Sodium-hydrogen exchanger inhibitory potential of Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale

DOI 10.1515/jbcpp-2013-0088 J Basic Clin Physiol Pharmacol 2014; 25(1): 99–108

Vivek Verma, Nirmal Singh and Amteshwar Singh Jaggi*

Sodium-hydrogen exchanger inhibitory potential of Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale

Abstract

Background: The involvement of sodium-hydrogen exchangers (NHE) has been described in the pathophysi-ology of diseases including ischemic heart and brain dis-eases, cardiomyopathy, congestive heart failure, epilepsy, dementia, and neuropathic pain. Synthetic NHE inhibi-tors have not achieved much clinical success; therefore, plant-derived phytoconstituents may be explored as NHE inhibitors.Methods: In the present study, the NHE inhibitory poten-tial of hydroalcoholic and alkaloidal fractions of Malus domestica, Musa × paradisiaca, Daucus carota, and Sym-phytum officinale was evaluated. The different concen-trations of hydroalcoholic and alkaloidal extracts of the selected plants were evaluated for their NHE inhibitory activity in the platelets using the optical swelling assay.Results: Among the hydroalcoholic extracts, the highest NHE inhibitory activity was shown by M. domestica (IC50 = 2.350 ± 0.132 μg/mL) followed by Musa × paradisiaca (IC50 = 7.967 ± 0.451 μg/mL), D. carota (IC50 = 37.667 ± 2.517 μg/mL), and S. officinale (IC50 = 249.330 ± 1.155 μg/mL). Among the alkaloidal fractions, the highest NHE inhibitory activity was shown by the alkaloidal frac-tion of Musa × paradisiacal (IC50 = 0.010 ± 0.001 μg/mL) followed by D. carota (IC50 = 0.024 ± 0.002 μg/mL), M. domestica (IC50 = 0.031 ± 0.005 μg/mL), and S. offici-nale (IC50 = 4.233 ± 0.379 μg/mL). The IC50 of alkaloidal fractions was comparable to the IC50 of synthetic NHE inhibitor, EIPA [5-(N-ethyl-N-isopropyl)amiloride] (IC50 = 0.033 ± 0.004 μg/mL).Conclusions: It may be concluded that the alkaloidal frac-tions of these plants possess potent NHE inhibitory activ-ity and may be exploited for their therapeutic potential in NHE activation-related pathological complications.

Keywords: alkaloidal fraction; Daucus carota; Malus domestica; Musa × paradisiaca; sodium-hydrogen exchanger; Symphytum officinale.

*Corresponding author: Dr. Amteshwar Singh Jaggi, Department of Pharmaceutical Sciences and Drug Research, Punjabi University Patiala, Patiala-147002, India, Mobile: +919501016036, E-mail: [email protected] Verma and Nirmal Singh: Department of Pharmaceutical Sciences and Drug Research, Punjabi University Patiala, Patiala, India

IntroductionNa+/H+ exchangers (NHEs) are ubiquitous integral mem-brane ion transporters and mediate the electroneutral exchange of H+ with Na+ or K+ to regulate pH, osmolarity, cell volume, and cell proliferation during physiological as well as in pathophysiological conditions. The Na+ and H+ ion gradients across cellular membranes is the major gov-erning factor for NHE activation, and in acidic intracellular conditions, NHEs are activated to restore the basal cellular pH levels by exchanging Na+ (extracellular) with H+ (intra-cellular) stoichiometrically (1:1) [1]. However, the exces-sive stimulation of NHE results in increased intracellular Na+ concentration and a subsequent activation of Na+/K+-ATPase, with a consecutive increase of energy consump-tion [2]. The high intracellular Na+ level also contributes to the activation of Na+/Ca2+ antiporter, which results in increased intracellular Ca2+ load [3] (Figure 1). The role of high intracellular Ca2+ levels is very well-defined in various pathophysiology of different diseases like ischemia-reper-fusion-induced cardiac and cerebral diseases [4, 5], and hence, NHE inhibition may be regarded as the central target in the diseases involving Ca2+ overload [6].

The major role of NHE has been described in patholo-gies of the heart, as its activation has been shown to be involved in cardiac hypertrophy, heart failure, and ischemia-reperfusion injury, and its inhibition is shown to attenuate diabetic cardiomyopathy, cardiac fibrosis, cardiac remodeling, contractile dysfunction, ventricu-lar hypertrophy, heart failure, and myocardial ischemic injury [7]. Because the heart and the brain are both prone to ischemia-induced injury, the role of NHEs in brain

Brought to you by | Brown University Rockefeller LibraryAuthenticated | 128.148.252.35

Download Date | 6/10/14 1:33 PM

mailto:[email protected]

100 Verma et al.: Medicinal plants as sodium-hydrogen exchanger inhibitors

pathologies, particularly in cerebral ischemia, has been investigated. The majority of studies have suggested that the activation of NHE is associated with development of mental disorders like epilepsy, Alzheimer’s disease, stroke, and neuropathic pain, and accordingly, its phar-macological inhibition has been shown to exert beneficial effects in the different brain diseases [8, 9]. Thus, the drugs targeting NHE may present important therapeutic alterna-tives for the diseases involving excessive NHE activation. The uncertainty about the use of synthetic inhibitors in clinical settings raised the demands for the development of alternatives. Phytochemicals, which were not explored for NHE inhibitory activity until now, can present a better alternative for synthetic inhibitors possibly due to their good safety and efficacy profiles. In the present study, four plants, Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale were tested for NHE inhibitory potentials.

The plants were mainly selected on the basis of the presence of alkaloidal constituents, which are structurally similar to synthetic NHE inhibitors. The majority of avail-able synthetic inhibitors including amiloride (1), EIPA [5-(N-ethyl-N-isopropyl)amiloride] (2), and cariporide (3) possess guanidine moiety as an important structural com-ponent that is shown to be responsible for their potent NHE inhibitory activity. S. officinale contains allantoin (9) as the major alkaloidal constituent, which is a guanidine-derived moiety [10]. M. domestica (shoot) and Musa × par-adisiaca (fruit) also contain guanidine compounds, i.e., γ-guanidinebutramide (6), γ-guanidinobutyric acid (7), and γ-guanidinopropionic acid (8) [11]. In the present

Na+

Na+

Ca2+

3Na+

3K+H+

Sodium-potassium

ATPase

NHE

NCX

Figure 1 Cellular ion transport is mainly mediated through concen-tration gradient-dependent sodium-hydrogen exchanger (NHE) and sodium-calcium exchanger (NCX) and the active transporter pump-like Na+/K+-ATPase.The functioning of these ion exchangers is influenced by each other and is important in physiological as well as in pathophysiological states.

study, we have evaluated the in vitro NHE inhibitory potential of hydroalcoholic and alkaloidal fractions of M. domestica, Musa × paradisiaca, D. carota, and S. officinale by platelet optical swelling assay.

Materials and methods

Chemicals and biological preparationsEIPA and HEPES buffer [4-(2-hydroxyethyl)-1-piperazineethanesul-fonic acid] (acid free) were from Sigma-Aldrich (St Louis, MO, USA). Sodium propionate, magnesium chloride, calcium chloride, glucose, sodium carbonate, methanol, ethyl acetate, acetic acid, and chlo-roform were from LOBA Chemicals Pvt Ltd (Mumbai, India). All the chemicals and solvents were of analytical grade and the solutions were freshly prepared. Fresh platelet-rich plasma (PRP) was collected from the blood bank of Govt. Rajindra Hospital, Patiala, Punjab. The PRP sample was stored at 22°C for a maximum of 2 days.

Plant materialsThe dried root of S. officinale (comfrey), fresh aerial part of D. carota (carrot), fresh unripe fruit of Musa × paradisiaca (banana), and shoot of young plant of M. domestica (apple) were obtained locally. The samples of M. domestica, Musa × paradisiaca, D. carota, and S. officinale were kept as voucher specimens (PuP-034/2012-2013, PuP-035/2012-2013, PuP-036/2012-2013, and PuP-037/2012-2013, respec-tively) at Punjabi University, Patiala, India.

Preparation of hydroalcoholic extractsThe respective plant parts of the selected plants were dried in the shade and ground to make a coarse powder. The powder was then extracted three times with methanol-water mixture (3:1) by stir-ring at room temperature for 1 h each time. The extraction from the plants was exhaustive and it was confirmed by the presence of no residue after evaporating a few drops of the extractive fluid. The extract was filtered and the solvent was completely removed at 50°C under reduced pressure. The yield of the different extracts was noted (% w/w) in terms of dried starting material [12].

Preparation of alkaloidal fractionsThe hydroalcoholic extract was further used for the preparation of alkaloid-rich fraction. The hydroalcoholic extract was treated with 10% aqueous acetic acid solution and subsequently washed with ethyl acetate. The acetic acid fraction was alkalinized with concen-trated sodium carbonate solution (2%) and the resulting basic solu-tion was exhaustively extracted with chloroform four to five times. The organic layer was subsequently evaporated at reduced pressure in a rota evaporator to obtain the alkaloid-rich fraction. The per-centage yield (w/w) was calculated in terms of dried hydroalcoholic



Verma et al.: Medicinal plants as sodium-hydrogen exchanger inhibitors 101

extract [13]. The quantification of total alkaloids was done by volu-metric titration method based on the reaction of alkaloidal bases with acids (acid-base titrations). The alcoholic solution of alkaloidal residue was titrated with 1% perchloric acid using methyl red as indi-cator.

Assay for sodium-hydrogen exchanger activity

Platelet count

The platelet count of PRP was done manually with a hemocytometer (Neubauer chamber). The concentration of platelets was adjusted to 1 × 108 cells/mL by diluting with physiological saline (0.9% w/v solu-tion of sodium chloride) [14].

Preparation of activating medium (propionate medium)

The platelet-activating propionate medium was prepared by dissolv-ing sodium propionate (140 mmol), HEPES (free acid, 20 mmol), glu-cose (10 mmol), KCl (5 mmol), MgCl2 (1 mmol), and CaCl2 (1 mmol) in distilled water. The pH was adjusted to 6.7 and temperature was maintained at 37°C.

Optical swelling (spectrophotometric) assay

The platelet NHE-1 activity was measured in terms of increase in light transmission of PRP due to platelet swelling using a spectrophoto-metric method [14]. Sodium propionate solution (1200 μL, main-tained at 37°C) was added to 400 μL of PRP in a cuvette and change in absorbance was measured over a period of 4 min at 550 nm. The difference in absorbance, i.e., immediately after addition of sodium propionate to PRP and after 4 min of addition, is directly related to NHE activation and is used for measuring platelet NHE activity [15]. Application of sodium propionate (pH 6.7) produces an acidic intra-cellular pH at which platelet NHE is activated, and the increase in Na+ influx associated with efflux of cytosolic H+ via NHE results in cellular swelling as a result of water accumulation in the cytoplasm. Light

Table 1 The percentage yield of hydroalcoholic and alkaloidal extracts in terms of starting dried material and hydroalcoholic extract, respectively.

Sample no. Name of the plant

Yield, %

Hydroalcoholic extract

Alkaloidal extract

1 S. officinale 14.92 0.7522 M. domestica 11.50 0.7363 Musa × paradisiaca 6.84 0.2424 D. carota 16.20 0.430

120

100

80

60

40

Per

cent

age

inhi

bitio

n (%

)

20

00.01 µg/mL 0.1 µg/mL 1 µg/mL 100 µg/mL10 µg/mL

Figure 2 The NHE inhibitory activity (%) of different concentrations of the standard drug, EIPA.The values are expressed as mean ± SD in triplicate.

transmission through PRP is increased due to decrease in the density of cellular components as a consequence of platelet swelling [14]. The standard NHE inhibitors EIPA (0.01–100 μg/mL) and the plant extracts (0.01–100 μg/mL) were added 3 min before the addition of sodium propionate solution. The percentage inhibition of NHE acti-vation and the IC50 values were calculated for different plant extracts and EIPA.

Statistical analysisThe results were expressed as mean ± standard deviation (SD).

ResultsThe yields of hydroalcoholic and alkaloidal fractions of these plants are shown in Table 1. EIPA, a synthetic NHE inhibitor, exhibited 100% inhibition against plate-let NHE activation (IC50 = 0.033 ± 0.004 μg/mL) (Figure 2). The hydroalcoholic extracts of four plants and their cor-responding alkaloidal fractions inhibited platelet NHE activity to various extents (Figures 3–10). The alkaloidal fractions of all the plants showed higher inhibitory activ-ity compared to those of the hydroalcoholic extracts. Among the hydroalcoholic extracts, M. domestica showed




90

80

70

60

50

40

30

20

10

0

Per

cent

age

inhi

bitio

n (%

)


Figure 3 The NHE inhibitory activity (%) of different concentrations of the hydroalcoholic fraction of M. domestica.The values are expressed as mean ± SD in triplicate.

120

100

80

60

40

Per

cent

age

inhi

bitio

n (%

)

20


Figure 4 The NHE inhibitory activity (%) of different concentrations of the alkaloidal fraction of M. domestica.The values are expressed as mean ± SD in triplicate.

80

70

60

50

40

30

20

10

0

Per

cent

age

inhi

bitio

n (%

)


Figure 5 The NHE inhibitory activity (%) of different concentrations of the hydroalcoholic fraction of Musa × paradisiaca.The values are expressed as mean ± SD in triplicate.

the highest NHE inhibitory activity (IC50 = 2.350 ± 0.132 μg/mL) followed by Musa × paradisiaca (IC50 = 7.967 ± 0.451 μg/mL), D. carota (IC50 = 37.667 ± 2.517 μg/mL), and S. offici-nale (IC50 = 249.330 ± 1.155 μg/mL) (Table 2). Among the alkaloidal fractions, the highest NHE inhibitory activ-ity was shown by Musa × paradisiaca (IC50 = 0.010 ± 0.001 μg/mL) followed by D. carota (IC50 = 0.024 ± 0.002 μg/mL), M. domestica (IC50 = 0.031 ± 0.005 μg/mL), and S. officinale (IC50 = 4.233 ± 0.379 μg/mL) (Table 2).

DiscussionIn the present study, the hydroalcoholic extracts of M. domestica, Musa × paradisiaca, D. carota, and S. offici-nale inhibited the platelet NHE activation (assessed in terms of IC50 value). The highest NHE inhibitory activ-ity was shown by M. domestica (IC50 = 2.350 ± 0.132 μg/mL) followed by Musa × paradisiaca (IC50 = 7.967 ± 0.451 μg/mL), D. carota (IC50 = 37.667 ± 2.517 μg/mL), and




120

100

80

60

40

Per

cent

age

inhi

bitio

n (%

)

20


Figure 6 The NHE inhibitory activity (%) of different concentrations of the alkaloidal fraction of Musa × paradisiaca.The values are expressed as mean ± SD in triplicate.

70

60

50

40

30

20

10

0

Per

cent

age

inhi

bitio

n (%

)


Figure 7 The NHE inhibitory activity (%) of different concentrations of the hydroalcoholic fraction of D. carota.The values are expressed as mean ± SD in triplicate.

120

100

80

60

40

Per

cent

age

inhi

bitio

n (%

)

20


Figure 8 The NHE inhibitory activity (%) of different concentrations of the alkaloidal fraction of D. carota.The values are expressed as mean ± SD in triplicate.

S. officinale (IC50 = 249.330 ± 1.155 μg/mL). However, all of these hydroalcoholic extracts were less potent compared to the standard NHE inhibitor, EIPA (IC50 = 0.033 ± 0.004 μg/mL). Among these hydroalcoholic extracts, M. domestica demonstrated the maximum NHE inhibition (78.90%) at 100 μg/mL (cutoff concentration beyond which the color of extract interfered with the test reading) followed by Musa × paradisiaca (67.75%), D. carota (57.22%), and S. officinale (39.70%). The maximum NHE inhibition with

these hydroalcoholic extracts was also less than that with EIPA (99.74%).

NHEs are the transporter proteins that play an impor-tant role in intracellular pH (pHi) regulation, osmolarity, cell differentiation, and cell volume, and their activation is associated with the development of [Na+]i-mediated increase in cellular Ca2+ overload and cell death. Phar-macological inhibition of these transport proteins pre-vents myocardial infarction and other heart diseases




like congestive heart failure, arteriosclerosis, pulmo-nary hypertension, insulin-dependent diabetes, tumor growth, fibrotic diseases, and organ/cell hypertrophy or hyperplasia in experimental animal models [16] as well as in clinical situations [17]. More recent studies have also implicated the role of these exchangers in the pathophysi-ology of brain diseases like epilepsy [18], dementia [19], stroke [20], and neuropathic pain [21]. Thus, the drugs targeting NHE may present important therapeutic alterna-tives for the diseases involving excessive NHE activation.

Phytopharmaceuticals, due to their high efficacy and safety profiles, have attracted worldwide attention

45

40

35

30

25

20

15

5

10

Per

cent

age

inhi

bitio

n (%

)


Figure 9 The NHE inhibitory activity (%) of different concentrations of the hydroalcoholic fraction of S. officinale.The values are expressed as mean ± SD in triplicate.

80

70

60

50

40

30

20

10

0

Per

cent

age

inhi

bitio

n (%

)


Figure 10 The NHE inhibitory activity (%) of different concentrations of the alkaloidal fraction of S. officinale.The values are expressed as mean ± SD in triplicate.

Table 2 The IC50 (μg/mL) values of hydroalcoholic and alkaloidal extracts of plants for NHE inhibitory action.

Sample no.

Plant

IC50, μg/mL

Hydroalcoholic extract

Alkaloidal extract

1. S. officinale 249.330 ± 1.155 4.233 ± 0.3792. M. domestica 2.350 ± 0.132 0.031 ± 0.0053. Musa × paradisiaca 7.967 ± 0.451 0.010 ± 0.0014. D. carota 37.667 ± 2.517 0.024 ± 0.002

of researchers, and plant-derived drugs have gained great importance in the past few years. Despite having large numbers of synthetic NHE inhibitors, until now no attention has been given to exploring plants for screen-ing natural NHE inhibitors. Our study presents the first report of plant-derived NHE inhibitors from four differ-ent plants: M. domestica, Musa × paradisiaca, D. carota, and S. officinale. The fruit of M. domestica is shown to be protective in experimental models of viral infections [22], ulcers [23], cancer [24], and heart diseases [25]. However, it is the first report showing the beneficial activity of the shoot portion of M. domestica. Musa × paradisiaca pro-duces significant hepatoprotective [26], antihypertensive [27], and leishmanicidal [28] activities in different experi-mental models. D. carota is reported to have antihyperten-sive [29], hepatoprotective [30], and anticancer activities [31]. It is also reported to possess antioxidant actions and improve kidney function, especially in ischemia-reperfu-sion injury in rats [32]. S. officinale has been mainly used for the topical treatment of painful muscle, low back pain, and joint complaints [33]. It is clinically proven to relieve pain, inflammation, and swelling of muscles and joints in the case of degenerative arthritis [34]. More recent in vitro




N

N

N

NH NH

HN

N

H

HNH

NH

NH

N

NHO

Cl

N

N

NHNCl

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

O2

HO HN

NH HN

NH

NH

OH

OH

OH

OH

OH

Na+ +

S

NH2

H2N

H2N

H2N

H2N

H2N

H2N

NH

O

O

O

O

O

O

O

O

O

O

O

Amiloride (1)

Cariporide (3) Trihydrated sodium ion (4)

Gamma-guanidino butyramide (6) Gamma-guanidino butyramide (7)

Gamma-guanidino propionic acid (8)

Carotamine (10)

Allantoin (9)

Guanidinium ion (5)

EIPA (2)

Figure 11 The alkaloidal constituents from M. domestica, Musa × paradisiaca, D. carota, and S. officinale showed structural similarities with the synthetic NHE inhibitors (amiloride, cariporide, EIPA, etc.).S. officinale contains allantoin as the major alkaloidal constituent, which is a guanidine-derived moiety. M. domestica (shoot) and Musa × paradisiaca (fruit) also contain guanidine compounds, i.e., γ-guanidinebutramide, γ-guanidinobutyric acid, and γ-guanidinopropionic acid.




experiments have also shown the MAO-B (monoamine oxidase involved in Parkinson’s disease) inhibitory poten-tial of S. officinale [35].

Previous data suggest that alkaloids are one of the major secondary metabolites of plants and hence the major contributor to the pharmacological activity of plants. The clinically employed plant-derived constituents are mainly alkaloidal in nature, and these include atropine in myopia, morphine in pain management, caffeine in treatment of migraine, vinca alkaloids (vincristine, vinblastine) in cancer treatment, ephedrine in asthma, and quinine as antimalarial agent. In the present study, the alkaloidal frac-tions of the selected plants were isolated and screened for NHE inhibitory activity. The different alkaloidal fractions exhibited potent NHE inhibitory activity, and among these alkaloidal fractions, the highest NHE inhibitory activity was shown by Musa × paradisiaca (IC50 = 0.010 ± 0.001 μg/mL) followed by D. carota (IC50 = 0.024 ± 0.002 μg/mL), M. domestica (IC50 = 0.031 ± 0.005 μg/mL), and S. officinale (IC50 = 4.233 ± 0.379 μg/mL). The alkaloidal fractions of Musa × paradisiaca, D. carota and M. domestica showed better NHE inhibition (in terms of IC50 value) than the standard NHE inhibitor EIPA (IC50 = 0.033 ± 0.004 μg/mL). Among the alkaloidal fractions of these plants, the alka-loidal fraction of M. domestica showed maximum NHE inhibition (99.35%) at 100 μg/mL followed by D. carota (96.75%), Musa × paradisiaca (94.96%), and S. officinale (78.42%).

Studies have shown that S. officinale produces liver toxicity and carcinogenicity, which is mainly due to pyr-rolizidine alkaloids [10]. The amount of total pyrrolizidine alkaloids in S. officinale is found to be 450–6000 μg/g [36]. The LD50 of most pyrrazilidine alkaloids (including symphytine and echimidine, the major contributor to S. officinale toxicity) is in the range of 34–300 mg/kg [37]. Furthermore, in vitro experiments employing the alkaloi-dal fraction of S. officinale showed no lymphocytic toxicity at 1.4 and 14 μg/mL, and chromosome aberrations were observed at much higher concentrations ranging from 140 to 1400 μg/mL [38]. The IC50 value (NHE inhibition) of the alkaloidal fraction of S. officinale in our study is 4.233 μg/mL; therefore, at such low concentration there may be very few chances of hepatotoxicity. A recent study by Gomes and coworkers also demonstrated that administration of 10% comfrey extract (three times a week, in a volume of 0.02 mL/kg) produces significant protection against the development of preneoplastic liver lesions [39].

The most important structural component that is shown to be highly linked with NHE inhibition is the guanidine group (Figure 11). Potent NHE inhibitors like amiloride (1), EIPA (2), and cariporide (3) possess the

guanidine moiety in their structure [40]. Recently devel-oped synthetic NHE inhibitors having the guanidino group have also shown potent NHE inhibitory action [41]. Natochin reported that in aqueous medium, the Na+ ions form trihydrated ionic moiety (4), which is similar to the guanidinium ion (5) in charge, shape, and size. Guanidine compounds, due to formation of guanidinium ions (5), may mimic the trihydrated Na+ ions (4). The inability of the binding site to distinguish between the drug and the Na+ ions may increase the binding of drug to the extracellular Na+ binding site of NHE, ultimately leading to significant NHE inhibition [40]. The fruit of Musa × paradisiaca and shoot of M. domestica contain guanidine moieties including γ-guanidinebutramide (6), γ-guanidinobutyric acid (7), and γ-guanidinopropionic acid (8) [11]. Therefore, the NHE inhibitory potential of these plants may be possibly attributed to these guani-dine alkaloids. The major alkaloid present in S. officinale is allantoin (0.6%–4.7%) (9) [42]. Allantoin possesses imidazolinecarbamide (urea-containing) moiety, which is very similar to the pyrazinecarboxamide (guanidine-containing part) moiety of amiloride, and hence these moieties may be regarded as bioisosteres. Therefore, the NHE inhibitory activity of the alkaloidal fraction of S. officinale may be possibly attributed to the presence of allantoin, a bioisostere of guanidine. Carotamine, the major alkaloidal constituent of D. carota, does not contain any guanidine group and is not structurally related to the synthetic inhibitors. Although the major-ity of the synthetic NHE inhibitors are guanidine deriva-tives, some non-guanidine-based compounds have also shown potent NHE inhibitory activity [43]. Therefore, the non-guanidine carotamine of D. carota may also contrib-ute to NHE inhibition.

The hydroalcoholic extract of M. domestica showed the highest NHE inhibitory potential, whereas the alkaloi-dal fraction of Musa × paradisiaca showed the most potent NHE inhibitory activity. However, phytochemical screen-ing of the individual plant components and evaluation of their NHE inhibitory potential can reveal the actual active constituent.

ConclusionsIt may be concluded that both hydroalcoholic and alka-loidal fractions of M. domestica, Musa × paradisiaca, D. carota, and S. officinale possess NHE inhibitory activi-ties. However, the alkaloidal fractions showed far better NHE inhibitory action as compared to corresponding




hydroalcoholic fractions and their potency was compara-ble to that of the synthetic NHE inhibitor, EIPA. However, the isolation and in vivo activity analysis of the active constituents responsible for the NHE inhibitory action of these plants need further research. The clinical implica-tions can be explored thereafter.

Acknowledgments: The authors are grateful to Depart-ment of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, India, and Dr. Kanchan, Head, Blood Bank, Govt. Rajindra Hospital, Patiala, Punjab, India, for providing platelet-rich plasma for the study.

Conflict of interest statement

Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article.Research funding: None declared.Employment or leadership: None declared.Honorarium: None declared.

Received July 12, 2013; accepted October 1, 2013; previously published online November 2, 2013

References1. Numata M, Orlowski J. Molecular cloning and characterization

of a novel (Na+, K+)/H+ exchanger localized to the trans-Golgi network. J Biol Chem 2001;276:17387–94.

2. Holthouser KA, Mandal A, Merchant ML, Schelling JR, Delamere NA, Valdes RR Jr., et al. Ouabain stimulates Na-K-ATPase through a sodium/hydrogen exchanger-1 (NHE-1)-dependent mechanism in human kidney proximal tubule cells. Am J Physiol Renal Physiol 2010;299:F77–90.

3. Shi Y, Kim D, Caldwell M, Sun D. The role of Na+/H+ exchanger isoform 1 in inflammatory responses: maintaining H+ homeostasis of immune cells. Adv Exp Med Biol 2013;961:411–8.

4. Qi H, Cao Y, Huang W, Liu Y, Wang Y, Li L, et al. Crucial role of calcium-sensing receptor activation in cardiac injury of diabetic rats. PLoS One 2013;8:e65147.

5. Bostanci MO, Bagirici F. Blocking of L-type calcium channels protects hippocampal and nigral neurons against iron neurotoxicity. Int J Neurosci 2013. Doi:10.3109/00207454.2013.813510. Epub ahead of print 2013 Jul 15.

6. Masereel B, Pochet L, Laeckmann D. An overview of inhibitors of Na+/H+ exchanger. Eur J Med Chem 2003;38:547–54.

7. Young M, Funder J. Mineralocorticoid action and sodium-hydrogen exchange: studies in experimental cardiac fibrosis. Endocrinology 2003;144:3848–51.

8. Park SL, Lee DH, Yoo SE, Jung YS. The effect of Na+/H+ exchanger-1 inhibition by sabiporide on blood-brain barrier dysfunction after ischemia/hypoxia in vivo and in vitro. Brain Res 2010;1366:189–96.

9. Cengiz P, Kleman N, Uluc K, Kendigelen P, Hagemann T, Akture E, et al. Inhibition of Na+/H+ exchanger isoform 1 is neuropro-tective in neonatal hypoxic ischemic brain injury. Antioxid Redox Signal 2011;14:1803–13.

10. Stickel F, Seitz HK. The efficacy and safety of comfrey. Public Health Nutr 2000;3:501–8.

11. Duke JA. Handbook of phytochemical constituents of GRAS herbs and other economic plants. USA: The CRC Press, 1992.

12. Singh K, Jaggi AS, Singh N. Exploring the ameliorative potential of Punica granatum in dextran sulfate sodium induced ulcerative colitis in mice. Phytother Res 2009;23:1565–74.

13. Takayama H, Matsuda Y, Masubuchi K, Ishida A, Kitajima M, Aimi N. Isolation, structure elucidation, and total synthesis

of two new Chimonanthus alkaloids, chimonamidine and chimonanthidine. Tetrahedron 2004;60:893–900.

14. Rosskopf D, Morgenstern E, Scholz W, Osswald U, Siffert W. Rapid determination of the elevated Na+/H+ exchange in platelets of patients with essential hypertension using an optical swelling assay. J Hypertens 1991;9:231–8.

15. Kusumoto K, Igata H, Abe A, Ikeda S, Tsuboi A, Imamiya E, et al. In vitro and in vivo pharmacology of a structurally novel Na+/H+-exchange inhibitor, T-162559. Br J Pharmacol 2002;135: 1995–2003.

16. Pedersen SF, O’Donnell ME, Anderson SE, Cala PM. Physiology and pathophysiology of Na+/H+ exchange and Na+/K+/2Cl– cotransport in the heart, brain, and blood. Am J Physiol Regul Integr Comp Physiol 2006;291:R1–25.

17. Rupprecht HJ, vom Dahl J, Terres W, Seyfarth KM, Richardt G, Schultheibeta HP, et al. Cardioprotective effects of the Na+/H+ exchange inhibitor cariporide in patients with acute anterior myocardial infarction undergoing direct PTCA. Circulation 2000;101:2902–8.

18. Ali A, Ahmad FJ, Pillai KK, Vohora D. Evidence of the antiepileptic potential of amiloride with neuropharmacological benefits in rodent models of epilepsy and behavior. Epilepsy Behav 2004;5:322–8.

19. Garbern JY, Neumann M, Trojanowski JQ, Lee VM, Feldman G, Norris JW, et al. A mutation affecting the sodium/proton exchanger, SLC9A6, causes mental retardation with tau deposition. Brain 2010;133:1391–402.

20. Ferrazzano P, Shi Y, Manhas N, Wang Y, Hutchinson B, Chen X, et al. Inhibiting the Na+/H+ exchanger reduces reperfusion injury: a small animal MRI study. Front Biosci (Elite Ed) 2011;3:81–8.

21. Muthuraman A, Jaggi AS, Singh N, Singh D. Ameliorative effects of amiloride and pralidoxime in chronic constriction injury and vincristine induced painful neuropathy in rats. Eur J Pharmacol 2008;587:104–11.

22. Hamauzu Y, Yasui H, Inno T, Kume C, Omanyuda M. Phenolic profile, antioxidant property, and anti-influenza viral activity of Chinese quince (Pseudocydonia sinensis Schneid.), quince (Cydonia oblonga Mill.), and apple (Malus domestica Mill.) fruits. J Agric Food Chem 2005;53:928–34.




23. Hamauzu Y, Irie M, Kondo M, Fujita T. Antiulcerative properties of crude polyphenols and juice of apple, and Chinese quince extracts. Food Chem 2008;108:488–95.

24. Olsson ME, Gustavsson KE, Andersson S, Nilsson A, Duan RD. Inhibition of cancer cell proliferation in vitro by fruit and berry extracts and correlations with antioxidant levels. J Agric Food Chem 2004;52:7264–71.

25. Lewis N, Ruud J. Apples in the American diet. Nutr Clin Care 2004;7:82–8.

26. Nirmala M, Girija K, Lakshman K, Divya T. Hepatoprotective activity of Musa paradisiaca on experimental animal models. Asian Pac J Trop Biomed 2012;2:11–5.

27. Sarkar C, Bairy KL, Rao NM, Udupa EG. Effect of banana on cold stress test and peak expiratory flow rate in healthy volunteers. Indian J Med Res 1999;110:27–9.

28. Accioly MP, Bevilaqua CM, Rondon FC, de Morais SM, Machado LK, Almeida CA, et al. Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions. Vet Parasitol 2012;187:79–84.

29. Gilani AH, Shaheen E, Saeed SA, Bibi S, Irfanullah, Sadiq M, et al. Hypotensive action of coumarin glycosides from Daucus carota. Phytomedicine 2000;7:423–6.

30. Singh K, Singh N, Chandy A, Manigauha A. In vivo antioxidant and hepatoprotective activity of methanolic extracts of Daucus carota seeds in experimental animals. Asian Pac J Trop Biomed 2012;2:385–8.

31. Butalla AC, Crane TE, Patil B, Wertheim BC, Thompson P, Thomson CA. Effects of a carrot juice intervention on plasma carotenoids, oxidative stress, and inflammation in overweight breast cancer survivors. Nutr Cancer 2012;64:331–41.

32. Afzal M, Kazmi I, Kaur R, Ahmad A, Pravez M, Anwar F. Comparison of protective and curative potential of Daucus carota root extract on renal ischemia reperfusion injury in rats. Pharm Biol 2013;51:856–62.

33. Giannetti BM, Staiger C, Bulitta M. Efficacy and safety of a comfrey root extract ointment in the treatment of acute upper or lower back pain: results of a double-blind, randomised, placebo controlled, multicentre trial. Br J Sport Med 2010;44:637–41.

34. Grube B, Grünwald J, Krug L. Efficacy of a comfrey root (Symphyti offic. radix) extract ointment in the treatment of patients with painful osteoarthritis of the knee: results of a double-blind, randomised, bicenter, placebo controlled trial. Phytomedicine 2007;14:2–10.

35. Mazzio E, Deiab S, Park K, Soliman K. High throughput screening to identify natural human monoamine oxidase B inhibitors. Phytother Res 2013;27:818–28.

36. Mroczek T, Glowniak K, Wlaszczyk A. Simultaneous determination of N-oxides and free bases of pyrrolizidine alkaloids by cation-exchange solid-phase extraction and ion-pair high-performance liquid chromatography. J Chromatogr A 2002;949:249–62.

37. Hirono I, Haga M, Fujii M, Matsuura S, Matsubara N, Nakayama M, et al. Induction of hepatic tumors in rats by senkirkine and symphytine. J Natl Cancer Inst 1979;63:469–72.

38. Behninger C, Abel G, Röder E, Neuberger V, Göggelmann W. Studies on the effect of an alkaloid extract of Symphytum officinale on human lymphocyte cultures. Planta Med 1989;55:518–22.

39. Gomes MF, de Oliveira Massoco C, Xavier JG, Bonamin LV. Comfrey (Symphytum officinale) and experimental hepatic carcinogenesis: a short-term carcinogenesis model study. Evid Based Complement Alternat Med 2010;7:197–202.

40. Natochin YV. Mechanism of drug action on ion and water transport in renal tubular cells. Progr Drug Res 1982;26: 87–142.

41. Bhatt HG, Patel PK. Pharmacophore modeling, virtual screening and 3D-QSAR studies of 5-tetrahydroquinolinylidine aminoguanidine derivatives as sodium hydrogen exchanger inhibitors. Bioorg Med Chem Lett 2012;22:3758–65.

42. Dennis R, Dezelak C, Grime J. Studies on Symphytum species – HPLC determination of allantoin. Acta Pharm Hung 1987;57:267–74.

43. Lorrain J, Briand V, Favennec E, Duval N, Grosset A, Janiak P, et al. Pharmacological profile of SL 59.1227, a novel inhibitor of the sodium/hydrogen exchanger. Br J Pharmacol 2000;131:1188–94.



Documents

Sodium-hydrogen exchanger inhibitory potential of Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale