Cardioprotective potential of a lanosteryl triterpene from Protorhus longifolia

Abstract

Context: The current rapid increase in the incidence of cardiovascular events indicates a need for the discovery of more effective cardioprotective agents.

Objective: This study evaluated the cardioprotective potential of a lanosteryl triterpene from Protorhus longifolia (Benrh.) Engl. stem bark.

Materials and methods: Spectroscopic data analysis was used to confirm the structure of methyl-3β-hydroxylanosta-9, 24-dien-21-oate (RA-3). The cardioprotective effect of RA-3 in isoproterenol-induced myocardial injury in hyperlipidemic rats was investigated. Rats were divided into the normal diet (ND) fed and high fat diet (HFD) fed groups. The HFD rats were further subdivided into three groups. The experimental group was orally administered with RA-3 (100 mg/kg) for 15 days. The rats were then injected with isoproterenol (85 mg/kg) to induce myocardial injury. At the end of the experiment, hearts and blood tissues were collected and used for histology and biochemical assays, respectively.

Results: RA-3 exhibited a cardioprotective effect as it minimized myocardial injury in HFD rats. Few lesions of acute hyaline degeneration and reduced fat deposition were observed in the heart tissue of the triterpene pretreated rats. Lactate dehydrogenase (LDH) activity was decreased in the blood of the RA-3 pretreated rats (44.1 mU/mL) compared to the untreated group (64.8 mU/mL). Increased glutathione (GSH) content and catalase (CAT) activity along with lower levels of malondialdehyde (MDA) in the triterpene pretreated animals (120.8 nmol/μL) than in the non-treated HFD fed rats (143.6 nmol/μL) were also observed.

Discussion and conclusion: The cardioprotective effect exhibited by RA-3 indicates its potential use in the management of cardiovascular diseases (CVD) and related health problems.

Keywords:

• Cardioprotective effect
• antioxidant
• hyperlipidemia

Introduction

Cardiovascular diseases (CVD) continue to be the leading cause of morbidity and mortality worldwide (Gersh et al. 2010; Vallabi & Elango 2015) with myocardial infarction and stroke being the current major public health threats (Upaganlawar et al. 2011; Zaafan et al. 2013). Early deaths due to cardiovascular events in the working age group have been predicted to increase by 41% between 2000 and 2030 (Leeder et al. 2004). Reports have shown that a larger percentage (about 80%) of all cardiovascular-related deaths occur in developing countries than in developed countries (Gersh et al. 2010). This could partly be attributed to increased urbanization and the related changing life style along with limited access to good healthcare systems.

Hyperlipidemia and resulting atherosclerosis are the major risk factors of myocardial ischemia and cardiac events. Interaction between atherosclerotic plaque and arterial thrombosis, which compromise efficient coronary blood flow and oxygen supply, underlies the majority of CVDs (Lee et al. 2009). During ischemic events, inflammation and oxidative stress are considered as the main cause of myocardial tissue damage (Marchant et al. 2012; Zaafan et al. 2013). Some preventive measures of CVDs include physical activity, reduction in consumption of high caloric food and salt. Therapeutic agents such as statins, receptor blockers, anticoagulants and antiplatelets aggregation agents are currently used against cardiovascular events. However, adverse effects such as myopathy, dizziness and gastrointestinal discomfort (Hernández-Díaz & García-Rodríguez 2006; Messerli et al. 2009; Rao et al. 2011) associated with these drugs limit their clinical use.

Natural products derived from medicinal plants are perceived to be more effective with improved safety profile when compared to their synthetic counterparts. Plant-derived triterpenes have been reported to possess bioactivities beneficial to CVD prevention. These benefits include vasorelaxing (Rodríguez-Rodríguez et al. 2006), anti-inflammatory (Márquez-Martín et al. 2007), antioxidant, antithrombotic (Allouche et al. 2010) and antiplatelet aggregation (Mosa et al. 2011) properties. A lanosteryl triterpene, methyl-3β-hydroxylanosta-9, 24-dien-21-oate (RA-3) (Figure 1), from Protorhus longifolia (Benrh.) Engl. (Anacardiaceae), has also been reported to possess anticoagulant, anti-inflammatory (Mosa 2014), antihyperlipidemic (Machaba et al. 2014; Mosa et al. 2014) and antihyperglycemic (Mosa et al. 2015) activities. The hypolipidemic activity of RA-3 has been reported to be accompanied by reductions in atherogenic index and coronary risk index (Machaba et al. 2014), thus indicative of its potential cardioprotective benefit.

Figure 1. Methyl-3β-hydroxylanosta-9,24-dien-21-oate (RA-3).

This study evaluated the in vivo cardioprotective effect of RA-3, the lanostane triterpene, from P. longifolia stem bark. To the best of the authors’ knowledge, this compound has not been isolated and reported from any other plants except the P. longifolia stem bark.

Materials and methods

Chemicals and reagents

Unless otherwise stated, all other chemicals and reagents used were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). Sunflower oil was purchased from the local Supermarket, Empangeni, South Africa.

Plant material

The stem bark of P. longifolia was freshly harvested from Hlabisa, KwaZulu-Natal, South Africa. The plant (voucher specimen number RA01UZ) was authenticated by Dr. N.R. Ntuli from Botany Department, University of Zululand, South Africa. The plant material was then air-dried and ground to powder.

Extraction and isolation

Extraction and isolation of the triterpene from P. longifolia stem bark was conducted following the method described by Machaba et al. (2014). Briefly, the powdered plant material was defatted with n-hexane and then extracted (1:5 w/v) with chloroform. Silica gel column chromatography (24 × 700 mm; silica gel 60; 0.063–0.2 mm; 70–230 mesh ASTM, Merck, Darmstadt, Germany) was used to isolate the triterpene from the chloroform extract. The column was eluted stepwise with an n-hexane-ethyl acetate solvent system (9:1–3:7). Thin-layer chromatography (silica gel 60 TLC aluminium sheets, F₂₅₄, Merck, Darmstadt, Germany) was used to analyze collected small fractions. RA-3 was obtained after recrystallization in ethyl acetate.

Compound identification

The physical and spectral data of methyl-3β-hydroxylanosta-9,24-dien-21-oate were compared to previously published data (Machaba et al. 2014; Mosa et al. 2014).

Animals

Ethical clearance for use of experimental animals and approval of procedures was obtained from the University of Zululand Research Ethics Committee (UZREC 171110–030 Dept. 2013/23). Sprague-Dawely rats (130–160 g) were obtained from the Department of Biochemistry and Microbiology animal house, University of Zululand, South Africa. Caring of the experimental animals was strictly adhered to the institutional guidelines. The animals were kept under standard conditions, maintained at room temperature (23 ± 2 °C) and 12 h light–dark cycle. The animals had free access to enough rat feed and drinking water. The rats were allowed to acclimatize for four days before the commencement of the experiment.

Induction of hyperlipidemia

The method of Machaba et al. (2014) was followed in preparing high fat diet (HFD) and inducing hyperlipidemia in rats. Briefly, rats were randomly divided into two groups of 30 rats per group; the HFD fed group and normal diet (ND) fed group. After 21 days on their respective diets, the HFD (hyperlipidemic) rats were subdivided into three groups of 10 rats per group. The experimental group was orally administered with RA-3 (100 mg/kg body weight) for 15 days while 2% Tween 20 and simvastatin (60 mg/kg body weight) were used as negative and positive controls, respectively. Four rats were then sacrificed from each group and the remaining animals were used in the cardioprotective study.

Cardioprotective effect of the triterpene

After 15 days of the drug administration (i.e., a total of 36 days on the HFD) the remaining rats in the respective groups received a single subcutaneous injection of isoproterenol (ISO, 85 mg/kg body weight) for two consecutive days at 24 h interval to induce myocardial injury (Ojha et al. 2012). The experimental design is shown below.

• Group I: normal control, received 2% Tween 20 (vehicle)

• Group II: ISO non-treated, received 2% Tween 20 (vehicle)

• Group III: ISO-treated, received RA-3 in 2% Tween 20 (100 mg/kg)

• Group IV: ISO-treated, received simvastatin in 2% Tween 20 (60 mg/kg)

At the end of the experiment, the animals were fasted for 8 h before being sacrificed. Hearts and blood samples were collected for histology and biochemical assays.

Collection of blood and heart samples for biochemical assays

The rats were euthanized and blood samples were collected from the abdominal aorta. The collected blood samples were centrifuged at 1200 rpm for 10 min and the serum was collected for biochemical assays. The serum was used for estimation of malondialdehyde (MDA), gluthathione content, lactate dehydrogenase (LDH) and some antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT) activities. All the biochemical parameters were estimated using respective commercial assay kits (Sigma Aldrich) following the manufacturer’s instructions.

Histological studies

Hearts were excised and preserved in 10% (v/v) neutral buffered formalin for histological studies. Tissue slides were prepared following standard procedures. The heart tissues were stained with hematoxylin and eosin (H & E) for histopathological analysis by photomicroscope (Vet Diagnostix Laboratories, Pietermaritzburg, RSA). The slides examination was performed by a qualified pathologist with no prior knowledge about the respective animal groups. This method allowed for unbiased description of the histological lesions which were present or absent in the samples.

Data analysis

Unless stated otherwise, all experiments were replicated at least three times. Data generated were reported as mean ± SEM. Outliers were excluded using Grubb’s test. Statistical differences were determined using one-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test (GraphPad Prism version 5.03, San Diego, CA). The values were considered statistically significant where p < .05.

Results

Serum LDH activity

The cardioprotective effect of the triterpene (RA-3) was determined by estimating serum LDH activity in the isoproterenol-induced myocardial injury in HFD fed rats. The results are shown in Table 1. The triterpene exhibited cardioprotective effect as it effectively decreased LDH activity (44.1 mU/mL) in the serum of the triterpene pretreated HFD fed rats. This LDH activity was lower than that observed in the untreated hyperlipidemic rats (64.8 mU/mL).

Table 1. LDH activity (mU/mL) in ISO-induced myocardial injury in rats.

Group	36 days	After myocardial injury induction (39 days)
ND +2% T20	35.5 ± 0.05	64.8 ± 0.03
HFD +2% T20		90.9 ± 0.02
HFD + RA-3 (100 mg/kg)		44.1 ± 0.06^a
HFD + Simvastatin (60 mg/kg)		56.8 ± 0.10^a

Values are expressed as mean ± SEM (n = 4), ^ap < .05 vs HFD 39 days.

Antioxidant capacity

Table 2 shows the effect of the triterpene on the antioxidant capacity in the myocardial injury-induced HFD fed animals. A decrease in antioxidant levels with consequent increase in lipid peroxidation end product, MDA, was observed in all groups following myocardial injury induction. However, a relatively higher glutathione (GSH) content and CAT activity along with lower levels of MDA in the triterpene pretreated animals (0.31 nmol/mL, 3.90 Units/mL and 120.8 nmol/μL, respectively) than in the non-treated HFD fed rats (0.22 nmol/mL, 1.27 Units/mL and 143.6 nmol/μL, respectively) were also observed. The antioxidant status in the RA-3 pretreated group was comparable to that of simvastatin, a standard hypolipidemic drug.

Table 2. Effect of RA-3 on the blood MDA level, GSH content, SOD and CAT activity.

Group	GSH (nmol/mL)	SOD (Units/mL)	CAT (Units/mL)	Antioxidant status (mM)	MDA (nmol/μL)
After 36 days of hyperlipidemia induction
ND +2% T20	0.39 ± 0.013	59.0 ± 0.001	3.41 ± 0.143	1.79 ± 0.074^a	24.4 ± 0.012^a
HFD +2% T20	0.32 ± 0.008	48.5 ± 0.012	2.80 ± 0.020	0.80 ± 0.044	38.2 ± 0.027
HFD + RA-3	0.36 ± 0.012	50.7 ± 0.018	4.36 ± 0.122	1.23 ± 0.161	27.4 ± 0.002^a
HFD + Simva	0.31 ± 0.012	25.5 ± 0.064	2.39 ± 0.124	1.73 ± 0.130^a	23.1 ± 0.007^a
After 3 days of myocardial injury induction (39 days)
ND/T20 + ISO	0.15 ± 0.004	36.7 ± 0.017	3.05 ± 0.122	0.70 ± 0.041	36.5 ± 0.001^b
HFD/T20 + ISO	0.22 ± 0.006	27.2 ± 0.021	1.27 ± 0.037	0.58 ± 0.008	143.6 ± 0.015
HFD/RA-3 + ISO	0.31 ± 0.030	26.9 ± 0.053	3.90 ± 0.01^b	1.18 ± 0.064^b	120.8 ± 0.015^b
HFD/Simva + ISO	0.21 ± 0.004	16.0 ± 0.008	2.10 ± 0.170	1.11 ± 0.162^b	45.6 ± 0.004^b

Values are expressed as mean ± SEM (n = 4), ^ap < .05 vs HFD 36 days, ^bp < .05 vs HFD 39 days.

Histological study

The results of the histological examination of heart tissues of the rats are shown in Figure 2. Hearts of the rats in the ND fed rats (after 36 days) showed normal architecture of myocardium, with clear striations of myocardial fibres and centrally placed nucleus, without any histological alterations (Figure 2(A)). Consumption of HFD (after 36 days) caused lipid accumulation, focal swelling, fragmentation and hyalinization of the myofibres in the hearts of untreated HFD fed rats (Figure 2(B)). However, upon pretreatment with RA-3 (100 mg/kg bw, for 15 days) the lipid content, swelling and fragmentation were reduced (Figure 2(C)). The isoproterenol injection (85 mg/kg) caused pathological changes in the heart tissues which resulted in myocardial fibre loss without replacement fibrosis along with inflammatory cells infiltration in the hearts of untreated animals (Figure 2(D,E)). A marked improvement was observed in the rats pretreated with the triterpene (Figure 2(F)). The hearts showed appearance comparable to that of the normal control accompanied by reduced fats deposition, thus indicating the apparent cardioprotective effect of the triterpene.

Figure 2. Histological changes (200× magnification) of the heart tissues from rats following ISO-induced myocardial injury. The rats were fed on (A) ND and (B,C) HFD for 36 days, with the (C) experimental group being pretreated with RA-3 for 15 days, (D–F) before they received a subcutaneous injection of ISO (85 mg/kg) to induce myocardial injury.

Discussion

The demand for alternative and more efficient forms of treatment against CVD cannot be over emphasized. Some triterpenes including methyl-3β-hydroxylanosta-9,24-dien-21-oate (RA-3) from Protorhus longifolia, have been reported to possess potential bioactivities (Allouche et al. 2010; Machaba et al. 2014; Mosa et al. 2015) beneficial to CVD prevention.

The present study evaluated the in vivo cardioprotective effect of RA-3 in ISO-induced myocardial injury in rats. ISO causes severe stress in myocardium, compromising cardiac membrane integrity (Zhou et al. 2008) and thus, consequent leakage of cardiac biomarker enzymes (LDH, CK and CK-MB) into the blood stream. Serum activity of these biomarker enzymes serves as an important diagnostic tool for cardiac damage. Various medicinal plants or plant-derived compounds have been demonstrated to exhibit cardiac protective effect, evidenced by among others, attenuation of serum activities of the cardiac biomarker enzymes (Senthil et al. 2007; Kamel et al. 2010; Yuan 2015). The observed decreased serum total LDH activity in the triterpene pretreated group (Table 1) could be associated with the compound’s ability to maintain myocardial cells membrane integrity, which is indicative of its cardioprotective effect.

Infarct like necrosis of heart muscle, resulting from ISO toxicity, is usually characterized by myofibrillar degeneration and myocardial lipids accumulation, typical signs of acute myocardial infarction in humans (Ojha et al. 2012, Radhiga et al. 2012). A marked improvement, few lesions of acute hyaline degeneration and reduced fat deposition, observed in the heart tissue of the triterpene pretreated rats (Figure 2(F)) confirmed the cardioprotective effect of RA-3. Elevated plasma levels of cholesterol and its cardiac tissue accumulation are well linked to cardiovascular damage (Slater & White 1996). The potential cardioprotective effect of some other triterpenes has been associated with their hypolipidemic and anti-inflammatory effects (Sudhahar et al. 2007; Radhiga et al. 2012; Camer et al. 2016). The reduced fat deposition observed in the triterpene treated group (Figure 2(C)) further support the hypolipidemic activity of the compound. The hypolipidemic activity of RA-3 has previously been reported to be accompanied by reductions in atherogenic index and coronary risk index (Machaba et al. 2014).

The myocardial necrotic effect of ISO is known to be mediated by oxidative stress and consequent lipid peroxidation (Kumar et al. 2001). In addition to hypolipidemic property, the cardioprotective effect of triterpenes has also been attributed to their ability to increase endogenous antioxidant capacity and prevent lipid peroxidation (Senthil et al. 2007; Haleagrahara et al. 2011; Radhiga et al. 2012). The observed increased serum GSH content and CAT activity, along with a decrease in MDA content (Table 2) in the triterpene pretreated group is indicative of the compound’s ability to exert its cardioprotective effect through increasing the tissue antioxidant capacity. The ability of RA-3 to increase endogenous antioxidant capacity has also been demonstrated in streptozotocin (STZ)-induced diabetic animals (Mosa et al. 2015).

Conclusion

Some reported bioactivities of RA-3, which include anticoagulant, anti-inflammatory (Mosa 2014), antihyperglycemic (Mosa et al. 2015) and antihyperlipidemic (Machaba et al. 2014) activities, are all indicative of the potential cardioprotective effect of the triterpene. The results obtained from this study confirmed that the lanosteryl triterpene [methyl-3β-hydroxylanosta-9, 24-dien-21-oate, RA-3] possesses the cardioprotective effect. In addition to its reduction of atherogenic and coronary risk indices (Machaba et al. 2014), the cardioprotective effect of this triterpene could also be linked to its ability to increase tissue antioxidant capacity and thus prevent oxidative tissue damage. The cardioprotective effect exhibited by RA-3 indicates its potential use in the prevention of CVD and related health problems.

Future study will evaluate the effect of the triterpene on CK-MB and LDH isoenzymes for greater specificity of the cardiac injury. Effect of the triterpene on cardiomyocytes is also recommended.

Disclosure statement

The authors declare no conflict of interest.

Funding

This work was financially supported by the South African Medical Research Council (SAMRC) and University of Zululand Research Committee.