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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                                                       Research Article 

In-Vitro Antidiabetic Effect of Ziziphus mucronata Leave Extracts

¹Ukwuani-Kwaja A.N., ¹Sani I., ²Sylvester M.N., and ^1* Abolaji Olaitan Kabir

¹ Department of Biochemistry, Faculty of Life Sciences, Kebbi State University of Science and Technology Aliero, Kebbi State, Nigeria

² Department of Pharmaceutics & Pharmaceutical Microbiology, Usmanu Danfodiyo University, Sokoto, Sokoto state, Nigeria

1.0 INTRODUCTION

A global prevalence of diabetes mellitus and impaired glucose tolerance in adults has been on the rise in recent years. The rate at which the prevalence of diabetes mellitus changes in many countries and regions has been enhanced by rapid urbanization and the increasing tendencies for sedentary lifestyle. According to the world health organization, about 108 million people were estimated to be diabetic in 1980. This estimate rose to 422 million individuals in 2014 (Cho et al., 2018). In Nigeria, the total pooled prevalence of diabetes mellitus was 5.77%. In the six geopolitical zones of Nigeria, the pooled prevalence of diabetes mellitus indicates that the north-west zone is placed at 3.0% (95% CI 4.3-7.7), the north-east zone at 5.9% (95% CI 2.9-9.4), the north-central zone at 3.8% (95% CI 4.0-7.1), the south-west zone at 5.5% (95% CI 2.4- 9.4), the south-east zone at 4.6% (95% CI 3.4-5.9), and the south-south zone at 9.8% (95% CI 7.2-12.4) (Uloko et al., 2018). There is an absence of effective cure for diabetes mellitus at the moment and available drugs and insulin therapy used for the management of the disease are associated with several adverse effects (Chikezie et al., 2015). Thus, the adverse effects and high cost of anti-diabetic drugs has led to the search for medicinal plants that exhibit hypoglycaemic property, with a view to applying them for the management of diabetes mellitus (Ali et al, 2006). 

Z. mucronata is an important multi-purpose plant. The genus Ziziphus comprise of approximately 135 plant species, appearing as a spiny shrubs or trees, mostly found in Indo-Malayan arid region, while few others are found in Africa, Australia, America and the subcontinent of South Asia (Mhaskar et al, 2000). Biologically, Ziziphus species are known to possess various important pharmacological activities including antimicrobial (Najafi et al, 2013), antioxidant and anti-inflammatory properties, antidiabetic, anti-malarial and anthelmintic properties, anticancer, antiulcer, analgesic, sedative and antipyretic effects amongst other recorded activities (Madara et al, 2010). The species are generally not toxic and safe for both human and animal consumption (Al-Saeedi et al, 2017). Members of the genus are known to possess a large number of cyclopeptide alkaloids, flavonoids, tannins, saponins, terpenoids, fatty acids, sterols and a wide variety of phenolic compounds (Modi et al, 2014). Thus, the long list of bioactivities reported for this plant as well as the folklore claims of antidiabetic properties in the leaves, prompted this study. Hence the need to further scientifically validates and establish it antidiabetic potency.

2.0 MATERIALS AND METHODS

2.1 Drug, Chemicals and Reagents

Gentamycin (Merck, Germany) was purchased from pharmaceutical store. Organic solvents used for extraction of plant material included n-hexane (C₆H₁₄), Acetone (C₄H₈₀₂), Methanol (CH₃OH), all are of analar grade (Sigma Aldrich, Germany). Enzyme (α-amylase) and substrates (Potato starch and p-nitro phenyl α-D glucopyranoside) were purchased from Sigma Chemical Co St. Louis M.O., (USA).

2.2 Apparatus and Equipment

General laboratory glass ware including volumetric flask, measuring cylinder, beakers, pestle and mortar, filter papers, test tubes, micro pipette, weighing balances and water bath, were used in the study. 

2.3 Plant Material

Fresh leave of Z. mucronata (Kurna) were collected from a farmland around Kwatarkwashi village in Zamfara state in January, 2020. The plant sample was authenticated at the Herbarium, Botany unit of Department of Biological science of the Usmanu Danfodiyo University, Sokoto, Nigeria. A voucher number, UDUSH/ANS/0831 was used and the plant specimen was deposited for referencing.

2.4 Successive extraction of Z. mucronate leaves.

Fresh leave of Z. mucronata were rinsed in tap water, air-dried at room temperature, and were crushed into small pieces, these were then grounded and homogenized into a fine powder using pestle and mortar. The finely ground air-dried leave (200 g) was successively extracted using solvents of increasing polarity (1500mls each) (Hexane, Acetone, Methanol and separately with Water). Thus, 200g of the fine powder was firstly extracted twice with hexane for 48 hours. The combine extracts were filtered and the filtrate concentrated using rotary evaporator. The mac obtained from this was then dried and further extracted twice (72 hours each) with acetone and again the combined extracts was filtered and concentrated. The mac obtained from filtration of the acetone was then extracted twice (72 hours each) with methanol and the combine extracts was filtered and concentrated  (John et al., 2018). The water extract was similarly prepared in the same way but using fresh powdered plant material of 200g. Consequently, four resulting extract residues were obtained and labelled as HE, AE, ME and WE for hexane extract, acetone extract, methanol extract and water extract respectively. These were kept separately at 4⁰C until when needed.

2.5 In vitro Antidiabetic effects of Z. mucronata leaves.

2.5.1 Alpha-amylase inhibitory activity (Enzymatic)

The effect of the HE, AE, ME, and WE of Z. mucronata extracts on alpha amylase activity (EC 3.2.1.1) was determined according to the method described by Rammohan et al, 2008. The enzyme solution was prepared by dissolving α-amylase in 20mM phosphate buffer (6.9) at the concentration of 5mg/ml. 1ml of the extract of various concentrations (5, 10, 15, 20 and 25) mg/ml and 1ml of enzyme solutions were mixed and incubated at 25°C for 10min. After incubation, 1ml of starch (0.5%) solution was added to the mixture and further incubated at 25°C for 10min. The reaction was stopped by adding 2ml of dinitrosalicylic acid (DNS, color reagent), and heating the reaction mixture in a boiling water bath for 5min. After cooling, the absorbance was measured calorimetrically at 565 nm and the inhibition percentage was calculated using the formula, below;

Percentage inhibition =   

2.5.2 Inhibitory Concentration (IC₅₀) of alpha-amylase by AE (most potent).

The concentration of the plant extracts required to inhibit 50% of enzyme (IC₅₀) was calculated by using the percentage scavenging activities at five different concentrations of the extract. Percentage inhibition (I %) was calculated by

I % =  , (Shai et al, 2010).

where Ac is the absorbance of the control and As is the absorbance of the sample.

2.5.3 Hemoglobin glycosylation (Non-enzymatic)

To measure hemoglobin glycosylation, 1ml each of Glucose (2%) hemoglobin (0.06%) and Gentamycin (0.02%) in phosphate buffer 0.01M at pH 7.4. were taken and mixed in a test tube. The methanol extract was weighed and dissolved in DMSO to obtain stock solutions of 5 μg/ml. Thereafter 1 ml of each concentration was added to the above mixture. The Mixture was incubated in dark at room temperature for 72 hrs. The degree of glycosylation of hemoglobin was measured calorimetrically at 520 nm (Adisa et al, 2004). Metformin was used as a standard drug for assay and % inhibition was calculated using the formula:

% inhibition of glycosylation =  

Where Abs control is the absorbance of the control reaction (containing all reagents except the test sample) and Abs sample implies absorbance of the test sample.

2.5.4 Inhibitory Concentration (IC₅₀) of hemoglobin glycosylation.

The concentration of the plant extracts required to inhibit 50% of glycosylation (IC₅₀) was calculated by using the percentage scavenging activities at five different concentrations of the extract. Percentage inhibition (I %) was calculated using the equation; 

I % = =   (Shai et al, 2010).

where Ac is the absorbance of the control and ‘As’ is the absorbance of the sample.

3.0 RESULTS AND DISCUSSION

The percentage yield of the extracts was found to be 1.3, 4.9, 8.8 and 6.4grams for HE, AE, ME and WE respectively. The AE of Z. mucronata (most potent) revealed the presence of phytochemicals such as flavonoids, tannins, terpenoids, phenols, anthraquinone and alkaloids.

3.1 Hemoglobin glycosylation effect of Ziziphus mucronata leave extracts. 

The plant extracts except n-hexane significantly inhibited the haemoglobin glycosylation (P<0.05) which is indicated by the presence of increasing concentration of haemoglobin (Table 1). Acetone extract of Ziziphus mucronata (AEZM) exhibited higher inhibition of glycosylation as compared with the standard Metformin. The plant extracts also displayed the inhibition of haemoglobin glycosylation at different physiological concentrations of the glucose over the period of 72 hours, indicating that the plant extracts decrease the formation of the glucose- haemoglobin complex and thus amount of free haemoglobin increases.

3.2 Alpha-amylase inhibitory Effect of Ziziphus mucronata leave extracts.

The administration of four extracts of Ziziphus mucronata indicated that; all four extracts except n-hexane exhibited a dose dependent increase in alpha-amylase inhibitory activity. At concentration of 1.0 mg/ml, AEZM (most potent extract), is 71.02% while Voglibose (standard drug), is 83.47 (Table 2). And also, the IC₅₀ of AEZM is 0.62 while that of voglibose is 0.42 (figure 1 and 2).

Table 1. Effect of EAZM on hemoglobin glycosylation.

Values are expressed as mean SEM, values having the same superscript are not significantly different at (P<0.05). analyzed using one-way anova of n=3

nEZM= n-hexane extract of Ziziphus mucronata, AEZM= acetone extract of Ziziphus mucronata, MEZM= methanol extract of Ziziphus mucronata and WEZM= water extract of Ziziphus mucronata

Table 2: Effect of AEZM on alpha-amylase inhibition % inhibition mean  

All determinations were carried out in triplicate manner and values are expressed as the mean ± SEM. The IC₅₀ value is defined as the concentration of inhibitor to inhibit 50% of its activity under the assayed conditions. AEZM= Acetone extract of Ziziphus mucronata, nEZM= n-hexane extract of  Ziziphus mucronate, MEZM= Methanol extract of  Ziziphus mucronate, WEZM= Water extract of  Ziziphus mucronata.

Figure 1: Alpha-amylase Inhibitory concentration (IC₅₀) of AEZM.

Figure 2: Alpha-amylase Inhibitory concentration (IC₅₀) of standard drug (Voglibose).

Table 3: Phytochemical constituents of Acetone leave extract of Ziziphus mucronata.

Keys: + = Present, ND = Not detected 

DISCUSSION

Metabolism of carbohydrates including fat and protein are affected when insulin is lacking, which can lead to homeostatic imbalance (Frier et al, 2006). Recent attempts to understand the activity of intestinal enzymes such as alpha-amylase which is important in digestion of carbohydrate and also in the absorption of glucose resulted to the development of recent pharmacological agents. Alpha-amylase inhibitors stop the digestion of carbohydrates and slow down the absorption. Acarbose is competitive inhibitor of alpha-amylase and slows down the absorption of starch and disaccharides (Hengameh et al, 2016). therefore, one of the therapeutic approaches for reducing blood glucose levels in diabetic patient is to inhibit the absorption of carbohydrate after meal. Inhibition of this enzyme (alpha-amylase) reduced the high blood glucose in diabetes (Conforti et al, 2005). Acarbose is a complex oligosaccharide that delays the digestion of carbohydrates. It inhibits the action of pancreatic amylase in breakdown of starch. The reaction mechanisms involved in inhibition of alpha-amylase enzymes by plant protein inhibitors are not clearly understood. However, there are some suggestions that the plant protein (flavanols) might cause conformational changes in structure of the enzyme (Hengameh et al, 2016). The maintenance of plasma glucose concentration for a long term under a variety of dietary conditions is one of the most important and closely regulated processes observed in human (Srividya et al., 2012). Increased concentration of plasma glucose leads to its binding to hemoglobin which may result in the formation of glycated heamoglobin. The in vitro assays of the present study indicated that all the plant extracts of Z. mucronata except hexane exhibited higher inhibition of alpha-amylase and glycosylation of heamoglobin as compared with the control. The acetone extract of Z. mucronata (AEZM) exhibited significantly higher activity than other plant extracts. And haemoglobin glycosylation at different concentrations of the glucose over the period of 72 hours, indicating that the plant extracts decrease the formation of the glucose- haemoglobin complex and therefore the concentration of free haemoglobin increases. Tetracyclic triterpenoids, such as dammarane, cucurbitane, and protostane groups, happens to be class of triterpenoids commonly found in several medicinal plants, especially those commonly used for the treatment of diabetes and its complications, such as Panax quinquefolium, Panax notoginseng, Gynostemma pentaphyllum, and Ganoderma lucidum (Kaiser et al, 2015). Flavonoids can inhibit some of the complication of diabetes mellitus (Havsteen et al., 2002). Several researches have been carried out to understand the potential role of flavonoid in the treatment of diabetes (Jung et al., 2006; Matsui et al., 2006; Qi et al., 2010). Flavonoid has been demonstrated using different experimental models of treatments and the drug candidates have been shown to possess beneficial effects against the disease, either through their ability to prevent glucose absorption or to promote glucose tolerance (Zhou et al, 2009). Phenolic compounds are commonly found in plants, and they have been reported to have several biological activities, including antioxidant activity. Several studies have showed that the phenolic content in plants can be linked to their antioxidant activity (Kujala, 2004; Lavelli, 2000). Any of these secondary metabolites either singly or in combination is capable of causing antidiabetic activity of the extract, and many of such compounds are known to possess potent antioxidant activity (Lee et al., 2004).

CONCLUSIONS

In conclusion, Z. mucronata possesses alpha-amylase inhibitory potential, preventing the digestion of starch and also inhibited glycosylation of hemoglobin and thereby prevented the formation of glycated end products. Such activities validates the use of Z. mucronata in traditional medicine. 

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