Evidence-Based Complementary and Alternative MedicineVolume 2011 (2011), Article ID 802937, 8 pagesdoi:10.1155/2011/802937

Research Article

Hemidesmus indicus and Hibiscus rosa-sinensis Affect Ischemia Reperfusion Injury in Isolated Rat Hearts

Vinoth Kumar Megraj Khandelwal,1,2 R. Balaraman,² Dezider Pancza,¹ and Táňa Ravingerová¹

¹Institute for Heart Research, Centre of Excellence for Cardiovascular Research, Slovak Academy of Sciences, 840 05 Bratislava, Slovakia²Pharmacy Department, Faculty of Technology & Engineering, M. S. University of Baroda, 390 001 Vadodara, Gujarat, India

Received 9 June 2009; Revised 29 December 2009; Accepted 10 July 2010

Copyright © 2011 Vinoth Kumar Megraj Khandelwal et al. Vinoth Kumar Megraj Khandelwal 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.

Abstract.

Hemidesmus indicus (L.) R. Br. (HI) and Hibiscus rosa-sinensis L. (HRS) are widely used traditional medicine. We investigated cardioprotective effects of these plants applied for 15 min at concentrations of 90, 180, and 360 g/mL in Langendorff-perfused rat hearts prior to 25-min global ischemia/120-min reperfusion (I/R). Functional recovery (left ventricular developed pressure—LVDP, and rate of development of pressure), reperfusion arrhythmias, and infarct size (TTC staining) served as the endpoints. A transient increase in LVDP (32%–75%) occurred at all concentrations of HI, while coronary flow (CF) was significantly increased after HI 180 and 360. Only a moderate increase in LVDP (21% and 55%) and a tendency to increase CF was observed at HRS 180 and 360. HI and HRS at 180 and 360 significantly improved postischemic recovery of LVDP. Both the drugs dose-dependently reduced the numbers of ectopic beats and duration of ventricular tachycardia. The size of infarction was significantly decreased by HI 360, while HRS significantly reduced the infarct size at all concentrations in a dose-dependent manner. Thus, it can be concluded that HI might cause vasodilation, positive inotropic effect, and cardioprotection, while HRS might cause these effects at higher concentrations. However, further study is needed to elucidate the exact mechanism of their actions.

1. Introduction

There is an increasing demand for the herbal drug treatment for various ailments, and many plant drugs from traditional medicine like Ayurveda are being explored globally.

Hemidesmus indicus (L.) R. Br. (Asclepiadaceae; HI) is a twining shrub used as folk medicine and an ingredient in Ayurvedic and Unani preparations. It is known as Indian Sarsaparilla (English), Ananta, Gopasuta, Sariva (Sanskrit), Anantamul (Hindi), Ushba Hindi (Urdu), Ushbahindi (Persian), and Irimusk (Sinhalese) [1]. The root extract of HI was used for preparing herbal soft drinks and as food during famine [2]. The plant has been used traditionally for the treatment of blood disorders, low digestion, anorexia, diarrhea, asthma, fever, cough, itching, and skin diseases including leprosy [1]. Various effects of HI, such as hypoglycemic [3], hypolipidemic [4], antioxidant, antithrombotic [5], antiinflammatory [6], antiulcerogenic [7], hepatoprotective [8], renoprotective [9], and neutralization of viper venom [10] have been reported. It mainly comprises saponins, tannins, hemidesmine, hemidesmol, hemidesterol, stearoptin, pregnane glycosides, β-sitosterol, indicusin, coumarin, volatile oils, triterpines, flavonoids, and so forth [1, 7].

Hibiscus rosa-sinensis L. (Malvaceae; HRS) is an ornamental plant native to China, and found in India and Philippines. It is called as Chinese rose, Shoe flower (English), Arkapriya, Japapushpa (Sanskrit), Jasund (Hindi), Angharee-hind (Persian), and Wadamal (Sinhalese) [1]. In some regions, the flowers of HRS are eaten raw or cooked [11] and made into a kind of pickle or used as a dye for coloring foods, such as preserved fruits and cooked vegetables [12, 13]. The young leaves are sometimes used as a substitute for spinach [12, 13], while the roots are also edible, but are fibrous, mucilaginous, and without very much flavor [14]. In addition to its traditional value as emollient, demulcent, emmenagogue, antiinflammatory, refrigerant, aphrodisiac, anodyne, and laxative, various researchers had described the use of the flower to treat heart disorders [1, 10, 15]. Sachdewa and Khemani [16] demonstrated the antidiabetic activity of HRS in diabetic rats and the effect was comparable with glibenclamide. It has been also shown to be beneficial in fever and bronchial catarrh [16]. It is known to possess various activities like antidiarrheal, antiphologistic, antispermatogenic, androgenic, antitumor [16], antiestrogenic [17], antiimplantation [18, 19], wound Healing [20], anticonvulsant [21], and so forth. It mainly consists of flavonoids, anthocyanins, quercetin, cyanidin, kaempferol, hydrocitric acid, and so forth [1, 22].

However, till date, no research work has been performed to study the effects of HI and HRS in isolated heart preparation. Hence, this study was initiated to evaluate the potential myocardial protective effect of both the drugs in the model of ischemia-reperfusion (I/R) injury in rat hearts in vitro.

2. Materials and Methods

2.1. Preparation of the Extract

Standardized dry extracts of HI and HRS (prepared as below) were kindly gifted by Rumi herbal research institute, Chennai, India. In brief, dried roots of HI were coarsely powdered and refluxed with 50% ethanol by hot percolation method and extracted. The yield was 23.18% of black-brown extractives containing 2.87 mg % of saponins and 1.62 mg % tannins, heavy metals—arsenic—not more than (NMT) 1 parts per million (ppm), lead NMT 1 ppm, and E. coli and Salmonella were absent. Dried flowers of HRS were coarsely powdered and refluxed with 80% ethanol by hot percolation method and extracted. It yielded 15.6% of dark-brown extractives containing 4.16 mg % hydrocitric acid, heavy metals—arsenic—NMT 1 ppm, lead NMT 1 ppm, and E. coli and Salmonella were absent.

The dry extract (600 mg) was added to 15 mL of boiling distilled water and boiled for 2 min. The decoction was cooled and centrifuged to separate any undissolved material and the supernatant was considered as a stock solution containing 40 mg/mL of the extract [23]. The decoction was prepared fresh every day.

2.2. Animals

Male adult Wistar rats (230–270 g) were used in this study. Rats were housed under standard conditions and supplied with drinking water and food ad libitum. All procedures and experimental protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and approved by the Animal Health and Animal Welfare Division of the State veterinary and Food Administration of the Slovak Republic.

2.3. Perfusion Technique

The rats were anesthetized (sodium pentobarbitone, 60 mg/kg, i.p.) and heparinized (500 IU, i.p.) [24]. Hearts were excised and rapidly mounted on the Langendorff perfusion apparatus. Retrograde perfusion in a nonrecirculating mode was established at a constant perfusion pressure of 70 mmHg and 37C. Krebs-Henseleit buffer (KHB) gassed with 95% O₂ and 5% CO₂ (pH 7.4) containing (mM) NaCl 118.0, KCl 4.7, MgSO₄·7H₂O 1.18, NaHCO₃ 25.0, KH₂PO₄ 1.18, CaCl₂·2H₂O 2.25, and glucose 11.1 was used as the perfusion medium. The perfusate was filtered through a 5-m porosity filter (Millipore), before it entered the heart. An epicardial electrogram was registered using two stainless steel electrodes, one attached to the apex of the heart and the other to the aortic cannula.

Left ventricular (LV) pressure was measured using a nonelastic water-filled balloon inserted into the left ventricle via the left atrium (adjusted to obtain end-diastolic pressure of 4–7 mmHg) and connected to a pressure transducer (MLP844 Physiological Pressure Transducer, ADInstruments). Left ventricular developed pressure (LVDP, systolic minus diastolic pressure), maximal rate of pressure development (+dP/dt) as an index of contraction, heart rate (HR; derived from electrogram), and coronary flow (CF) were monitored continuously. The hearts were allowed to stabilize (15 min) before further interventions. Baseline values of functional parameters were recorded after stabilization and recording of the data was performed until the end of an experiment, except for the contractile function, as the balloon was deflated after 40 min of R. Heart function and arrhythmias were analyzed using PowerLab/8SP Chart 5 software (ADInstruments).

Recovery of function was expressed as a percentage of preischemic baseline values.

2.4. Experimental Protocol

The experimental protocol consisted of a stabilization period (15 min), perfusion with drugs dispersed in KHB at the required concentrations for 15 min, global ischemia (25 min), and reperfusion period (120 min).

All animals were randomly divided to the following groups (seven rats per group).(1)Control (C): hearts were perfused with KHB throughout the experiment. (2)HI 90: hearts were perfused with HI extract at a concentration of 90g/mL in KHB for 15 min, prior to ischemia and reperfusion with KHB.(3)HI 180: hearts were perfused with HI extract at a concentration of 180g/mL in KHB for 15 min, prior to ischemia and reperfusion with KHB.(4)HI 360: hearts were perfused with HI extract at a concentration of 360g/mL in KHB for 15 min, prior to ischemia and reperfusion with KHB.(5)HRS 90: hearts were perfused with HRS extract at a concentration of 90g/mL in KHB for 15 min, prior to ischemia and reperfusion with KHB.(6)HRS 180: hearts were perfused with HRS extract at a concentration of 180g/mL in KHB for 15 min, prior to ischemia and reperfusion with KHB.(7)HRS 360: hearts were perfused with HRS extract at a concentration of 360g/mL in KHB for 15 min, prior to ischemia and reperfusion with KHB.

2.5. Quantification of Arrhythmias

Susceptibility to ventricular arrhythmias was analyzed from the electrogram recording during the first 10 min of R, as per the guidelines for the study of ischemia- and reperfusion-induced arrhythmias, known as the Lambeth conventions [25]. We focused on the measurement of the total number of ventricular premature beats (VPB), as well as on the total duration of the episodes of ventricular tachycardia (VT), which was defined as a run of four or more consecutive ectopic beats.

2.6. Infarct Size Determination

The measurement of infarct size using triphenyl tetrazolium staining was essentially identical to that described by Ravingerová et al. [26]. In brief, at the end of R, the hearts were stained with 1% 2,3,5-triphenyl tetrazolium chloride (Sigma, USA) dissolved in 0.1 M phosphate buffer (pH 7.4). The hearts were then stored overnight in 10% neutral formaldehyde solution and cut perpendicularly to the long axis of the ventricle into 1-mm thick slices. The infarct area (IA) and the area at risk (AR), which in the setting of global ischemia was the whole mass of the left ventricle, were measured by a computerized planimetric method. The infarct size was normalized to the size of the area at risk (IA/AR).

2.7. Statistical Evaluation

The data were expressed as mean ± S.E.M. The statistical analysis was performed with one-way ANOVA followed by Newman-Keuls multiple comparison test or two-way ANOVA followed by Bonferroni post tests. Differences were considered significant when .

3. Results

3.1. Preischemia

After 15-min stabilization of all the hearts with KHB, the perfusion was switched to the drug-containing KHB solution. During perfusion with HI, we observed a transient increase in LVDP that occurred during perfusion with HI 90 (32%), HI 180 (52%), and HI 360 (75%) when compared with predrug values. Table 1 shows that at the end of 15 min perfusion with HI, LVDP and HR were similar to those of pre-drug values at all doses, while CF was significantly increased by HI 180 () and HI 360 () when compared with pre-drug values (8.8 ± 0.3 mL/min).

Table 1: Effect of 15-min perfusion with HI and HRS on hemodynamic parameters of the isolated rat heart. LVDP: left ventricular developed pressure (LV systolic minus diastolic pressure); HR: heart rate; CF: coronary flow; BD: Before Drug (pre-drug values). , versus BD (baseline).

BD
HI
BD
HRS

pre-ischemia
pre-ischemia

HI 90
HI 180
HI 360
HRS 90
HRS 180
HRS 360

LVDP (mmHg)
89.5 ± 9.1
83.9 ± 2.2
84.8 ± 1.9
82.2 ± 7.5
83.9 ± 4.0
77.6 ± 3.5
82.6 ± 3.2
80.7 ± 2.8

HR (BPM)
302.6 ± 22.6
290.1 ± 31.5
286.6 ± 9.7
299.4 ± 33.1
305.8 ± 11.4
284.1 ± 9.3
290.0 ± 23.8
292.1 ± 13.8

CF (mL/min)
8.8 ± 0.3
14.1 ± 2.8
17.7 ± 2.5
20.6 ± 2.7
8.8 ± 0.4
10.5 ± 0.9
11.0 ± 2.2
12.4 ± 1.6

When the hearts were perfused with HRS, a transient increase in LVDP at HRS 180 (21%) and HRS 360 (55%) was observed when compared with pre-drug values. At the end of 15-min perfusion with HRS, there was only a tendency to increase CF, and no significant changes in LVDP and HR at all doses (Table 1).

3.2. Post-Ischemic Recovery of Function

3.2.1. LVDP

Figure 1(a) shows the time course of post-ischemic recovery of LVDP. At 40 min of R, HI 180 and HI 360 significantly ( and , resp.) improved the recovery of LVDP to 52.7 and 81.2%, respectively, when compared with 19.4% in the nontreated C group. The changes in the LVDP recovery induced by HI 90 were not significant at any time point.

(a)

(b)

Figure 1: Effect of HI (a) and HRS (b) on the time course of LVDP recovery after I/R expressed as a percentage of the baseline values. , , versus C.

Figure 1(b) depicts the time course of post-ischemic LVDP recovery in the presence of HRS. HRS 90 did not exert any effect when compared with C. However, HRS 180 exerted a more pronounced effect on recovery of LVDP and was significant at few time points. HRS 360 showed a significant recovery at all time points after 20 min of R. At the end of 40 min of I/R, both HRS 180 and HRS 360 induced almost similar recovery which was significantly () better when compared with C.

3.2.2. +dP/dt

Figure 2(a) shows the recovery of +dP/dt at 40 min of I/R. When compared with C, the recovery was significant when the hearts were perfused with HI 90 (), HI 180 (), and HI 360 (). The recovery of +dP/dt was dose-dependent, that is, HI 360 induced a significantly higher recovery () than HI 90 and HI 180.

(a)

(b)

Figure 2: Effect of HI (a) and HRS (b) on +dP/dt at 40 min of R. , , , .

The recovery of +dP/dt was significantly () better when compared with C, when the hearts were perfused with HRS 180 and HRS 360. HRS 180 and HRS 360 showed a significantly stronger effect () than HRS 90 (Figure 2(b)).

3.2.3. LVEDP

Figure 3 shows the recovery of LVEDP, which was significantly lowered by all concentrations of HI, HI 90 (), HI 180 (), and HI 360 (). Furthermore, perfusion with HI 360 led to a significantly better recovery of LVEDP when compared with HI 90 and HI 180 ( and , resp.).

Figure 3: Effect of HI on LVEDP at 40 min of R. , , .

HRS did not cause an improvement of LVEDP recovery (in mmHg) at any concentration HI 90 (77.8 ± 3.5), HI 180 (68.2 ± 1.8), HI 360 (67.3 ± 2.4), in comparison to C (78.6 ± 4.7).

3.2.4. Arrhythmias

HI exerted a significant antiarrhythmic protection at HI 90, HI 180, and HI 360 manifested by a reduced number of PVB (, , and , resp.). The protection was also dose dependent (Figure 4(a)), as perfusion with HI 360 resulted in a significantly lower number of PVB than HI 90 () and HI 180 (), while HI 180 induced a significantly () lower number of PVB than HI 90. There was also a significantly () shorter duration of episodes of VT at HI 180 (9.8 ± 3.0 s) and HI 360 (5.3 ± 2.8 s), but not at HI 90 (28.5 ± 9.7 s), when compared with 39 ± 6.5 s in C.

(a)

(b)

Figure 4: Effect of HI (a) and HRS (b) on arrhythmias (PVB) during the first 10 min of R. , , .

Figure 5: Potential mechanisms for the beneficial effects of HI and HRS in I/R rat hearts.

Interestingly, HRS 90, HRS 180, and HRS 360 significantly () reduced the number of PVB and decreased the duration of VT () to 19.8 ± 6.8, 13.7 ± 5.6, and 13.2 ± 5.1 s, respectively, when compared with nontreated C (Figure 4(b)).

3.2.5. Infarct Size

The size of infarction (percentage of the risk area; IA/AR) was significantly reduced only after administration of HI 360 (20.3 ± 1.4%; ) and not at HI 90 (33.5 ± 5.3%) and HI 180 (30.1 ± 4.9%), when compared with C (43.2 ± 2.4%).

The infarct size was significantly () smaller at HRS 90 (29.4 ± 4.7%), HRS 180 (24.8 ± 3.6%), and HRS 360 (22.5 ± 2.4%), when compared with C.

4. Discussion

The extracts of HI and HRS were tested for their potential protective effect on I/R-induced lethal injury and functional deterioration. The effects of the extracts were evaluated before I and during R. The widely used model of 25-min global I for optimum functional deterioration [27], followed by 120 min of R for sufficient development of necrosis and infarct size determination in the Langendorff setup [28–30] was utilized.

HI had a dose-dependent effect on the recovery of LVDP and +dP/dt. HRS had a similar effect on the recovery of LVDP and +dP/dt at HI 180 as in HRS 360 and no protection was observed at HRS 90, suggesting that HRS 180 is the minimum dose required to increase the recovery of contractile function. The significantly better recovery of LVEDP and attenuation of post-ischemic diastolic dysfunction at all three doses of HI infers that HI could improve myocardial relaxation and may reduce the edema caused by I/R injury [31]. In contrast, HRS did not have any significant activity towards the relaxation of the cardiac muscle at any dose.

HI could protect the heart from arrhythmias at all doses in a dose-dependent manner manifested by a reduced number of PVB (extra heart beat caused by abnormal electrical activity). In addition, a significantly lower total duration of episodes of VT (rapid heart rhythm) was observed at higher doses. Interestingly, HRS at all doses had a significant protection against arrhythmias. The size of infarction (death of a macroscopic area of cardiac tissue) was significantly reduced by HI at the highest dose, while HRS significantly lowered the infarct size at all the doses. In comparison, after 25-min I, HI exerted a higher protective activity against functional deterioration and a moderate protection against arrhythmias and infarct size, while HRS had a moderate effect on functional recovery and a stronger protection in terms of antiarrhythmic effect and infarct size limitation.

In two different studies recently performed in our laboratory in a similar model, N-acetylcysteine (4 mM) [27] and quercetin (15M) [32] were found to protect the myocardium against I/R. In the present study, the recovery of various parameters in the presence of both the extracts was comparable with that of N-acetylcysteine (LVDP—50%) and quercetin (LVDP—39.4%, +dP/dt—30.9%, IA/AR—14.3%). At higher concentrations, the extracts showed a better effect than quercetin [27, 32]. Functional deterioration and severe arrhythmias upon reperfusion were found to be related, to a certain extent, to an excessive generation of reactive oxygen species (ROS) during prolonged I/R [33–36]. ROS may also participate in I/R injury through the depression of sarcoplasmic reticulum (SR) Ca²⁺ handling by modulating gene expression in the I/R heart [37]. This has been verified by the efficacy of antioxidants and scavengers in the experimental settings of acute I/R [38–41]. Similarly, pre-treatment with antioxidants, such as melatonin [38, 39] and N-acetylcysteine [27] prior to I reduced the severity and duration of R-induced ventricular arrhythmias in isolated perfused rat hearts, attenuated calcium overload of the heart [40, 42], and improved post-ischemic recovery of the contractile function [38].

The previously reported antioxidant effect of HI [5, 43] may be associated with tannins, one of the main constituents [44]. Likewise, saponins have also been shown to have beneficial effects on cardiovascular diseases [45]. Flavonoids produce vasodilation by regulating endothelial nitric oxide (NO) production [46] and interaction with ion channels [47]. Moreover, flavonoids are known to protect the I/R-induced myocardial injury by their multifaceted properties, such as antioxidant, antiinflammatory, vasodilatory, and antiplatelet aggregation [47]. Therefore, it is conceivable that the cardioprotective effect of HI may be related to the combined effects of saponins, tannins, and flavonoids. HRS has been shown to enhance the endogenous antioxidant activity and protect the heart from isoproterenol-induced injury [48]. Quercetin has been shown to reduce blood pressure and exhibit endothelium-dependent vasodilation by enhancing eNOS activity [46, 49–51]. In addition, cyanidin and quercetin are known to possess antioxidant activity [48, 49]. Kim et al. [52] have shown that kaempferol protected the cardiac muscle cells against I/R-induced damage by increasing the expression of antiapoptotic protein and downregulating the expression of endoplasmic reticulum stress proteins. Thus, the combined effect of constituents of HRS, such as quercetin, cyanidin, and kaempferol might be responsible for the beneficial effects (Figure ).

In conclusion, HI might cause vasodilation, positive inotropic effect, and cardioprotection, while HRS might cause these effects at higher concentrations. In addition, based on the drug effects observed at lower doses, it could be suggested that the suppression of arrhythmias results in a smaller size of infarction than that achieved by the protection against contractile dysfunction. However, further study is required to explore the in vivo activity of both the plants.

Acknowledgments

The study was supported by the Grant VEGA SR 2/0173/08. The authors acknowledge SAIA for providing scholarship to V. K. M. Khandelwal and Rumi herbal research institute for providing the extracts.

References

J. Anjaria, M. Parabia, G. Bhatt, and R. Khamar, Nature Heals, a Glossary of Selected Indigenous Medicinal Plants of India, SRISTI Innovations, Ahmedabad, India, 2002.

A. H. Rajasab and I. Mahamad, “Documentation of folk knowledge on edible wild plants of north Karnataka,” Indian Journal of Traditional Knowledge, vol. 3, pp. 419–429, 2004.

S. Murshed, B. Rokeya, N. Nahar, et al., “Hypoglycemic and hypolipidemic effect of Hemidesmus indicus root on diabetic model rats,” Diabetes Research, vol. 39, pp. 15–23, 2005.

K. N. Bopanna, N. Bhagyalakshmi, S. P. Rathod, R. Balaraman, and J. Kannan, “Cell culture derived Hemidesmus indicus in the prevention of hypercholesterolemia in normal and hyperlipidemic rats,” Indian Journal of Pharmacology, vol. 29, no. 2, pp. 105–109, 1997.

N. K. Mary, C. R. Achuthan, B. H. Babu, and J. Padikkala, “In vitro antioxidant and antithrombotic activity of Hemidesmus indicus (L) R.Br,” Journal of Ethnopharmacology, vol. 87, no. 2-3, pp. 187–191, 2003.

I. Lampronti, M. T. H. Khan, M. Borgatti, N. Bianchi, and R. Gambari, “Inhibitory effects of Bangladeshi medicinal plant extracts on interactions between transcription factors and target DNA sequences,” Evidence-Based Complementary and Alternative Medicine, vol. 5, no. 3, pp. 303–312, 2008.

A. Anoop and M. Jegadeesan, “Biochemical studies on the anti-ulcerogenic potential of Hemidesmus indicus R.Br. var. indicus,” Journal of Ethnopharmacology, vol. 84, no. 2-3, pp. 149–156, 2003.

M. Prabakan, R. Anandan, and T. Devaki, “Protective effect of Hemidesmus indicus against rifampicin and isoniazid-induced hepatotoxicity in rats,” Fitoterapia, vol. 71, no. 1, pp. 55–59, 2000.

M. S. Kotnis, P. Patel, S. N. Menon, and R. T. Sane, “Renoprotective effect of Hemidesmus indicus, a herbal drug used in gentamicin-induced renal toxicity,” Nephrology, vol. 9, no. 3, pp. 142–152, 2004.

M. I. Alam, B. Auddy, and A. Gomes, “Viper venom neutralization by Indian medicinal plant (Hemidesmus indicus and Pluchea indica) root extracts,” Phytotherapy Research, vol. 10, no. 1, pp. 58–61, 1996.

R. N. Chopra, S. L. Nayar, and I. C. Chopra, Glossary of Indian Medicinal Plants, National Institute of Science Communication, New Delhi, India, 1956.

S. Facciola, Cornucopia II: A Source Book of Edible Plants, Kampong Publications, Vista, Calif, USA, 1998.

G. Kunkel, Plants For Human Consumption: An Annotated Checklist of the Edible Phanerogams and Ferns, Koeltz Scientific Books, 1984.

A. B. Cribb and J. W. Cribb, Wild Food in Australia, Fontana, Sydney, Australia, 1982.

J. A. Vinson, Y. A. Dabbagh, M. M. Serry, and J. Jang, “Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease,” in Cure of Heart Diseases with Ayurvedic Drugs, U. D. Sharma, Ed., pp. 95–96, Sachitra Ayurved, 1994.

A. Sachdewa and L. D. Khemani, “Effect of Hibiscus rosa sinensis Linn. ethanol flower extract on blood glucose and lipid profile in streptozotocin induced diabetes in rats,” Journal of Ethnopharmacology, vol. 89, no. 1, pp. 61–66, 2003.

S. D. Kholkute and K. N. Udupa, “Antiestrogenic activity of Hibiscus rosa sinensis Linn. flowers,” Indian Journal of Experimental Biology, vol. 14, no. 2, pp. 175–176, 1976.

D. R. K. Murthy, C. Madhusudana Reddy, and S. B. Patil, “Effect of benzene extract of Hibiscus rosa sinensis on the estrous cycle and ovarian activity in albino mice,” Biological and Pharmaceutical Bulletin, vol. 20, no. 7, pp. 756–758, 1997.

N. Vasudeva and S. K. Sharma, “Post-coital antifertility activity of Hibiscus rosa-sinensis Linn. roots,” Evidence-Based Complementary and Alternative Medicine, vol. 5, no. 1, pp. 91–94, 2008.

B. Shivananda Nayak, S. Sivachandra Raju, F. A. Orette, and A. V. Chalapathi Rao, “Effects of Hibiscus rosa sinensis L (Malvaceae) on wound healing activity: a preclinical study in a Sprague Dawley rat,” International Journal of Lower Extremity Wounds, vol. 6, no. 2, pp. 76–81, 2007.

V. S. Kasture, C. T. Chopde, and V. K. Deshmukh, “Anticonvulsive activity of Albizzia lebbeck, Hibiscus rosa sinesis and Butea monosperma in experimental animals,” Journal of Ethnopharmacology, vol. 71, no. 1-2, pp. 65–75, 2000.

H. Yamasaki, H. Uefuji, and Y. Sakihama, “Bleaching of the red anthocyanin induced by superoxide radical,” Archives of Biochemistry and Biophysics, vol. 332, no. 1, pp. 183–186, 1996.

S. Suchalatha and C. S. Shyamala Devi, “Effect of arogh—a polyherbal formulation on the marker enzymes in isoproterenol induced myocardial injury,” Indian Journal of Clinical Biochemistry, vol. 19, no. 2, pp. 184–189, 2004.

A. Adameová, T. Ravingerová, P. Švec, V. Faberová, and M. Kuželová, “The myocardial infarct size-limiting and antiarrhythmic effects of acyl-CoA: cholesterol acyltransferase inhibitor VULM 1457 protect the hearts of diabetic-hypercholesterolaemic rats against ischaemia/reperfusion injury both in vitro and in vivo,” European Journal of Pharmacology, vol. 576, no. 1–3, pp. 114–121, 2007.

M. J. A. Walker, M. J. Curtis, D. J. Hearse, R. W. F. Campbell, M. J. Janse, D. M. Yellon, S. M. Cobbe, S. J. Coker, J. B. Harness, D. W. G. Harron, A. J. Higgins, D. G. Julian, M. J. Lab, A. S. Manning, B. J. Northover, J. R. Parratt, R. A. Riemersma, E. Riva, and D. C. Russell, “The Lambeth conventions: guidelines for the study of arrhythmias in ischaemia, infarction, and reperfusion,” Cardiovascular Research, vol. 22, no. 7, pp. 447–455, 1988.

T. Ravingerová, J. Neckář, and F. Kolář, “Ischemic tolerance of rat hearts in acute and chronic phases of experimental diabetes,” Molecular and Cellular Biochemistry, vol. 249, no. 1-2, pp. 167–174, 2003.

J. Matejíková, J. Kucharská, M. Pintérová, D. Pancza, and T. Ravingerová, “Protection against ischemia-induced ventricular arrhythmias and myocardial dysfunction conferred by preconditioning in the rat heart: involvement of mitochondrial channels and reactive oxygen species,” Physiological Research, vol. 58, no. 1, pp. 9–19, 2009.

P. Pasdois, C. L. Quinlan, A. Rissa, L. Tariosse, B. Vinassa, A. D. T. Costa, S. V. Pierre, P. Dos Santos, and K. D. Garlid, “Ouabain protects rat hearts against ischemia-reperfusion injury via pathway involving src kinase, mito, and ROS,” American Journal of Physiology, vol. 292, no. 3, pp. H1470–H1478, 2007.

S. Bae and L. Zhang, “Gender differences in cardioprotection against ischemia/reperfusion injury in adult rat hearts: focus on akt and protein kinase C signaling,” Journal of Pharmacology and Experimental Therapeutics, vol. 315, no. 3, pp. 1125–1135, 2005.

A. Hobbs, P. Foster, C. Prescott, R. Scotland, and A. Ahluwalia, “Natriuretic peptide receptor-C regulates coronary blood flow and prevents myocardial ischemia/reperfusion injury: novel cardioprotective role for endothelium-derived C-type natriuretic peptide,” Circulation, vol. 110, no. 10, pp. 1231–1235, 2004.

H. V. Schaff, V. L. Gott, R. A. Goldman, J. W. Frederiksen, and J. T. Flaherty, “Mechanism of elevated left ventricular end-diastolic pressure after ischemic arrest and reperfusion,” The American journal of physiology, vol. 240, no. 2, pp. H300–307, 1981.

M. Barteková, S. Čarnická, D. Pancza, M. Ondrejčáková, A. Breier, and T. Ravingerova, “Acute treatment with polyphenol quercetin improves postischemic recovery of isolated perfused rat hearts after global ischemia,” Canadian Journal of Physiology and Pharmacology, vol. 88, no. 4, pp. 465–471, 2010.

T. Ravingerová, A. Džurba, N Vrbjar, N. Tribulová, A. Ziegelhöeffer, and J. Slezák, “Free oxygen radicals-induced impairment of heart function: the role of Na, K-ATPase,” Pharmacol (Life Sci Adv), vol. 13, pp. 133–140, 1994.

T. Ravingerová, J. Slezák, N. Tribulová, A. Džurba, B. Uhrík, and A. Ziegelhöffer, “Free oxygen radicals contribute to high incidence of reperfusion-induced arrhythmias in isolated rat heart,” Life Sciences, vol. 65, no. 18-19, pp. 1927–1930, 1999.

P. S. Ray, G. Maulik, G. A. Cordis, A. A. E. Bertelli, A. Bertelli, and D. K. Das, “The red wine antioxidant resveratrol protects isolated rat hearts from ischemia reperfusion injury,” Free Radical Biology and Medicine, vol. 27, no. 1-2, pp. 160–169, 1999.

P. Kaplan, E. Babusikova, J. Lehotsky, and D. Dobrota, “Free radical-induced protein modification and inhibition of -ATPase of cardiac sarcoplasmic reticulum,” Molecular and Cellular Biochemistry, vol. 248, no. 1-2, pp. 41–47, 2003.

J. W. Hoffman Jr., T. B. Gilbert, R. S. Poston, and E. P. Silldorff, “Myocardial reperfusion injury: etiology, mechanisms, and therapies,” Journal of Extra-Corporeal Technology, vol. 36, no. 4, pp. 391–411, 2004.

O. Szárszoi, G. Asemu, J. Vaněček, B. Ošt'ádal, and F. Kolář, “Effects of melatonin on ischemia and reperfusion injury of the rat heart,” Cardiovascular Drugs and Therapy, vol. 15, no. 3, pp. 251–257, 2001.

R. Važan, D. Pancza, I. Béder, and J. Styk, “Ischemia-reperfusion injury—antiarrhythmic effect of melatonin associated with reduced recovering of contractility,” General Physiology and Biophysics, vol. 24, no. 3, pp. 355–359, 2005.

R. Važan, J. Styk, I. Béder, and D. Pancza, “Effect of melatonin on the isolated heart in the standard perfusion conditions and in the conditions of calcium paradox,” General Physiology and Biophysics, vol. 22, no. 1, pp. 41–50, 2003.

J. Matejíková, J. Kucharská, D. Pancza, and T. Ravingerová, “The effect of antioxidant treatment and NOS inhibition on the incidence of ischaemia-induced arrhythmias in the diabetic rat heart,” Physiological Research, vol. 57, no. 2, pp. S55–S60, 2008.

E. Sahna, H. Parlakpinar, Y. Turkoz, and A. Acet, “Protective effects of melatonin on myocardial ischemia-reperfusion induced infarct size and oxidative changes,” Physiological Research, vol. 54, no. 5, pp. 491–495, 2005.

G. M. M. Rao, Ch. Venkateswararao, A. K. S. Rawat, P. Pushpangadan, and A. Shirwaikar, “Antioxidant and antihepatotoxic activities of Hemidesmus indicus R. Br,” Acta Pharmaceutica Turcica, vol. 47, no. 2, pp. 107–113, 2005.

C.-Y. Hong, C.-P. Wang, S.-S. Huang, and F.-L. Hsu, “The inhibitory effect of tannins on lipid peroxidation of rat heart mitochondria,” Journal of Pharmacy and Pharmacology, vol. 47, no. 2, pp. 138–142, 1995.

H. Matsuura, “Saponins in garlic as modifiers of the risk of cardiovascular disease,” Journal of Nutrition, vol. 131, no. 3, pp. 1000S–1005S, 2001.

C. A. Schmitt and V. M. Dirsch, “Modulation of endothelial nitric oxide by plant-derived products,” Nitric Oxide, vol. 21, no. 2, pp. 77–91, 2009.

M. Akhlaghi and B. Bandy, “Mechanisms of flavonoid protection against myocardial ischemia-reperfusion injury,” Journal of Molecular and Cellular Cardiology, vol. 46, no. 3, pp. 309–317, 2009.

K. K. Gauthaman, M. T. S. Saleem, P. T. Thanislas, V. V. Prabhu, K. K. Krishnamoorthy, N. S. Devaraj, and J. S. Somasundaram, “Cardioprotective effect of the Hibiscus rosa sinensis flowers in an oxidative stress model of myocardial ischemic reperfusion injury in rat,” BMC Complementary and Alternative Medicine, vol. 6, article no. 32, 2006.

J. Duarte, R. Pérez-Palencia, F. Vargas, M. Angeles Ocete, F. Pérez-Vizcaino, A. Zarzuelo, and J. Tamargo, “Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats,” British Journal of Pharmacology, vol. 133, no. 1, pp. 117–124, 2001.

M. Ibarra, L. Moreno, R. Vera, A. Cogolludo, J. Duarte, J. Tamargo, and F. Perez-Vizcaino, “Effects of the flavonoid quercetin and its methylated metabolite isorhamnetin in isolated arteries from spontaneously hypertensive rats,” Planta Medica, vol. 69, no. 11, pp. 995–1000, 2003.

A. Machha, F. I. Achike, A. M. Mustafa, and M. R. Mustafa, “Quercetin, a flavonoid antioxidant, modulates endothelium-derived nitric oxide bioavailability in diabetic rat aortas,” Nitric Oxide, vol. 16, no. 4, pp. 442–447, 2007.

D.-S. Kim, K.-C. Ha, D.-Y. Kwon, M.-S. Kim, H.-R. Kim, S.-W. Chae, and H.-J. Chae, “Kaempferol protects ischemia/reperfusion-induced cardiac damage through the regulation of endoplasmic reticulum stress,” Immunopharmacology and Immunotoxicology, vol. 30, no. 2, pp. 257–270, 2008.