{"id":60342,"date":"2024-06-30T11:18:36","date_gmt":"2024-06-30T11:18:36","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=60342"},"modified":"2025-07-14T11:45:35","modified_gmt":"2025-07-14T11:45:35","slug":"antidiabetic-and-hypolipidemic-effect-of-ethanolic-seed-extract-of-prosopis-juliflora-in-fructose-induced-hyperglycemia-in-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/antidiabetic-and-hypolipidemic-effect-of-ethanolic-seed-extract-of-prosopis-juliflora-in-fructose-induced-hyperglycemia-in-rats\/","title":{"rendered":"Antidiabetic and Hypolipidemic Effect of Ethanolic Seed Extract of Prosopis juliflora in Fructose Induced Hyperglycemia in Rats"},"content":{"rendered":"\n

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

\u201cDiabetes mellitus (DM)\nis a chronic heterogeneous metabolic disorder characterized by persistently\nelevated blood glucose levels (hyperglycemia) with disturbances in\ncarbohydrate, protein and fat metabolism as a result of defect in insulin\nsecretion, insulin action or both\u201d. The chronic hyperglycemia in diabetes can\ncause damage, dysfunction and failure of various organs particularly the\nkidneys, eyes, heart, nerves and blood vessels.1<\/sup><\/p>\n\n\n\n

Diabetes can be\nclassified based on its etiology into type I and type II. Type I diabetes\nmellitus (T1DM) occurs as a result of absolute insulin deficiency due to\nautoimmune destruction of pancreatic beta cells. It is commonly seen among the\nchildren and adolescents.2<\/sup> Type II diabetes (T2DM) occurs as a\nresult of impaired insulin secretion or increased insulin resistance due to\nbeta cell dysfunction or insulin resistance due to reduced responsiveness of\nthe target tissue like liver, skeletal muscle and adipose tissues towards\ninsulin. It is commonly seen among the older individuals.3<\/sup> Insulin\nresistance in T2DM causes increased production as well as reduced clearance of\ntriglyceride rich lipoproteins thereby causing altered lipid metabolism and\ndiabetic dyslipidemia.4<\/sup><\/p>\n\n\n\n

Diabetes can be managed\nwith both pharmacological agents and non-pharmacological approaches such as\nlike lifestyle modifications. Proper glycemic control helps in preventing and\ndelaying complications due to diabetes. T1DM can be managed with insulin\nreplacement therapy, whereas T2DM can be managed with a combination of oral\nhypoglycaemic agents, dietary changes and exercise. In spite of the\navailability of several groups of drugs, most of the patients do not respond to\nmono drug therapy and might require a combination of drugs for optimal glycemic\ncontrol.5<\/sup> Most of the conventional antidiabetic drugs are not devoid\nof adverse effects. Hence there is a constant search for newer agents including\ntraditional medicines. <\/p>\n\n\n\n

Currently many studies\nare being conducted on a large number of medicinal plants and some of these\nmedicinal plants have proven their efficacy in treatment of DM. Prosopis<\/em> species are commonly used in\nfolk medicine for treatment of various diseases including DM.6<\/sup><\/p>\n\n\n\n

Prosopis\njuliflora<\/em> (Sw) DC belongs to the family-fabacea and subfamily-mimosoideae.\nIt is commonly called as mesquite.It is an aggressive invader and propagates\nthrough the seeds. Various parts of P.juliflora<\/em> like the seeds, leaves\nand the fruits have been reported to have to have antibacterial7-10<\/sup>,\nantifungal11-14<\/sup>, antioxidant activity15<\/sup>, anticancer16-18<\/sup>\nand antidiabetic properties.19,20 <\/sup>Though the effect of P.juliflora<\/em>\nseeds on blood sugar levels has already been reported by literature, there are\nno data available regarding the effect of P.juliflora<\/em> seeds on insulin\nlevels and lipid profile, which usually get altered in patients with diabetes.\nIn this study we used fructose to induce diabetes and diabetic dyslipidemia in\nwistar rats and planned to evaluate the anti-diabetic and hypolipidemic effect\nof P.juliflora<\/em> seeds. Fructose is a lipogenic sugar. Increased\nconsumption of fructose causes hepatic lipogenesis, which raises plasma\ntriglyceride levels, lowers VLDL clearance, activates genes involved in hepatic\nde novo lipogenesis, and promotes triglyceride accumulation in hepatocytes and\nskeletal muscle. Ectopic triglyceride accumulation in tissues eventually leads\nto dyslipidaemia and insulin resistance.21<\/sup><\/p>\n\n\n\n

In our study we used\nfructose solution to induce T2DM in wistar albino rats as this model will\ninduce features of T2DM along with metabolic symptoms like obesity,\ndyslipidemia and hyperinsulinemia. Also, patients with T2DM often present with\nobesity, reduced insulin sensitivity and beta cell compensatory mechanisms like\nexcess basal insulin secretion and hyperinsulinemia. Diabetes and metabolic\nsyndrome are often interrelated and many times, metabolic disorders may coexist\nalong with T2DM.<\/p>\n\n\n\n

Also, this model\novercomes the disadvantages of chemically induced diabetic models by\nstreptozotocin and alloxan as they cause selective loss of pancreatic beta\ncells and hyperglycemia occurs predominantly due to the cytotoxic effects on\nbeta cells.22<\/sup> Because of the spontaneous regeneration of beta cells,\nchemically induced diabetic models are often less stable and occasionally\nreversible. In addition to their cytotoxic effects on beta cells, these\nchemicals can also have toxic effects on other organs, especially the liver and\nthe kidney. Despite being the most widely used model, chemical induction of\nT2DM has recently been criticized because it causes beta cell toxicity, which\nleads to rapid and accelerated destruction of beta cells causing insulin\ndeficiency more than insulin resistance thus mimicking T1DM rather than T2DM.23<\/sup>\nDue to the limitations of chemically induced diabetic models, we attempted to\ninduce insulin resistance along with dyslipidemia in wistar rats by\nsubstituting the regular drinking water with 20% fructose solution and the\nmodel\u2019s stability was determined by monitoring the fasting glucose and insulin\nlevels. We further evaluated the antihyperglycemic and hypolipidemic effect of P.juliflora<\/em> seed extract in wistar rats\nwith fructose induced hyperglycemia as this model induced diabetic dyslipidemia\nalong with insulin resistance. <\/p>\n\n\n\n

Objectives<\/strong><\/p>\n\n\n\n

To assess the antidiabetic effect of ethanolic seed extract of Prosopis juliflora<\/em> with the following outcome measures:<\/p>\n\n\n\n

Fasting blood sugar (FBS), Homeostatis model assessment- estimated insulin resistance (HOMA-IR).<\/p>\n\n\n\n

To assess the hypolipidemic effect of ethanolic seed extract of Prosopis juliflora<\/em> with the following outcome measures:<\/p>\n\n\n\n

Total cholesterol (TC), Triglycerides (TG), LDL cholesterol (low-density lipoprotein), VLDL cholesterol (very-low density lipoprotein) and HDL cholesterol (high-density lipoprotein).<\/p>\n\n\n\n

Materials\nand Methods<\/strong><\/p>\n\n\n\n

Seeds\ncollection and extract preparation<\/strong><\/p>\n\n\n\n

P.juliflora\n<\/em>pods were collected near Madipakkam, Chennai, Tamil Nadu, and the\nseeds were separated. The plant and the seeds were identified and certified by\nan authorized botanist before preparation of the extract. Seeds of P.juliflora<\/em> (500g) were washed, shade\ndried, powdered and soaked in 1 liter of ethanol (99.9 % v\/v) for 72 hours with\nperiodic shaking. The supernatant was filtered using whatman filter paper no 1.\nThe filtrate obtained was evaporated using water bath for 48 hours. Finally, a\nbrown sticky extract was obtained. The percentage yield obtained was 3.9%.\nExtract is stored in airtight container in refrigerator at 2-8for further use. <\/p>\n\n\n\n

Phytochemical\nscreening<\/strong><\/p>\n\n\n\n

Phytochemical analysis\nof the seed extract was performed to identify different phytochemicals such as\nsaponins, alkaloids, glycosides, terpinoids, flavanoids and steroids using the\nmethods described in literature. 24-28<\/sup><\/p>\n\n\n\n

Animals<\/strong><\/p>\n\n\n\n

This study was carried\nout with Institutional Animal Ethics Committee (IAEC) approval (Ref no: IAEC\n1\/Proposal:58\/A.Lr:41\/Dt:05.03.2021). 30 male Wistar albino rats weighing\napproximately 180-200g were housed in individual clean polypropylene cages. They\nwere maintained at a temperature of 23-25  humidity 50-60% in alternate light and dark\ncycle with food and water.<\/p>\n\n\n\n

Induction of hyperglycemia\nalong with dyslipidemia<\/strong><\/p>\n\n\n\n

Hyperglycemia and dyslipidemia were induced by administering 20%\nfructose water daily for 16 weeks.29-33<\/sup> Each rat was housed in a\nseparate cage. 20% fructose solution was prepared daily by dissolving 2g of\nfructose powder in 10 ml of drinking water. Fructose water was provided daily\nthrough animal feeding bottles which were fit to the cage and the amount of fructose\nwater consumed by the animals was monitored daily. If the animals exhausted all\nthe fructose water, then regular drinking water was provided for the rest of\nthe day. Animals with FBS level > 150mg\/dl were included for the experiment.<\/p>\n\n\n\n

Dosage selection of interventions<\/strong><\/p>\n\n\n\n

The dose of Metformin\nwas calculated based on the formula given in FDA draft guidance for calculating\nanimal dose from the human equivalent dose. The maximum recommended dose of\nMetformin is 2000 mg\/day in humans. For a 60 kg human, the dose will be 33.3\nmg\/kg\/day. The animal equivalent dose is calculated by multiplying the factor\n6.2 for rats (FDA).34<\/sup> The calculated animal dose of Metformin is\n206.7 mg\/kg\/day. The value was rounded off to 200 mg\/kg\/day for Metformin in\nthis study. The doses of the P.juliflora<\/em>\nseed extract 400 mg\/kg and 600 mg\/kg was decided based on previous studies. 19,35<\/sup>\nThe interventions were be dissolved in 2 ml of distilled water and administered\nvia orally using gavage.<\/p>\n\n\n\n

Drugs and Reagents<\/strong><\/p>\n\n\n\n

D-fructose was purchased\nfrom Rankem laboratory India ; Metformin was obtained from Intas\nPharmaceuticals Ltd, India; Enzyme Linked Immunosorbent Assay kit for\nestimating rat fasting insulin levels was purchased from Bioassay technology\nPvt. Ltd; Ethanol (99.9%) was purchased from Changshu Hongsheng Fine chemicals\nCo.Ltd; FBS levels were measured using glucometer- Contour plus, Ascensia\nDiabetes Care India Pvt Ltd; Lipid profile parameters (TC, TG, LDL, VLDL and\nHDL) were assessed using auto-analyser. Ketamine was purchased from Neo\nlaboratories, India and Halothane was purchased from Raman & Weil Pvt Ltd,\nIndia.<\/p>\n\n\n\n

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

30\nmale wistar albino rats divided into 5 groups and each group had 6 animals. The\nday of induction (starting fructose) was taken as day \u20180\u2019 (baseline). <\/p>\n\n\n\n

Table 1: Details of the grouping and study interventions<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

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

\n

No of rats<\/strong><\/p>\n<\/td>\n

\n

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

\n

0 to 8 weeks<\/strong><\/p>\n<\/td>\n

\n

9 to 16 weeks<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 I<\/p>\n

(control)<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

Regular drinking water<\/p>\n<\/td>\n

\n

Regular drinking water<\/p>\n<\/td>\n<\/tr>\n

\n

II<\/p>\n

(Hyperglycemia control)<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

20% fructose solution in drinking water<\/p>\n<\/td>\n

\n

20% fructose solution in drinking water + 2 ml distilled water<\/p>\n<\/td>\n<\/tr>\n

\n

III<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

20% fructose solution in drinking water<\/p>\n<\/td>\n

\n

20% fructose solution in drinking water + Metformin 200 mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

IV<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

20% fructose solution in drinking water<\/p>\n<\/td>\n

\n

20% fructose solution in drinking water + P.juliflora<\/em> seed extract 400mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

V<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

20% fructose solution in drinking water<\/p>\n<\/td>\n

\n

20% fructose solution in drinking water + P.juliflora<\/em> seed extract 600mg\/kg<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Study\nassessments<\/strong><\/p>\n\n\n\n

All the parameters were assessed at baseline, 8 weeks and 16 weeks. For assessing all the biochemical parameters mentioned below except FBS, blood was collected via retro orbital route after administering ketamine anesthesia intra peritoneally (50mg\/kg). For measuring FBS level blood samples were obtained from tail tip. <\/p>\n\n\n\n

FBS levels were measured after overnight fasting using glucometer.<\/p>\n\n\n\n

For the assessment of beta cell function and insulin resistance, serum insulin levels were measured after overnight fasting with rat insulin ELISA kit and HOMA-IR was calculated using the formula (fasting insulin (micro U\/L) * fasting glucose (nmol\/L)\/22.5 <\/p>\n\n\n\n

Lipid profile (TC, TG, LDL, VLDL and HDL) using auto-analyser.<\/p>\n\n\n\n

Body weight was measured using automatic weight scale.<\/p>\n\n\n\n

All the rats were sacrificed using high dose\nhalothane anesthesia at the end of 16 weeks and histopathological examination\n(HPE) of pancreas was done.<\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

Graphpad instat version\n3.0 was used for performing statistical analysis. All the continuous variables\nwere summarized as mean  Standard deviation. For comparing the data\nwithin the groups and between the groups, inferential statistical tests such as\npaired t test& repeated measures ANOVA (for within group analysis) and one\nway ANOVA with Tukey\u2019s post hoc test (for between group analysis) were used. P\nvalue less than 0.05 was considered as statistically significant.<\/p>\n\n\n\n

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

All the animals in the groups II to V\ndeveloped features of T2DM and insulin resistance evidenced by increase in FBS\nlevels and HOMA-IR. There was no mortality throughout the experimental\nduration. <\/strong><\/p>\n\n\n\n

Phytochemical screening<\/strong><\/p>\n\n\n\n

The phytochemical screening of the extract\nshowed the presence of alkaloids, flavanoids and steroids.  <\/p>\n\n\n\n

Results of in vivo\nstudy<\/strong><\/p>\n\n\n\n

Body weight (BW)<\/strong><\/p>\n\n\n\n

All groups had similar\nBW at baseline. At 8 weeks all groups gained significant weight. At 16 weeks,\nthe normal and disease control groups (I and II) gained significantly more\nweight compared to the baseline and 8 weeks, whereas the treatment groups (III\nto V) lost weight significantly when compared to 8 weeks (within group\nanalysis, P<0.0001). Intergroup comparison of BW showed that, although all\nthe groups gained weight after 8 weeks, animals in fructose supplemented groups\n(II to V) had significantly higher weight gain than the normal control group\n(I) (P<0.0001). At 16 weeks, Metformin and extract treated groups (400mg\/kg\nand 600mg\/kg) lost weight significantly when compared to 8 weeks. On the other\nhand, the normal and disease control groups (I and II) continued to gain\nweight. Nevertheless, group II gained significantly more weight than group I\n(P<0.001), and weight loss was similar and not statistically significant\namong groups III to V (P>0.05). Data on BW is showed in figure 1.
\n<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Effect on body weight.<\/strong><\/p>\n

<\/p>\n

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

Fasting Blood sugar<\/strong><\/p>\n\n\n\n

All the groups (I-V) had similar FBS at baseline. In fructose fed\ngroups (II to V) FBS increased at 8 weeks (all rats had FBS>150mg\/dl). At 16\nweeks, Metformin and extract treated groups (III to V) showed significantly\nreduced FBS compared to 8 weeks (within group analysis, P<0.0001) while the\ndisease control group did not show any difference and the FBS remained elevated.\nWhen the reduction in FBS was compared among the groups, Metformin and extract\n(600mg\/kg) showed similar reduction in FBS. Group IV (400mg\/kg), though reduced\nthe FBS levels before and after treatment, it was not comparable to Metformin\nand 600mg\/kg extract. Although the FBS level decreased with treatment in the\nMetformin and extracts treated groups (III to V), their levels were still\nhigher when compared to the normal control group (group I). The data of fasting\nblood sugar levels is shown in table 2.<\/p>\n\n\n\n

Table 2: Fasting blood sugar<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

\u00a0<\/strong>Groups<\/strong><\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n<\/td>\n

\n

Fasting blood Sugar (mg\/dl)<\/strong><\/p>\n

mean\u00a0SD<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

\u00a0<\/strong><\/p>\n

8 Weeks<\/strong><\/p>\n

\u00a0<\/p>\n<\/td>\n

\n

\u00a0<\/strong><\/p>\n

16 Weeks<\/strong><\/p>\n

\u00a0<\/p>\n<\/td>\n

\n

Within group comparison<\/strong><\/p>\n

(Repeated measures ANOVA)<\/strong><\/p>\n<\/td>\n

\n

Between <\/strong>
groups<\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

I<\/p>\n<\/td>\n

\n

Normal control<\/p>\n<\/td>\n

\n

86 \u00b1 10.431<\/p>\n<\/td>\n

\n

87.66 \u00b1 10.78<\/p>\n<\/td>\n

\n

87.5 \u00b1 9.50<\/p>\n<\/td>\n

\n

0.2771<\/p>\n<\/td>\n

\n

< 0.0001 *<\/p>\n<\/td>\n<\/tr>\n

\n

II<\/p>\n<\/td>\n

\n

Disease control<\/p>\n<\/td>\n

\n

85.66 \u00b1 5.95\u00a0\u00a0<\/p>\n<\/td>\n

\n

158.16 \u00b1 7.05<\/p>\n<\/td>\n

\n

178.66 \u00b1 7.36<\/p>\n<\/td>\n

\n

< 0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

III<\/p>\n<\/td>\n

\n

Standard drug
(Metformin)<\/p>\n<\/td>\n

\n

87 \u00b1 5.17\u00a0\u00a0<\/p>\n<\/td>\n

159.66 \u00b1 6.31<\/td>\n127.16 \u00b1 5.52<\/td>\n\n

< 0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

IV<\/p>\n<\/td>\n

\n

P.juliflora
(400mg\/kg)<\/p>\n<\/td>\n

\n

84.83 \u00b1 5.45\u00a0\u00a0<\/p>\n<\/td>\n

\n

163.33 \u00b1 5.46<\/p>\n<\/td>\n

\n

150.5 \u00b1 6.38<\/p>\n<\/td>\n

\n

< 0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

V<\/p>\n<\/td>\n

\n

P.juliflora
(600mg\/kg)<\/p>\n<\/td>\n

\n

84.66 \u00b1 8.73\u00a0\u00a0<\/p>\n<\/td>\n

\n

160.66 \u00b1 5.46<\/p>\n<\/td>\n

\n

131.66 \u00b1 4.84<\/p>\n<\/td>\n

\n

< 0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

Post hoc analysis:Group I (16weeks) Vs Groups III, IV and V (16 weeks)- P<0.001*, Group II (16 weeks) Vs groups III, IV and V (16 weeks)-P<0.001*, Group III (16weeks) Vs Group IV (16 weeks)-P<0.001*, Group III (16weeks) Vs Group V (16 weeks)- P>0.05, Group IV (16weeks) Vs Group V (16 weeks)-P<0.01*<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

HOMA-IR<\/strong><\/p>\n\n\n\n

All the groups (I-V) had\nsimilar HOMA-IR levels at baseline. At 8 weeks, the HOMA-IR level increased\nsignificantly in the fructose supplemented groups (II to V). At 16 weeks,\nMetformin and extract treated groups (III to V) showed significant reduction in\nthe HOMA-IR levels compared to 8 weeks (within group analysis, P<0.0001).\nHowever, HOMA-IR remained elevated in disease control group (II) till the end\nof the study (within group analysis, P< 0.0001). Intergroup comparison of\nthe reduction in HOMA-IR levels showed that, group III and V treated with\nMetformin and extract (600mg\/kg) showed similar reduction in HOMA-IR level.\nEven though group IV (400mg\/kg) reduced the HOMA-IR levels before and after\ntreatment, it was not comparable to Metformin and 600mg\/kg extract. Although\nHOMA-IR levels decreased with treatment in the Metformin and extracts treated\ngroups (III to V), it still remained higher than the normal control group\n(group I). Data is shown in figure 2.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Homeostasis model assessment-estimated insulin resistance<\/strong><\/p>\n

<\/p>\n

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

HDL cholesterol<\/strong><\/p>\n\n\n\n

HDL level in the normal control (group I) remained constant\nthroughout the duration of the study. At 8 weeks, HDL levels decreased\nsignificantly in the fructose supplemented groups (II to V). At 16 weeks, HDL\nlevels increased significantly in metformin and extract treated groups (III to\nV) when compared to 8 weeks due to the effect of the treatment, whereas it\nremained low in the disease control animals due to the progression of the\ndisease (group II) (Within group analysis, P<0.0001). When the HDL levels\nwere compared among the different groups, it was observed that the Metformin\nand extracts 400mg\/kg and 600mg\/kg treated groups (III to V) had significantly\nhigher HDL levels at 16 weeks when compared to the disease control group (II).\nAlso, the increase in the HDL levels in these groups were similar and\ncomparable. However, when compared to the normal control group, HDL levels were\nlower in these groups. Data of HDL levels are shown in table 3.<\/p>\n\n\n\n

Table 3: Effect on HDL cholesterol<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

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

\n

HDL cholesterol (mg\/dl)<\/strong><\/p>\n

mean SD<\/strong><\/p>\n<\/td>\n

\n

 P value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 Baseline<\/strong><\/p>\n<\/td>\n

\n

8 Weeks<\/strong><\/p>\n

<\/p>\n<\/td>\n

\n

16 Weeks<\/strong><\/p>\n

<\/p>\n<\/td>\n

\n

Within group comparison (Repeated measures ANOVA)<\/strong><\/p>\n<\/td>\n

\n

Intergroup comparison (One way ANOVA)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

I<\/p>\n<\/td>\n

27.83\u00b11.60<\/td>\n27.5\u00b11.64<\/td>\n27.6\u00b11.21<\/td>\n0.59<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

II<\/p>\n<\/td>\n

27.5\u00b11.87   <\/td>\n22.5\u00b11.37<\/td>\n19.5\u00b11.37<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

III<\/p>\n<\/td>\n

27.66\u00b11.50  <\/td>\n20.5\u00b11.87<\/td>\n23.5\u00b11.37<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

IV<\/p>\n<\/td>\n

\n

27.33\u00b11.96 <\/p>\n<\/td>\n

20.3\u00b12.33 <\/td>\n23\u00b11.89<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

V<\/p>\n<\/td>\n

\n

27.5\u00b11.64  <\/p>\n<\/td>\n

20.1\u00b12.04<\/td>\n23.3\u00b11.36<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

Post hoc analysis: Group I (16weeks) Vs Groups III, IV and V (16 weeks)- P<0.01*, Group II (16 weeks) Vs Groups III, IV and V (16 weeks)- P<0.01*, <0.05* and <0.05* respectively. Group III (16 weeks) Vs Group IV and V (16 weeks)- P>0.05, Group IV (16 weeks) Vs Group V (16 weeks)- P>0.05<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

TC,\nTG, LDL and VLDL cholesterol<\/strong><\/p>\n\n\n\n

In the normal control\nanimals (group I) TC, TG, LDL and VLDL levels remained constant throughout the\nexperimental duration. These levels, on the other hand, were significantly\nelevated in the fructose fed groups (II to V) at the end of 8 weeks, and\ndecreased after treatment in the metformin and extract treated groups (III to\nV) but remained elevated in the disease control animals (group II) (within\ngroup analysis, P<0.0001). Intergroups comparison showed that at the end of\n16 weeks TC, TG, LDL and VLDL levels decreased significantly in the Metformin\nand extract treated (400mg\/kg and 600mg\/kg) groups when compared to the disease\ncontrol animals (group II). But, when compared to the normal control animals\n(group I), the levels remained elevated. Furthermore, in the Metformin and\nextract treated (400mg\/kg and 600mg\/kg) groups the reduction in the levels of\nthese parameters in these groups were almost similar and comparable. Data of TC\nand LDL cholesterol is shown in figure 3, 4 and data on TG and VLDL cholesterol\nis shown in table 4 and 5.<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 3: Effect on Total cholesterol<\/strong>

<\/p>\n

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

Table 4: Effect on Triglycerides<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 Groups<\/p>\n<\/td>\n

\n

Triglycerides (mg\/dl)<\/p>\n

mean SD<\/p>\n<\/td>\n

\n

 P value<\/p>\n<\/td>\n<\/tr>\n

\n

 Baseline<\/p>\n<\/td>\n

\n

 <\/p>\n

8 Weeks<\/p>\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n

16 Weeks<\/p>\n

 <\/p>\n<\/td>\n

\n

Within group<\/p>\n

(Repeated measures ANOVA)<\/p>\n<\/td>\n

\n

Between groups<\/p>\n

(One way ANOVA)<\/p>\n<\/td>\n<\/tr>\n

\n

I<\/p>\n<\/td>\n

60.5\u00b11.87  <\/td>\n60.66\u00b12.06<\/td>\n61.16\u00b12.04<\/td>\n\n

0.10<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

II<\/p>\n<\/td>\n

60.16\u00b13.06 <\/td>\n73.5\u00b13.45 <\/td>\n78.83\u00b12.92<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

III<\/p>\n<\/td>\n

60.66\u00b11.86 <\/td>\n73.66\u00b11.50 <\/td>\n70.16\u00b11.47<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

IV<\/p>\n<\/td>\n

60.33\u00b12.25<\/td>\n73.33\u00b11.50<\/td>\n70\u00b11.67<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

V<\/p>\n<\/td>\n

60.5\u00b11.37 <\/td>\n74.16\u00b11.16 <\/td>\n70.1\u00b11.16<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

Post hoc analysis: Group I (16 weeks) Vs Groups III, IV and V (16weeks)- P<0.001*, Group II (16 weeks) Vs Groups III, IV and V (16weeks)- P<0.001*, Group III (16 weeks) Vs Group IV and V (16 weeks)- P>0.05 and Group IV (16 weeks) Vs Group V (16 weeks)-P>0.05<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 4: Effect on LDL cholesterol.<\/strong><\/p>\n

<\/p>\n

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

Table 5: Effect on VLDL cholesterol<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n

 <\/p>\n

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

\n

VLDL (mg\/dl)<\/strong><\/p>\n

mean SD<\/strong><\/p>\n<\/td>\n

\n

P value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

 <\/p>\n

8 Weeks<\/strong><\/p>\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n

16 Weeks<\/strong><\/p>\n

 <\/p>\n<\/td>\n

\n

Within group<\/strong><\/p>\n

(Repeated measures ANOVA)<\/strong><\/p>\n<\/td>\n

\n

Between groups<\/strong><\/p>\n

(One way ANOVA)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

I<\/p>\n<\/td>\n

12.1\u00b10.37  <\/td>\n12.13\u00b10.41<\/td>\n12.23\u00b10.40<\/td>\n\n

0.10<\/p>\n<\/td>\n

\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

II<\/p>\n<\/td>\n

12.03\u00b10.61 <\/td>\n14.7\u00b10.68 <\/td>\n15.76\u00b10.58<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

III<\/p>\n<\/td>\n

12.13\u00b10.37  <\/td>\n14.73\u00b10.30<\/td>\n14.03\u00b10.29<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

IV<\/p>\n<\/td>\n

12.06\u00b10.45 <\/td>\n14.66\u00b10.30 <\/td>\n14\u00b10.33<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

V<\/p>\n<\/td>\n

12.1\u00b10.27<\/td>\n14.83\u00b10.23<\/td>\n14.03\u00b10.23<\/td>\n\n

<0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

Post hoc analysis: Group I (16 weeks) Vs Groups III, IV and V (16weeks)- P<0.001*, Group II (16 weeks) Vs Groups III, IV and V (16weeks)- P<0.001*, Group III (16 weeks) Vs Groups IV and V (16 weeks)- P>0.05 and Group IV (16 weeks) Vs Group V (16 weeks)-P>0.05<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Histopathology<\/strong><\/p>\n\n\n\n

HPE of pancreas showed the following changes:<\/p>\n\n\n\n

In the normal control group (group I), HPE of pancreas did not show any significant changes (Figure 5). Whereas, in the disease control group (group II) islet cells were surrounded with inflammatory infiltrates such as lymphocytes, macrophages, and plasma cells (Figure 6). The metformin and P.juliflora<\/em> extract 600mg\/kg treated groups (group III and V) showed normal islets cells with large, pale and ovoid beta cells (Figure 7 and 9). While the P.juliflora<\/em> extract 400mg\/kg treated group showed some degenerative changes (Figure 8). These findings indicate that the islet cells may have recovered after treatment with P.juliflora <\/em>extract 600mg\/kg.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 5: Group I- Normal islet cells<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 6: Group II- Presence of inflammatory infiltrates <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 7: Group III- Islet cells with pale large to ovoid beta cells with regenerative changes <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 8: Group IV- Islet cells with some degenerative changes <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 9: Group V- Islet cells with pale and ovoid beta cells
with regenerative changes <\/strong>

<\/p>\n

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

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

Lipid abnormalities such\nhypertriglyceridemia and reduced HDL cholesterol levels as are commonly\nobserved in patients with T2DM. Diabetic dyslipidemia can be treated with\nstrict glycemic control and weight loss. Majority of the conventional\nanti-diabetic drugs are associated with unavoidable side effects.36<\/sup>An\nideal anti-diabetic drug should be safe, effective in maintaining optimal blood\nglucose levels and also prevent long term complications. Alternative system of\nmedicine is practiced by a large population for treatment of various diseases\nand ailments including DM.37<\/sup> Herbal medicine & plant components\nhave relatively low toxicity and fewer side effects and therefore, they can be\nconsidered as a therapeutic option for DM.38<\/sup><\/p>\n\n\n\n

In this study, male\nwistar albino rats were fed with 20% fructose water daily for a period of 8\nweeks presented with features of T2DM like fasting hyperglycemia, insulin\nresistance. In addition to T2DM, fructose supplementation also induced some of\nthe features of metabolic syndrome like weight gain and altered lipid profile\n(dyslipidemia).<\/p>\n\n\n\n

The anti-diabetic and\nhypolipidemic activity of the seed extract was compared with metformin. It was\nobserved that both the doses of extract (400mg\/kg and 600mg\/kg) reduced the\nblood glucose levels. However, the dose 600mg\/kg showed maximum reduction which\nwas comparable to the effect produced by metformin. Therefore, it is clear that\nthe ethanolic seed extract of P.juliflora<\/em>\nsignificantly reduced the blood glucose levels in a dose-dependent manner. Similar\nfindings were observed in another study where methanolic seed extract of P.juliflora<\/em>\nin the dose 600mg\/kg reduced the blood sugar levels in streptozotocin induced\ndiabetic rat model.19<\/sup><\/p>\n\n\n\n

Phytochemical analysis\nshowed the presence of alkaloids, flavonoids, terpenoids &steroids. Based\non this, it is proved that P.juliflora<\/em>\nseeds are rich in polyphenolic compounds like flavonoids. Flavonoids are\nbiologically active secondary metabolites in the plants.39<\/sup> They have\nnumerous beneficial effects on metabolic disorders such as cardiovascular\ndisease, obesity, cancer and DM.40<\/sup>Chronic hyperglycemia in T2DM\nraises inflammatory cytokine levels which may cause endoplasmic reticulum\nstress, oxidative stress and lysosomal destabilization, which ultimately leads\nto beta cell death through apoptosis.41<\/sup> Flavonoids promote beta cell\nproliferation and decrease apoptosis, regulate glucose metabolism in the liver,\nreduce the hepatic glucose output, regulates the enzymes involved in the\ncarbohydrate metabolism (inhibits -glucosidase), exhances\nthe expression of glucose transporters, promote insulin secretion and reduce\ninsulin resistance.42,43<\/sup> According to many studies, flavonoids also\nact as insulin mimetics and insulin secretagogues.44-46<\/sup> Therefore,\nthe hypoglycaemic effect of ethanolic seed extract of P.juliflora <\/em>might be due to the presence of bioactive\nphytoconstituents like flavonoids that act separately or synergistically and\neither stimulates or mimics the action of insulin.<\/p>\n\n\n\n

In our study, we\ndiscovered that animals in the fructose fed groups gained significantly more\nweight at the end of 8 weeks, when compared to the animals in the normal\ncontrol group. Similar findings were observed in another study where wistar\nrats fed on 20% fructose water for 8 weeks had significantly higher body weight\nwhen compared to the control group.47 <\/sup>Fructose promotes de novo\nlipogenesis in animals, resulting in increased body weight and adipocity.\nObesity develops as a result of the dyslipidemia and increased body fat stores.\nInsulin resistance and obesity frequently coexist. This could explain the\nincreased body weight and insulin resistance observed in our study48-50<\/sup><\/p>\n\n\n\n

We also observed that\nthe levels of TG, LDL, VLDL and TC increased while HDL cholesterol decreased\nafter the induction of diabetes at 8 weeks. Similar findings were reported in\nsome of the previous studies where wistar rats fed on 20% fructose water for 8\nweeks and 21 weeks respectively, developed signs of dyslipidemia as well as\ninsulin resistance.29,30<\/sup><\/p>\n\n\n\n

\u201cDyslipidemia is\ncharacterized by elevated levels of TC, LDL, VLDL and TG, as well as a decrease\nin HDL cholesterol\u201d51<\/sup>. In our study, the lipid parameters remained\nunchanged in the disease control group at the end of treatment. In contrast,\nthere was a significant increase in HDL levels and a decrease in the levels of\nTG, LDL, VLDL and TC in the metformin and extracts treatment groups (400mg\/kg\nand 600mg\/kg). Moreover, both the doses of extract were having anti\ndyslipidemic effect similar to metformin<\/p>\n\n\n\n

Hyperglycemia is\nfrequently associated with dyslipidemia in T2DM. Insulin activates the enzyme\nlipoprotein lipase, which causes triglyceride hydrolysis. Lipoprotein lipase is\nnot activated in T2DM due to insulin resistance, and catabolism of\ntriglyceride-rich lipoproteins is decreased, resulting in\nhyper-triglyceridaemia and other lipoprotein alterations.52 <\/sup>Furthermore,\nthe hypolipidemic effect of the P.juliflora<\/em>\nseed extract can be attributed to the presence flavonoids and terpenoids which\nmight cause inhibition of the enzyme lipoprotein lipase and thereby decreasing\nthe levels of triglycerides, LDL, VLDL and total cholesterol.53,54<\/sup><\/p>\n\n\n\n

Biochemical and\nhaematological assessments were done at baseline and at 16 weeks to assess\nsafety of the interventions and no significant abnormality was observed. <\/p>\n\n\n\n

HPE of pancreas showed regenerative changes\nin the metformin and extract 600mg\/kg treated groups. These findings indicate\nthat the damaged islet cells may have recovered after treatment with the plant\nextract.<\/p>\n\n\n\n

One of the major goals in the management of\ndiabetes is to maintain optimal blood glucose levels to prevent complications.55\n<\/sup>considering the current study\u2019s findings as well as the available\nevidence for anti-diabetic and hypolipidemic activity, P.juliflora<\/em> seed extract can be used to treat hyperglycemia and\ndyslipidemia associated with T2DM. However, further studies are needed to\ndetermine the exact mechanism of such activity by isolating bioactive compounds\nand assessing their effect.<\/p>\n\n\n\n

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

P.juliflora\n<\/em>seed extract in the doses 400 mg\/kg and 600mg\/kg exhibited\nantidiabetic and hypolipidemic activity in terms of reduction in the fasting\nblood sugar, HOMA-IR and lipid levels. Both the doses, 400 and 600 mg\/kg showed\nhypolipidemic activity similar to Metformin whereas the antidiabetic activity\nshowed a dose dependent response i.e., 600 mg\/ kg was having better antidiabetic effect compared to 400 mg\/ kg and the\nreduction in blood sugar was similar to that of Metformin. Both the\ndoses of the extract were safe as there were no significant abnormality\nobserved in the hematological and biochemical parameters. Further, regenerative\nchanges were noted in the pancreas of the animals treated with Metformin and\n600 mg\/kg of the extract. All these findings suggest that the seed extract of P.juliflora<\/em> has a potential therapeutic\nbenefit in diabetes and hyperlipidemia.  <\/p>\n\n\n\n

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

We thank Chettinad\nAcademy of Research and Education for permitting and supporting us to conduct\nthis study. <\/p>\n\n\n\n

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

The author(s) declares\nno conflict of interest<\/p>\n\n\n\n

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

The author(s) received no financial support\nfor the research, authorship, and\/or publication of this article <\/p>\n\n\n\n

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

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