Anti-diabetic Effect of Ethanol Extract of Carica Papaya leaf in Alloxan induced Diabetic
Mice
Adebiyi Olubodun1, Aluko Olayemi2, Banigbe Shade3, Shotayo Abidat1, Dada Odunayo3, Abdulrasheed Amina3 and Bala Ahmad4
1Food Technology Department, Federal College of Agricultural Produce Technology, PMB 3013 Kano, Nigeria.
2Science Laboratory Technology Department, Federal College of Animal Health and Production Technology, Moor Plantation, PMB 5029 Ibadan, Nigeria. 3Science Laboratory Technology Department, Federal College of Agricultural Produce Technology PMB 3013 Kano, Nigeria.
4Pharmaceutical Technology Department, Federal College of Agricultural Produce Technology, PMB 3013 Kano, Nigeria.
Article Received: 22 March 2018 Article Accepted: 29 July 2018 Article Published: 09 September 2018
1.INTRODUCTION
Diabetes Mellitus is a disease of carbohydrate metabolism characterized by abnormal increase in glucose and lipid levels. The incidence of this disease has gone up astronomically in recent times by the reason of change in lifestyle and feeding habits, from consumption of wholesome food to consumption of more refined products. Treatment of this ailment in the developing nations has been very difficult and frustrating because of the high cost of drugs, unavailability of the drugs in rural areas and low patient to doctors’ ratio. This has led to untold suffering of the patients when the condition is not well managed
leading to vascular and non-vascular complications. In fact there is no organ that could not be affected by the disease ranging from nephropathy, neuropathy, reticulopathy, retinopathy to retrograded ejaculation [1].
The use of medicinal plants as a source for relief from illness can be dated back to the premedeaval period an art as old as mankind. Even today plants are the most exclusive source of drugs for the majority of the world’s population. Substances derived from higher plants constitutes about 25% of prescribe medicines
[2]. Plant based indigenous knowledge was passed down from generation to generation in various parts
A B S T R A C T
The present study was aimed at evaluation of anti-diabetic effect of leaf extract of Carica papaya (Linn) in alloxan-induced diabetic mice. The phytochemicals screening of the extract and acute toxicity study and were conducted. Thirty mice weighing 23.0±3.0g were divided into 5 groups of 6 animals each. Group 1, normal control received 0.1 ml vehicle, Group II, diabetic untreated (received 0.1 ml vehicle), Group III; diabetic control (received 600µg/kg body weight of glibenclamide), Group IV and V received 250 and 500mg/kg of extract respectively. Diabetes was induced by intraperitoneal injection of mice with alloxan monohydrate 150mg/kg body weight. All the animals received the respective treatment for 28 days. Blood glucose level was measured by using glucometer on days 0, 7, 14, 21 and 28. Phytochemical analyses revealed both male and female species contain Tannins, Flavonoids, Anthraquinones, Cardiac glycoside, Steroids, Triterpenes, Phlobatannins and Phenolic compounds while Saponins, Reducing sugar and Cyanogenic glycoside were absent in the male leaf extract. The estimated LD50 of the male leaf extract was >5,000 mg/kg body weight. After twenty eight days of treatment, result showed significant (P< 0.05) fall in blood glucose, lipid profiles, biochemical parameters like Atherogenic and Coronary Risk indices while eliciting significant increase (p<0.05) in the serum level of high density lipoprotein cholesterol. The observation from this study showed that Carica papaya leaf extract has anti-hyperglycemic and beneficial effects on blood lipid profile, thus, justifying the use of the plant by traditional medicine practitioners for the treatment of diabetes mellitus.
medicine. The use of plants as medicine or pharmacological agent involves extraction and isolation of active compounds. The potential for finding new compound is enormous as till date only a meager percentage of the tropical species have been screened for physiochemical constituents and studied for biological activity. However, a lot of work has started in different laboratories all over the world (the developing nations inclusive) by the reason of the 1994 declaration of WHO emphasizing the need to develop and evaluate better Pharmacological agents for improving insulin secretion, enhancing insulin sensitivity, preventing beta – cell destruction, promoting beta – cell regeneration or repair and interrupting pathways leading to the various complication of diabetes mellitus. These recommendations, the cost and side effects of most orthodox hypoglycemic agents, stimulated an increased demand for natural product with anti – diabetes activities that have fewer side effects. The most promising of such product are of plant origin [3].
The hypoglycemic and indeed the medicinal properties of plants used by traditional medical practitioners as phyto-therapy of diabetes mellitus may be due to one or more of the many arrays of chemical constituent of the plant material (i.e. seed, leave or stem etc.). These phytochemicals include saponins, alkaloids, flavonoids, tannins, terpenoids, glycosides, cyanogen, complex carbohydrates compounds and inorganic ions among numerous others. Some of these compounds might be toxic and thus, plant
containing them, when consumed could confer varied levels of toxicity, often with cytotoxic, carcinogenic effect or some other toxic properties [4].
typhoid fever, wound infection, eye illness and so on [7]. The latex of Carica papaya is used for the treatment of diarrhea, boils, hypertension, and malaria [8]. In some part South East of Africa Carica papaya leaves are still employed as natural remedy today, when taken internally is ease, when gastro
intestinal disorders such as ulcers when applied tropically to problems area. And its dried leaves have been smoked to relieve asthma [9]. Some believed that juice made from Carica papaya contains papain.
This powerful enzymes, can aid digestion by breaking down carbohydrate, proteins and wheat gluten Health food stores sells papain as a digestive aid. It is available as a pill and extract and it can also be ingested as a tea. The leaves have been also poultice into nervous pains, elephantoid growths [10]. Recently, anti-fertility, anti-helminthic and anti-inflammatory activity has been reported from Ethno botanical. Carica papaya seed possess moisture, proteins, fatty acids and phosphatide iodine and cardiolipin. Other compound present in seed and some other part are carparine, benzylisothiocynate, benzyglucosinolate, beta-sitosterol, caricain, and enzyme myrosin. The most well-studies proteinases from Carica papaya are papain [11], chymopapain, and caricain and glyclendo peptidase [12]. Papain occurs in all part of the tree except the root [10]. Fruit and seed extract have antibacterial activity against Staphylococcus aureus, Bacillus cereus, E. coli, Shigella and Pseudomonas aeruginosa [13]. While
several independent animal studies [14] have reported the anti-diabetic effect of the unripe mature fruits
of Carica papaya, there is a dearth reports on the anti- hyperglycemic & hypolipidemic effect of the leaves of this plant.
2. MATERIALS AND METHOD
2.1 Sample collection and extraction
Male and female leaves of Carica papaya were collected from Kundila Estate, Zaria road of Kano metropolis. The leaves were washed and shade dried for about two weeks after which it was pulverized using the mortar and pestle. 100g of the powdered leaf sample were soaked in 400ml of 70% Ethanol for about 72hours with regular shaking using a shaker. The content was filtered using a Whatman No1 filter paper and the filtrate was allowed to evaporate at room temperature in vacuum desiccators.
2.2 Phytochemical screening of Carica papaya leaf extract
Test for Saponin: The ability of Saponins to produce frothing in aqueous solution was used as screening test for these compounds as described by [17]. About 0.5g of each plant extract was shaken with water in a test tube. Frothing which persists on warming was taken as a preliminary evidence for the presence of Saponins.
Test for Flavonoid: To test for the presence of flavonoid, the Shinoda’s test and Sodium hydroxide test
were carried out. About 0.5g of extract was dissolved in 1-2ml of 50% methanol in the heat. Metallic magnesium and five drops of conc. HCl were added. A red or orange colour indicates the presence of flavonoid. Also few drops of aqueous NaOH were added to 5ml of extract, a yellow colouration shows the presence of flavonoid [15].
Test for Alkaloid: Few drops of picric acid solution were added to the solution of the extract. Formation
of a yellow coloured solution indicates the presence of alkaloids [16].
Test for Tannin: About 0.5g of extract was dissolved in 10ml of distilled water, and then filtered. Few
drops of ferric chloride solution were added to the filtrate. Formation of a blue-black precipitate indicates hydrolysable tannins and green precipitate indicates the presence of condensed tannin
Test for Cardiac Glycoside: The Salkowsky’s test was used for the determination of Cardiac glycosides.
0.5g of the extract was dissolved in 2ml of chloroform H2SO4 acid was carefully added to form a lower
layer. A reddish brown colour at the interface indicated the presence of cardiac glycoside [16].
Test for Anthraquinones (Borntrager’s test): 0.5g of each plant extract was shaken with 10ml benzene
filtered and 5ml of 10% ammonia solution added to the filtrate. The mixture was shaken and the presence of a pink red or violet colour in the ammoniacal (lower) phase indicated the presence of free hydroxyl anthraquinone [15].
Test for Cyanogenic Glycosides: To test for the presence of Cyanogenic glycosides, powdered plant
materials moistened with water was placed in a test tube and sodium picrate paper is suspended above by trapping the top edge between the cork and the tube and allowed to stand for about 30 minutes [18].
Test for Reducing Sugars: To the clear solution of the extract dissolved in water was heated with 5ml
equal volumes of Fehling solution A and B. formation of a red precipitate of Cu2O indicate the presence
Test for Steroid & Triterpenes (Liberman-Burchards test): Equal volume of acetic anhydride was
added to the extract, 1ml of concentrate H2SO4 acid was added down side the tube. The colour change was
observed immediately and later red, pink or purple colour indicates the presence of triterpenes while blue or blue-green indicates steroids [15].
Test for Phlobatannins: Deposition of a red precipitate when an aqueous extract of the plant part was
boiled with 1% aqueous HCl acid was taken as evidence for the presence of phlobatannins [15].
Test for Phenolic Compound: 0.5g of plant materials was boiled in5ml of water and filtered. To the
filtrate, a few drops of ferric chloride were added. A green or blue-green precipitate shows the presence of phenolic compounds [15].
2.3 Animals
Mice of weight between 20 – 26g were procured from National Veterinary Research Institute Vom, Jos Nigeria. The animals were allowed to acclimatize to laboratory conditions of Kano, Nigeria for a period of two weeks before the commencement of the experiment.
2.4 Acute toxicity study
The method of Lorke [19] was used. Nine mice divided into three groups of three animals each were administered with Carica papaya ethanol leaf extract (CPELE) intra peritoneal with doses 10mg/kg, 100kg/mg and 1000mg/kg respectively in the first phase of the experiment and observed for 24 hours for signs of toxicity and death. In the absence of toxic signs, the second phase is made up of another three groups with one mouse per group unto which the extract was administered at dose levels 1600mg/kg, 2700mg/kg and 5000mg/kg. The animals were also monitored for signs of toxicity for 24 hours. The volume of the CPELE administered was determined by its weight and required dose as follows:
Volume administered in ml = Weight of animal (Kg) × Targeted dose (mg/kg) Concentration of the extract (mg/ml)
2.5 Diabetic study
Twenty five albino mice were divided into five equal groups. Groups I was taken as the normal control. The other groups were given intraperitoneal injection of Alloxan monohydrate (150mg/kg) each and watched for 72 hours for development of diabetes which was confirmed by measuring the blood glucose from the tail vein using a Glucometer. Any mice with a blood glucose level of 200mg/dl was confirmed and included in the study. Animals in Group II are the diabetic control, Group III (diabetic treated with standard drug glibenclamide), Group IV (diabetic treated with 250mg/kg CPELE) and Group V (diabetic treated with 500mg/kg CPELE).
The animals were given feed and water ad libitum with CPELE administration for 28 days while the weight and the blood sugar level taken on weekly bases using a Metlar balance and Glucometer (AccuCheck) respectively. The animals were sacrificed under light anesthesia and blood collected from the heart into a plain specimen bottle, centrifuged to harvest the serum for biochemical analysis.
2.6 Statistical analysis
The results are expressed as a mean ± standard deviation. The statistical comparisons were made using Student t’ test and p-value <0.05 was considered significant.
3. RESULT AND DISCUSSION
Table 2 (a-b) shows the result of the first and second phase of the acute toxicity study of the male Carica papaya extract (CPELE). The result indicates the safety of the extract and so could be administered in
therapeutic dose without the fear of any form of damage to the tissues or any side effects. The LD50 is
estimated to be greater than 5000mg/kg body weight. According to Saidu et al [21], any compound or drug with the intra-peritoneal LD50 estimate greater than 1000mg/kg could be considered of low toxicity
and safe. Arising from this documented fact, Carica papaya extract at dose of 5000mg/kg could considered relatively safe on acute intra-peritoneal exposure.
Table 1: Result of Phytochemical Screening of Carica papaya leaves extract
Phytochemical constituent Males leaf Extract Female leaf Extract
Saponins - +
Flavonoids ++ +
Alkaloids + +
Tannins ++ +
Cardiac Glycoside + +
Steroids + +
Triterpenes + +
Cyanogenic glycosides
Reducing Sugar
- -
+ +
Yield (%) 67 70
Key: + = Present; ++ = Highly Present; - = Absent
Table 2a: Acute toxicity (LD50) determination of ethanol extract of male Carica papaya leaf (Phase 1).
Dose (mg/kg) Mortality Index (n = 3)
10 0/3 100 0/3 1000 0/3
Key: Numerator = Number of death
Table 2b: Acute toxicity (LD50) determination of ethanol extract of male Carica papaya leaf (Phase 2).
Dose (mg/kg) Mortality Index (n = 1)
5000 0/1
Key: Numerator = Number of death
Table 3 presents the effect of Carica papaya extract on the blood glucose level of experimental mice over a period of 4weeks. There is a significant reduction (P<0.05) in the blood glucose level of Groups III, IV and V when compared to the Diabetic Untreated mice (Group II). It could be noticed that the group treated with 250mg/kg dose of Carica papaya extract (CPELE) exhibited the most steady decrease and potency in reducing the blood sugar level of the mice even much better than the standard drug glibenclamide. The effect of the ethanol leaf extract on the lipid profile of alloxan-induced diabetic mice is presented (Table 4). Serum Total Cholesterol (TC) and Triglyceride (TG), LDL and VLDL of the treated groups (Group III, IV and V) shows a very significant reduction (P<0.05) compares with the diabetic untreated group while the HDL shows a significant increase. All these parameters fall in agreement with experimental anti-diabetic models [22, 23].
The observed significant reduction in serum concentration of triglycerides, total cholesterol and cholesterol fraction could also be due to depressed hepatic gluconeogenesis by CPELE, although this claim remains a speculation until studies are carried out on the pathway. A positive relationship between gluconeogenesis and lipogenesis has been well documented in literature [24]. Any drug that interferes with gluconeogenesis has also been reported to also interfere with lipogenesis.
There is also a significant difference (P<0.05) in the artherogenic and coronary risk indices of the
different treatment groups compares to the diabetic control group (Group II). The Artherogenic Index shows the likelihood of manifestation of artherogenic development and /or complication. Coronary Risk Index is the propensity or potency of development of heart related complication. The lower values of these indices in the groups treated with different doses CPELE shows that it has a very good hypolipidemic effect also better than the control and the glibenclamide treated groups; the higher dose (500mg/kg) manifest this effect more.
Table 3: Effect of Carica papaya extract on Blood Glucose Level of experimental mice
Treatment Groups
(N=5)
Blood Glucose Level (mg/dl)
Week 0 Week 1 Week 2 Week 3 Week 4
Group I Normal Control
Group II Diabetic Untreated
222.3 ± 4.5 240.0 ± 8.5 263.5 ± 10.5 266.0 ± 8.5 279.5 ± 8.6
Group III
Diabetic + Glibenclamide
226.7 ± 5.2 191.7 ± 12.1a 158.7 ± 9.3b 134.4 ± 6.2b 115.2 ± 9.4c
Group IV Diabetic+250mg/kg
CPELE
221.4 ± 6.4 180.5 ± 10.4b 158.4 ± 5.0b 125.5 ± 4.5c 106.1 ± 3.0c
Group V Diabetic +500mg/kg
CPELE
224.8 ± 4.6 179.7 ± 7.8c 159.7 ± 8.0c 121.3 ± 9.3c 101.7 ± 8.9c
Values are expressed as Mean ± SD of 5 mice in each group
Significantly different compared to the diabetic untreated group a (p<0.05), b (P<0.01) and c (0.001).
Table 4: Effect of Carica papaya extract on Lipid profile in Alloxan induced diabetic mice
Parameter (mg/dl) Experimental Groups
I II III IV V
Total Cholesterol 100.1±0.3b 138.7±0.5 90.2±3.9a 71.0±1.0b 62.7±2.3c
Triglycerides 100.5±1.5a 129.0±2.1 81.9±9.9a 73.7±4.8a 60.5±3.4c
LDL-Cholesterol 56.8±7.2a 78.9±3.5 51.7±3.2a 19.7±4.6c 12.3±3.6c
HDL-Cholesterol 25.1±0.8a 20.2±3.1 29.0±2.3a 39.1±5.1b 48.4±5.2c
VLDL 19.1±1.2c 42.9±4.9 13.2±2.8c 12.3±1.6c 9.6±1.2c
Atherogenic Index (AI) 2.26±1.4a 3.95±0.6 1.79±0.2b 0.53±0.16c 0.37±0.03c
Coronary Risk Index
(CRI)
4.11±0.1a 6.93±0.1 3.11±0.2c 1.82±0.04c 1.12±0.05c
Values are expressed as Mean ± SD of observations. a, b and c are statistical significant considerate at (P<0.05), (P<0.01) and (P<0.001) respectively when compared with diabetic untreated group.
Coronary Risk Index (CRI) = Total- Cholesterol HDL-Cholesterol
4.CONCLUSIONANDRECOMMENDATION
This study has been able to demonstrate the anti-diabetic potentials of Carica papaya in diabetic mice. Hence, the use of male Carica Papaya leaves for the control of diabetes has been validated by this approach, which seems to yield promising value for the development of potent phyto-therapy for diabetes mellitus without the side effects or toxicity of hypoglycemic orthodox drugs. Further scientific evaluation is needed to elucidate the mechanism of action, particularly the bioactivity guided transaction, isolation, identification and enzymatic study of constituent of the plant extract responsible for the observed pharmacological activities. The effects of this plant on the organs (histopathological studies) need to be explored on scientific base.
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