{"id":27723,"date":"2019-06-25T11:34:16","date_gmt":"2019-06-25T11:34:16","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=27723"},"modified":"2020-04-23T03:52:57","modified_gmt":"2020-04-23T03:52:57","slug":"antidiabetic%e2%80%8e-activity-of-terfezia-claveryi-an-in-vitro-and-in-vivo-study","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol12no2\/antidiabetic%e2%80%8e-activity-of-terfezia-claveryi-an-in-vitro-and-in-vivo-study\/","title":{"rendered":"Antidiabetic\u200e Activity of Terfezia Claveryi; An In Vitro and In Vivo Study"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Diabetes is considered one of the world\u2019s largest endocrine disease, that characterized by an increased blood glucose level (hyperglycemia). Clinically, Diabetes is classified as type-1(T1DM) characterized by insulin deficiency and type-2 (T2DM) characterized by insulin inefficiency. Uncontrolled diabetes could lead to severe complications to the cardiovascular system 1<\/sup>. Natural products have aided humans since long ages. They are considered as sources of important active ingredients. In comparison with synthetic drugs, synthetic one may cause many drawbacks such as vomiting, diarrhea, fluid retention, allergic reaction2<\/sup>. Recently, the International Diabetes Federation (IDF) 7th edition of the Diabetes Atlas specified that 415 million people worldwide is diabetics3<\/sup>. T2DM represents about 90-95% of all cases of diabetes 4<\/sup>. T2DM is considered one of the main international health concerns. T2DM affects around 422 million people all over the world5<\/sup>. Prediabetes and diabetes prevalence and complications are growing in a bothersome way. By year of 2035, it is anticipated that about 592 million people will suffer from DM6<\/sup>. The treatment of T2DM is currently achieved through the usage of conventional drugs that are effective in treatment of diabetes but to some extents still accompanied by some undesirable effects7<\/sup>. The management of diabetes is considered a global problem and the search for a definite therapy is still ongoing.\u00a0 Truffle is a fungus, which grows wildly in desert regions depending on water rainfall8<\/sup>. In addition, many researches stated that truffle can be used in many purposes such as source of energy, activation of sex hormones, and as antibiotics against gram positive bacteria including Bacillus subtilis<\/em> and Staphylococcus aureus<\/em> 9-11<\/sup>. Terfezia boudieri<\/em> ethanol extract showed anti-hyperglycemic effect \u200e on streptozotocin (STZ) induced-diabetic rats 8<\/sup>. \u00a0Currently, there are no research studies were conducted to investigate the in vitro and in vivo <\/em>antidiabetic potential of Terfezia claveryi<\/em> \u200e<\/em>. The previously mentioned data provoked us to assess \u200e the \u03b1-amylase inhibitory activity and effect on inhibition of glycosylation of hemoglobin as well as in vivo<\/em> studies in streptozotocin-induced diabetic rats to evaluate and confirm its potential hypoglycemic effect.<\/p>\n

Material and Methods<\/strong><\/p>\n

Plant Material<\/strong><\/p>\n

Terfezia claveryi (T. claveryi)<\/em> was purchased from a local folk market in spring season, Al-Hasa, eastern region of Saudi Arabia. The fungus was subjected to air-drying according to the standard protocols. T. claveryi<\/em> \u200e was kindly identified by Dr. Mamdouh Shokry, director of El-Zohria botanical garden, Giza, Egypt. A voucher specimen was kept in Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Hasa, Saudi Arabia (03-17-Apr-TC).<\/p>\n

Extraction and Fractionation of Different Plant Organs Extracts<\/strong><\/p>\n

The air dried powdered material (500.0 g) was exhaustively extracted three times at room temperature (for 5 days) using 3l of 70 % MeOH\/H2<\/sub>O applying cold maceration technique at room temperature to protect the potential active ingredients from being decreased or destroyed. The solvent mixture was removed through distillation under vacuum using Rota vapor and dried extracts were directly freeze-dried to give the total methanol extract weighting 60.2 g that were kept in -20o<\/sup>C for the next steps 12<\/sup>.<\/p>\n

Animals<\/strong><\/p>\n

Male Wistar albino rats having a weight of 150 \u2013 210 g were kept in quarantine for 2 weeks under standard husbandry conditions (27o<\/sup>, Relative humidity 65\u00b110 %) for 12 h in dark and light cycle, respectively, and were given standard food and water ad libitum <\/em>13<\/sup>. All of the experiments were done in this study according to the Animal Ethics Committee of King Faisal University.<\/p>\n

Chemicals<\/strong><\/p>\n

Acarbose, glibenclamide, streptozotocin, metformin, gentamycin, \u03b1-amylase from porcine pancreas, hemoglobin porcine and alpha-tocopherol were purchased from Sigma Aldrich (ST. Louis. Mo, USA). Solvents used for extraction and assays were all of analytical grade.<\/p>\n

In Vitro<\/em><\/strong> Anti-Diabetic Models<\/strong><\/p>\n

\u03b1-Amylase Inhibitory Activity <\/strong><\/p>\n

The assay mixture was prepared to contain 0.02M sodium phosphate buffer (200 \u03bcl), \u03b1-amylase enzyme \u200e(20 \u03bcl, 2 unit\/ml) \u200e together with different plant extracts in the range of concentrations 20-100 \u03bcg\/ml. Then, it was incubated for 10 min at room temperature followed by the addition of 200 \u03bcl of 1 % starch suspension to all the tubes containing reaction mixture. The reaction was later terminated by the addition of 400 \u03bcl of 3, 5 di-nitro salicylic acid (DNSA) color reagent. Then the tubes were kept in boiling water bath for 5 minutes, and later were kept till being cooled at room temperature and diluted with 15 ml of distilled water. The absorbance of each reaction mixture was measured at 540 nm. Control mixture reactions were also prepared accordingly without addition of extracts of plant under investigation and were compared with the test samples containing concentration of different plant extracts (20-100 \u03bcg\/ml) freshly prepared in DMSO. The results were indicated as % of inhibition of activity using the following formula:<\/p>\n

\"Formula<\/p>\n

where; Abs (control) is the absorbance of the control reaction (containing all reagents except the test sample) and Abs (sample) is the absorbance of different plant extracts14,15<\/sup>. The IC50<\/sub> values (inhibitory concentration which will produce 50 % inhibition of the enzyme activity) of the plant extracts were determined. Acarbose which is a well-known and safe anti-diabetic drug used to treat T2DM, was applied as a positive control in the concentrations ranged from 20 to100 \u200e\u00b5g\/ml\u200e16<\/sup>. Experiments were achieved in triplicates<\/p>\n

Non-Enzymatic Glycosylation of Hemoglobin Assay <\/strong><\/p>\n

Solutions of glucose (2 %), hemoglobin (0.06 %) and gentamycin (0.02 %), were freshly prepared in phosphate buffer (0.01 M, pH 7.4). One ml of each of above mentioned solution was mixed. One ml of each concentration of different plant extracts (20-100\u03bcg\/ml) was added to the prepared mixture. Then, the test tubes containing reaction mixture were incubated in dark place at room temperature for three days. After, the degree of glycosylation of hemoglobin was obtained colorimetrically at 520 nm where the percentage of inhibition was calculated applying this formula:<\/p>\n

\"Formula<\/p>\n

where; Abs (control) is the absorbance of the control reaction (containing all reagents except the test sample) and Abs (sample) is the absorbance of different plant extracts. The IC50<\/sub> values (inhibitory concentration which will produce 50% inhibition of the enzyme activity) of the plant extracts were determined. Alpha-Tocopherol was used as a standard drug14-16<\/sup>. Experiments were carried out in triplicates<\/p>\n

In Vivo<\/em><\/strong> Anti-Diabetic Model<\/strong><\/p>\n

Acute Toxicity Testing<\/strong><\/p>\n

Acute toxicity testing was performed for T. claveryi<\/em> total methanol extract, were studied where the rats took ascending oral doses up to 2000\u2009mg\/kg of each extract, and signs and symptoms of toxicity were observed for the next 48 h 17<\/sup>.<\/p>\n

Induction of Diabetes<\/strong><\/p>\n

Diabetes was induced by intraperitoneal (i.p.) injection of streptozotocin (STZ) dissolved in 0.1\u00a0M cold citrate buffer (pH=4.4) at a dose of 60\u00a0mg\/kg body weight. On the third day after STZ injection, fasted blood glucose levels were measured by hand-held glucose monitoring (BAYER Contour). Only rats with serum glucose levels of 190-200\u2009mg\/dl were selected and considered diabetic animals 18<\/sup>.<\/p>\n

Experimental Design<\/strong><\/p>\n

The animals were segregated into five groups of five rats each. Group I served as normal control rats, administered drinking water and 0.1\u00a0M cold citrate buffer (pH=4.4) daily for 12 d; Group II had diabetic control rats, administered drinking water daily for 12 days; Group III diabetic rats were administered T. claveryi<\/em> total methanol extract (200 mg\/ kg) for 12 d; and Group IV diabetic rats were administered standard drug glibenclamide (0.25 mg\/kg) for 12 d. The fasting glucose levels were determined on days 1, 5, and 12 of extracts administration 3, 17, 18<\/sup>.<\/p>\n

Statistical Analysis<\/strong><\/p>\n

Values were expressed as mean\u00b1SE\u200e (Standard Error) \u200e. To analyze the differences between groups, statistical analysis was performed by one-way ANOVA followed by post-hocTukey using a computer soft program SPSS v.20. Significance was considered at a p value <0.05.<\/p>\n

Results<\/strong><\/p>\n

\u03b1-Amylase Inhibitory Activity <\/strong><\/p>\n

The in vitro<\/em> \u03b1-amylase\u200e<\/em> inhibitory measurements demonstrated that T. clavery<\/em> total methanol extract has potential of \u03b1- amylase inhibitory possessions. \u03b1-amylase inhibitory activities were compared based on the calculated IC50<\/sub> values (Table 1). The observed \u03b1-amylase inhibitory activity of T. claveryi<\/em> total methanol extract was (\u200e<\/em>38.7 \u00b5g\/ml). Acarbose was used as the positive standard. It showed IC50<\/sub> value of \u200e<\/em>45.3\u200e<\/em>\u00b5g\/ml\u200e<\/em> under similar conditions.<\/p>\n

Table 1: \u03b1- amylase inhibitory effect of T. claveryi<\/em> total methanol extract.\u200e<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
<\/td>\npercentage of inhibition<\/strong><\/td>\n<\/tr>\n
conc. \u00b5g\/ml<\/strong><\/td>\nT. <\/em>claveryi<\/em> \u00a0methanol\u200e extract<\/strong><\/td>\nstandard (Acarbose)\u200e<\/strong><\/td>\n<\/tr>\n
20<\/td>\n\u200e17.1\u00b1\u200e0.9<\/td>\n\u200e32.2\u00b1\u200e1.1<\/td>\n<\/tr>\n
40<\/td>\n\u200e28.0\u00b11.1<\/td>\n\u200e43.8\u00b1\u200e1.3<\/td>\n<\/tr>\n
60<\/td>\n\u200e54.2\u00b1\u200e\u200e 1.3<\/td>\n\u200e64.9\u00b1\u200e2.3<\/td>\n<\/tr>\n
80<\/td>\n\u200e59.5\u00b1\u200e1.1<\/td>\n\u200e75.5\u00b1\u200e1.4<\/td>\n<\/tr>\n
100<\/td>\n\u200e68.4\u00b11.7<\/td>\n\u200e81.1\u00b11.3<\/td>\n<\/tr>\n
IC50<\/sub> \u00b5g\/ml<\/td>\n38.7<\/td>\n45.3<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Values were expressed as mean \u00b1 SE (Standard Error) n=3 independent experiments.<\/p>\n

Non-Enzymatic Glycosylation of Hemoglobin Assay<\/strong><\/p>\n

The inhibitory activities of T. claveryi<\/em> total methanol extracts were recorded (Table 2). <\/em>T. claveryi<\/em> total methanol extract showed almost the same value of IC50<\/sub> (33.1 \u00b5g\/ml) to the positive control, alpha-tocopherol (\u200e35.4 \u200e\u00b5g\/ml).<\/p>\n

Table 2: Non-enzymatic glycosylation of hemoglobin effect by T. claveryi<\/em> total methanol extract.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
<\/td>\npercentage of inhibition<\/strong><\/td>\n<\/tr>\n
conc. \u00b5g\/ml<\/strong><\/td>\nleaves methanol extract<\/strong><\/td>\nstandard (alpha-Tocopherol)\u200e<\/strong><\/td>\n<\/tr>\n
20<\/td>\n24.4\u00b11.2<\/td>\n38.8\u00b10.5<\/td>\n<\/tr>\n
40<\/td>\n28.5\u00b10.3<\/td>\n49.3\u00b10.6<\/td>\n<\/tr>\n
60<\/td>\n34.6\u00b11.3<\/td>\n71.6\u00b10.6<\/td>\n<\/tr>\n
80<\/td>\n44.2\u00b11.5<\/td>\n81.0\u00b11.0<\/td>\n<\/tr>\n
100<\/td>\n50.5\u00b10.5<\/td>\n82.7\u00b11.6<\/td>\n<\/tr>\n
IC50<\/sub> \u00b5g\/ml<\/td>\n33.1<\/td>\n35.4<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Values were expressed as mean \u00b1 SE (Standard Error, n=3 independent experiments.<\/p>\n

Acute Toxicity Study<\/strong><\/p>\n

No toxicity or death was observed in the experimental rats. Hence 200\u2009mg\/kg (1\/10 of the 2000\u2009mg\/kg) was selected as a maximum safety dose.<\/p>\n

In vivo<\/em><\/strong>\u00a0antidiabetic activity<\/strong><\/p>\n

The effect of T. claveryi<\/em> total methanol extract \u200e <\/em>on fasting blood glucose levels of diabetic rats was presented in table 3. In diabetic rats, as shown in table 3\u200e<\/em>, T. claveryi<\/em> total methanol extract and glibenclamide had a significant time dependent hypoglycemic activity, compared with the diabetic control group at each time point (p<0.001).<\/p>\n

Table 3: Results of the in vivo<\/em> study on STZ-induced diabetic rats by T. claveryi <\/em>total methanol extract.<\/strong><\/p>\n\n\n\n\n\n\n\n\n
Groups<\/strong><\/td>\nFasting plasma glucose concentration (mg\/dl)<\/strong><\/td>\n<\/tr>\n
Day 1<\/strong><\/td>\nDay5<\/strong><\/td>\nDay12<\/strong><\/td>\n<\/tr>\n
I- Normal control \u200e<\/td>\n\u200e79.9\u00b11.2 \u200e \u200e\u200e<\/td>\n80.9\u00b10.8<\/td>\n\u200e81.6\u00b11.1<\/td>\n<\/tr>\n
II- Diabetic control\u200e (streptozotocin)\u200e \u200e(55\u2009mg\/kg)\u00a0 \u200e<\/td>\n196.8\u00b12.4<\/td>\n\u200e198.18\u00b11.6<\/td>\n200.3\u00b12.5<\/td>\n<\/tr>\n
III- Diabetic + leaves methanol extract \u200e(200 mg\/kg)<\/td>\n197.9\u00b11.9<\/td>\n138.6\u00b11.6*<\/td>\n122.1\u00b13.0*<\/td>\n<\/tr>\n
IV- Diabetic + standard glibenclamide (0.25 mg\/kg)<\/td>\n196.5\u00b11.5<\/td>\n91.38\u00b11.1*<\/td>\n79.4\u00b11.4*<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Values were expressed as mean \u00b1 SE (Standard Error), (n=6), *significantly different from diabetic control (p<0.001).<\/p>\n

Discussion<\/strong><\/p>\n

\u03b1-Amylase enzyme is one of the enzymes responsible for the hydrolysis of \u03b1-oriented bond polysaccharides and oligosaccharides such as starch, glycogen and other macromolecules of \u03b1-bond linked monosaccharides to disaccharides and finally to glucose19-22<\/sup>. T. claveryi<\/em> total methanol extract \u200e<\/em> showed promising result in \u03b1-amylase inhibition assay, suggesting that T. claveryi <\/em>might be effective in slowing down hydrolysis of starch to minimized glucose availability.<\/p>\n

In vitro<\/em> non-enzymatic glycosylation of hemoglobin method is one of important assays to judge the control of diabetes. The hemoglobin present in RBCs has an affinity to bind to glucose. The greater the glucose level in blood, more amount of glucose-bound (called glycosylated) hemoglobin will be formed. Such glucose hemoglobin association is to some extent stable and stays for1-2 months (the life-span of red blood corpuscles) 22, 23<\/sup>. Consequently presence of higher concentration of glycosylated hemoglobin is a sure guide to the higher concentration of glucose in the blood. Normally, the percentage of glycated hemoglobin should not be exceeding 12%. The current study demonstrated good activity of T. claveryi<\/em> total methanol (almost the same that of positive control, alpha-tocopherol) in preventing such binding of glucose to surface proteins of erythrocytes.<\/p>\n

The fundamental mechanism underlying elevated blood sugar in diabetes mellitus involves over-production and decreased utilization of glucose by the tissues. In the current study, the difference observed between the initial and final fasting plasma glucose levels of different groups under investigation, revealed a significant elevation in blood glucose in the diabetic control group as compared to normal animals, at the end of the twelve-day experimental period. When T. claveryi<\/em> total methanol was administered to diabetic rats, a decrease in plasma glucose level was observed after 12 days. T. claveryi<\/em> total methanol reduced plasma glucose (Table 3). During the study it was found that T. claveryi<\/em> total methanol significantly controlled the blood glucose level in Streptozotocin-induced diabetic rats as compared to the diabetic control group (Table 3).<\/p>\n

Conclusion<\/strong><\/p>\n

The above conducted in vitro<\/em> examinations depict a substantial \u03b1-amylase inhibitory and percentage of inhibition glycosylation of hemoglobin of T. claveryi<\/em> total methanol. Which was further confirmed by in vivo<\/em> studies that showed T. claveryi<\/em> total methanol significantly controlled the blood glucose level diabetic rats. It could be therefore conclude from this study that T. claveryi <\/em>can serve as a therapeutic agent and can be used as a potential source of new antidiabetic product.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

There is no conflict of interest.<\/p>\n

References<\/strong><\/p>\n

    \n
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    Introduction Diabetes is considered one of the world\u2019s largest endocrine […]<\/p>\n","protected":false},"author":9,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[69],"tags":[],"class_list":["post-27723","post","type-post","status-publish","format-standard","hentry","category-vol12no2"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/27723","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/9"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=27723"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/27723\/revisions"}],"predecessor-version":[{"id":32065,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/27723\/revisions\/32065"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=27723"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=27723"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=27723"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}