High Resolution GC/MS Analysis of the Holoptelea Integrifoli's Leaves And

Biocatalysis and Agricultural Biotechnology 22 (2019) 101405

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Biocatalysis and Agricultural Biotechnology

High resolution GC/ MS analysis of the Holoptelea integrifoli's leaves and their medicinal qualities

Vijay Kumar a , *, 1, Simranjeet Singh b, c, d, 1, Sapna Avinash Kondalkar e, 1, Bhavana Srivastava a , Brijesh Singh Sisodia f, Bandana Barthi a , Ravindara Singh g, Om Prakash h a Department of Chemistry, Regional Ayurveda Research Institute for Drug Development, Aamkho, Gwalior, M. P, 474009, India b Department of Biotechnology, Lovely Professional University, Jalandhar, Punjab, 144111, India c Regional Advanced Water Testing Laboratory, Mohali, Punjab, 160059, India d Punjab Biotechnology Incubators, Mohali, Punjab, 160059, India e Department of Pharmacognosy, Regional Ayurveda Research Institute for Drug Development, Aamkho, Gwalior, M. P, 474009, India f Department of Biochemistry, Regional Ayurveda Research Institute for Drug Development, Aamkho, Gwalior, M. P, 474009, India g Department of Chemistry, Central Council for Research in Ayurvedic Sciences (CCRAS), New Delhi, 110058, India h Department of Ayurveda, Regional Ayurveda Research Institute for Drug Development, Aamkho, Gwalior, M. P, 474009, India

ARTICLE INFO ABSTRACT

Keywords : Plants and their sec ondary mol ecules are con sid ered as unique gift of God to humans. Secondary mol ecules of Holoptelea integrifolia plants are con sid ered as major thera peu tic agents to cure diverse classes of dis ease. The aim of the present Antioxidant activity study was to iden tify the sec ondary mol ecules of the leaf ex tract of Holopte lea inte gri fo lia (HI). Iden tifi ca tion of Anti -diabetic activity bio mol ecules us ing the fourier trans forma tion in frared (FTIR) and gas chromatog raphy cou pled with high res- Anti -inflammatory activity Antimicrobial activity olu tion mass spectrom e ter (GC - HRMS) have con firmed the presence of seven prominent mol ecules in clud ing four new mol ecules. The four new mol ecules iden tified though GC- HRMS were (Z)- octadeca - 9,17 - dienal (13.17%), (9Z,12Z) - octadeca - 9,12 - dienoyl chlo ride (3.22%), 1,3 - dihydroxypropan - 2- yl palmi tate (6.01%), and 17 - acetyl - 14 - hydroxy - 10,13 - dimethyl - decahydro - 2H- cyclopenta [α]phenanthrene - 3,12,15 (4H,14H,16H) - trione. The aque ous ex tract of HI leaves ex hibited the an tiox idant poten tial (>59%), anti - diabetic (78.45 ± 5.55%), anti - inflammatory (value of bind ing con stants (k) was 1.83 ± 0.06 × 10 −4 μM −1) and an - timicro bial (MIC val ues 20 –30 mg/ml) qual ities. Moreover, the observed val ues of var ious physic ochem i cal para me ters in clud ing pH, loss on drying, to tal ash, acid in sol u ble ash, water ex trac tive val ues, and al cohol ex - trac tive stud ies were 7.07 ± 0.37, 8.55 ± 0.75%, 18.12 ± 1.21%, 10.07 ± 0.87%, 37.97 ± 2.31%, and 29.36 ± 1.27% respec tively. Also, pharma cog nos tic analy ses were performed. The results of present study could con tribute to the iso la tion of new compounds those may have medi c inal qual ities.

1. Introduction genus Holoptela and family ( Ulmaceae ) of al mond plants. It is a tree with height 15 –25 m having yellow ish and light gray col ored bark Holoptelea inte gri fo lia (HI) is a well doc u mented plant in Ayurvedic ( Benjamin and Christopher, 2009 ; Kumar et al., 2012a & 2012b; System of Medi cine ( Padmaa and Durga, 2011 ; Reddy et al., 2008 ; Mahmud et al., 2010 ). Offen sive odour and irri ta tive taste are the ma- Sahoo et al., 2010 ; Srinivas et al., 2008 ; Vinod et al., 2010 ). It is con - jor traits of HI. Asia and Africa are the major culti va tors and dis trib- sumed as folk medi cine to treat arthritis, inflam ma tion, anaemia, skin uter of HI. In India, it is abundantly found throughout the whole Hi- dis eases, liver dis orders etc ( Ahmad et al., 2012 ; Ganie and Yadav, malayan belt ( Kumar et al., 2012a & 2012b; Mahmud et al., 2010 ; 2014 , 2015 ; Joshi et al., 2013 ; Kavitha and Narasu, 2014 ). In India, it Padmaja et al., 2009 ; Prajapati and Patel, 2010 ; Sharma et al., 2010a, is well known plant for its pollen al lergenic proper ties ( Acharya, b). 2008 ; Sharma et al., 2005 ; Singh and Kumar, 2003 ). It is from the

* Corresponding author. E- mail address: vijaychem99@ gmail. com, vijay. kr1984@ gov. in (V. Kumar). 1 Equal con tribu tion. https://doi.org/10.1016/j.bcab.2019.101405 Received 16 November 2018; Received in revised form 1 March 2019; Accepted 26 October 2019 Available online 31 October 2019 1878-8181/© 2019 Elsevier Ltd. All rights reserved. V. Kumar et al. Biocatalysis and Agricultural Biotechnology 22 (2019) 101405

HI is renowned for its medi c i nal proper ties like antimi cro bial (SA), napthyldiamine dichloride (NED), bovine serum al bumin (BSA), ( Ahmad et al., 2012 ; Ganie and Yadav, 2014 & 2015; Joshi et al., sodium carbon ate, alu minium chloride salt, phospho molyb de num 2013 ; Kavitha and Narasu, 2014 ; Padmaa and Durga, 2011 ; Reddy et reagent, ferrozine, ferrous ammo nium sul phate salt, α- amylase, and al., 2008 ; Sahoo et al., 2010 ; Srinivas et al., 2008 ; Vinod et al., 2010 ), dinitro -salicylic acid were of AR grade and used without any purifi ca - antivi ral, antiox i dant ( Ganie and Yadav, 2014 & 2015; Kavitha and tion. Narasu, 2015 ; Reddy et al., 2008 ; Saraswathy et al., 2008 ; Srinivas et al., 2008 ; Srivastava et al., 2013 ), anti- obesity ( Bambhole and 2.2 . Pharmacognostical analysis Jiddewar, 1985 ; Ganie and Yadav, 2014 ; Kumar et al, 2012b ), wound- healing ( Reddy et al., 2008 ; Srinivas et al., 2008 ), hypolip i daemic Organolep tic (color, odour, taste etc.) and micro scopic (cuti cle, ( Subash and Augustine, 2013 ), analgesic ( Rizwani et al., 2012 ), anti- epider mis, trichomes, stom ata etc. i.e. histo log ical study) para me ters diabetic ( Sharma et al., 2010a, b & 2012 ), anti- inflammatory ( Kalpana were performed as per reported methods ( Kumar et al., 2012a & and Upadhyay, 2010 ; Sharma et al., 2009 ), antitumour ( Lakshmi et 2012b). al., 2010 ; Soujanya et al., 2011 ), antitubercular ( Joshi et al., 2013 ), larvi ci dal ( Singha et al., 2012 ), anthelmintic ( Durga and Paarakh, 2.3 . Physicochemical analysis 2010 ; Kaur et al., 2010 ), antineoplastic ( Guo et al., 2013 ), and adap- to genic activ i ties ( Puri et al., 2011 ). Physico chemi cal proper ties like pH, loss on drying, to tal ash, acid It is well estab lished fact that, the medi c i nal activ i ties of medi c i nal insol u ble ash, water extrac tive values, and al cohol extrac tive values plants depend on the matrix of sec ondary metabolites present in were tested accord ing to reported methods ( Kumar et al., 2018c ). plants ( Padmaa and Durga, 2011 ; Reddy et al., 2008 ; Sahoo et al., Stud ies were performed in tripli cates. 2010 ; Srinivas et al., 2008 ; Vinod et al., 2010 ). Qual ita tive phyto - chemi cal analy sis revealed that, HI con tained sev eral classes of chemi - 2.4 . Extraction cal con stituents like phenol, terpenoids, gly cosides, flavanoids, sterols and saponins ( Benjamin and Christopher, 2009 ; Padmaja et al., 2009 ; Exper i ment was performed in two phase, in first phase, 4 gm of Prajapati and Patel, 2010 ). The isolated and doc u mented compounds powder was put into 100 ml dis tilled water, shaken mechan ically and of HI are 1, 4 -naphthalenedione, hexacosanol, stig masterol, β- amyrin, the su pernatant was filtered after 24 h by us ing the Whatman filter betulin, betulinic acid, epifriedlin, octa cosanol, friedlin, holoptelin -A paper No. 1. A green color so lu tion was obtained and su pernatant of and holoptelin -B ( Ganie and Yadav, 2014 ; Kumar et al., 2012a & first exper i ment was stored at 4 –10 °C for biochem i cal analy sis. 2012b ; Sutar et al, 2014a, b; Vinod et al., 2010 ). In sec ond phase of exper i ment, initially above steps were repeated To identify the sec ondary metabolites of HI, vari ous researchers and volume of pure aqueous extract of HI was reduced to semi solid have performed HPTLC analy sis ( Reddy et al., 2008 ), where they have mass by evapo rating at 60 –65 °C. Further, semisolid mass was recon - quan tified the phytos terols ( Sutar et al, 2014a, b), flavonoids ( Sutar et sti tuted with ethanol for GC -HRMS quan tifica tion and antimi cro bial al, 2014a, b), al kaloids ( Sutar et al., 2016 ), and 1,4 - naphthalene dione as say. To perform the FTIR study, a disc was prepared by mixing ( Vinod et al., 2010 ). Subash and Augus tine have applied liquid chro- semisolid mass with KBr. matographic mass spectro scopic (LC -MS) technique for the identi fi ca - tion and isola tion of chemi cal compound, 3 -(7 -ethoxy - 4 -methyl - 2 -oxo - 2.5 . Phytochemical analysis 2H- chromen - 3 -yl) propanoate having signif i cant hypolip i daemic effect ( Subash and Augustine, 2013 ). Methanol, ethyl acetate, ethanol and Qual ita tive analy sis of phyto chemi cals includ ing carbo hy drates, hexane extracts of HI have been stud ied by appling the gas chromato - gly cosides, proteins, amino acids, al kaloids, terpenoids, flavanoids, graphic mass spectrom e try (GC -MS) technique, where n- hexadecanoic saponins, tan nins, pheno lics and steroids, present in the aqueous ex- acid, phytols and some other fatty acids were observed and reported tract of HI were performed as per the reported methods ( Kumar et al., ( Kavitha and Narasu, 2014 ). 2018c ). Moreover, aqueous and hydro - alcoholic extracts are accept able for the drug formu la tion(s). The quan tifica tion of volatile compounds 2.6 . Functional groups identification present in aqueous extract is very signif i cant to check the presence of volatile thera peutic metabolites. To the best of our knowledge, no one Functional groups of the sec ondary metabolites present in HI ex- have analysed aqueous extract of HI by apply ing the fourier transfor - tract were identi fied by us ing FTIR spectroscopy. FTIR (Make: Shi- mation infrared (FTIR) and gas chromatog raphy coupled with high madzu- 8400; range 500 –4000 cm− 1) was used to charac ter ize the HI. reso lu tion mass spectrom e try (GC -HRM) techniques. Here, we have KBr was used to prepare the disc of HI. designed and exe cuted a study, where GC -HRMS analy sis of leaf extract of Holoptelea inte gri fo lia (HI) was performed. Nutri ent and medi c - 2.7 . GC- HRMS determination inal proper ties like antiox i dant poten tial, to tal pheno lic con tent (TPC), to tal flavonoids con tents (TFC), antidiabetic, protein binding The GC -MS analy sis was performed at SAIF, IIT – Bombay, India inter ac tions, anti- inflammatory and antimi cro bial qual ities were by us ing GC -HRMS (GC Make: Agi lent 7890 with FID detec tor; MS checked. Make: Jeol) sys tem. The detailed speci fi ca tions of instru ment are avail able at link http://www. rsic. iitb.ac. in/gc - hrms. html . For GC, 2. Materials and methods quartz capil lary col umn of dimen sions 30 mm × 0.25 mm × 0.25 μm was used. The col umn temper a ture and flow rate were 60 °C and 2.1 . Plant materials and chemicals 1.0 ml/min respec tively. The inlet temper a ture and heating rate were 250 °C and 6 °C/min respec tively. Pre- column pressure and split ratio The leaves of HI were col lected from the garden of Regional Re- were 80 kPa and 40:1 respec tively. The helium gas was used as car- search Insti tute for Drug Devel opment, Gwalior (M.P.), India. The rier gas. The con ditions for MS were as; EI as ion ization mode with shade dried leaves were powdered and kept at 5 °C for further use. TOF ana lyzer, electron energy 70 Ev, source temper a ture was 230 – Gal lic acid, quercetin, ascor bic acid (AA), butylhy drox ytoluene (BHT), 250 °C, mass reso lu tion 6000 A.U., mass range 10 –2000 amu. The sys - Folin Ciocal teau reagent, ascor bic acid, 2,2 -diphenyl - 1 -picrylhydrazyl tem was equipped with nat ural product library of stan dard spectrum (DPPH), Ethyl enedi amine tetra acetic acid (EDTA), sul fanilic acid and computer was used to quan tify the compo nents.

2 V. Kumar et al. Biocatalysis and Agricultural Biotechnology 22 (2019) 101405

2.8 . Antioxidant, anti- inflammatory, antidiabetic qualities Table 1 Physic o chem i cal, Phar ma cog nos ti cal pa ra me ters and phy to chem i cals pre sent Total pheno lic con tent (TPC) and to tal flavonoids con tent (TFC) in the leaves (aque ous ex tract) of Holopte lea In te gri fo lia. were analysed by us ing Folin Ciocal teau reagent and AlCl as says as 3 Physicochemical Parameters per recent reports ( Kumar et al., 2018c ). Spectropho to metric methods were used to check antiox i dant qual ity us ing 2,2 -diphenyl - 1 - Parameter Results picrylhydrazyl (DPPH), metal chelat ing (MC), and nitric oxide (NO) pH 07.07 ± 0.37 as says ( Kumar et al. 2018a, b,c ; Kumar et al. 2015 , 2016 , 2017 ). Anti- Loss on Drying (%) 08.55 ± 0.75 inflammatory qual ity (by denat u ration as say), BSA proteins binding Total Ash (%) 18.12 ± 1.21 (in terms of binding con stant (k)), and antidiabetic qual ity (us ing α- Acid Insoluble Ash 10.07 ± 0.87 amylase as say) of HI extract were as sayed ( Kumar et al. 2018a, b,c ; (%) Water Extractive 37.97 ± 2.31 Kumar et al. 2015 , 2016 , 2017 ). UV– visible spectropho tome ter (Shi- Values (%) −1 madzu- 1800; range 190 –1100 cm ) was used to deter mine the bio- Alcohol Extractive 29.36 ± 1.27 chemi cal proper ties. Values (%) Pharmacognostical parameters 2.9 . Antimicrobial assay Characteristic Observation Organoleptic Parameters In present study, aqueous extract of HI was also checked for its an- Colour Ventral - dark green, dorsal - pale green timicro bial effect against two bacte r ial ( Escherichia coli NCIM 2501 Odour Characteristic, herbaceous and Sal monella typhimurium NCIM 2563) and two fungal ( Aspergillus Taste Characteristic, herbaceous, slight pungent to bitter aftertaste fumi gates NCIM 902 and Pencil lium chrysogenum NCIM 738) strains by Size Length - 5.1 – 14.4 cm L, width - 4.2 – 8.5 cm us ing the disc diffu sion method. Zone of inhi bi tion (di ame ter in mm) Shape Ovate, acute, cordate, entire, pinnate, petiolate was measured. To check the mini mum inhibitory con centra tion (MIC) Surface Glabrous ser ial dilu tion method was used as per our recent report ( Kumar et al., Powder Microscopy Analysis 2018a , 2018b & 2018c ; Kumar et al., 2015 ). To perform the antimi - Cuticle Outermost shiny crobial activ i ties, semisolid mass of HI extract was dis solved in Epidermis Flattened, polygonal, straight - double walled cells without ethanol. intercellular spaces Palisade Longitudinal, isobilateral Parenchyma Polygonal, thin walled 3. Results and discussions Stomata Anomocytic Trichomes Covering, unicellular, uniseriate with narrow lumen, pointed 3.1 . Pharmacognostical and physicochemical analyses apex and broad base Xylem vessels Spiral Pharma cog nos tical charac ter is tics includ ing organolep tic and mi- Crystals of Ca Flat and prismatic oxalate croscopic stud ies are given under Table 1 & Fig. 1 . In organolep tic Starch Simple, spherical eval u a tion, leaves were found to have charac ter is tic, herbaceous Others Spherical bases of trichomes, cells with pigment odour and taste, with slight pungency to bitter after taste. Vari ous Phytochemical Analysis (+ + + = excellent, + + = good, + = average, and – morpho log ical charac ter is tics were also deter mined. The leaves were not present). found to be ovate to cordate with acute apex, entire, pinnate, petio - Phytochemicals Results late with glabrous surface. Ventral surface was dark green, while dor- Glycosides + + sal surface was pale green with about 5.1 –14.4 cm length and 4.2 – Terpenoids + 8.5 cm width. Proteins - Powder microscopy is neces sary for initial identi fi ca tion, identi fi - Amino Acids - cation of crude drugs as fragments or powder and for detec tion of Alkaloids - Carbohydrates + adulter ants ( Mahmud et al., 2010 ; Padmaja et al., 2009 ). The powder Flavonoids + microscopy revealed the cel lu lar compo nents and their dis trib u tion. Phenols + + As the leaves are the out growth of stems con sist of three layer sys tem, Saponins + + the out ermost shiny layer was of cuti cle fol lowed by flattened, polyg- Steroid - o nal, straight- double walled epider mal cells without inter cel lu lar Tannins + + spaces, with anomocytic stom ata and cov ering, unicel lu lar, unis eri ate, pointed trichomes with narrow lu men and broad base. There were polar compounds may be present in these polar extracts. Researchers longi tu di nal, isobi lat eral palisade cells beneath the epider mis. Polygo - have identi fied non- volatile polar compounds from the al coholic ex- nal, thin walled parenchyma tous cells con sti tuted the body. Spiral tracts of HI ( Sutar et al, 2014a , 2014b , 2016 ; Vinod et al., 2010 ). xylem ves sels, flat and prismatic crystals of calcium oxalate, simple, Moreover, the identi fi ca tion and quan tifica tion of volatile polar com- spheri cal grains of starch, spheri cal bases of trichomes, pigmented pounds present in polar extract (s) is an excit ing and impor tant as pect, cells, and spiral xylem ves sels were clearly vis ible. The observed re- which is performed in present study with aqueous extract of HI sults of pharma cog nos tic stud ies were very con sistent with recent leaves. stud ies ( Benjamin and Christopher, 2009 ; Kumar et al., 2012a & 2012b ; Mahmud et al., 2010 ; Padmaja et al., 2009 ; Prajapati and 3.2 . Qualitative and quantitative analysis of phytochemicals Patel, 2010 ; Sharma et al., 2010a, b). The results for physico chemi cal stud ies are tabu lated under Table Aqueous extract of HI was analysed to check the presence of phy- 1 . The observed values for pH, loss on drying, to tal ash, acid insol u ble to chemi cals. In qual ita tive analy sis, diverse classes of sec ondary ash, water extrac tive values, and al cohol extrac tive stud ies were metabolites were observed includ ing gly cosides, terpenoids, tan nins, 7.07 ± 0.37, 8.55 ± 0.75%, 18.12 ± 1.21%, 10.07 ± 0.87%, flavonoids, phenols and saponins ( Table 1 ). Same results have been 37.97 ± 2.31%, and 29.36 ± 1.27% respec tively. The percent age of reported by vari ous researches ( Benjamin and Christopher, 2009 ; extrac tive values were more than 30%, which means good quan tity of Ganie and Yadav, 2014 ; Padmaja et al., 2009 ; Prajapati and Patel,

3 V. Kumar et al. Biocatalysis and Agricultural Biotechnology 22 (2019) 101405

Fig. 1. Morpho log ical and Powder micro scop ically char ac teris tics of leaf of HI. (a - Leaves, b- cross sec tional view from powder, c -mag ni fied xylem ves sels, d- leaf surface with stomata, e- crys tals of Ca - oxalate, f- cells with starch grains, g- tri chomes, h -bases of trichomes and i- pig mented cells).

2010 ). To con firm the presence of above mentioned sec ondary 2915 ± 10 cm− 1 (cor respond ing to asymmet ric stretch of –CH), metabolites FTIR and GC -HRMS analy ses were performed. FTIR study 2360 ± 10 cm− 1 (cor respond ing to symmet ric stretch of –CH), and was performed to con firm the functional groups of leaves extract of 1654 ± 10 cm− 1 (cor respond ing to –CH of benzene ring and –NH). HI. Fig. 2 depicted peaks at 3420 ± 10 cm− 1 (cor respond ing to –OH The peaks at 1437, 1410, 1315, 1024, 953, 705, and 671 cm− 1 corre - and –NH groups of phenols and amines), 3000 ± 10 cm− 1 and spond ing to the basic functional groups of extract ( Kumar et al., 2012a ; Kumar et al., 2018a , 2018b & 2018c). The GC -HRMS chromatogram is given under Fig. 3 & 4 and S1, where ten chemi cal con stituents were noticed at differ ent reten tion time (RT). The observed chemi cal con stituents at differ ent RT and oc- currence percent age are given under Table 2 . The spectrum of each peak was retrieved by us ing library search (wiley7n.1). The observed compounds include one each of acid, alde hyde, ketones (cyclic and non- cyclic), terpenoids, ester, and phenols (aliphatic) cat egory. The observed chemi cal con stituents (with occur rence percent age) were n- hexadecanoic acid (22.04%), (Z)- octadeca -9,17 -dienal (13.17%), (9Z,12Z) -octadeca -9,12 -dienoic acid (36.11%), (9Z,12Z) -octadeca - 9,12 -dienoyl chloride (3.22%), 1,3 -dihydroxypropan - 2 -yl palmitate (6.01%), 17 -acetyl -14 -hydroxy - 10,13 -dimethyl - decahydro -2H - cyclopenta [α]phenanthrene - 3,12,15 (4H,14H,16H) -trione (6.70%). Three compounds viz hexade canoic acid, (Z)- octadeca -9,17 -dienal, and (9Z,12Z) -octadeca -9,12 -dienoic acid were found at good percent - age ( Table 2 ). Detailed mass fragmen ta tions and library search proba - bility are given under supple men tary data S2 –S10. Out of to tal identi fied chemi cal compounds, n- hexadecanoic acid (or Palmitic Acid) and (9Z,12Z) -octadeca -9,12 -dienoic acid (or Fig. 2. FTIR Spec trum of leaf ex tracts HI. Linoleic Acid) are well known and remain ing are reported first time.

Fig. 3. GC- HRMS Chromatogram of leaf ex tracts HI.

4 V. Kumar et al. Biocatalysis and Agricultural Biotechnology 22 (2019) 101405

Fig. 4. GC- HRMS Chromatogram of the main compounds of the aque ous ex tract of leafs of HI (In tensity on Y - axis and mass to charge ratio (m/z) on X - axis).

Table 2 Com pounds ob served in GC - MS analy sis of HI.

Structure Component RT Peak Area Ref. (min) (%)

n - Hexadecanoic acid (or Palmitic Acid) 16.16 22.04 CAS:57 - 10 - 3 NIST:335494

(Z) - octadeca - 9,17 - dienal 21.66 13.17 CAS:56554 - 35 - 9 NIST:35789

(9Z,12Z) - octadeca - 9,12 - dienoic acid 18.30 36.11 CAS:60 - 33 - 3 NIST:27829 (or Linoleic Acid)

(9Z,12Z) - octadeca - 9,12 - dienoyl chloride 24.18 3.22 CAS:7459 - 33 - 8 NIST:76312

1,3 - dihydroxypropan - 2 - yl palmitate 19.84 6.01 CAS:23470 - 00 - 0 NIST:15400

17 - acetyl - 14 - hydroxy - 10,13 - dimethyl - decahydro - 2H - cyclopenta [α] phenanthrene - 3,12,15 33.48 6.70 CAS:16396 - 79 - 5 (4H,14H,16H) - trione NIST:16181

Palmitic acid and linoleic acid are known for their physi o log ical and percent age, which means that aqueous extract of HI may be used as bio log ical activ i ties. They have shown anti- obesity ( Gilbert et al., effec tive medi cine in future. 2011 ; Kim et al., 2010 ), anti- cancerous ( Arab et al., 2016 ; Lau and Archer, 2010 ), antidiabetic ( Castro -Webb et al., 2012 ; Olson et al., 3.3 . Antioxidant potential of aqueous extract of HI leaves 2017 ; Yuce et al., 2017 ), anti- inflammatory ( Jaudszus et al., 2005 ), bone strength ening, and ather o scle rosis proper ties ( Kim et al., 2014 ). Antiox i dant activ ity refers to the forma tion of non- reactive sta ble Both con stituents are more effec tive when used in combi na tion with radi cals through the inhi bi tion of the oxi da tion of mole cules by pre- each other and other fatty acids ( De Moraes C et al., 2017 ; Kim et al., vent ing the initi a tion step of the oxida tive chain reac tion ( Kumar et 2010 ; Martins et al., 2017 ). GC -HRMS results of present stud ies has al., 2012a & 2012b ; Kumar et al., 2015 ; Reddy et al., 2008 ; revealed the presence of both palmitic acid and linoleic acid at good Saraswathy et al., 2008 ). The antiox i dant poten tial depends on the

5 V. Kumar et al. Biocatalysis and Agricultural Biotechnology 22 (2019) 101405 proper ties of vari ous phyto chemi cals present in the sample ( Ganie and times more effi ciency to reduce the heat induced effect on BSA pro- Yadav, 2014 & 2015; Kavitha and Narasu, 2015 ; Srinivas et al., 2008 ; tein. In previ ous stud ies, aqueous and ethanolic extracts of HI have Srivastava et al., 2013 ). Here, to tal pheno lic con tent (TPC) and to tal shown good anti- inflammatory activ i ties on rats (through car- flavonoids con tent (TFC) values of the HI were checked and reported rageenan- induced paw edema test) as compared to stan dard in- as gal lic acid equiva lent (GAE/g) and quercetin equiva lent (QE/g). To domethacin ( Kalpana and Upadhyay, 2010 ; Sharma et al., 2009 ). In calcu late the TPC and TFC, stan dard curves of gal lic acid and present study, the excel lent effi cacy of HI to wards the inhi bi tion of in- quercetin were plotted by taking con centra tion on X- axis and ab- flamma tion may be attrib uted to the inter ac tions of sec ondary sorbance at Y -axis. The observed equation of line for TPC and TFC metabolites of HI with complex protein at higher temper a ture 51 ○C was Y = 0.009X + 0.005 with R2 = 0.999 and Y = 0.011X + 0.028 ( Kalpana and Upadhyay, 2010 ; Sharma et al., 2009 ). with R2 = 0.998 respec tively. The observed values of TPC and TFC Protein binding ability of HI extract was tested by us ing the UV– for the HI were 116.37 ± 8.22 mg GAE/g and 8.19 ± 1.17 mg QE/g visible spectropho to metric method at 278 nm, where the complex a - respec tively. The observed TPC and TFC values were signif i cant which tion behav iour of the HI was stud ied as per the proto col described by attrib uted to the presence of sec ondary metabolites of HI as con firmed Kumar et al., (2018c) . The binding ability of HI with BSA was re- by phyto chemi cal, FTIR, and GC -HRMS analy ses. ported in terms of binding con stant (k). The observed values of the Total antiox i dant capac ity of stan dard (ascor bic acid) as well as HI binding con stants (k) for the stan dard (acetyl sal icylic acid) and HI extract was calcu lated by us ing Phospho molyb de num As say. The ob- were 2.60 ± 0.05 × 10 −4 μM− 1 and 1.83 ± 0.06 × 10 −4 μM− 1 reserved antiox i dant activ ity for BHT (stan dards), and HI were spectively. Both, stan dard as well HI showed al most simi lar values 98.31 ± 5.17 and 85.23 ± 4.01% respec tively. Clearly, HI exhib ited which highlights the util ity of HI as a potent drug in future ( Kalpana very good to tal antiox i dant capac ity. In DPPH as say, con centra tion and Upadhyay, 2010 ; Kumar et al., 2015 ; Kumar et al., 2018a , 2018b depended effect was noticed for the HI. At 100 μL, the observed % age & 2018c; Sharma et al., 2009 ). inhi bi tion value of the aqueous extract of HI and con trol (ascor bic acid) was 61.07 ± 2.11 and 94.12 ± 4.07%. The results of present 3.5 . Anti- diabetic qualities of aqueous extract of HI leaves study exhib ited the close resem blance with recent study where ethanolic extract of stem bark of HI has shown al most very close an- To eradi cate the dia betes, world is still looking for effec tive medi - tioxi dant value (65%) in DPPH as say as compared that of con trol - cine or therapy ( Castro -Webb et al., 2012 ; Olson et al., 2017 ; Yuce et tocopherol ( Ganie and Yadav, 2014 ). In recent stud ies, for DPPH an- al., 2017 ). It is lifestyle based dis order and its level vary from person tioxi dant as say, the % inhi bi tion of vari ous extracts of leaves of HI to person. Plants having antidi a betic qual ities can con tribute as signif - were 75.78% (methano lic), 89.21% (ace tone), and 87.49% (ethyl ac- icant source for the devel opment of secure and eco nomi cal antidi a - etate) ( Ganie and Yadav, 2014 & 2015; Kavitha and Narasu, 2015 ; betic drugs ( Sharma et al., 2010a, b & 2012 ). Here, antidi a betic qual - Reddy et al., 2008 ; Saraswathy et al., 2008 ; Srinivas et al., 2008 ; ity of the HI extract was tested by us ing the α- amylase as say. Colori - Srivastava et al., 2013 ). metric as say (at 540 nm) was used, where acar bose was used as a Detailed liter a ture revealed that no antiox i dant study has been stan dard. The antidi a betic property of HI was found increase in a dose done by means of MC and NO as says. In MC as say, as like DPPH as - depen dent manner. At lowest con centra tion (25 μL), HI and stan dard say, con centra tion depended effect was noticed. At higher con centra - has shown 51.21 ± 3.57 and 72.22 ± 4.31% of inhi bi tion. At the tion (at 100 μL), the observed inhi bi tion (%) values for the HI and highest con centra tion (100 μL), the observed % of inhi bi tion of HI EDTA (con trol) were 59.75 ± 3.33 and 94.24 ± 5.17% respec tively. and acar bose was 78.45 ± 5.55% and 95.01 ± 5.41% respec tively. The MC as say is depends upon the presence of chelat ing agent like Clearly, HI exhibits very good antidi a betic activ i ties and could be 1,3 -dihydroxypropan - 2 -yl palmitate as noticed by GC -HRMS. In case used as antidi a betic agent. In liter a ture, vari ous extracts of HI have of NO antiox i dant as say, HI have shown simi lar trend as like that of shown antidi a betic appli ca tions through the inhi bi tion of the ATP- DPPH and MC as says. At 100 μL, the observed inhi bi tion (%) values sensitive potas sium chan nels in pancre atic beta cells mecha nism for the HI, AA and BHT were 88.22 ± 5.14, 94.44 ± 4.88 and ( Sharma et al., 2010a, b & 2012 ). 91.23 ± 4.53% respec tively. Here, good NO activ ity was noticed as compared to above mentioned two as says viz DPPH and MC. These 3.6 . Antimicrobial qualities of aqueous extract of HI leaves varia tions may be attrib uted to the presence of sec ondary metabolites having differ ent mecha nisms for differ ent as says. Differ ent bio- Plant extracts are known for their bio log ical proper ties, espe cially ingredients have differ ent antiox i dant proper ties with in differ ent an- antimi cro bial proper ties. In present study, HI extract has shown an- tioxi dant as says, which help in reduc ing oxida tive stress in cells timicro bial effect against two bacte r ial and two fungal strains by us - ( Reddy et al., 2008 ; Saraswathy et al., 2008 ). Present study con firms ing the disc diffu sion method ( Table 3 ). Zone of inhi bi tion (di ame ter the useful ness of HI as an anti- antioxidant agent. Aqueous extract of in mm) was con centra tion depen dent ( Table 3 ). The observed MIC HI could serve as a free radi cal scav enger, possi bly act ing as a pri- values were between 20 and 30 mg/ml. HI extract exhib ited good an- mary antiox i dant having health bene fi cial proper ties includ ing vari - timicro bial proper ties attrib uted to the presence of sec ondary metabo- ous phyto chemi cals. It may be a good al ternate against syn thetic an- tioxi dants like butylated hydroxyl toluene, butylated hydroxyl anisole, Table 3 and propyl gal late having adverse health effects ( Ganie and Yadav, An timi cro bial as says of HI. 2014 & 2015; Kavitha and Narasu, 2015 ; Reddy et al., 2008 ; Saraswathy et al., 2008 ; Srinivas et al., 2008 ; Srivastava et al., 2013 ). Strain Zone of Inhibition at different concentration of MIC HI extract (mg/ml)

3.4 . Anti- inflammatoryand protein binding qualities of aqueous extract of 10 mg/ml 20 mg/ml 40 mg/ml 80 mg/ml HI leaves Aspergillus ND 3 mm 8 mm 11 mm 24 fumigates Aqueous extract of HI was tested for its anti- inflammatory proper - Penicillium ND 5 mm 7 mm 14 mm 22 ties by employ ing the heat induced action on BSA protein at opti mum chrysogenum con ditions. The UV– visible spectro scopic study was performed at Escherichia coli ND 4 mm 6 mm 9 mm 23 660 nm. HI has shown con centra tion depen dent effects. At 100 μL, as Salmonella ND 2 mm 4 mm 7 mm 27 typhimurium compared to stan dard (acetyl sal icylic acid), HI extract has shown 1.5 ND = Not de tected.

6 V. Kumar et al. Biocatalysis and Agricultural Biotechnology 22 (2019) 101405 lites of HI. In recent reports, vari ous extracts of HI have been tested rats . Arch. Endocrinol. Metab. 61 , 45 – 53 . Durga , N. , Paarakh , P. M. , 2010 . Evaluation of anthelmintic activity of stem bark of against diverse classes of patho genic microor gan isms where the MIC Holoptelea integrifolia (Roxb) Planch . Int. J. Res. Ayurveda Pharm. 1 (2 ), 637 – 641 . value was between the 5 –10 mg/ml ( Kavitha and Narasu, 2014 ; Ganie , S. A. , Yadav , S. S. , 2015 . FT- IR spectroscopic analysis of Holoptelea integrifolia Padmaa and Durga, 2011 ; Reddy et al., 2008 ; Sahoo et al., 2010 ; (roxb.) planch seed extracts and their antibacterial activity . Res. J. Med. Plant 9 (8 ), Srinivas et al., 2008 ; Vinod et al., 2010 ). In present study, except E. 417 – 426 . Ganie , S. A. , Yadav , S. S. , 2014 . Holoptelea integrifolia (roxb.) planch: a review of its Coli strain, all the strains were new one. The observed MIC value for ethnobotany, pharmacology, and phytochemistry . BioMed Res. Int. Article ID E. Coli was as per the recent reports. Moreover, mini mum or aver age 401213 . https:// doi. org/ 10. 1155/ 2014/ 401213 . activ ity of any drug against one or two strains does not mean that it is Gilbert , W., Gadang , V., Proctor , A. , Jain , V., Devareddy , L., 2011 . Trans -trans conjugated linoleic acid enriched soybean oil reduces fatty liver and lowers serum non- effective against other patho genic microor gan isms ( Kavitha and cholesterol in obese Zucker rats . Lipids 46 , 961 – 968 . Narasu, 2014 ; Padmaa and Durga, 2011 ; Reddy et al., 2008 ; Sahoo et Guo , H. , Wang , D. S. , Rizwani , G. H. , 2013 . Antineoplastic activity of Holoptelea al., 2010 ; Srinivas et al., 2008 ; Vinod et al., 2010 ). Medi c i nal proper - integrifolia (Roxb.) Planch bark extracts (in vitro) . Pak. J. Pharm. Sci. 26 , 1151 – 1156 . ties and thera peutic effects are drugs specific and the con centra tion Jaudszus , A. , Foerster , M. , Kroegel , C. , Wolf , I., Jahreis , G. , 2005 . Cis -9, trans -11 -CLA level of sec ondary metabolites could intend the effi ciency and potency exerts anti -inflammatory effects in human bronchial epithelial cells and eosinophils: of herbal drug ( Ahmad et al., 2012 ; Ganie and Yadav, 2014 & 2015; comparison to trans -10, cis -12 -CLA and to linoleic acid . BBA Mol. Cell Biol. 1737 , 111 – 118 . Joshi et al., 2013 ). Joshi , S. D. , Hallikeri , C. S. , Kulkarni , V.H. , 2013 . Evaluation of antibacterial and antitubercular activities of Holoptelea integrifolia (roxb) planch bark . Univ. J. 4. Conclusion Pharm. 2 , 87 – 90 . Kalpana , K. , Upadhyay , A. , 2010 . Anti -inflammatory evaluation of ethanolic extract of leaves of Holoptelea integrifolia Planch . Sch. Res. Libr. 1 , 185 – 195 . Plant based medi cines are con sid ered as cost effec tive and good al - Kaur , S. , Kumar , B. , Puri , S. , Tiwari , P. , Divakar , K. , 2010 . Comparative Study of ternate of syn thetic drugs and supple ments (like BHT) having su per- anthelmintic activity of aqueous and ethanolic extract of bark of Holoptelea fluous side effects. Vari ous plant mate r ial and herbal drugs have been integrifolia . Int. J. Drug Dev. Res. 2 (4 ) , 758 – 763 . Kavitha , C. A. , Narasu , M. L., 2015 . HPLC analysis and antioxidant activities of explored as poten tial thera peutic agents. In the present study, sec - Holoptelea Integrifolia . Int. J. Chem. Sci. 13 (2 ), 1014 – 1026 . ondary metabolites of aqueous extract of leaves of HI were exam ined Kavitha , C. A. , Narasu , M. L., 2014 . Comparative evaluation of antimicrobial activities of by GC -HRMS and FTIR techniques. GC -HRMS analy sis has sug gested root, stem and leaves of Holoptelea integrifolia against pathogenic bacteria . Asian J. Microbiol. Biotechnol. Environ. Sci. 16 , 145 – 154 . presence of new mole cules. Vari ous medi c i nal qual ities were checked. Kim, L., Park , Y. , Park , Y. , 2014 . Trans -10, cis -12 CLA promotes osteoblastogenesis via The bio log ical stud ies of HI extract sug gests that it can serve as mediated mechanism in bone marrow mesenchymal stem cells . J. Funct. Foods 8 , promising anti rad i cal, anti- inflammatory, antimi cro bial, and antidi a - 367 – 376 . Kim, J.H. , Pan , J.H. , Park , H. G. , Yoon , H. G. , Kwon , O. J., Kim, T. W., 2010 . Functional betic agent. However, further research may be required to screen the comparison of esterified and free forms of conjugated linoleic acid in high -fat -diet - antimi cro bial activ ity of HI against other microor gan isms. Moreover, induced obese C57BL/ 6J Mice . J. Agric. Food Chem. 58 , 11441 – 11448 . to develop the novel drug from the extract of HI, there is need to de- Kumar , V., Chawla , M. , Cavallo , L., Wani , A. B. , Manhas , A. , Kaur , S. , Poater , A. , Chadar , termine the thera peutic index of HI and further pharma co ki net ics H. , Upadhyay , N. , 2018 . Complexation of trichlorosalicylic acid with alkaline and first row transition metals as a switch for their antibacterial activity . Inorg. Chim. stud ies are desir able. Acta 469 , 379 – 386 . Kumar , V., Singh , S. , Singh , R. , Upadhyay , N. , Singh , J., Pant , P. , Singh , R. , Shrivastava , Declaration of competing interest B. , Singh , A. , Subhose , V., 2018 . Spectral, structural and energetic study of acephate, glyphosate, monocrotophos and phorate: an experimental and computational approach . J. Taibah Univ. Sci. 12 , 69 – 78 . There is no state ment on con flictof inter est. Kumar , V., Singh , S. , Singh , A. , Dixit , A. K. , Srivastava , B. , Sidhu , G. K. , Singh , R. , Meena , A. K. , Singh , R. P. , Subhose , V., Prakash , O. , 2018 . Phytochemical, antioxidant, antimicrobial, and protein binding qualities of hydro -ethanolic extract of tinospora cordifolia . J. Biol. Act. Prod. Nat. 8 , 192 – 200 . Acknowledgement Kumar , V., Singh , S. , Singh , R. , Upadhyay , N. , Singh , J., 2017 . Design, synthesis, and characterization of 2, 2 -bis (2, 4 -dinitrophenyl) -2 -(phosphonatomethylamino) acetate as a herbicidal and biological active agent . J. Chem. Biol. 11 , 1 – 12 . Authors are thankful to Direc tor General, CCRAS and Insti tute In- Kumar , V., Kumar , V., Kaur , S. , Singh , S. , Upadhyay , N. , 2016 . Unexpected formation of charge of RARIDD, Gwalior for their moti va tion and guid ance. At the N- phenyl -thiophosphorohydrazidic acid O, S -dimethyl ester from acephate: chemical same time RARIDD, Gwalior and SAIF- IIT, Bombay are highly ac- biotechnical and computational study 3 . Biotech 6 , 1 – 11 . knowledged for the instru menta tions facil ities. Kumar , V., Upadhyay , N. , Manhas , A. , 2015 . Designing syntheses characterization computational study and biological activities of silver -phenothiazine metal complex . J. Mol. Struct. 1099 , 135 – 140 . Kumar , D. , Kumar , K. , Gupta , J., Bishnoi , N. , Kumar , S. , 2012 . A mini review on Appendix A. Supplementary data chemistry and biology of Holoptelea integrifolia Roxb. Planch (Ulmaceae) . Asian Pac. J. Trop. Biomed. S1200 –S1205 . Kumar , D. , Kumar , K. , Kumar , S. , Kumar , T. , Kumar , A. , Prakash , O. , 2012 . Supple men tary data to this arti cle can be found online at https:// Pharmacognostic evaluation of leaf and root bark of Holoptelea integrifolia Roxb . doi. org/10. 1016/ j. bcab.2019. 101405 . Asian Pac. J. Trop. Biomed. 169 – 175 . Lakshmi , K. S. , Sharma , S. S. , Rajesh , T. , Chitra , V., 2010 . Antitumour activity of ethanolic extract of leaves of Holoptelea integrifolia on Dalton’s ascitic lymphoma in Swiss albino mice . Int. J.Green Pharm. 4 , 44 – 47 . References Lau , D. S. , Archer , M. 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