Hypoglycemic activity of endophytic extract of Senna Alata in STZ-induced diabetic mice model

Ogechukwu Lucy Nwankwo; Samuel J Bunu; Omoirri Moses Aziakpono

doi:10.4103/jihs.jihs_25_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 9 | Issue : 2 | Page : 75-80

Hypoglycemic activity of endophytic extract of Senna Alata in STZ-induced diabetic mice model

Ogechukwu Lucy Nwankwo¹, Samuel J Bunu², Omoirri Moses Aziakpono³
¹ Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Anambra State, Nigeria
² Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria
³ Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Federal University of Oye-Ekiti, Ekiti State, Nigeria

Date of Submission	07-Sep-2021
Date of Decision	20-Dec-2021
Date of Acceptance	20-Jan-2022
Date of Web Publication	15-Mar-2022

Correspondence Address:
Dr. Ogechukwu Lucy Nwankwo
Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka
Nigeria

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jihs.jihs_25_21

Abstract

Background: Senna alata belongs to the Senna family and is known to contain several bioactive constituents that contribute to its therapeutic properties. A variety of medicinal and pharmacological effects have been reported, including antidiabetic, antiasthma, anthelmintic, and antiplasmodium infection effects. Objectives: The goal of the study was to determine whether the extract of S. alata can reduce blood sugar levels in streptozotocin (STZ)-induced diabetic mice. Materials and Methods: S. alata leaves were collected from the Department of Botany, University of Nigeria Nsukka, Nigeria, authenticated, and validated. Phytochemical screening was conducted. Specifically, leaf blades were extracted in 70% ethanol for 3 min, transferred to 500 ml of water for 5 min, then cut into small pieces, and then inoculated onto malt extract agar, and local rice was used to inoculate the fermentation medium. 25 male albino mice of 30–35 g weight, average weight of 30–35 g were used in the study. Streptozotocin (STZ; 65 mg/kg) was injected intravenously to induce type 2 diabetes. Results: The crude extract significantly (P < 0.05) reduced the fasting blood glucose levels in STZ-induced diabetic mice. The two doses (250 and 500 mg/kg) of the ethanol extract and metformin 500 mg/kg caused a significant (P < 0.05) reduction in the fasting blood glucose levels from 0 h to the 10^th h of treatment. The extract displayed a dose-related reduction in blood sugar level concerning time. The highest reduction rate was found in 500 mg/kg 409.66 ± 1.92. The fasting blood glucose reduction was dose-dependent. The highest reduction rate was found in 500 mg/kg 80.00 ± 4.00 (80.47%) being the highest dose so far. There was a reduction in the bodyweight of the animals induced with STZ. Conclusion: The endophytic extract of S. alata displayed useful pharmacological properties and can be used to manage diabetes and its complications.

Keywords: Diabetes, endophyte, hypoglycemia, Senna alata, streptozotocin

How to cite this article:
Nwankwo OL, Bunu SJ, Aziakpono OM. Hypoglycemic activity of endophytic extract of Senna Alata in STZ-induced diabetic mice model. J Integr Health Sci 2021;9:75-80

How to cite this URL:
Nwankwo OL, Bunu SJ, Aziakpono OM. Hypoglycemic activity of endophytic extract of Senna Alata in STZ-induced diabetic mice model. J Integr Health Sci [serial online] 2021 [cited 2023 Jun 10];9:75-80. Available from: https://www.jihs.in/text.asp?2021/9/2/75/339652

Introduction

Diabetes is a metabolic disorder that causes high blood sugar levels, altered lipid, carbohydrate, and protein metabolism, which adversely affects the patient's physical and psychological health.^[1] Both types of diabetes (Types 1 and 2) are characterized by hyperglycemia, but their pathogenesis is different. Hyperglycemia in Type 2 diabetes is caused by impaired insulin secretion and/or impaired insulin action.^[2] The WHO reported that diabetes is rife among all global populations. Southeast Asians and Western Pacificians are more susceptible to diabetes, and most of them have Type 2 diabetes. It is common for insulin resistance to precede Type 2 diabetes and to be associated with other cardiovascular risk factors, such as dyslipidemia, hypertension, and prothrombotic factors.^[3] It has been shown that diabetics are 2–4 times more likely to suffer a stroke due to atherosclerosis resulting from altered lipoprotein metabolism.^[4] The development of a better molecule without unwanted side effects than existing drugs is a challenging task, despite multiple classes of drugs available for the treatment of type 2 diabetes. Many medicinal plants have been used in Nigeria traditional medicine systems since ancient times to effectively treat diabetes.^[5] The antidiabetic activity of medicinal plants was documented by multiple mechanisms due to their phytoconstituents. Hence, the characterization of the chemical constituents of antidiabetic medicinal plants is being applied in the pharmaceutical research program to produce a lead molecule that can treat diabetes.^[6]

Fabaceae comprises the flowering shrub Senna alata (L.) Because of the structure of its inflorescences, it is called a candle bush. Herbs of average height between one and four meters, growing in warm, humid climates, it is usually annual and occasionally biannual. A thick, oblong plant is usually found with five to fourteen leaflets, a strong petiole (2–3 mm), caduceus bracts (2 × 3 by 1 cm × 2 cm), and dense flowers (20 × 50 by 3 cm × 4 cm). Flowers with zygomorphic flowers are bright yellow, have seven stamens, and have pubertal ovaries.^[7] When the fruit is ripe, it has brown wings with many diamond-shaped brown seeds in a tetragonal pod that is 10–16 cm in size. A seed spreads the plant and disperses it every 1,500 m above sea level.^[7] Precious studies have reported the antidiabetic properties of S. alata leaves, using through α-glucosidase inhibition model.^[8] It is, therefore, possible for the endophyte of S. alata to possess some pharmacological properties, especially as those related to antdiabetic, since it survives on the nutritional strength of the plant.^[7],[8] In an experiment, the antidiabetic properties of the endophyte Aspergillus striatus were assessed.

Materials And Methods

Collection of plant materials

S. alata leaves were collected in the Ufuma Orumba region of the state of Anambra, North. Mwafor Felix of the Botany Department at the University of Nigeria, Nsukka, authenticated, and validated the samples' botanical identities.

Phytochemical analysis

Qualitative phytochemical and quantitative phytochemical analyses of samples of the powdered plant materials were performed to detect the presence of alkaloids (Mayer's reagent and Wagner's reagent), cyanogenic glycosides, steroids (Libermann–Burchard's test), terpenoids (Salkowski's test), flavonoids (alkaline reagent test), phenols (ferric chloride test), saponins, and tannins (gelatin test).

Surface sterilization of the leaves of Senna alata

Before transferring the leaves into a beaker containing 2% hydrochloric acid, they were first washed under running tap water to reduce soil particles. After the leaves were submerged into 70% ethanol for 3 min, and then in 500 ml of water for 5 min, they were transferred onto an aseptic aluminum foil sheet on the laboratory table, from where they were picked, cut, and inoculated onto malt extract agar.

Isolation and purification of endophytic fungi

The sterile forceps were used to inoculate the cut leaf blades and midribs onto the solidified malt extract agar after it had been prepared and autoclaved as directed. Masking tape was used to seal the Petri dish ^{More Details}es and they were incubated at room temperature for 5 days. A variety of endophytic fungi colonies began to form on the 5^th day. To obtain pure cultures of specific fungal endophytes, the colonies were continuously subcultured using malt extract. Fermentation was performed on these pure endophytic fungi.

Fermentation of endophytic fungi

To aid in differentiation, these pure cultures were labeled (Sa-LB2 and Sa-MR3). 100 g of local rice was weighed into conical flasks with 200 ml of water to prepare the food for fermentation. After autoclaving for 30 min, the flasks were allowed to cool maximally for 24 h. To introduce pure cultures and agar into the conical flasks containing the cooled autoclaved rice, well-famed spatulas were used to cut both into bits. Fermentation took place for 21 days after the flasks were plugged back and sealed well with foil.^[9],[10]

Postfermentation

A sterile glass rod was used to turn the rice using ethyl acetate on the 21^st day to halt fermentation. 3 days of continuous shaking was sufficient for homogenizing the mixture instead of using an electric shaker for a day.^[11] As the mixtures were homogenized for 3 days, sterile beakers were decanted into them (Sa-LB2 and Sa-MR3). After 5–7 days of exposure to room temperature, the filtrates were concentrated again. To make further use of the extracts, they were stored at 40°C.

Animals

The Laboratory Animal Facility of the Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, provided a total of 25 albino mice, average weight of 30–35 g, for this study. In the animals' house at the School of Pharmacy, Agulu, the animals were housed in clean metal cages, supplied with clean drinking water, and fed pelleted commercial food (Guniea Feed®, Nigeria). Guidelines for laboratory animal care and use were followed by the National Institutes of Health.

Induction of experimental diabetes

An experiment to induce type 2 diabetes: Type 2 diabetes was induced by injection of freshly prepared streptozotocin (STZ - 65 mg/kg; i.p.) in cold citrate buffer (0.1 M, pH: 4.5), 15 min after the administration of nicotinamide (NIC - 110 mg/kg; i.p.) in overnight-fasted rats.^[1] We determined the induction of diabetes by measuring blood glucose levels with the glucose meter (Glucocard 01-mini, Arkray Factory Inc., Japan) after 72 h. During up to 14 days of standard laboratory conditions, diabetic rats were kept under conditions that stabilized their blood glucose levels. To evaluate the antidiabetic activity of endophytic fungus extract, blood glucose was measured again after 14 days in diabetic rats with blood glucose >200 mg/dL.^[12],[13]

Experimental design

Comparative studies on crude extract of endophytic extract in mice model

A total of 25 mice were studied, 20 diabetic mice, and five nondiabetic mice, which were randomized into the following five groups:

Group 1: Normal control to be given water only
Group 2: Diabetic control nontreated
Group 3: Diabetic + metformin, 500 mg/kg
Group 4: Diabetic + crude extract, 250 mg/kg
Group 5: Diabetic + crude extract, 500 mg/kg.

Statistical analysis

An analysis of the study's results was performed using the Statistical Package for the Social Sciences (SPSS-20). The results were presented as mean ± standard error of the mean for replicates of the samples. One-way analyses of variance was performed on raw data and a post hoc Turkey's test was performed after. Statistical significance was determined by P < 0.05.

Results

The endophyte extracted from S. alata showed the presence of alkaloids, steroidal nucleus, terpenoids, flavonoids, phenols, saponins, and tannins [Table 1].

Table 1: Result of the phytochemical screening

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Effect of endophytic extract on blood glucose level (acute or hourly study)

In STZ-induced diabetic mice, the crude extract significantly reduced fasting blood glucose levels (P < 0.05). In the fasting blood glucose levels of the rats from 0 h to the 10^th h of the study, the two doses of ethanol extract and metformin (250 and 500 mg/kg) led to a significant (P < 0.05) reduction in fasting blood glucose. Blood sugar levels decreased dose-dependently with time following intake of the extract. The highest reduction rate was found in 500 mg/kg 409.66 ± 1.92 (16.28%) being the highest dose so far. This is shown in [Table 2] and [Figure 1].

Table 2: Effect of various treatments on blood glucose level (hourly study)

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Figure 1: Effect of various treatments on blood glucose level (Hourly study). NC: Normal control, DC: Diabetic control, CE: Crude extract, D.H20: Distilled water

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Effect of various daily treatments of endophyte Aspergillus striatus on blood glucose level

The two doses (250 and 500 mg/kg) of the extract, as well as metformin, produced a significant (P < 0.05) reduction in the fasting blood glucose levels when compared with the untreated control from day 0 to 14^th day of treatment [[Table 3] and Figure 2]. The fasting blood glucose reduction was dose-dependent. The highest reduction rate was found in 500 mg/kg 80.00 ± 4.00 (80.47%) being the highest dose so far.

Table 3: Effect of various treatments in blood glucose level (daily study)

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Figure 2: Effect of various treatments on blood glucose level (daily study). NC: Normal control, DC: Diabetic control, CE: Crude extract, D.H20: Distilled water

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Change in bodyweight

With STZ, the animals' body weights were reduced. Following treatment with various extracts, body weight increased significantly. When compared to diabetic control, the controls and treated groups gained weight more [Table 4] and [Figure 3].

Table 4: Effect of various treatments on body weight

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Figure 3: Effect of various treatments on body weight. Key: NC: Normal control, DC: Diabetic control, CE: Crude extract, D.H20: Distilled water, WBT: Weight before induction, WAT: Weight after treatment, WAI: weight after induction

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Discussion

The endophyte showed the presence of alkaloids, steroidal nucleus, terpenoids, flavonoids, phenols, saponins, and tannins. Various methods are employed to induce diabetes in experimental animals to screen natural and synthetic chemicals for antidiabetic activity. STZ -nicotinamide is commonly used to induce noninsulin-dependent diabetes in animals, resulting in moderate hyperglycemia with clinical symptoms similar to type 2 diabetes.^[14] STZ activates poly ADP-ribosylation, causing cell-dependent depletion of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate. Consequently, pancreatic cell necrosis is caused by free radical generation.^[15] Niacinamide and STZ can act as weak inhibitors of poly (ADP-ribose) polymerase, preventing poly ADP-ribosylation, and precursors to NAD+, which are needed by cells for metabolism and function. Hence, nicotinamide protects the pancreas from the effects of STZ-mediated cytotoxicity and produces a diabetic state in rats that resembles Type 2 diabetes.^[16] Compared with control rats, rats incubated with STZ were shown to have significantly raised blood glucose levels and decreased body weight and insulin levels, which confirm the induction of diabetes, possibly due to partial necrosis of pancreatic cells. In addition, diabetic rats' body weights were reduced, and this may be the result of reduced insulin. The function of insulin in skeletal muscle is to regulate protein synthesis and proteolysis.^[17] When diabetic rats were administered orally A. striatus extract and metformin (250 and 500 mg/kg dose), blood glucose levels were significantly reduced and body weight and insulin levels were significantly higher than in diabetic control rats. Endophytic extract, therefore, causes bodyweight improvement in diabetic rats by reducing STZ-induced cell damage and inhibiting muscle proteolysis. These two processes may result in improvement in body weight in diabetic animals treated with the endophytic extract. Lipoprotein abnormalities are frequently observed in type-2 diabetic patients due to abnormal insulin metabolism, and atherogenic dyslipidemia is associated with cardiovascular diseases. Continual hyperglycemia causes glycosylation of all proteins, specifically collagen cross-linking in the arterial wall, which results in incomplete endothelial cell function and advances atherosclerosis [Figure 4]. Diabetic mellitus is associated with 95% dyslipidemia, which is a significant risk factor for coronary heart disease.^[18] The pathogenesis of diabetic dyslipidemia is associated with insulin resistance, which leads to an increase in the free fatty acid release from insulin-resistant fat cells and an increase in triglyceride production.^[19]

Figure 4: Pancreas histology architecture of various treatment (a) This image shows a large pancreatic islet and a secretory acini (long arrow). At low power, the intralobular duct can be seen in the histology (short arrow). (b) The cytoplasm is arranged with acinar pattern structure (short arrow), with pyknotic nuclei of some acinar cells apparent. Strongly stained cells of the acinar region have prominent nuclei arranged in lobules. An arrow points out how the islets are embedded within the acinir cells and surrounded by a fine capsule (long arrow). (c) This image shows a large pancreatic islet and a secretory acini (long arrow). Pyknotic nuclei are evident (d) Staining on acinar cells reveals lobulated cells with prominent nuclei. In this image, we can see the islet cells embedded within the acinar cells and surrounded by fine capsules (long arrow). (e) These pancreatic features show acinar cells mixed with islet-cells showing lymphocytic infiltrates (long arrow) with mildly deranged pyknotic nuclei (short arrow)

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The regenerative effect of the endophytic extract on the pancreatic cells indicates positive effects of the extract on insulin production. The regeneration of the Islet of Langerhans may be due to the effect of the extract on the pancreatic cells, which resulted in the production of insulin.

Conclusion

From the results obtained, the endophytic extract of S. alata possess a number of phytochemicals present on S. alata and further displayed remarkable hypoglycemic effects on the test animals, hence can be used as an alternative in the treatment and management of diabetes and its complications, after thorough standardization and formulation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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