Introduction

A group of metabolic illnesses known as diabetes is defined by hyperglycemia developed by alterations in insulin function, insulin release, or both. Long-term problems, dysfunction, and organ collapse caused by this continuous hyperglycemia are a concern, especially for the blood vessels, kidneys, nerves, heart and eyes. The problems brought on by diabetes are a major contributor to chronic morbidity and disability in working-age people. A phase of insulin resistance and increased pancreatic insulin production precedes the onset of type 2 diabetes mellitus. As the illness worsens, pancreatic functionalities are reduced to the point that they can no longer support peripheral needs1.

Type 2 diabetes has grown to be a major global health problem in recent years. According to estimates from the World Health Organization, this ailment affects over 176 million people globally. Every 10 s, a person with diabetes dies2. As estimated by the WHO, 70% of people worldwide utilize medicinal plants to treat their illnesses. A total of 1200 plant species have been tested for their ability to treat various hyperglycemia symptoms. There are many widely distributed families. Plant-based medications have emerged as a significant alternative to current treatments because to its accessibility, affordability, convenience of use, and minimal risk of adverse effects, especially in rural and/or developing regions. The most frequently used antidiabetic plants are Aloe vera (high sensitivity of blood glucose towards insulin)3 Momordica charantia (recovery of beta cells)4 and Carthamus oxycantha (peripheral uptake of glucose)5. Many synthetic molecules including iminosugars and glycosidase inhibitors have been reported as a novel approach to treating diabetes6,7,8,9,10. However, diabetes treatment may be conventional, experimental, or coincidental. Locally, Berberis orthobotrys is known as Ishkeen. It is a member of the Berberidaceaefamily. It is a brown-yellow shrub that grows in India, Pakistan, China and Afghanistan. Berberis orthobotryshas numerous well-known medical benefits. The seeds of Berberis orthobotrys produce linoleic oil and oleic oil, two different types of oils. The seeds of this plant have been used by many local healers in Pakistan as anti-diabetic therapy, therefore the current study was conducted to learn more about the anti-diabetic properties of Berberis orthobotrys seeds.

Materials and methods

Acquisition of Berberis orthobotrys seeds

Ishkeen seeds were amassed from forests of Gilgit-Baltistan, Pakistan. This specimen of the plant was recognized and authenticated by the botanical department of the University of Lahore. The herbarium section of the institution additionally received a voucher for the plant specimen (No. DBS09/BSP/ID/2020/67). The Berberis orthobotrys seeds were separated, then thoroughly cleaned with distilled H2O and rinsed with tap H2O. They took ten to twelve days to dry in the shade. To extract the seeds, they were dried and then crushed into a coarse powder using a Chinese herbal grinder. To protect them from the sun, the processed samples were stored in a sealed container.

Preparation of seeds’ extract

The ground seeds for 72 h, 2.05 kg of Berberis orthobotrys were imbibed in an aqueous methanolic combination of 70% methanoland 30% distilled H2O and stirred periodically. Extraction was carried out 3 times, followed by muslin cloth filtration and finally Whatmannfilter paper (Merck) filtration at ambient temperature (Khan et al., 2011). To obtain the extracts, all of the filtrateswas collected and evaporated in a rotary evaporator. The rotating evaporator’s temperature was kept at 51 °C, and a pressure of −760 mm Hg was created. The extract had a dark green color to them. To obtain the solid bulk, the aqueous extract was dried at room temperature. The residual extract was stored in the refrigerator (2–8) for future studies.

Screening of phytochemicals in seeds’ extract

For preliminary screening of phytoconstituents detected in the seeds extract, standard analytic-cal procedures were used. The qualitatively detected phytochemicals in seeds extract seeds extract have been mentioned in the following (Table 1).

Table 1 Initial phytochemical examination.
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Selection of rats for experiment

Male, young and healthy under regular circumstances, 150–250 g Sprague-Dawley rats were housed. The “Sprague-Dawley rats” used in the experiment were purchased from University of Lahore’s animal facility. With unrestricted access to water and pellet food, all rats, aged four to five months, were brought up to the optimal temperature of 25 degrees Celsius and humidity of 40 to 10%. In accordance with globally recognized guidelines for the use and care of lab animals, the research was conducted using the following environmental conditions: a 12-hour light-dark cycle, 22 °C ± 02, and 55% humidity. The University of Lahore’s animal facility housed all the animals. All the tests carried out in this work were authorized by the University of Lahore, Pakistan’s Ethics Committee (UOL-REC/2020/08–15). The National Research Council issued uniform recommendations for the treatment of all animals11. Blood samples were taken for blood glucose monitoring in both types of investigations. Every technique was carried out in compliance with the ARRIVE standards. Additionally, every technique was used in compliance with the applicable rules and regulations.

Grouping of rats

Three groups of five rats each were used for the two tests (glucose-loaded and normoglycemic). Groups 2 and 3 received the extract, while group 1 was the control group. Both short- and long-term experiments included randomly assigning rats with alloxan-induced diabetes to five groups, each with five animals. The non-diabetic group received a physiological solution consisting of 1.5 milliliters of normal saline (normal control). Group 2 received glibenclamide (0.5 mg/kg orally) as a usual therapy for diabetes, whereas Group 3 received no medication at all. For Groups 4 and 5, the obtained product to be examined was inserted in a NaCl vehicle at two different doses: 151 mg/kg and 301 mg/kg, correspondingly.

Histological examination of pancreas

1 cc of Lidocaine (5%) solution was injected in each rat via intraperitoneal route before dissection. The animals were sacrificed and then had their abdomens dissected after blood was taken for biochemical tests. After quickly removing each animal’s pancreas, any blood that might have hampered the fixation procedure was removed by rinsing it in a physiological saline solution (0.9% NaCl). Samples were allowed to soak in fixative for twenty-four hours (10% Formalin). The slices underwent a thickness of about 5 microns using a microtome. They were then placed onto sterile glass slides, deparaffinized twice for a duration of five minutes each, rehydrated using graded alcohol, and stained using eosin (H&E) dye and hematoxylin. A digital microscope (Tucsen ISH1000) at a magnification power of 100x was used to view and take a photomicrograph of the stained pancreas. The islet of Langerhans from the diabetes group was compared to the islet of Langerhans from the various treatment groups.

Statistical analysis

The results were shown using a one-way analysis of variance (ANOVA) as the mean standard error of the mean (SEM). Statistical significance was determined by data with p-values of 95% (P < 0.05).

Results

Effect of aqueous methanolic extract of Berberis orthobotrys seeds in normoglycemic rats

In normoglycemic rats, the AMEBO also found a decrease in blood glucose. The hypoglycemia potential was investigated using two different dosages of 151 mg/kg and 301 mg/kg. A considerable drop in blood glucose occurred after the seventh hour (p < 0.05), but no significant reduction occurred after the first, third, or fifth hours at this concentration. After 5 and 7 h, the 300 mg/kg concentration of AMEBO had extremely significant outcomes (p < 0.01) (Table 2; Fig. 1).

Table 2 Effect of Berberis orthobotrys in normoglycemic rats.
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Impact of Berberis orthobotrys seed aqueous methanolic extract on rats with glucose-induced hyperglycemia

All of the groups were given glucose (1 g/kg) dissolved in distilled water orally in this sort of experiment. The goal of this experiment was to see whether AMEBO could lower blood glucose levels after the rats were given glucose orally. The rats were given a plant extract shortly after receiving the glucose loading dosage. After the first hour of glucose treatment (Fig. 2), blood glucose levels increased in all of the experimental groups (At 150 mg/kg dose, AMEBO exhibited only a highly substantial drop (p < 0.01) after 7 h. After the third and fifth hours, it was unable to lower blood glucose levels. The second dose of AMEBO (300 mg/kg) has a greater potential for blood glucose lowering. The table shows that it began to decrease significantly (p < 0.05) at the third hour. From the 5th to the 7th hour, the 300 mg/kg dose of AMEBO continued to lower hyperglycemia, with extremely significant outcomes (p < 0.01) (Table 3).

Table 3 Effect of Berberis orthobotrys on glucose-Induced Hyperglycemic rats.
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Examining the antidiabetic effects of Berberis orthobotrys seed aqueous methanolic extract in alloxan-induced diabetic models

Long-term and short-term research were the two kinds of investigations carried out in alloxan-induced diabetes mice. This brief study showed that AMEBO had an impact on diabetic mice as early as the seventh hour. However, in long-term research, AMEBO was given orally daily for up to seven days. These tests involved a total of five groups (n = 5). One group was normal (no diabetes induction), while the other four had diabetic rats. The second group functioned as the diabetic control group throughout the research, receiving no treatment and continuing to raise blood glucose levels due to insulin shortage induced by beta cell death. The third group received glibenclamide as the conventional medicine (0.5 mg/kg daily) dissolved in normal saline. The remaining members of the two groups (4 and 5) were given AMEBO at doses of 151 mg/kg and 301 mg/kg, correspondingly. The AMEBO was essentially non-significant in short-term research at 150 mg/kg doses, but in a long-term study, it demonstrated a substantial drop in blood sugar on the 5th and 7th days. However, AMEBO concentrations of 300 mg/kg were effective in reducing hyperglycemia in both short and long-term studies, with very significant outcomes on the 5th and 7th days (Table 4; Fig. 1).

Fig. 1
Fig. 1
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Effect of Berberis orthobotrys in (A) Normoglycemic Rats (B) Glucose Loaded Rats (C) Alloxan Induced Diabetic Rats.

Table 4 Impact of Berberis orthobotrys on rats with Alloxan-Induced diabetes.
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Effect of AMEBO on serum triglycerides and total cholesterol in diabetic rats

Blood triglycerides and total cholesterol were tested 1, 07, and 15 days after extract administration. The mice that received a methanolic extract of seeds in water had substantially reduced triglyceride levels at day 15 compared to the diabetic controls (p < 0.01). Table 5 displays the effect of AMEBO on total cholesterol in hypercholesterolemic rats. A 300 mg/kg AMEBO extract throughout this study caused a notable decrease in total cholesterol after seven days; however, the significant reduction was much higher after fifteen days.Reduced triglycerides and total cholesterol are likely to reduce insulin resistance or improve glucose uptake by muscle cells in this study (Table 6), especially when the beta cells are entirely dysfunctional.

Table 5 Plant extract’s impact on rats’ serum triglycerides.
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Table 6 Plant extract’s impact on rats’ blood total cholesterol levels.
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Detection of Berberine in blood and urine by GC-MS

Specimens of blood and urine were collected from the animals of groups 4 and 5 at the end of the long-term study. The liquid-liquid extraction was performed at alkaline pH for the detection of basic alkaloids from biological matrices. The alkaloid under investigation was back extracted into acid. The final step included the re-extraction of ammonium hydroxide and dichloromethane. The extracted alkaloid was reconstituted into isopropyl alcohol and analyzed using GCMS. A gas chromatograph equipped with an auto-sampler, auto-injector in splitless mode, and an inert mass selective detector in positive electron impact (EI) mode supplied by Agilent Technologies (Melbourne, Australia) was operated in SCAN mode for qualitative screening of the extract. Using GC-MS for basic alkaloidal screening, berberine was found in both urine and blood. Retention duration and comparing produced mass spectra with library spectra allowed for its identification (NIST match was greater than 90%).

Fig. 2
Fig. 2
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Gas chromatograms for determination of Berberine in blood and urine.

Effect of Berberine on islets of Langerhans

The pancreatic structural changes play a vital role in the metabolic regulations of hormonal secretion along with the release and sensitivity of insulin. Atrophic changes in the islet of Langerhans and decreased number of beta cells are the prominent features of the pancreatic destruction induced by alloxan resulting in the end stages of diabetes (Fig. 3). The islet of Langerhans was equally divided among the normal study group in the current investigation. In contrast, the diabetic group’s islets showed lymphocyte infiltration and atrophic reduction in size. Although atrophic alterations were also seen in the groups of rats treated with regular medication and plant extracts (both dosages), the islets in these groups were like the normal rat group. The extract from the seed or the metabolite Berbereine found in the blood may be responsible for this pancreatic-protective action. To validate the signaling pathway in charge of this defense, further research is necessary.

Fig. 3
Fig. 3
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Histological changes in islets of Langerhans. (A) Normal group, (B) Diabetic group, (C) Glibenclamide 0.5 mg/kg treated group, (D) AMEBO 150 mg/kg treated group, (E) AMEBO 300 mg/kg treated group.

Discussion

Diabetes is expected to be common among people of all ages. In the year 2000, there was a prevalence of 2.8%. It will continue to climb until it reaches 4.4% in 2030. Males are more likely than females to get diabetes. People over the age of 65 have a higher risk of having diabetes, which is a significant demographic shift12.Long-term diabetic problems might also cause sexual dysfunction13. Endothelial dysfunction and atherosclerosis in the circulatory system may be exacerbated by insulin resistance14. The duration of diabetes is the single most important factor in coronary artery arteriosclerosis15. Sulfonylureas, metformin, and insulin reduce the onset of microvascular diseases such as neuropathy, retinopathy, and nephropathy in addition to improving blood sugar control16.α-glucosidase inhibitors, biguanides, thiazolidinediones and sulfonylureasare the most commonly utilized treatments17. However, these medications have side effects18so plants are also a large cause of drugs to manage disorders to overcome these side effects19. Aqueous methanolic extract of Berberis orthoboric (AMEBO) was used in a recent investigation at doses of 151 mg/kg and 301 mg/kg. Both dosages of the plant had significant effects in rats with normoglycemia, glucose overload, and alloxan-induced diabetes. This decrease in blood glucose may be related to peripheral glucose absorption or insulin secretion in normoglycemic and glucose-loaded animals. In diabetic animals, the lower blood glucose levels may be due to either the beta cells’ recovery from the original damage or their defense against the toxic effects of the alloxan.Some of the active chemical components were identified by doing a phytochemical investigation using the AMEBO. The presence of biologically active chemicals such as flavonoids, terpenoids, alkaloids, tannins, glycosides and steroids in the crude extract were discovered.Different types of active principles, such as steroids20and flavonoids21, may be accountable for reducing blood glucose stages. Flavonoids have also been studied for their potential to alleviate diabetes consequences such as heart issues22, retinopathy23, and neuropathy24.

In comparison to normoglycemic models and glucose-loaded animals, diabetic rats had a stronger response to AMEBO. Berberine was found in blood and urine during GC-MS-based basic alkaloidal screening. More research is needed, however, to determine the exact mechanism by which this alkaloid lowers blood glucose levels, to establish potential signaling pathways for producing a more potent antidiabetic active agent.A rise in free fatty acid levels has been seen in diabetic patients25. Free radical generation26, protein kinase C establishment27, and an boost in the difficulty of dyslipidemia28,29are all processes and mechanisms through which flowing free fatty acids have a negative impact on endothelial functioning. Oxidative stress is brought on by the body producing more free radicals30,31,32, which leads to micro33and macro34vascular complications in diabetes.The consequences and progression of diabetes and insulin resistance are strongly linked to oxidative stress35. Insulin action and secretion are negatively impacted by elevated oxidative stress, which is linked to elevated blood glucose and free fatty acid levels36. Reduced triglycerides in rats indicated improved insulin action and reduced oxidative stress.The lipid-lowering effects of AMEBO in rats were confirmed in this investigation. AMEBO at 300 mg/kg was effective in lowering total cholesterol levels in the plasma (Table 6). Further research is needed to establish the exact component of AMEBO that causes the observed effect, as this component could be a candidate for use as an anti-hypercholesterolemia.

Conclusion

It is reasonable that Berberis orthoboric seeds have historically been used as a hypoglycemic drug. An anti-diabetic effect like that of the prescription drug glibenclamide is also shown by the aqueous methanolic extract of seeds taken from this plant. Furthermore, Total cholesterol and triglycerides were significantly lower in the serum lipid parameters. The islets in the pancreas were protected by the extract, according to the histological analysis. The major metabolite Berberine, which was qualitatively detected by GC-MS in blood and urine, might be the alkaloid controlling the release of insulin or the restoration of beta cells following alloxan destruction.