1. Introduction
Diabetes mellitus (DM) is a persistent metabolic disorder that is delineated by hyperglycemia and pancreatic β-cell dysfunction, due to which β-cells cannot produce sufficient insulin, ultimately leading to impaired insulin secretion and inappropriate utilization of carbohydrate metabolism disorder in the body [
1]. Recent research has reported that oxidative stress is a prime cause of diabetes and its complications. Due to oxidative stress, the number of free radicals increases in the body, eventually resulting in cell death. All these complications lead to various types of abnormalities, including cardiomyopathy, hepatopathy, nephropathy, neuropathy, and retinopathy [
2].
Diabetic cardiomyopathy is described as a disease in which irregular myocardial structure and function occurs in diabetic patients, while other risk factors such as hypertension, significant valvular disease, and coronary artery disease are absent. If systolic dysfunction happens due to diabetes, then there is an imperilment for idiopathic cardiomyopathy (ICM), a condition in which systolic function decreases with an enlarged left or right ventricle. The exact cause remains unknown initially in almost 50% of patients; this condition is known as idiopathic dilated cardiomyopathy (DCM). The pathology provides no indication; the heart becomes dilated and the size of the heart also increases, but the thickness of the ventricular walls remains the same [
3].
The use of plants and herbal medications is a common practice for the treatment of several ailments such as diabetes mellitus and its complications [
4].
Jasminum sambac is an ornamental plant that is cultivated all over Asia and it belongs to the
Oleaceae family. The existence of glycosides, proteins, and coumarins; flavonoids such as rutin, quercetin, kaempferol, and apigenin; phenolics such as caffeic acid, gallic acid, and chlorogenic acid; ascorbic acid, resin, terpenes, saponins, steroids, salicylic acid, and essential oils was confirmed by phytochemical analysis of
Jasminum sambac. The extract obtained from
Jasminum sambac has therapeutic activities such as antimicrobial, anti-oxidant, and anti-inflammatory properties. This plant has also been proven to be curative and efficacious in the treatment of several infectious diseases because it contains phenols, cardiac glycosides, carbohydrates, alkaloids, terpenes, and some other constituents [
5].
The extract from the leaves of
Jasminum sambac produces a lowered plasma glucose level, serum urea, and improved hyperlipidemia in hyperglycemic rats [
6]. The pharmacological basis for the therapeutic uses of
Jasminum sambac in relation to cardiovascular diseases was investigated in a research study. According to the study,
Jasminum sambac crude leaf extract demonstrated ex vivo vasorelaxant effects in an endothelium-intact aorta ring preparation, and hypotensive effects were also noted with the aid of the Power Lab. During an ex vivo study, it was discovered that the
Jasminum sambac leaf extract caused a vasorelaxant/hypotensive action via activating muscarinic receptors and/or producing the local vasodilator nitric oxide [
7]. Keeping in view the various therapeutic roles of
Jasminum sambac, the current experimental study aimed to evaluate the pharmacological effect of phenols extracted from
Jasminum sambac leaves on diabetes-induced cardiomyopathy in alloxanized hyperglycemic rats.
3. Discussion
Medicinal plants have been used for years to medicate many health-related complaints and meet patients’ nutritional requirements. Herbal medicines have also been used for decades for the treatment of diabetes, along with its complications. These drugs contain many phytochemical components, each with a distinct structure, which work together to cure various illnesses [
8]. The most important phytochemical group with anti-inflammatory, antistress, antioxidant, and antihyperglycemic characteristics is the phenolic group. In our study, the phenolic concentration in the JSP extract was 129.6 ± 3.0 mg GAE/g. Because of their antioxidant and antihyperglycemic properties, phenolic extracts are accountable for therapeutic efficacy against diabetes and its complications. These phenolic compounds influence the glucose metabolism through various mechanisms, including activation of insulin release from pancreatic cells, insulin receptor activation, glucose uptake in insulin-sensitive cells, increased glucokinase activity, and glucose release control from the liver [
9].
In a recent study, alloxan monohydrate was used to produce diabetes, resulting in hyperglycemia [
10]. Under diabetic conditions, the body mass of the diabetic rats was considerably decreased because there was a decrease in the level of insulin in the body, and the body’s ability to absorb glucose was diminished [
11]. In this context, the body starts to burn fats, thus resulting in overall weight loss. Oral administration of the JSP extract gradually increased (
p < 0.05) body weight, as well as lowered the fasting blood glucose level depending on the time and dose of the drug. In a hyperglycemic state, due to the eradication of glucose homeostasis, the levels of Hb1Ac and insulin in the body are disturbed [
12]. The results of this study showed that the JSP extract considerably reverted (
p < 0.05) the serum glucose, HbA1c, and insulin levels in the treated groups. Compared to the alloxan-induced diabetic rats, JSP extract therapy reduced the rise in cardiac parameters, hyperlipidemia, and hyperglycemia, with a comparative reduction in body weight [
13,
14].
The results revealed that the levels of cardiac troponins I, pro-BNP, and IMB were elevated effectively (
p < 0.05) in the diabetic rats. An increase in troponin in diabetic individuals with numerous cardiovascular risk factors is linked to chronic coronary artery disease. Troponin might be used as a tracer in high-risk groups [
15]. The levels of brain natriuretic peptides (Nt-proBNP and BNP) increase in left ventricular dysfunction. A high production of these peptides has been linked to coronary artery disease [
16]. Administration of graded doses of the JSP extract in the third and fourth groups of hyperglycemic rats gradually lowered (
p < 0.05) the cardiac troponins I, pro-BNP, and IMB levels. These improved cardiac troponins I, pro-BNP, and IMB levels predict the antioxidant properties of the JSP extract. These findings are in agreement with a previous study, which also identified the effects of a
Marigold hydroalcoholic extract on diabetes-induced cardiotoxicity with a remarkable reduction (
p < 0.05) in cardiac biomarkers [
17]. An upraised serum level of CK-MB is a possible and sensitive sign to determine the likelihood of cardiac problems [
18]. The current work also showed that hyperlipidemia, another component attributed to cardiac dysfunction and cell death in diabetes, is related to increased CK-MB and troponin I in the serum of rat models of myocardial degeneration. The link between hyperlipidemia and increased triglyceride and cholesterol deposition produces toxicological effects on the heart [
19].
Diabetes mellitus is frequently accompanied by hyperglycemia, insulin resistance, inflammation, and dyslipidemia, all of which promote the production of reactive oxygen species (ROS), which are recognized to be a factor in the development of diabetic ailments such as cardiomyopathy [
20]. The buildup of ROS and superoxide produces oxidative stress, which can cause DNA damage, cardiac cell death, myocardial contractility loss, and cardiac fibrosis. Additionally, the buildup of ROS/RNS causes degradation of the antioxidant defense system, which is made up of scavenger antioxidating GPx, SOD, and catalase [
21]. Our findings show that the GPx, SOD, and catalase levels were remarkably lower (
p < 0.05) after the induction of diabetes. In contrast, continuous use of the JSP extract had a dose-dependent ability to restore depleted antioxidant enzymes to their normal levels. Strong markers of antioxidants’ preventive effect include the recovery of antioxidant enzymes (GPx, CAT, and SOD) and the lowering of antioxidants. These findings are consistent with earlier literature [
22].
Numerous cytoprotective proteins, such as heme oxygenase-1 (HO-1), an enzyme that catalyzes the breakdown of heme into the antioxidant biliverdin, carbon monoxide (an anti-inflammatory and antioxidant), and ferrous iron, may be induced by Nrf2 activation [
23]. In response to stress factors like oxidative stress, heavy metals, nitric oxide, hypoxia, and cytokines, HO-1 is highly induced. In the antioxidant reaction, the Nrf2/HO-1 pathway always plays a crucial role [
24]. To examine the therapeutic role of the JSP extract against induced oxidative stress, the expression of Nrf2 and HO-1 was observed. In line with prior research [
25], JSP extract therapy increased the expression of HO-1 and Nrf2 in the treated groups, encouraging the restoration of antioxidant enzymes (GPx, SOD, and CAT). The NO synthase (NOS), which consists of endothelial NOS, neuronal NOS, and iNOS, is responsible for producing nitric oxide (NO). Depending on the disease risk factors, NO-mediated actions might be advantageous or detrimental [
26]. Only specific stresses, such as those present in obesity and diabetes, can induce inducible NOS (iNOS) [
27]. It has been previously noticed that the proliferation of cardiac fibroblasts is promoted by heart-specific iNOS overexpression in female rats [
28]. One of the primary causes of diabetic myopathy is mitochondrial dysfunction, which is encouraged by oxidative stress and results in the activation of pro-apoptotic proteins Bax and caspase-3 and a decrease in anti-apoptotic proteins. Cell survival or death in response to apoptotic stimuli is determined by the Bax/Bcl-2 ratio. It had been demonstrated that increasing Bax expression and decreasing Bcl-2 both encourage cell death [
29]. The ratio of Bax/Bcl-2, considered an index of apoptosis, was higher in the diseased group of rats, and JSP extract treatment significantly prevented (
p < 0.05) this increases in Bax/Bcl-2 ratio in the treated groups.
It is widely acknowledged that a significant increase in the levels of circulating cardiac damage indicators like troponin and CK-MB serves as a reliable predictor of increased cardiac problems [
30]. The alloxanized hyperglycemic rats in this study showed a considerable increase in heart injury indicators, as seen by the degeneration of myocardial tissue, vacuolation, apoptotic nuclei, and necrosis. The JSP extract significantly regularized (
p < 0.05) the histoarchitecture of pancreatic tissues, according to histopathological examination. In those rats treated with JSP, a dose-dependent increase in the pancreatic beta cell population and a decrease in necrotic tissue were seen. Apoptosis caused by oxidative stress has been shown to be reduced by the phenols found in traditional plants, which could be the reason for pancreatic B-cell regeneration [
31]. Administration of the JSP extract to the diabetic rats reduced heart damage and restored the heart cells and nuclei’s histological organization.
4. Methods
4.1. Extraction Procedure of Phenols
The leaves of
Jasminum sambac were used for this experiment.
Jasminum sambac leaves were collected from the University of Agriculture, Faisalabad (UAF) botanical garden, and verified by the department of botany. Following verification, the plant material was stored in the UAF, Faisalabad, herbarium with voucher number 21,189. After cleaning with water to remove dirt and other unwanted materials, the gathered plant matter (leaves) was dried in the shade. The dried
Jasminum sambac leaves were powdered and extraction was carried out with 70% ethanol. To acquire residue, the extract was evaporated until it was completely dry. The acquired residue was then treated with petroleum ether and diethyl ether, forming two layers. The aqueous layer containing phenols was collected and utilized for further research [
32].
4.2. Qualitative Phytochemical Analysis of Phenols and Flavonoids
For the detection of phenols, a few drops of 10% FeCl
3 were added to the extract, and the appearance of a green color indicated the presence of phenols. For further confirmation of phenols, the extract was dissolved in 10% lead acetate, and bulky white preparation stated the presence of phenols [
33]. To detect flavonoids, a few drops of dilute NaOH were added to 1 mL of extract, and a yellow color indicated the presence of flavonoids [
34].
4.3. Quantitative Phytochemical Analysis of Phenols and Flavonoids
The entire phenolic content in the extract was achieved using the Folin–Ciocalteu method. The absorbance at 765 nm was monitored using a UV-visible spectrometer to achieve determination. The outcomes were compared using gallic acid as the reference component, and are shown as milligram Gallic acid equivalent per gram of extract (mg GAE/g) [
35]. The total flavonoid content was calculated by using the aluminum chloride calorimetric technique. The absorbance was determined at 510 nm by spectrophotometer, and the results were demonstrated as milligram catechin equivalent per gram of the extract (mg CE/g).
4.4. Quantification of the Phenols in the Jasminum sambac Extract
High-performance liquid chromatography–mass Spectrometry (HPLC/MS, Agilent Technologies, Santa Clara, CA, USA) was used to analyze the
Jasminum sambac polyphenol (JSP) extract. A C-18 column (4.6 × 250 mm, as well as 5 µm Agilent, USA) was used to separate the different components of the extract. Glacial acetic acid at a concentration of 2% (
v/
v) was used to mobile phase A, while mobile phase B was made up of acetonitrile. The analytical conditions were: 10 B, 10 min; 25% B, 15 min; 50% B, 45 min; 75% B, 65 min; 100% B, 75 min. The column temperature was 28 °C, the injection volume was 20 µL, and the flow rate was 1 mL/min. The absorbance wavelength was 280–310 nm for identifying phenolic content using a UV detector. At least three separate phenolic extractions were carried out and evaluated using HPLC/MS. Phenolic substances were measured by comparing each molecule to the standard [
36].
4.5. In Vitro Antioxidant Analysis of the JSP Extract
4.5.1. Total Antioxidant Capacity (TAC)
The procedure described by Prieto et al. [
37] was used to calculate the total antioxidant capacity of the JSP extract with slight modifications. A 0.5 mL extract was mixed with 3 mL of the reagent mixture (28 mM sodium phosphate, 0.6 M sulfuric acid, and 4 mM ammonium molybdate), incubated at 95 °C for 90 min, and the results were compared to the blank at 695 nm. Ascorbic acid was employed as the calibration standard. The results are presented in terms of milligrams of AA per gram of wet weight.
4.5.2. DPPH Assay
For the 1,1 diphenyl-2-picryal hydrazyl (DPPH) assay of the JSP extract, 900 µL of DPPH was added under dark light with 0.1 mL of JSP at various concentrations. The reaction mixtures were kept at room temperature. The prepared sample was incubated for 40 min, and the absorbance of the sample was recorded at 517 nm. The assays were all run in triplicate, and the ascorbic acid equivalent was used to calculate the parentage inhibition [
38].
By using the following equation, the radical scavenging activity of the phenolic extract was determined:
4.5.3. ABTS•+ Radical Scavenging Assay
The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•
+) radical cation-based assay was employed to assess the antioxidant capacity of the JSP extract [
39]. The reaction between 7 mM ABTS in water and 2.45 mM potassium persulfate (1:1) created the ABTS•+ cation radical, which was then kept at room temperature for 12–16 h in the dark. The ABTS•+ solution was diluted with methanol to produce an absorbance of 0.700 at 734 nm. Absorbance was measured 30 min after mixing 4 mL of the diluted ABTS
·+ solution and 5 μL of the extract. Trolox was used as the standard substance and the results are presented in terms of mole Trolox/gram.
4.6. Experiment Animals
Young Wistar albino rats in good health, weighing 180–200 g, were acquired from the animal house of the Department of Pharmacy, University of Faisalabad, Pakistan. The rats were retained in the animal room situated in the Department of Pharmacy. The animals were housed for a two-week acclimation period before the experiment. They were housed in four cages with a consistent 12 h cycle of light and darkness and they received a normal basal diet and water ad libitum. The rats were fed a typical standard diet in the form of 50 g pellets composed of maize (48%), soyabean (20%), corn bran (6.8%), palm kernel cake (5%), fish and bone meal (4% and 3%, respectively), ground nut (12%), and salt (0.25%), with crude proteins (23%) and crude fiber (3%) and balanced with vitamins and minerals. The University of Faisalabad’s institutional review board (IRB) provided ethical approval (TUF/IRB/180/23) to follow the experimental protocols. The research study was conducted in accordance the Guide for the Care and Use of Laboratory Animals by National Research Council (NRC).
4.7. Acute Oral Toxicity Test
The selection of doses of the JSP extract was based on the acute oral toxicity test. The method of OECD Guidelines [
40] was used for determining acute oral toxicity in the rats. The rats were divided into five groups, with three animals in each, along with one control group. The animals were kept on water only for a whole night before receiving doses of 1000, 1500, 2000, and 2500 mg/kg of the JSP extract in groups II, III, IV, and V, respectively, for the determination of safe doses. The rats were observed for 14 days after dosage for lethargy, jerkiness, and mortality. The safe dose was determined to be between 1/5th and 1/10th of LD
50. In the acute oral toxicity dose test, no death or morbidity was seen at any dose. Therefore, the LD
50 may be greater than 2500 mg/kg. As a result, the maximal safe dosages of 250 and 500 mg/kg were chosen for pharmacological tests [
41].
4.8. Experimental Design
Following the adaption phase, the animals were split into four groups, each with 20 rats. According to previous studies conducted regarding diabetes induction in rats through alloxan monohydrate, a single shot of alloxan monohydrate (150 mg/kg) mixed with 0.9% normal saline was provided intraperitoneally for diabetes induction in the overnight-fasted rats, except in the normal group. The most important problem associated with use of alloxan is triphasic blood glucose response in animals with initial hyperglycemia due to the apoptosis of beta cells, profound hypoglycemia for approximately 6–12 h, and persistent hyperglycemia after 24 h. Therefore, the rats were given a 5% glucose solution orally during the first 24 h to prevent any negative effects of hypoglycemia. Animals can exhibit signs of illness such as reduced mobility or lethargy due to triphasic response. Thereby, the physical activity of the diseased rats was also continuously monitored for 48 h in order to reduce the chances of morbidity or mortality.
The blood sugar of all groups of rats was measured using a glucometer via the tail vein one week after the injection. In the study, the diabetic rats were determined to have blood glucose levels higher than 300 mg/dL. During this time period, the animals were given access to water and their regular meal. After confirmation of disease induction (seven days after alloxan induction), treatment of the diseased rats with the test formulation was started by dividing them in groups, and the blood glucose level of all groups was monitored by checking the glucose level of the rats in order to observe the glycemic status and effect of the JSP extract in the treated groups up until six weeks of study. The rats were organized into four groups as follows:
- -
Group I: Untreated normal control group on a routine diet;
- -
Group II: Untreated diabetic (positive control) given alloxan (150 mg/kg);
- -
Group III: Diabetic group treated with the JSP extract (250 mg/kg) for six weeks;
- -
Group IV: Diabetic group treated with the JSP extract (500 mg/kg) for six weeks.
4.9. Physical Parameters
4.9.1. Body Weight
Each rat’s body weight was measured weekly until six weeks into the experimental study.
4.9.2. Feed and Water Intake
Each rat’s feed and water consumption were measured every day up until six weeks of the experimental study. From the data, the average feed and water intake of each group was calculated.
4.10. Blood Sampling
After the six-week period of observation, the overnight-fasted rats were sedated with 3% intraperitoneal sodium pentobarbital and then slaughtered. Blood samples were taken from every rat. The gathered blood samples were centrifuged at 3000 rpm at 4 °C for 15 min. The plasma and serum were separated and kept at −20 °C for hormonal and biochemical testing. The heart and pancreas were kept in a 10% buffer formalin solution for histopathological assessment.
4.11. Preparation of Tissue Homogenate
The tissues of the rats from each group were kept in 10% (
w/
v) buffer (50 mM Tris-HCL, 1.15% KCL, and pH 7.4) and centrifuged at 9000 rpm at 4 °C for 3 min. The obtained tissue homogenate was used for the biochemical analysis. According to the procedure [
42], the obtained homogenate’s protein concentration was examined.
4.12. Biochemical Analysis
The fasting blood glucose level was measured by glucometer weekly through the tail prick method up to six weeks of the experiment. The serum glucose and insulin concentrations were estimated by a rat glucose assay kit (81693, crystal chem, Elk Grove Village, IL, USA) and an insulin ELIZA kit (ELR-insulin, RayBio®, Peachtree Corners, GA, USA). Glycosylated hemoglobin was estimated using a rat hemoglobin Hb1Ac assay kit (MBS2033689, My BioSure, Grass Valley, CA, USA). The serum triglyceride, high-density lipid (HDL), low-density lipid (LDLs), and cholesterol levels were calculated using commercially available kits (ab65359, ab65336, and ab65390, respectively; Abcam, Cambridge, UK). The serum level of liver function enzymes, i.e., alanine aminotransaminase (ALT) and aspartate aminotransferase (AST), were determined through the kit assay method (ab105134/k752-100 and ab105135, respectively; Abcam, Cambridge, UK).
4.13. Oxidative Stress and Antioxidant Enzyme Measurements
A thiobarbituric acid reactive substances (TBARS) assay kit was used to measure lipid peroxidation in the form of malondialdehyde (MDA) using tissue homogenate, according to the method of [
43]. The resultant TBARS absorbance was calibrated at 532 nm using a spectrophotometer, and the outcomes were presented as nmol MDA/mg protein. Assays for antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) were performed on the tissue homogenate via a catalase activity assay kit (ab83464, Abcam, UK), a SOD assay kit (706002, Cayman chemical, Ann Arbor, MI, USA), and a glutathione peroxidase assay kit (ab102530, Abcam, UK), respectively.
4.14. Determination of Cardiac Biomarkers
The cardiac troponin I level was determined using a commercially available ELISA kit (Cardiac troponin ELISA Kit, ab223860, Abcam, UK). To determine natriuretic peptide (pro-BNP), an ELISA kit (ab263877, Abcam, UK) was used as per the manufacturer’s instructions. The ischemic-modified albumin (IMA) level was analyzed through an IMA ELISA kit (MBS263569) based on IMA’s ability to bind to cobalt. Lactate dehydrogenase (LDH) was investigated through the kit method (MBS269777, MyBioSource, San Diego, CA, USA) and the serum creatinine kinase-MB fraction (CK-MB) level was determined using a CK-MB ELISA kit (CSB-E14403r, CUSABIO, Houston, TX, USA).
4.15. Histopathological Examination
The animals were slaughtered at the end of the research session, and organs from the body were separated. The hearts and pancreases of the rats were placed in 10% buffered formalin. The hearts were cut into 3 mm pieces. The organs were sliced perpendicularly, starting from the apex and leading to the base. Sliced heart and specimens from the pancreas were put into paraffin, stained with hematoxylin and eosin, and examined beneath a light microscope [
44].
4.16. RNA Extraction and Real-Time Quantitative PCR
The TRIzol technique (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the RNA, which was then modified and measured using the nanodrop technique [
45]. A RevertAid cDNA synthetic kit (Thermo Fisher Scientific) was used to reverse transcribe the total extracted mRNA to cDNA, as specified by the manufacturer’s instructions. The maxima SYBR Green/ROX qRT-PCR Master Mix (Thermo Fisher Scientific) performed real-time qPCR (RT-qPCR) on the iQ5 Bio-Rad equipment. Rat primers for gene expression of the Nrf-2, Bcl-2, Bax, Caspase-3, HO-1, and β-Actin genes (
Table 7) were used [
46] as per the manufacturer’s guidelines.
The PCR conditions were 40 cycles for cDNA denaturation at 95 °C for 15 s, 25 s of annealing at 52 °C, and 20 s of extension at 72 °C. The relative expression of the genes was determined using the 2−ΔΔCT technique.
4.17. Statistical Analysis
The results are presented as the mean and SD. The data were statistically analyzed using Duncan’s multiple range test (DMR) and ANOVAs.
p < 0.05 was used to determine statistical significance [
47].