Abstract

Secondary metabolites of medicinal plants constitute a real alternative treatment for diabetes mellitus. They are also sources of natural compounds that could lead to the discovery of new drugs. In this study, the anti-amylase and antioxidant activities of alkaloids and flavonoids extracted from Mitracarpus hirtus leaves were evaluated. DPPH radical and DNS inhibition methods were used for antioxidant activity and anti α-amylase activity, respectively. The results obtained showed that di- and tri-glycoside flavonoids and monoglycosides are the most present with contents of 796 and 522 mg RQE/gES while alkaloids are present at 203 mg RQQ/gES. The DPPH radical inhibitory activity test gives IC₅₀ of 187.99; 236 ug/ml for the di and tri glycosides and monoglycosides flavonoids respectively. Alkaloids show IC₅₀ of 815ug/ml and ascorbic acid used as reference has IC₅₀ of 110 ug/ml. The result of the anti α-amylase activity shows that the monoglycoside flavonoids are more active with an IC₅₀ of 74 ug/ml while the alkaloids have an IC₅₀ of 228 ug/ml. We note that in all the studied parameters, flavonoids are more active than alkaloids. However, the activity of flavonoids is not polarity dependent. The fractions rich in flavonoid mono, di and tri glycosides present rather interesting activities and deserve to be tested in vivo in order to propose them as an alternative treatment for diabetes mellitus.

1. Introduction

Mitracarpus hirtus is a seasonal plant belonging to the Rubiaceae family. It is widely distributed in tropical and subtropical regions. In Senegal this plant grows wild only during the rainy season. Phytochemical screening carried out on the leaves of this plant showed the presence of several families of chemical compounds including flavonoids and alkaloids ¹.

Alkaloids are a large group of phytochemical compounds containing one or more nitrogen atoms (heterocycles) and widely distributed in the plant kingdom. Based on their biosynthetic precursor and heterocyclic ring system, alkaloids have been classified into different categories including indole, tropane, piperidine, purine, imidazole, pyrrolizidine, pyrrolidine, quinolizidine and isoquinoline type alkaloids ². These compounds have shown a wide spectrum of therapeutic utility in several diseases ³. Thus miglitol (anti-diabetic); galantamine and rivastigmine (anti-alzheimer); apomorphine (anti-parkinson); capsaicin (analgesic) are all derived from alkaloid-based metabolites ^{4, 5, 6}.

While flavonoids are part of the polyphenol group and play an important role in antioxidant activity. Indeed, antioxidants inhibit free radicals generated in many pathologies such as cancer, inflammatory diseases, diabetes, cardiovascular diseases and Alzheimer's etc ⁷. Flavonoids are highly present in plants and so far, more than 9000 flavonoids have been reported and their daily intake varies between 20 and 500 mg depending on the source of the food supplement ⁸. They have a basic skeleton consisting of two 6-carbon rings (rings A and B) connected by an oxygenated heterocyclic of three carbon atoms (ring C). The main subgroups are flavanols, flavonols, flavones, isoflavones, flavanones, flavanonols and anthocyanins which are defined by the functional groups attached to the basic structure of flavonoids ⁹. They are often in glycosylated or esterified form showing their extreme diversity. According to the literature, more than 800 medicinal plants have a potential to be used in the management of diabetes and the antidiabetic properties of these plants are confirmed in more than 450 experimental bioassays ¹⁰. The bioactive molecules isolated from these plants consist largely of alkaloids and polyphenols. The latter in the context of diabetes management present several modes of action. Thus, three main mechanisms are described for flavonoids, according to their actions on islet β-cells. Firstly, they play a protective role against β-cells ¹¹, secondly, they participate in the increase of islet β-cell proliferation ¹² and finally they participate in the preservation of insulin signaling by increasing insulin secretion ¹³. While synthetic or natural alkaloids with antidiabetic activity act mainly by increasing insulin secretion in the pancreas, decreasing glucose release from the liver, reducing gastrointestinal absorption of carbohydrates, and improving peripheral glucose disposal ¹⁰. For example, acarbose, voglibose, and miglitol are widely used for inhibition of intestinal α-glucosidase, whereas these drugs have no direct effect on insulin production. In contrast, other sulfonylurea-based antidiabetic drugs such as glimepiride and glyburide directly increase insulin hormone secretion while having no or minor effects on α-amylase activity ³.

The aim of this work was to study in vitro the anti-α-amylase and antioxidant activity of alkaloid and flavonoid extracts isolated from Mitracarpus hirtus leaves. Based on the results to be obtained in this study, the isolation of active compounds and the analysis of their modes of action on diabetes and associated parameters will be considered.

2. Materials and Methods

The method of isolation of flavonoids depends on the type of flavonoids present in the plant material. The protocol used to extract flavonoids was carried out according to the method described by SAIDI et al ¹⁴ with some modifications. A mass 25.2g of Mitracarpus hirtus leaf powder was macerated in 100 mL of a hydro-ethanol mixture (30/70). The obtained mixture was stirred for 72 hours before being filtered to recover the organic substances and especially the flavonoids. The volume of the filtrate obtained is adjusted to 290 mL with boiling water. The mixture is left to settle for 24 hours and then filtered to remove impurities. This final mixture is then extracted with organic solvents of different polarity.

• Petroleum ether (3 times 100ml) is used to extract flavonic aglycones.

• Ethyl acetate (3 times 100ml) to extract mono glycosides.

• Butan-1-ol (3 times 100ml) allows to extract di and tri glycosides and C-glycosides.

The different organic phases thus obtained are evaporated to dryness using a rotary evaporator.

A mass of 14.3g of Mitracarpus hirtus leaf powder was first wetted with 22 mL of NaOH (0.5N) for 24 h at room temperature. Then maceration was performed with 300 mL of chloroform for 48 hours at room temperature. After vacuum filtration, the filtrate was stirred with an acidified aqueous solution (5% dilute sulfuric acid), the alkaloids solubilized in the aqueous phase as salts while the neutral impurities remained in the organic phase. After basification of the aqueous solution at pH 9-10 with NaOH solution (0.5 N), it was extracted with chloroform (3 times 100 mL). The resulting organic phase was washed with distilled water to neutral pH, dried with potassium sulfate (K2SO4) and then concentrated to dryness under reduced pressure to give the crude.

For the identification of the different chemical groups by thin layer chromatography (TLC), the method described by Bekro et al was used ¹⁵. The identification of the two families of chemical compounds present in the leaves of M. hirtus was done by staining and precipitation tests.

For flavonoids, a Methanol/Water (20/80) mixture was used as eluent. The stationary phase was silica gel. The revelation is done with aluminum chloride and the observation under UV at 254 nm. The yellow coloration indicates the presence of flavonoids. Thus, several luminescences with various colorations at room temperature under protected from light could be observed. The Rutin molecule was used as a reference against the different flavonoid samples obtained (Butanol, Ethyl acetate and Petroleum ether).

To identify alkaloids from M. hirtus leaves, silica gel is used as stationary phase and chloroform/ethyl acetate (30/7) mixture as eluent. The control used is quinidine. The revelation is done with Dragendorff's reagent. The red and orange-red coloration shows the presence of alkaloids in the extracts.

The quantification of flavonoids in the extracts of M. hirtus leaves was done according to the colorimetric method of aluminum trichloride ¹⁶.

The principle of this colorimetric assay is based on the nitration with NaNO₂ in alkaline medium of the aromatic ring bearing a catechol group. Therefore¸ after the addition of aluminum trichloride (AlCl₃), a yellow colored complex is formed, which later immediately turns red after the addition of sodium hydroxide (NaOH).

This assay was carried out by adding a volume of 250µl of the extract solution with a concentration of 1000µg/ml in 75µl of a NaNO2 solution (5%). The mixture was incubated for 6 minutes at room temperature, to which 150µl of a 10% aluminum chloride (AlCl3) solution was then added. A second resting time (5 minutes) is necessary before adding 500µl of NaOH (1M). The final volume is brought to 2500µl with distilled water. Then the final mixture obtained is incubated for 30min before reading in the spectrophotometer. The blank is made by replacing the extract with distilled water. The absorbances are measured at 510 nm using a UV-visible spectrophotometer. The results are expressed in mg rutin equivalent/g of dry extracts by referring to the calibration curve of rutin.

The determination was done by the spectrophotometric method described by Sreevidya et al ¹⁷ with some modifications. An amount of 1mL of extract solution was taken and the pH was maintained between 2 and 2.5 with dilute HCl. Then 0.4mL of Dragendorff’s reagent was added to it and the precipitate formed was centrifuged (3000 rpm) until complete settling. The precipitate was washed with methanol and then treated with 0.4mL of di-sodium sulfate solution (0.5M). The brownish-black precipitate formed was then centrifuged. Completion of the precipitation was checked by adding 2 drops of disodium sulfate. The residue was dissolved in 0.4mL of concentrated nitric acid, warming if necessary and then diluted to 10mL with distilled water. A volume of 0.5mL of this diluted solution was taken and 2.5mL of thiourea solution (5g/l) was added. The absorbances were read with a spectrophotometer at 435 nm against the blank tube prepared under the same conditions by replacing the extract with distilled water. The standard curve was made from a stock solution of quinidine at 5mg/L with a concentration range from 0.3125 to 5 mg/mL.

The measurement of the anti-radical activity of flavonoids and alkaloids extracted from the leaves of Mitracarpus hirtus is performed by the 2,2'-diphenyl-1picrylhydrazyl (DPPH) assay following the method described in our previous study ¹. The activity of the polar extracts was evaluated by determining the IC₅₀ of the samples which was compared to that of ascorbic acid, used as a reference sample. The percentage of DPPH radical scavenging is calculated by the following formula.

Where: %. I: percentage of inhibition; Ac: control absorbance; Ae: absorbance of extracts.

It is possible to deduce from the IC₅₀ values, the effective concentration (EC₅₀) and the anti-free radical power (ARP). The EC₅₀ or 50% effective concentration is defined as the quantity of antioxidant necessary to reduce the initial DPPH concentration by 50%. The EC₅₀, expressed in gram of extract per mole of DPPH was calculated according to the following formula.

"EC50 = IC50 (µg/mL) / MDPPH (µmol/mL) MDPPH = molarity of the DPPH solution. The anti-radical power (ARP) corresponds to the inverse of the effective concentration "ARP = 1 / EC₅₀". It measures the anti-free radical effectiveness of the product concerned. The higher its value, the greater the anti-free radical power of the product.

The anti-α-amylase activity of flavonoid and alkaloid extracts from M. hirtus was performed using a modified procedure of Waleed et al ¹⁸ and described in our previous study ¹. The inhibitory activity of α-amylase was calculated by considering the following equation.

3. Results

The different results obtained during this study are given below. They are respectively the results of phytochemical characterization, determination of alkaloid and flavonoid contents in M. hirtus leaves, antioxidant and anti-α-amylase activities of alkaloids and flavonoids extracted from M. Hirtus leaves.

On the left, flavonoids (R = rutin; Bu= n-butanol fraction; AE: ethyl acetate fraction; EP: petroleum ether fraction), on the right, alkaloids (Quin = quinidine, Ext = extract).

3.2.2. Alkaloid Content Determination of M. hirtus Leaves

IC₅₀ values were determined from plots of percent inhibition versus concentration of extracts using their respective linear regression equations. All tests were performed in triplicate.

4. Discussion

Diabetes mellitus is a chronic disease of glucose metabolism and affects a significant proportion of the world's population. A significant reduction in hyperglycemia would significantly reduce the complications associated with this disease. Conventional treatments play an important role in the management of diabetes, but unfortunately, they are often not accessible to everyone and also cause numerous side effects. This is why plant-based treatments are becoming more and more popular. It is often less toxic and available with satisfactory results if followed by professionals. The present study aimed to evaluate the antioxidant and anti-α-amylase effect of flavonoids and alkaloids extracted from M. hirtus leaves. The presence of these two secondary metabolites in M hirtus leaves was demonstrated using specific reagents for each compound family (Table 1) in comparison to commercial reference compounds rutin and quinidine respectively.

Table 2 and Table 3 show the content of these compounds in the leaves of M. hirtus. Thus, the different flavonoids presented in Table 2 show a strong presence of di and tri glycosides (796 mg RQE) while flavonoid mono glycosides and aglycones are present at 522 and 288 mg RSE respectively. On the other hand, alkaloids are present at 203 mg QQE in M hirtus leaves. These results show that the leaves of M. hirtus are richer in flavonoids, these are 8.3 times more important than alkaloids. And that di and tri glycosides are more present among the extracted flavonoids.

The results of the antioxidant activity show that flavonoids are more active in the inhibition of the DPPH radical than alkaloids (Table 3). Indeed, di and tri glycosides (IC₅₀=187 µg/ml) and monoglycosides (IC₅₀=236 µg/ml) are respectively 4 times and 3 times more active than the alkaloids extract. The ascorbic acid used as reference has an inhibition concentration of 115 µg/ml. Flavonoids belonging to the polyphenol family have the ability to easily release a hydrogen from their hydroxyl group to scavenge the DPPH radical ¹⁹. This could explain the higher antioxidant activity of flavonoids compared to alkaloids. It has been described that the pain experienced during many pathologies is partly related to the production of reactive oxygen species (ROS), oxidative stress and inflammatory factors. Antioxidants are one of the most important biological compounds that protect the body from endogenous and exogenous oxidant hazards ²⁰. According to the recorded results, flavonoid glycosides have the most interesting antiradical powers among the tested compounds. Their EC₅₀ are 4.69 and 5.92 relatively lower than that of ascorbic acid, whose EC₅₀ is equal to 2.76.

The results of anti-α-amylase activity of alkaloids and flavonoids extracted from M. hirtus leaves are given in Table 5. They show that alkaloids (IC₅₀=4379 µg/ml) are significantly less active on the in vitro inhibition of α-amylase activity. The monoglycoside flavonoids were the most active with an IC₅₀=74 µg/ml followed by the di and tri glycoside extract (IC₅₀=228 µg/ml). On the whole on the two parameters studied in vitro, we note that flavonoids are more active than alkaloids. And that the activity of flavonoids is not always polarity dependent. For the flavonoids of the more polar butanol extract are less active on α-amylase inhibition than the flavonoids of the ethyl acetate extract.

Two enzymes play an important role in the increase of blood glucose in diabetics. It is the α-glucosidase which catalyzes the digestion of polysaccharides and oligosaccharides in monomers and thus causes a hyperglycemia. While α-amylase plays an important role in the digestion and absorption of starch and glycogen in the small intestine ²¹. One of the strategies in the herbal treatment of type 2 diabetes is the management of hyperglycemia by inhibiting these two enzymes.

Thus, some flavonoids may act indirectly on diabetes, for example those of Citrus are described among others as compounds able to modulate lipid metabolism and adipocyte differentiation, suppress inflammation and apoptosis and improve endothelial dysfunction, indicating potential antidiabetic effects ²². Glucose absorption from the intestine is mediated by two specific transporter enzymes (SGLT1 and GLT2) ²³. Some authors have shown that ferulic, tannic and chlorogenic acid polyphenols were able to inhibit the activity of the SGLT1 enzyme ²⁴ while others have demonstrated in vitro inhibition of the GLT2 enzyme by quercetin, myricetin and apigenin ²⁵. Inhibition of the activity of these transport enzymes would reduce blood glucose levels and consequently the development of type 2 diabetes and its complications.

Natural alkaloids of plant origin marketed for the treatment of pathologies are estimated to be more than fifty and present variable bioavailability parameters depending on their nature ²⁶. In addition to the effect on carbohydrate digestive enzymes, plant alkaloids have also shown inhibitory activity against the enzyme "human aldose reductase". This enzyme is a monomeric cytosolic NADPH-dependent oxidoreductase that catalyzes the reduction of a variety of aldehydes and/or carbonyls and has been implicated in diabetic neuropathy ²⁷. Dineshkumar et al showed that mahanimbine, koenimbine and kurryam alkaloids have direct effects on blood glucose levels through a stimulatory effect on insulin secretion by pancreatic cells ²⁸. According to Morrish et al ²⁹, diabetes has become a major risk factor for cardiovascular disease and in turn CVD has become the leading cause of death in people with diabetes.

5. Conclusion

Diabetes is a real public health problem in all countries of the world. The use of medicinal plants as an alternative treatment for diabetes is gaining momentum in developing countries where access to conventional treatments is not available to everyone. Alkaloids and flavonoids extracted from the leaves of Mitracarpus hirtus with high contents, presented in vitro interesting activities on the inhibition of DPPH radical and α-amylase, hyperglycemic enzyme. Further studies on the isolation and characterization of flavonoid compounds more active than alkaloids on α-amylase and antioxidant activity are now warranted. This will also allow the continuation of the bioguided study already started since the evaluation of noisy extracts and fractions on the same targets.

Competing Interests

Authors have declared that no competing interests exist.

References