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.

KEYWORDS:

• Rumex
• Proximate
• Functional food
• Anti-diabetic
• Fluorescence
• Sensory evaluation
• Saponins
• Flavonoids

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.^[6–10] But the difference is that each plant species differs in the amount and concentration of these substances. The plants rich in primary metabolites are usually used for nutritional purposes, while secondary metabolites are preferred for medicinal purposes.^[6,11,12] The plants may possess fats, protein, carbohydrates, vitamins and minerals in such quantity that are considered sufficient for our daily requirements. One can use specific species of plant as food that possess required amount of primary metabolites and low amount of secondary metabolites. But the plants that possess high amount of secondary metabolites than the primary metabolites can cause serious health complications.^[13,14] So this is necessary to figure out the amount of nutritional and medicinal elements in those plants, which are being used as food by people. The nutritional values of various plants have been reported by different investigators.^[15,16]

Diabetes mellitus is a metabolic disorder characterized by increase in blood glucose level. It can affect individuals at any stage of life but the frequency of diabetes is considerably high among the obese and aged people.^[17] Various therapeutic measures are employed to alleviate the symptoms of this disease. One of the effective therapeutic measures is to decrease the absorption of glucose from the intestine. Therefore, the absorption of glucose from the intestine can be decreased effectively by α_glucosidase inhibition. Various plants have been reported to possess the α_glucosidase inhibition potential.^[18]

Rumex hastatus belongs to the family Polygonaceae that consist of 48 genera and 1,200 species.^[19] The Rumex genus consists of about 200 species, among which a wide variety has been used for medicinal purposes.^[20] The R. dentatus, R. maritimus, R. bucephalophorus, R. nervosus, R. abyssinicus and many more have been used for medicinal purposes including diabetes mellitus.^[18,21–25] The R. vesicarius has been analyzed for nutritional value and recommended good and safe for nutritional purposes.^[26] R. crispus, R. obtusifolius and R. acetosa has been used as vegetable and fodder since long.^[27–29] In the same way R. hastatus has been used ethnomedicinally for various ailments including GIT ailments, cuts, wounds, bleedings, as appetizer, as anthelmintic, in snake bites, blood pressure, tonsillitis, sore throat, as flavoring agent, carminative and diuretic.^[30–35] R. hastatus has been reported by several investigators to possess high quantity of flavonoids.^[36] Flavonoids consist of wide variety of compounds with excellent anti-diabetic potential in vivo as well as in vitro.^[37–39] R. hastatus has also been reported to possess anthelmintic, cytotoxic, phytotoxic, antibacterial, anticholinesterase and antioxidant potentials.^[36,40] The R. hastatus has also been used as fodder for animals. In Pakistan R. hastatus has been used as vegetable in the northern Himalayan areas.^[41]

Going to the detailed literature survey of R. hastatus, it is obvious that this plant has been used for multiple purposes. So the current study was arranged to evaluate the R. hastatus for its nutritional value and sort out bioactive compounds and provide an implication regarding the use of this plant for nutritional purpose.

Materials and methods

Plant’s collection

The aerial part of R. hastatus was collected from the hilly area of Gorha Gat in the proximity of University of Malakand, Chakdara, Dir (L), KPK, Pakistan. The plant identification was performed by Dr. Ali Hazrat (plant taxonomist) and deposited in the Department of Botany, Shaheed Benazir Bhutto University, Sheringal, Dir upper, KPK, Pakistan, with voucher number (1015SJ). All the extra particles were carefully removed from the plant material and were spread on neat paper in a room in the shade and appropriately dried for two weeks. The paper was changed daily to avoid fungal growth on plant material. After shade drying the plant was pulverized using cutter mill.^[42]

Extraction and fractionation

The powdered plant sample was soaked in 80% methanol for a period of 8 days followed by filtration. The filtrate obtained was evaporated at low temperature under reduced pressure using rotary evaporator. The evaporation resulted in a semisolid mass, i.e., crude methanolic extract (Rh.Cr).

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.

$\mathrm{\%}\mathrm{f}\mathrm{i}\mathrm{b}\mathrm{r}\mathrm{e}=\frac{lossofweightonignition}{weightofsampleused}\times 100$

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.

Secondary metabolites

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 NH₄OH 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;

$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\frac{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}-\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}\times 100$

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 IC₅₀ values. The standard error mean (SEM) were calculated at 95% confidence intervals.

Results

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.

Table 1. Proximate and phytochemical analysis of samples of Rumex hastatus.

S. no.	Sample	Percent (Mean ± SD)	mg/g
1.	Protein	13.4 ± 0.7	133.9
2.	Carbohydrates	43.2 ± 1.8	432.4
3.	Fats	2.6 ± 0.1	25.6
4.	Fiber	12.4 ± 0.9	124.4
5.	Moisture	22.9 ± 1.4	–
6.	Ash	5.5 ± 0.6	54.5
7.	Saponins	6.5 ± 0.2	65.5
8.	Flavonoids	8.5 ± 0.3	84.5
9.	Alkaloids	4.9 ± 0.2	49.5

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 shows that the flavonoids are comparatively in greater amount, i.e., 84.3912 (8.4391%), 81.8865 (8.1886%) and 87.3657 (8.7365%) mg/g in test 1, 2 and 3, respectively. Similarly, the saponins were 6.57321%, 6.31794% and 6.75507% in test 1, 2, and 3, respectively. As far as the alkaloids are concerned, this plant possess normal amount of them, i.e., 4.7973%, 5.1443% and 4.9001% in test 1, 2 and 3, respectively. The percent of secondary metabolites present in R. hastatus is highest for flavonoids following saponins and alkaloids.

Table 2. Fluorescence analysis of powdered sample of Rumex hastatus under visible and UV lights

S. no.	Treatments	Visible light	Ultraviolet light
1	Only Powder	Grayish brown	Brown
2	Powder + FeCl3	Grayish black	Yellowish gray
3	Powder + Conc. HCl	Yellowish red	Yellowish gray
4	Powder + 10% HNO3	Pinkish yellow	Greenish yellow
5	Powder + 10% K2Cr2O7	Yellowish brown	Greenish brown
6	Powder + 1 M NaOH	Brownish black	Grayish brown
7	Powder + AgNO3	Pinkish red	Bluish green
8	Powder + Conc. HNO3	Blackish brown	Grayish brown
9	Powder + Conc. H2SO4	Brown	Grayish brown
10	Powder + Br2 water	Light brown	Yellowish brown
11	Powder + 5% H2O2	Blackish brown	Greenish brown
12	Powder + CCl4	Brownish black	Greenish black
13	Powder + CH3OH	Blackish brown	Yellowish brown
14	Powder + CH3COOH	Pinkish brown	Blackish green
15	Powder + Xylene	Grayish brown	Reddish green
16	Powder + NH3	Grayish brown	Yellowish brown
17	Powder + I2	Deep black	Grayish black

Fluorescence analysis

The fluorescence characteristics of powdered sample of R. hastatus were studied under ordinary visible light and ultraviolet light. The powdered sample was treated with various reagents in on a slide on bench top and studied for the color under UV and visible lamps. The powdered sample upon treatment with any reagent gave grayish brown color in visible light and brown color under UV lamp. The powdered sample treated with FeCl₃ exhibited grayish black color in visible light and yellowish gray color under UV lamp. Similarly, the plant sample was treated with variety of chemical reagents, i.e., HCl, HNO₃, K₂Cr₂O₇, H₂SO₄, Br₂, H₂O₂, CCl₄, CH₃OH, CH₃COOH, xylene, NH₃ and I₂. All the reagents displayed different colors in different environments. All the results obtained have been summarized in Table 3.

Table 3. α-Glucosidase inhibitory potential of various samples of Rumex hastatus.

Samples	Conc. 31. 25 µg/mL	Conc. 62.5 µg/mL	Conc. 125 µg/mL	Conc. 250 µg/mL	Conc. 500 µg/mL	Conc. 1,000 µg/mL	IC50 µg/mL
Rh.Cr	32.33 ± 0.49***	41.33 ± 0.33***	51.00 ± 1.15***	52.43 ± 0.97***	62.17 ± 1.40***	71.34 ± 1.30***	108.83
Rh.Hex	29.67 ± 0.89***	35.61 ± 1.70***	38.00 ± 0.58***	44.90 ± 0.52***	47.96 ± 1.01***	51.33 ± 1.20***	978.35
Rh.Chf	49.61 ± 1.70**	55.17 ± 1.40***	61.90 ± 0.52***	64.00 ± 1.15***	71.43 ± 0.97***	77.17 ± 1.40**	42.09
Rh.EtAc	39.30 ± 0.43***	46.33 ± 1.20***	51.73 ± 0.78***	57.87 ± 0.26***	64.13 ± 0.20***	69.50 ± 0.58***	111.84
Rh.Aq	28.33 ± 0.68***	36.61 ± 1.70***	41.40 ± 0.33***	46.34 ± 1.30***	49.86 ± 1.04***	56.20 ± 1.11***	534.61
Rh.Sp	42.53 ± 1.07***	43.86 ± 1.39***	58.66 ± 1.20***	61.00 ± 1.73***	65.16 ± 1.58***	70.66 ± 2.02***	104.96
Rh.Fl	39.17 ± 1.33***	46.73 ± 0.78***	58.33 ± 1.20***	63.90 ± 0.52***	67.96 ± 1.01***	73.87 ± 0.26***	88.73
P.cont	57.90 ± 0.52	65.96 ± 1.01	73.87 ± 0.26	79.56 1.27	81.83 ± 1.09	86.23 ± 0.39	13.92

Key: Rh.Cr: Crude methanolic extract, Rh.Hex: n-hexane fraction, Rh.Chf: chloroform fraction, Rh.EtAc: ethyl acetate fraction, Rh.Aq: aqueous fraction, Rh.Sp: saponins, Rh.Fl: Flavonoids, P.cont: Acarbose

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.

Figure 1. Sensory evaluations of Rumex hastatus on hedonic scale

α-Glucosidase inhibitory effect

The α-glucosidase inhibition assay revealed marked activity of various samples of R. hastatus (Table 4). Among the test samples Rh.Chf exhibited highest activity exhibiting 49.61 ± 1.70, 55.17 ± 1.40, 61.90 ± 0.52, 64.00 ± 1.15, 71.43 ± 0.90 and 77.17 ± 1.40 at the concentrations of 31.25, 62.5, 125, 250, 500 and 1,000 µg/mL, respectively with IC₅₀ value of 42.09 µg/mL. Similarly, flavonoids and saponins also demonstrated significant enzyme inhibition with IC₅₀ values of 88.74 and 104.96 µg/mL, respectively. The rest of sample also demonstrated enzyme inhibition potential up to considerable extent. The overall activity was recorded as dose dependent response.

Table 4. List of compounds in GC (FID-MS) of crude methanolic extract of Rumex hastatus.

S.No	Compound	RT	Common name	Formula	Hits (DB)
1	GUANIDINE (CARBONATE)	6.011	Guanidine	CH5N3	10
2	(2S*,3R*)-2-tert-Butyl-3-ethyloxtane	6.1	NF	C8H16O	1
3	Benzene, 1-methyl-4-(1-methylethyl)	6.281	p-Cymol	C10H14	10
4	Thiophene, 2-methoxy-5-methyl-	6.708	NF	C6H8OS	8
5	3-Methyl-2-cyclohexen-1-one	6.946	Seudenone	C7H10O	10
6	(E)-1-Ethoxyhex-1-ene	7.154	NF	C8H16O	2
7	8-Hydroxy-2-octanone	7.291	NF	C8H16O2	10
8	r-1-Fluoro-t-2-iodo-1-methylcyclohexane	7.554	NF	C7H12FI	9
9	Benzene, (2-methyl-1-propenyl)-	7.639	NF	C10H12	10
10	1,2,4,4-Tetramethylcyclopentene	7.936	NF	C9H16	10
11	1-methyl-3-hepten-2-one	8.412	NF	C8H14O	4
12	5,6-Dimethylundecane	8.456	NF	C13H28	10
13	(2S,3R)-2-ethylthreonine hydrate	8.859	NF	C6H13NO3	8
14	2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one	9.038	NF	C6H8O4	10
15	Cpd 18: 4-Azidohept-1-ene	9.236	4-Azidohept-1-ene	C7H13N3	2
16	1,2,3-Trimethylcyclopentene	9.408	NF	C8H14	10
17	omega.-Isonitrosoacetophenone	9.522	Oximinoacetophenone	C8H7NO2	10
18	3-Methylbenzamide	9.763	m-Toluamide	C8H9NO	10
19	2-(2-Butoxyethoxy)ethanol	9.791	BuCb	C8H18O3	10
20	iso-Butylamine	10.028	Valamine	C4H11N	10
21	Bicyclo[3.1.0]hex-2-ene, 2-methyl-5-(1-methylethyl)-	10.187	alpha-Thujene	C10H16	10
22	Dihydrobenzofuran	10.579	Dihydrocoumarone	C8H8O	10
23	5-Formyl-2-furfurylmethanoate	10.695	NF	C7H6O4	10
24	(2R)-1-(Tetrahydro-2H-2-pyranyloxy)-2-butanol	11.142	NF	C9H20O3	10
25	2-Furancarboxaldehyde, 5-(hydroxymethyl)	11.315	NF	C6H6O3	10
26	Geranyl acetate, 2,3-epoxy-	11.568	NF	C12H20O3	10
27	Nonanoic acid	11.732	Pelargic acid	C9H18O2	1
28	R-(+)-METHYL-3-ISOPROPYL-6-OXOHEPTANOATE	11.798	NF	C11H20O3	6
29	Cyclohexene, 1-acetyl-2-(1-hydroxyethyl)-	11.883	NF	C10H16O2	10
30	3-Buten-2-one, 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)	11.943	Ionone	C13H20O	10
31	2,3,5-Trimethylanizole	12.153	NF	C10H14O	10
32	Phenol, 5-methyl-2-(1-methylethyl)	12.378	Thymol	C10H14O	10
33	2,4-Pentadien-1-ol, 3-propyl-, (2Z)-	12.476	NF	C8H14O	10
34	2-Methoxy-4-vinylphenol	12.655	p-Vinylguaiacol	C9H10O2	10
35	trans-3-methyl-4-hexenal	12.861	NF	C7H12O	10
36	Tricyclo [4.2.1(4,7).0(3,8)]nona-5-en-2-one	12.973	NF	C9H10O	10
37	3-Methyl-4-methylamino-1,2,4-triazole-5-thiol	13.05	NF	C4H8N4S	2
38	3-Oxatricyclo[3.2.1.0(2,4)]octane, (1.alpha.,2.beta.,4.beta.,5.alpha.)-	13.289	NF	C7H10O	10
39	Phenol, 2,6-dimethoxy-	13.446	Syringol	C8H10O3	10
40	5-Allyl-2-Methoxyphenol	13.58	Chavibetol	C10H12O2	10
41	(2E,6E)-2,6-Dimethylocta-2,6-dien-1,8-dial	13.935	NF	C10H14O2	6
42	Benzenemethanol, .alpha.-(aminomethyl)-4-hydroxy	14.098	Norden	C8H11NO2	10
43	1,8-Cyclopentadecadiyne	14.254	NF	C15H22	10
44	3-Hexyne-2,5-diol, 2,5-dimethyl	14.408	Kemitracin-50	C8H14O2	10
45	4-Fluorobenzoic acid, 3,5-dimethylcyclohexyl ester	14.538	NF	C15H19FO2	7
46	2,5-Dimethyl-2,3,4-hexatriene	14.602	NF	C8H12	10
47	2-(3,5-Dimethylphenyl)propan-2-ol	15.081	NF	C11H16O	2
48	Benzene, 1-(bromomethyl)-3-nitro	15.689	NF	C7H6BrNO2	10
49	Hydroxy-.alpha.-terpenyl acetate	15.916	NF	C12H20O3	10
50	(-)-trans-Pinocarvyl acetate	16.106	NF	C12H18O2	9
51	4-(2,6,6-Trimethylcyclohexa-1,3-dienyl)but-3-en-2-one	16.238	NF	C13H18O	8
52	(E)-4-(1'-Formyl-1'-Cyclopentyl)-3-Buten-2-One	16.25	NF	C10H14O2	10
53	2-Propenoic acid, 1,7,7-trimethylbicyclo[2.2.1]hept-2-yl ester, exo-	16.56	NF	C13H20O2	10
54	1,5,5-Trimethyl-6-[3-acetoxybutyl]-3,6-epidioxycyclohexene	16.674	NF	C15H24O4	10
55	7-Oxabicyclo[4.1.0]heptan-3-ol, 6-(3-hydroxy-1-butenyl)-1,5,5-trimethyl	16.962	NF	C13H22O3	10
56	4-(4-Methylphenyl)pentanal	17.528	NF	C12H16O	10
57	n-Octoic acid	17.806	Octanoic Acid	C8H16O2	10
58	2,6 - di - methoxy - 4 - vinyl – phenol	17.881	4 - vinyl – syringol	C10H12O3	10
59	3,6-Dimethyl-3-octene-2,7-dione	17.977	NF	C10H16O2	5
60	Bicyclo[5.2.0]nonane, 2-methylene-4,8,8-trimethyl-4-vinyl-	18.19	NF	C15H24	10
61	Benzoic acid, 4-hydroxy-3-methoxy-	18.365	Vanillic acid	C8H8O4	10
62	1,2-Benzenedicarboxylic acid, diethyl ester	18.406	Anozol	C12H14O4	10
63	2-(1-Cyclohexen-1-Yl)Cyclohexyl Acetate	18.862	NF	C14H22O2	10
64	o-Toluic acid, 3-chloropen-2-enyl ester	18.919	NF	C11H11ClO2	4
65	5-endo-(Phenylsulfonyl)-5-exo-methylbicyclo[2.2.2]oct-2-ene	18.963	NF	C15H18O2S	2
66	3,5,7-trimethyl-2E,4E,6E,8E-undecatetraene	19.11	NF	C14H22	10
67	Bisabolol oxide A	19.203	Bisabolol oxide A	C15H26O2	8
68	N-(2-methylpropyl)indoline	19.283	NF	C12H17N	2
69	2-Hexanol, 3,3,5-trimethyl-2-(3-methylphenyl)-	19.348	NF	C16H26O	10
70	1-Phenylcyclohexylamine	19.532	NF	C12H17N	10
71	Tridecanedial	19.632	Tridecanedial	C13H24O2	10
72	endo-(2R/S,4R/S,4'S)-2,4-Diethoxy-6-(carbonyl-4'-tert-butyloxazolodin-2'-one…	19.755	NF	C17H27NO6	10
73	Nerolidoloxide	19.833	Nerolidoloxide	C15H26O3	10
74	alpha.-Bisabolol $$ 5-Hepten-2-ol, 6-methyl-2-(4-methyl-3-cyclohexen-1-yl)-	20.19	alpha.-Bisabolol	C15H26O	10
75	5-Methyl-3,5-octadien-2-one	20.298	NF	C9H14O	10
76	Acetyl bromide	20.472	Acetyl bromide	C2H3BrO	10
77	2-Cyclopentene, 4-(hydroxymethyl)-1,1,2,3-tetramethyl-	20.835	NF	C10H18O	10
78	(2S*,6R*)-6-Allyl-2-hydroxy-2-vinyl-1-cyclohexanone	20.97	NF	C11H16O2	10
79	(Z)-4-Methoxy-2-(N-methylanilino)penta-2,4-dienenitrile	21.132	NF	C13H14N2O	1
80	2-Methoxy-5-(acetoxymethyl)phenol	21.391	NF	C10H12O4	4
81	Tetradecanoic acid	21.925	Myristic acid	C14H28O2	10
82	3-Cyclohexene-1-acetaldehyde, .alpha.,4-dimethyl-	22.008	NF	C10H16O	10
83	3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-	22.337	NF	C15H22O	10
84	4,4-Dimethyladamantan-2-ol	23.363	NF	C12H20O	10
85	7,11-Hexadecadienal	23.555	NF	C16H28O	1
86	2,5-Dimethyl-2,4-hexadiene	23.706	Biisocrotyl	C8H14	10
87	cis-11-Hexadecen-1-ol	24.055	NF	C16H32O	10
88	6-Methyl-2-tridecanone	24.275	NF	C14H28O	10
89	Benzenemethanol, 3,4,5-trimethoxy-	24.445	NF	C10H14O4	10
90	cis-11-Hexadecen-1-ol	24.872	NF	C16H32O	10
91	1,2-Benzenedicarboxylic acid, dibutyl ester	25.087	Butyl phthalate	C16H22O4	10
92	3-Eicosyne	25.429	3-Icosyne	C20H38	10
93	4,6-di-tert-Butyl-m-cresol	26.66	NF	C15H24O	3
94	N,N,N',N'-Tetramethyl-1,2-di-p-tolyl-ethane-1,2-diamine	26.94	NF	C20H28N2	6
95	cis-9-Hexadecenoic acid	27.3	NF	C16H30O2	6
96	Hexadecanoic acid	27.904	Palmitic acid	C16H32O2	10
97	Hexadecanoic acid, ethyl ester	28.44	Ethyl hexadecanoate	C18H36O2	10
98	2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]-	31.04	Phytol	C20H40O	10
99	1-methylene-3-propylcyclobutane	31.552	NF	C8H14	10
100	Hexadecanoic acid, 2-methylpropyl ester	31.619	NF	C20H40O2	1
101	9,12,15-Octadecatrienoic acid, methyl ester	31.693	Methyl linolenate	C19H32O2	10
102	Linoleic acid ethyl ester	31.947	Mandenol	C20H36O2	10
103	9-Octadecenoic acid (Z)	32.057	Oelsauere (Red oil)	C18H34O2	10
104	Cis-8-methyl-exo-tricyclo[5.2.1.0(2.6)]decane	32.075	NF	C11H18	10
105	Undecanoic acid, ethyl ester	32.529	Ethyl undecanoate	C13H26O2	3
106	Propanamide, N-(2-fluorophenyl)-3-(4-morpholyl)-	32.995	NF	C13H17FN2O2	1
107	trans-Caryophyllene	33.999	Caryophyllene	C15H24	10
108	2-methylbut-2-enoic acid, 3-hydroxy-3-isopropyl-6,8a-dimethyl-8-oxo-1,2,3,3a…	34.324	NF	C20H30O4	8
109	1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester	38.001	DNOP	C24H38O4	10
110	(E)-3,3'-Dimethoxy-4,4'-dihydroxystilbene	39.5	NF	C16H16O4	10
111	n-Hexadecane	39.891	Isohexadecane	C16H34	10
112	Xanthanin	40.827	Xanthanin	C18H20O5	4
113	(Z)-13-Docosenamide	40.945	Erucyl amide	C22H43NO	10
114	Octadeamethyl-cyclononasiloxane	41.736	Siloxane	C18H54O9Si9	10
115	n-Tetracosane	42.425	n –tetracosane	C24H50	10
116	4H-1-Benzopyran-4-one, 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)-	43.277	NF	C17H14O5	10
117	Hexadecamethylheptasiloxane	43.698	Heptasiloxane	C16H48O6Si7	10
118	4H-1-Benzopyran-4-one, 5-hydroxy-6,7-dimethoxy-2-(4-methoxyphenyl)	46.833	NF	C18H16O6	10
119	(-)-.alpha.-Selinene	53.791	Selinene	C15H24	10
120	N-Butyl-N-[2-(9H-9-carbazolyl)propyl]amine	74.013	NF	C19H24N2	1

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.

Figure 2. Chromatogram of methanolic extract of R. hastatus representing different peaks

Figure 3. Structures of bioactive compounds sorted out from the GC(FID-MS) analysis

Figure 4. (a–h) Mass spectra of bioactive compounds of Rumex hastatus.

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.^[59–62] 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.^[63–65] In the same way, the deficiency of protein intake can cause various pathological disorders, including lung diseases, cardiac disorders, neurological disorders and cancer.^[66–68] 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.^[70–72] Nonetheless, these secondary metabolites have also been reported to possess antimicrobial, antioxidant and other beneficial effects.^[36,73–76] 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.

Acknowledgments

The authors are thankful to Dr. Ali Hazrat for identification of the plant. The authors declare that they have no competing interests.