Nutritional and medicinal aspects of Rumex hastatus D. Don along with in vitro anti-diabetic activity

ABSTRACT Rumex hastatus being used for medicinal and nutritional purposes as a functional food in various countries is hereby evaluated by the gas chromatography-mass spectroscopy, proximate analysis, physicochemical (fluorescence) analysis, quantitative analysis of secondary metabolites and sensory evaluations studies. Various samples of R. hastatus were also evaluated for in vitro anti-diabetic potential. The investigational study demonstrated that R. hastatus is a rich source of carbohydrate, i.e., 432.4 mg/g. Moisture content, protein, fiber, ash content and fats were recorded as 22.8%, 133.9 mg/g, 124.4 mg/g, 54.5 mg/g and 25.6 mg/g, respectively. In the same way, the secondary metabolite displayed a relatively greater amount of flavonoids (84.5 mg/g) followed by saponins (65.5 mg/g) and alkaloids (49.5 mg/g). Similarly, the GC (FID-MS) analysis of R. hastatus revealed the detection of 120 compounds. Out of those identified compounds, selected anti-diabetic compounds were sorted out, viz butyl phthalate, phytol, ethylthreonine, dihydrobenzofuran, indoline, guanidine, nerolidol, myristic acid, palmitic acid, caryophyllene, anozol. In physicochemical fluorescence analysis and the sensory evaluation, data were also recorded along with the anti-diabetic with IC50 value of 42.09 µg/ml. The overall investigational analysis of R. hastatus obviously demonstrated that this plant was a rich source of primary and secondary metabolites. It may be concluded from the GC (FID-MS) analysis that R. hastatus is a potential source of anti-diabetic constituents, which may confer hypoglycemic potential. Based on the recorded data it may also be inferred that R. hastatus is among safe and nutritious herbs, which can be used in lieu of green vegetables and functional food with anti-diabetic potential.


Introduction
Globally the advanced researchers are in continuous struggle to combat with various challenging diseases, to fabricate more and more nutritional commodities, to figure out novel resources and to facilitate the mankind. [1,2] One of the most critical and focused issue is the nutritional crunch throughout the world. A wholesome portion of population goes into the famine, malnutrition and drought calamities each year. In the same way people get various physiological anomalies due to starvation and nutritional deficiency. [3] To cope with such calamities, the agronomists are getting focused on various procedures to enhance the agricultural outcome. [4] Plants are considered as staple by majority of world's population. The people of those areas wholly and solely depend on herbs, where the access of sophisticated techniques of getting multiple nutritional products is not developed. [5] As reported by several investigators, the plants consist of primary metabolites, secondary metabolites and micronutrients. [6][7][8][9][10] But the Some of the Rh.Cr was kept for activities and the remaining was suspended in sufficient amount of water followed by fractionation with various solvents in separating funnel. The fractionation was started with less polar n-hexane (500 mL × 3), then chloroform (500 mL × 3), ethyl acetate (500 mL × 3) and final fraction obtained was aqueous. [9] Similarly, the fractions obtained were n-hexane (Rh. Hex), ethyl acetate (Rh.EtAc), chloroform (Rh.Cf) and aqueous fraction (Rh.Aq).

Gas chromatography-flame ionization detector (GC-FID) analysis
The GC-FID analysis of Rh.Cr was carried out with the help of gas chromatograph Agilent USB-393752 (Agilent Technologies, Palo Alto, CA, USA) via HHP-5MS (5%) phenylmethylsiloxane capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Restek, Bellefonte, PA) attached with FID detector. The oven was allowed to set at temperature of 70°C for a minute and then augmented to 180°C at the rate of 6°C/min for the period of five minutes and lastly to 280°C at the rate of 5°C/min for a period of 20 min. The temperature of detector and injector were maintained at 290°C and 220°C correspondingly. The flow rate of carrier gas (Helium) was maintained as 1 mL/min and the diluted samples (1/1,000 in n-pentane, v/v) of 1 μL were injected manually in the split-less mode.

Gas chromatography-mass spectrometry (GC-MS) analysis
The -F/MS of Rh.Cr was performed via USB-393752 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with a HHP-5MS 5% phenylmethylsiloxane capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Restek, Bellefonte, PA) outfitted with an Agilent HP-5973 mass selective detector in the electron impact mode (Ionization energy: 70 eV) working under the experimental conditions as those maintained for GC.

Identification of components
All the major constituents of Rh.Cr were identified by the comparison of their retention time with the literature of genuine compounds. The identification of compounds was further carried out with the help of the spectral data obtained from the libraries of Wiley and NIST as well as their fragmentation patterns and comparisons of the mass spectra with data reported in literature or with those of mass spectra from literature. [43,44] Each process was carried twice.

Proximate analysis
Proximate analysis of powdered plant sample was carried out following the standard procedure of Association of Official Analytical Chemist. [45] Moisture content Loss on drying (LOD) method was followed for the determination of moisture content of the plant sample. A weighed quantity of powdered plant sample was taken in a suitable container and allowed to dry at 105°C in oven till the achievement of constant weight. Thus the amount of moisture present in the powdered plant sample was figured out from the difference of dried weight of sample and the total weight of the sample.

Ash content
Incineration procedure was followed for determination of ash content of powdered plant sample. A weighed amount of sample was put in a crucible and transferred into the muffle furnace and allowed to incinerate at 550°C for 24 h. Similarly, total ash content was figured out after conversion of dried mass of powdered plant sample into ashes.

Crude fats
Soxhlet method was followed for the determination of total fats in the sample. Briefly, 2 g of dried powdered plant sample was transferred into a Soxhlet extractor and petroleum ether was added to the flask of the extractor. The extraction was carried out for 6 h till the exhaustion of sample from fat content. The obtained petroleum ether was filtered and the filtrate obtained was allowed to be evaporated in a weighed beaker. The total fats were calculated as the total increase in weight of the beaker.

Crude fibers
The value of crude fiber was figured out from the data of loss in weight after the ignition of dried samples remaining after digestion of fat-free samples with 1.25% each of sulfuric acid and sodium hydroxide solution under specified conditions. % fibre ¼ loss of weight on ignition weight of sample used Â 100

Crude protein
For the determination of crude proteins, the method of micro Kjeldahl nitrogen method was followed. This method involved the digestion of plant sample with concentrated sulfuric acid and catalyst for the conversion of organic nitrogen into ammonium sulfate in the solution. After which the decomposition of ammonium sulfate was carried out via NaOH. The liberated ammonia was distilled into 5% boric acid. After this the titration of trapped ammonia was carried out with 0.05 N HCl for the deduction of nitrogen from ammonia. The indicators used were methylene red and blue both. The percent proteins were calculated from the value of nitrogen obtained multiplied by 6.25.

Carbohydrate content
The total crude carbohydrate content was determined by the subtraction formula. In short, the total protein, total fiber, ash content, moisture content and total lipids were subtracted from the dried mass and the total carbohydrates were calculated.

Alkaloids
The alkaline precipitation gravimetric method was followed to find out alkaloids. A weighed powdered plant sample was taken in a beaker and 10% acetic acid solution in ethanol was transferred into the beaker. The mixture was incubated at 28°C for 4 h. Then it was filtered through Whatman No. 42 filter paper. The filtrate was allowed to be evaporated to one quarter of its original volume followed by the addition of drop-wise NH 4 OH for the precipitation of alkaloids. The precipitated alkaloids were received on filter paper and washed with 1% ammonia solution and then dried in an oven at 80°C. So the amount of alkaloids was calculated per gram of the dried powdered sample of R. hastatus.

Flavonoids
For the extraction of flavonoids, the procedure of Harborne was followed. [46] Plant sample (5 g) was taken and boiled in 50 mL of 2 M HCl under reflux for 30 min. It was cooled and filtered using Whatman No. 42 filter paper. The extract was treated with equal volume of ethyl acetate. The flavonoids present in the extract were precipitated which were recovered with the help of weighed filter paper. The amount of flavonoids recovered on filtered paper was calculated.

Saponins
For the determination of crude saponins in powdered sample of R. hastatus, 20 g of powdered sample was put in a conical flask and 100 mL of 20% ethanol was added to the conical flask. The sample allowed to heat at 55°C in the water bath for 4 h with continuous stirring. After 4 h the sample was filtered and the residue was re-extracted with 200 mL of 20% ethanol. The sample after extraction was allowed to heat until a concentrated volume of 40 mL was obtained. The sample obtained was shifted into a separating funnel and 20 mL of diethyl ether was added to it. After vigorous shaking, the separating funnel was put in a stand to get two layers. The lower aqueous layer was collected while the upper diethyl ether layer was discarded. The aqueous layer obtained was diluted with 60 mL of n-butanol and the combined n-butanol extract was washed with 10 mL of 5% saline. The final solution obtained was kept in a hot water bath until complete evaporation and the saponins obtained were dried in an oven and the saponins per gram of powdered plant sample was calculated. [40] Fluorescence analysis The powdered plant sample was treated with different chemical reagents for the determination of fluorescence characters. The plant sample was put in small quantity on glass slide and treated with various reagents followed by the determination of color under the visible light and ultraviolet light.

Sensory evaluation
The sensory evaluation of R. hastatus was performed based on the Hedonic Scale following AOAC procedure. [47] Various parameters were assayed such as color, flavor, taste, general appearance and texture. The evaluation was performed based on the questionnaire to score out of seven points of hedonic scale.
α-Glucosidase inhibition assay α-Glucosidase inhibitory activity was carried out following chromogenic assay. [48] Enzyme solution was prepared having 0.5 unit/mL and 20 μL of this solution was mixed 120 μL of phosphate buffer having pH 6.9. p-nitrophenyl-α-D-glucopyranoside solution (5 mM) was prepared in the same buffer that was employed as substrate. Briefly, 10 μL of test samples having various concentrations (31.25-1,000 μL) were added to them and kept for 15 min at 37°C. After incubation, 20 μL of substrate solution was added to all of them and incubated for further 15 min. Sodium carbonate solution (0.2 M) having volume of 80 μL was added to them to terminate the reaction. Absorption of each sample was measured at 405 nm via double beam spectrophotometer (Thermo electron corporation USA). The reaction mixture with the test sample served as control while acarbose served as positive control. The percent enzyme inhibitory potential was calculated as; Percent enzyme inhibition ¼ Control absorption À Sample absorption Control absorption Â100

Statistical analysis
The statistical analysis was carried out by Two-way ANOVA followed by Bonferroni post test, in which the positive control was compared with the groups of test samples. P values less than or equal to 0.05 were considered as significant statistically. GraphPad Prism and XL sheet were employed to draw the graphs and IC 50 values. The standard error mean (SEM) were calculated at 95% confidence intervals.

Proximate composition
In the proximate analysis, the powdered sample of R. hastatus exhibited high percentage of carbohydrate as compared to the protein, fats, fibers and minerals. The proximate analysis has been summarized in Table 1. Table 1 represents the percent of protein along with various parameters in the powdered sample of R. hastatus. Each of the analysis was performed in triplicates and the percentage of each ingredient is approximately the same in the results.
The percent proteins were detected as 13.4 ± 0.7% (133.9 mg/g), which demonstrate that R. hastatus is rich in proteins. Similarly, in Table 1 the percent ash content is summarized. The ash content shows adequate amount of minerals in the plant sample, i.e., 54.5 mg/g. In the same way, the percent fats analysis was also performed, which is summarized in Table 1. The percentage of fats is comparatively less from the other components, i.e., 25.6 mg/g. The powdered sample was also assessed for the percent moisture content, which is summarized in Table 1. Though the powdered sample was dried for 2 weeks prior to Loss on drying procedure, but still the amount of moisture trapped in the tissues of plant was considerably sufficient, i.e., 22.9 ± 1.4%. Moreover, the percent fiber and carbohydrates are summarized in Table 1, which represent a wholesome amount of carbohydrates in the sample of R. hastatus. As far as the percent fiber is concerned, it also goes parallel with the percent proteins, i.e., 133.9 mg/g. The proximate analysis shows that the carbohydrate was in high quantity, i.e., more than 40% followed by the moisture content, proteins, fibers, ashes and then fats.

GC (FID-MS) analysis
The GC (FID-MS) analysis of R. hastatus revealed the identification of 120 compounds. The literature review of the identified compounds was performed, in which several compounds previously reported to possess anti-diabetic potential were sorted out. The bioactive compounds sorted out were butyl phthalate, phytol, ethylthreonine, dihydrobenzofuran, indoline, guanidine, nerolidol, myristic acid, palmitic acid, caryophyllene, anozol. The anti-diabetic literature of these compounds is summarized in the discussion section.

Saponins, flavonoids and alkaloids
The total saponins, flavonoids and alkaloids have been summarized in Table 2. The data is represented in the terms of percentage and mg/g of the powdered sample. The  Table 3.

Sensory evaluation
The results of sensory evaluations have been summarized in Figure 1. The highest value was shown by the color, i.e., 6.5 followed by the 5.8 that of flavor and the least value has been shown by texture, i.e., 5.3. The overall sensory evaluation reveals the mean value of 5.74 ± 0.211.

α-Glucosidase inhibitory effect
The α-glucosidase inhibition assay revealed marked activity of various samples of R. hastatus (Table 4)

Discussions
The GC (FID-MS) analysis of methanolic extract of R. hastatus revealed the presence of 120 compounds summarized in Table 4 and the chromatogram in Figure 2. The literature survey of these compounds showed the presence of several reported anti-diabetic compounds given in Figure 3.
The bioactive compounds sorted out were butyl phthalate, phytol, ethylthreonine, dihydrobenzofuran, indoline, guanidine, nerolidol, myristic acid, palmitic acid, caryophyllene, anozol. Butyl phthalate is one of the constituent identified in the sample of R. hastatus, and one of the closely similar structure, i.e., butyl iso-butyl phthalate has previously been published with significant α-glucosidase inhibition potential. [49] R. hastatus also reveals the presence of medicinally important compound, i.e., phytol, which possess antidiabetic effect due activation of nuclear receptors and heterodimerization with PPARγ. [50] This plant also possess guanidine derivatives that are potentially antidiabetic compounds. [51] Insulinotropic polypeptides contains threonine and thylthreonine is also an identified constituent in the GC (FID-MS) analysis of R. hastatus. [52] Benzoic acid derivatives have been reported with antidiabetic potential and benzamides are getting fame with good results. [53] Dihydrobenzofuran is also one of the constituent of R. hastatus that possess antidiabetic properties. [54] In the same way, indoline derivatives are also effective antidiabetic compounds, which have been identified in the sample of R. hastatus. [55] The Momordica charantia (bitter gourd) has been demonstrated with significant antidiabetic activity and it has been reported with high percentage of Nerolidol. [56] The GC (FID-MS) analysis of R. hastatus also reveals the presence of fatty acids, i.e., Myristic acid and palmitic acid and GPR-40 "fatty acid receptors" have been identified on the B-cells of pancrease that   are responsible for insulin secretion. [57] Caryophyllene has also been reported with notable antidiabetic activity. [58] Benzoic acid derivatives have also been reported with significant hypoglycemic effect and the GC (FID-MS) of R. hastatus also contains Anozol, which is benzoic acid ester. The MS spectra of bioactive compounds of R. hastatus have been given in Figure 4.
To evaluate the nutritional value of certain green vegetables, the sensory evaluation of that specific food should be performed on priority basis. The sensory evaluation can give a specific value from hedonic scale, which represents the mean acceptance and nutritional esthetics of certain vegetables. Going to the results of hedonic scale evaluation, the mean value calculated for R. hastatus was 5.74 ± 0.211 out of 7.00, which represents that this plant possesses an acceptable place in the group of green vegetables. Similarly, the proximate value of certain species can decide their use as food based on their nutritional value. Proximate analysis is performed for dietary fiber, carbohydrates, fats, protein, moisture content and ash content. Each of the parameter has its one vital function and nutritional benefits for human beings and other animals. Dietary fiber can absorb huge   amount of water and make the stool soft to get easily out of alimentary canal, so in this way dietary fiber can relieve hemorrhoids and constipation. Fiber has also been reported to alleviate obesity, diabetes and cancer. [59][60][61][62] As far as the total fats are concerned, this plant contains 2.5% of fats and can be considered as balanced when taken in lieu of milk that contain 3.5-5% fats sufficient for daily requirements as excess cause atherosclerosis other complications. [63][64][65] In the same way, the deficiency of protein intake can cause various pathological disorders, including lung diseases, cardiac disorders, neurological disorders and cancer. [66][67][68] The carbohydrates, being the major portion of plant and the most abundant biomolecule in the universe help in the energy production and some percent of our body mass. It is obvious from the results that R. hastatus is a good source of carbohydrates, i.e., > 40% which meet the daily body requirement. In the same way, though the moisture content is high, i.e., above 20% and high moisture content degrade the bioactive compounds by increasing the microbial growth but still this plant can be protected from microbial growth by various other factors like viscosity, micronutrients, etc. [69] As far as the secondary metabolite are concerned, it is obvious from the results that R. hastatus is also a good source of secondary metabolites, especially the flavonoids, alkaloids and saponins. The percent of these secondary metabolites cannot affect the physiological processes in the human body, because this plant is also cooked as a vegetable and the majority of alkaloids and saponins are degraded by high temperature. [70][71][72] Nonetheless, these secondary metabolites have also been reported to possess  antimicrobial, antioxidant and other beneficial effects. [36,[73][74][75][76] Moreover, the florescence analysis of the samples of R. hastatus revealed various colors under different wavelengths. The fluorescence analysis is a significant system of identification of powdered drugs with their particular references. [77] α-Glucosidase enzyme is responsible for the breakdown of large molecules of carbohydrate to glucose units. To target this enzyme may alleviate the blood glucose level in diabetic patients by decreasing the absorption of glucose from the intestine. Plethora of plants have been reported to possess multiple compounds responsible for α-glucosidae inhibition, which may be used to alleviate the symptoms of diabetes mellitus. [39] As discussed earlier, the R. hastatus has been used by multiple communities for variety of ailments and various species of this genus has been reported to possess anti-diabetic potential. [18,25] The current investigational study reveals that R. hastatus contains variety of compounds responsible for in vitro inhibition of α-glucosidase.

Conclusion
It may be inferred from the current investigational studies that Rumex hastatus is a good source of basic nutritional components along with secondary metabolites. It can meet the basic needs of human body for energy production, growth and other vital functions. Furthermore, this plant possesses marked in vitro anti-diabetic potential, so it may be a possible remedy for the management of diabetes mellitus. R. hastatus can also be used as vegetable when conventional vegetables are scarce, unavailable or expensive.