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Evaluation of the Antidiabetic Activities of Crude Extracts of Phoenix Dactyliferaseedsin Alloxan-Diabetic Rats
Johnson-Ajinwo OR1 and Kuebari Penuel B2*
1Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Nigeria
2Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Nigeria
Received Date: 30/09/2020; Published Date: 30/10/2020
*Corresponding author: Johnson-Ajinwo Okiemute Rosa, Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Nigeria. E-mail: okiemute_2002@yahoo.co.uk
Abstract
Diabetes Mellitus (DM) is a chronic endocrine disorder characterized by glucosuria and hyperglycemia and has been ranked the 7th leading cause of death in the US and globally. In 2019, DM gulped over 760 billion dollars in US health care expenditure. The disease is linked to other terrible health conditions such as kidney failure, stroke, cardiovascular diseases, lower limb amputation and blindness.A number of anti-diabetic medications are in use, but these are not without undesirable setbacks, which leaves the search for better therapeutic agents worthwhile and expedient. Phoenix dactylifera popularly called dates is a sweet-tasting fruit, found in the Mediterranean and North of Africa. Much attention has been given to the fruit, while the pits or seeds are considered a waste. In this research work, the antidiabetic activities of the or ganic extracts of P.dactylifera (seeds) at 200mg/kg, 300mg/kg and 400mg/kg were screened for their antidiabetic activities in normoglycemic and alloxan-induced diabetic rats(150-200g) at 1h, 2h, 4h, 6h, 8h, and the 12th hour. DMSO (0.3ml of 33.3%v/v stock) and Glibenclamide 10mg/kg were administered to the control and reference groups respectively. Organic extracts of P. dactylifera seeds at 400mg/kg significantly decreased FBGL in both normoglycemic and hyperglycaemic groups by 82.8% and 78.3% respectively at the 12th hour (P<0.05). The activity of P.dactylifera seeds extract at 400mg at the 12th hour showed comparable antidiabetic activity to that of the reference -glibenclamide at 10mg/kg (with 83.2% and 80.7% activity) in the normoglycemic and hyperglycaemic groups respectively. This study disclosed that the seeds of P.dactylifera possess significant antidiabetic effect.
Keywords: Phoenix Dactylifera; Hyperglycemia
Introduction
According to the annual report of International Diabetes Federation (IDF), 463 million people were living with diabetes mellitus (DM) in 2019 of which 79% of these people live in low and middle-income countries. IDF predicts this number to rise to 700 million people by 2045 [1]. Generally, more men develop DM than women. But given that most communities have a large population of older women than men; this leaves more women living with DM, [2]. Currently, over one million children and adolescents have type 1 diabetes, with some 20 million live births affected by gestational diabetes [1]. In the United States, an estimated 10.5% of the population has diabetes with some 2.8% of individuals remaining undiagnosed in 2018 [3]. Traditional rural and urban communities in Africa have prevalence in diabetes of 1-l2% and 1-13% respectively [4]. For instance in Enugu, Nigeria the prevalence of diabetes among pregnant women was found to be 1.7% [5]. A report from the WHO has it that the number of adults suffering from diabetes has almost quadrupled since 1980 mainly due to the increased number of people living with diabetes mellitus type 2 and related contributing factors including overweight and obesity. In 2016, 1.6 million deaths were attributed to DM, which placed DM as the seventh leading cause of death. Further more, the disease claimed 2.2 million lives indirectly in 2012 and has been associated with kidney failure, stroke, cardiovascular diseases, lower limb amputation and blindness. A worrisome development is the unprecedented rise in DM among under-developed and developing countries in comparison to developed countries [6].
In the light of pharmacoeconomics, the cost of managing diabetes can be catastrophic in low-income populations. There is therefore a need to seek other possible means of managing diabetes in order to reduce the high mortality rate. According to WHO, a greater percentage of the world's populations rely on herbal medicines due to their cost-effectiveness and accessibility [7].The value of Traditional Chinese medicine, TCM was one-third the turnover from the Chinese pharmaceutical industries in 2012 [8]. This is not yet the case with Africa, where 80% of the populations depend on herbal medicine [9]. The WHO has since recognized the important role of traditional
and complementary medicine in prevention and in the man agreement of most life-style diseases, which includes diabetes mellitus. Presently, 88% of the member states of WHO have embraced Traditional and complementary medicine, TC&M in their healthcare programs [10,11,12].
According to Kinghorn et al. [13], natural products and their derivatives represent more than 50 % of all drugs in clinical use in the world and higher plants contribute no less than 25% of the total. The world is currently undergoing a rapidly growing interest in natural and traditional herbal remedies as a source of new commercial products in health shops and pharmacies. Several people all over the world still rely on medicinal plants or plant-derived medicines for daily healthcare needs as natural substitute to synthetic chemicals. Approximately 80% of the populations in Africa still rely on traditional medicinal plants to cure many kinds of ailments especially in rural areas. These herbal remedies are an important part of African cultural heritage transmitted from one generation to the next based on trial and error over many centuries [14]. DM affects millions of people worldwide which has resulted in numerous amputations and massive fatalities, such that medical researchers are still working on the management of DM which is still a big challenge for them. The management of this condition has raised the demand for safe and cost effective remedial measures be cause of several side effects associated with the present use of modern medicines [15].
Thus, it is crucial to explore other options for diabetes man agreement such as the use of medicinal plants. Traditional plant based remedies are still the first choice in the developing countries because of their cost effectiveness, easy availability and minimum or no side effects [16,17]. P.dactylifera L., generally called the date palm, is a valuable plant that is grown in the Southwest Asia and North Africa and in the wild in some countries. Based on the phytochemical studies, date fruits contain anthocyanins, phenolics, sterols, carotenoids, procyanidinsand flavonoids. Date fruits have been shown to possess antioxidant, anticancer, anti-inflammatory, hepatoprotective and nephro protective activities [18]. Seeds of date palm (pits) are waste products of many industries that are added to the domesticated animals’ foods. The toxicity of date seeds was studied recently. The oral acute toxicity investigations recorded that at a dose of 3000 mg/kg, no fatality was sustained in the animals [19]. Date seeds are rich in protein (5.1 g/100 g), fat (9.0 g/100 g), dietary fiber (73.1 g/100 g), phenolics (3942 mg/100 g), and antioxidants [20].
Although, the date palm fruit served as the low cost food for millions of people around the world for several centuries, studies on its benefits are inadequate and hardly recognized as a healthy food by the health professionals and the public. It has been reported that the total phenolic content of seeds of several fruits such as mango, avocado, and jackfruit, was higher than their edible flesh. Date seeds can also be considered as a valuable source of phenolic compounds and an inexpensive rich source of natural dietary fiber and antioxidants [20]. Studies have shown that dates are loaded with a plethora of activities such as antioxidants, antihyperlipidemic, antidiabetic, antimicrobial and anticancer. Previous research has also proven that dates seed extract can reduce inflammation, through its role as an antioxidant to reduce oxidative stress. Research on the use of dates flesh has been done, but the seeds still need to be studied further [18].
The chronic nature and complications of diabetes are some vital elements that potentiates it's socioeconomic and health implications. That over 463 million people in the world live with diabetes of which majority live in low and middle-income countries, traditional medicines would offer an opportunity for novel drug discovery to meet the demand for readily available and cost-effective antidiabetic medications in such areas. Hence, this research work on the antidiabetic activities of crude extracts of P. dactylifera was carried out to further profer additional therapeutic options for DM through medicinal plants.
Materials & Methods
Plant Materials and Reagents
Seeds of P. dactylifera were collected, from a local vendor, who specializes in preparing a local drink, called “Tigernut”. The date fruits are used while the seeds are thrown away as waste products. The seeds were air-dried under shade before being milled into fine particles. Methanol, Dichloromethane, Chloroform, Diethyl Ether, Acetic Anhydride, Glacial acetic acid, Sodium picrate, 2% 3,5-Dinitrobenzoic acid, Picric acid, Iodine solution, Dimethylsulfoxide, (JHD company, Guang dong. GuanghuaSci-Tech. Co. Ltd. China), Hydrochloric acid, Million's reagent, Benedict's solution, Wagner's reagent, Sodium Hydroxide, Ferric chloride solution, Saturated lead acetate solution, Dragendorff’s reagent, Kedde reagent, Ammonia solution, 7.5% Potassium Hydroxide, Fehling’s solution A and B (Sigma Aldrich Chemicals, St Louis, USA), Distilled water, Deionized water (Pharmaceutical Chemistry Lab, University of Port Harcourt).
Assay Materials
Alloxan monohydrate (Sigma-Aldrich Merck Germany), Glib enclamide (Actiza Pharmaceutical Private Limited, Surat), ACCU-CHECK glucometer.
Extraction of Plant Materials
The 2 Kg of the pulverized P. dactylifera seeds were extracted according to the American National Cancer Institute (NCI) method of extraction (21). The pulverized plant material was macerated in a 1:1 mixture of dichloromethane and methanol for 24 h to obtain the extracts. The procedure was repeated thrice. The ratio of plant material to solvent used was 1:5. The residue was further macerated in methanol for another 24 h to yield the methanol extract, which was combined with the dichloromethane/methanol extract to yield the total organic extract, which was henceforth referred to as date seed extract (DSE). The obtained dry extracts were further dried in a desiccator to remove any trace of solvent.
Phytochemical Screening
Phytochemical tests were carried out on the plants’ extract by the method described by Trease and Evans (22), highlighted below:
Test for Flavonoids
Test for Flavonoids Shinoda Reduction Test:
100mg of the plant extract was dissolved in 5ml of hydrochloric acid, a few pieces of magnesium metal was added to 5ml of each plant extract solution. The formation of orange; i.e.
a reddish crimson colour signified the presence of flavonoids. Sodium Hydroxide Test for Flavonoids: A small amount of each of the portions was dissolved in water and filtered; to this, 2ml of 10% aqueous sodium hydroxide was added to produce a yellow colouration. Upon addition of dilute hydrochloric acid, a change in colour from yellow to colourless indicated the presence of flavonoids.
Test for Tannins Ferric Chloride Test:
100mg of plant extract was boiled with 5ml of distilled water for 5 minutes, cooled and filtered, few drops of 5% ferric chloride reagent was added. The formation of green to blue black precipitate indicated the presence of tannins.
Test for Alkaloids: 100mg of the plant extracts were heated for about 2 minutes with 5ml 2M HCl on the steam bath, the mixture was filtered and to 1ml of the filtered mixture. Thereafter, 2 drops of the following reagents were added and observed. Dragendorff’s reagent; where a reddish precipitate indicates the presence of alkaloid. Wagner’s reagent, development of yellow precipitate indicates the presence of alkaloid.
Test for Saponins Frothing Test:
100mg of the plant extract was mixed with 5ml of water and shaken vigorously for persistent foam, then warmed and observed persistent foaming on warming indicated the presence of saponins.
Emulsion Test:
100mg of plant extract was dissolved in 10ml of water and mixed with 3 drops of olive oil and shaken vigorously then observed for the formation of emulsion which signifies a posi tive test for saponins.
Test for Phlobatannins:
Hydrochloride Acid Test 100mg of plant extract was dissolved in water and filtered. The filtrate was boiled with 1% hydro chloric acid. Deposition of a red coloured precipitate indicates a positive test
Test for sterols/Triterpenoids Liebermann-Buchard Test:
100mg of plant extract is dissolved in chloroform and acetic anhydride is added followed by sulphuricacid. Green colour indicates the presence of steroids and pink colour indicates terpenoids.
Salkwoski’s Test:
100mg of plant extract was dissolved in 2ml chloroform, then conc. H2SO4 was carefully added to form a layer. The forma tion of a reddish brown colour at the interface is indicative of a steroidal nucleus.
Test for Cardiac glycosides Keller Killiani Test:
100mg of plant extract was dissolved in about 5ml of water, then glacial acetic acid, one drop of 5% FeCl3 and conc. H2SO4 were added. Reddish brown colour that appears at the junction of the two liquid layers and an upper layer that appears to be bluish-green confirms the presence of glycosides. Kedde Test: About 5ml of dissolved extract was treated with a small amount of Kedde reagent (which was prepared by combining the same volumes of 2% solution of 3, 5 dinitrobenzoic acid in menthol and a 7.5% aqueous solution of KOH). The formation of a blue or violet colour that fades out in 1-2 hours indicates the presence of cardiac glycoside.
Test for Carbohydrates Fehling’s Test:
100mg of the plant extract was mixed with few drops of Fehling’s solution A + B and boiled for a few minutes. A deep blue to green; yellow to red indicates a positive reduction test. Molish’s Test: 100mg of plant extract was mixed with 5ml of distilled water. The mixture was boiled for 15 minutes and filtered. 1ml of Molish reagent was added to 1ml of the filtrate and shaken; 1ml of concentrated H2SO4 was added and observed. The presence of reddish ring indicates the presence of carbohydrate.
Test for proteins:
About 100mg of the sample was extracted with distilled water by boiling then filtering.
Million's Test:
To a little portion of the filtrate in a test tube, 2 drops of Mil lion's reagent was added. Formation of white Precipitate which indicates the presence of protein was watched out for.
Picric acid test:
To a little portion of the filtrate were added drops of Picric acid solution. A yellow Precipitate indicates the presence of protein. Tests for Fixed oils and Fats
Oil Stain test:
The extracts were placed between two filter papers and pressure exerted on the filter papers. The detection of oil stains on the papers indicated the presence of fixed oils.
Experimental Design
This study was designed in line with the ethically approved experimental protocols adopted by the department of Experimental Pharmacology and Toxicology, of the Faculty of Pharmaceutical Sciences, University of Port Harcourt. Adult mixed-gender wistar rats weighing between 150-200g, bred by the animal house unit of the department of Experimental Pharmacology and Toxicology were used for the study. The rats were housed in spacious cages, to allow for free movement at room temperature, sufficient humidity and 12 hourly cycles of light and darkness. The animals, had access to standard laboratory animal feed and water prior to the commencement of the experiment.
The rats were randomly divided into 12 groups of 6 rats each: 6 test groups and 6 control groups and were fasted overnight. The 12 groups were sub-divided into two categories, A and B. Category A consist of normoglycemic rats that received the extracts and the controls. While category B were alloxan-induced rats that were treated with the extracts and the controls respectively.
Freshly prepared alloxan solution (in 0.9%w/v normal saline) was administered at a dose of 150mg/kg Intraperitoneally to normoglycemic rats. Those with FBGL>200mg/dL after 72hrs were considered to have developed experimental diabetes. The FBGL of the animals were determined to ensure that they were not diabetic before carrying out the actual experiment. ACCU CHECK glucometer was used in BGL determination. The blood analyzed was gotten by pricking the tail of the animals. 6 of the 12 test groups were treated with intraperitoneal injection of P. dactylifera organic extract, one dose per group (200mg/kg, 300m/kg and 400mg/kg) respectively. Of the 6 control groups: one was untreated, 0.3ml of normal saline was administered to 1 group; two other groups were treated with I. P injection of 0.3 ml of the DMSO stock while the last two group were treated with Glibenclamide 10mg/kg (I.p) The Maximum Reduction was calculated using the formula: (Initial reading after Induction) – (Lowest reading obtained after treatment)
The Maximum Reduction was calculated using the formula: (Initial reading after Induction) – (Lowest reading obtained after treatment) While the percentage Maximum Reduction was calculated us ing the formula:
Statistical Analysis
The analytical tool used was Graph pad Prism version 8 for calculation of the mean values ± standard error of mean (SEM). The one-way ANOVA was used to determine statistical difference between means. A p<0.05 was considered statistically significant.
Results
Results from the Phytochemical Screening
Table 1: The results of the qualitative phytochemical tests of extracts of the plant materials.
Key: - = Absent; + = Slightly present; ++ = Moderately present; +++ = Heavily present
The results presented in Table 1 showed that tannins and carbohydrates were the most abundant constituents in the DSE. The extracts also contained moderate amount of flavonoids, alkaloids and fats and oils. Previous studies reported that the seed of P.dactylifera constitutes 5-14% of the total weight of the fruit. The proximate analysis of the seed as documented showed that carbohydrate made up 20.95%, with the protein content being 2.63% while the fat content was 8.55% [23]. The same researchers noted that the flavonoid content was 45.28+0.32 mg/100 g. The phenolic content which was lesser was determined as 28.22+0.43 g/100 g. Similarly, other workers have reported that the seed is rich in unsaturated fatty acids, which were non-toxic [24].
Result of Antidiabetic studies
Effect of P.dactylifera seeds extracts (DSE), on blood glucose levels (mg/kg) in normoglycemic and
alloxan-treated diabetic rats. The results disclosed that the extract significantly reduced the blood glucose levels by 52.2%, 71.8%, and 82.8%, while its effect in diabetic rats displayed in Table 3 below revealed significant reduction in FBGL by 48.0%, 61.4% and 78.3% after 12 hours post administration of (200, 300 and 400) mg/kg doses respectively.
The observations above depicts that the hyperglycemic activity of the extract was more pronounced in normoglycemic rats than in the hyperglycemic rats. These activities are in a dose dependent manner. Comparing the effects of P. dactylifera extracts at the 12th hour post administration, to that of the reference drug Glibenclamide, dosed at 10mg/kg at the same hour, similarities in activity where observed in normoglycemic group(82.8%) and hyperglycemic group (78.3%) at 400mg/kg as against glibenclamide 83.2% and 80.7% in normoglycemic and diabetic groups respectively. The antidiabetic activity of P. dactylifera can be attributed to its high concentration in phenolic compounds -Tannins and flavonoids. This observation is consistent with those of other researchers who reported flavonoids, alkaloids, tannins and saponins present in Anacardiumoccidnetale L stem-bark to have blood glucose reduction effect. The antioxidants and anti-hypercholesterolemic activities of P. dactylifera has been reported by Mohammed R. T et al [25]. These activities are believed to be beneficial to the Beta cells of the pancreas by increasing insulin absorption in tissues which could be partly responsible for the antidiabetic activity of the date plant.
Table 2: Shows the effect of organic extract on FBGL±SEM on normoglycemic rats.
* = p<0.05 ≥ 200mg/dLFBGL = Diabetic. Lo = too low to be detected.
Table 3: Effect of P. dactylifera seeds organic extract on blood glucose levels (mg/dl) in diabetic rats.
Discussion
The organic extract of P. dactylifera indicated the Presence of flavonoids, tannins, Lipids and carbohydrates heavily. This is consistent with the findings by El-Fouhil et al (26),that the phytochemical constituents of P. dactylifera are rich in phenolic compounds. It has been observed that plant extracts with moderate to heavy Presence of tannins, flavonoids and alkaloids showed relatively high antidiabetic activities. This is affirmed by several research works including those by Yogesha Mohan et al [27].
In 2015, an investigation of the potentials of P.dactylifera seeds in ameliorating early diabetic complications in streptozotocin- induced diabetic rats was carried out. The oral administration of the aqueous suspension of P. dactylifera seeds (aqPDS) (1 g/kg/d) was implemented for 4 weeks. The results showed a significant reduction in the blood glucose levels of the diabetic rats [28].
Also, studies on the antidiabetic activities of the ethanol ex- tracts of P dactyliferous leaves, showed significant activity on alloxan-induced male rats (18). A similar study on the ethanolic extracts of the date seeds on alloxan-induced diabetic rats also demonstrated significant reduction in blood glucose in a 14 days study [19]. In this present study, an attempt has been made to evaluate the date seed extracts, DSE on both normoglycemic and alloxan-induced rats, in comparison with 10 mg/kg Glibenclamide, a standard antidiabetic drug in clinical use. The results of this study showed that there was no statistically significant difference between treatment with 400g/ kg of date seed extract and 10 mg/kg Glibenclamide from the fourth hour of treatment in the normoglycemic rats. While in the diabetic rats the activity of the DSE was comparable to that of indomethacin at the 12th hour. This implies that date seed possess significant anti-diabetic activities. Also from the results obtained, the activities of the DSE and Glibenclamide were more pronounced in the normoglycemic rats than in the diabetic rats. El-Fouhil et al., 2011 documented the safety of date seed extract at a dose of 10 ml administered in rats for a period of 30 days. The researchers however lamented their in- ability to explore the extracts at higher doses and for a longer period [29]. Our findings suggest that the seeds of dates should be consumed in moderation by non-diabetic subjects, to avoid the possibility of developing hypoglycemia.
Conclusion
According to the National vital statistics Reports in 2019, DM was the seventh leading cause of death in the US in 2017 [30]. This is again re-echoed by WHO, whose most recent rating, ranked DM as the World’s seventh killer disease [6]. Given that 374 million people are at a higher risk of developing type 2 diabetes [1], the need to seek cost effective remedies from nature cannot be over-emphasized. P. dactylifera seeds are common waste products on the streets of Nigeria owing to the use of the fruits in a local beverage “tiger nut milk”, withthe seeds been discarded as less consideration of possible pharmacological activity is given to these seeds. Currently the seeds find use as an additive in some beverages such as “date seed coffee beverages”. The study has shown that the seeds have significant antidiabetic activity and could be further explored as treatment option for Type 2 diabetes mellitus.
Conflict of Interest
None
Acknowledgement
Our sincere appreciation goes to MrNwanosike A. Okeosisi for his seasoned advice in the pharmacological assays.
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