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BY 4.0 license Open Access Published by De Gruyter Open Access May 23, 2023

Anti-diabetic potentials of Sorbaria tomentosa Lindl. Rehder: Phytochemistry (GC-MS analysis), α-amylase, α-glucosidase inhibitory, in vivo hypoglycemic, and biochemical analysis

  • Falak Naz , Muhammad Zahoor EMAIL logo , Muhammad Ayaz EMAIL logo , Muhammad Ashraf , Asif Nawaz and Amal Alotaibi
From the journal Open Chemistry

Abstract

This study aimed to examine the anti-diabetic effects of an unexplored medical plant Sorbaria tomentosa Lindl. Rehder using in vitro and in vivo approaches. The extracts were tested as inhibitors of α-glucosidase and α-amylase following standard protocols. Methanolic extract was analyzed via gas chromatography-mass spectrometry (GC-MS) for tentative identification of the secondary metabolites. GC-MS analysis revealed the presence of several compounds. α-Amylase was more potently inhibited by chloroform and methanolic extracts (27 and 40 µg mL−1, respectively), whereas α-glucosidase was more potently inhibited by methanolic extract (IC50 = 530 µg mL−1). Methanolic extract was also subjected to in vivo studies using an alloxan-induced diabetes rat model. Diabetic animals treated with 150 mg kg−1 body weight dose of methanolic extract cause a steady decrease in blood glucose levels (529.16, 446.66, 348.00, 269.33, and 165.5 mg dL−1, respectively, on days 1, 7, 14, 21, and 28). At 300 mg kg−1 dose, the blood glucose level was decreased to 111.83 mg dL−1 on day 28. Blood biochemistry results indicated that treatment with methanolic extract normalized the elevated parameters including cholesterol, triglycerides, low-density lipoprotein, serum creatinine, blood urea, uric acid, serum glutamate pyruvate transaminase, bilirubin, alkaline phosphatase, and aspartate aminotransferase in diabetic animals.

1 Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder of insulin insufficiency due to a gradual decrease in its production or tissue resistance to its action [1,2]. The deficiency of insulin leads to an increase in the concentration of blood glucose level, which gradually compromises the physiology of vital organs [3]. The disease has a high global prevalence with about 422 million people affected, and is expected to reach 492 million by 2035. Presently, DM has been reported to affect about 8.5% of adults and the number of cases is rapidly increasing, especially among low- and middle-income countries [4].

There are two categories of DM, type 1 DM (insulin-dependent DM) and type 2 DM (noninsulin-dependent DM). Among all individuals with DM, 95% of individuals have type 2 DM [5]. The characteristics of type 1 DM is absolute deficiency of insulin due to the destruction of pancreatic β cells [6], while type 2 DM is caused by a gradual decrease in the secretion of insulin as well as tissue resistance to its action [7]. Persistent high levels of blood sugar if not controlled can cause injury to various organs and long-term complications that include nephropathy, retinopathy, neuropathy as well as cerebrovascular and cardiovascular complications [8,9]. In individuals with impaired glucose tolerance, the development of DM can be delayed or prevented through lifestyle modifications including weight loss, dietary restrictions, and the use of some anti-diabetic agents [10]. But the use of anti-diabetic drugs is associated with some undesirable side effects like fluid retention, hypoglycemia, heart failure, and osteoporosis [1113]. Therefore, the development of new anti-diabetic drugs is required to control hyperglycemia with fewer side effects.

For thousands of years, natural products and their extracts have been utilized for the treatment of various diseases in humans. These agents have more proven efficacy, fewer side effects, and low cost [1416]. Therefore, research on these natural traditional medicinal plants is of great importance for the development of new drugs. A large number of bioactive agents derived from plants revealed anti-diabetic potentials [17,18]. The principal active constituents of these medicinal plants include alkaloids, glycosides, steroids, terpenoids, carbohydrates, galactomannan gum, amino acids, polysaccharides, glycopeptides, peptidoglycan, inorganic ions, guanidine, and hypoglycans [19]. Various metabolic cascades are affected by these active constituents, thus affecting the level of blood glucose in the human body.

The plant of Sorbaria tomentosa Lindl. Rehder belongs to the Rosaceae family. In Pakistan, it is present in Swat and Murree Valley; locally, it is called as Kathi or Bakre Jar [20,21]. The plant is a shrub and is 1.5–3 m tall. The leaves are short-petiolate and 1–8 cm long and 2–4 cm wide. The buds are 2 mm long and ovoid, obtuse, and brown [22]. The flowering season is from July to September. The flowers are many and small, which are 25 cm long, 4 mm in diameter, and 7 cm wide. The color of the petal is pale pink to rose-purple and has pubescent calyx. The fruit comprises five hairy follicles, each having 2–10 winged seeds [23]. S. tomentosa are used traditionally for the treatment of lung infection, asthma, newborn skin rashes, bronchitis, diarrhea, indigestion, and rheumatism and also as anti-inflammatory, antifungal, antitumor, and antiseptic agent [24]. The plant is not scientifically tested for various diseases and thus the current project was designed to assess the plant phytochemistry and evaluate it for efficacy against type 2 DM using in vitro and in vivo approaches.

2 Materials and methods

2.1 Plant collection, recognition, and processing

Aerial plant parts of S. tomentosa at the flowering stage were collected from Murree, Pakistan, and were verified by Dr. Ali Hazrat, a botanical taxonomist at the Botany Department, University of Malakand. The plant was dried in the shade, and a voucher sample at the herbarium of the University of Malakand was deposited with voucher number I.UOM. St.112.

2.1.1 Extraction

The shade-dried plant was cut down into small pieces and subsequently it was crudely crushed to a powder form. The powdered plant material was then soaked in 80% methanol in a stainless-steel container for 10–14 days with occasional shaking to completely dissolve soluble phytochemicals. The solvent was filtered through muslin cloth and then through the filter paper [25]. After filtration, the filtrate was evaporated at 40°C under low pressure, utilizing a rotary evaporator. Finally, a reddish-black semisolid mass of methanolic extract was obtained. A dried mass of the crude methanolic extract was processed for fractionation.

2.1.2 Fractionation

The crude methanolic extract obtained from S. tomentosa was subjected to the fractionation process using a separating funnel. Briefly, 500 mL of distilled water was used for dilution of the crude extract followed by the addition of 500 mL of n-hexane. The mixture was stirred vigorously and then allowed to stand for some time to form two layers. The n-hexane layer was isolated from the mixture by the addition of 500 mL of n-hexane, and the same process was repeated three times. The n-hexane extracts were merged and concentrated at reduced pressure with the help of a rotary evaporator; finally, the concentrated extract of n-hexane was obtained. The above-mentioned protocol was sequentially followed for the fractionation using various polarity solvents like ethyl acetate, chloroform, n-butanol, and aqueous fraction using a separating funnel. All sub-fractions/extracts were collected in separate containers and finally, the solvents were removed using a rotary evaporator.

2.2 GC-MS-based analysis of phytochemicals

The methanolic plant extract prepared was analyzed using GC-MS. About 2 µL of this solution was used for analysis using a GC-MS analyzer (GC Clarius 500 Perkin Elmer). The data were received on an Elite-1 (100% dimethyl polysiloxane) column (30 × 0.25 mm 1 µm df). The carrier gas used was helium (99.99%) having a flow rate of 1 mL min−1 in a split mode (10:1). Subsequently, 2 µL of the ethanolic sample solution was introduced into the column by an injector at a temperature of 250°C. The GC oven was started at a temperature at 110°C and maintained for 2 min, then it was increased at a rate of 10°C min−1 up to the temperature of 200°C, without holding. Holding was permitted at a temperature of 280°C for 9 min with 5°C min−1 of the program rate. The temperature of the injector was adjusted to 250°C and that of the detector was adjusted to 280°C. The ion source temperature was maintained at 200°C. An electron ionization of 70 eV was used to obtain the mass spectrum of compounds present in samples, and the detector was operated in a scan mode from 45 to 450 amu (atomic mass units). A scan interval of 0.5 s was maintained, and the mass of the fragments ranged from 45 to 450 Da; the total running time was 36 min [26].

2.3 Identification of components

The identification of components was done based on the molecular mass, molecular structure, and calculated fragments. The interpretation of the mass spectrum of GC-MS was performed using the National Institute of Standards and Technology (NIST) database which has more than 62,000 patterns. The name, molecular weight, and structure of the test material components were determined. The average peak area was compared to the total area to calculate the relative percentage quantity of each component. However, the unknown component spectrum was compared with the spectrum of the component saved in the NIST library version (2005), software, Turbomas 5.2. This was performed to find whether this plant extract contained a single compound or group of compounds, which may confirm its present commercial as well as its traditional use as an herbal medicine. Along with this, it can play a significant role in determining the most suitable techniques for the isolation of these compounds [26].

2.4 In vitro anti-diabetic studies

2.4.1 Inhibition of α-amylase enzymes

The test sample and the standard drug (total 500 µL) having a concentration of 100–1,000 mcg mL−1 were added to a tube containing 0.20 mM phosphate buffer (500 µL) at pH 6.9 and a solution of α-amylase (concentration, 0.5 mg mL−1). The solution was incubated for 10 min at 25°C. Then, a solution of 1% starch (500 µL) prepared in 0.02 M sodium phosphate buffer was added to each tube. Then, the reaction was stopped by adding 1 mL of 3,5-dinitro salicylic acid reagent. All of the test tubes were then incubated for 5 min in a boiling water bath before being cooled to room temperature. The resulting mixture was further diluted with 10 mL of distilled water. The absorbance was measured at 540 nm. The percent enzyme inhibition was determined as

% Inhibition = 1 Δ A Samples Δ A Control × 100 .

2.4.2 Inhibition of α-glucosidase enzymes

To determine the enzyme inhibitory activity, various concentrations of plant samples were mixed with 50 µL of enzyme solutions (0.5 U mL−1) in phosphate buffer; the pH was adjusted to 6.8. The solution mixture was mixed and incubated for 15 min at 37°C. Thereafter, a 3 mM pNPG solution (100 µL) was added to it and was incubated for 10 min at 37°C. A 0.1 M Na2CO3 solution was finally added to stop the reaction. Absorption was observed at 405 nm, and percent inhibition was calculated as

% Inhibition = 1 Absorbance of s ample Abosrbance of b lank Abosrbance of control × 100 .

2.5 In vivo anti-diabetic activities

2.5.1 Experimental animals

Animals used in this study were rats weighing 145–180 g with an average weight of 162.5 g. The animals were placed in an animal house under standard laboratory conditions of 25°C temperature and 12 h light and dark with the supply of water ad libitum during the whole experiment period.

2.5.2 Ethical committee approval

The animal work of this project was examined and approved by the Departmental Research Ethics Committee at the Department of Pharmacy, University of Malakand, under reference number DREC/ST/Diabetes/Pharm-2020.

2.5.3 Acute toxicity studies

Acute toxicity studies of solvent extracts were carried out in experimental animals. Animals were given higher dosages of crude extracts orally, and they were monitored for 24 h for any abnormal behavior, such as convulsions, respiratory distress, or changes in reflex activities [27].

2.6 Diabetes induction

Experimentally, i/p alloxan monohydrate (10%) was administered at a dose of 160 mg kg−1 body weight to cause hyperglycemia in mice. Alloxan monohydrate was obtained from Sigma Aldrich (Steinhein, Switzerland) [28]. The animals were kept on a fasting mode for 8–12 h but were allowed freely to water before bioassay. The blood glucose level was recorded after 48 h of alloxan administration using a glucometer. Only those rats were considered diabetic whose blood glucose levels measured above 200 mg dL−1 and were further selected for the study [29].

2.7 Experimental design

The anti-diabetic potential of S. tomentosa in alloxan-induced diabetic rats was studied. All animals were grouped into four groups having six rats in each group.

Group I: Control (normal/non-diabetic) group received i/p only normal saline.

Group II: Diabetic group received i/p alloxan and Tween 80.

Group III: Treatment group received po glibenclamide (5 mg kg−1).

Group IV: Treatment group received i/p test compounds (150 and 300 mg kg−1).

2.8 Oral glucose tolerance test (OGTT)

An OGTT, after diabetes induction, was performed in order to confirm hyperglycemia. Briefly, pre-confirmed diabetic animals (control and treated animal) were kept on fasting overnight, and an oral glucose dose of 3 g kg−1 was administered to each animal. Using heparin capillary tubes, blood samples were collected and the subsequent blood glucose level was monitored 30 min before the test and at 30, 60, and 120 min intervals after administration of glucose.

2.9 Sampling of blood and in vivo hypoglycemic assay

The tails of the animals were sterilized, and then the tail was gently “milked” from the body towards the tip; then, blood was obtained from the tail by nipping to begin bleeding. Repeated blood sampling was done at 1, 2, 3, 4, 7, and 24 h post-administration using a glucometer, and the blood glucose level was noted. The tails of the rats were sterilized after the operation using a 70% ethanol swab [29].

2.10 Biochemical analysis of blood samples

Under a light anesthetic, blood samples were collected using capillary tubes from the retro-orbital plexus of each animal, and biochemical tests were performed.

2.10.1 Lipid profile

Serum triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and serum cholesterol were analyzed using standard methods. Briefly, 10 mL of serum sample and 1,000 mL of the triglyceride solution were mixed in a tube and incubated at 37°C for 10 min. Then, absorbances were measured at 546 nm. Furthermore, diagnostic kits were used for monitoring blood cholesterol following the manufacturer’s specifications. In brief, 1,000 µL of cholesterol solution was mixed with 10 µL of the serum sample and incubated at 37°C for 10 min. The absorbance at 546 nm against the blank was noted. The concentration of HDL was determined by mixing 500 µL of the HDL solution with 200 µL of serum sample and then allowed to stand at room temperature for 5 min. The resulting solution was mixed again and subjected to centrifugation for 5 min, and the supernatant was collected. Then, 500 µL of cholesterol was mixed with 50 µL of the HDL supernatant and incubated at 37°C for 5 min. After incubation, the absorbance of the sample was measured at a wavelength of 546 nm.

In all groups, LDL was also quantified following previously reported protocols and specifications of manufacturers:

LDL = Total cholesterol + HDL Triglycerides/5 .

2.10.2 Renal function tests (RFTs)

RFTs involve determining both serum creatinine and blood urea. The serum creatinine test was handled very carefully because it is a highly temperature-sensitive reaction. To start, 50 µL of the serum was added to 500 µL of the reagent and mixed. It was then incubated at 37°C for 1 min and then the absorbance was recorded at 500 nm. Similarly, blood urea was also determined. Briefly, 1,000 µL of enzyme reagent 1 was added to serum (10 µL) and incubated at 25°C for 5 min. Then, 1,000 µL of reagent 2 was added to it and the absorbance of the sample against the reagent blank was observed after 5 min at 578 nm.

2.10.3 Liver function tests (LFTs)

LFTs involved determining alkaline phosphatase (ALP), serum glutamate pyruvate transaminase (SGPT)/ALT, and bilirubin following the standard protocols using Tecno plus 786 biochemistry analyzer and Micro lab 300 biochemistry analyzers. For determining SGPT/ALT, the serum sample (50 µL) was added to 500 µL of the reagent (R1, 400 µL; R2, 100 µL) and the mixture was incubated for 30 s at 37°C. The absorbance was recorded at 340 nm. Similarly, ALP was determined using a kit, following the specifications of the manufacturer. Briefly, 10 µL of the serum sample and 500 µL of the reagent (R1, 400 µL; R2, 100 µL) were mixed and incubated at 37°C for 30 s. The absorbance was noted at 405 nm. Moreover, the bilirubin concentration was determined following the standard protocols. Four different reagents were utilized including 100 µL of R1 to which 25 µL of R2 was added; then, 100 µL of the serum sample and 500 µL of R3 were mixed. The solution, after mixing, was allowed to stand for 5 min at 25°C. Finally, R4 (500 µL) was added to it and incubated at 25°C for 5 min. After 5 min, the sample absorbance was measured at a wavelength of 546 nm.

2.11 Data analysis

One-way ANOVA and post-ANOVA (Tukey’s post hoc test) were used to compare the mean of the untreated group of rats with the diabetic groups of rats treated with plant extracts. The findings of the statistical analysis were shown as mean ± SEM. Statistical significance was defined as P ≤ 0.05.

3 Results

3.1 GC-MS analysis

In GC-MS analysis of various samples of S. tomentosa, about 35 compounds were recognized by the mass spectra produced by GC-MS. The various compounds present in various samples of S. tomentosa were found by GC-MS, as shown in Figures 1 and 2. The most abundant compounds were oleic acid (20.52%), α-sitosterol (10.44%), 8-octadecenoic acid, methyl ester, (E) (9.11%), trichothec-9-en-8-one, 4-(acetyloxy)-12,13-epoxy-3,7,15-trihy droxy- (3α,4α,7α), (7.57%), phthalic acid, 6-ethyloct-3-yl 2-ethylhexyl ester (7.55%), 9,12,15-octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z) (7.44%), lupeol (6.02%), α-d-galactopyranose, 6-O-(trimethylsilyl)-cyclic 1,2:3,4-bis(butylboronate) (5.15%), and α-sitosterol trimethylsilyl ether (5.06%). Apart from these compounds, a number of other compounds having area % of less than 5 were also identified.

Figure 1 
                  GC-MS chromatogram of the crude methanolic extract of S. tomentosa.
Figure 1

GC-MS chromatogram of the crude methanolic extract of S. tomentosa.

Figure 2 
                  Major compounds identified in GC-MS of S. tomentosa.
Figure 2

Major compounds identified in GC-MS of S. tomentosa.

3.2 Inhibition study of α-amylase

The results of inhibitory studies of α-amylase are shown in Figure 3. The most potent extract was chloroform, which exhibited an inhibition of 84.4 ± 1.12% of the enzyme at a concentration of 1,000 µg mL−1. The chloroform extract showed inhibitions of 82.5 ± 0.76, 77.2 ± 0.9, 66.3 ± 1.32, and 58.2 ± 1.12% at concentrations of 500, 250, 125, and 62.5 µg mL−1, respectively, with an IC50 of 27 µg mL−1. The positive control (acarbose) demonstrated an IC50 of 7.6 µg mL−1 at the same tested concentrations. The methanolic extract was the second most active sample exhibiting inhibitions of 81.23 ± 0.88, 78.45 ± 0.98, 71.5 ± 1.76, 66.6 ± 1.43, and 53.2 ± 1.32% at concentrations of 500, 250, 125, and 62.5 µg mL−1, respectively, with an IC50 of 40 µg mL−1. Among the other extracts, n-hexane showed inhibitions of 51.34 ± 1.55, 48.3 ± 1.43, 46.4 ± 1.76, 45.23 ± 1.76, and 42.2 ± 1.13%, whereas ethyl acetate showed inhibitions of 51.7 ± 1.98, 49.4 ± 1.87, 47.6 ± 1.56, 46.2 ± 1.65, and 44.2 ± 1.43%, respectively, at the same tested concentrations. The IC50 values for n-hexane and ethyl acetate extracts were 843 and 576 µg mL−1, respectively.

Figure 3 
                  Inhibitory potentials of α-amylase in various solvent extracts and standard drugs. Statistical significance was set as p < 0.05. * p < 0.01 and *** p < 0.001.
Figure 3

Inhibitory potentials of α-amylase in various solvent extracts and standard drugs. Statistical significance was set as p < 0.05. * p < 0.01 and *** p < 0.001.

3.3 Inhibition study of α- glucosidase

In the inhibition study of α-glucosidase, the crude methanolic extract exhibited concentration-dependent moderate enzyme inhibitory potentials. The methanolic extract at concentrations of 1,000, 500, 250, 125, and 62.5 µg mL−1 showed inhibitions of 60.29, 52.33,48.00, 34.66, and 18%, respectively. The IC50 for the crude methanolic extract was 530 µg mL−1 (Figure 4). The standard drug acarbose revealed enzyme inhibitions of 90.21, 85.94, 82.11, 79.75, and 67.7% at the same tested concentrations, respectively, with an IC50 value of 20 µg mL−1. Further, α-glucosidase inhibitions were 49.79, 44.5, 32.33, 30, and 17% for the n-hexane extract at concentrations of 1,000, 500, 250, 125, and 62.5 µg mL−1, respectively, with an IC50 of >1,500 µg mL−1. Among other samples, ethyl acetate showed inhibitions of 55.14, 52.40, 47.25, 45.23, and 41.64%, respectively, at the same tested concentrations; whereas the chloroform extract showed inhibitions of 56.58, 44.66, 31.64, and 4.25% at the same concentrations, respectively. The IC50 values for ethyl acetate and chloroform extracts were 830 and >1,500 µg mL−1, respectively (Figure 4).

Figure 4 
                  Inhibitory potentials of α-glucosidase of the crude sample from Sorbaria tomentosa. Statistical significance was set as p < 0.05. *** p < 0.001.
Figure 4

Inhibitory potentials of α-glucosidase of the crude sample from Sorbaria tomentosa. Statistical significance was set as p < 0.05. *** p < 0.001.

3.4 In vivo studies

3.4.1 Acute toxicity study

No morbidity or mortality was observed. Likewise, no aberrant behavior was observed during the acute toxicity studies at the tested doses.

3.4.2 In vivo anti-hyperglycemic effect

In the diabetes group maintained on a normal diet without treatment, a hyperglycemic state was observed with blood glucose levels of 527.33, 545.33, 523.16, 535.83, and 533.16 mg dL−1 at days 1, 7, 14, 21, and 28, respectively (Figure 5, diabetes group). In normal group animals, average blood glucose levels were 107.16, 105.16, 115, 114.66, and 114.16 mg dL−1 on days 1, 7, 14, 21, and 2, respectively. Diabetic animals treated with the standard drug exhibited a gradual decrease in blood glucose during therapy. The blood glucose level of diabetic animals treated with the standard drug was 516.16 mg dL−1 on day 1, 413.66 mg dL−1 on day 7, 305.16 mg dL−1 on day 14, 205.16 mg dL−1 on day 21 and 110.33 mg dL−1 on day 28 of treatment (Figure 5, standard group). The treatment of diabetic animals with 150 mg kg−1 dose of crude methanolic extract also showed a steady decrease in blood glucose levels. On day 1, the blood glucose level was 529.16 mg dL−1, with a gradual decrease on day 7 (446.66 mg dL−1), on day 14 (348.00 mg dL−1), on day 21 (269.33 mg dL−1), and on day 28 (165.5 mg dL−1), respectively. Furthermore, the crude methanolic extract at a dose of 300 mg kg−1 exhibited blood glucose levels of 528.33, 44.83, 342.66, 204.66, and 111.83 mg dL−1 on days 1, 7,14, 21, and 28, respectively (Figure 5).

Figure 5 
                     Results of in vivo anti-diabetic potentials of S. tomentosa.
Figure 5

Results of in vivo anti-diabetic potentials of S. tomentosa.

3.4.3 Changes in the weight of vital organs

Table 1 shows the weight variations of the vital organs in several groups of animals. In normal group animals, the average weights of the liver, heart, pancreas, and kidney were 5.02 ± 0.08, 0.77 ± 0.05, 0.54 ± 0.05, and 0.52 ± 0.02 g, respectively. However, in untreated diabetic animals, the average weights of the liver, heart, pancreas, and kidney were 4.9 ± 0.23, 0.685 ± 0.05, 0.49 ± 0.06, and 0.48 ± 0.01 g, respectively. Among diabetic animals treated, the weights of these vital organs with the positive control drug were 5.31 ± 0.09 g (liver), 0.77 ± 0.03 g (heart) 0.63 ± 0.03 g (pancreas), and 1.06 ± 0.07 g (kidney). The average weights of the liver, heart, pancreas, and kidney in diabetic mice treated with methanolic extract at 150 mg dose were 5.52 ± 0.16, 0.55 ± 0.01, 0.59 ± 0.05, and 0.68 ± 0.06 g, respectively. The same pattern was recorded for the animal groups treated with the methanolic extract (300 mg kg−1). Overall, no major changes were recorded during therapy in the weight of these organs.

Table 1

Effects of the crude extract treatment on the weights (g) of vital organs in fasting rats

Organ Animal number and organ weight Average SD SEM
Normal animal group
Liver 4.775 5.331 4.876 5.231 4.956 4.988 5.02 0.21 0.08
Kidney 0.525 0.542 0.601 0.432 0.544 0.532 0.52 0.05 0.02
Pancreas 0.451 0.782 0.643 0.432 0.511 0.444 0.54 0.14 0.05
Heart 0.831 0.675 0.885 0.932 0.634 0.673 0.77 0.12 0.05
Diabetic animal group
Liver 5.729 4.765 4.453 4.211 5.453 4.906 4.91 0.58 0.23
Kidney 0.512 0.433 0.466 0.489 0.499 0.501 0.48 0.02 0.01
Pancreas 0.662 0.342 0.324 0.546 0.425 0.648 0.49 0.14 0.06
Heart 0.655 0.643 0.832 0.745 0.433 0.802 0.685 0.14 0.05
Standard drug-treated diabetic animal group
Liver 5.445 5.654 5.23 5.432 5.124 4.987 5.31 0.24 0.09
Kidney 1.222 0.998 1.332 0.845 0.988 1.006 1.06 0.17 0.07
Pancreas 0.546 0.656 0.723 0.648 0.535 0.712 0.63 0.08 0.03
Heart 0.845 0.752 0.876 0.771 0.665 0.721 0.77 0.07 0.03
Methanol extract (150 mg kg −1 )-treated diabetic animal group
Liver 5.975 5.729 5.136 5.344 5.922 5.015 5.52 0.41 0.16
Kidney 0.626 0.507 0.711 0.654 0.631 0.998 0.68 0.16 0.06
Pancreas 0.452 0.692 0.543 0.664 0.435 0.765 0.59 0.13 0.05
Heart 0.54 0.517 0.564 0.533 0.567 0.621 0.55 0.03 0.01
Methanol extract (300 mg kg −1 )-treated diabetic animal group
Liver 5.842 5.729 5.876 5.432 5.988 5.754 5.77 0.18 0.07
Kidney 0.532 0.511 0.567 0.655 0.588 0.798 0.60 0.10 0.04
Pancreas 0.512 0.711 0.822 0.688 0.932 0.675 0.72 0.14 0.05
Heart 0.61 0.613 0.566 0.597 0.654 0.543 0.59 0.03 0.01

3.5 Analysis of blood biochemistry

3.5.1 Lipid profile

The results of the lipid profile among different groups of animals are summarized in Table 2. Major blood biomarkers of the lipid profile including cholesterol, triglycerides, HDL, and LDL were determined for comparison among different groups. In normal group animals, the average cholesterol was 68.83 ± 6.04 mg dL−1, whereas its concentrations among other groups were 208.83 ± 6.00 mg dL−1 (diabetic untreated), 79.16 ± 3.60 mg dL−1 (diabetic animals treated with positive control), 86.66 ± 5.57 mg dL−1 (diabetic animals treated with 150 mg kg−1 of crude methanolic extract), and 80.16 ± 8.83 mg dL−1 (diabetic animals treated with 300 mg kg−1 of crude methanolic extract). So, the test samples showed a decrease in the cholesterol level and it was comparable with the standard drug. Likewise, the average triglyceride concentrations were 96.66 ± 4.97 mg dL−1 in normal animals, 281.50 ± 35.78 mg dL−1 in untreated diabetic animals, 94.66 ± 6.49 mg dL−1 in diabetic animals treated with the standard drug, whereas it was decreased to 115.16 ± 8.88and 102.83 ± 2.53 mg dL−1 among the diabetic animals treated with 150 and 300 mg kg−1 of methanolic extract, respectively. Among the other parameters, the normal group animals exhibited 28.66 ± 1.42 mg dL−1 (HDL) and 22.16 ± 3.76 mg dL−1 (LDL), respectively, whereas the average HDL (41.50 ± 3.25 mg dL−1) and LDL (108.66 ± 7.74 mg dL−1) were quite elevated among the diabetic untreated group animals. In diabetic animals treated with the standard drug, the HDL was 34.16 ± 2.25 mg dL−1 and LDL was 25.16 ± 3.47 mg dL−1. Diabetic animals treated with 150 and 300 mg kg−1 of crude methanolic extract normalized the HDL and LDL profile (150 mg kg−1: HDL = 32.83 ± 1.60 mg dL−1, LDL = 31.66 ± 9.39 mg dL−1) and (300 mg kg−1: HDL = 34.33 ± 1.90 mg dL−1 and LDL = 22.66 ± 3.95 mg dL−1), respectively (Table 2).

Table 2

Effect of various treatments on the lipid profile among various groups of the treated animals

Test name Animals number and observations Average SD SEM
Normal animal group
Cholesterol (mg dL−1) 94 60 69 54 77 59 68.83 14.79 6.04
Triglycerides (mg dL−1) 113 88 96 79 98 106 96.66 12.19 4.97
HDL (mg dL−1) 33 28 30 23 31 27 28.66 3.50 1.42
LDL (mg dL−1) 39 18 20 16 26 14 22.16 9.21 3.76
Diabetic animal group
Cholesterol (mg dL−1) 204 222 199 231 205 192 208.83 14.71 6.00
Triglycerides (mg dL−1) 416 314 196 277 310 176 281.50 87.65 35.78
HDL (mg dL−1) 38 55 44 42 39 31 41.50 7.96 3.25
LDL (mg dL−1) 82 104 115 140 102 109 108.66 18.97 7.74
Standard drug-treated diabetic animal group
Cholesterol (mg dL−1) 75 79 77 66 88 90 79.16 8.84 3.60
Triglycerides (mg dL−1) 74 98 86 109 85 116 94.66 15.92 6.49
HDL (mg dL−1) 28 28 38 34 42 35 34.16 5.52 2.25
LDL (mg dL−1) 32 31 21 10 26 31 25.16 8.51 3.47
Methanolic extract (150 mg kg −1 )-treated animal group
Cholesterol (mg dL−1) 102 80 65 90 78 69 86.66 13.64 5.57
Triglycerides (mg dL−1) 106 97 89 132 146 121 115.16 21.75 8.88
HDL (mg dL−1) 32 32 37 38 28 30 32.83 3.92 1.60
LDL (mg dL−1) 48 17 14 71 24 16 31.66 23.00 9.39
Methanolic extract (300 mg kg −1 )-treated animal group
Cholesterol (mg dL−1) 95 72 89 77 91 93 80.16 9.38 3.83
Triglycerides (mg dL−1) 103 99 106 93 111 105 102.83 6.21 2.53
HDL (mg dL−1) 30 30 41 39 32 34 34.33 4.67 1.90
LDL (mg dL−1) 39 22 27 21 16 11 22.66 9.68 3.95

3.5.2 Effect on renal function markers

Changes in the markers of kidney function including serum creatinine, blood urea, and uric acid are summarized in Table 3. The average serum creatinine was 0.56 ± 0.04 mg dL−1 among normal animals, whereas it was 2.91 ± 0.43 mg dL−1 among untreated diabetic animals, 0.716 ± 0.07 mg dL−1 among diabetic animals treated with the standard drug, 0.76 ± 0.04 and 0.72 ± 0.04 mg dL−1 for diabetic animals treated with 150 and 300 mg kg−1, respectively, of the plant sample. Blood urea was also quite elevated in untreated diabetic animals (115.33 ± 47.92 mg dL−1) as compared to normal animals (19.83 ± 0.60 mg dL−1). Diabetic animals treated with the standard drug (20.33 ± 1.08 mg dL−1), 150 mg kg−1 of the plant sample (22.16 ± 1.42 mg dL−1), and 1,300 mg kg−1 of the plant sample (21.33 ± 1.22 mg dL−1) cause a considerable decrease in blood urea. Uric acid was not found to be elevated in any of the groups.

Table 3

Effect of various treatments on kidney function markers

Test name Animals number and observations Average SD SEM
Normal animal group
Serum creatinine (mg dL−1) 0.6 0.5 0.7 0.7 0.5 0.4 0.56 0.12 0.04
Blood urea (mg dL−1) 19 22 18 20 21 19 19.83 1.47 0.60
Uric acid (mg dL−1) 2 2.2 2.3 2.7 2.5 2.6 2.38 0.26 0.10
Diabetic animal group
Serum creatinine (mg dL−1) 4.9 2.1 1.9 3.1 2.7 2.8 2.91 1.070 0.43
Blood urea (mg dL−1) 351 57 43 107 66 68 115.33 117.40 47.92
Uric acid (mg dL−1) 4.1 2.8 2.6 3.3 2.4 2.5 2.95 0.64 0.26
Standard drug-treated diabetic animal group
Serum creatinine (mg dL−1) 0.5 0.6 0.9 0.8 0.6 0.9 0.716 0.17 0.07
Blood urea (mg dL−1) 17 23 18 22 19 23 20.33 2.65 1.08
Uric acid (mg dL−1) 2.7 2.3 1.9 2.4 1.8 2.9 2.33 0.43 0.17
Methanolic extract (150 mg kg −1 )-treated animal group
Serum creatinine (mg dL−1) 0.8 0.9 0.8 0.7 0.8 0.6 0.76 0.10 0.04
Blood urea (mg dL−1) 21 27 19 18 25 23 22.16 3.48 1.42
Uric acid (mg dL−1) 2.1 2.9 1.9 2.6 2.9 2.7 2.51 0.42 0.17
Methanolic extract (300 mg kg −1 )-treated animal group
Serum creatinine (mg dL−1) 0.7 0.8 0.8 0.6 0.7 0.7 0.72 0.81 0.04
Blood urea (mg dL−1) 22 24 19 21 25 17 21.33 3.01 1.22
Uric acid (mg dL−1) 2.9 2.6 3.1 2.9 2.7 3.4 2.93 0.28 0.11

3.5.3 Estimation of liver function biomarkers

The results of the effect of various treatments on liver function enzymes including bilirubin, SGPT, ALP, and AST among various animal groups are summarized in Table 4. Serum bilirubin was 0.53 ± 0.04 mg dL−1 for normal, 1.98 ± 0.16 mg dL−1 for untreated diabetic, 0.65 ± 0.07 mg dL−1 for diabetic animals treated with the standard drug, 0.71 ± 0.04 mg dL−1 for 150 mg kg−1 crude extract-treated diabetic animals, and 0.71 ± 0.047 mg dL−1 for diabetic animals treated with 300 mg kg−1 of the plant crude extract.

Table 4

Effect of various treatments on liver enzymes and other biochemical parameters of treated animals

Test name Animals number and observations Average SD SEM
Normal animal group
Bilirubin (mg dL−1) 0.6 0.5 0.7 0.6 0.4 0.4 0.53 0.12 0.04
SGPT (U L−1) 22 18 17 25 14 26 20.33 4.76 1.94
ALP (U L−1) 186 112 104 101 105 125 122.16 32.43 13.23
AST (U L−1) 35 39 32 40 34 44 37.33 4.45 1.81
Diabetic animal group
Bilirubin (mg dL−1) 2.3 1.5 1.8 2.4 1.6 2.3 1.98 0.39 0.16
SGPT (U L−1) 86 61 58 70 48 57 63.33 13.17 5.37
ALP (U L−1) 388 210 234 199 175 197 233.83 77.93 31.81
AST (U L−1) 48 57 65 68 51 62 58.5 7.91 3.23
Standard drug-treated diabetic animal group
Bilirubin (mg dL−1) 0.4 0.7 0.9 0.6 0.5 0.8 0.65 0.18 0.07
SGPT (U L−1) 20 28 25 27 28 32 26.83 3.98 1.62
ALP (U L−1) 112 108 122 118 97 114 111.83 8.7 3.56
AST (U L−1) 41 48 62 52 65 48 52.66 9.15 3.73
Methanolic extract (150 mg kg −1 )-treated animal group
Bilirubin (mg dL−1) 0.6 0.7 0.9 0.6 0.7 0.8 0.71 0.11 0.04
SGPT (U L−1) 21 29 30 26 28 27 26.83 3.18 1.30
ALP (U L−1) 132 100 122 115 95 103 111.16 14.27 5.82
AST (U L−1) 39 50 61 51 65 53 53.16 9.131 3.72
Methanolic extract (300 mg kg −1 )-treated animal group
Bilirubin (mg dL−1) 0.6 0.5 0.7 0.4 0.6 0.7 c 0.11 0.047
SGPT (U L−1) 35 29 27 32 21 26 28.33 4.88 1.99
ALP (U L−1) 140 165 123 135 156 117 139.33 18.55 7.57
AST (U L−1) 39 45 51 35 43 47 43.33 5.71 2.33

Among the liver function enzymes, SGPT was 20.33 ± 1.94 (U L−1) (normal animals), 63.33 ± 5.37 (U L−1) (untreated diabetic animals), 63.33 ± 1.62 (U L−1) (standard drug-treated animals), 26.83 ± 1.83 (U L−1) (150 mg kg−1 crude extract-treated animals), and 28.33 ± 1.99 (U L−1) (300 mg kg−1 crude extract-treated animals), respectively. The average concentrations of ALP were 122.16 ± 13.23 U L−1 in normal animals and 233.83 ± 31.81 U L−1 in untreated diabetic animals. The enzyme blood concentrations were 111.83 ± 3.56 U L−1 in the control-treated group, 111.16 ± 5.82 U L−1 in animals treated with 150 mg kg−1 of methanolic extract, and 139.33 ± 7.57 U L−1, treated with 300 mg kg−1 of methanolic extract, respectively. Likewise, serum AST was 58.5 ± 3.23 U L−1 among normal animals, 58.5 ± 3.23 U L−1 in untreated diabetic animals, 52.66 ± 3.73 U L−1 in standard treated animals, 53.16 ± 3.72 U L−1 in animals treated with 150 mg kg−1 of methanolic extract, and 43.33 ± 2.33 U L−1 in animals treated with 300 mg kg−1 of methanolic extract (Table 4).

4 Discussion

For thousands of years, natural products have been used for the treatment of various diseases. Especially those plants that are ethnomedicinally used by local communities are of great scientific interest in the development of novel agents as they are safe and have known efficacy. The chances of drug discovery from ethnomedicinally important plants are extremely high in comparison to randomly synthesized compounds. As we know, metformin, which is used for 60 years as the first-line drug for the treatment of type 2 diabetes, was obtained from a French lilac called Galega officinalis [30]. Several plant extracts have been found to be effective during preclinical studies against diabetes. For instance, Spatholobus suberectus can help to ameliorate type 2 diabetes by in vitro activation of GLUT4 translocation as well as blocking in vivo hepatic gluconeogenesis. Therefore, research on these natural traditional medicinal plants is of great importance for the development of new drugs.

In the GC-MS analysis, 35 various peaks were observed, among which some compounds were likely to be present in large quantities whose medicinal activities have been reported previously. For instance, the anti-diabetic activity of lupeol has been previously reported. One study reported the significant anti-diabetic potential of synthetic lupeol analogues and lupeol isolated from Crataeva nurvala [31]. α-Sitosterol is known to control inflammation and benign prostrate hypertrophy and to reduce cholesterol [32]. The oriental melon seed hexane extract contains oleic acid, linoleic acid, and palmitic acid, 17.3, 29.6, and 6.5%, respectively, and has been reported to exhibit potent inhibitory activities against α-amylase (61.8%) and α-glucosidase (35.3%) and hence can be utilized for controlling and treating type 2 diabetes [33]. For the replacement of synthetic drugs that possess a number of side effects, the phenolic compounds of Stevia rebaudiana are considered as natural anti-diabetic alternatives. The major compounds shown by GC-MS analysis were 1-heptatriacotanol, lupenone, γ-sitosterol, duvatrienediol, β-amyrin, agatholic acid dihydroxanthin, phytol, and fatty acids. The in vitro α-glucosidase and α-amylase potential of the extracts was investigated [34]. The GC-MS analysis of Melilotus indicus exhibited the presence of important compounds that are effective in the management of DM [35]. The presence of these bioactive compounds in our tested plants might play an important role in the anti-diabetic potentials of the plant extracts.

In 2017, IDF (International Diabetes Federation) estimated that globally more than 96,000 per year new cases of type 1 diabetes are diagnosed in children and adolescence (age < 15 years). Countries with highest number of cases include USA, Brazil, India, China, Russia, UK, Saudi Arabia, Nigeria, Germany, and Algeria, which account for almost 60% of new cases. In 2017, about 425 million adult people (age: 20–79 years) globally were estimated to have diabetes. The highest prevalence is in certain ethnic groups, especially in obese people. The difference in the prevalence of diabetes in different genders is very small, which in 2017 was estimated to be about 17 million more in men than in women. But, the prevalence sharply increases with aging among both genders [36]. Subsequently, there is a dire need for the discovery of more safe and effective anti-diabetic agents from natural sources.

During the in vitro studies, we observed that plant samples were effective in inhibiting the activity of two vital enzymes including α-amylase and α-glucosidase. Results of the in vitro studies revealed that chloroform and methanolic extracts exhibited inhibitions of 84.4 ± 1.12 and 81.23 ± 0.88%, respectively, against α-amylase enzyme at 1,000 µg mL−1. The IC50 values for these extracts were 27 and 40 µg mL−1, respectively. However, the acarbose showed an IC50 of 7.6 µg mL−1 at the same tested concentrations. Both extracts need further evaluation for the isolation of bioactive anti-α-amylase compounds. Further, various extracts exhibited concentration-dependent inhibition against α-glucosidase but were less potent, and the crude methanolic extract showed IC50 of 530 µg mL−1.

Our OGTT indicated the confirmation of diabetes in the animals after treatment with alloxan, and in the untreated disease group, blood glucose levels of 527.33, 545.33, 523.16, 535.83, and 533.16 mg dL−1 were observed at days 1, 7, 14, 21, and 28, respectively. Treatment with 150 and 300 mg kg−1 of the crude methanolic extract showed a considerable decrease in the blood glucose level of diabetic animals and was comparable with the standard drug-treated diabetic animals. For instance, diabetic animals treated with 150 mg kg−1 dose of the crude methanolic extract showed a decrease in blood glucose levels, i.e., 529.16 mg dL−1 on day 1, 446.66 mg dL−1 on day 7, 348.00 mg dL−1 on day 14, 269.33 mg dL−1 on day 21, and 165.5 mg dL−1 on day 28 of therapy. Moreover, the crude methanolic extract at a dose of 300 mg kg−1 exhibited blood glucose levels of 528.33, 44.83, 342.66, 204.66, and 111.83 mg dL−1 on days 1, 7,14, 21, and 28, respectively.

After the completion of experiments, we isolated vital organs for changes in their weight during the treatment process and a detailed lipid profile, kidney function profile, and the biochemistry of liver enzymes were performed for all groups of animals. No obvious changes in the average weight of the organs, including liver, kidney, pancreas, and heart, were observed among all groups. However, a considerable decrease in the lipid profile including cholesterol, triglycerides, HDL, and LDL was observed in the diabetic animals treated with 150 and 300 mg kg−1 of the crude methanolic extract (Table 2). Similarly, the kidney function markers including serum creatinine, blood urea, and uric acid were elevated in diabetic animals and were significantly reduced after treatment with 150 and 300 mg kg−1 of the test sample. The results were well comparable with diabetic animals treated with glibenclamide (positive control) (Table 3). In the liver profile, a steady decrease in the serum level of vital hepatic function markers including bilirubin, SGPT, ALP, and AST was observed, which was again comparable with the standard group (Table 4). In vitro results coupled with in vivo data revealed the efficacy of the plant in the management of some pathological targets of diabetes and thus might be subjected to further detailed studies including the isolation of bioactive moieties and molecular studies for potential use in the treatment and management of DM.

5 Conclusion

The results of the phytochemical analysis of our study revealed that plants contain various metabolites, which might be responsible for the anti-diabetic potentials of the plant. Various extracts exhibited concentration-dependent inhibition of key enzymes implicated in postprandial hyperglycemia and type 2 diabetes. The crude methanolic extract showed considerable in vivo efficacy against alloxan-induced diabetes in rats. Lipid profile was observed to be normalized among the diabetic animals treated with plant samples. Further, liver function enzymes were elevated among the untreated diabetic animals, and the crude methanolic extract exhibited a normogenic effect toward these enzymes and other blood markers. Further studies include activity-guided isolation of bioactive metabolites, and molecular studies are required for further results.

List of Abbreviations

AST

aspartate aminotransferase

ALP

alkaline phosphatase

DM

diabetes mellitus

GC-MS

gas chromatography-mass spectrometry

IDF

International Diabetes Federation

LDL

low-density lipoprotein

LFTs

liver function tests

NIST

National Institute of Standards and Technology

OGTT

oral glucose tolerance test

RFTs

renal function tests

SGPT

serum glutamate pyruvate transaminase

Acknowledgments

The authors wish to thank Princess Nourah bint Abdulrahman University Riyadh, Saudi Arabia, for the financial support (Project number: PNURSP2023R33).

  1. Funding information: This work was financially supported by Princess Nourah bint Abdulrahman University Researchers Support, Riyadh, Saudi Arabia (Project number: PNURSP2023R33).

  2. Author contributions: Falak Naz, Muhammad Zahoor, and Muhammad Ayaz conceptualized the project; Falak Naz, Asif Nawaz, Muhammad Ashraf, and Muhammad Ayaz, carried out the experimental work and data analysis; Falak Naz, Muhammad Zahoor, Muhammad Ayaz, Asif Nawaz, Amal Alotaibi, and Muhammad Ashraf wrote and edited the manuscript. All authors read and agreed with the submission of the current manuscript.

  3. Conflict of interest: The authors declare no conflict of interest.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-03-31
Revised: 2023-05-05
Accepted: 2023-05-06
Published Online: 2023-05-23

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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