Physicochemical characterization, phytochemical analysis, and pharmacological evaluation of Sambucus wightiana

2.5.1 Aerial plant material extraction

The plant material after being dried in shadow and ultimately was powdered using ball mills. The powdered material was allowed to go through mesh no.40, subsequently weighed & employed for extraction. An amount of approximately 200 gms in the powder form was consecutively extracted in the Soxhlet apparatus with petroleum ether, chloroform, ethyl ethanoate, and methanol. The final marc was then extracted with water using the decoction method. Individually, the weighed powder was extracted with different solvents using the soxhlet and maceration methods (Tzanova et al., 2020).

2.5.2 Fluorescence analysis

Many plants fluoresce when their powder or cut surface is exposed to UV light, making it an important tool for identifying them. Using a variety of reagents, including sodium hydroxide, picric acid, acetic acid, hydrochloric acid, nitric acid, iodine, ferric chloride, and others, plant powders (mesh 40) were investigated for their fluorescence properties in daylight and UV light (254 and 366 nm) as well as after treatment (Dinesh et al 2011).

2.6.1 Determination of fat content

Anhydrous ether was applied to a known quantity of the sample (3 g) in an extraction device for six hours, followed by filtration of the extract. Utilizing a process developed by Aziz et al., (Aziz and Mitu, 2019), the solvent was evaporated and dried to a consistent weight at 105 °C.

2.6.2 Saponin determination

20 g of plant material that had been powdered and 100 ml of 20% aqueous ethanol were put to a conical flask. After 4 h of continuous stirring at 55 °C in a hot water bath, the sample was filtered and subjected to a second extraction with 200 ml of 20% ethyl alcohol. The extract was then reduced to a volume of 40 ml in a water bath that was 90 °C or thereabouts. A 250 ml separating funnel was filled with the concentrated extract, and 20 ml of diethyl ether was added while the mixture was being violently shaken. The ether layer was removed, but the aqueous layer was retained. 60 ml of n-butyl alcohol was used to repeat the purification process. Two times, 5% aqueous sodium chloride solution was used to wash the extract. The sample was oven dried to a consistent weight before the saponin concentration was determined, and the leftover solution was boiled in a water bath to remove the solvent (Obadoni and Ochuko, 2001).

2.6.3 Thin layer chromatography (TLC) profile

Prepared TLC plates were procured from the Sigma Pvt. Ltd. (Mumbai, India) and used for study. When not in use the plates were kept in a desiccator (Stahl and Sies, 2003). The sample was applied to TLC plates using glass capillaries. The spots were placed with a minimum distance of 1 cm between adjacent spots. They were also marked to determine their identity. The glass chromatogram chamber (16.5 cm × 29.5 cm) was employed for carrying out the experiments. The chamber was given 24 hrs for saturation hours before use. The different solvent combinations were tried for different extracts and the process was continued till the satisfactory resolution system was developed. The different components that were present on the TLC plates were identified using a compressed air sprayer. The detecting reagent was then diluted to a volume of about 50 ml, and the sprayer with the air compressor was put to work. Following each spray, the sprayer was cleaned separately with water, chromic acid, distilled water, and acetone. The fluorescent substances were also tested in a UV chamber. The results were obtained at two different UV wavelengths of 254 nm and 366 nm.

2.8.1 Processing of plant material for determination of lead, cadmium, and mercury

Accurately weighed 0.2 g of powdered drug was taken into a clean silica crucible. To this, 1 ml of digestion mixture containing one part by weight of concentrated nitric acid and one part by weight of concentrated perchloric acid was added. Crucible was placed in oven and the temperature raised slowly up to 100 ^°C. This temperature was maintained for 3 hrs. Temperature was then raised up to 120 ^°C and the same maintained for 2 hrs. Again, the temperature was raised very slowly up to 450 ^°C and maintained for 4 hrs. Then, the contents of crucible were allowed to cool. 2.5 ml of 0.5% v/v nitric acid was added to the crucible and suitably diluted with double distilled water. The contents were filtered through membrane filters. The above processed sample was subjected to atomic absorption spectrometer (AAS) analysis for determination of lead, cadmium, and mercury. Three determinations were done under each analysis to get concordant results (Rajan, 2018).

2.8.2 Processing of plants material for determination of Arsenic

50 g of powdered drug was taken into a 1 L Kjeldhal flask. 25 ml of double distilled water, 50 ml of nitric acid, and 20 ml of sulphuric acid were added to the flask. Contents of the flask were heated slowly and carefully to avoid excess foaming. Nitric acid equivalent to 1000 g/l was added drop by drop until all the organic matter got destroyed. This was identified by no darkening of the solution by further addition of nitric acid and continued heating. The flask was then allowed to cool. 75 ml of distilled water and 25 ml of ammonium oxalate (25 g/l) were added to the flask to expel nitrogen oxide from the solution. Heated again and cooled. The contents were transferred into a 250 ml volumetric flask and made-up the final volume with double distilled water. The solution was filtered through membrane filters. The processed sample was subjected to AAS analysis for determination of Arsenic. Three determinations were done under each sample to get concordant results (Rajan, 2018).

2.9.1 Isolation of flavonoids from whole plant of S. Wightiana

Before being ground into powder, the complete aerial plant material was shade-dried for a week. Using a Soxhlet system and 95% ethanol, powdered material (5 kg) was extracted over the course of around 72 h. A rotating evaporator was used to filter and concentrate the extract while operating under reduced pressure and a vacuum. It was feasible to produce a 400 g (8.0% w/w) dark brown viscous mass. The extract (200 g) was subjected to lead acetate treatment, producing a yellow precipitate that was then suspended in methanol, subjected to lead removal using hydrogen sulfide gas, and finally filtered. After the filtrate was evaporated, the residue was heated in boiling water and extracted with ether. Before being acidified, the concentrated ether fraction was extracted using sodium hydrogen carbonate, sodium hydroxide, and hydrochloric acid. A reddish brown amorphous solid (500 mg), a cream-colored solid (300 mg), and a white-colored solid (200 mg) were generated by recrystallization from alcohol:water and subsequently analyzed spectroscopically (Bhujbal et al., 2009). After being isolated, the compound (C-1) was characterized.

2.9.2 Isolation of rutin from S. swightiana

500 g of plant powder and water (100 ml) were combined. The pH was changed to 6–8 using 1 M NaOH, and the mixture was then boiled at a low temperature. The mixture was filtered, and the filtrate was then treated with 1 M HCl to make it acidic (pH 5–6) in order to precipitate the rutin. The precipitate was purified by vacuum filtering, dissolved in water, and then adjusted to a pH range of 6 to 8. The solution was then heated for 30 min, filtered, and then dried at 700 °C in an oven. The compound was characterised to confirm its identity as rutin (C-2).

2.11.1 Total flavonoid content

A spectrophotometric technique was used to determine the flavonoids content of the plant extract (Quettier-Deleu et al., 2000). The sample included 1 ml of a 3 to 15 mg/ml methanol extract solution as well as 1 ml of a 2% AlCl₃ solution dissolved in methanol. The samples were incubated for one hour at room temperature. Using a spectrophotometer, the absorbance was measured at a wavelength of 415 nm. Triplicate samples were generated for each analysis, and the absorbance mean value was computed. The same process was carried out again using the rutin standard solution to create the calibration curve. The quantity of flavonoids in extracts was represented in terms of rutin equivalent (mg of rutin/g of extract), and the concentration of flavonoids was obtained (mg/ml) from the calibration curve based on measured absorbance.

2.11.2 Total phenolic content

The crude extracts of S. wightiana were determined using a reported method (Kupina et al 2008) The total phenolic content of extracts was calculated using the Folin-Ciocalteu technique. The extracts were oxidized with Folin-Ciocalteu reagent and then neutralized with sodium carbonate. The resulting blue color's absorbance was measured at 760 nm after 60 min using gallic acid as a reference. The amount of total phenol in each gram of extract was calculated as mg gallic acid equivalent.

2.11.3 Total antioxidant capacity

With some modifications, the phosphomolybdenum method was used to determine total antioxidant activity (Prieto et al 1999). This method's fundamental idea is based on the extract converting Mo (VI) to Mo (V), which it then does at an acidic pH to generate a green phosphate Mo (V) complex. 3 ml of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) were combined with 1 ml of extract in varied concentrations (between 5 and 30 mg/ml). The tubes containing the reaction solution were then sealed and incubated at 95 °C for 90 min. The samples were cooled to room temperature before measuring the solution's absorbance at 695 nm in comparison to a blank. Instead of extract, methanol was used as a blank. The samples were prepared in triplicate for each assay, and the mean absorbance value was calculated. The antioxidant activity is measured in mg of ascorbic acid equivalents (mg AAE)/g of extract.

2.11.4 DPPH radical scavenging activity

The 1,1-diphenyl-2-picryl-hydrazyl (DPPH) compound's capacity to scavenge free radicals was used to measure the antioxidant activity of plant extracts (Kedare and Singh, 2011). The methanolic extract (0.3 ml) was combined with 2.7 ml of DPPH (6 × 10^-5 mol/l) at various concentrations. After being vortexed, the mixture was left in the dark for 60 min. The DPPH radical's reduction was measured by the drop in absorption at 517 nm. 1 ml of 0.1 mM DPPH solution was combined with 1 ml of methanol as a control. The values for IC₅₀ were calculated. This formula was used to compute the % inhibitory activity: $$\%\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\frac{A0-A1}{A0}x100$$ where, A₁ is the extract/standard absorbance and A₀ is the control absorbance. The IC₅₀ values were computed and the inhibition curves were produced. Methanol (1 ml) and extract solution (2.5 ml) were employed as a negative control. The positive control was rutin solution.

2.11.5 Reducing power

The approach outlined in the literature by (Bhalodia et al 2013) was used to examine the extracts' reduction power. The methanolic extract (2.5 ml) was combined with 2.5 ml of 1% potassium ferricyanide and 200 mmol/l sodium phosphate buffer (pH 6.6). The mixture was incubated at 50 °C for 20 min. The mixture was centrifuged at 650 rpm for 10 min after adding 2.5 ml of 10% trichloroacetic acid (w/v). After adding the upper layer (2.5 ml) to the 2.5 ml distilled water and 0.5 ml of FeCl₃ (0.1%), the absorbance at 700 nm was measured. A higher reducing power is indicated by a higher absorption. The experiments were conducted three times, and the outcomes were presented as mean values and standard deviations.

2.12.1 Gulcose tolerance test of extract/fraction/molecules

Rats that had been fasted normally were placed into ten groups of six each. As the control group, group I received saline solution as usual. Groups III and IV received a methanol extract of the aerial part of S. wightiana (SW 200 & 400 mg/kg/day), while groups V and VI received compounds isolated from S. wightiana (rutin and quercetin 5 mg/kg/day). Group II received the reference medication, glibenclamide, at a dose of 5 mg/kg body weight. The rats in all groups received 2 g after 30 min. Before and after the delivery of glucose, as well as 30, 90, and 150 min later, blood samples were obtained from the tip of the tail. The glucose oxidase method was used to assess blood glucose levels after serum had been isolated (Yashwant et al., 2011).

2.12.2 Alloxan induced antidiabetic activity of extract/fraction/molecules

Six rats from each of six groups had the following course of treatment:

Group 1: Vehicle control group, in which rats were given vehicle on a daily basis i.e. saline (normal control).

Group 2: Diabetic control group in which alloxan (150 mg/kg i.p.) was administered to the rats.

Group 3: The rats in the standard control group also received alloxan (150 mg/kg i.p.) before receiving glibenclamide (5 mg/kg/day).

Group 4 & 5: Test group in which the rats received alloxan (150 mg/kg i.p.) and a methanolic extract of the entire aerial part of S. wightiana (200 & 400 mg/kg/day).

Group 5 & 6: Alloxan (150 mg/kg i.p.) had previously been given to the rats in the test group, who also received daily doses of isolated pure components from S. wightiana (qurecetin and rutin, each at 5 mg/kg/day).

Through a feeding cannula, plant extracts, the standard drug glibenclamide (5 mg/kg), and saline were given. The normal control group (group 1), received saline for 14 days. Group 2 contains the diabetic control rats. Group 3 served as the standard control group, which received glibenclamide (5 mg/kg) for 14 days. Groups 4 to 5 (previously received alloxan) received a constant dose of SW (200 & 400 mg/kg, p.o.), were administered daily with pure isolated compounds 5 mg/kg. On the first day, a homogeneous suspension of the extracts and the standard drug glibenclamide (5 mg/kg) was freshly prepared individually using saline and alloxan, and the test compounds were administered for 14 days. Instead of drugs, the normal control group was given a vehicle.

2.12.3 Induction of diabetes in experimental rats

Rats developed diabetes after receiving a single i.p. injection of alloxan monohydrate (150 mg/kg) (Guruvayoorappan and Sudha, 2008; Rathnaker et al, 2011). Each animal's dosage of alloxan was determined individually based on body weight before being dissolved in 0.2 ml of saline (154 mM NaCl) right before injection. Two days following alloxan injection, rats having plasma glucose levels of 200 mg/dl were enrolled in the study. 48 h after the alloxan injection, the animals started receiving treatment with plant extracts.

2.12.4 Blood sample collection for the measurement of glucose

Up to the end of the study, blood samples were taken from the rat's tip of the tail on a weekly basis (i.e., 2 weeks). On the first, seventh, and fourteenth study days, measures of body weight and fasting blood glucose were taken. Using a one-touch electronic glucometer and glucose test strips, blood sugar levels were tested using a procedure based on the glucose dehydrogenase method. Days 0, 7, and 14 after treatments were used to measure blood glucose levels. Under light ether anaesthesia, blood was taken from the retro-orbital plexus of overnight fasting rats on day 14. A blood sample without anticoagulant was spun at 3,000 × g for 5 min to extract serum for biochemical parameter measurement. Until biochemical parameters were assessed, serum was stored at −20 °C. Utilizing the A15 Analyzer Automatic Clinical Chemistry (Biosystems S.A., Spain), the serum was examined. Glucose, total cholesterol, triglycerides, creatinine, serum HDL, LDL, urea, and alkaline phosphatase were the biochemical markers that were assessed.

3.2.1 Phytochemical analysis of multiple extracts

The results for different fractions of S. wightiana fractions, such as PE, chloroform, EA, methanol, and aqueous fractions are summarized in Table S2. Different group of compounds was detected in different fractions of S. wightiana.

3.2.2 Phytochemical screening of methanol extract of S. wightiana

The whole plant of S. wightiana was extracted with methanol in a Soxhlet system and examined for phytochemicals. Table S3 provides a summary of the findings.

3.2.3 Fat and volatile content of S. wightiana

S. wightiana has been found to have fat and volatile levels of 2.05% and 0.5% (flowers), respectively.

3.2.4 Saponin content of S. wightiana

The saponin content of S. wightiana was determined as 48.4 mg/g.

3.2.5 Thin layer chromatography profile

The results for TLC profile of different fractions of S. wightiana are summarized in Table S4. Different phytoconstituents were separated with different R_f values.

3.5.1 Total flavonoid content

A spectrophotometric technique was used to measure the flavonoid content using an AlCl₃ solution. The quantity of flavonoids in extracts was then quantified in terms of rutin equivalent (mg rutin/g of extract), depending on the concentration of flavonoid that was read (mg/ml) on the calibration line based on the observed absorbance. Rutin made up 113.74 mg of the total flavonoid content per gram of extract.

3.5.2 Total phenolic content

The total phenolic content was ascertained using the Folin-Ciocalteu colorimetric technique. As mg GAE/g, the total phenolic content was measured. 249.15 mg GAE/g of phenols were discovered to be present overall.

3.5.3 Total antioxidant capacity

Using AA as a reference, the total antioxidant capacity (TAC) was calculated. TAC mg of AA/g was used to calculate the TAC. The TAC for AA was determined to be 195.54 mg/g.

3.5.4 DPPH radical scavenging activity

As a recognized benchmark for free radical scavenging activity, rutin was chosen. Table 3 lists the findings for DPPH scavenging activity. Standard rutin and S. wightiana extract both had IC₅₀ values of 269.46 and 549.36 g/ml, respectively.

3.5.5 Reducing power

BHT was chosen as a standard for reducing power assessment. The results for reducing power assessment are summarized in Table 4. Standard showed excellent reducing power. Good reducing power potential was also seen in S. wightiana extract.

3.6.1 Compound 1: Quercetin 3-O-β-D-glucopyranoside (C-1)

Quercetin 3-O-β-D-glucopyranoside (C-1) was obtained as creamish powder. The compound also produced color reactions for a hydroxyl flavone with a variety of reagents. The following physicochemical properties were found in compound C-1.

Chemical formula: C₂₁H₂₀O₁₂.

Melting point = 190–260 °C.

UV–Vis: (ethanol) λ_max; 245, 345 nm.

HR Mass: Molecular weight: 464.09, M⁺¹ (465.24) (Figure S1).

NMR: ¹H NMR (chemical shift δ in ppm, coupling constant J in Hz) 6.20 (1H, d, J = 2 Hz, C6-H), 6.42 (1H, d, J = 2 Hz, C8-H),7.69 (1H, d, J = 2.2 Hz, C2′-H), 6.90 (1H, d, J = 8.5 Hz,C5′-H),7.56 (1H, dd, J = 8.5,2.2 Hz, C6′-H),9.41(1H, s, C4′-OH), 9.35 (1H, s, C3′OH), 9.63 (1H, s, C3-OH), 12.52 (1H, s, C5-OH), 10.82 (1H, s, C7-OH) (Figure S2).

¹³C NMR (chemical shift δ in ppm) 148.40 (C-2),134.84 (C-3), 178.26 (C-4), 157.13 (C-5), 98.40 (C-6), 164.48 (C-7), 93.30 (C-8), 161.83 (C-9), 104.5 (C-10), 121.57 (C-1′), 115.53 (C-2′), 145.02 (C-3′), 148.40 (C-4′), 114.9 (C-5′), 121.4 (C-6′) (Figure S3). The final molecular structure of compound C-1 is presented in Figure S4. And these data were matched with references (Mir et al., 2017).

3.6.2 Compound 2: Rutin

Rutin was obtained in the form of a yellowish needle/amorphous solid. The compound also produced colour reactions for a hydroxyl flavone with a variety of reagents. The TLC of rutin in solvent system i.e. ethyl acetate: glacial acetic acid: formic acid: water (100:11:11:25) showed single spot at R_f = 0.42. The following physicochemical properties were recorded in compound C-2.

Chemical formula: C₂₇H₃₀O₁₆.

Melting point: 190–195 °C.

UV (ethanol) ƛ_max = 252, 358 nm.

FT-IR (KBR, cm⁻¹): 3434.56, 2938.92, 2087.17, 1647.38, 1504.88, 1458, 1363, 1235, 1204, 1132, 1063, 969, 826, 728, 656, 631, 596, 453 (Figure S5).

HR Mass: Molecular weight: M/Z 610.52 (M + ) (Figure S6).

¹H NMR: (CD₃OD; Chemical shift δ in ppm, coupling constant J in Hz) 6.21 (1H, d, J = 2, C6-H), 6.40 (1H, d, J = 2, C8-H),7.63 (1H, d, J = 2.1, C2′-H),6.86 (1H, d J = 9, C5′-H),7.66 (1H, dd, J = 9,2.1, C6′-H),8.05 (1H, s, C4′-OH), 8.07 (1H, s, C3′-OH), 12.34 (1H, s, C5-OH), 10.86 (1H, s, C7-OH), 5.23– (1H, d, J = 7.4, H1-G), 5.11 (1H, d, J = 6.45, H1-R), 1.11 (3H, d, J = 6.74, CH3-R) (Figure S7).

¹³C NMR: (chemical shift δ in ppm) 157.94 (C-2), 134.22 (C-3), 178.03 (C-4), 157.9 (C-5), 98.54 (C-6), 164.64 (C-7), 93.45 (C-8), 161.6 (C-9), 104.21 (C-10), 122.14 (C-1′), 114.65 (C-2′), 144.45 (C-3′), 148.41 (C-4′), 116.28 (C-5′), 121.7 (C-6′),103.30 (C1-G), 74.33 (C2-G), 76.79 (C3-G), 72.54(C4-G), 75.84 (C5-G), 67.15 (C6-G), 101.02 (C1-R), 68.31 (C2-R), 70.84 (C3-R), 70.7 (C4-R), 70.0 (C5-R), 16.47 (C6-R) [R and G represent signals from rhamnose and glucose moieties, respectively] (Figure S8). The final molecular structure of compound C-2 is presented in Figure S9. And these data were matched with references (Mir et al., 2017).

3.7.1 Effect on total cholesterol

The effect of methanol extract and isolated compounds of S. wightiana on total cholesterol levels in alloxan induced diabetic albino rats after 14 days of treatment is shown in (Fig. 1). The result exhibits that alloxan induced diabetic control (DC) group albino rats show increase in total cholesterol levels (271.16 ± 10.5 mg/dl) compared to the normal control (NC) group (145.36 ± 3.2 mg/dl) given only vehicle. However, the standard glibenclamide (67.33 ± 1.856 mg/dl) significantly decreased the serum total cholesterol levels compared to DC indicating the anti-hyperglycemic activity (p < 0.05). The methanol extract of S. wightiana also decreased the serum total cholesterol levels compared to the DC group in the ascending order as SW-200 mg/Kg (158.46 ± 5.6 mg/dl) and SW-400 mg/Kg (138.46 ± 4.6 mg/dl) (p < 0.05). In case of isolated compounds, the order follows as rutin (5 mg/Kg) (148.32 ± 5.3 mg/dl) < qurecetin (5 mg/Kg) (144.38 ± 4.1 mg/dl) (p < 0.05).

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Fig. 1

Effect of methanolic extract and isolated compound on total cholesterol level in alloxan-induced diabetic rats. All values are presented as mean ± SEM, n = 3. Data was analysed by ANOVA followed by Tukey’s multiple comparisons' test; ^ap < 0.001 compared' to NC and ^bp < 0.001 compared to DC.

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3.7.2 Effect on high density lipoprotein (HDL) cholesterol

The effect of alloxan (150 mg/kg, i.p.) administration on HDL levels in DC produced significantly lower levels of HDL cholesterol in serum when compared to the normal control group (p < 0.05). The effect of methanol extract and isolated compounds of S. wightiana on HDL levels in alloxan induced diabetic albino rats is shown in (Fig. 2). The result reveals that alloxan induced DC group rats shows decrease in HDL level (30.0 ± 1.9 mg/dl) compared to NC group (36.83 ± 2.5 mg/dl) given only vehicle. However, the standard glibenclamide (36.73 ± 1.5 mg/dl) significantly increased the HDL levels compared to DC indicating the anti-hyperglycemic activity (p < 0.05). From both the methanol extracts of S. wightiana, highly significant activity was observed in SW-200 mg/Kg (36.63 ± 2.1 mg/dl) (p < 0.05), while in case of isolated compounds, highly significant increase in serum HDL level was observed by qurecetin (5 mg/Kg) (39.71 ± 1.8 mg/dl) (p < 0.05).

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Fig. 2

Effect of methanolic extract and isolated compound on HDL level in alloxan-induced diabetic rats. All values are presented as mean ± SEM, n = 3. Data was analysed by ANOVA followed by Tukey’s multiple comparisons' test; ^ap < 0.05 compared to NC, ^bp < 0.05 compared to DC, ^cp < 0.05′ compared to alloxan + glibenclamide (5 mg/kg), ^dp < 0.05 compared' to alloxan + SW (200 mg/kg), ^ep < 0.05 compared to alloxan + SW (400 mg/kg).

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3.7.3 Effect on low density lipoprotein (LDL) cholesterol

After 14 days of treatment, the effect of methanol extract and isolated compounds of S. wightiana on LDL levels in alloxan-induced diabetic albino rats is shown in Fig. 3. The result showed that alloxan induced DC group in rats shows increase in LDL levels (189 ± 12.4 mg/dl) compared to the NC (91.32 ± 1.2 mg/dl) given only vehicle. However, the standard glibenclamide (92.35 ± 3.1 mg/dl) significantly decreased LDL levels compared to DC indicating the anti-hyperglycemic activity (p < 0.05). The methanol extracts of S. wightiana showed highly significant anti-hyperglycemic activity (p < 0.05). From the methanolic extracts, alloxan/SW-400 mg/Kg (89.55 ± 2.5 mg/dl) showed highly significant decrease in serum LDL level (p < 0.05). Thus, authenticating the traditional claim of the studied crude drug as potent anti-hyperglycemic agent.

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Fig. 3

Effect of methanolic extract and isolated compound on LDL level in alloxan-induced diabetic rats. All values are presented as mean ± SEM, n = 3. Data was analysed by ANOVA followed by Tukey’s multiple comparisons' test; ^ap < 0.001 compared to NC, ^bp < 0.001 compared to DC, ^cp < 0.001 compared to alloxan + glibenclamide (5 mg/kg), ^dp < 0.001 compared to alloxan + SW (200 mg/kg), ^ep < 0.001 compared to alloxan + SW (400 mg/kg), ^fp < 0.001 compared to alloxan + qurecetin (5 mg/Kg).

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3.7.4 Effect on creatinine levels

After 14 days of treatment, the effect of methanol extract and isolated compounds of S. wightiana on creatinine levels in alloxan-induced diabetic rats is shown in Fig. 4. The result showed that alloxan induced DC group shows increase in creatinine levels (2.4 ± 0.1 mg/dl) compared to the NC group (0.54 ± 0.3 mg/dl) given only vehicle. However, the standard glibenclamide (0.58 ± 0.1 mg/dl) significantly decreased creatinine levels compared to DC indicating the anti-hyperglycemic activity (p < 0.05). The methanol extracts of S. wightiana showed highly significant anti-hyperglycemic activity (p < 0.05). Thus, authenticating the traditional claim of the studied crude drug as potent anti-hyperglycemic agents.

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Fig. 4

Effect of methanolic extract and isolated compound on creatinine level in alloxan-induced diabetic rats. All values are presented as mean ± SEM, n = 3. Data was analysed by ANOVA followed by Tukey’s multiple comparisons' test; ^ap < 0.001 compared to NC and ^bp < 0.001 compared to DC.

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3.7.5 Effect on urea levels

After 14 days of treatment, the effect of methanol extract and isolated compounds of S. wightiana on urea levels in alloxan-induced diabetic rats is shown in Fig. 5. The result showed that alloxan induced DC group shows increase in urea levels (62.6 ± 1.8 mg/dl) compared to the NC group (31.83 ± 2.2 mg/dl) given only vehicle. However, the standard glibenclamide (31.24 ± 4.0 mg/dl) significantly decreased urea levels compared to DC indicating the anti-hyperglycemic activity (p < 0.05). S. wightiana methanol extracts demonstrated highly significant anti-hyperglycemic activity (p < 0.05). From the methanolic extracts of S. wightiana at a dose of alloxan/SW-400 mg/Kg (30.33 ± 1.2 mg/dl) showed highly significant decrease in serum urea level (p < 0.05). Thus, authenticating the traditional claim of the studied crude drug as potent anti-hyperglycemic agent.

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Fig. 5

Effect of methanolic extract and isolated compound on urea level in alloxan-induced diabetic rats. All values are presented as mean ± SEM, n = 3. Data was analysed by ANOVA followed by Tukey’s multiple comparisons' test; ^ap < 0.001 compared to NC, and ^bp < 0.001 compared to DC.

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3.7.6 Effect on ALP levels

After 14 days of treatment, the effect of methanol extract and isolated compounds of S. wightiana on serum ALP levels in alloxan-induced diabetic rats is shown in Fig. 6. The result showed that alloxan induced DC rats shows increase in serum ALP levels (276 ± 3.6 mg/dl) compared to the NC group (120 ± 3.2 mg/dl) which were given only vehicle. However, the standard glibenclamide (130.75 ± 2.9 mg/dl) significantly decreased ALP levels compared to DC indicating the anti-hyperglycemic activity (p < 0.05). The methanol extracts of S. wightiana showed highly significant anti-hyperglycemic activity (p < 0.05). From the methanolic extracts of S. wightiana, a dose of alloxan/SW-400 mg/Kg (111.55 ± 6.9 mg/dl) also decreased significantly the serum ALP level (p < 0.05). Thus, authenticating the traditional claim of the studied crude drug as potent anti-hyperglycemic agent.

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Fig. 6

Effect of methanolic extract and isolated compound on ALP level in alloxan-induced diabetic rats. All values are presented as mean ± SEM, n = 3. Data was analysed by ANOVA followed by Tukey’s multiple comparisons' test; ^ap < 0.001 compared to NC and ^bp < 0.05 compared to DC.

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3.7.7 Anti-Hyperglycemic activity

The purpose of this study was to investigate the efficacy of a methanolic extract of S. wightiana and its isolated compounds against alloxan-induced diabetes, as well as their effect in a glucose tolerance test (GTT). The activity was found dose dependent in the extracts of both the plants while isolated compound at dose level of 5 mg/kg showed the protection and antidiabetic potential as compared to the standard glibenclamide (5 mg/kg). Therefore, both the plants have potential antidiabetic activity.

3.7.8 Effect of glucose tolerance test

The data shown in (Fig. 7 A-D) indicates that S. wightiana and isolated compounds remained persistently euglycemic throughout the experimental period. The initial plasma glucose level in DC group was greater compared to methanolic extract of S. wightiana, and isolated compounds treated groups. During the experimental period in DC group, the glucose level was elevated from 67 to 98 mg/dl. Whereas, in the methanolic extract of S. wightiana (glucose + SW 400 mg/kg, p.o) group, the elevated glucose level was gradually decreased from 67 to 60 mg/dl. Whereas, in the DC group treated with S. wightiana, a significant antihyperglycemic effect was evident from the 7th day, and the decrease in glucose was maximum and reached near normal levels by the 14th day of treatment (p < 0.05).

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Fig. 7

Graphical representation of effect of methanolic extract of S. wightiana on glucose level at (A): 0 min; (B): 30 mins; (C): 90 mins; (D): 150 mins in alloxan induced diabetic rats after 14 days of treatment. All values are presented as mean ± SEM, n = 3. Data was analysed by ANOVA; all groups were compared with the NC group followed by Tukey’s test. No significance difference was found (A); *p < 0.05; **p < 0.01; ***p < 0.001 (B); *p < 0.05; ***p < 0.001 (C); *p < 0.05; ***p < 0.001 (D).

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Chronic treatment of diabetic rats with methanolic extract of S. wightiana for 14 days reduced plasma glucose levels to near normal levels (Fig. 8). The plasma glucose levels in the NC and DC groups during the experiment clearly show that studied crude drug had antihyperglycemic activity.

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Fig. 8

Graphical representation of effect of methanolic extract/isolated compounds of S. wightiana on glucose level on 1st, 7th^, and 14th day in alloxan induced diabetic rats after 14 days of treatment.

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3.7.9 Effect of glucose on body weight

The data given in Fig. 9 revealed a gradual increase in the body weight of DC rats at 1, 7 and 14 days of experimental period when compared to corresponding initial weights. Thus, the results revealed the beneficial effect of methanolic extract of S. wightiana and isolated compounds treatment against the weight loss observed under diabetic conditions.

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Fig. 9

Graphical representation of effect of methanolic extract/isolated compounds of S. wightiana on glucose level on 1st, 7th and 14th day in alloxan induced diabetic rats after 14 days of treatment.

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