PHYTOCHEMICAL SCREENING AND ANTIDIABETIC ACTIVITY OF METHANOLIC EXTRACT OF CAYLUSEA ABYSSINICA LEAVES  

Ali Gamal Al-Kaf1image, Nwali  Onubuiwe Nelson2image, Ugwudike Patrick O2image, Amadi Peace N3image, Egbuji Jude Victor4image, Sandra Oluchi Okolie3image, Idoko Alexander2image 

1Sana'a University-Faculty of Pharmacy, Yemen.

2Department of Biochemistry, Faculty of Natural Sciences, Caritas University, Amorji – Nike, P.M.B. 01784, Enugu, Nigeria. 

3Department of Microbiology, Faculty of Natural Science, Caritas University, Amorji Nike P.M.B. 01784, Enugu, Nigeria. 4Biology Unit, Faculty of Natural Sciences, Caritas University, Amorji – Nike, P.M.B. 01784, Enugu, Nigeria.

 

ABSTRACT 

Introduction:  Controlling type 2 diabetes with a treatment having no side effects remains a challenge for researchers even if the side effects are reduced and there may be a chance for reduced adverse reactions or severe side effects due to drug interaction. These interactions could result from concurrent use of dietary supplements or pharmacological therapy in addition to the medications. The current reading's objective was to assess the qualitative and quantitative phytochemical analyses, and antidiabetic potentials of methanol leaf extract of Caylusea abyssinica (C. abyssinica) in diabetic rats. 

Methods:  The qualitative and quantitative phytochemical analyses of total alkaloids phenol, and flavonoids were determined using the well-known test procedure outlined in the literature. The obtained C. abyssinica methanol leaf extract was used to examine in vitro anti-diabetic activity (α-Amylase inhibition), oral acute toxicity, and in vivo anti-diabetic activity against streptozotocin-induced diabetes rat model. Alkaloids, carbohydrates, reducing sugars, saponins, phenolic compounds, glycosides, tannins, and flavonoids were all established in the samples after phytochemical examination.

Results: The amount of phenolics in the methanol leaf extract was (295.50 mg/g), with flavonoids coming in second (136.66 mg/g) and alkaloids coming in third (11.23 mg/g). The extract's ability to inhibit α-amylase was investigated. The study's findings show that the chosen plants had significant in vitro anti-diabetic effect. Up to 2000 mg/kg given over 14 days, the methanol extract's acute toxicity trials did not reveal any harmful effects. Rats were given streptozotocin (60 mg/kg; i.p.) to induce diabetes, and glibenclamide (500 mcg/kg body weight) was utilized as the usual medication. Body weight, HDL, total protein, SGOT, SGPT, cholesterol, blood sugar levels, and triglycerides were all assessed in this study. Comparing diabetic rats to normal (control) rats, blood sugar and total protein, SGOT, SGPT, cholesterol levels, and triglyceride concentrations were all considerably (p<  0.001) reduced after oral administration of methanol extract of C. abyssinica at doses of 100 and 200 mg/kg and increased the level of HDL and body weight. 

Conclusion: As a result of the aforementioned findings, it can be said that C. abyssinica methanol leaf extract significantly reduces blood sugar levels in streptozotocin-induced diabetic rats.

Keywords: Antidiabetic activity, α- Amylase inhibition, Caylusea abyssinica, Glibenclamide, Phytochemical analysis, Streptozotocin.

 

INTRODUCTION

 

Diabetes mellitus is the primary basis of, blindness, renal failure, strokes, lower limb and heart attacks amputations in adults around the world. In most developed countries, it ranks as the fourth leading cause of mortality. According to the International Diabetes Federation, 330 million people worldwide are expected to have diabetes by the year 2025, with Africa and Asia potentially experiencing the biggest increases. Developing nations will see this numerical growth. By 2025, more than 75% of diabetes would reside in developing countries, up from 62% in 19951. Diabetes is treated using insulin as well as a number of oral antidiabetic drugs such glinides biguanides, and sulfonylureas. One of the key areas of research is the hunt for more powerful and secure hypoglycemic agents because many of them have a lot of major side effects2. Orally consumed herbs and plants by experimental animals have been said to have hypoglycemic properties3,4,5. According to the WHO, more than 1200 plant species are used to treat diabetes mellitus worldwide, and a sizable number of these plants shown beneficial hypoglycemic action during laboratory testing6. Certain medicinal plants have recently been shown to be effective in treating diabetes worldwide and have been empirically used in anti-hyperlipidemic7 and anti-diabetic therapy. Finding new anti-diabetic drugs made from natural plants is still intriguing even if there are more than 400 plant species with hypoglycemic activity described in literature.This is because these plants include chemicals that have various and safe effects on diabetes mellitus. Alkaloids, terpenoids, glycosides, carotenoids, and flavonoids, among other compounds, are present in the majority of plants and well acknowledged to have antidiabetic properties8. However, research into plants hypoglycemic, antioxidant, and hypolipidemic, qualities may lead to the creation of fresh pharmaceutical treatments for the treatment of diabetes mellitus9. Streptozotocin (STZ) one of the most widely used diabetogenic drugs is, an antibiotic produced by Streptomycetes achromogenes that is inexpensive and has few side effects. When coupled with nicotinamide (NA), it is used to cause both type 1 and type 2 diabetes10. Pancreatic cells are highly selectively poisonous to it. The hexose moiety, which aids it in crossing the cell membrane via GLUT211, is responsible for selectivity. DNA is alkylated by STZ by the transfer of the CH3 group to the DNA particle, which fragments the DNA and contributes to its toxicity. This excessively activates poly (ADP-ribose) polymerase-1 (PARP-1), which reduces cellular NAD+ (the substrate of PARP), ATP, and ultimately leads to beta cell necrosis. This plant primarily rises between 1500 and 2750metres above sea level in open grasslands, meadows, byways, and rocky regions. It is found throughout Northern and Eastern Africa as well as the Mediterranean region. The plant is widespread in some Eastern African countries, including Kenya, Uganda, Rwanda, Burundi, Tanzania, Malawi, Sudan, and Ethiopia12. For instance, in Ethiopia, and Tanzania its leaves and stems are eaten alone or as vegetables. People who live in the places where the plant grows are also familiar with using it as medicine. Its leaves are used to cure amoebiasis, diabetes mellitus, and skin conditions [17-19].

Similar to this, its roots are used to treat erectile dysfunction, diarrhoea from Scabies, and gastrointestinal pain in people13. The effectiveness of C. abyssinica crude extracts against several human ailments has been the subject of some investigation. There have been reports of certain chemicals being isolated from various morphological areas of the plant14. The leaves of C. abyssinica have been shown to contain saccharopine [(2S, 2′S)-N6- (2-glutaryl) lysine], 2-aminoadipic acid and several-glutamyl peptides15. The aim of this study was to assess the quality (types), quantity (amount), and antidiabetic potential of the methanolic extract of C. abyssinica leaves in diabetic rats16.

 

MATERIALS AND METHODS

 

Plant material

Fresh undamaged leaf and fruit samples were harvested from several parts of the innermost canopies of fruiting plants  from  Emene,  Enugu,  Enugu  State  Nigeria  in June,  2022. 

Extraction 

Plant material fattening

Plant matter from C. abyssinica was crushed up and allowed to air dry at ambient temperature. Soxhlation was used to remove the substance from the shade-dried plants using petroleum ether after it had been coarsely crushed up. The substance was extracted repeatedly until it had been adequately fatted.

Extraction by soxhlation process

C. abyssinica powder that has been defatted was thoroughly extracted with methanol using the soxhlation process. The extract evaporated beyond their boiling points. The dried crude concentrated extract was weighed in order to calculate the extractive yield. When ready for analysis, it was then put into glass vials (6 x 2 cm) and stored in a refrigerator (4 °C)[26].

Phytochemical screening 

According to the protocols described, phytochemical screening was done to find any bioactive compounds. By visually seeing a colour change or the production of precipitates following the addition of specific reagents to the solution, the tests were recognized17. 

Total phenol measurement

The Folin Ciocalteu reagent was employed to calculate the total phenolic substance of the extracts. Gallic acid concentration (20-100μg/ml) was produced in CH3OH. 100μg/ml plant extract concentrations were likewise made in CH3OH, and 0.5 ml of every sample was added to the test along with 4 ml of 7.5% sodium carbonate and 2 ml of a 10 fold diluted folin Ciocalteu reagent. After parafilming the tubes, they were keep warmed at RT for 30 minutes with periodic shaking. The absorbance at 765 nm was calculated against CH3OH as a vacant. Gallic acid's conventional regression curve was utilized to calculate the content of phenol overall, and the results were given in milligrammes per gramme (mg/gm) of gallic acid [29].

Total flavonoids measurement

Rutin (20 to 100μg/ml) was produced in CH3OH at various concentrations. Test samples with a polarity of 100μg/ml or close to it were created. A sample was diluted to 0.5 ml and then added to 0.15 ml of a 5% NaNO2 solution along with 2 ml of distilled H2O. A 10% AlCl3 solution was added after 6 minutes had passed. The combination was then given 5 minutes to stand before receiving 2 ml of a 4% NaOH solution. With distilled water, the final volume was adjusted to 5ml, and then it was left to stand for an additional 15 minutes. At 510 nm, the absorbance was calculated using H2O as the reference. Rutin's standard regression curve was employed to calculate the whole flavonoid substance18.

Total alkaloids measurement

Dimethyl sulphoxide (DMSO) was employed to liquefy the plant extract (1 mg), and then 1 ml of 2 N HCl was added and drinkable. This solution was reassigning to a separating funnel, and then 5ml of phosphate buffer and 5ml of bromocresol green solution were included. The mixture was vigorously agitated with 1, 2, 3, and 4ml CHCl3 before being collected in a 10ml volumetric flask and CHCl3 was added to dilute to the volume. Atropine reference standard solutions ranging from 50to250μg/ml were created. The absorbance of the test and standard solutions in relation to the reagent blank at 470 nm was measured using a UV/Vis spectrophotometer. Mg of AE/g of extract was used to express the overall alkaloid content19.

In-vitro Anti-diabetic activity 

α- Amylase inhibition activity

Polysaccharides like starch, carbohydrates, and other polysaccharides are metabolized by the alpha amylase enzyme. Four test tubes containing a prepared (1:1) amylase solution were used. 1 cc of the solution was taken from the test tube above and kept in another test tube for testing. Four rows, one for each tube, of two drops of iodine solution were placed in the spot plate. This is then combined with 0.5 cc of 1% starch solution. The first tube received a drop of solution right away, while the second tube received a second drop of solution one minute later. The sampling was carried out every min awaiting every starch had been broken down and the tube's colour had turned light brown or vanished. Because large concentrations of amylase will digest the starch quickly and provide results, amylase concentration increases along with a corresponding rise in response rate and a corresponding decline in reaction time. As the concentration of the medication grows, so does the time it takes for a reaction, therefore glibenclamide was used as a standard and methanolic extracts of C. abyssinica served as an amylase inhibiting agent20.

Animals  

Wistar rats (180–250 g) were residence in groups of six (n=12) under regulated humidity and temperature settings (25±2 °C, 55-65%). Rats were given regular rodent food and unlimited amounts of water. Prior to the experiments, rats spent 7 days becoming used to the lab environment. Between 8:00 and 15:00 hours, all studies were conducted in a room with no background noise. Each set of studies used a different group of rats (n=6). The Institutional Animal Ethics Committee (IAEC), established by the India's Ministry of Environment and Forests, located in New Delhi to oversee and supervise the use of experimental animals, gave its approval to the animal experiments21.

Acute oral toxicity

According to Organization for Economic Co-Operation and Development (OECD) Guidelines-423, an acute toxicity investigation of the produced extracts was conducted. Animals were starved for 4 hours, but were given unrestricted access to water during that time. The beginning dose level should, in accordance with OECD standards, be the one that will most likely result in animal mortality in some of the dosed animals. When this information regarding a chemical being tested is not yet available, one of three fixed starting dose levels—5, 50, 300, or 2000 mg/kg body weight—is chosen21. 

Induction of experimental diabetes in rats

Six rats were placed in each of the different groups of separated animals. After an overnight fast, group II-V received an IP injection of STZ at a dose of 60 mg/kg body weight, melted in an O.1M sodium citrate buffer at pH 4.5. (Divest of food for 16 hours but agreed unrestricted access to H2O). The identical quantity of 0.1 M sodium citrate buffer was given to the control rats. To treat the hypoglycemia brought on by drugs, over the duration of the night, the animals had admission to 5% glucose solution to drink. By assessing blood glucose levels 72 hours after STZ injection, diabetes status was established. The study only included animals with levels of fasting blood sugar more than 250 mg/dl. Rats' body weight was recorded by an electronic balance on the first and last days of post-treatment, or pre- and post-treatment. Rats' fasting blood sugar levels were checked before and after the treatment, or on the eighth and twenty-first days following treatment.

In the current investigation, 5 groups of 5 rats every containing 6 animals were used.

Group I: Normal

Group II: Diabetes management only received STZ (negative control)

Group III: Received Glibenclamide orally for 14 days at a dose of 500 mcg/kg body weight.

Group IV: Diabetes-prone rats were given C. abyssinica leaf extract in methanol (100 mg/kg/day p.o.)

Group V: Diabetes-prone rats were given 200 mg/kg/day of C. abyssinica leaf extract in methanol.

Taking blood samples and measuring blood sugar

To assess blood glucose, blood was drawn using the tail-snipping technique. Blood was taken from a heart puncture, which is an appropriate approach for estimating various lipid profiles and biochemical markers. All through the research period, blood samples from all the (16–20 hours) overnight-fasted animals were taken in plain micro centrifuge tubes every other week while they were under anaesthesia. Centrifugation was used to extract the serum from the blood sample for 10 minutes at 4000 rpm. The automated biochemistry analyzer Hitachi-902 was used to study biochemical parameters22.

Estimation of oral glucose tolerance test

In the presence of glucose oxidize, oxygen and glucose combine to form gluconic acid and hydrogen peroxide. The dyes turn blue when H2O2 oxidizes them in a mechanism mediated by peroxide reductases. However, it was found that the glucometer was acceptable for diagnosing conditions in terms of test accuracy, requiring only a drop of blood to obtain results, and being simple to use at temperatures above 95° F. It was utilized in place of the customary and drawn-out process of other diagnostic kits23.

Statistical analysis

The mean and SEM were employed to state all the data. One-way analysis of variance was employed to evaluate the groups' statistical implication before Dunnett's t-test post-hoc analysis. P values below 0.5 were regarded as noteworthy.

 

RESULTS

 

After completing each consecutive soxhlation extraction process, the crude extracts were focused on a bath of water by totally evaporating the solvents to achieve the real extraction capitulate. Petroleum ether and methanol were found to produce extracts from plant portions called leaves with yields of 2.56 and 9.51%, respectively. Table 1 shows the results of a qualitative phytochemical analysis of the raw leaves of C. abyssinica. Alkaloids, reducing sugars, saponins, phenolic and steroidal chemicals, glycosides, tannins, and flavonoids were all detected in methanolic plant extracts, whereas protein and carbohydrates were detected in petroleum ether extracts24.

Calculating the total phenolic content in milligrammes of gallic acid equivalents per 100 milligrammes of dry sample weight. The percentage of rutin equivalent per 100 mg dry weight of sample used to represent the percentage of total flavonoids in the extracts. The percentage of atropine equivalent per 100 mg dry weight of sample used to express the total amount of alkaloids present in the extracts. 

The C. abyssinica plant's methanol extract has a high level of total phenolic and flavonoid content (Table 2). Since there is a high concentration of α-amylase present, the starch will be broken down quickly, as the concentration of α-amylase rises, so does the pace of reaction (Table 3). As glibenclamide concentration rises, the time it takes for a reaction to take place likewise rises because there are insufficient amounts of the enzyme molecules needed to break down starch (Table 4). The current work focuses on how methanolic extracts of C. abyssinica inhibit α-amylase. Increase in reaction time, or the time required by α-Amylase to digest the starch, is a sign that an extract has α-amylase inhibitory activity. According to the observations, the time it takes for a reaction to occur grows as the concentration of the extract does as well, however they are less active than regular medications. As the concentration of α-amylase rises, the rate of reaction likewise rises while the time of reaction decreases because a high concentration of amylase will swiftly digest the starch25.

The methanolic extracts of C. abyssinica were observed to have α-amylase inhibitory activity, but this activity was determined to be less than that of a conventional medication (Table 5). Up to 2000 mg/kg, the methanolic extracts of C. abyssinica did not exhibit any mortality or adverse events. As a result, the study was conducted at doses of 100 and 200 mg/kg26. When compared to the control mice throughout the research, the diabetic animals displayed a considerable loss in body weight. However, C. abyssinica methanol extract plus glibenclamide prevented the body weight loss caused by diabetes Table 6. Compared to normal animals, diabetic control animals had significant hyperglycemia. On day 0 and day 21, the average diabetic control group's blood sugar level was 290.40±7.20mg/dl and 405.20± 10.20mg/dl, respectively. On the other hand, as compared to the diabetic control group, the methanol extract of C. abyssinica at 100 mg/kg and 200 mg/kg considerably (p<0.001) lowered the fasting blood serum glucose level in the diabetic rats on the 7th, 14th, and 21st days.

The serum glucose level was shown to be dramatically decreased by the widely used medication glibenclamide, returning it to a level that was close to normal. The results are shown in Table 7. At the conclusion of the experiment, the levels in the treatment group were still within normal ranges while those in the diabetic rats' blood lipid profiles dramatically (p<0.001) rose when compared to the control animals, with the exception of HDL. Table 8 shows the impact of glibenclamide and a methanol extract of the C. abyssinica plant on the lipid profile of diabetic pigs27. Total protein levels in diabetic mice were significantly lower than in control animals, although levels were normal and total protein in C. abyssinica and glibenclamide-treated animals. Table 9 shows the effects of glibenclamide and C. abyssinica methanol extract on the liver and kidney indicators of diabetic rats.

 

DISCUSSION

 

An essential technique in the investigation of bioactive compounds is the phytochemical screening test. The process is easy, rapid, and affordable, and it provides the researcher with an immediate response to the many phytochemical kinds included in a mixture. Due to the presence of numerous components that are essential for good health, these phytochemicals looked to have the potential to serve as a source of helpful medications and to improve the health condition of the consumers.

The results of a qualitative phytochemical analysis of C. abyssinica leaves that were not treated are shown in Table 1. The human digestive tract contains a variety of enzymes that aid in food digestion. α-amylase catalyses the conversion of polysaccharides into mono-saccharides, because the stomach can only absorb foods in their monosaccharide form. It is well known that the digestive tract quickly breaks down starch into glucose. Shortly after consuming starch, a pronounced hyperglycemia that results in hyperinsulinemia is seen. Both of these undesirable processes in the patient's GIT are caused by the α-amylase enzyme, which is distributed throughout the GIT and is in charge of the metabolism or digestion of starch and carbohydrate into glucose molecules28.

The methanolic extracts of C. abyssinica were observed to have α-amylase inhibitory activity, but this activity was determined to be less than that of a conventional medication (Table 3- Table 5). The OECD criteria were followed when conducting the toxicity studies. Up to 2000 mg/kg, the methanolic extracts of C. abyssinica did not exhibit any mortality or adverse events. As a result, the study was conducted at doses of 100 and 200 mg/kg. A gram-positive bacterium called Streptomyces achromogenes produces the poisonous glycoside known as STZ. Through the glucose transporter 2 (GLUT2), it builds up in pancreatic cells and lowers their expression. Insulin-dependent diabetes is brought on by the alkylating activities of the STZ, which alter biological macromolecules, break up DNA, and kill cells29.

The diabetes control group saw a considerable decrease of body weight, which may be due to an increase in muscle wasting and a loss of tissue proteins24,30. In the current investigation, the treatment groups significantly reduced their body weight, which suggests that glibenclamide and methanolic extracts of C. abyssinica stop the muscle loss caused by hyperglycemia. Increased plasma insulin levels or improved blood glucose transport in peripheral tissue may be to blame for the drop in glucose levels26,27. Current findings demonstrate that methanolic extracts of C. abyssinica increase plasma insulin levels and have potential anti-diabetic effects. The STZ-induced diabetic hyperglycemia increases plasma levels of SGPT and SGOT, which are important indicators of liver impairment. The consequence of STZ on the liver markers was reversed in rats known methanolic extracts of C. abyssinica. This might be because the different biomarkers found in the methanolic extracts of C. abyssinica have hepatoprotective properties28. In STZ diabetic rats, there are more lipid peroxides and reactive oxygen species, which cause hyperglycemia. The continual generation of free radicals might harm tissue through the peroxidation of unsaturated fatty acids29,30. The ability of the biomarkers to scavenge free radicals may explain why animals treated with methanolic extracts of C. abyssinica showed less hyperglycemia than those treated with STZ.

 

CONCLUSION

 

In order to find and screen the phytochemical components that are essential for the creation of novel medications, medicinal plants are used. Due to the existence of the phytochemical elements, the results of the current study and the prior phytochemical examination are remarkably similar. The anti-hyperglycemic properties of C. abyssinica leaves extract were comparable to those of the standard medication used in this investigation; this might be brought about by a rise in insulin production from the pancreatic beta cells that have grown again and a reduction in α-amylase. Through a rise in HDL cholesterol, the plant also demonstrated cardio protective effects. The outcomes of this research, however, support the plant's conventional use in the management of diabetes. 

 

CONFLICT OF INTEREST 

 

No conflict of interest associated with this work. 

 

ACKNOWLEDGEMENTS

 

The authors would like to present thanks to Caritas University, Enugu, Nigeria to provide necessary facilities for this work.

 

REFERENCES 

 

  1. Eseyin O, Ebong P, Eyong E, Awofisayo O, Agboke A. Effect of Telfairia occidentalis on oral glucose tolerance in rats. Afr J Pharm Pharmacol 2010; 4: 368-372.
  2. Idoko A, Ikpe VPO, Rita ON, et al. Hypoglycemic and lipid lowering effect of aqueous fresh leaf extract of Chromolaena odorata (linn) in albino wistar rats fed different concentrations of cholesterol enriched diet. Universal J Pharm Res 2018; 3(1):37-42.http://dx.doi.org/10.22270/ujpr.v3i1.R7
  1. Abubakar SM, Umar SA, Idoko A, Abubakar N, Abdulazeez MA, Sule MS. Evaluation of hypoglycaemic, hypolipi-daemic and non toxic effect of hydro-methanolic extracts of Ziziphus mauritiana, Ziziphus spina christi fruit and glibenclamide on alloxan induced diabetic rats. J Drug Deliv Therap 2018; 8(3):82-92.https://doi.org/10.22270/jddt.v8i3.1711
  1. Idoko A, Ikpe VPO, Nelson NO, et al.  Effects of lime juice and honey on lipid profile of cholesterol enriched diet fed rat model. Annual Res Rev Biol 2017; 20(3):1-10.http://dx.doi.org/10.9734/ARRB/2017/37213
  1. Malviya N, Jain S, Malviya S. Antidiabetic potential of medicinal plants. Acta Pol Pharm 2010; 67: 113-118.PMID: 20369787
  1. Yalniz M, Pour PM. Toxicology of the endocrine pancreas. Taylor and Francis group, Boca Raton, Florida, USA, 542-545; 2006.
  2. Ghasemi A, Khalifi S, Jedi S. Streptozotocin-nicotinamide-induced rat model of type 2 diabetes (review). Acta Physiol Hung 2014; 101: 408-420.
  3. Eleazu CO, Eleazu KC, Chukwuma S, Essien UN. Review of the mechanism of cell death resulting from streptozotocin challenge in experimental animals, its practical use and potential risk to humans. J Diabetes Metab Disord 2013; 12: 60.
  4. Mwai GN, Schippers RR. Solanum scabrum. In: Grubben GJH, Denton OA, editors. Plant Resources of Tropical Africa 2: Vegetables, (PROTA). The Netherlands: Buckuys Publishers; 2004:493–501.
  5. Grubben GJH, Denton OA. Phytochemical studies revealed the presence of plant resources of tropical Africa. Vegetables. alkaloids, terpenes, flavonoids, polyphenols, saponnins, PROTA Foundation, Wageningen; Backhuys, steroids and tannins and absence of anthraquinones and Leiden. Wageningen: CTA; 2004:165.
  6. Martin-Bravo S, Meimberg H, Luceno M, et al. Molecular systematics and biogeography of Resedaceae based on ITS and trnL-F sequences. Mol Phylogenet Evol 2007; 44:1105–20.
  7. Etana B. Ethnobotanical study of traditional medicinal plants of Goma wereda, Jimma Zone of Oromia Region, Ethiopia (M. Sc Thesis). Ethiopia: Addis Ababa University; 2010.
  8. Abebe D, Debela A, Uga K. Medicinal plants and other useful plants of Ethiopia. Singapore: Camerapix; 2003.
  9. Chidi E, Nwobodo NN, Paul NAC. Anti-hyperglycemic and anti-oxidant activities of methanol extract of Gongronema latifolium. Universal J Pharm Res 2017; 2(2): 12-15.http://doi.org/10.22270/ujpr.v2i2.R4
  1. Tolossa K, Etana D, Spiridoula A, Adugna T, Gebeyehu G, Jos GMH. Ethno-medicinal study of plants used for treatment of human and livestock ailments by traditional healers in South Omo Southern Ethiopia. J Ethnobiol Ethnomed 2013; 9:32.http://dx.doi.org/10.1186/1746-4269-9-32
  1. Osman OA, Saeed AA, Mousnad MA, HamidA. Assessment of the risk of type 2 diabetes among healthy without diabetes in Sudan using the findrisc tool. Universal J Pharm Res 2020; 5(4):37-41. http://dx.doi.org/10.22270/ujpr.v5i4.438
  2. Mesfin F, Demissew S, Teklehaymanot T. An ethnobotanical study of medicinal plants in Wonago Woreda, SNNPR, Ethiopia. J Ethnobiol Ethnomed 2009; 5:5–28.
  3. Mahadeva Rao US. Phytochemical screening and in vitroantioxidant and anti-diabetic potentials of Persea Americana (lauraceae) fruit extract. Universal J Pharm Res 2018;3(5):34-41. https://doi.org/10.22270/ujpr.v3i5.200
  4. Celine T, Yee CJ, Aslam MS. The relationship between diabetes mellitus and tuberculosis in review of prevalence, diagnostics and prevention. Universal J Pharm Res 2019; 4(4):43-46. https://doi.org/10.22270/ujpr.v4i4.300
  5. Olsen O, Sørensen H. 4-Carboxy-4-hydroxy-2-aminoadipic acid and other acidic amino acids in Caylusea abyssinica. Phytochem 1980; 19:1717–21.
  6. Baljic R, Velic A, Muhic A, Gojak R, Gazibera B, Bajramovic N, Hadzovic–Cengic M, Huric I. Clinical course and disease outcome in covid-19 patients with diabetes mellitus. Universal J Pharm Res 2021; 6(2):42-45.https://doi.org/10.22270/ujpr.v6i2.567  
  1. Gadekar S, Goyal S, Khan A, Jha M. Chemopreventive action of Sphaeranthus indicus on dmba-induced skin papillomagenesis in mice. J Adv Sci Res 2020; 11(3):161-167.
  2. Ngounou EMD, Mang YD, Dongmo F, Malla OWI, Dongmo SS, Yanou NN. Effect of the aqueous extract of Clerodendrum thomsoniaelinn (verbenaceae) leaves on type 2 diabetic wistar rats induced by the MACAPOS1 type diet and dexamethasone. Universal J Pharm Res 2021; 6(3):9-16. https://doi.org/10.22270/ujpr.v6i3.601
  3. Salim Mirza, Sayed Ayaz Ali, Indrajeet Sanghvi. Evaluation of methanolic extract of Euphorbia neriifoliastem bark on blood sugar levels, serum and tissue lipids in a preclinical model. Universal J Pharm Res 2017; 2(3): 1-5.http://doi.org/10.22270/ujpr.v2i3.R1
  1. Al-Baoqai N, Al-Mahbashi H, Al-Adhal A. Antidiabetic and antihyperlipidemic activity of Dracaena cinnabari Resin ethanolic extract of Soqatra Island in Experimental Animals. Universal J Pharm Res 2018; 3(5):1-10.https://doi.org/10.22270/ujpr.v3i5.194
  1. Guideline Document on Acute oral Toxicity Testing, Series on Testing and Assessment No. 423. Paris: Organization for Economic Co-Operation and Development, OECD Environment, Health and Safety Publications; 1996.
  2. Jonsson M, Jestoi M, Nathanail AV, Kokkonen UM, Anttil, M, Koivisto P, Peltonen K. Application of OECD Guideline 423 in assessing the acute oral toxicity of moniliformin. Food and chemical toxicology 2013; 53: 27-32.
  3. Annadurai T, Muralidharan AR, Joseph T, Hsu MJ, Thomas PA, Geraldine P. Antihyperglycemic and antioxidant effects of a flavanone, naringenin, in streptozotocin‑nicotinamide‑ induced experimental diabetic rats. J Physiol Biochem 2012; 68:307‑18.
  4. Cheng D, Liang B, Li Y. Antihyperglycemic effect of Ginkgo biloba extract in streptozotocin‑induced diabetes in rats. Biomed Res Int 2013; 2013:162724.http://dx.doi.org/10.1155/2013/162724
  1. Wilcox G. Insulin and Insulin Resistance. Clin Biochem Rev 2005; 26:19‑39.