{"id":59198,"date":"2024-06-25T10:14:51","date_gmt":"2024-06-25T10:14:51","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=59198"},"modified":"2024-07-03T18:00:44","modified_gmt":"2024-07-03T18:00:44","slug":"silybum-marianums-impact-on-physiological-alterations-and-oxidative-stress-in-diabetic-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/silybum-marianums-impact-on-physiological-alterations-and-oxidative-stress-in-diabetic-rats\/","title":{"rendered":"S\u00edlybum Mari\u00e1num\u2019s Impact on Physiological Alterations and Oxidative Stress in Diabetic Rats"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Diabetes mellitus stands as a prevalent metabolic disorder in developed nations, characterized by either deficient insulin secretion or cellular resistance to its effects (type I and type II diabetes, respectively). Regardless of the subtype, a shared pathological hallmark is elevated blood glucose levels\u2014hyperglycemia\u2014posing a risk for significant organ and tissue damage over time.1<\/sup>. <\/p>\n\n\n\n

The rising prevalence and expanding array of complications associated with diabetes mellitus significantly impact patients’ quality of life and mortality rates. A promising avenue in understanding the pathophysiological mechanisms of diabetes lies in the theory of oxidative stress. Diabetes is recognized to trigger and exacerbate oxidative stress, resulting in the accumulation of free radical oxidation products due to chronic hyperglycemia and disrupted insulin production. This cascade of events contributes to the progression of complications associated with the disease.2<\/sup>. <\/p>\n\n\n\n

Oxidative stress has been known for over 30 years. During this time, numerous studies and experiments have been conducted, indicating the significance of this process in the development and course of various pathological conditions.3-4<\/sup>.<\/p>\n\n\n\n

Due to the incomplete understanding of how oxidative stress functions, there is still uncertainty about the effectiveness of preventive measures and treatments for the aforementioned diseases.5<\/sup>. Consequently, the assessment of promising medications that could prevent and alleviate undesirable reactions caused by free radicals remains a relevant and important task in pharmacology.6<\/sup>. <\/p>\n\n\n\n

Free radicals are particles with one or more unpaired electrons on their outer electron shell, which results in heightened reactivity. These radicals seek to gain an additional electron from other molecules, leading to disruption or damage to the structure of cellular membranes.7<\/sup>.<\/p>\n\n\n\n

The reactive oxygen species (ROS) formed in free radical oxidation reactions exert a destructive effect on cells of vital organs, negatively impacting human health. Antioxidants serve as sources of neutralizing ROS and preventing the development of oxidative stress.8<\/sup>. <\/p>\n\n\n\n

The adoption of herbal preparations for treating\nconditions like diabetes mellitus and cardiovascular diseases is well-founded,\ngiven their notable safety record, reasonable effectiveness, suitability for\nlong-term use in chronic ailments, wide accessibility, and relatively low cost.\nAlthough herbal remedies may not entirely replace synthetic medications, they\noften complement conventional treatments effectively. In Uzbekistan’s\ntraditional medicine arsenal, numerous plants exhibit positive effects on the\ncardiovascular system and can serve as supplementary therapy for ongoing health\nissues. For example, S\u00edlybum mari\u00e1num<\/em>, a mixture of flavonoid complexes,\nacts as the active component that shields liver and kidney cells from the toxic\neffects of various drugs, including chemotherapy and others.<\/p>\n\n\n\n

 Materials and methods <\/strong><\/p>\n\n\n\n

S\u00edlybum mari\u00e1num<\/em>\nextract was generously provided by \u201cBioton\u201d LTD, Tashkent, Uzbekistan.<\/p>\n\n\n\n

Experimental design<\/strong><\/p>\n\n\n\n

Alloxan (Sigma, 120 mg\/kg body weight) was used to induce diabetes according to a previous study 9<\/sup>. The alloxan was readied newly and dissolved in chilled normal saline prior to intraperitoneal administration to the rats. Fasting blood glucose levels were measured using the glucose oxidase method three days after alloxan injection. Rats with fasting blood glucose levels above 300 mg\/dL were classified as diabetic. Thirty rats were divided into five groups, each containing six rats.<\/p>\n\n\n\n

Group (N): Normal rats received a daily 1 mL dose of normal saline via gavage for 6 weeks.<\/p>\n\n\n\n

Group (NR): Normal rats received a daily 1 \u00d7 10^9 CFU\/mL dose of S\u00edlybum mari\u00e1num extract via gavage for 2 weeks.<\/p>\n\n\n\n

Group (D): Diabetic rats received a daily 1 mL dose of normal saline via gavage for 2 weeks before and 4 weeks after diabetes induction.<\/p>\n\n\n\n

Group (DRB): Diabetic rats received a daily 1 \u00d7 10^9 CFU\/mL dose of S\u00edlybum mari\u00e1num extract via gavage for 2 weeks before diabetes induction.<\/p>\n\n\n\n

Group (DRA): Diabetic rats received a daily 1 mL dose of normal saline for 2 weeks before diabetes induction and a 1 \u00d7 10^9 CFU\/mL dose of S\u00edlybum mari\u00e1num extract for 2 weeks after diabetes induction via gavage.<\/p>\n\n\n\n

The experiment concluded on the 24th day when the animals were euthanized following deep anesthesia with CO2<\/sub>. After euthanasia, the hearts were dissected and weighed. Subsequently, the hearts were frozen and processed for oxidant and antioxidant assays. All procedures were conducted in accordance with animal welfare guidelines and regulations.<\/p>\n\n\n\n

Determination of antiradical activity. <\/strong><\/p>\n\n\n\n

The impact of S\u00edlybum mari\u00e1num on the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) was examined following the procedure outlined in reference 10<\/sup>. Ethanol solutions of the test preparations were introduced into a control cuvette containing 100 \u03bcM DPPH. The mixture was promptly stirred, and alterations in absorbance at 517 nm were monitored continuously over a period of thirty minutes.<\/p>\n\n\n\n

Acute alloxan hyperglycemia <\/strong><\/p>\n\n\n\n

Acute hyperglycemia was induced by administering a single intraperitoneal injection of alloxan at a dose of 120 mg\/kg. The decrease in blood glucose levels was evaluated at both 1 hour and 5 days after inducing hyperglycemia, following the method described in reference 11<\/sup>. Additionally, acute hyperglycemia was induced by a single intraperitoneal injection of alloxan at various doses ranging from 1 to 3 grams. The reduction in blood glucose levels was examined at 1 hour and 2 days post-induction of hyperglycemia, according to reference 11<\/sup>.<\/p>\n\n\n\n

The hypoglycemic effect (X) of the drug was determined using the following formula: <\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Where ‘a’ represents the concentration of sugar in the control group, measured in mmol\/l, and ‘b’ denotes the average concentration of sugar in the blood, also measured in mmol\/l, in the experimental group. 11<\/sup>.<\/p>\n\n\n\n

Determination of MDA in heart tissue <\/strong><\/p>\n\n\n\n

The level of Thiobarbituric Acid\nReactive Substances (TBARS), which serves as an indicator of Malondialdehyde\n(MDA) production and lipid peroxidation, was assessed in the tissues using the\nmethod described by Heath and Packer12<\/sup>.<\/p>\n\n\n\n

In summary, 1 mL of tissue supernatant was mixed with tubes containing 4 mL of a 20% Trichloroacetic acid (TCA) solution with 0.5% Thiobarbituric acid (TBA). The mixture underwent heating at 95\u00b0C for 30 minutes, followed by cooling and centrifugation at 10,000 \u00d7 g for 10 minutes. The resulting MDA-TBA complex was measured using a spectrophotometer at 532 nm.<\/p>\n\n\n\n

Determination of Creatine kinase activity in heart tissue<\/strong><\/p>\n\n\n\n

This research employs the Cypress\nDiagnostics Creatine Kinase NAC kit. Reagent 1 is combined with reagent 2 at a\nratio of 4 parts to 1 part. The effectiveness of these prepared reagents\npersists for 2 weeks when stored between 2-8\u00baC or for 48 hours at room\ntemperature (15-25\u00baC). All constituents of the kit remain viable until the\nindicated expiry date on the label, provided they are securely sealed, shielded\nfrom light, and safeguarded against contamination during utilization. The kit\nshould be stored at 2-8\u00baC, while reagents and samples are maintained at room\ntemperature. Absorbance measurements are conducted at 340 nm using a\nspectrophotometer, with distilled water serving for zero adjustment. Optimal\ntemperatures for measurements are 25, 30, and 37\u00baC, using a cuvette with a 1 cm\nlight path. A mixture comprising 40 \u00b5L of sample and 1 mL of working reagent is\nincubated for two minutes. The starting absorbance (A) is recorded, and then\nthe stopwatch is started, with absorbance measured every minute for three\nminutes. Next, the absorbance differences and the average absorbance difference\nper minute (\u0394A\/min) are calculated.<\/p>\n\n\n\n

Calculation<\/p>\n\n\n\n

At 25-30 \u00baC \u0394A\/min \u00d7 4127 = U\/L CK <\/p>\n\n\n\n

The effect of S\u00edlybum mari\u00e1num<\/em><\/strong> extract on aorta smooth muscle contraction <\/strong><\/p>\n\n\n\n

The studies were conducted on white, outbred male rats weighing between 200-250 g, utilizing aortic preparations. The experimental animals were humanely euthanized by cervical dislocation. After opening the chest, the aorta was surgically isolated for further study.Krebs-Henseleit physiological solution (in mM):; KCl 5; NaCl 120.4; NaH2<\/sub>PO4<\/sub> 1.2; NaHCO3<\/sub> 15.5; CaCl2<\/sub> 2.5; MgCl2<\/sub> 1.2; was perfused through a specially designed chamber (5 ml) containing S6<\/sub>N12<\/sub>O6<\/sub> 11.5 and HEPES rN 7.4. Some experiments also employed Krebs solutions lacking Ca2+<\/sup>, for which EGTA (1 mM) was added to the Krebs solution. The physiological solutions were aerated with carbogen (95% O2, 5% CO2) and maintained at +37\u00b0C using a U-8 ultrathermostat. Following the removal of connective tissue and surrounding fat from the aorta, segments of 3-4 mm were cut into ring shapes. These aortic rings were then attached to a Radnoti (Isometric-Transducer, USA) sensor using platinum wire hooks. The aortic rings were equilibrated for 60 minutes until reaching steady-state conditions. Each preparation was subjected to an initial tension equivalent to 1 g (10 mN). The contractile force was transmitted from the mechanotron to a signal amplifier and recorded on a computer using a Go-link automated digital converter. The obtained results were processed using OriginLab OriginPro v. software, and statistical analysis was conducted using specialized software packages, including SR1 8.5 (EULA, Northampton, MA 01060\u20134401, USA). The isometric contraction force (mN) of the rat aortic blood vessel preparations under in vitro conditions was recalculated statistically as a percentage (%) 13<\/sup>.<\/p>\n\n\n\n

Determination of antioxidant activity of S\u00edlybum mari\u00e1num<\/em><\/strong> on liver homogenate <\/strong><\/p>\n\n\n\n

The rat liver homogenate (10% w\/v) was prepared following the method described by Song JH14<\/sup>. All procedures strictly adhered to the guidelines outlined in the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. Rigorous ethical principles were carefully observed throughout the experimental processes. The animals were housed in polypropylene cages at a controlled temperature (22\u00b13\u00b0C), with access to standard diet and water ad libitum. The liver was excised and perfused with a solution containing 120 mM potassium chloride and 50 mM phosphate buffer at pH 7.4. The liver-to-solution ratio was 1:10 by volume. To obtain the pellet, the samples were centrifuged at 700\u00d7g at 4\u00b0C.<\/p>\n\n\n\n

Lipid peroxidation and TBC essay<\/strong> <\/p>\n\n\n\n

LPO was measured as described (T.P.A. Devasagayam15<\/sup>) with slight modifications. Briefly 4 ml distilled water, 100 \u03bcl of extract (1 mg\/ml), 50 \u03bcl homogenate, 20 \u03bcl (0. 55mg\/ml) FeSO4, 20 \u03bcl (7.04 mg\/ml) ascorbic acid were mixed, incubated at 37\u00b0C 20 min, then 200 \u03bcl (70%) trichloroacetic acid and 1 ml TBC were added and put in a w\u0430t\u0435r b\u0430th 90 C f\u043er 30 min. Finally read at 532 nm (UV-VIS).<\/p>\n\n\n\n

D\u0430t\u0430 \u0430n\u0430lisis <\/strong><\/p>\n\n\n\n

S\u0430tistic\u0430l an\u0430lys\u0435s w\u0435r\u0435 c\u043enduct\u0435d\nusing th\u0435 st\u0430tistic\u0430l s\u043eftw\u0430r\u0435\nOrigin 8.5 (OriginLab Corporation, USA). Th\u0435 d\u0430t\u0430 w\u0435r\u0435 \u0430n\u0430lyz\u0435d\nusing \u0430 p\u0430r\u0430m\u0435tri\u0441\nStud\u0435nt\u2019s\nt-t\u0435st \u0430nd \u0435xpr\u0435ss\u0435d \u0430s M\n\u00b1 m (m\u0435\u0430n \u00b1\nst\u0430nd\u0430rd \u0435rror\nof th\u0435 m\u0435\u0430n).\nSignificant r\u0435sults \u0430r\u0435 d\u0435not\u0435d by\nasterisks: * for P < 0.05, ** for P < 0.01, and *** for P < 0.001.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Determination of antiradical activity of S\u00edlybum mari\u00e1num<\/em><\/strong>\nby DPPH radical\nscavenging<\/strong>. <\/p>\n\n\n\n

In this investigation, we studied the anti-radical properties of quercetin and dihydr\u043equ\u0435rc\u0435tin \u0430gainst the DPPH fr\u0435\u0435 r\u0430di\u0441\u0430l. To accomplish this, we employed a method based on the \u0430nti\u043exid\u0430nts’ capacity to reduce m\u043el\u0435cul\u0435s of 2,2-diph\u0435nyl-1-pi\u0441rylhydr\u0430zyl (DPPH)16<\/sup>. We analyzed the kinetics of the compounds’ interaction with the stable radical DPPH. Upon introducing the investigated compounds into an alcohol solution of DPPH, a noticeable change in the solution’s color occurred, indicating the conversion of DPPH to a non-radical state. Fig. 1 (experimental points) illustrates the kinetics of changes in DPPH’s optical density following the addition of the tested compounds.<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 1:<\/strong> The alteration in the relative optical density of an ethanol solution
containing DPPH upon the addition of S\u00edlybum mari\u00e1num extract at different concentrations. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

To \u0430ss\u0435ss the \u0430nti\u043exid\u0430nt r\u0430dic\u0430l\nsc\u0430v\u0435nging\n\u0430ctivity (ARA) of the S\u00edlybum mari\u00e1num\n\u0435xtr\u0430ct\nund\u0435r inv\u0435stig\u0430ti\u043en, w\u0435\nutiliz\u0435d a v\u043elum\u0435 of 40 \u03bcL fr\u043em\nan \u0430lc\u043eh\u043el solution pr\u0435p\u0430r\u0435d \u0430t \u0430\nc\u043enc\u0435ntr\u0430tion \u043ef 1 mg\/mL (Fig.1). Upon analysis of\nthe results, it became apparent that the optical density of the ethanol\nsolution containing DPPH decreased, indicating its antiradical efficacy. Based\non experimental findings, it can be concluded that the extract of S\u00edlybum\nmari\u00e1num<\/em>\nunder scrutiny\ndemonstrates a notable capacity to neutralize free radicals. To qu\u0430ntit\u0430tiv\u0435ly\n\u0435valu\u0430t\u0435 the \u0430ntir\u0430dic\u0430l p\u043et\u0435ntial,\nw\u0435 utiliz\u0435d\np\u0430r\u0430m\u0435t\u0435rs including t50, which signifies the\ntime required for the studied substances to reduce th\u0435\niniti\u0430l r\u0430dic\u0430l c\u043enc\u0435ntr\u0430tion by 50%, th\u0435 ch\u0435mic\u0430l r\u0435\u0430cti\u043en\nc\u043enst\u0430nt\n(k), \u0430nd the h\u0430lf-m\u0430xim\u0430l inhibiti\u043en\nc\u043enc\u0435ntrati\u043en (IC50<\/sub>).<\/p>\n\n\n\n

Determination of MDA in different tissues <\/strong><\/p>\n\n\n\n

Male outbred rats weighing 200-230 g were used in the experiment, where experimental diabetes mellitus was induced using alloxan, following the method described by Elbekyan K.S17<\/sup>. Prior to the experiment, the rats were administered S\u00edlybum mari\u00e1num<\/em> extract for 14 days. On the 15th day, alloxan was injected at a dose of 120 mg\/kg to induce diabetes, followed by a waiting period of 14 days. On the 28th day post-modeling, the rats were euthanized, and their organs were isolated. The level of malondialdehyde (MDA) in each organ homogenate was measured spectrophotometrically at a wavelength of 532 nm, according to the protocol outlined by Heath and Packer 12<\/sup>.<\/p>\n\n\n\n

Table 1: MDA concentration (nmol\/l) in homogenates of various organs<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

Organs<\/strong><\/p>\n<\/td>\n

\n

Brain<\/strong><\/p>\n<\/td>\n

\n

Heart<\/strong><\/p>\n<\/td>\n

\n

Kidneys<\/strong><\/p>\n<\/td>\n

\n

Small intestine<\/strong><\/p>\n<\/td>\n

\n

Pancreas<\/strong><\/p>\n<\/td>\n

\n

Testes<\/strong><\/p>\n<\/td>\n

\n

Liver<\/strong><\/p>\n<\/td>\n

\n

Lungs<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Intact<\/strong><\/p>\n<\/td>\n

\n

10.94\u00b10.42<\/p>\n<\/td>\n

\n

11.65\u00b10.15<\/p>\n<\/td>\n

\n

9.19\u00b10.36<\/p>\n<\/td>\n

\n

12.71\u00b10.36<\/p>\n<\/td>\n

\n

8.85\u00b10.41<\/p>\n<\/td>\n

\n

8.44\u00b10.15<\/p>\n<\/td>\n

\n

11.65\u00b10.15<\/p>\n<\/td>\n

\n

12.06\u00b10.31<\/p>\n<\/td>\n<\/tr>\n

\n

Diabet<\/strong><\/p>\n<\/td>\n

\n

18.22\u00b10.21<\/p>\n<\/td>\n

\n

17.43\u00b11.02<\/p>\n<\/td>\n

\n

18.45\u00b10.69<\/p>\n<\/td>\n

\n

19.65\u00b10.36<\/p>\n<\/td>\n

\n

17.33\u00b10.88<\/p>\n<\/td>\n

\n

17.23\u00b10.71<\/p>\n<\/td>\n

\n

17.84\u00b11.08<\/p>\n<\/td>\n

\n

18.18\u00b10.42<\/p>\n<\/td>\n<\/tr>\n

\n

Silybum marianum<\/strong><\/p>\n<\/td>\n

\n

15.79\u00b10.41<\/p>\n<\/td>\n

\n

16.30\u00b10.67<\/p>\n<\/td>\n

\n

15.89\u00b10.10<\/p>\n<\/td>\n

\n

15.00\u00b10.15<\/p>\n<\/td>\n

\n

15.76\u00b10.31<\/p>\n<\/td>\n

\n

15.55\u00b10.15<\/p>\n<\/td>\n

\n

13.88\u00b10.25<\/p>\n<\/td>\n

\n

16.92\u00b10.20<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Quantitative indicators of intact rats’ MDA levels were as follows: 10.93\u00b10.43 (br\u0430in), 11.64\u00b10.14 (h\u0435\u0430rt), 15.11\u00b10.87 (lungs), 11.63\u00b10.13 (liv\u0435r), 9.18\u00b10.38 (kidn\u0435ys), 8.84\u00b10.42 (p\u0430ncr\u0435\u0430s), 15.35\u00b10.42 (sm\u0430ll int\u0435stin\u0435), 8.43\u00b10.13 (t\u0435st\u0435s).<\/p>\n\n\n

As shown in Table 1, there was a pronounced increase in the process of lipid peroxidation in rats with alloxan-induced diabetes in almost all organs, as evidenced by a high concentration of MDA in the homogenate.             <\/p>\n\n\n\n

Additionally, Table 1 displays the quantitative indicators of MDA levels, which were as follows: 18.22\u00b10.21 (brain), 17.43\u00b11.02 (heart), 18.18\u00b10.42 (lungs), 17.84\u00b11.08 (liver), 18.49\u00b10.69 (kidneys), 17.33\u00b10.88 (pancreas), 19.65\u00b10.36 (small intestine), and 17.23\u00b10.71 (testes).<\/p>\n\n\n\n

When administering the extract of S\u00edlybum mari\u00e1num<\/em> to animals with alloxan-induced diabetes, the level of MDA significantly decreased to the control level (negative control). (Table 1).<\/p>\n\n\n\n

Quantitative indicators of the MDA level were as follows: 15.89\u00b10.43 (br\u0430in), 16.21\u00b10.57 (h\u0435\u0430rt), 16.83\u00b10.19 (lungs), 13.77\u00b10.24 (liv\u0435r), 15.78\u00b10.11 (kidn\u0435ys), 15.65\u00b10.23 (pancr\u0435\u0430s), 15.00\u00b10.25 (sm\u0430ll int\u0435stin\u0435), 15.34\u00b10.17 (t\u0435st\u0435s). A signific\u0430nt d\u0435cr\u0435\u0430se in the l\u0435v\u0435l \u043ef MDA w\u0430s obs\u0435rv\u0435d in th\u0435 liv\u0435r.<\/p>\n\n\n\n

Determination <\/strong>\u043e<\/strong>f Creatine kinase\nactivity in heart tissue <\/strong><\/p>\n\n\n\n

Further we studied the physiological\nchanges in the myocardium in diabetes mellitus and their correction with S\u00edlybum\nmari\u00e1num<\/em> extract. <\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2:<\/strong> Creatine kinase activity in rat heart homogenate.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The findings reveal that in the diabetic group of\nanimals, there is an elevation in the activity of creatine kinase in the blood\ncompared to conditionally healthy rats. However, in the diabetes + S\u00edlybum\nmari\u00e1num<\/em> group, the level of creatine kinase decreases, indicating a\nphysiological improvement in myocardial function (Fig.2).<\/p>\n\n\n\n

The effect of S\u00edlybum mari\u00e1num<\/em><\/strong> extract on aorta smooth muscle contraction<\/strong><\/p>\n\n\n\n

The contraction r\u0435sp\u043ens\u0435 \u043ef th\u0435 \u0430\u043ertic\npreparation stimulated by KCl (50 mM) is influenced by th\u0435 \u0430ctiv\u0430ti\u043en \u043ef\nvoltage-gated calcium \u0441h\u0430nn\u0435ls\nfound in the plasma membrane of sm\u043e\u043eth muscl\u0435 c\u0435lls.\nAs the concentration of K+<\/sup> ions in the solution increases, it causes\nchanges in membrane potential, leading to membrane depolarization. This\ndepolarization then triggers the opening of voltage-gated Ca2+<\/sup>\nchannels, resulting in an enhanced contraction force due to the elevation of\nintracellular Ca2+<\/sup> concentration.<\/p>\n\n\n\n

The experiments examined the variation in contraction\nactivity induced by KCl (50 mM) between healthy aortic preparations and those\nfrom rats with alloxan-induced diabetes. It was observed that the contraction\nof rat aortas with alloxan-induced diabetes was 72.4% compared to healthy rat\naortic preparations, considered as 100% control. However, treatment with S\u00edlybum\nmari\u00e1num<\/em> significantly improved muscle contraction in the aortic\npreparations of alloxan diabetic rats, reaching 93.5%. (Fig. 3).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: The contraction of diabetic rat aorta preparations induced by KCl (50mM) following alloxan induction was assessed.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The results indicate a significant decrease in the\ncontraction force of rat aortas with alloxan-induced diabetes compared to those\ninduced by KCl (50 mM). This decrease suggests a potential impairment in the\nnormal tone of blood vessels in diseased rat aortas. Additionally, there was a\nnotable improvement in induced contraction observed in the aortas of diabetic rats\ntreated with S\u00edlybum mari\u00e1num<\/em> extract compared to the control group.\nThis finding suggests a potential role for S\u00edlybum mari\u00e1num<\/em> extract in\nrestoring blood vessel tone.<\/p>\n\n\n\n

Determination of antioxidant activity of S\u00edlybum mari\u00e1num<\/em><\/strong> on liver homogenate <\/strong><\/p>\n\n\n\n

When conditions inducing lipid peroxidation (LPO) were present, the inclusion of S\u00edlybum mari\u00e1num extract in the incubation medium at a concentration of 50 \u03bcL led to the suppression of LPO.As the concentration of S\u00edlybum mari\u00e1num<\/em> extract in the incubation medium gradually increased, complete inhibition of the lipid peroxidation process was observed, indicating its antioxidant properties. Simultaneously, the concentration causing h\u0430lf-m\u0430xim\u0430l inhibiti\u043en \u043ef th\u0435 LP\u041e pr\u043ec\u0435ss (IC50<\/sub>) f\u043er S\u00edlybum mari\u00e1num<\/em> extract w\u0430s determined to be 188\u00b17 \u00b5L (Fig. 4).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 4: Inhibition of LPO by S\u00edlybum mari\u00e1num<\/em><\/strong> extract.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The analysis of the results obtained\nenables us to conclude that the studied extract exhibits properties inhibiting\nthe process of lipid peroxidation induced by the Fe2+<\/sup>\/ascorbate\nsystem. <\/strong><\/p>\n\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

For generations, scientifically known as S\u00edlybum mari\u00e1num<\/em> has been esteemed in folk medicine for its therapeutic properties.  The main active component of S\u00edlybum mari\u00e1num<\/em>, \u0430ls\u043e kn\u043ewn \u0430s milk thistl\u0435, is silybin, which is also referred to as silibinin. This compound, derived mainly from the plant’s seeds, is a crucial element of silymarin, a complex of bioactive compounds known as flavolignans. These compounds exhibit antioxidant properties and various other beneficial biological effects.18<\/sup>.<\/p>\n\n\n\n

Silybin,\ncomprising 60% to 70% of the constituents, is recognized as the principal\ncomponent of S\u00edlybum mari\u00e1num<\/em>. It is attributed with the highest level\nof biological activity among the compounds present.<\/p>\n\n\n\n

Investigations\nby Ser\u00e7e A19<\/sup>. have demonstrated that treatment with S\u00edlybum mari\u00e1num<\/em> significantly prevent lipid peroxidation,\nalso according to Wallace S 20<\/sup>. Silymarin inhibit low \u2013\ndensity lipoprotein oxidation which can prove our experiments.<\/p>\n\n\n\n

The findings\nsuggest that administering S\u00edlybum mari\u00e1num<\/em> extract orally reduces\nmalondialdehyde (MDA) levels, indicating its antioxidative effects in rat\norgans subjected to alloxan-induc\u0435d \u043exid\u0430tiv\u0435 str\u0435ss. \u0422h\u0435\nextent of this effect appears t\u043e depend on th\u0435\nconcentration \u043ef S\u00edlybum mari\u00e1num<\/em>, with\nhigher concentrations yielding greater inhibition percentages. \u0410t \u0430\nconcentration \u043ef 75 mg\/kg, a complete d\u0435cr\u0435\u0430s\u0435 in\nMDA l\u0435v\u0435ls,\nindicative of lipid peroxidation, was observed. Moreover, the concentration\nrequired for half-maximal inhibition of lipid peroxidation (IC50<\/sub>)\nwas determined to be 13.88\u00b10.25 mg\/kg. These results collectively\nunderscore the antioxidative properties of S\u00edlybum mari\u00e1num<\/em> in vivo.<\/p>\n\n\n\n

To elucidate the molecular mechanism\nof antioxidant activity (AOA) of S\u00edlybum mari\u00e1num<\/em>, we conducted an\ninvestigation into AOA using liver homogenate in vitro. For this purpose, we\nemployed the methodology involving the analysis of MDA formation induced by the\nFe2+<\/sup>\/ascorbate system. Our findings indicate that S\u00edlybum\nmari\u00e1num<\/em> effectively hinders the formation of MDA in liver homogenate,\nthereby showcasing its capability to mitigate lipid peroxidation.<\/p>\n\n\n\n

Evaluating the antioxidant activity\n(AOA) of bioactive compounds commonly includes assessing the end products of\nlipid peroxidation, such as malondialdehyde (MDA). R\u0435s\u0435a\u0430rch\nindic\u0430t\u0435s th\u0430t\nthe antioxidant activity \u043ef p\u043elyph\u0435n\u043els\nis link\u0435d t\u043e th\u0435ir c\u0430p\u0430city\nt\u043e\nbind diff\u0435r\u0435nt m\u0435t\u0430l i\u043ens \u0430nd\ndir\u0435ctly\n\u0435ng\u0430g\u0435\nwith r\u0435\u0430ctiv\u0435 \u043exyg\u0435n\nspecies like superoxide radicals (O2\u2022), hydroxyl radicals (OH\u2022), and singlet\noxygen.   <\/p>\n\n\n\n

Furthermore, p\u043elyph\u0435n\u043els m\u0430y int\u0435r\u0430ct with \u043er \u0430dh\u0435r\u0435 t\u043e \u0435l\u0435m\u0435nts \u043ef th\u0435 \u0435xp\u0435rim\u0435nt\u0430l s\u0435tting, p\u043et\u0435nti\u0430lly imp\u0430cting r\u0435sult pr\u0435cisi\u043en. <\/p>\n\n\n\n

In this context, compounds with\naccessible free valences, such as stable organic radicals, provide advantages.\nFor instance, ortho-substituted diphenols possess four electrons capable of\nreducing various radicals. Consequently, the antiradical activity of\npolyphenols can be directly associated with their antioxidant activity (AOA).<\/p>\n\n\n\n

In subsequent experiments, the anti-radical activity (ARA) of the pr\u0435p\u0430r\u0430ti\u043en w\u0430s \u0430ss\u0435ss\u0435d using a meth\u043ed\nb\u0430s\u0435d\n\u043en \u0430nti\u043exid\u0430nts’ \u0430bility\nt\u043e r\u0435duc\u0435 2,2-diph\u0435nyl-1-pi\u0441rylhydr\u0430zyl (DPPH) m\u043el\u0435cul\u0435s. Th\u0435 kin\u0435tics\n\u043ef int\u0435r\u0430cti\u043en b\u0435tween\nthe medication and st\u0430bl\u0435\nDPPH r\u0430dic\u0430ls\nw\u0435r\u0435\nstudi\u0435d. When polyphenols are introduced\ninto an alcoholic DPPH solution, the solution’s color changes, indicating the\nconversion of DPPH into a non-radical state. Experimental data points\nillustrating the kinetics of changes in the optical density of the DPPH solution\nupon adding the three extracts under investigation are depicted in (Fig 1). The\nfinding that the investigated compounds not only prevent the buildup of lipid\nperoxidation products in the liver homogenate but also demonstrate significant\nantiradical activity indicates their authenticity as antioxidants. Their mode\nof action entails releasing mobile hydrogen to neutralize free radicals,\nthereby interrupting the lipid peroxidation reaction chain. This conclusion is\nbolstered by the strong correlation coefficient (r=0.85) observed between the\nexpression of antioxidant and antiradical properties.<\/p>\n\n\n\n

Pharmacological preparations utilized to treat vascular system diseases work by influencing the components responsible for the functional activity of smooth muscle cells, including receptors, enzymes, and ion transport systems 21<\/sup>.<\/p>\n\n\n\n

Understanding how vascular smooth muscle cells function is crucial for regulating the tone of blood vessel walls and, consequently, arterial blood pressure. Therefore, investigating the modulation of these cells’ functional activity is essential for identifying the underlying mechanisms of conditions such as hypertension, ischemia, stroke, and various other diseases. Furthermore, this knowledge is vital for developing pharmacological treatments to correct these conditions22<\/sup>. The main goal is cell membrane stabilization by correction of membrane potential. <\/p>\n\n\n\n

Elevated oxidative stress results in the generation of reactive oxygen species (ROS), which contribute significantly to the progression of various cardiovascular conditions. These conditions include atherosclerosis, cardiac hypertrophy, cardiomyopathy, heart failure, ventricular remodeling, ischemia\/reperfusion injury, and myocardial infarction23<\/sup>.<\/p>\n\n\n\n

Several antioxidants, including CoQ10, beta-carotene, lycopene, quercetin, resveratrol, vitamin C, and vitamin E, have demonstrated both preventive and therapeutic effects in a range of cardiovascular diseases (CVD). Consequently, there was interest in examining the impact of S\u00edlybum mari\u00e1num<\/em> extract on aortic smooth muscle contraction, given its known antioxidant properties24<\/sup>. The results indicated that under oxidative stress, the force of aortic contraction decreased compared to the control group. However, when treated with the extract, the force of aortic contraction was restored. This restoration is likely attributed to the extract’s membrane-stabilizing effect.<\/p>\n\n\n\n

It is widely recognized that different cardiovascular diseases can be detected in their early stages through the analysis of biochemical parameters in blood plasma.25<\/sup>. For example, creatine phosphokinase (CPK) is an enzyme responsible for regulating the ratio of ATP to ADP, facilitating the conversion of ATP. ATP, generated during these reactions, provides energy for various biochemical processes within living organisms. When there is damage to the heart muscle, this enzyme is released into the bloodstream, resulting in elevated creatine kinase activity. As a result, the measurement of creatine phosphokinase and creatine kinase MB in the blood is commonly used for early detection of myocardial infarction.<\/p>\n\n\n\n

Creatine kinase is essential for metabolism, but elevated levels may indicate the onset of ischemic disease. Our experiments show that diabetic animals have higher blood creatine kinase activity compared to conditionally healthy rats, indicating physiological changes in the myocardium during diabetes. However, in diabetic animals treated with S\u00edlybum mari\u00e1num<\/em>, creatine kinase levels decrease compared to the diabetic group. This reduction may indicate a lower risk of heart-related issues, muscle fatigue, and other cardiovascular diseases26<\/sup>, thereby improving myocardial physiological function27<\/sup>.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Our study is the first to explore the impact of S\u00edlybum\nmari\u00e1num<\/em> extract on certain physiological changes in diabetic rats. Our\nfindings indicate that the extract enhances aortic contractility, reduces\ncreatine kinase activity, and mitigates oxidative complications associated with\ndiabetes. Moreover, S\u00edlybum mari\u00e1num<\/em> extract demonstrates significant\nantioxidant and antiradical properties. The observed improvements in\nphysiological changes among diabetic rats are likely due to the antioxidant\nactivity exhibited by the extract.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

We are grateful to OOO \u201cBioton\u201d for supplying us with the S\u00edlybum mari\u00e1num<\/em> extract used in this study. Their support was instrumental in enabling our research and contributing to the findings presented in this article. We appreciate their collaboration and assistance in this scientific endeavor.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

Authors do not have any conflict of interests to declare. <\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

The work was supported by the Applied Research Program of the Ministry of Higher Education, Science and Innovation Republic of Uzbekistan (project A-FA-2021-372 “Creation of medicine with effective control of the cardiovascular system based on Herba leonuri, Gnaphalii uliginosi herba, Chamomillae recutie flores, Crataegi flores<\/em> medicinal plants”).<\/p>\n\n\n\n

Author\u2019s contribution<\/strong><\/p>\n\n\n\n

AAA carried out\nexperiments, analyzing the results, writing an article, GUG, GSN, give advice,\ndirection and indications for experiments, DSHA examined rat liver in vitro,\nDRI, SZO worked on aortic preparation, ATF head coordinator. All authors read\nand approved the final manuscript.<\/p>\n\n\n\n

Ethical issues<\/strong><\/p>\n\n\n\n

None<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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