High performance liquid chromatographic profiling of antioxidant and antidiabetic flavonoids purified from Azadirachta indica (neem) leaf ethanolic extract

Introduction

Neem is an evergreen, temperature tolerant, flowering plant, native to the whole Indian subcontinent, which can be found growing in a lot of tropical and semi-tropical countries located in the equatorial belt. Botanically it is classified as a member of the family Meliaceae, under the latinised name Azadirachta indica A. Juss (A. indica). This name derives from the Persian words “azad” (free), “darakht” (tree) and “i-hind” (of Indian origin), which literally mean “the free tree of India” [1]. All part of the neem tree (root, bark, leaves, flowers, seeds and fruits) are used to treat or prevent a wide range of diseases [2]. Neem leaves are considered the most versatile part of the plant and contain several natural compounds, showing many biological activities, exploited to treat various diseases and disorders, by including sexually transmitted disease pathogens, HIV/AIDS and cancer [2], [3].

Flavonoids have existed for over one billion years in vegetables and possess wide spectrum of biological activities [4]. These polyphenolic compounds are present both in the aqueous and ethanolic leaf extract of neem [5]. In particular, the content of total flavonoids in different crude extracts prepared from neem leaves is in the range of 61.5–529.5 mg/100 g powder sample [6]. Some studies show that the ingestion of flavonoids reduces the risk of cardiovascular diseases, metabolic disorders, and certain types of cancer. These effects are due to the physiological activity of flavonoids in the reduction of oxidative stress, inhibiting low-density lipoproteins oxidation as well as platelet aggregation, and acting as vasodilators in blood vessels [4]. Dini et al. found that the hydro-alcoholic extract of A. indica leaves prevents streptozotocin-induced intestinal ileal and pancreatic islet lesions, ameliorating the hepatic glycogenosis, pancreatic and liver oxidative status of diabetic rats [7], [8], [9]. These in vivo-observed biological activities could be due to the presence of flavonoid compounds in the extract, especially those exhibiting antioxidant and antidiabetic properties. Therefore, with the prospect to produce drugs, food supplements, cosmetics and other products based on the purified neem leaves extract enriched with a specific composition of these flavonoids, the ethanolic leaf extract of A. indica underwent further liquid-liquid extractions by using three different organic solvents with an increasing Rohrschneider and Snyder solvent polarity parameter, i.e. dichloromethane<n-butanol<ethyl acetate [10], chosen among those exhibiting no safety concerns when used as a flavouring agents in food and beverage industries [database of FAO/WHO Expert Committee on Food Additives (JECFA); PubChem database of National Institutes of Health (NIH)]. Qualitative and quantitative analyses were carried out on each extract by using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The chemical characteristics and main biological activities of the investigated flavonoids are summarized in Table 1.

Table 1:

Chemical characteristics and main biological activities of antioxidant and antidiabetic flavonoids investigated.

Name		Formula		Mol. weight (g/mol)	Main biological activities
IUPAC	Trivial	Chemical	Structural
2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one	Quercetin	C15H10O7		302.238	Protective abilities against several degenerative diseases by preventing lipid peroxidation and against tissue injury induced by various drug toxicities [4]
3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)chromen-4-one	Myricetin	C15H10O8		318.237	Carbohydrate metabolic enzymes and insulin signalling molecules enhancing in streptozotocin-cadmium induced diabetic nephrotoxic rats [11]
5,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one	Astragalin	C21H20O11		448.380	Anti-inflammatory, antioxidant, neuroprotective, cardioprotective, antiobesity, antiosteoporotic, anticancer, antiulcer and antidiabetic properties [12]
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-one	Quercitrin	C21H20O11		448.380	Plasma glucose decrement, insulin levels increment, and antioxidant status improving in streptozotocin-induced diabetic rats by decreasing lipid peroxidative products and increasing enzymic and nonenzymic antioxidants [13]
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one	Isoquercitrin	C21H20O12		464.379	Chemoprotective effects both in vitro and in vivo, against oxidative stress, cancer, cardiovascular disorders, diabetes and allergic reactions [14]
5,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(2S, 3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R, 4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one	Nicotiflorin	C27H30O15		594.522	Antioxidant and immunoregulation properties protecting the liver against acute immunological and chemical injury [15]
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one	Rutin	C27H30O16		610.521	Decrement in plasma glucose, augmentation in insulin levels, and restitution of glycogen content and glycolytic enzymes in streptozotocin-induced diabetic rats [16]

Materials and methods

Results and discussion

The TLC running with the hydro-alcoholic A. indica extract and pure standard flavonoids in co-chromatography indicated that astragalin, quercitrin, isoquercitrin, nicotiflorin and rutin were the only flavonoids found in the hydro-alcoholic extract among those screened (Table 3; [17]).

Flavonoids were identified by matching both HPLC retention times (t_R) and their spectral characteristics against those of standard references. As shown in Table 4, the differences (%) between the t_R mean value of standard references vs. the t_R mean value of different extracts never exceed 1.15%, therefore fully in accordance with the European Commission Decision 2002/657/EC implementing Council Directive 96/23/EC about the performance of analytical methods and the interpretation of results, which suggests that “The ratio of the chromatographic t_R of the analyte to that of the internal standard, i.e. the relative t_R of the analyte, shall correspond to that of the calibration solution at a tolerance of ±2.5% for liquid chromatography”.

The content of each flavonoid has been determined by using calibration curves (not shown). By comparing the HPLC chromatograms of purified extracts obtained with the different solvents, it was found that the qualitative-quantitative composition of flavonoids is dependent on extracting solvent (Fig. 1). This outcome is in accord with the finding of Anokwuru et al. [18], who showed as the amount of flavonoids is dependent on the solvent used for extraction. The flavonoid concentrations in ppm for each extracting solvent used are reported in Table 5. The total content of all screened flavonoids, such as found in the ethanolic extract prepared from neem leaves, is 15.9 mg/ml/g powder sample. As expected, this concentration decreased and varied as phytochemicals composition in all different extracts obtained. In particular, dichloromethane allows extraction of 89.5% quercitrin, 5.3% isoquercitrin, 5.2% rutin (total content of all screened flavonoids=0.2 mg/ml/g powder sample); n-butanol allows extraction of 6.0% isoquercitrin, 6.2% nicotiflorin, 87.8% rutin (total content of all screened flavonoids=7.5 mg/ml/g powder sample); ethyl acetate allows extraction of 4.2% astragalin, 12.0% quercitrin, 50.3% isoquercitrin, 6.7% nicotiflorin, 26.9% rutin (total content of all screened flavonoids=4.3 mg/ml/g powder sample). Each of these flavonoids is an antioxidant and/or antidiabetic agent (Table 1). Since for each extract varies both the concentration and the type of flavonoid present, the extent of these biological activities is predicted to vary accordingly.

Fig. 1:

Representative HPLC chromatograms (at 366 nm) of the initial hydro-alcoholic extract of A. indica (a) and the relative dichloromethane (b), n-butanol (c), ethyl acetate (d) organic, and the remaining aqueous (e) phases obtained from it.

For the same ethanolic extract of neem leaves herein prepared, both antidiabetic and antioxidant properties have been already demonstrated in mice [7], [8], [9]. In 2011 Bhat et al. [19] found that the chloroform extract of A. indica, when administered for up 21 days to diabetic mice, induces: (i) a gradual decrease in fasting glucose level and a significant weight gain comparable to the control, indicating an increase in muscle mass; (ii) normalizes both the plasma insulin and c-peptide levels; (iii) as also happens with the aqueous extract, inhibits intestinal glucosidases; (iv) an increase in both liver and muscle glycogen content, as well as of the activity of islets producing insulin. In addition, is well tolerated when administered orally, showing an IC₅₀ of 7.5 mg/ml. Flavonoids and other phytochemicals like alkaloids, sesquiterpene and saponins, polysaccharides, dietary fibers, ferulic acid, tannins, limonene, and oleuropeoside have direct or indirect effect in diabetes pathways as enzyme inhibitors. The most involved mechanisms are inhibition of intestinal α-glucosidase and α-amylase, lens aldose reductase, oxidative stress protection, inhibition of formation of advanced glycation end products, inhibition of aldose reductase, lowering plasma glucose levels, altering enzyme activity of hexokinases and glucose-6-phosphate (G6P), synthesizing and releasing of insulin, postprandial hyperglycemia inhibition, stimulation of the glucose transporter type 4 (GLUT-4), decreasing activity of G6P, lowering the level of skeletal hexokinases, etc. [17], [20], [21]. The molecular bases of many diseases, by including diabetes, are known to involve oxidative stress caused by free radicals. Most of the studies reveal the inference of oxidative stress in diabetes pathogenesis by the alteration in enzymatic systems, lipid peroxidation, impaired glutathione metabolism and decreased vitamin C levels [22]. Oxidative stress plays also a role in the development of complications of both types of diabetes mellitus [23]. Sithisarn et al. [24], [25] assessed neem leaves, fruits, flowers and stem bark extracts for their antioxidant activity and inhibition of lipid peroxidation in ChaGo K-1 cancer cells for up 24 h. Leaf aqueous, flower and stem bark ethanol extracts exhibited higher free radical scavenging effect on the 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay with 50% scavenging activity at 26.5, 27.9 and 30.6 μg/ml, respectively; the total antioxidant activity of these extracts was found to be 0.959, 0.988 and 1.064 mM of standard 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), respectively. At 100 μg/ml, the flower ethanol and leaf aqueous extracts significantly decreased malondialdehyde (MDA) levels (46.0 and 50.6%, respectively), such as detected by the thiobarbituric acid reactive substances (TBARS) method. The same authors speculated that flavonoids might play a role in all these observed antioxidant activities. In 2011, Choudhary and Swarnkar [26], by analysing both leaves and stem bark of A. indica, have provided evidence-based data that the DPPH radical scavenging activity is positively correlated with the total phenolic content and that superoxide radical scavenging activity increases with increasing flavonoids content. The underlying molecular mechanism remains poorly understood, therefore needs further investigation.

Conclusions

Azadirachta indica can be regarded as a valuable plant source in paving a frontier in the field of drug discovery [27]. Even though different parts of this plant have been studied and reported to contain many bioactive compounds that are exploited to treat various diseases and disorders [2], [3], the current research shows that:

1. in accord to the chemical analysis performed by Chattopadhyay in 1999 [17], astragalin, quercitrin, isoquercitrin, nicotiflorin and rutin contained in the ethanolic extract of A. indica could contribute to its antihyperglycemic/hypoglycemic effect. Thus, these flavonoids, in association with their antioxidant property, could be employed as an alternative source of medicine to mitigate/counteract the pathogenesis of diabetes and its complications;

2. by depending on specific purposes and needs, the organic solvents dichloromethane, n-butanol and ethyl acetate could be used like extracting solvents to prepare drugs, food supplements, cosmetics and other products that are based on the purified neem leaves extract enriched with the most suitable flavonoids formulation.

In addition, by considering the huge market of the formulations based on the neem leaves in the world, HPLC could be used as elective analytical technique to unmask herbal formulations containing A. indica of poor quality if already on the market, as well as to perform qualitative investigations regarding other products still to be marketed [28].