Antidiabetic Effects of Crude Flavanoid and Alkaloid of Abrus precatorius Linn Seed in Alloxan Diabetic Rabbits.

Monago Comfort C.¹* and Nwodo O. Fred C.²

¹Department of Biochemistry, Faculty of Sciences, University of Port Harcourt, Choba, Rivers State, Nigeria

²Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria

ABSTRACT:

The antidiabetic effects of the crude flavonoid (FV) and alkaloid (AK) of Abrus precatorius seed were studied in alloxan diabetic rabbits. The effects were compared to that of chlorpropamide (CP) a known antidiabetic drug and a diabetic control group (CO). Blood glucose level drawn before the alloxan injection was 1.11+ 0.04, 1.33+0.06, 1.14 + 0.04, and 1.24+0.02 x 10² mg/dl for FV, AK, CP and CO groups respectively. When 50mg/kg body weight of FV, AK, CP and 5ml of normal saline for CO were given orally, blood glucose level decreased in FV, AK and CP groups but not in CO group. The percentage reductions in blood glucose levels were 35.3, 44.1, 72.0, 69.7, 56.1, 67.7 and 61.6% for FV and 53.40, 116.4, 165.6, 116.4, 104.4, 125.20 and 116.4% for AK after 05, 10, 20, 30, 40, 60 and 168 hrs of oral administration of FV and AK respectively. The percentage increase in glycemic level in CO was in the range of 0.8 and 4.0%. When the blood glucose reduction of FV was compared to that of CP at p<0.05, significant differences were observed after 05, 10, 30, 60 and 168hrs of oral administration. Significant induction of diabetes by alloxan at p<0.05 was observed for all the groups used in the study. The flavoniod and alkaloid of Abrus precatorius seed were able to reduce the blood glucose level in alloxan diabetic rabbit and could be tested further for implementation in herbal formulations.

KEYWORDS: Alloxan; Diabetes mellitus; Alkaloids; Flavonoids; Abrus precatorius.

INTRODUCTION:

Diabetes mellitus is a global problem. The world health organization has predicted that the numbers of diabetic patients will double 143 million in 1997 to about 300 million in 2025 largely because of dietary and other lifestyle factors (WHO, 1998; Siedell, 2000). Type 2 diabetes is associated with many other diseases like renal, coronary, eye, neurological and vascular diseases (Miller, 1991). Also diabetes leads to obesity and other genetic problems (Wolf and Colditz, 1998).

Many flavonoids and alkaloids from plants have been used in treatment of diabetes. These are epicatechin from Pterocarpus marsupium (Manickan et al., 1997): ginkgo-flavone consisting of ginkgolides and bilobalides (Kleijnen and Knipschild, 1992) and a sulphur containing flavone from Allium cepa (Festrow and Avila, 1999). Also flavonoid extracts of plants like Carqueja (Dickel, 2007), Bridelia ferruginea leaves, roots and bark (Iwu, 1983), and Vaccinium mytillus (blueberry) (Festrow and Avila, 1999).

Cinnamon has been in the news lately because of its effects on improving insulin, blood sugar and blood lipid metabolism for the past 20 years. In 2004, cinnamon’s bioactive compound polyphenol type-A polymer was identified (Anderson, 2004). Daily amounts of 1, 3, or 6 grams of cinnamon reduced glucose by 18-29% following 40 days of treatment; a continued reduction in glucose levels even at the 60-day mark with 16% reduction was observed (khan, 2003).

Hence treatment with herbal drugs has an effect on protecting β-cells and smoothing out fluctuation in glucose levels (Jia et al., 2003; Elder, 2004). In general, there is very little biological knowledge on the specific modes of action in the treatment of diabetes, but most of the plants have been found to contain substances like glycosides, alkaloids, terpenoids, flavonoids etc., that are frequently implicated as having antidiabetic effects (Loew and Kaszkin, 2003). Members of fabeceae have been used in management of glycemic index and various diseases, we, therefore deem it necessary to extract the flavoniods and the alkaloids of this plant and see how it can be used in management of diabetes mellitus.

MATERIALS AND METHODS:

Sample Preparation: Fresh seeds of Abrus were collected from Nsukka, Nigeria. The seeds were cleansed and ground with a high speed blender (Philips - Mexico) . The ground seed was stored dry and used throughout the work. A known weight of the seed was soaked in chloroform-methanol (2:1) and extracted for 18 hrs in a container on a flask shaker (Gallen Kamp). The mixture was filtered and the filtrate was extracted with equal volume of water and evaporated to dryness. A known weight of the extract was subjected to column and thin layer chromatography using Sephadex LH₂₀ (Sigma) and silica gel (sigma) respectively. Elution was done with a mixture of chloform:methanol (2:1and 1:1) and finally with 95% methanol at a flow rate of 2mls/5mins and 150 fractions were collected. The elution pattern was monitored using thin layer chromatography. Fractions were tested for the presence of flavonoid and alkaloid using UV light, ammonium solution and Draggendoff’reagent.

The fractions that showed the presence of flavonoid were pulled together while those that showed positive alkaloidal test were pulled together and used as crude flavonoid (FV) and alkaloidal (AK) extracts respectively(figs. 1.0 , 2.0 and 3.0). Chlorpropamide(CP) (Pz) a known antidiabetic drug was bought from the pharmaceuticals and used as control. The drug was ground into powder and 50mg/kg body weight was given orally.

Treatment of the Animal: The rabbits were treated according to the Ethical Guidelines of the Animal Center, University of Nigeria, Nsukka and the experimental protocol was approved by the Animal Studies Committee of University of Nigeria, Nsukka.

Four groups of male healthy rabbits (n=3) with average weight of 1.6kg were used. Food, water, ambient temperature and proper ventilation were allowed throughout the work. Normal glucose level of all the rabbits was determined before alloxan injection. Alloxan (120mg/kg body weight) was injected intraperitoneally to all the rabbits. They were allowed for 72 hrs for full development of diabetes. After 72 hrs hyperglycemic glucose levels were determined. Then oral administration of 50mg/kg body weight of the FV, AK and CP were given to groups A, B and C respectively while group D served as control and received 5ml of normal saline instead of the extract.

Determination of Blood and Determination of Blood Glucose Levels:

Blood was drawn after 05, 10, 20, 30, 40, 50, 60 and 168hrs of oral administration of FV, AF, CP and CO. The blood was drawn from the ear vein of the rabbit and transferred to NaF/Oxalate bottles. Blood was centrifuged at 2000g for 10 minutes. Blood glucose levels were determined using O’ toliudine method of Frings et al. (1970).

Statistical Analysis:

Statistical analysis was done using a two way ANOVA with SPSS. Values were considered significant at p<0.05.

RESULTS:

Table 1.0: Antidiabetic Effects of flavonoid (FV) and Alkaloidal (AK) Extracts of Abrus precatorius seed in Alloxan Diabetic Rabbit.

	Blood Glucose Conc. Before Alloxan Injection (mg/dl)	Hyperglycemic Conc. After 3 Days of Alloxan Injection. (mg/dl)	Hours after Oral Administration of AF.
			05	10	20	30	40	60	168
GRP A -FV Ext Blood Glucose Levels (x 10^2 mg/dl)	1.11+ 0.04^f	2.86+ 0.13^f	1.85+ 0.04^fa	1.60+ 0.13^f	0.77+ 0.08 ^fab	0.86+ 0.07^fab	0.98+ 0.05^fab	1.00+ 0.09^fab	1.19+ 0.06^fb
Reduction from Hyperglycemic level (x 10^2 mg/dl)	1.74	0	1.01	1.26	2.08	1.99	1.60	1.84	1.75
% Reduction (%)	61.2	0	35.5	44.1	72.9	69.7	56.1	67.7	61.6
GRP B AK- Ext. Blood Glucose Conc. (x 10^2 mg/dl)	1.33 +0.06^f	2.46 +0.01^f	1.92 +0.07^f	1.29+ 0.05^fa	0.80+ 0.05^fab	1.29+ 0.03^fa	1.41+ 0.02^fa	1.20+ 0.05^fa	1.29+ 0.01^fa
Reduction from Hyperglycemic level (x 10^2 mg/dl)	1.13	0	0.53	1.16	1.65	1.16	1.04	1.25	1.16
% reduction (%)	51.1	0	21.7	47.3	67.3	47.3	42.4	51.2	47.3
GRP C- CP (Drug) Blood Glucose Conc. (x 10^2 mg/dl)	1.14 + 0.04^f	2.34 + 0.02^f	2.02+ 0.05^fa	1.58+ 0.04^f	0.92+ .09^fab	1.08+ .03^fab	1.24+ .02^fab	1.26 + .03^fab	1.26+ .02^fab
Reduction from Hyperglycemic level (x 10^2 mg/dl)	1.19	0	0.32	0.75	1.41	1.25	1.10	1.08	1.08
% Reduction (%)	50.5	0	13.8	32.3	60.3	53.5	46.8	46.2	46.2
GRP D- CO (Normal Saline) Blood Glucose Conc. (x 10^2 mg/dl)	1.24 +0.02^f	2.38+0.07^f	2.35+ 0.03^a	2.28 +0.12	2.37 +0.10^a	2.35 +0.02^a	2.30 +0.04^a	2.32 + 0.07^a	2.30+ 0.05^a
Decrease in Hyperglycemic level (x 10^2 mg/dl)	1.14	0	2.8	9.99	0.01	1.80	7.20	5.20	7.60
% Decrease in hyperglycemia	48.0	0	1.2	4.0	0.1	0.8	3.0	2.3	3.2

Values represent Mean+ Standard error of mean of three samples in a group (n=3), ^arepresents significant difference (p<0.05) when GRP D was compared to GRPs A, B and C, vertically, ^brepresents significant difference (p<0.05) when GRP C was compared to GRPs A and B, vertically, ^f represents significant difference (p<0.05) when the hyperglycemic level was compared to other glucose levels horizontally.

Table 1.0 shows the blood glucose levels before and after alloxan induced diabetes in male rabbits. The normal blood glucose levels before alloxan injection were 1.11±0.04, 1.33±0.06, 1.14±0.04 and 1.24±0.02 x 10² mg/dl for FV, AK, CP and CO respectively. Alloxan diabetes significantly increased the blood glucose level from a normal blood glucose range of 1.11±0.04 - 1.33±0.06 x 10² mg/dl to a hyperglycemic range of 2.34±0.07-2.86±0.13 x 10² mg/dl. After 5 hrs of administration of FV, AK and CP the blood glucose level significantly reduced (p<0.05). The reduction in glucose level continued till 10 hrs for Fv extract but not for CP. The reduction continued after 20, 30, 40, 60 and 168 hrs for all the groups. Group A pattern of reduction of blood glucose significantly resembled that of chlorpropamide except after 12 and 168hrs which were not significant. AK significantly reduced the hyperglycemic level after 20hrs of oral administration. The normal blood glucose level was quickly attained by FV than AK and CP. The reduction of glucose in the control group was not significant as the glycemia continued to increase. The percentage increase was in the range of 0.1-4.0%. When FV group was compared to CO group at p<0.05, significant difference was observed at 05, 20, 30 and 60hrs of FV administration. The highest percentage reduction of blood glucose (72.9%) was attained by FV, followed by AK (67.3%) and CP (60.3%). These high percentage reductions in glucose level were coincidentally achieved at the same time, after 20hrs of each administration.

DISCUSSION:

Many botanical supplements have been used as therapeutic agents in the management of diabetes. The FV and Ak of Abrus precatorious were able to reduce blood glucose in alloxan diabetic rabbit. The highest glucose reduction for FV, AK and CP were 72.9, 67.3 and 60.3% respectively, after 20hrs of oral administration of 50mg/kg body weight of each sample. Many flavonoids extracted from plants have been used in the treatment of diabetes. The Carqueja plant has been used in South America as a natural aid for diabetes, and several studies confirm its blood sugar-lowering effect in mice, rats, and humans (in both normal and diabetic subjects) (Dickel, 2007). The major blood glucose lowering effect is as a result of the flavoniod content. Carqueja has been documented to lower blood glucose levels in human and animal studies (Oliveria, 2005). A standard infusion is prepared with 5 g (about a teaspoon) of dried herb to 4-6 ounces water and infused for 10 minutes. Another plant with flavonoind as its active ingredient against diabetes is Ginkgo. Extracts from dried leaves of Ginkgo are used in complementary therapies. Active ingredients include flavonoids (ginkgo-flavone glycosides) and terpenoids, consisting of ginkgolides and bilobalides (Kleijnen and Knipschild, 1992)

Ginkgo biloba is one of the most widely used drugs in Germany. In diabetes, ginkgo biloba is use in ameliorating peripheral circulatory problems, such as intermittent claudication (Pittler and Ernst, 2000).

Flavonoids and alkaloids are phenolic compounds. The possible mechanism of action of these extracts may be in their effects on receptors and blood vessels. They may have the ability to bind to these receptors or active sites of glucose regulating enzymes. Epicatechin of Pterocarpus marsupium was able to decrease fasting blood glucose, postprandial blood glucose and glycated hemoglobin by 0.4%. This flavonoid was proved to have effect on lipid levels, gastrointestinal glucose absorption and insulin like glucose action (Menickam et al., 1997). In the current study, it was observed that flavoniods had significant reduction in blood glucose resembling that of chlorpropamide a first generation sulfornylurea. The reduction in glucose was such that the reduction was significantly below the normal glucose level entering into hypoglycemia which is one of the adverse effects of sulfornylureas. The concentration of CP used in the study seems adequate and did not develop into hypoglycemia ie reducing the blood glucose below the normal level.

Bilberry is another plant whose flavonoids is already used in preparation of antidiabetic drugs (Jellin et al., 1999). In folk medicine, it is used as a "blood sugar–reducing" drug, and is therefore a common constituent in "antidiabetic" teas, (Wichtl, 1994). It decreases vascular permeability and redistribute microvascular blood flow (Jellin, et al., 1999). The bioflavonoids are the chemical constituents in bilberry fruit thought to be responsible for some of its vascular effects. Vascular disease associated with altered vascular reactivity is a major complication of diabetes. Several reports have shown that both endothelium-dependent and -independent vasodilation are impaired in this disease (Yugar-Toledo et al., 2004). This reduction in vascular reactivity has been attributed to decreased endothelial nitric oxide (NO) synthesis (Yugar-Toledo et al., 2004), increased NO degradation (Bagi et al., 2003), and/or abnormalities in vascular smooth muscle (Yugar-Toledo et al., 2004). The flavone glycosides, including quercetin, kaempferol, and isorhamnetin, are thought to have antioxidant activity and inhibit platelet aggregation. The ginkgolides are thought to improve circulation and inhibit the platelet-activating factor. The bilobalides are thought to have neuroprotective properties (Jellin et al., 1999; Kleijnen and Knipschild , 1992).

Alloxan, a beta cytotoxin, induces "chemical diabetes" (alloxan diabetes) in a wide variety of animal species by damaging the insulin secreting pancreatic β-cell, resulting in a decrease in endogenous insulin release, which paves the ways for the decreased utilization of glucose by the tissues (Omamoto et al., 1981). Similar observation was made in the present study. Alloxan increased the blood glucose level by 48-62%.

The possible mechanism of action of extract could be correlated with the reminiscent effect of the hypoglycemic sulphonylureas that promote insulin secretion by closure of K+-ATP channels, membrane depolarization and stimulation of Ca2+ influx, an initial key step in insulin secretion. In this context, number of other plants has also been reported to have antihyperglycemic and insulin stimulatory effects (Venkateswaran and Pari, 2002; Latha and Pari, 2003). Like the plant extract, glibenclamide also produced significant reduction in blood glucose levels of alloxan diabetic rats.

Since alloxan is known to destroy pancreatic β-cells, the present findings appear to be in consonance with the earlier findings that sulphonylureas have extra- pancreatic antihyperglycemic mechanism of action secondary to their insulin secreting effect and the attendant glucose uptake into, and utilization by, the tissues.

The effects of the flavonoid and alkaloid of Abrus compare well with other active constituents of different plants, like alkaloid and pectins from Coccinia indica (Hossain et al., 1992) alkaloids from Tinospora cordifolia (Prince et al., 2002), trigonelline and scopoltin from Trigonella foenum graecum (Jachak, 2002), alkaloid-6-methoxybenzoxazolinone and terpenoids such as scoparic acids A,B,C and scopadulcic acid A and B from 'scoparia dulcis (Pari and Latha , 2004), which may be responsible for scavenging free radicals liberated by alloxan in diabetic rats.

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Received on 07.03.2010

Accepted on 17.04.2010

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Research Journal of Pharmacognosy and Phytochemistry. 2(4): July-Aug. 2010, 331-335