Research ArticleCardioprotective and Metabolomic Profiling of Selected MedicinalPlants against Oxidative Stress

Nadia Afsheen,1 Khalil-ur-Rehman ,1 Nazish Jahan,2 Misbah Ijaz,3 Asad Manzoor,3

Khalid Mahmood Khan,1 and Saman Hina1

1Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan2Department of Chemistry, University of Agriculture, Faisalabad, Pakistan3Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan

Correspondence should be addressed to Khalil-ur-Rehman; khaliluaf@yahoo.com

Received 28 April 2017; Revised 13 July 2017; Accepted 23 August 2017; Published 14 January 2018

Academic Editor: Patricia Morales

Copyright © 2018 Nadia Afsheen et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In this research work, the antioxidant and metabolomic profiling of seven selected medicinally important herbs including Rauvolfiaserpentina, Terminalia arjuna, Coriandrum sativum, Elettaria cardamom, Piper nigrum,Allium sativum, and Crataegus oxyacanthawas performed. The in vivo cardioprotective potential of these medicinal plants was evaluated against surgically induced oxidativestress through left anterior descending coronary artery ligation (LADCA) in dogs. The antioxidant profiling of these plants wasdone through DPPH and DNA protection assay. The C. oxyacantha and T. arjuna showed maximum antioxidant potential,while the E. cardamom showed poor antioxidative strength even at its high concentration. Different concentrations of extracts ofthe said plants exhibited the protection of plasmid DNA against H2O2 damage as compared to the plasmid DNA merely treatedwith H2O2. The metabolomic profiling through LC-MS analysis of these antioxidants revealed the presence of active secondarymetabolites responsible for their antioxidant potential. During in vivo analysis, blood samples of all treatment groups weredrawn at different time intervals to analyze the cardiac and hemodynamic parameters. The results depicted that the grouppretreated with HC4 significantly sustained the level of CK-MB, SGOT, and LDH as well as hemodynamic parameters near tonormal. The histopathological examination also confirmed the cardioprotective potential of HC4. Thus, the HC4 being safe andinexpensive cardioprotective herbal combination could be considered as an alternate of synthetic drugs.

1. Introduction

Oxidation is a natural phenomenon that leads to the forma-tion of free radicals known as reactive oxygen species(ROS) [1]. Some of the ROS are very important in cell metab-olism including intercellular signaling, phagocytosis, andenergy production [2]. However, overproduction of ROSduring biological processes resulted in extensive pathologicalalterations like DNA damage and various degenerative disor-ders. Humans are constantly exposed to natural DNA-damaging agents such as UV light, dietary agents, and endog-enously formed free radicals. Damaged DNA accumulates inthe brain, muscle, liver, kidney, and in long-lived stem cell,which causes aging, decline in gene expression, and loss offunctional capacity [3].

Antioxidants are compounds that slow down or delay theoxidation process by obstructing the initiation of a series ofoxidizing reactions [4]. Owing to the presence of antioxi-dants, medicinal plants have a shielding effect against variousdiseases, thus emerging as substantial therapeutic agents.Medicinal plants are a time-honored medicine used sincethe ancient era for treatment of various ailments in humanbeings [5]. Herbal medicines, in addition to their traditionalvalues, also hold great public and medical interest worldwideas sources of novel lead compounds for drug development.Hence, the medicinal plants will be natural protective strat-egy and would be freely available with low cost as comparedto synthetic drugs [6].

Pakistan is bestowed with a wide range of plant specieswith unique biodiversity in different climatic zones [7]. These

HindawiOxidative Medicine and Cellular LongevityVolume 2018, Article ID 9819360, 17 pageshttps://doi.org/10.1155/2018/9819360

medicinal plants have been used in scientific research forvarious cardiovascular disorder in human beings [8, 9].Currently available synthetic cardioprotective drugs exhibita number of side effects and are out of reach for poor com-munities. Cardioprotective effects of some medicinal plants,which are safe and inexpensive, have already been explored[10–13]. Therefore, the green products having cardioprotec-tive and antioxidative potential have attracted manyresearchers towards metabolomic profiling and phytother-apy. The antioxidative strength of medicinal plants is becauseof the secondary metabolites present in it [11].

An LC-MS-based metabolomic study has become a pow-erful analytical tool for assessment of various secondarymetabolites in herbal medicine [14]. These secondary metab-olites have been found to possess a broad range of therapeuticproperties, including antioxidant, cardioprotective, and anti-hypertensive potential [15]. A thorough integration of infor-mation from metabolomics is expected to provide solidevidence-based scientific rationales for the development ofmodern phytomedicines [16]. Therefore, in this research,the antioxidant potential, metabolomic profiling, andin vivo cardioprotective evaluation of Rauvolfia serpentina,Terminalia arjuna, Coriandrum sativum, Elettaria carda-mom, Piper nigrum, Allium sativum, and Crataegus oxya-cantha was done to get the potent role of these naturalantioxidants in health. All these medicinal plants wereselected as these plants have already been reported to possesscardiotonic, antioxidant, and antilipidemic potential [4, 17].Moreover, the previous literature and the knowledge ofCAM practitioners also endorsed the cardioprotective effectof these selected parts of the plants.

2. Materials and Methods

2.1. Preparation of Herbal Extract. Different parts of themedicinal plant like the roots of R. serpentina, bark of T.arjuna, seeds of C. sativum and E. cardamom, leaves of P.nigrum, and fruit of A. sativum, and C. oxyacantha were col-lected from the Botanical Garden of the University of Agri-culture, Faisalabad, Pakistan and from the local herbalmarket. All the selected parts of the plants were identifiedby the plant taxonomist in the Department of Botany, Uni-versity of Agriculture, Faisalabad, Pakistan. These parts ofthe plants were washed and pulverized to get fine powder.The powdered plants (5 g of each) were macerated in metha-nol (50mL). The macerate was kept in an orbital shaker forfour days. The supernatant was decanted and the residuewas remacerated with methanol. The pooled supernatantswere combined and filtered through Whatman’s filter papernumber 1. The rotary evaporator was used to concentratethe filtrate, and subsequently the filtrate was lyophilized [17].

2.2. Antioxidant Assay

2.2.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Free RadicalScavenging Assay. The antioxidant potential was determinedby using 1,1-diphenyl-2-picrylhydrazyl as a free radical. Themethanolic solution of DPPH (0.1mM) and plant extract ofdifferent concentrations (20, 40, 60, 80, and 100μg/mL) were

mixed in equal volume. The mixtures was left for 30 minutesin the dark, and the absorbance was noted at 517nm.Ascorbic acid was used as a standard. The percentageDPPH inhibition of plant extract was calculated as follows:

DPPH inhibition % = 1 − A1A0

× 100, 1

whereA1 is the absorbance of the sample, and A0 is the absor-bance of control [4, 18].

2.2.2. DNA Protection Assay. The DNA protection assay ofextracts of different concentrations (100, 500, and 1000μg/mL) of selected plants was performed by using the pBR322plasmid DNA. pBR 322 DNA plasmid (0.5μL) was dilutedby using 50mM sodium phosphate buffer (pH7.4). Afterdilution, pBR 322 DNA (3μL) was treated with 5μL ofselected concentrations of plant extracts. 4μL of 30% H2O2was added to it, and sodium phosphate buffer (pH7.4) wasused to make the volume up to 15μL. The difference inmigration between the oxidized and native DNA wasobserved on 1% agarose by horizontal DNA gel electrophore-sis using a wide mini system (Techview, Singapore). 1% aga-rose was prepared by dissolving 1 g agarose in 100mL of1X×TAE buffer and placed in a microwave oven for twominutes. It was cooled and poured in a casting plate. Aftersolidification, the gel was kept in the sodium phosphatebuffer and samples were loaded in the wells one by one.The gel was stained with ethidium bromide and documentedby Gene NuGenius unit Syngene model (Cambridge, UK).DNA protection was observed from DNA band corre-sponding to that of native in the presence and absence ofvarious concentrations (100, 500, and 1000μL) of eachplant’s extract [19].

2.3. Metabolomic Profiling. Metabolomic profiling of all theselected medicinal plants was performed through liquidchromatography-mass spectrometry (LC-MS) analysis.

2.4. Liquid Chromatography-Mass Spectrometry (LC-MS).The selected medicinal plants were analyzed by using liquidchromatography combined with electrospray ionizationmass spectrometry (LC-ESI-MS). The plant extracts werefiltered through a 0.45μm syringe filter before analysis.Separation was performed on Surveyor plus HPLC Systemequipped with Surveyor auto (Thermo Scientific, San Jose,CA, USA). The pump was equipped with a Luna ReversePhase C-18 analytical column (4.6× 150mm, 3.0μm particlesize) (Phenomenex, USA). Solvent elution consisted of LC-MS grade methanol and acidified water (0.5% formic acidv/v) as the mobile phase A and B, respectively. Solvent elu-tion consisted of gradient system which runs at a flow rateof 0.3mL/min. The gradient elution was programmed asfollows: from 5min in 15% A to 20min in 25% B andmaintained it till the end of the analysis. A 20 minute re-equilibration time was used after each analysis. The columnwas maintained at 25°C and the injection volume was5.0μL. The effluent from the HPLC column was directed toan electrospray ionization mass spectrometer (LTQ XL™ lin-ear ion trap, Thermo Scientific, River Oaks Parkway, USA).

2 Oxidative Medicine and Cellular Longevity

Parameters for analysis were set using negative ion modewith spectra acquired over a mass range from 260 to2000m/z. The optimum values of the ESI-MS parameterswere spray voltage +4.0 kV, sheath gas and auxiliary gas were45, and 5units/min, respectively, capillary temperature320°C, capillary voltage −20V, and tube lens −66.51V.sThe accurate mass spectra data of the molecular ions wasprocessed through the Xcalibur software (Thermo Fisher Sci-entific Inc, Waltham, MA, USA) [20].

2.5. Selection of Animals. The eighteen male stray dogs of 1-2years were selected as experimental animals and acclimatizedfor one week under laboratory conditions (27°C in 12 hrdark/light cycle). All the animals were housed in the AnimalHouse, Department of Clinical Medicine and Surgery, Uni-versity of Agriculture, Faisalabad. They were fed with stan-dard feed. The experiment was performed by taking theapproval from the institutional ethics review board in thepresence of a licensed veterinarian.

2.6. Herbal Combination Therapy. The four different herbalcombinations of selected plant extracts were formed as givenin Table 1. These herbal combinations were evaluated fortheir synergistic cardioprotective potential. The dogs weredivided into three groups. The first group of dogs was thecontrol group, to which normal diet was fed for 23 days.The second group was the positive control group, in whichthe dogs were treated with normal diet for 22 days, and afterthat, the ligation of the left anterior descending coronaryartery (LADCA) was performed on the 23rd day. The thirdgroup was the treatment group which was further dividedinto four subgroups. Each subgroup was pretreated with itsrespective herbal combination (Table 1) for 22 days. Onday 23, all the dogs of the treatment group underwentLADCA ligation. After completion of the surgical procedure,the blood samples were taken at various time intervals (0 to48 hr) to analyze the cardiac markers (CK-MB, SGOT, andLDH). At the end of the experiment, the dogs were anesthe-tized by Sodium Pentothal and the hearts were excised forhistopathological studies.

2.7. Surgical Induction of Myocardial Infarction. The dogswere anesthetized with Sodium Pentothal (20mg/kg). Atro-pine was administered subcutaneously at a dose of 0.1mg/kg once before the surgery to keep the heart rate elevated dur-ing the surgical procedure and to reduce the bronchotrachealsecretions. The body temperature was monitored and main-tained at 37°C. The animals were ventilated with room airfrom a positive pressure by using compressed air at the rate

of 90 stroke/min and tidal volume of 10mL/kg. The left jug-ular vein was cannulated with polyethylene tube for adminis-tration of supplemental anesthetic and saline (0.9%) infusion.The neck was opened and left thoracotomy was performed toopen the thoracic cavity. Anatomy of the left anteriordescending coronary artery (LADCA) was examined visuallyand then ligated 4-5mm from its origin and the end of thisligature was passed through polyethylene tube to form asnare. The thoracic cavity was covered with saline-soakedgauze to prevent the heart from drying. After completion ofthe surgical procedure, the heart was returned to its normalposition in the thoracic cavity [21, 22].

2.8. Estimation of Hemodynamic Variables. The mean arte-rial pressure (MAP) and heart rate of dogs in all the groupswere calculated. The left thoracic cavity was opened by anincision at the fifth intercostal space and the heart wasexposed. A sterile metal cannula was introduced in the cavityof the left ventricle from the posterior apical region of theheart for measuring left ventricular dynamics at preset timethroughout the surgical procedure [23].

2.9. Biochemical Analysis. The blood sampling was per-formed at different time intervals (0, 12, 24, and 48 hr) duringthe experimental period. The cardiac biomarkers includingcreatine kinase-MB (CK-MB), serum glutamic-oxaloacetictransaminase (SGOT), and lactate dehydrogenase (LDH)were analyzed by using “BioMed kits” having patch numbersMBS705376, BGO094144, and LDHK0103016, respectively.All the kits were purchased by “UH Analytics Pakistan.”

2.10. Statistical Analysis. The data was statistically analyzedby using two-way ANOVA and Turkey’s multiple compari-son tests with the help of GraphPad Prism version 7.00, sup-plied by developer GraphPad software, Inc. [24]. The resultshave been presented as Mean± SD.

3. Results

3.1. Antioxidant Assay

3.1.1. DPPH Free Radical Scavenging Activity. The DPPH freeradical scavenging activity (in terms of % age inhibition) of R.serpentina, T. arjuna, C. sativum, P. nigrum, E. cardamom, A.sativum, and C. oxyacantha at various concentrations (20, 40,60, 80, and 100μg/mL) was examined (Figure 1). The T.arjuna and A. sativum showed higher antioxidant potentialeven at least concentration of 20μg/mL as compared to thesame concentrations of other selected plants. On the otherhand, the E. cardamom presented relatively low antioxidant

Table 1: Different herbal combinations given to treatment group.

Groups R. serpentina E. cardamom P. nigrum A. sativum T. arjuna C. oxyacantha C. sativum

Herbal ratio

HC1 1 0.5 1 0.5 — — 0.5

HC2 1 0.5 1 0.25 — 1 0.5

HC3 1 1 0.5 — 1 — 0.5

HC4 0.5 — — 0.5 1 0.5 1

3Oxidative Medicine and Cellular Longevity

potential even at its higher concentration of 100μg/mL.In case of C. oxyacantha, the concentration of 20 and40μg/mL showed low antioxidative strength but it rapidlyincreased with further increase in concentration from 60 to100μg/mL. All the said medicinal plants depicted the dose-dependent response for free radical scavenging potential, thatis, the activity of plant extracts in terms of % age inhibitionincreased with respect to concentrations (Figure 1). Theselected medicinal plants could be beneficial to mankind byvirtue of their effective antioxidant activity which may ableto impart therapeutic role against various diseases.

3.2. DNA Protection Assay. The effect of varying concentra-tions of medicinal plants on DNA damage along with posi-tive controls (30% H2O2, 2mM FeSO4) has been presentedin Figures 2 and 3. The free radicals produced in responseto O2 and FeSO4 caused the strand cleavage of pBR322 plas-mid DNA and resulted in DNA band streaking (Figure 2). Allthe plants exhibited protection of plasmid DNA againstH2O2 damage as compared to the plasmid DNA merelytreated with H2O2. The DNA protective potential of all con-centrations of said medicinal plants was in concentration-dependent manners which revealed that higher concentra-tions of extracts are more protective against H2O2-induceddamage. The least concentration (100μg/mL) of P. nigrumshowed the band streaking (lane 7 of Figure 2) while theconcentration of 500 and 1000μg/mL of P. nigrum exhibiteda good protection of pBR322 plasmid DNA as presented inthe corresponding lanes 8 and 9. However, in case of C. oxy-acantha, the concentrations of 100μg/mL and 500μg/mL(lanes 4 and 5 of Figure 2) showed noticeable DNA protec-tion. Minor strand breaks were observed with low concen-tration (100μg/mL) of A. sativum (Figure 2, lane 13), whileits higher concentration (500 and 1000μg/mL) showedpromising protection against DNA damage (Figure 2, lanes14 and 15).

3.3. Metabolomic Profiling. Metabolomics approaches usingLC-MS-based techniques are a useful technique in evaluatingthe secondary metabolites of medicinal plants. LC-MS-basedmetabolomics is a powerful new tool for mechanistic studiesof drug metabolism.

3.4. Terminalia Arjuna. LC-MS analysis of T. arjuna wasperformed to evaluate the phytoconstituents including phe-nolics, flavonoids, and alkaloids. The full mass spectrumobtained by LC-MS analysis was presented in Figure 4. Themass spectrum depicted the high peaks at 413.42, 511.50,321.33, 589.33, and 685.58. The CIDMS-MS-ESI fragmentsion of 685.58 peak resulted in three abundant peaks at667.50, 523.33, and 457.25. The peak of 667.50 indicatedthe presence of termiarjunoside 1,3,9,22-tetraol-12-en-28-oicacid-3-D-glucopyranoside. The presence of termiarjuno-side I from the bark of T. arjuna was also reported in a studyby Ali et al. 2006. The fast atom bombardment mass spec-troscopy (FABMS) of T. arjuna also displayed a molecularion peak at m/z= 666 [M]+ indicating the presence of ter-miarjunoside I, with a molecular formula of C36H58O11,which was also supported by 13C and distortionless enhance-ment by polarization transfer (DEPT) NMR spectra. Themass spectrum revealed the highest peak at 301.08, 317.25,and 169.08 which indicate the presence of quercetin, myrice-tin, and gallic acid in T. arjuna. The presence of gallic acidwas further confirmed by MS-MS using CID (30.00). Thepeak at 125.08 is the consequence of the removal of COO−

from gallic acid. The MS-MS of the peak 317.25 by CID(21.00) showed the highest peaks at 302.08, 241.08, and179.06. However, the peaks at 193 and 289 may indicate thepresence of ferulic acid and catechin, respectively.

3.5. Crataegus oxyacantha. The LC-MS analysis of C. oxya-cantha was executed to assess the phytoconstituents. Themass spectrum of C. oxyacantha showed the peak at 593.17

0

10

20

30

40

50

60

70

80

90

100

R. serpentina T. arjuna C. sativum P. nigrum E. cardamom A. sativum C. oxyacantha

Inhi

bitio

n (%

)

Medicinal plants

20 �휇g/ml

40 �휇g/ml

60 �휇g/ml

80 �휇g/ml

100 �휇g/ml

Figure 1: Graphical presentation of antioxidant potential of selected medicinal plants through DPPH radical scavenging activity.

4 Oxidative Medicine and Cellular Longevity

which indicated the presence of bioactive compoundsproanthocynidine with positive mode of ESI (Figure 5). TheMS-MS of peak 593 gave the highest peaks at 429.25, 457.17,411.25, and 401.17, where the peak at 457.17 might indicatethe presence of ursolic acid. The LC-MS-ESI also revealedthe presence of cratagolic acid at peak of 417 (m/z). TheCID MS-MS of the peak 381 of C. oxyacantha showed thepeak at 301.17 may give the idea of the presence of quercetin.

3.6. Rauwolfia serpentina. The LC-MS analysis of rootsextract of R. serpentina was performed. The full mass spec-trums along with the highest peaks at 327.25 and 355.33 indi-cated the presence of ajmaline and yohimbine, respectively(Figure 6). The MS-MS with CID of 25.00 at peak 327 pro-duced different fragment ion peaks. Among these peaks, thepeak at 353.25m/z may indicate the presence of ajmailacine.The mass spectrum of R. serpentina also depicted the pres-ence of serpentine at the peak 349.52.

3.7. Allium sativum. A. sativum was subjected to LC-MSanalysis to evaluate the presence of phytoconstituents thatmight be responsible for cardiovascular diseases, dyslipid-emia, and hypertension. The LC-MS analysis of A. sativumdepicted the highest peaks at 896.92, 917.75, and 782.58(Figure 7). The MS-MS of 896.92 with CID (25.00) gave thepeak at 319.25, which indicated the presence of myricetin.The mass spectrum also showed the presence of apigenin atpeak 269.08 with negative mode of ESI.

3.8. Coriandrum sativum. LC-MS analysis of the seed extractof C. sativum was performed to evaluate the active phytocon-stituents including phenolics, flavonoids, and alkaloids. Thefull mass spectrum indicated the existence of caffeic acid atpeak 179.08 and isorhamnetin-3-O-glucoside at 478.17m/z.The mass spectrum of C. sativum also showed apigenin-6-C-glucoside at peak 593.25m/z with negative mode ofelectrospray ionization (Figure 8).

3.9. Elettaria cardamom. The mass spectrum obtained byLC-MS analysis of E. cardamom represented the high peaksat 195.17133.06 and 333.33. The peak at 195.17 indicatedthe presence of terpinyl acetate. The mass spectrum alsodepicted the presence of sebinen at 137.08 peak (Figure 9).

3.10. Piper nigrum. The methanolic extract of P. nigrum issubjected to LC-MS analysis to determine its bioactivecompounds that impart crucial role in cardioprotection.The pippercide, an active ingredient of P. nigrum, showedits peak at 219.08 (Figure 10).

3.11. In Vivo Analysis

3.11.1. Effect of Herbal Combinations on Cardiac Markers. Toinvestigate whether the combinations of herbal extracts underinvestigation would offer any added advantage over individ-ual herbal treatment, the effects of HCs were compared withnormal and the surgically induced MI group. The potentialof herbal combinations was evaluated by analysing the car-diac markers including CK-MB, SGOT, and LDH.

Figure 2: Agarose gel electrophoresis pattern of pBR322 plasmid DNA treated with 30mM H2O2 in the presence and absence of differentplants extract. [lane 1: pBR322 DNA+30mM H2O2 +P1 (100 μg/mL), lane 2: pBR322 DNA+30mM H2O2 +P1 (500 μ/mL), lane 3:pBR322 DNA+30mM H2O2 +P1 (1000 μg/mL), lane 4: pBR322 DNA+30mM H2O2 +P2 (100 μg/mL), lane 5: pBR322 DNA+ 30mMH2O2 +P2 (500 μg/mL), lane 6: pBR322 DNA+30mM H2O2 +P2 (1000 μg/mL), lane 7: pBR322 DNA+ 30mM H2O2 +P3 (100 μg/mL),lane 8: pBR322 DNA+30mM H2O2 + P3 (500 μg/mL), lane 9: pBR322 DNA+ 30mM H2O2 +P3 (1000 μg/mL), lane 10: pBR322DNA+ 30mM H2O2 + P4 (100 μg/mL), lane 11: pBR322 DNA+ 30mM H2O2 + P4 (500 μg/mL), lane 12: pBR322 DNA+ 30mMH2O2 +P4 (1000 μg/mL)].

Figure 3: Lane13: pBR322 DNA+30mMH2O2 + P5 (100 μg/mL), lane 14: pBR322 DNA+30mMH2O2 +P5 (500 μg/mL), lane 15: pBR322DNA+ 30mMH2O2 + P5 (1000 μg/mL), lane 16: pBR322 DNA+30mMH2O2 +P6 (100 μg/mL), lane 17: pBR322 DNA+30mMH2O2 +P6(500 μg/mL), lane 18: pBR322 DNA+30mM H2O2 +P6 (1000 μg/mL), lane 19: pBR322 DNA+30mM H2O2 +P7 (100 μg/mL), lane 20:pBR322 DNA+30mM H2O2 + P7 (500 μg/mL), lane 21: pBR322 DNA+30mM H2O2 + P7 (1000 μg/mL). P1 =T. arjuna; P2 =C.oxyacantha; P3 = P. nigrum; P4 =R. serpentina; P5 =A. sativum; P6 =C. sativum; P7 = E. cardamom.

5Oxidative Medicine and Cellular Longevity

The effect of different herbal combinations on CK-MBlevel against surgically induced MI has been presented inFigure 11(a). The normal control group showed the normalCK-MB level (173± 3.51 IU/L) throughout the experimen-tal period. There was a considerable increase in the levelof CK-MB in the positive control group after 12 hr of leftanterior descending coronary artery ligation while the levelof enzyme was further raised up to 294.3± 1.53 IU/L after24 hr. The first herbal combination (HC1) did not signifi-cantly (p > 0 05) restored the CK-MB level after 12 and24 hr of ligation as compared to the normal control group.In comparison of HC1, the group pretreated with HC2showed better maintenance of CK-MB level after 12 and24 hr of ligation. A decrease in CK-MB level was observedin group pretreated with HC4 after 12 hr of ligating leftanterior descending coronary artery. After 24 hr of liga-tion, this group showed considerable decline in the levelof CK-MB that was very close to the control group.The prior administration of HC4 depicted the bettermaintenance of the serum CK-MB as compared to otherherbal combinations.

The effect of different herbal combinations on the levelof SGOT has been presented in Figure 11(b). In the normalcontrol group, the SGOT level was 43± 2 and 46± 1.05 IU/Lwith time intervals of 12 and 24 hr, respectively. The SGOTlevel was 115± 1.527 IU/L and 123± 1.154 IU/L after thecorresponding time intervals of 12 and 24hr of LADCAligation in the positive control group. The HC1 showedthe SGOT level with a value of 94± 1.53 IU/L after 12 hrand 74± 1 IU/L after 24 hr of ligation. The pretreatment ofHC2 significantly (p > 0 05) maintained at the level of SGOTafter 24 hr of ligation in LADCA as compared to the positivecontrol group. There was no considerable variation in theoutcomes of HC1 and HC3 preventive treatment. However,the pretreatment of HC4 showed maximum potentialagainst myocardial infarction as it upholds the SGOT level73± 1 IU/L after 12 hr and 53± 1.53 IU/L after 24 hr ofLADCA ligation.

The preventive treatment of herbal combinations againstsurgically inducedMI on the level of LDH has been presentedgraphically in Figure 11(c). The serum analysis of the normalcontrol group revealed 223± 1.15 to 235 IU/L of LDH from 0to 48 hr, respectively. The LDH level in the positive controlgroup was considerably higher as compared to the normalcontrol group. The group of dogs pretreated with HC1showed 382.33± 1.53 IU/L of LDH after 12hr and 283±1.15 IU/L after 24 hr of ligation. In dogs treated with HC2,the LDH level was 291.67± 1.15 IU/L and 264± 2.08 IU/Lat corresponding time intervals of 12 and 24 hr after LADCAligation. While the pretreatment of HC3 showed 343±1.53 IU/L level of LDH after 12 hr and maintained at thelevel of 250± 1 IU/L after 24 hr of ligation. The preventivetreatment of HC4 revealed significant maintenance of LDHlevel after 12 hr of ligation (Figure 11(c)).

The HC4 showed the prominent cardioprotective poten-tial by maintaining the cardio-specific markers near thenormal against surgically induced myocardial infarctionafter 24 hr of LADCA ligation. Although the precise mech-anism of the cardioprotective potential of HCs in surgically

Full mass spectrum of Terminalia arjuna

MS-MS of 685.58 with CID showing termiarjunoside I at 667.50 m/z

200018001600140012001000800600400200(m/z)

(m/z)

(m/z)

(m/z)

(m/z)

(m/z)

(m/z)

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

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0102030405060708090

100

Rela

tive a

bund

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0102030405060708090

100

Rela

tive a

bund

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139.08

321.33

203.08

413.42511.50

589.33

685.58701.58

917.75955.92

1075.58 1173.581395.92 1570.92 1664.08 1791.67 1970.58

750700650600550500450400350300

500450400350300250200

18001400 2000160012001000800600400200

200 2201801601401201008060

OH

OHOH

OHOH

OHHOHO

COOH

H

O O

Termiar junoside1,3,9,22-tetreaol-12-en-28-oicacid-3-Dghicopyranoside

OHOH

OHOH

OH

OH

OH

O

OHO

Gallic acid

HO

O

Quercetin

Mass spectrum of T. arjuna showing quercetin at 301.08 m/z

Mass spectrum of T. arjuna showing gallic acid at 169.08 m/z

MS-MS CID (30.00) of peak 169 m/z

Mass spectrum indicating the presence of myricetin

MS/MS of T. arjuna peak 317 at CID (21.00) showing ferulic acid at 193 m/z and catechin at 289 m/z

500450400350300250200150

360280 300 320 340260240220200180160140120100

HO

OHOH

OH

OHOH

O

OMyricetin

0102030405060708090

100

Rela

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0102030405060708090

100

Rela

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Ferulic acidOH

OH

OHOH

HO O

Catechin

O

O

OH

HO

319.08

317.08

302.08

299.08

289.00

273.00

245.08

241.08

227.08207.08193.00

179.08

169.08165.08

151.08

136.92124.9297.08

485.33459.33443.33

413.33

391.33337.25

321.33

317.25

286.17279.17

249.08

239.08219.08

203.08

141.08151.08

186.92171.08169.08

157.33151.08141.08127.00

125.08

121.08109.0897.1781.1769.0057.17

153.08

169.08

183.08

301.08

315.17325.25339.33

523.42685.50

611.42734.67 878.75

892.671011.42 1187.50 1349.58 1443.001559.581692.001835.92

0102030405060708090

100

Rela

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150

183.08

191.08 215.08 247.17

301.08

315.17

325.33

383.42 441.25497.25

317.08 364.92407.33429.33

445.42

457.25

485.33 513.42

523.33

541.50 552.25597.67 641.67653.50

667.50

685.58

702.67725.17

Figure 4: LC-MS analysis of T. arjuna.

6 Oxidative Medicine and Cellular Longevity

100221.08

201.08247.08

259.08 377.25 591.33 685.58 813.58 998.17 1076.75 1314.25 1411.75 1529.75 1724.92 1842.50 1945.75

200018001600140012001000(m/z)

800600400200

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Full mass spectrum of C. oxyacantha

Mass spectrum of C. oxyacantha showing proanthocynidine 593.17 m/z

MS-MS with CID (20.00) of peak 591.42 showing ursolic acid at 457.25 m/z

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40487.17 521.17

553.42

591.33

539.25561.42

579.17

593.17

595.17621.50

625.25 685.58

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HOOH

OH

Proanthocynidine

OH

O

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191.25

203.08

232.00248.92

269.08

309.17351.08

381.25401.17

411.25

429.25

457.17

475.17501.17

531.42 561.42573.25

Ursolic acid

HOH

H

O

OH

591.42

612.08 629.00

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784.83773.42

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411.25

439.25 459.35471.08

491.33

519.00

543.25

555.58577.33

597.25625.08

643.08653.33

681.08 699.33738.83 753.50

785.08

Crateagolic acid

817.17 836.08

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Mass spectrum of C. oxyacantha showing crateagolic acid at 471.08 m/z

MS-MS of 381 of C. oxyacantha with CID (20.00) showing quercetin at 301.17 m/z

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0111.17 155.08 173.17

207.08

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OH

OH

OH

HO O

O

191.08

217.00

249.17

Quercetin

277.17301.17

321.17 345.08363.25

381.25

383.92

100 150 200 250 300 350 400 450 500

OH

Figure 5: LC-MS analysis of C. oxyacantha.

7Oxidative Medicine and Cellular Longevity

100 191.08 281.33

133.08

339.33383.42

397.42413.50

461.25

327.25

355.33

383.25

503.42 609.50717.25818.83 989.50 1211.3312.97.421442.421577.751681.921847.251966.08

311.25219.08

110.05138.08

144.08

158.17182.17

184.08

194.08 220.17

238.08

264.17

291.25

309.17

Ajmailicine

Serpentine

327.25

353.25262.17

170.08

132.00

311.25

355.25

327.25

381.25383.33

399.33 415.33 429.25 444.33453.33 473.33 489.50503.33513.25 531.42

349.255

600.50734.58

748.67804.58 980.831068.67 1304.75 1433.25 1583.00 1709.92 1842.17 1974.58

200018001600140012001000(m/z)

800600400200

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800600400200

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H

H

HAjmaline

Yohimbine

HN

N N

N

H

N

N

H

H

H

H

H

H

OHO

O

O

O

OO

O

OH

N

N

Full mass spectrum of R. serpentina

Mass spectrum of R. serpentina showing yohimbine at 355.33 and ajmaline at 327.25 m/z

Mass spectrum of R. serpentina showing serpentine at 349.25 m/z

MS2 of peak 327 with CID (25.00) showing ajmailicine at 353.25 m/z

Figure 6: LC-MS analysis of R. serpentina.

8 Oxidative Medicine and Cellular Longevity

induced myocardial injury is not fully understood, it may beattributed to its favorable myocardial adaptogenic proper-ties. Furthermore, this herbal combination might have thepotential for the management of patients at risk of myocar-dial infarction.

3.12. Effect of Herbal Combinations onHemodynamic Variable

3.12.1. The Mean Arterial Pressure. Measurement of thehemodynamic variables was also incorporated into the exper-imental design for better understanding and more precise

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115.08

137.08

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183.08

184.92205.17

215.08

217.17 243.08255.33

269.08

290.17311.25

325.33339.25

341.25355.33 369.17297.17

279.25133.00

153.00

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OH

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OH

OH O

O

OH

OH

OHOH

O

OHO

863.50733.33599.58

613.58

639.58

453.33359.17

303.00

319.25

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1259.421157.42

Myricetin

1400

Full mass spectrum of A. sativum

MS2 CID 25.00 of 896 of A. sativum showing myricetin at 319.25 m/z

Mass spectrum of A. sativum apigenin at 327.25 m/z

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1897.251796.251593.751494.251291.171076.67995.58

896.92

782.58655.50527.25

429.33381.17

365.25

336.25

286.25

175.17116.08

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Figure 7: LC-MS analysis of A. sativum.

9Oxidative Medicine and Cellular Longevity

information of the correlation between biochemical andfunctional changes in the myocardium subjected to surgicallyinduced damage. The normal control group depicted the85± 6.81 mean arterial pressure (MAP) mmHg while the

positive control group showed the decline in MAP (33±4.35mmHg) after occlusion in LADCA (Figure 12). Thepretreatment of HC1 tried to sustain the level of MAP up to52± 5.13mmHg. However, the group treated with HC2 and

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133.08

161.08209.17

255.33

297.33

281.33

HO

HO

Caffeic acid

Isorhamnetin −3−O-glucoside

HO

OH OO

O

OH3O OH

OH

OHOH

HO

OH

O

311.33339.33

337.17 393.25

466.17

461.33 477.17179.08

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156.08

221.08

337.25

381.25

659.67

923.83

429.33

543.33

643.75 796.67895.83

939.92

955.83985.83 1220.25

1279.331515.92

1543.08

1780.421806.42

1864.67

50

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297.42513.42537.25

535.33

539.33

541.25

561.25

563.25

579.25

603.58

593.25

619.58639.33

HO

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HO

HOO

O

O

OH

OOHOH

623.33655.33

671.50675.58 691.58

Apigenin 6-O-Glucoside

697.58 719.08 737.08

520 540 560 580 600 620(m/z)

640 660 680 700 720 740

100 150 200 250 300(m/z)

350 400 450 500

400 600 800 1000(m/z)

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Mass spectrum of C. sativum showing caffeic acid at 179.08 m/z and isorhamnetin-3-O-glucoside at 477.17 m/z

Mass spectrum of Coriandrum sativum showing apigenin-6-C-glucoside at 593.25 m/z

Full mass spectrum of C. sativum

Figure 8: LC-MS analysis of C. sativum.

10 Oxidative Medicine and Cellular Longevity

HC4 substantially maintained the MAP 76± 4.04mmHg and77± 5.13mmHg, respectively, as compared to other groups.

Similarly, in the positive control group, there was anabrupt increase in heart rate (HR) beats/min (277± 8.02) as

compared to the normal control group (186± 4.04 beats/min). On the other hand, the pretreatment of surgicallyinducedMI groups with herbal combinations revealed signif-icant (p > 0 05) maintenance of HR as compared to the

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Full mass spectrum of E. cardamom

Mass spectrum of E. cardamom showing terpinylacetate at 195.17 m/z

Mass spectrum of E. cardamom showing sabinene at 137.08 m/z

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195.17

311.25

399.33 537.25 719.25831.58

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219.17 255.33237.17 267.17

279.33

Sabinene

Terpinylacetate

290.17

325.33339.33

349.25

377.17

379.17383.33

399.33369.25

311.25

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1858.501715.501611.331475.921336.921251.001045.67959.75820.67655.50

597.50

419.33

381.17

293.25196.00

251.17

267.17117.00

H3C

OH3

CH2

(m/z)

Figure 9: LC-MS analysis of E. cardamom.

11Oxidative Medicine and Cellular Longevity

positive control group. Among all the treatment groups, thegroup pretreated with HC2 and HC4 showed significant(p > 0 05) restoration of HR.

3.12.2. Effect of Herbal Combinations on Ventricular Function.A significant decline in left ventricular end-diastolic pressure(LVEDP) (9±3.05) marked the onset of myocardialinfarction in surgically induced MI group which remaineddecreased throughout the experimental period in comparisonto the normal control group (32±5.51) (Figure 12). Thepretreatment withHC4 andHC2 significantly (p > 0 05)main-tained the LVEDP level as compared to the surgically inducedischemic group. The HC1 and HC3 also tried to sustain theLVEDP with corresponding values 12±4.04 and 08±1.53.

The positive control group showed the significantdecrease in left ventricular systolic pressure (LVSP) as com-pared to the normal control group. The LADCA ligationresulted in significant cardiac dysfunction evidenced byreduced MAP and increased HR. The left ventricular con-tractile function was also altered. The pretreatment of HC4showed the marked restoration as compared to other groupsas it maintained the level of LVSP near to the normal control

group. It is materialized that the HC4 is more potent in pre-venting the hemodynamic deteriorations observed in thepositive control group.

3.13. Histopathological Examination. The histopathologicalfindings of myocardial tissue in the normal control groupillustrated clear integrity of the myocardial cell membrane.The myofibrillar structure was normal with no inflammatorycell infiltration. The nuclei were also normal without anypyknotic changes (Figure 13(a)). The histopathologicalexamination of the surgically induced MI group showedextensive myofibrillar degeneration related to infiltrationand disruption of cardiac myofibers. There was markednecrosis in the ventricular region. Pyknotic changes in nucleiwere also observed (Figure 13(b)).

The treatment of HC1 prior to ligation showed myofibri-lation (Figure 13(c)) while the pretreatment with HC2demonstrated marked improvement in surgically inducedalterations, but there was cellular infiltration at few places.The nuclei were also normal (Figure 13(d)). The grouptreated with HC3 did not protect the cardiac dysfunctionsas compared to the other groups. Myocardial fibrillation as

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Full mass spectrum of P. nigrum

Mass spectrum of P. nigrum showing pipercide at 219.08 m/z

800600400

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Pipercide

853.67 937.75 1037.83 1163.92 1320.75 1468.92 1578.75 1693.75 1799.75 1934.00

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259.080

Figure 10: LC-MS analysis of P. nigrum.

12 Oxidative Medicine and Cellular Longevity

well as some pyknotic changes in the nuclei were also seen inthe group treated with HC3 (Figure 13(e)). The histopatho-logical examination of the group treated with HC4 showedthat there was no myofibrilation, and the cardiac paren-chyma was also normal. This confirmed the potential ofherbal combination (HC4) over oxidative stress related tocardiac ailment (Figure 13(f)).

4. Discussion

The evidence-based study about metabolomes of medicinalplants is an emerging approach to develop a new group ofphytotherapeutics [16]. The therapeutic potential of plantsecondary metabolites has augmented an interest in pharma-ceutical research for the development of novel therapeuticagents. The antioxidant profiling of the said medicinal plantswas explored through DPPH and DNA protection assay. Theantioxidative potential of these medicinal plants was found tobe dose-dependent. This dose-dependent response of variousmedicinal plants for antioxidative potential has already beenreported by many researchers [18, 25, 26]. The increasedantioxidant potential with high dose of medicinal plantsmay be due to positive correlation with high quantity ofpowerful chain-breaking antioxidants like phenolics andother phytoconstituents [27]. Different mechanisms likescavenging of free radicals, chelation of metal ions, andinhibition of enzymes may be responsible for good thera-peutic antioxidant potential of medicinal plants [28].

In HPLC, the extremely narrow peaks are generated;thus, the high-speed data handling performance demands ablend of MS segment [29]. LC-MS has such features that

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###

### #

## # # # # # ##

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0 12Time (hr)

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(a) Effect of herbal combinations of plant extracts on the level of

CK-MB (IU/L)

(b) Effect of herbal combinations of plant extracts on the level of

SGOT (IU/L)

(c) Effect of herbal combinations of plant extracts on the level of

LDH (IU/L)

Figure 11: (a–c) Effect of herbal combinations of plant extracts on cardiac markers in the serum of all experimental groups. ∗∗ indicatessignificance (p < 0 0001) compared to the normal control, # indicates significance (p < 0 001) compared to the positive control, and ##indicates significance (p < 0 0001) compared to the positive control (ANOVA, Turkey’s multiple comparison test). Values are presented asthe mean± SEM (n = 3).

050

100150200250300350

Control Surgery HC1 HC2 HC3 HC4Hem

odyn

amic

par

amet

ers

Treatment groups

MAPHR

LVEDPLVSP

Figure 12: Hemodynamic parameters of various groups treatedwith different herbal combinations.

13Oxidative Medicine and Cellular Longevity

make it applicable for metabolomic profiling of a wide rangeof low to high polarity metabolites, including nonvolatilecompounds. It also covers a broad range of metabolites, sinceit operates ionization in negative and positive modes [30].Hence the LC-MS-based metabolomics is a powerful tool inorder to evaluate the important active secondary metaboliteswhich play a vital role to prevent oxidative stress by scaveng-ing free radicals.

The LC-MS analysis of T. arjuna revealed the presenceof some important phytoconstituents like termiarjunosideI, quercetin, ferulic acid, and gallic acid which wereresponsible for its antioxidative strength. The HPLC analysisof the T. arjuna bark by Jahan et al. [17] also exhibited theexistence of polyphenols and phenolic acids including ferulicacid, gallic acid, caffeic acid, and catechin. The fast atombombardment mass spectroscopy (FABMS) and distortion-less enhancement by polarization transfer (DEPT) NMRspectra of T. arjuna also displayed a molecular ion peak atm/z= 666 [M]+ indicating the presence of termiarjunosideI, with a molecular formula of C36H58O11 (Ali et al. 2006).The quercetin and gallic acids are strong antioxidantswhich play a crucial role in a number of biological andpharmacological activities and also protect DNA damage[31]. The ferulic acid present in T. arjuna is not only agood antioxidant in various biological systems but alsohas the potential to protect the DNA against H2O2-induceddamage [32].

The metabolomic profiling of C. oxyacantha depicted thepresence of procynidine, crateagolic acid, ursolic acid, andquercetin. These major phytoconstituents are mainly respon-sible in curing various diseases like myocardial infarction,coronary heart diseases, hypertension, and diabetes-relatedcomplications owing to their antioxidant potential [33].

The presence of ursolic acid in C. oxyacantha has also beenreported to have angiotensin-converting enzyme-inhibitingand cardioprotective potential (Lacaille et al. 2001). R. ser-pentina has been a popular field of research for decades,and several researchers have explored its excellent phyto-chemical properties [34, 35]. Various secondary metabolitessuch as yohimbine, ajmaline, serpentine, and ajmalicine pres-ent in the roots of R. serpentina contribute for its antiox-idant potential [36]. Ajmaline is a sodium channel blockerthat illustrated the instant therapeutic potential whengiven intravenously. It has also been claimed to stimulate res-piration and intestinal movements. Serpentine is useful toprevent the oxidative stress-induced DNA damage, hyper-tension, cardiovascular, and neurological diseases [37]. R.serpentina is a hopeful herbal option in the pharmaceuticalworld due to the existence of considerable bioactive com-pounds in the roots [38]. The LC-MS analysis of A. sativumindicated the existence of myricetin and apigenin. The myr-icetin due to its specific chemical structure counteracts oxi-dative stress generated as a result of reactive oxygen species[39, 40]. The hydroxylated apigenin is found to inhibittumor cell proliferation and angiogenesis. Caffeic acid is apotent antioxidant and has several therapeutic propertiesincluding antioxidants, anti-inflammatory, and anticarcino-genic. It has been reported that caffeic acid inhibits bothlipoxygenase activity and suppresses lipid peroxidation thuscompletely blocks the production of ROS [41]. Cardamomfruit is used against vesicular calculi, dyspepsia, debility, hal-itosis, and gastrointestinal disorders [42]. Phytochemicalinvestigation of cardamom has revealed highly bioactivecomponents. High-phenolic compounds, in extracts of allplants, could be considered as the key reason behind theantioxidant potential of the said medicinal plants [43, 44].

(a) Normal control group (b) Positive control group

(c) HC1 group (d) HC2 group

(e) HC3 group (f) HC4 group

Figure 13: (a–f) The histopathological representation of cardiac tissue of all treatment groups.

14 Oxidative Medicine and Cellular Longevity

During in vivo trial, the increased cardiac markers in thepositive control group are due to the ligation of the coronaryartery. The ligation imparts an additional workload on theremaining viable myocytes that may be unbearable, resultingin pathological alterations [11]. Alterations in integrity, fluid-ity, and permeability of the myocardial membrane due toligation have been believed to be a reason for the leakage ofcardiac markers [21]. The treatment with HCs might salvageviable myocytes, which are at risk of injury, thus preventingcell loss induced by necrosis [45]. The HC4 showed theprominent cardioprotective potential by maintaining thecardio-specific markers near the normal against surgicallyinduced myocardial infarction. The better maintenance ofthe cardiac markers with HC4 as compared to other herbalcombinations might be due to the presence of synergism ofsome specific phytoconstituents like crateagolic acid, ter-miarjinoside-I, ajmaline, and serpentine and antioxidantslike quercetin, gallic acid, ferulic acid, and myricetin in it.This may render the myocytes less leaky by preventing myo-cardial membrane destruction [46]. A considerable fall inMAP and increased HR in the surgically induced MI groupindicated hemodynamic impairment and ventricular dys-function due to increased generation of ROS [22]. A fall inMAP due to coronary occlusion is expected to increase HRand myocardial contractility by activating the baroreceptorreflex, which may subsequently result in reflex vasoconstric-tion and thus worsening the imbalance, between myocardialoxygen demand and supply [47]. The increase in blood flowthrough the subendocardial region of the left ventricularmuscle is the major consequence of the reduction in LVEDPin the surgically induced infarction group [48]. The thera-peutic efficacy of HC4 might be due to the improvement inboth inotropic and lusitropic function of the heart and con-siderable maintenance of antioxidant defense capacity ofthe myocardium [48].

5. Conclusion

The HC4 (T. arjuna, R. serpentina, E. cardamom, and C. oxy-acantha) considerably ameliorated cardiotoxicity by keepingthe levels of biochemical parameters near to normal. Theantioxidants property and phytoconstituents of medicinalplants present in this herbal combination might be responsi-ble for its cardioprotective potential. On the basis of thisevidence-based study, it can be concluded that the HC4 canbe safely used as an alternative product for the managementof cardiovascular diseases.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by the Higher Education Commis-sion, Pakistan, under the Indigenous 5000 PHD FellowshipProgram pin no. 112-24406-2BM1-387.

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