodegenerative diseases, diabetes, hypertension, and skin problems is amongst the most investigated matters in the scientific manifesto. Towards that end, we examined enzyme inhibition potential of E. milii extracts against acet-ylcholinesterase, butracet-ylcholinesterase, α-amylase, α-glucosidase and tyrosinase enzymes (Bender et al., 2018).

Alzheimer’s disease (AD) is one of the well-known neurodegenera-tive disorder and cholinesterases enzyme inhibition is one of the most effective strategy for treatment of Alzheimer’s disease (Asghari et al., 2018). As indicated inTable 5, the aerial-DCM was the most active one against AChE (5.37 ± 0.49 mg GALAE/g extract) while root-DCM showed maximum BChE inhibition (5.00 ± 0.43 mg GALAE/g extract). Overall, the DCM extracts exhibited higher AChE and BChE inhibition as compared to methanolic extracts. Although the DCM extracts showed higher inhibition against cholinesterases, but these extracts contained least phenolic and flavonoid contents, which indicates the presence of other non-phenolic compounds responsible for modulating the ob-served inhibition. Similarly, as shown inFig. 2, a negative correlation was observed between total bioactive contents of the tested extracts and Table 2

UHPLC-MS secondary metabolites identified in E. milli aerial methanol extract.

S.no RT (min) B. peak m/z Compound identified MFG Formula Comp. class Mol. mass

1 8.555 363.18 Eremopetasitenin A1 C20 H28 O6 Sesquiterpene 364.18

2 8.583 441.18 Lusitanicoside C21 H30 O10 Phenolic 442.18

3 9.486 207.03 Fraxetin C10 H8 O5 Coumarin 208.03

4 15.485 330.21 Megastachine C20 H29 N O3 Alkaloid 331.21

5 17.857 547.29 Peruvoside C30 H44 O9 Glycoside 548.29

RT: retention time; B.peak: base peak. Table 3

UHPLC-MS secondary metabolites identified in E. milli root methanol extract.

S.no RT (min) B. peak m/z Compound identified MFG Formula Comp. class Mol. mass

1 7.817 177.02 7,8-Dihydroxycoumarin C9 H6 O4 Coumarin 178.02

2 8.118 395.21 Isopetasoside C21 H32 O7 Glycoside 396.21

3 8.56 363.18 Eremopetasitenin A1 C20 H28 O6 Sesquiterpene 364.18

4 8.562 477.181 Dichotosinin C24 H30 O10 Flavonoid 478.18

5 8.95 275.03 Dihydroferulic acid 4-sulfate C10 H12 O7 S Phenolic 276.03

6 9.029 389.13 Abruquinone B C20 H22 O8 Flavonoid 390.13

7 9.067 301.0 Ellagic acid C14 H6 O8 Phenol 302.0

8 9.651 651.16 Kaempferol 3-(6”-acetylglucoside)-7-glucoside C29 H32 O17 Flavonoid 652.16

9 9.784 637.14 Kaempferide 5-glucoside-7-glucuronide C28 H30 O17 Flavonoid 638.148

10 12.29 343.05 Herbacetin 8-acetate C17 H12 O8 Flavonoid 344.05

11 15.251 337.15 Licochalcone A C21 H22 O4 Phenol 338.15

RT: retention time; B.peak: base peak. Table 4

Antioxidant properties of E. milii extracts.

Extracts Radical Scavenging activity Reducing power Total antioxidant capacity (TAC) Ferrous chelating DPPH (mgTE/g

extract) ABTS (mgTE/gextract) FRAP(mgTE/g extract) CUPRAC (mgTE/gextract) Phosphomolybdenum(mgTE/g extract) Metal Chelating(mgEDTAE/g) Aerial-MeOH 11.50 ± 1.06 28.36 ± 1.70 40.38 ± 3.02 88.94 ± 1.94 0.38 ± 0.06 23.51 ± 3.02 Aerial-DCM 7.23 ± 0.43 22.71 ± 0.64 24.47 ± 0.47 46.93 ± 0.72 1.78 ± 0.08 49.92 ± 1.23 Root-MeOH 58.54 ± 0.66 89.14 ± 2.62 87.73 ± 1.85 136.44 ± 6.41 0.78 ± 0.04 23.36 ± 0.25 Root-DCM 17.55 ± 0.85 36.79 ± 1.88 41.61 ± 2.08 82.02 ± 3.02 1.91 ± 0.10 na TE: trolox equivalent; EDTAE: EDTA equivalent. All values expressed are means ± S.D. of three parallel measurements.

their AChE and BChE inhibition (R values in the range -0.19 to -0.89). This indicates the presence of other compounds modulating the ob-served activities as previously mentioned. Moreover, this obob-served higher inhibition by DCM extracts can be ascribed to the existence of non-phenolic secondary metabolites like alkaloids which have been previously reported for cholinesterase activities (Parveen et al., 2001; Yan et al., 2011) and these findings are in accordance with previous reports which also presented the considerable cholinesterase inhibition of other Euphorbia species (Anuradha et al., 2010;Pintus et al., 2013; Pisano et al., 2016).

Additionally, a copper containing enzyme, namely tyrosinase, is involved in the melanin biosynthesis (Lai et al., 2017) which has a prime role in skin protection against ultra-violet ionizing radiations (Liu et al., 2017). Kojic acid and arbutin are common tyrosinase inhibitors but they exert some adverse effects on human health (Chen et al., 2015). In this sense, the discovery of effective and safer tyrosinase in-hibitors from natural plant products is of significant interest (Deri et al., 2016). In this research work, tyrosinase inhibitory capacity of E. milii

extracts was determined in vitro and the results are presented inTable 5. The aerial-DCM extract (140.52 ± 0.60 mg KAE/g extract) showed strongest tyrosinase inhibition followed by root-MeOH (124.64 ±

0.22 mg KAE/g extract). However, the root-DCM and aerial- MeOH extracts also exhibited considerable inhibition with values of 122.82 ± 0.95 and 122.59 ± 1.09 mg KAE/g extract. Previously, Pintus et al. (2015)had described the significant tyrosinase inhibition potential of different solvent extracts from E. characias different parts (leaves, stems, and flowers) (Pintus et al., 2015). In another study, the

E. supina extracts were reported to produce skin-whitening effects by

the suppression of melanin production in B16F10 melanoma cells and also showed tyrosinase inhibition and decreased melanin content (Kang et al., 2018). However, as shown in Fig. 2, a negative correlation is observed between TPC (R = −0.95), TFC (R = −0.51) and tyrosinase inhibition and the observed findings are in line with some previous reports already presenting no correlation for total phenolic/flavonoid contents and tyrosinase inhibition (Chiocchio et al., 2018;Zengin et al., 2018a). At this point, non-phenolic inhibitors could be attributed to the Fig. 2. Statistical evaluations (A and B: distribution of all tested extracts on the factorial plan and representation of biological activities on the correlation circle based on PCA; C: Correlation coefficient between total bioactive contents and biological activities (Pearson correlation coefficient (R), p < 0.05), EmA-M: E. milii aerial methanol extract; EmA-D: E. milii aerial DCM extract; EmR-M: E. milii root methanol extract; EmR-D: E. milii root DCM extract; TPC: total phenolic contents: TFC: total flavonoid contents; PPBD: Phosphomolybdenum; MCA: Metal chelating assay: TYR: Tyrosinase inhibition.

Table 5

Enzyme inhibition effects of E. milii extracts. Extracts AChE inhibition (mg GALAE/g

extract) BChE inhibition (mg GALAE/gextract) Tyrosinase(mg KAE/g extract) Amylase (mmol ACAE/gextract) Glucosidase (mmol ACAE/gextract)

Aerial-MeOH 3.98 ± 0.21 3.41 ± 0.08 122.59 ± 1.09 0.48 ± 0.02 1.94 ± 0.01

Aerial-DCM 5.37 ± 0.49 4.14 ± 0.19 140.52 ± 0.60 0.62 ± 0.02 1.77 ± 0.09

Root-MeOH 3.63 ± 0.16 3.30 ± 0.02 124.64 ± 0.22 0.49 ± 0.02 1.79 ± 0.02

Root-DCM 4.59 ± 0.63 5.00 ± 0.43 122.82 ± 0.95 0.55 ± 0.01 1.70 ± 0.14

observed tyrosinase inhibition activity.

Diabetes mellitus is categorized as a complex metabolic disease which is associated with the irregular production of insulin, developing insulin resistance and β-cell malfunction. This disorder ultimately results in impaired metabolism for glucose, lipids and proteins which conse-quently leads towards oxidative damage and inflammation (Farzaei et al., 2017,2015). The intestinal absorption and breakdown of carbo-hydrates are majorly regulated by the enzymes α-amylase and α-gluco-sidase. The most common available marketed medicines which are used clinically for treating hyperglycemia and Type 2 diabetes are metformin, miglitol, and acarbose. But, as these drugs have reported some side ef-fects, thus, there is a major intention for discovery of natural α-amylase and α-glucosidase enzyme inhibitors (Asghari et al., 2018). In the current research work, we probed into the α-amylase and α-glucosidase inhibi-tion capabilites of E. milii extracts and the results are presented in Table 5. For α-glucosidase inhibition, both aerial-MeOH (1.94 ± 0.01 mmol ACAE/g extract) and root- MeOH (1.79 ± 0.02 mmol ACAE/ g extract) extracts exhibited higher inhibitory potential than DCM ex-tracts (Table 5). This higher α-glucosidase activity by methanolic ex-tracts might be due to their higher phenolic and flavonoid contents and this fact is also supported byGulati et al., 2012, who also correlated the α-glucosidase inhibitory activity of E. drummondii, with its highest amount of phenolic compounds (Gulati et al., 2012). In another report, E.

dioeca aqueous and methanol extracts were screened for their oral starch

tolerance test and α-glucosidase inhibition potential and both the ex-tracts presented moderated α-glucosidase inhibition with IC50values of

0.55 and 0.85 mg/mL, respectively (Cristians et al., 2015). However, in case of α-amylase, opposite results were obtained and the DCM extracts showed higher inhibition than methanolic extracts. Generally, all the extracts showed relatively higher α-glucosidase inhibition as compared to α-amylase and these findings are in agreement with an earlier study conducted on E. characias, also reporting the higher α-glucosidase in-hibition (Fais et al., 2018). Similarly, another study reported the me-thanolic extract of E. hirta whole plant to show stronger α-glucosidase inhibition and mild α-amylase inhibitory activity (Sheliya et al., 2016). Our results are also in accordance with Pearson correlation analysis (Fig. 2) which showed a strong positive association between TPC (R = 0.08), TFC (R = 0.85) and α-glucosidase inhibition. Whereas a negative relationship was observed for α-amylase inhibition TPC (R = −0.86) and TFC (R = −0.87).

4. Conclusion

This is the first endeavor to provide an in-depth study of the chemical composition and biological (antioxidant and enzyme inhibition) prop-erties of E. milii aerial and root parts. We noticed that the polar metha-nolic extracts from both parts contained higher amounts in terms of phenolic and flavonoids and also presented better free radical scaven-ging, reducing power and α-glucosidase inhibition potential. Contrarily, the intermediate polar DCM extracts showed higher total antioxidant capacity, AChE, BChE and tyrosinase inhibition as compared to the methanol extracts. Data presented by PCA analysis showed a strong correlation among total bioactive contents and DPPH, ABTS, FRAP, CUPRAC and α-glucosidase assays. Moreover, UHPLC analysis identified the presence of flavonoids phenolics, glycosides, coumarins and alkaloids as important secondary metabolites. Current findings suggest that E. milii aerial and root parts had a great potential to isolate antioxidant and enzyme inhibitors of natural origin and may be regarded as a natural remedy to overcome various public health related problems. However, further research is recommended regarding toxicity studies.

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