1Department of Biochemistry, Shrimati Indira Gandhi College, Tiruchirappalli, Tamil Nadu, India;
2Department of Food Science and Biotechnology, College of Life Science, Sejong University, Seoul, South Korea;
3Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India;
4Department of Zoology, School of Biosciences, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, Tamil Nadu, India;
5Department of Life Sciences, CHRIST Deemed to be University, Bengaluru, India;
6Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India;
7Transcription Regulation Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India;
8Rashtriya Uchchatar Shiksha Abhiyan (RUSA) Scheme, Madurai Kamaraj University, Madurai, Tamil Nadu, India
†Equally contributed as first author.
Cardiovascular disease (CVD) and its complications have been regarded as the leading cause of morbidity and mortality. The drugs available in the market are effective to treat CVD, but with many adverse reactions. Nowadays, herbal products are the attention of researchers because of their less adverse effects. In this study, the cardioprotective effects of ethanolic leaves extract of Psidium guajava Linn. (Guava) (P. guajava) were evaluated in streptozotocin (STZ)-treated animal models. Mice acquired for the study were divided into five groups, each consisting of six mice. The toxin-induced mice were treated with the ethanolic leaves extract of P. guajava (300 mg/kg body weight [b.w.]). The results were compared to the standard drug (glibenclamide)-treated mice (3 mg/kg b.w.). The following parameters were considered for further investigations: creatine kinase-muscle brain (CK-MB), creatine kinase (CK), troponin, lysosomal, and mitochondrial enzymes. Then the docking study was accomplished. The levels of cardiac marker enzymes and lysosomal enzymes increased significantly in the toxin-induced mice, while the level of mitochondrial enzyme decreased significantly. During treatment with the ethanolic leaves extract of P. guajava, the levels of all parameters were notably reversed to normal range (P < 0.05). Further, in docking analysis, the interaction of compounds, such as alpha-terpineol, cyclopentanecarboxamide, guaiol (a sesquiterpenoid alcohol), 1H-cyclopropanaphthalene, tetracyclotridecan-9-ol, dormin/abscisic acid, and epiglobulol, with the respective protein molecules, evidenced the cardioprotective effect of P. guajava leaves. Hence, it was concluded that the ethanolic leaves extract of P. guajava leaves have a cardioprotective effect.
Key words: Psidium guajava, lysosomal enzymes, mitochondrial enzymes, cardiac markers
*Corresponding Authors: Ramasamy Manikandan, Department of Biochemistry, Shrimati Indira Gandhi College, Tiruchirappalli, Tamil Nadu, India. Email: [email protected]; Balasubramanian Balamuralikrishnan, Department of Food Science and Biotechnology, College of Life Science, Sejong University, Seoul, South Korea. Email: [email protected]; Arumugam Vijaya Anand, Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, India. Email: [email protected]
Received: 31 December 2022; Accepted: 21 March 2023; Published: 27 April 2023
© 2023 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)
India is known as the global capital of diabetes mellitus. Diabetes mellitus causes several secondary disorders; CVD is one of the diabetes-related disorders. The end products of glycation make for an important role in the cardiac disorder associated with diabetes (Buynes and Thorpe, 2000). According to the World Health Organization (WHO), an estimated 17.3 million individuals died due to CVD in 2008, and it may increase to 23 million by 2030. In India in 1990, nearly 2.6 million people died due to CVD (Kumar et al., 2011; Sivakumar and Dhanarajan, 2001). CVD is a substantial health burden with an ever-increasing prevalence, and has remained the foremost reason for morbidity and mortality worldwide. Some allopathic drugs, such as aspirin, anti-arrhythmic drugs, and receptor blocker, are available for myocardial infarction, but these drugs are quite expensive and have a number of adverse reactions (Vishal et al., 2010, Xie et al., 2022).
Nowadays, scientists are exploring to develop drugs from plants because of their positive effects to treat diseases (Panche et al., 2016). Medicinal plant Psidium guajava Linn (guava; P. guajava) belongs to family Myrtaceae. P. guajava, a tropical plant, is used globally as a food as well as medicine because of its various medicinal properties. Although P. guajava has a variety of medicinal benefits, it is the most common and classic therapy for gastrointestinal infections, including diarrhea, dysentery, stomach ache, and indigestion (Gutiérrez et al., 2008).
P. guajava is cultivated throughout India, mostly in the states of Andhra Pradesh, Assam, Bihar, Maharashtra, Uttar Pradesh, and West Bengal (Daswani et al., 2017). Leaves of the plant contain a large number of phytoconstituents, such as alkaloids, terpenoids, phenols, flavonoids, and ethanol extracts (Manikandan et al., 2013, 2016; Manikandan and Vijaya Anand, 2015). Zakaria and Mohd (1994) established that the leaves of P. guajava contained caryophyllene, alpha-pinene, quercetin, and limonene compounds. Its leaves are used to study medicinal properties in the treatment of malaria (Gessler et al., 1994), diarrhea (Lutterodt et al., 1992), diabetes (Manikandan et al., 2018), and bacterial diseases (Canceras et al., 1993). Therefore, the present investigation intended to find changes in cardiac marker, lysosomal, and mitochondrial enzymes in the STZ)-induced and P. guajava leaves’ extract-treated mice with in silico analysis. The analysis was done to analyze the binding affinity of phytoconstituents of P. guajava leaves with marker enzymes.
All the chemicals and reagents used were of higher analytical grade required for the assays and were obtained from Sigma-Aldrich (Bangalore, India), unless otherwise noted specifically. The biochemical analysis was done with the help of the autoanalyzer Turbochem 100 and the reagents as well as calibrators were obtained from iChem™ 100.
Fresh leaves of P. guajava were obtained from Tiruchirappalli, Tamil Nadu, India, and authenticated and deposited at Rapinat Herbarium, St. Joseph College, Tiruchirappalli, India. The leaves were shade-dried, powdered, and stored till further use. Extract of P. guajava leaves was obtained by the hot percolation method using Soxhlet apparatus, with ethanol used as a solvent.
In the present investigation, male albino mice, aged 6–8 weeks and weighing approximately 150–180 g, were used as the study model. The mice were kept in clean and dry plastic cages. All the animals were fed with a commercially available standard diet (Sai Durga Feeds and Foods, Bengaluru, India). The study was conducted at Srimad Andavan Arts and Science College (SAASC), Tiruchirappalli, Tamil Nadu, India, and ethical clearance to conduct the study with mice model was obtained from the Institutional Animal Ethics Committee, SAASC, Tiruchirappalli, India. (CPCSEA approval No. 790/03/ac/CPCSEA).
The animals were split into the following five groups, G1–G5, each with six mice: Control G1 group was given a standard diet and saline water; negative control G2 group: diabetic mice induced with STZ (60 mg/kg body weight [b.w.]); G3 group was treated with 300-mg/kg b.w. of ethanolic leaves extract of P. guajava; G4 group comprised G2 mice treated with 300-mg/kg b.w. of ethanolic leaves extract of P. guajava; and G5 group comprised G2 mice treated with glibenclamide, 3-mg/kg b.w., for 45 days. The animals were fasted overnight (for about 12–18 h) on the last day of the experiment prior to the induction of anaesthesia and collection of blood samples. The animals were sacrificed at the end of experimental period by cervical dislocation under diethyl ether anesthesia. Blood samples were collected from the heart of animals for different biochemical assays. All biochemical parameters were quantified and determined by using commercial kits.
In this study, we evaluated cardiac marker, lysosomal, and mitochondrial enzymes. The cardiac marker enzymes included were CK-MB (Apple et al., 1988), CK (Okinaka et al., 1961), and troponin (Burtis and Ashwood, 1996). The lysosomal enzymes included were acid phosphatase (ACP; King, 1965), β-D-glucuronidase (Kawai and Anno, 1971), β-N-acetyl glucosaminidase (Moore and Morris, 1982), cathepsin D (Sapolsky et al., 1973); and mitochondrial enzymes evaluated were isocitrate dehydrogenase (ICH; Bell and Baron, 1960), α-ketoglutarate dehydrogenase (KDH; Reed and Mukherjee, 1969), succinate dehydrogenase (SDH; Slater and Bonner, 1952), malate dehydrogenase (MDH; Mehler et al., 1948), nicotinamide adenine dinucleotide (NAD)+hydrogen (H) (NADH) dehydrogenase (Tsoo et al., 1967), and cytochrome-C-oxidase (Pearl et al., 1963).
The Protein Data Bank (PDB) was used for retrieving the three dimensional structure of cardiac markers were CK-MB (PDB ID 3B6R), CK (PDB ID 1I0E), and troponin (PDB ID 1J1D). Similarly, for evaluation of lysosomal enzymes were ACP (PDB ID 1RPA), β-D-glucuro-nidase (PDB ID 6LEJ), β-N-acetyl glucosaminidase (PDB ID4GVF), and cathepsin D (PDB ID 4OBZ). Same as for evaluation of mitochondrial enzymes were ICH (PDB ID 3LC6), KDH (PDB ID 7WGR), SDH (PDB ID 1NEK), MDH (PDB ID 2PWZ), NADH dehydrogenase (PDB ID 5XTC), and cytochrome-C-oxidase(P DB ID 5Z62).
All the selected target proteins were imported by the maestro platform of the Schrodinger software, using appropriate PDB Id and the protein preparation wizard module. The retrieved three-dimensional (3D) structures were pre-processed by following addition of hydrogen, zero-order bonds to metals, converting selenomethionine (SeMet) to methionine (Met), creating disulfide bonds, and filling in missing side chains. In addition, inhibitors were removed, H-bonds were optimized, and energy minimization was applied by using the OPLS4 force field.
Previously we found GC-MS analysis was used to determine 21 phytoconstituents in ethanolic leaves extract of P. guajava (Manikandan et al., 2018). The PubChem database 3D structures were obtained in the structure data file (SDF) format. In addition, the LigPrep module was applied to refine the structure. In all, 32 various states of stereoisomerism were obtained after applying the OPLS4 force field (Schrödinger software 2021-2: LigPrep, Schrödinger, LLC, New York, NY, 2021).
Phytoconstituents in ethanolic leaves extract of P. guajava were investigated for molecular docking analysis using the “extra precision” (XP) mode of Glide (a ligand docking program for predicting protein-ligand binding modes) module. A pose viewer was used to investigate the docked ligand interaction with protein for ideal conformation. Both protein and ligand complex interaction modules generated 2D interactions (Leslie et al., 2021).
The present investigation was conducted by using one-way analysis of variance (ANOVA). Statistical testing was done using Statistical Package of Social Science (SPSS) version 14.0 for Windows. The data were represented as mean ± standard deviation (SD), and P < 0.05 was considered statistically significant.
Based on the previous results, the phytoconstituents from P. guajava were docked with targeted proteins. The top interacting molecules were highlighted among the docked 21 phytoconstituents. The levels of cardiac markers, such as CK, CK-MB, and troponin, are shown in Table 1. These markers were notably increased in the STZ-treated diabetic animals, compared to the control group. However, the increased markers reverted to normal levels following the treatment with ethanolic extract of P. guajava leaves, compared to the standard drug-treated and control groups. No notable changes were observed in the plant extract-treated mice group. Table 2 shows the molecular interaction of cardiac target proteins with P. guajava phytoconstituents. The top compounds were bound at the same location and their 2D interactive structures were observed (Figure 1). Further, the molecular interactions are presented in Table 2. Among cardiac target proteins, PG9, PG15, and PG19 were observed as being the top-ranked molecules.
Table 1 Phytochemicals profiling of P. guajava using GC-MS analysis.
|Compounds ID||Compound name||Pub Chem ID|
Table 2 Quantity of cardiac marker enzymes creatine kinase-myoglobin binding (CK-MB), creatine kinase (CK), and troponin in an animal model.
Values are given as mean ± SD of five experimental groups for each rat.
Values marked with superscript a, b, c differ significantly at P ≤ 0.05 (Duncan’s multiple range test [DMRT]).f
Figure 1 The 21 phytochemicals extracted from P. guajava leaves were docked with cardiac marker target proteins. The three-dimensional docked complex structure of (A,B) creatine kinase (CK) from the brain, and (E,F) troponin. The two-dimensional structure of CK from the brain with (C) PG15 and (D) PG19; and that of troponin with (G) PG9 and (H) PG15.
The results of ACP, cathepsin D, β-D-glucuronidase, and β-N-acetyl glucosaminidase in control group, toxin-treated group, and ethanolic leaves extract of P. guajava group are shown in Table 3. Compared to the control group, the concentration of lysosomal enzymes was augmented in the toxin-induced diabetic mice but decreased in the ethanolic leaves extract of P. guajava group. No notable changes were observed in the plant alone-treated group.
Table 3 Molecular interaction of cardiac marker enzymes with P. guajava phytochemicals.
|Target/ Compounds ID||Compounds Pubchem ID||Amino acid interaction(3-letter code)||Bond length
|Creatine kinase from brain|
|PG15||5375199||ARG74, ARG79||(2.17, 3.61, 1.84, 3.06, 2.26) (2.06, 2.16)||–2.609|
|PG15||5375199||TYR269, LYS156,||1.80 (1.85, 4.02)||–2.868|
|PG19||11858788||GLU261, LYS265||1.58, 1.91||–2.772|
Among 21 phytoconstituents of P. guajava docked with target proteins, the 3D interaction of five compounds is shown in Figure 2. Figure 3 shows the 3D interaction of compounds with specific ligands. We observed that all top five compounds were docked in the same region. Further, the molecular interaction was studied for top three compounds as shown in Table 4. Among lysosomal-targeted proteins, PG5, PG7, PG9, PG11, and PG15 were observed as the top-ranked molecules.
Figure 2 The 21 phytochemicals extracted from P. guajava leaves were docked with lysosomal target proteins. The three-dimensional docked complex structure of (A,B) acid phosphatase (ACP), (C,D) β-D-glucuronidase, (E,F) β-N-acetyl glucosaminidase, and (G,H) cathepsin D.
Figure 3 The two-dimensional structure of acid phosphatase (ACP) with (A) PG7, (B) PG9, (C) PG11, and (D) PG15; that of β-D-glucuronidase with (E) PG5, (F) PG7, and (G) PG15; that of β-N-acetyl glucosaminidase with (H) PG9, (I) PG7, and (J) PG11; and that of cathepsin D with (K) PG7, (L) PG9, and (M) PG11.
Table 4 Effect of P. guajava leaves extract on acid phosphatase (ACP), cathepsin D, β-D-glucoronidase, and β-N-acetyl glucosaminidase proteins in control and STZ-induced rat models.
The effect of P. guajava leaves extract on lysosomal enzymes ACP, cathepsin D, β-D-glucuronidase, and β-N-acetyl glucosaminidase are expressed in μmol/h/100 mg.
Values are given as mean ± SD of five experimental groups, each with six mice.
Values marked with superscript a, b, c differ significantly at P ≤ 0.05 (DMRT).
The levels of ICH, KDH, SDH, MDH, NADH dehydrogenase, and cytochrome-C-oxidase in the study groups are shown in Table 5. Compared to the control group, the enzymes levels of tricarboxylic acid cycle (TCA), including ICH, KDH, SDH, and MDH, as well as respiratory enzymes, including the levels of NADH-dehydrogenase and cytochrome-C-oxidase, were significantly decreased in the toxin-induced group. However, the levels were significantly increased to the normal range following treatment with ethanolic extract of leaves of P. guajava. No change was observed in the plant extract-treated group.
Table 5 Molecular interaction of lysosomal target proteins with P. guajava phytochemicals.
|Target/ Compounds ID||Compounds Pubchem ID||Amino acid interaction(3-letter code)||Bond length(Å)||Glide score(kcal/mol)|
|Acid phosphatase (ACP)|
|PG15||5375199||SER175, ARG127||1.88 (1.81, 1.85, 3.32)||–5.118|
|PG9||318639||LEU124, ARG11, ASP258, ARG15||2.71, 2.41, 2.05 (1.84, 2.09, 2.76)||–4.052|
|PG7||226274||ARG15, HIE257, HIS12||1.81, 2.01, 1.93||–3.403|
|PG15||5375199||ASN308, LYS13, ASP307||1.90, 1.93, 1.66||–4.884|
|PG7||226274||PHE306, PRO48||1.70, 2.08||–3.958|
|PG9||318639||ARG222, SER268||(1.61, 1.96, 4.78), 2.79||–5.639|
|PG7||226274||GLH263, ILE257||1.95, 2.00||–4.337|
|PG7||226274||PRO173, ASP174||2.02, 2.22||–3.187|
|PG9||318639||LYS8, TYR16||(1.89, 2.85), 1.84||–2.884|
Among 21 phytoconstituents of P. guajava docked with target proteins, the top-ranked compounds for 3D interaction are shown in Figure 4. All compounds were docked in the same region. Further, molecular interaction was studied, which is given in Table 6. Among mitochondrial-targeted proteins, PG5, PG7, PG9, and PG8 were observed as the top-ranked molecules. Figure 5 presents the interaction of the binding of ligands to specific phytoconstituents determined in P. guajava leaves extract.
Figure 4 The 21 phytochemicals extracted from P. guajava leaves were docked with mitochondrial target proteins. The three-dimensional docked complex structure of (A,B) α-ketoglutarate dehydrogenase, (C,D) succinate dehydrogenase, (E,F) malate dehydrogenase, and (G,H) cytochrome-C-oxidase.
Figure 5 The two-dimensional docked complex structure of α-ketoglutarate dehydrogenase with (A) PG5 and (B) PG7; that of succinate dehydrogenase with (C) PG5 and (D) PG7; that of malate dehydrogenase with (E) PG8; and that of cytochrome-C-oxidase with (F) PG9.
Table 6 Effect of the ethanolic extract of P. guajava leaves on ICH, KDH, SDH, MDH, NADH, and cytochrome oxidase in a rat model.
|Isocitrate dehydrogenase (ICH)||612.31±16.72a||201.43±20.32b||613.46±21.32a||602.63±17.65c||604.89±20.23c|
|Alpha-keto dehydrogenase (KDH)||141.62±9.62a||90.43±8.82b||140.87±10.36a||134.78±9.61c||136.31±11.62a,c|
|Mitochondrial succinate dehydrogenase (SDH)||215.31±16.81a||107.61±15.4b||216.68±11.32a||200.68±10.56c||201.73±9.65c|
|Mitochondrial malate dehydrogenase (MDH)||299.61±11.83a||170.78±10.53b||300.60±10.82a||278.72±19.61c||288.63±10.57c|
|Nicotinamide adenine dinucleotide (NADH) dehydrogenase||32.36±1.16a||15.32±0.95b||32.01±1.29a||29.31±0.81c||30.37±1.57c|
The effect of P. guajava leaves extract on mitochondrial enzymes ICH, KDH, SDH, MDH, NADH, and cytochrome-C-oxidase are expressed in μmol/h/100 mg.
Values are in given as mean ± SD of five studied groups for each rat.
Values marked with superscript a, b, c differ significantly at P ≤ 0.05 (DMRT).
Table 7 Molecular interaction of mitochondrial target proteins with P. guajava phytochemicals.
|Target/Compounds ID||Compounds Pubchem ID||Amino acid interaction(3-letter code)||Bond length(Å)||Glide score(kcal/mol)|
|α-Ketoglutarate dehydrogenase (KDH)|
|PG7||226274||THR753 (A & B chain)||(2.08 & 2.12)||–4.067|
|Succinate dehydrogenase (SDH)|
|PG7||226274||ARG180, GLN151||2.07, 1.97||–4.422|
|Malate dehydrogenase (MDH)|
|PG9||318639||ARG438, ARG439, TRP126||(2.32, 4.91) (1.72, 2.38, 3.77) 2.20||–8.629|
The extract of P. guajava leaves was obtained with ethanol, as it has a polarity index of 5.2 and is safer and found to have advantages over other organic solvents (Hikmawanti et al., 2021). Ethanol is considered as a suitable solvent for recovering most of phytoconstituents and extraction of antioxidant components and foods derived from plants (Sultana et al., 2009). In our previous study, extraction done with ethanol demonstrated to have more phytoconstituents than other extraction solvents, such as aqueous chloroform, petroleum, ether, and hexane (Manikandan et al., 2016).
The CK-MB has a vital role in identifying patients with myocardial infarction and other cardiovascular complications. The CK-MB enzyme is present in the myocardial cells of the heart. In myocardial infarction, cells of the myocardium are damaged due to the toxin, which releases CK-MB in the bloodstream. The present study is focussed on the STZ-induced diabetes and its effect on the myocardial cells of the heart. A previous study conducted by Seager et al. (1984) proved that myocardial complications happen in STZ-treated chronic diabetic animals. Findings of the present study also demonstrate the same results. The administration of ethanolic extract of P. guajava leaves revealed a considerable reduction in glucose and lipid peroxidation levels. The treatment also increased significantly the levels of glutathione, glutathione peroxidase, superoxide dismutase, and catalase in the liver, compared to the levels in diabetic mice (Manikandan et al., 2016; Sinha, 1972).
Following the treatment with ethanolic extract of P. guajava leaves, the increase in chemical compounds was reversed to normal levels. This happened because the leaves extract restored myocardial cell damage and lead to decrease in CK-MB release in the bloodstream.
A similar effect was observed in the doxorubicin-induced mice treated with Grewia unbellifera (family Malvaceae) and Gmelina arborea or gamhar (family Lamiaceae) (Arafa et al., 2014). In myocardial infarction, the level of CK enzyme is increased in circulation (Okinada et al., 1961). In the current study, STZ induced myocardial damage and favored the release of CK in the blood. Following the treatment with ethanolic extract of P. guajava leaves, the level of CK decreased in the blood, because the secondary metabolites found in the leaves regenerated myocardial cells and prevented the release of CK enzyme.
In the control group, no significant changes were observed in mice when treated with the plant extract. In recent years, death of cardiac cells is determined by using the serum troponin level. In animals, the increased concentration of troponin indicates myocardial infarction (O’Brien et al., 2006). This was also proved in the present investigation, wherein the level of troponin was increased in the blood due to the toxin STZ-induced myocardial infarction. However, treatment with P. guajava leaves extract reduced the level of troponin in the blood. This effect was due to the prevention of leakage of troponin enzyme from myocardial cells. Significant changes were observed, compared to the negative control group. Vijayakumar et al. (2018) demonstrated that the leaves extract of P. guajava and its isolated quercetin fraction significantly controlled the lipid profile, which is one of the major risk factors for CVD pathogenesis in carbon tetrachloride-toxicated mice.
Phytoconstituents have been widely and effectively used in the treatment of various diseases, and the phytophysical properties of these compounds have been extensively studied and followed by in silico studies against diabetic targets. In the present investigation, phytoconstituents, such as abscisic acid, cyclopropanaphthalene, and epiglobulol, showed affinity toward CK and troponin. The abscisic acid and cyclopronaphthalene compounds adhered to CK in brain proteins and epiglobulol, and abscisic acid adhered to troponin. The target phytochemical compounds are evaluated with the binding affinity with the cardiac markers and further can be selected as hits.
Acid phosphatise is one of the important lysosomal enzymes. Myocardial injury because of toxin causes the release of ACP enzyme from lysosome to cytosol (Decker and Wildentha, 1980). The release of ACP enzyme may cause cell injury and cell death because of the attacking of alternative pathway in cardiac cells (Hoffstein et al., 1975). ACP level was increased in the present study because of necrosis in cardiac cells.
In diabetic condition, the renin-angiotensin-aldosterone system (RAAS) system leads to oxidative damage and activates necrosis (Frustaci et al., 2000). This was observed in the present investigation. However, the level of ACP reverted to normal if treated with the plant leaves extract. The phytoconstituents present in the extract may protect cardiac cells from necrosis and reduces ACP level in the serum.
Karthikeyan et al. (2007) proved that the treatment with grape seeds reduced ACP level in the serum. This was proved in the present study too. The animal cells contain cathepsin D, a lysosomal enzyme (Sudharsan et al., 2006). Cathepsin D may disturb the oxygen radical, which could lead to cardiac tissue damage. The level of cathepsin D is increased in STZ-induced mice, but treatment with the plant leaves extract may reverse the cathepsin D level to normal. The phytoconstituents may protect cellular membranes from damage and inhibit the release of lysosomal enzymes.
The free fatty acid level is increased in diabetes. These free fatty acids are deposited in the blood to reduce the cell membrane stability of cardiac cells. This damage caused to cellular membranes releases lysosomal enzymes, which include β-D-glucuronidase and β-N-acetyl glucosaminidase. In the STZ-induced mice, the levels of these two enzymes were significantly increased, compared to the control group. However, treatment with the plant leaves extract decreased the levels of these enzymes. This was due to the decreased concentration of free fatty acid by the plant leaves extract, which reduced injuries to the cellular membrane and subsequently decreased the levels of lysosomal enzymes.
The results of the present study concluded that compounds such as abscisic acid, cyclopropanaphthalene, and cyclopenanecarboxamide bind to ACP; compounds such as abscisic acid, cyclopentanecarboxamide, and alpha-terpineol bind to β-D-glucuronidase; compounds such as cyclopropanaphthalene, tetra cyclotridecan-9-ol, and cyclopentanecarboxamide bind to beta-N-acetyl glucosaminidase; and cathepsin-D binds to the compounds such as cyclopentanecarboxamide, cyclopropanaphthalene, and tetra cyclotridecan-9-ol. These compounds are found to have higher binding affinity and glide scores with respective lysosomal enzymes.
Prolonged oxidative stress releases free radicals, which affect and alter the structure and functioning of mitochondrial membrane (Subashini and Sumathi, 2012). Normally, TCA cycle enzymes are found in the matrix of the mitochondria. Damage to the membrane may reduce ICH, KDH, SDH, and MDH levels. In this study, owing to increased oxidative stress in toxin-treated mice, the levels TCA cycle enzymes were reduced. Nevertheless, treatment with the plant leaves extract significantly increased the levels of TCA cycle enzymes because of the antioxidant disposition of P. guajava.
NADH dehydrogenase and cytochrome-C-oxidase enzymes are found in the inner membranes of mitochondria. The levels of these enzymes are directly proportional to the levels of phospholipids (Subashini and Sumathi, 2012). In the present investigation, the levels of phospholipids were reduced in the toxin-treated animals. This decreasing concentration of phospholipids may reduce levels of both enzymes. However, the levels of these enzymes were significantly increased when animals were treated with the plant leaves extract. This could be due to the inhibition of degradation of phospholipids. A similar effect was also observed in the study conducted by Subashini and Sumathi (2012). Vijayakumar et al. (2020) established that isolated quercetin fractions exhibited more reasonable activity than that of the ethanolic extract of P. guajava leaves.
Following the above experiments, an in silico approach was carried out on phytoconstituents against cardiac target myocardial enzymes, which include KDH, SDH, MDH, and cytochrome-C-oxidase. Virtual screening of phytoconstituents are found to have binding affinity towards the myocardial enzymes, such as cyclopentanecarboxamide, and alpha-terpineol which are able bound to the KDH enzyme, cyclopentanecarboxamide and alpha-terpineol able to tightly bound to the SDH, guaiol (a sesquiterpenoid alcohol) compounds are able to bind to the MDH and 1H-cyclopropanaphthalene to cytochrome-C-oxidase enzyme. The phytoconstituents were screened for their binding affinity and the selected compounds were investigated for further in silico cardioprotective effects by docking with selected target proteins.
Based on docking energy and good interaction with the active site, ligand molecules were selected for further investigation. The docking studies confirmed the inhibition of cardiac target proteins to show the cardioprotective activity of various phytoconstituents discovered in P. guajava.
The present study concluded the positive effects of ethanolic extract of P. guajava leaves on the cardiac marker and mitochondrial enzymes of STZ-induced diabetic mice. We studied the phytoconstituents of P. guajava leaves against multiple target proteins, and observed that alpha-terpineol, cyclopentanecarboxamide, guaiol, 1H-cyclopropanaphthalene, tetracyclotridecan-9-ol, dormin/abscisic acid, and epiglobulol were the top-ranked molecules of cardiac, lysosomal, and mitochondrial target proteins. The limitation of the present investigation was that it did not cover molecular-level parameters to confirm overall medicinal benefits of P. guajava, which in the future could be a potential drug for diabetes-related cardiac complications.
Conceptualization of the study was accomplished by Ramasamy Manikandan, Balasubramanian Balamuralikrishnan, and Arumugam Vijaya Anand. Methodology, data curation, and formal analysis were done by Ramasamy Manikandan, Arthi Boro, Pushparaj Karthika, Arunkumar Malaisamy, Shanmugam Velayuthaprabhu, and Rengasamy Lakshminarayanan Rengarajan. Software and bioinformatics analysis was done by Pushparaj Karthika and Meyyazhagan Arun. Writing of original draft was done by Ramasamy Manikandan and Balasubramanian Balamuralikrishnan. Working group coordination was done by Balasubramanian Balamuralikrishnan. Reviewing and editing was done by Arumugam Vijaya Anand, Arunkumar Malaisamy, Shanmugam Velayuthaprabhu, and Rengasamy Lakshminarayanan Rengarajan. Reviewing and interpretation was done by Arumugam Vijaya Anand and Balasubramanian Balamuralikrishnan. All the authors read and finalized the published version of the manuscript.
The authors are grateful to the authorities for their support. The authors thank the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India, for computational studies, using the Schrodinger software 2021-2.
The authors declared to have no conflict of interest.
All the authors confirmed that the data supporting this study are accessible upon request.
Apple, F.S., Preese, L., Bennett, R. and Fredrickson, A., 1988. Clinical and analytical evaluation of two immunoasays for direct measurement of creatine kinase MB with monoclonal anti-CK-MB antibodies. Clinical Chemistry 34: 2364–2367. 10.1093/clinchem/34.11.2364
Arafa M.H., Mohammad N.S., Atteia H.H. and Abd-Elaziz H.R., 2014. Protective effect of resveratrol against doxorubicin-induced cardiotoxicity and fibrosis in male experimental rats. Journal of Physiology and Biochemistry 70: 701–711. 10.1007/s13105-014-0339-y
Bell, J.L. and Baron, D.N., 1960. A colorimetric method for determination of isocitrate dehydrogenase. Clinica Chimica Acta 5: 740–747. 10.1016/0009-8981(60)90017-6
Burtis, C.A. and Ashwood, E.R., 1996. Tietz fundamentals of clinical chemistry, 4th edition. W.B. Saunders, London, 881 p.
Buynes, J.W. and Thorpe, S.R., 2000. Glyoxidation and lipoxidation in atherogenesis. Free Radical Biology and Medicine 28: 1708–1716. 10.1016/S0891-5849(00)00228-8
Canceras, A., Figeroa, L., Taracena, A.M. and Samayoa, B.E., 1993. Plant used in Guatemala for the treatment of respiratory disease II. Evaluation of activity of 16 plants against Gram-positive bacteria. Journal of Ethnopharmacology 39(1): 77–82. 10.1016/0378-8741(93)90053-8
Daswani, P.G., Gholkar, M.S. and Birdi, T.J., 2017. Psidium guajava: A single plant for multiple health problems of rural Indian population. Pharmacognosy Reviews 11(22): 167. 10.4103/phrev.phrev_17_17
Decker RS, Wildenthal K., 1980. Lysosomal alterations in hypoxic and reoxygenated hearts. I. Ultrastructural and cytochemical changes. The American journal of pathology. 1980 Feb 98(2):425.
Frustaci, A., Kajastura, J., Chimenti, C., Jakonick, I., Leri, A., Maseri, A., et al. 2000. Myocardial cell death in human diabetes. Circulation Research 87: 1123–1132. 10.1161/01.RES.87.12.1123
Gessler, M.C., Nkunyak, M.H.H., Mwasumbi, L.B., Heinrich, M. and Tanner, M., 1994. Screening Tanzanian plants for antimalarial activity. Acta Tropica 56(1): 65–77. 10.1016/0001-706X(94)90041-8
Gutiérrez, R.M., Mitchell, S. and Solis, R.V., 2008. Psidium guajava: A review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology 117: 1–27. 10.1016/j.jep.2008.01.025
Hikmawanti N.P., Fatmawati S. and Asri A.W., 2021. The effect of ethanol concentrations as the extraction solvent on antioxidant activity of Katuk (Sauropus Androgynus (L.) Merr.) leaves extracts. In: IOP conference series: Earth and environmental science, Vol. 755, No. 1. IOP Publishing, Bristol, UK, p. 12060. 10.1088/1755-1315/755/1/012060
Hoffstein, S. and Weissmann, G., 1975. Mechanisms of lysosomal enzyme release from leukocytes. Arthiritis and Rheumatism 18(2): 153–165. 10.1002/art.1780180213
Karthikeyan, K., Sarala Bai, B.R. and Niranjali Devaraj, S., 2007. Cardioprotective effect of grape seed proanthocyanidins on isoproterenol-induced myocardial injury in rats. International Journal of Cardiology 115: 326–333. 10.1016/j.ijcard.2006.03.016
Kawai, Y. and Anno, K., 1971. Mucopolysaccharide-degrading enzymes from the liver of the squid, Ommastrephessloanipacificus I. Hyaluronidase. Biochimica et Biophysica Acta 242: 428–436. 10.1016/0005-2744(71)90234-8
King, J., 1965. In: Van, D. (ed.) Prac Clini Enzymology. Nostrand, London, Pp. 83–93.
Kumar, A., Khan, S.A., Parvez, A., Zaheer, M.S., Rabbani, M.U. and Zafar L., 2011. The prevalence of hyperhomocysteinemia and its correlation with conventional risk factors in young patients with myocardial infarction in a tertiary care centre of India. Biomedical Research 22: 225–229.
Leslie, V.A., Alarjani, K.M., Arunkumar, M., Balamuralikrishnan, B., 2021. Bacteriocin producing microbes with bactericidal activity against multidrug resistant pathogens. Journal of Infection and Public Health 14: 1802–1809. 10.1016/j.jiph.2021.09.029
Lutterodt, G.D., 1992. Inhibition of microlax induced experimental diarrhoea with narcotics like extracts of Psidium guajavain rats. Journal of Ethnopharmacology 37(2): 151–157. 10.1016/0378-8741(92)90073-Z
Manikandan, R. and Vijaya Anand, A., 2015. Evaluation of antioxidant activity of Psidium guajava Linn. in streptozotocin-induced diabetic rats. Free Radicals and Antioxidants 6(1): 72–76. 10.5530/fra.2016.1.9
Manikandan, R., Vijaya Anand, A. and Durai Muthumani, G., 2013. Phytochemical and in vitro anti-diabetic activity of methanolic extract of Psidium guajava leaves. International Journal of Current Microbiology and Applied Sciences 2(2): 15–19.
Manikandan, R., Vijaya Anand, A., Sampathkumar, P. and Manoharan, N., 2018. Protective effect of Psidium guajava leaf ethanolic extract against streptozotocin induced diabetes and lipidosis in rats. Indian Journal of Animal Research 52(8): 1198–1205. 10.18805/ijar.B-3337
Manikandan, R., Vijay Anand, A., Sampathkumar, P. and Pushp, 2016. Phytochemical and in vitro antidiabetic activity of Psidium guajava leaves. Pharmacognosy Journal 2016,8,4,392-394. 10.5530/pj.2016.4.13
Mehler, A.H., Kornberg, A., Grisolia, S. and Ochoa, S., 1948. The enzymatic mechanism of oxidation-reductions between malate or isocitrate or pyruvate. Journal of Biological Chemistry 174: 961–977. 10.1016/S0021-9258(18)57306-3
Moore, J.C. and Morris, J.E., 1982. A simple automated colorimetric method for determination of N-acetyl β-D glucosaminidase. Annals of Clinical Biochemistry 19: 157–159. 10.1177/000456328201900305
O’Brien, P.J., Smith, D.E., Knechtel, T.J., Marchak, M.A., Pruimboom-Bress, I., Bress, D.J., et al. 2006. Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals. Laboratory Animals 40(2): 153–171. 10.1258/002367706776319042
Okinada, S., Kumagai, H. and Ebashi, S.H., 1961. Serum creatine phosphokinase. Activity in progressive muscular dystrophy and neuromuscular diseases. Archives of Neurology 4: 520–525. 10.1001/archneur.1961.00450110050006
Panche A.N., Diwan A.D. and Chandra S.R., 2016. Flavonoids: an overview. Journal of Nutritional Science 5: e47. 10.1017/jns.2016.41.
Pearl, W., Cascarano, J. and Zweifach, B.W., 1963. Micro determination of cytochromeoxidase in rat tissues by the oxidation on N-phenyl-p-phenylene diamine or ascorbic acid. Journal of Histochemistry and Cytochemistry 11: 102–104. 10.1177/11.1.102
Reed, L.J. and Mukherjee, R.B., 1969. Α-ketoglutarate dehydrogenase complex from Escherichia coli. Methods Enzymology 13: 55-51. 10.1016/0076-6879(69)13016-5
Sapolsky, A.I., Altman, R.D. and Howell, D.S., 1973. Cathepsin-D activity in normal and osteoarthritic human cartilage. Federation Proceedings 32: 1489–1493.
Seager, M.J., Singal, P.K., Orchard, R., Pierce, G.N. and Dhalla, N.S., 1984. Cardiac cell damage: a primary myocardial disease in streptozotocin-induced chronic diabetes. British Journal of Experimental Pathology 65(5): 613–623.
Sinha, A.K., 1972. Colorimetric assay of catalase. Analytical Biochemistry 47: 389–394. 10.1016/0003-2697(72)90132-7
Sivakumar, V. and Dhana Rajan, M.S., 2011. Standardization & characterization of Tinospora cordifolia (Willd.) Miers ex Hook. F. & Thoms. Plant stem extract in different solvent fractions. Asian Journal of Biochemical and Pharmaceutical Research (AJBPR) 1(4): 105–112.
Slater, E.C. and Bonner, W.D., 1952. The effect of fluoride on succinic oxidase system. Biochemical Journal 52: 185–196. 10.1042/bj0520185
Subashini, R. and Sumathi, P., 2012. Cardioprotective effect of Nelumbo nucifera on mitochondrial lipid peroxide enzymes and electrolytes against isoproterenol-induced cardiotoxicity in Wistar rats. Asian Pacific Journal of Tropical Disease 2(2): S588–S591. 10.1016/S2222-1808(12)60227-8
Sudharsan, P.T., Mythili, Y., Selvakumar, E. and Varalakshmi P., 2006. Lupeol and its ester exhibit protective role against cyclophosphamide-induced cardiac mitochondrial toxicity. Journal of Cardiovascular Pharmacology 47(2): 205–210. 10.1097/01.fjc.0000200658.89629.ba
Sultana, B., Anwar, F. and Ashraf, M., 2009. Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules 14(6): 2167–2180. 10.3390/molecules14062167
Tsoo, E., King, R. and Howard, L., 1967. Preparations and properties of soluble NADH dehydrogenases from cardiac muscle. Methods in Enzymology 275–294. 10.1016/0076-6879(67)10055-4
Vijayakumar, K., Rengarajan, R.L., Radhakrishnan, R. and Anand, A.V., 2018. Hypolipidemic effect of Psidium guajava leaf extract against hepatotoxicity in rats. Pharmacognosy Magazine 14(53): 4–8. 10.4103/pm.pm_167_17
Vijayakumar K., Arumugam V.A., Ramasamy M., Natesan M., Palanisamy S., Thajuddin NB., et al. 2020. Hepatoprotective effects of Psidium guajava on mitochondrial enzymes and inflammatory markers in carbon tetrachloride-induced hepatotoxicity. Drug Development and Industrial Pharmacy 46(12): 2041–2050. 10.1080/03639045.2020.1843474
Vishal, D. Joshi, Akash, P. Dahake and Ashok, P. Suthar., 2010. Adverse effects associated with the use of antihypertensive drugs: an overview. International Journal of PharmTech Research 1 & 2: 10–13.
Xie, F., Wang, H., Cao, Q., Chen, Q. and Lin, F. 2022. The effects of Oldenlandia diffusa water extract on glucose metabolism and inflammation level in rats with streptozotocin-induced gestational diabetes mellitus. Quality Assurance and Safety of Crops & Foods 14(1): 24–30. 10.15586/qas.v14i1.970
Zakaria, M. and Mohd, M.S., 1994. Traditional Malay medicinal plants. Penerbit Fasar Bakti Suden Berhard, Selangor, Malaysia, pp. 129–132.