Background
Diabetes mellitus, one of the major public health problems worldwide, is a metabolic disorder of multiple etiologies distinguished by a failure of glucose homeostasis with disturbances of carbohydrate, fat and protein metabolism as a result of defects in insulin secretion and/or insulin action [
1,
2]. According to International Diabetes Federation (IDF) report, elevated blood glucose is the third uppermost risk factor for premature mortality, following high blood pressure and tobacco use globally [
2].
Cardiovascular diseases, neuropathy, nephropathy, and retinopathy are among the major risks that are associated with diabetes. These chronic complications may lead to hardening and narrowing of arteries (atherosclerosis) that could advance to stroke, coronary heart disease, and other blood vessel diseases, nerve damage, kidney failure, and blindness with time [
3].
In 2015, according to IDF report, 415 million (8.8%) adults (aged 20–79) worldwide were estimated to have diabetes; this number is expected to rise to 642 million (10.4%) by 2040 or one adult in ten people. An estimated 14.2 million adults aged 20–79 had diabetes in the Africa Region that represents a regional prevalence of 3.2% (2.1–6.7%) in 2015, which can be projected to 3.7%(2.6–7.3%; 34.2 million) by 2040. South Africa (2.3 million), Democratic Republic of Congo (1.8 million), Nigeria (1.6 million) and Ethiopia (1.3 million) are among the highly populated African countries containing the highest number of people living with diabetes [
2].
In Ethiopia, even though the occurrence of diabetes has not been made nationally, hospital-based studies indicate that its prevalence has increased from 3.32% in 2012 [
4] to 3.4% in 2015 and the number of diabetic cases is expected to increase to 10.6 million by 2040 [
2].
Diabetes mellitus can be managed by diet, physical exercise, and modern drugs (insulin and/or oral hypoglycemic drugs such as sulfonylureas and biguanides) [
5]. Different extracts from medicinal plants have also been used traditionally to manage diabetes globally, and these are considered as relatively inexpensive, less toxic and with relatively little or no side effects [
6]. There are also medicinal plants that contain some toxic constituents such as the cytotoxic anti-cancer plant-derived drugs, digitalis; however, the side effects of the phytotherapeutic agents are less common compared with synthetic drugs [
7]. Management of diabetes without any side effect is still a challenge and the available modern antidiabetic agents produce serious side effects such as hypoglycemia (Sulphonylureas), lactic acidosis and folate and B12 malabsorption (Metformin), gastrointestinal symptom (Acarbose), weight gain (Sulphonylureas and Thiazolidinediones), and edema (Thiazolidinediones) [
8]. Hence, the search for safer and more effective hypoglycemic agents has continued.
Globally, medicinal plants have been used as a source of medicine and 80–85% of populations rely on these medicinal plants using the extracts or their active components as traditional medicine to meet their primary health care needs [
9,
10]. A number of active components were isolated from medicinal plants for direct use as drugs, or act as a lead compound or pharmacological agents. Metformin, for example, is an oral hypoglycemic agent isolated from medicinal plant
Galega officinalis that was used historically in medieval Europe for the treatment of diabetes [
11‐
13].
Ajuga remota Benth (synonyms:
Ajuga integrifolia Buch.-Ham, Ajuga bracteosa Wall ex Benth.) [
14], is a shrub in the Lamiaceae family that grows widely in East Africa, at an altitude of 1500–3400 m above sea level in Saudi Arabia, Yemen and Afghanistan to East Asia [
15,
16]. In the Ethiopian traditional health system,
A.remota is commonly used for the treatment of various diseases including diabetes, malaria, toothache, skin disease, high blood pressure, stomach pain, pneumonia, liver problem and swelling of legs. Due to the bitter taste of the extract, sometimes honey is added to the preparation so as to make it palatable and for longer period storage for later use. In Ethiopia, the vernacular names for
A.remota include ‘Harmagussa’ (Ormigna), ‘Akorarach’ (Amharic); ‘Etse-Libawit’ (Ge’ez), ‘Akembiye’ (Guragegna), and ‘Tale’ (Welaytigna) [
15‐
20].
The plants of the genus
Ajuga have been assessed for different activities such as antiviral activity against Human Immunodeficiency Virus type 1 (HIV-1) and Type 2 (HIV-2) [
21], antipyretic [
22], diuretic [
23], anti-inflammatory, antidepressant, anticoagulant [
24], analgesic [
24,
25], antiarthritic [
26], antifeedant, antifungal, antihypertensive, insecticidal [
18,
27,
28], antimicrobial [
29], antioxidant [
24,
29,
30], hypoglycemic [
31‐
35], antinociceptive [
36], hypolipidemic [
37], antimycobacterial [
38], and antimalarial/antiplasmodial activities [
18,
19,
27,
28,
39,
40].
To verify the traditional uses of the plant Ajuga genus, various in vitro and in vivo studies have been conducted on different extracts of the species of the genus Ajuga using animal models, such as mice or rats; as their genetic, biological and behavior characteristics closely resemble human beings. A.remota, A.bracteosa, A.integrifolia, and A.iva are some of the species of the genus Ajuga that are ecologically related and were evaluated for various pharmacological applications.
Different extracts of
A.bracteosa wall ex. Benth was evaluated for its analgesic effect using Swiss albino mice with acetic acid-induced writhing and tail immersion test. A dose-dependent analgesic effect was observed at 200 and 400 mg/kg from the water and chloroform extracts [
25]. In another study that used
A.bracteosa Wall ex Benth, its antiarthritic effect was evaluated in albino rats using a 70% ethanolic extract and dose-dependent activities with a better effect than that of aspirin was observed after six h treatment on acute non-immunological arthritis and complete Freund’s adjuvant (CFA)-induced chronic immunological arthritis. The antiarthritic effect was reported to be due to the presence of the active constituents’ ajugarin I, lupulin A, withaferin A, reptoside and 6-deoxyharpagide that were isolated from the plant [
26].
In Taiwan, in vitro and in vivo hypoglycemic effect was evaluated on the 70% ethanol extracts of five
Ajuga species namely;
A.decumbens,
A.nipponensis,
A.pygmaea,
A.taiwanensis and
A.dictyocarpa on streptozotocin-induced diabetic mice. Among them,
A.nipponensis showed a superior effect in α-glucosidase inhibition (28.62 ± 1.56%) and glucose uptake (54.15 ± 2.56%) and somewhat postprandial blood glucose levels reduction. It also contains the highest content of flavonoids and ecdysterone as compared with the other species [
32].
A phytoecdysteroids rich aqueous extract from
A.iva on alloxan-induced diabetic rats significantly (
p < 0.01) decreased the level of blood glucose level and increased hepatic glycogen levels [
34]. In another study, the aqueous extract of whole parts of
A.iva was examined for hypolipidemic and hypoglycemic effect on streptozotocin-induced diabetic rats and a significant reduction in plasma cholesterol and triglyceride levels were observed by 35% (
P < 0.01) and 13% (
P < 0.05) [
35] and 44% (
P < 0.01) and 30% (
P < 0.01) [
37], respectively while the plasma levels of glucose was reduced by 24% (
P < 0.05) [
35].
A.remota and the other species of the genus Ajuga have a number of common phytochemical compounds. Although ample ethnobotanical, in vitro and in vivo evidence exists for the use of Ajuga species in the management of diabetes, the claim of A.remota has not been substantiated scientifically. Therefore, the aim of this study was to find out the scientific basis of the use A.remota in the management of diabetes used by traditional practitioners using aqueous and 70% ethanol extracts on alloxan-induced diabetic mice.
Methods
Collection of plant material
The leaves of Ajuga remota Benth were collected from Lebu, a few kilometers Southwest of Addis Ababa, Ethiopia in January 2008. Taxonomic identification was made by Mr. Melaku Wondafrash at the National Herbarium, College of Natural Sciences, Addis Ababa University and a voucher specimen (Voucher Specimen number T001) was preserved.
Chemicals and instruments
Alloxan monohydrate (Sigma Chemical Company, USA) was used to induce diabetes in mice and Glibenclamide (Hoechst Pharmaceuticals, Mumbai) was used as a standard hypoglycemic drug. Ethanol (BDH Ltd., England) and distilled water were used for extraction of the plant materials. ACCU CHEK Performa Glucometer (Roche Diagnostics India Pvt. Ltd., India) was used to measure the blood glucose level. For evaporating the solvents, BUCHI Rotavapour R-200, Switzerland and Lyophilizer (freeze dryer) (type: Heto power dry LL3000 Wag tech) was used. The following chemicals were used for phytochemical screening test: Chloroform and Ethyl acetate (ACS, Merck); Hydrochloric acid, Ferric sulphate, Lead acetate and Potassium ferrocyanide (BDH Ltd., England); Petroleum ether 60-80 °C (Labmerk Chemicals LTD India); Sulphuric acid (Farm Italia Carrloerba, Italy); Acetic anhydride and Methanol HPLC grade (Techno Pharmchem, Bahadurgarm, India); n-Hexane (Rathburn Chemicals Ltd., England); Acetonitrile (Sigma Aldrich, Germany) and Ferric chloride (FISHER Scientific Company, USA). All the chemicals were of analytical grades.
Experimental animals
Adult male Swiss albino mice bred in the animal house of Ethiopian Health and Nutrition Research Institute, Addis Ababa, Ethiopia, with weights ranging from 24 to 35 g and eight weeks of age were used for the experiment. The animals were kept in cages made of polypropylene (5 mice per cage randomly) at 23 ± 2 °C with 12 h/12 h light/dark cycle [
41‐
44]. Standard pellet and water were allowed to the animals throughout the experiment, except the fasting period. The care and handling of animals were in accordance with internationally accepted ethical guidelines for use of laboratory animals [
44] and the study protocol was approved by the School of Pharmacy Ethics Committee, Addis Ababa University, Ethiopia.
The leaves of A.remota were air-dried for one week under the shade at room temperature at Essential Oils Research Center laboratory, Addis Ababa. The dried plant material was manually powdered finely and used for extraction.
One hundred gram of the dried and powdered leaves of A.remota was kept in a thimble and extracted with 70% ethanol in a Soxhlet extractor. The extraction process was continued until the color of the final drop of the extract became colorless. Then, ethanol was removed from the extract using a rotavapor (BUCHI Rotavapor R-200, Switzerland) at 60 °C and the remaining 30% water was removed using Lyophilizer (freeze dryer) (type: Heto power dry LL3000 Wag tech). The resulting dry hydroalcoholic extract has a percentage yield of 10.95% (w/w). The extract was kept in a refrigerator until used for the experiment.
Twenty gram of the dried and powdered leaves of A.remota was added to 100 mL hot distilled water (60oc), mixed thoroughly and heated for 20–30 min. on a water bath with continuous stirring as traditionally done and cooled to room temperature. The decoction obtained was filtered under suction and Lyophilizer (freeze dryer) (type: Heto power dry LL3000 Wag tech) was used to dry the aqueous extract. The dried aqueous extract has 13.5% (w/w) percentage yield. The freeze-dried aqueous extract was kept in a refrigerator until used for the experiment and then the dried plant extract was reconstituted with distilled water for oral administration.
Acute toxicity test
Acute toxicity test were done on both plant extracts after the animals had been fasted overnight while only taking water [
45]. The weight of each mouse was recorded before administering the extract. Randomly the animals were divided into a control and three treatment groups (separately for both extracts), each group consisting of five mice. The control group received only the vehicle (1% Tween 80) and each treatment group received orally the 70% ethanol and aqueous extracts of
A.remota in a dose of 1000, 2000 and 5000 mg/kg [
46]. Animals were kept under close observation for explicit toxicities and/or behavioral changes like restlessness, tremor, diarrhea, sluggishness, loss of weight, and paralysis at regular intervals for the first four h after administering the extract [
47], and then they were observed daily for two weeks for any change in general behavior and/or other physical activities. Food was available after four h of administration of the extracts.
Induction of experimental diabetes
Male Swiss albino mice were fasted overnight (12–14 h) and their weight and fasting blood glucose level recorded with a glucometer and then made diabetic by a single intraperitoneal injection (a volume of 1 mL/kg) of freshly prepared alloxan monohydrate solution (20 mg/kg body weight). Alloxan was prepared by weighing according to individual animal weight and solubilized with 0.5 mL sodium citrate at pH 4.5 before injection. Food and water were presented to the animals 30 min. after administration of alloxan [
48,
49]. After 48 h of alloxan injection, plasma blood glucose level of each animal was determined by taking the blood from the tail and animals with a fasting blood glucose level above 200 mg/dL [
50,
51] were included in the study.
Experimental design
The animals were divided into seven groups for the evaluation of fasting blood glucose level and oral glucose tolerance test with five animals in each group. They were treated with the plant extracts two days after alloxan injection excluding the diabetic control groups. Blood samples were drawn for measuring blood glucose levels from each group on day 1, 7 and 14 during the study period [
43]. Changes in body weight were also recorded. Groups 1 and 2 served as normal and diabetic controls (receiving only the vehicle, i.e. 1% Tween 80), respectively. Group 3 received standard drug (glibenclamide, 10 mg/kg per day orally) [
45]. Groups 4 & 5 received the 70% ethanol extract at a dose of 300 & 500 mg/kg, respectively and groups 6 & 7 received aqueous extract at a dose of 300 & 500 mg/kg, respectively daily in one mL aqueous solution using oral gavage for two weeks.
Oral glucose tolerance test
After two weeks of treatment with the plant extracts, the animals were made to fast for 12–14 h but had free access of water and their fasting blood glucose level was measured four times. Glucose solution (2 g/kg of body weight) was administered orally in a volume of 1 mL/kg. Blood samples were collected at the time interval of 30, 60 and 120 min. after administration of glucose [
41].
Phytochemical screening
Preliminary phytochemical screening of the plant extracts was carried out using standard procedures [
52], to check for the presence or absence of secondary metabolites such as alkaloids, steroidal compounds, phenolic compounds, flavonoids, saponins, tannins and anthraquinones.
Statistical analysis
Data are expressed as a mean ± standard deviation. Differences among treatment group means were assessed by two-way analysis of variance (ANOVA) and group means were considered to be significantly different at P < 0.05. Data were statistically evaluated using Statistical Package for the Social Sciences (SPSS) version 20.0 software. Bar and line charts were drawn using Excel 2007 software.
Acknowledgements
We would like to acknowledge Addis Ababa University, School of Graduate Studies, for supporting the project, Essential Oils Research Center for providing the chemicals, reagents and laboratory space during extraction, Department of Biology for providing animal house and Department of Chemistry, Addis Ababa University, for providing necessary chemicals. We also acknowledged colleagues and all those who assisted in conducting the study.