2.1 Preparation of C. ternatea extract, LC-MS/MS assay

The telang flower plant were collected from Kampung Herbal, Pasuruan, East Java, Indonesia. CTE was processed by PT FAST (Depok, Indonesia) based on Good Manufacturing Practices (GMP) with CoA No. Batch 00103211072. The butterfly flower peas were extracted using 70 % ethanol, then it added lactose (Widowati et al., 2021). The bioactive component was analyzed and identified qualitatively using a liquid chromatography mass spectrometer (LC-MS/MS). Hypersil Gold column with 150 mm × 2.1 mm × 1.9 mm was used for the analysis (Gondokesumo et al., 2019a; Widowati et al, 2022b).

2.2 Induction of Diabetes Mellitus and dyslipidemia in rats

The DM and dyslipidemia research protocols have been approved by Maranatha Christian University Ethical Committee, Bandung, Indonesia (147/KEP/VI/2021). Male rats of Sprague Dawley species aged 6 weeks and weighed 120–140 g were obtained from IratCo Laboratory, Bogor, Indonesia. The rats were kept in individual cages in 25 °C room temperature and a 12-h dark/12-h light cycle. The rats were given standard diet and water ad libitum for 7 days (Widowati et al, 2022b). After acclimatization, the rats were administered High Fat Diet (HFD) with 5.5 % crude fiber, 18 % crude protein, 50 % crude fat (PT Indoofeed) and 0.01 % Propylthiouracil (PTU, Dexa Medica) in drunk water (Kurniati et al., 2021), while standard diet had 7.37 % crude fat (Widowati et al., 2013). The induction of dyslipidemia to rats was done by giving HFD and PTU for 28 days. Dyslipidemia of rats was confirmed by serum cholesterol level using Cholesterol Kit (Elabscsi, E-BC-K109-M) ≥ 200 mg/dL. Single intraperitoneal injection of streptozotocin (STZ) for inducing DM (Sigma Aldrich SO130) 60 mg/kg BW 60 min after intraperitoneal (ip) administration of nicotinamide (NA, Sigma Aldrich-N3376) 120 mg/kg of BW. After five days, 12 h Fasting Blood Glucose (FBG) was evaluated using Autocheck blood glucose, DM rats have glucose level > 250 mg/dL (Elamin et al., 2018). After the rats were confirmed to have DM and dyslipidemia, the rats were treated with CTE (200, 400, 800 mg/kg BW), glibenclamide 0.45 mg/kg of BW (Generic, GKL9520905004A2), simvastatin 0.9 mg/kg BW (Generic, GKL131670271A), combination of glibenclamide and simvastatin while distilled water for negative control was given for 28 days (Florence et al., 2014).

2.3 Oral glucose tolerance test

Oral Glucose Tolerance Test (OGTT) was managed as a modification of the tests conducted by Anusooriya et al. (2014), Elamin et al. (2018). The fasted rats were treated for 12 h with unrestricted access to water and glucose given orally 2 g/kg BW, and then their glucose levels were tested in the coccygeal vein blood at 0, 2, 4, and 6 h after glucose loading using Glucose Kit (Elabsci, E-BC-K234-M) (Elamin et al., 2018).

2.4 Serum Collection, rats termination, liver collection

On day 28 after treating CTE, blood samples were taken from plexus retro-orbitalis in 12 h-fasted rats before anesthesia. The blood samples were kept at 4 °C for 2 h and centrifuged at 3,500 g for 10 min to obtain the serum (Widowati et al., 2022b). Rats were administered 100 mg/kg BW ketamine HCl (Ikapharmindo Putramas), xyla at 10 mg/kg BW (Interchemie, 361453), and then liver and pancreas were collected (Widowati et al., 2022b). The liver and pancreas were preserved at −80 °C and the remaining liver and pancreas were fixed in formalin (10 %) for immunohistochemistry (IHC), histopathological assay (Florence et al., 2014; Gondokesumo et al., 2019b; Widowati et al., 2022b).

2.5 Serum glucose, insulin assay

The glucose, insulin levels of the serum were measured using Glucose Colorimetric Kit. Rat Insulin Elisa Kit (Elabsci, E-EL-R3034), according to the manufacturer protocol (Elamin et al., 2018).

2.6 CAT, SOD, MDA, IL-18 assay

The CAT, SOD, MDA, IL-18 levels of pancreas were assessed using Kit respectively CAT Kit (Elabsci, E-BC-K031-S), SOD Kit (Elabsci, E-BC-K020), MDA Kit (Elabsci, E-EL-0060), Rat IL-18 Elisa Kit (Elabsci, E-EL-R0567) at 405, 450, 450, 450 nm respectively and Microplate reader (Multiskan^TM Spectrophotometer, Thermo Scientific) was utilized to measure absorbance respectively of CAT, SOD, MDA, IL-18 (Widowati et al., 2019; Widowati et al., 2022b).

2.7 Glycogen gene expression assay

Total RNA of rat pancreas was isolated and purified using Direct-zol RNA Miniprep Plus Kit (Zymo, R2073). Complementary DNA synthesis was carried out using the iScript Reverse Transcription Supermix for RT-PCR (Bio-Rad, 170–8841). Quantitative gene expression was performed using the AriaMx 3000 Real-Time PCR System (Agilent). Evagreen master mix was performed as the q-RTPCR reaction mixture (Bio-Rad, 1725200) (Afifah et al., 2019). Table 1 shows the Glycogen and GAPDH sequences primer.

2.8 Protein assay

The 20 µL of standard solutions of Bovine Serum Albumin (BSA) stocks (Sigma Aldrich, A9576) in 1000 µL ddH2O, samples (serum, pancreas homogenate), each well plate was filled with Quick Start Dye Reagent 1X (Biorad, 5000205) 200 µL at room temperature, incubated for five minutes. After the incubation, Microplate reader was used for measure the sample with absorbance at 595 nm (Widowati et al., 2019).

2.9 Histopathological assay

Liver specimens were fixed in formalin (10 %) and examined by Haematoxylin Eosin (HE). Graded alcohols were used to dry the liver, which was then cleaned with xylene before being embedded in paraffin wax. The microtome was used to cut four or five micrometer (μM) thickness slices, which were subsequently stained with HE and coated with a cover glass. A light microscope was used to observe the samples and ImageJ was used to do quantitative data analysis. The quantitative data of the liver included necrosis, inflammation, and lipide degeneration (Elamin et al., 2018; Gondokesumo et al., 2019b; Rosni et al., 2021).

2.10 Immunohistochemistry assay

Cut paraffin blocks 4–5 μM (pancreas, liver), antigen (Abcam, ab208572) in pH 6.0, 121 °C for 10 min, 3 % of H₂O₂ in methanol for 15 min, 37 °C for blocking endogenous peroxidase. Then, 5 % BSA for pre-incubating the samples for 10 min. The primary antibody reaction using GSK-3β Polyclonal (Elabsci, E-AB-70043) for liver, IL-6 Polyclonal (Elabsci, E-AB-65801) for pancreas were done overnight at 37 °C. The HRP/DAB (ABC) rabbit specific detection IHC kit was used to visualize the protein target (Abcam, ab64261). Two-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System (with DAB solution) (Elabsci, E-IR-R217) was used for visualizing the protein target (Gondokesumo et al., 2019b; Rosni et al., 2021; Widowati et al., 2022b).

2.11 Statistical analysis

The data were examined using One Way ANOVA, and a post-hoc analysis was performed using the Tukey's HSD test. Significant at P 0.05.

3.1 Compound analysis in CTE by LC-MS/MS

The various compounds were described by the chromatogram peak with various molecular weights. In our investigation, the results of LC-MS/MS analysis were compared to the standard 2-hydroxycinnamic acid which had a molecular weight of 164.16 g/mol, inositol 180.16 g/mol, (+) catechin 7-O-β-glucoside 160.2 g/mol, (+)-catechin 7-O-β-glucoside 452.13 g/mol, and delphinidin-3-O-(6-O-p-coumaroyl) glucoside-pyruvic acid 679.12 g/mol. This result indicated that CTE contained: 1). 2-hydroxycinnamic acid, 2). inositol, 3). (+) catechin 7-O-β-glucoside, 4). delphinidin-3-O-(6-O-p-coumaroyl) glucoside-pyruvic acid (Fig. 1).

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Fig. 1

LC-MS/MS Spectrum of CTE. Mass spectra 100–200 m/z (165.029): 2-hydroxycinnamic acid with molecular weight of 164.16 g/mol. Mass spectra 100–200 m/z (181.109): inositol with molecular weight of 180.16 g/mol. Mass spectra 400–500 m/z (181.109): (+)-catechin 7-O-β-glucoside with molecular weight of 452.13 g/mol. Mass spectra 400–500 m/z (181.109): delphinidin-3-O-(6-O-p-coumaroyl) glucoside-pyruvic acid with molecular weight of 680.119 g/mol.

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3.2 Effect of CTE toward OGTT, serum Glucose, insulin levels

The effect of CTE toward serum OGTT in DM rats can be seen in Fig. 2A. Diabetic rats model showed an increase glucose levels at 0–4 h, then decreased at 6 h. The CTE treatment lowered blood glucose in DM rats. The blood glucose level at 0 h after CTE treatment did not show a decrease. Treatment CTE 800 mg/kg BW was the most effective to decrease OGTT.

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Fig. 2

Effect of CTE toward OGTT, Serum Glucose, and Serum Insulin in DM Rats Model. *Data provided as mean and standard deviation. I: negative control; II: positive control (DM rats); III: DM rats + CTE 200 mg/kg BW; IV: DM rats + CTE 400 mg/kg BW; V: DM rats CTE + 800 mg/kg BW; VI: DM rats + Glibenclamide 0.45 mg/kg BW; VII: DM rats + Simvastatin 0.9 mg/kg BW; VII: DM rats + Glibenclamide 0.45 mg/kg BW and Simvastatin 0.9 mg/kg BW *A. Effect CTE toward OGTT. *B. Various letters (a, b, c, d, e, f, g) differences between treatments toward glucose level (Tukey’s HSD test, p < 0.05). *C. Various letters (a, b, bc, cd, de, e) significantly differences among treatments toward insulin level (Tukey’s HSD test, p < 0.05).

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The effect of CTE toward serum glucose, insulin levels in DM rats on day 28^th can be seen in Fig. 2B and C. The CTE decreased glucose level (p < 0.05), increased insulin level of DM rats (p < 0.05). The effective CTE to decrease glucose level, increase insulin level was CTE 400 mg/kg BW.

3.3 Effect of CTE toward CAT, SOD, MDA, IL-18 levels of pancreas

The effect CTE toward levels of serum CAT, SOD, MDA, IL-18 in DM rats on day 28^th can be seen in Fig. 3. The results show that CTE increased CAT, SOD levels and lowered MDA, IL-18 levels (p < 0.05). The effective CTE to increase CAT, SOD levels, to lower MDA, IL-18 levels was CTE 400 mg/kg BW.

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Fig. 3

Effect of CTE toward CAT, SOD, MDA, IL-18 Level in DM Rats Model. *Data provided as a mean and standard deviation, I: negative control; II: DM rats; III: DM rats + CTE 200 mg/kg BW; IV: DM rats + CTE 400 mg/kg BW; V: DM rats CTE + 800 mg/kg BW; VI: DM rats + Glibenclamide 0.45 mg/kg BW; VII: DM rats + Simvastatin 0.9 mg/kg BW; VII: DM rats + Glibenclamide 0.45 mg/kg BW and Simvastatin 0.9 mg/kg BW. *A. Various letters (a, ab, bc, c) significantly differences between treatments toward CAT level (Tukey’s HSD test, p < 0.05). *B. Various letters (a, ab, b, c) significantly differences among treatments toward SOD level (Tukey’s HSD test, p < 0.05). *C. Various letters (a, b, bc, c, d) significantly differences among treatments toward MDA level (Tukey’s HSD test, p < 0.05). *D. Various letters (a, b, bc, c, d, e, f) significantly differences between treatments toward IL-18 level (Tukey’s HSD test, p< 0.05).

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3.4 Effect of CTE toward protein level of pancreas

The effect of CTE toward pancreatic protein level in DM rats on day 28^th can be seen in Fig. 4. The STZ induction decreased protein level (p < 0.05), CTE treatment increased protein level (p < 0.05). The most effective CTE to increase pancreatic protein was CTE 400 mg/kg BW.

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Fig. 4

Effect of CTE toward Protein Level on DM Rats Model. *Data provided as a mean and standard deviation I: negative control; II: positive control; III: CTE 200 mg/kg of BW + positive control; IV: CTE 400 mg/kg of BW + positive control; V: CTE 800 mg/kg of BW + positive control; VI: positive control + Glibenclamide 0.45 mg/kg of BW; VII: Simvastatin 0.9 mg/kg of BW + positive control; VIII: Glibenclamide 0.45 mg/kg of BW and Simvastatin 0.9 mg/kg of BW + positive control. Various letters (a, b, bc, bcd, cd, d) reveal substantially differences between treatments toward protein level (Tukey’s HSD test, p < 0.05).

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3.5 Effect of CTE toward IL-6 protein expression of pancreas

The effect of CTE toward IL-6 protein expression in DM rats on day 28^th can be seen in Fig. 5. The CTE treatment decreased IL-6 expression (p < 0.05), the effective CTE to lower IL-16 expression were CTE 400, 800 mg/kg BW.

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Fig. 5

Effect of CTE toward IL-6 Expression of Pancreas in DM Rats Model. Hystopathology of pancreas tissues, IL-6 positive cells are distinguishable by their brown cytoplasmic staining and hemotoxylin eosin counterstain (magnification x40). I: negative control; II: DM rats; III: DM rats + CTE 200 mg/kg BW; IV: DM rats + CTE 400 mg/kg BW; V: DM rats CTE + 800 mg/kg BW; VI: DM rats + Glibenclamide 0.45 mg/kg BW; VII: DM rats + Simvastatin 0.9 mg/kg BW; VII: DM rats + Glibenclamide 0.45 mg/kg BW and Simvastatin 0.9 mg/kg BW. *A. Various letters (a, ab, b, c) significantly differences between treatments toward IL-6 expression (Tukey’s HSD test, p < 0.05).

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3.6 Effect of CTE toward glycogen gene expression of pancreas

The effect of CTE toward pancreatic glycogen gene expression in DM rats on day 28^th can be seen in Fig. 6. The CTE treatment decreased glycogen gene expression (p < 0.05), the most effective of CTE to decrease glycogen gene expression was CTE 400, 800 mg/kg BW.

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Fig. 6

Effect of CTE toward Glycogen Gene Expression of Pancreas in DM Rat Model. *Data provided as a mean and standard deviation. I: negative control; II: DM rats; III: DM rats + CTE 200 mg/kg BW; IV: DM rats + CTE 400 mg/kg BW; V: DM rats CTE + 800 mg/kg BW; VI: DM rats + Glibenclamide 0.45 mg/kg BW; VII: DM rats + Simvastatin 0.9 mg/kg BW; VII: DM rats + Glibenclamide 0.45 mg/kg BW and Simvastatin 0.9 mg/kg BW. *Various letters (a, b, c, cd, de, e) significantly differences between treatments toward glycogen gene expression of pancreas (Tukey’s HSD test, p < 0.05).

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3.7 Effect of CTE toward GSK-3β protein expression of liver

The effect of CTE toward liver GSK-3β protein expression in DM rats on day 28^th can be seen in Fig. 7. Based on the result, CTE was able to lower GSK-3β expression in DM rats, the effective CTE to lower GSK-3β expression were CTE 400, 800 mg/kg BW.

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Fig. 7

Effect of CTE toward GSK-3β Expression of Liver in DM Rats Model. Hystopathology of liver tissues, GSK-3β cells are distinguishable by their brown cytoplasmic staining and hemotoxylin eosin counterstain (magnification x40) I: negative control; II: DM rats; III: DM rats + CTE 200 mg/kg BW; IV: DM rats + CTE 400 mg/kg BW; V: DM rats CTE + 800 mg/kg BW; VI: DM rats + Glibenclamide 0.45 mg/kg BW; VII: DM rats + Simvastatin 0.9 mg/kg BW; VII: DM rats + Glibenclamide 0.45 mg/kg BW and Simvastatin 0.9 mg/kg BW. *A Various letters (a, ab, abc, bc, cd, d) significantly differences between treatments toward GSK-3β expression (Tukey’s HSD test, p < 0.05).

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3.8 Effect of CTE toward histopathology of liver

The effect of CTE toward liver histopathology in DM rats on day 28^th can be seen in Fig. 8. Liver histopathology was performed using HE staining, the scoring of the inflammation, necrotic cell, and lipid accumulation of liver. The result shows that STZ induction triggered liver inflammation, necrotic cell, and lipid degeneration. The CTE treatment lowered the inflammation, necrotic cell, and lipid degeneration in the liver of DM rats. The CTE 800 mg/kg BW was the most effective to reduce the inflammation, necrotic cell, and lipid accumulation in liver rats.

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Fig. 8

Effect of CTE toward Liver Histopathology in DM Rats Model. Positive control over lipid degradation, necrotic cells, and inflammation. The inflammatory infiltration was initially noticed near the portal triad, where there was more severe inflammation that could be observed in the central vein (x10 magnification). I: negative control; II: DM rats; III: DM rats + CTE 200 mg/kg BW; IV: DM rats + CTE 400 mg/kg BW; V: DM rats CTE + 800 mg/kg BW; VI: DM rats + Glibenclamide 0.45 mg/kg BW; VII: DM rats + Simvastatin 0.9 mg/kg BW; VII: DM rats + Glibenclamide 0.45 mg/kg BW and Simvastatin 0.9 mg/kg BW. A* The data is provided as a mean and the scores of inflammation, necrotic cell, lipid degeneration of liver are based on category 0: normal (no change), 1: mild, 2: moderate, 3: severe.

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