SYNCHRONIZATION IN CHAOTIC SYSTEMS

Abstract - In current study, in vivo antidiabetic activity of methanolic extract of Taverniera nummularia and Iflago spicata was investigated in mice. The T. nummularia and I. spicata plants were collected, shade dried and extracted (5 Kg) in methanol. The Albino mice (male) four weeks old weighing 24±4 g were fasted for 8-12 hours and subjected to hyperglycemia by a single intraperitoneal administration of 150 mg/kg body weight of a freshly prepared 10% alloxan monohydrate. Mice above 200 mg/dL blood glucose levels were considered diabetic and used for further study. The T. nummularia and I. spicata methanolic extracts were fed to diabetic mice at 150 mg/kg and 300 mg/kg body weight. The mice treated with alloxan exhibited a significant increase in blood glucose level. The most effective dose of Taverniera nummularia for reducing blood glucose level was 150 mg/kg body weight after 10 and 15 days of alloxan treatment whereas, Ifloga spicata was significantly more effective at 300 mg/kg body weight. Treatment with methanolic leaf extract of T. nummularia and Ifloga spicata significantly decreased blood glucose level and resulted in increase in body weight under hyperglycemic conditions. The alloxan treated mice also exhibited a significant increase in total bilirubin, ALT, ALP, urea, creatinine, total proteins, triglycerides and LDL cholesterol content as compared to the control group (p<0.05). Moreover, increases in the content of bilirubin, ALT, ALP, urea, creatinine, total proteins, triglycerides and LDL cholesterol due to hyperglycemia was normalized by treatment with methanolic extract of both the plant species. Methanolic leaf extract of T. nummularia and I. spicata showed good antidiabetic properties and could be exploited as sources of novel compounds for treatment of diabetes mellitus.

Keywords - Phytochemicals, antioxidant, blood sugar, creatinine, cholesterol

I. INTRODUCTION

Throughout the world, the use of medicinal plants as drug sources against diseases has increased significantly. The use of medicinal plants in developing countries shows the beneficial prevalence of medicinal plants (Arunkumar and Muthuselvam, 2009). Beside this, there is an urgent need to upturn the studies on exploring plants for further exploring potentials of medicinal plants in improving overall health of humans.

Diabetes mellitus is a metabolic disorder in which there is a high blood sugar level that results from either acquired or congenital deficiency in the insulin production by the pancreatic islet cells of Langerhans (Arunkumar and Muthuselvam, 2009; Edoga et al., 2005). According to WHO (2015), about 1.6 million people were suffering from diabetes in 2016, and this number could total 366 million by 2030 (Njoku and Obi, 2009; Forni et al., 2019). In 2012, 1.5 million deaths were reported to be directly caused by diabetes (Arika et al., 2015). There are three main types of diabetes. Type I or insulin-dependent diabetes mellitus" (IDDM) or "juvenile diabetes results from the pancreas's failure to produce enough insulin. The cause of Type 1 DM is unknown. Type 2 DM or adult-onset diabetes begins with insulin resistance, a condition in which cells fail to respond to insulin properly. This form was previously referred to as "non-insulin-dependent diabetes mellitus" (NIDDM) (Murugi et al., 2012). The most common cause is excessive body weight and insufficient exercise. Type 3 is Gestational diabetes occurs when pregnant women without a previous history of diabetes develop high blood sugar levels (Babu et al., 2013). Polyuria (excessive excretion of urine), polydipsia (thirst), polyphagia (constant hunger), fatigue and blurred vision (Arika et al., 2015). Diabetic mellitus if persists for a long term it produced complication such as retinopathy (potential loss of vision), nephropathy (renal failure), peripheral neuropathy (foot ulcers), amputations (Charcot joints), autonomic neuropathy, genitourinary, sexual dysfunction and cardiovascular symptoms (Babu et al., 2013).

Blood plasma glucose levels in fasting condition, normally ranges between 3.5-6.7 mmol/L (63-120.6 mg/dl). After a carbohydrate rich meal, the blood glucose level rises to approximately 8 mmoles/L and rarely exceeds this level. Plants are a good source of biologically active compounds with various pharmacological applications with no side effects. Recently it has been shown that methanol extracts of medicinal plants significantly decrease blood sugar level in patients with hyperglycemia. Similarly, it was observed that treatment with plant extracts did not influence liver and kidney function in experimental mice. Pakistan has diverse climatic conditions supporting the growth of more than 600 highly medicinal plants (Yudofsky and Hales, 2008).

Taverniera nummularia and Ifloga spicata are plant species belonging to family Asteraceae with strong ethno medicinal properties. Both the plant species have been reported for their use in the cure of stomach disorders, as expectorants and wound healing. Therefore, in the current study, we have determined antidiabetic potential of Taverniera nummularia and Ifloga spicata in alloxan induced diabetic mice. Selection of these two plant species for this studies was based on our previous published data about pharmacological activities of these plant species which indicted strong antioxidant and antimicrobial activity and this led to the current findings reported in this manuscript (Khan et al., 2018; 2021).

II. METHODS

A. Extraction and Final Product Preparation

The plants materials i.e., T. nummularia and I. spicata were collected from Bannu District KPK Pakistan. The collected plant name was identified by Dr. Faizan Ullah, a taxonomist at Department of Botany, University of Science and Technology, Bannu Pakistan. The voucher specimen for I. spicata (Is-I-1) and T. nummularia (Tn-T-1) has been deposited at herbarium of department. The plant material was thoroughly washed under running tap water and dried under shade for seven days. The dried I. spicata and T. nummularia (5 Kg) was ground to fine powder and was dipped in methanol for seven days. It was then filtered via Whatman filter paper No.1. The same process was repeated for residue remains a few times. The filtrates were combined and methanol was evaporated under vacuum, using rotary Evaporator (Buchi Rota vapor R-200) at 45°C.

B. Phytochemical characterization of extracts

For the presence of bioactive constituents various chemical tests were performed for the presence of bioactive constituents in each extract of both plants by using standard methods (Khan et al., 2018; 2021).

C. Terpenoids

Cloroform (CH₂Cl₂) of 2ml was added to (0.5g) of each extract along with 3ml of Conc H₂SO₄. Redish-Browm coloration of the interface indicated the presence of terpenoids.

D. Tannins

The extract (0.5g) was boiled in 10ml of H₂O in test tube and then filtered. Few drops0.1%Fecl₃was added and brwnish green or blue-black coloration was observed which indicates the presence of Tannins.

E. Saponins

Take 0.5g of crude extract along with water .fist allow the water to boil in a test tube and allow to cool with continuous shaking persistent forth appeared which indicates the presence of saponins.

F. Sterols

Take extract (1ml) in a test tube add 1ml Conc H₂SO_4.Red color appearance indicate that Sterols is present.

G. Cardiac glycosides

The extract (0.5g) was diluted by (5ml) of D/H₂O Glacial Acetic acid (2ml) was added along with one drop of FeCl₃solution. The brown ring at the interface designates cardiac glycosides is present.

H. Anthraquinones

Take 0.5g crude extract with 10% HCL and boil for few minutes using hot water bath, filtered and allowed to cool. Volume of CHCL₃ in equal amount was added to filtrate along with few drops of Liq NH₃(10%). The entire mixture was then heated. Formation of pink rose color indicates presence of anthraquinones.

I. Experimental Animals

Albino mice (male) having four weeks of age with weight 24±4 g were used. They were kept under control temperature (25-27 ºC) in darkness photoperiod. Approval from ethical committee (EC. 72) of the Department of Biotechnology, University of Science and Technology Bannu.

J. Induction of Hyperglycemia

Experimental animals were fasted for 8-12 hours but allowed openly to water until the end of this experiment. Hyperglycemia was induced experimentally by a single intra peritoneal administration of 150 mg/kg body weight of a freshly prepared 10% alloxan monohydrate obtained from Sigma (Steinhein, Switzerland) ( Njagi et al., 2015). The 10 g powder of alloxan monohydrate was dissolved in distilled water to a volume of 100 ml. Blood glucose level was checked after 48 hrs alloxan administration using a glucometer. Mice above 200 mg/dL blood glucose levels were considered diabetic and used for further study (Arika et al., 2015)

K. Experimental Design

Animals were randomly divided into seven groups of five each. Group I consisted of normal mice orally administered with 0.1 ml physiological saline; Group II consisted of alloxan induced diabetic mice (150mg/Kg) orally administered with 0.1 ml physiological saline; Group III consisted of alloxan induced diabetic mice orally administered with 10 mg glibenclamide. Group 1V and Group V consisted of extract of 150mg/kg and 300mg/kg (T. nummularia). For testing of I. spicata separate experiment was performed having separate groups of animals in same way as described for experiment having T. nummularia. Group 1V and Group V in I. spicata experiment consisted of extract of 150mg/kg and 300mg/kg (I. spicata ). The animals in Groups I-III were kept separately for the two tested plant extracts.

The data was analyzed statistically via two-way ANOVA and means were compared by using least significant difference test (P < 0.05). Statistical package STATISTIX-10 (USA) was used for data analysis.

III. RESULTS AND DISCUSSION

A. Qualitative analysis of Tarvenaria numularia and Ifloga spicata.

Table1.1 Composition of both extracts of Tarvenaria numularia and Ifloga spicate.

Constituents	T.numularia extract	I.spicata extract
Sterols	-	+
terpeniods	-	+
tannins	+	+
glycosides	+	+
saponins	+	+
Anthraquinon	-	-

+ represents presence and,^_represents absence

Present investigation reveal various bioactive constituents are present in both these plants Tarvenaria numularia and Ifloga spicata extracts. Table 1 shown different constituents of both plants. In order to check the effect of T. nummularia and I. spicata extract on alloxan induced diabetic mice, various parameters were evaluated. We noted that alloxan considerably decreased body weight of mice compared to the control group measured at an interval of 5 days for 15 days. Co administration of different doses of both plant extracts, rescued the body weight significantly. Both plant species minimize adverse effects on body weight when supplement at 300 mg/kg body weight (Figs. 1 and 2).

Glucose level was significant and continuous increase in alloxan treated group animals after 15 days of the treatment (Figs. 3 and 4). Groups of mice treated with T. nummularia, I. spicata and glibenclamide, exhibited significant recovery in the level of blood glucose than diabetic control measured at 0th day-15st days of the experiment. The most effective dose of T. nummularia and I. spicata for reducing blood glucose level was 300 mg/kg body weight after 10 and 15 days of alloxan treatment.

In alloxan induced mice several organs such as liver and kidney were also affected by diabetes mellitus. The alloxan treated mice exhibited a significant increase in total bilirubin, ALT, and ALP compared to the control group. Second treatment with extracts of T. nummularia and I. spicata significantly decreased total bilirubin, ALT, and ALP level caused by alloxan (Fig. 5). The results showed the elevated serum level of total bilirubin (1.81±0.7), ALP (203.7±9.7) and ALT (49±4.1) when

Results presented in Fig, 7 showed that alloxan treated group exhibited significantly higher triglycerides and LDL cholesterol level as compared to untreated control group (p<0.05). On the other hand, alloxan treatment significantly decreased the level of HDL cholesterol than untreated control group. The application of T. nummularia and I. spicata extracts also notably ameliorated adverse effects of alloxan on serum lipid profile. Extracts of both of the plant species significantly decreased level of triglycerides and LDL while increasing the level of HDL cholesterol under hyperglycemic conditions. Effectiveness of both the extracts was higher when used at 300 mg/kg body weight.

The body weight and glucose concentrations of mice treated with alloxan and T. nummularia and I. spicata extract are presented in (Figs. 1-4). The result indicated significant (**p<0.05) increase in the body weight of mice treated with extract, while there was a decrease in the body weights of diabetic mice. The increase in body weight indicated the effectiveness of the extract in controlling diabetes. Diabetic Control group showed a significant increase in blood glucose level compared to the normal control. The difference observed between the initial and final fasting serum glucose levels of extract treated diabetic mice revealed anti-diabetic effect of T. nummularia and I. spicata . Both plant extracts also showed reduction in the blood glucose levels of the non-diabetic mice, thus revealing the natural anti-diabetic nature of the extract. Methanol extract of T. nummularia and I. spicata significantly (**p<0.05) attenuated the elevated blood glucose. The effect of the extract was compared to that of reference standard, glibenclamide and was found to be less significant. These effects might be achieved by facilitating insulin release from pancreatic ß-cells, inhibiting glucose absorption in gut, stimulating glycogenesis in liver and/ or increasing glucose utilization by the body. Our results are consistent with a previous study reporting Chenopodium quinoa seeds can reduce most of the adverse effects exerted by fructose on lipid profile and glucose level. In case of the effect of T. nummularia and I. spicata extract on liver enzymes (ALT and ALP) and total bilirubin. The extract showed significantly (**p<0.05) lower levels of ALT and ALP in comparison to the diabetic control group. The improvements noticed in the levels of these enzymes are a consequence of an improvement in the carbohydrate, fat and protein metabolism. The restoration of ALT levels after treatment also indicates a revival of insulin secretion. Elevation of ALP has been reported in diabetic mice (Karau et al., 2012). Individuals facing type 2 diabetes are reported with liver function disorders like high levels of ALT, AST, and GGT (Mandal et al., 2018). Antidiabetic drugs cause reduction in the level of transaminases resulting decline in blood sugar level (Elizabeth and Harris 2005). This increase in ALP was significantly reversed by the extract of T. nummularia and I. spicata .

The status of kidney function may be provided by analysis of blood (Szkudelski 2001). During normal condition the serum level of urea and creatinine remains at normal unless there is pathogenesis. The high levels of urea and creatinine indicate the kidney injuries induced through chemical treatment (Jha, 2017). Our results of diabetic group showed significant increase in blood urea and creatinine indicating renal injuries. The data showed that both of the plants extracts significantly (**p<0.05) restored urea and creatinine and increased the level of total proteins in blood. The extract treatment showed more significant (**p<0.05) effects in lowering urea concentration both in diabetic and non-diabetic mice. Failure of prolonged diabetics results in elevated level of creatinine in the body which indicates abnormal function of kidneys (Dabla, 2010).

Of importance, our study is the first such report on any species of T. nummularia and I. spicata up till now. The increased glucose level in diabetic mice was associated with a high serum concentration of total cholesterol and triglycerides as present in the normal diabetic conditions (Gallwitz., 2009; Khan et al., 2009). The extract of T. nummularia and I. spicata at a dose level of 150mg/kg and 300mg/kg reversed the diabetes induced hyperlipidemia compared to the diabetic control group. In extract (150mg/kg b. wt.) treated diabetic group the levels of high-density lipoproteins, low density lipoproteins and total cholesterol were found significant (**p<0.05) and triglyceride was found to be less significant. In extract (300mg/kg b. wt.) treated rats’ reduction in the levels of serum lipids were found significant (**p<0.05). In non-diabetic extract treated group the levels of serum lipids were found more significant (**p<0.05) as compared with the normal control group showing the hypolipidemic effect of T. nummularia and I. spicata plant. Similar findings were obtained from other studies (Ahmed et al., 2010; Mia, 2007). The hypolipidemic effect may be due to inhibition of fatty acid synthesis. The significant reduction of serum lipid levels in diabetic mice after treatment with extract of T. nummularia and I. spicata may be directly attributed to improvements in insulin level. The results suggested that this plant extract can be used for treatment diabetes and its complications. It can also be used to control abnormal levels of lipids, renal biochemical marker and liver enzymes both in diabetic and non-diabetic individuals

IV. CONCLUSIONS

Alloxan treatment reduced body weight and increased blood glucose level, total bilirubin, ALT, ALP, urea, creatinine, total proteins, triglycerides and LDL cholesterol content in rats. Treatment with methanolic leaf extract of T. nummularia and I. spicata (300 mg/kg body weight) significantly decreased blood glucose level and resulted in a better body weight under hyperglycemic conditions. It was inferred from findings of the present investigation that methanolic extract of leaves of T. nummularia and I. spicata could be administered for treatment of diabetes mellitus. Further studies to isolate and purify antidiabetic compounds from both the tested plant species are recommended.

ACKNOWLEGEMENT

The authors are thankful to University of Science and Technology Bannu Pakistan for provision of research facilities.

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Received: February 2, 2021

Sent to Subject Editor: April 14, 2021

Accepted: February 28, 2022

Recommended by Subject Editor Diego Lomonaco