Background
Cosmos caudatus belongs to the plant family Asteraceae. It is a popular local salad vegetable in Malaysia and is commonly known as “
ulam raja”. Traditionally, young leafy shoots of the plant are recommended for the alleviation of diabetes, high blood pressure, arthritis and fever [
1‐
3]. Other uses of the plant include a digestive aid and longevity [
4,
5]. This plant has also been reported to have antioxidant and antidiabetic properties, which were concluded to be due to the presence of phenolic compounds [
6‐
10].
Diabetes mellitus is a serious, debilitating disease that has become an increasing health burden to most of its sufferers. According to the International Diabetes Federation (IDF) [
11], approximately 415 million people worldwide suffer from diabetes. This number is expected to rise to 642 million by 2040. The present trend indicates that more than 60% of the world’s diabetic population will be from Asia [
12]. In Malaysia, an alarming 3.6 million adults older than 18 years are estimated to be affected by diabetes [
13]. Currently, six classes of oral antidiabetic drugs comprising biguanides (e.g., metformin), sulfonylureas (e.g., glimepiride), meglitinides (e.g., repaglinide), thiazolidinediones (e.g., pioglitazone), dipeptidyl peptidase IV inhibitors (e.g., sitagliptin) and α-glucosidase inhibitors (e.g., acarbose, miglitol, voglibose and nateglinide) are in clinical use, either alone or together with insulin, to treat this disease [
14,
15]. Unfortunately, these medications are also associated with various side effects and high secondary failure rates [
14‐
16]. Therefore, there is an increased demand for safer and more effective alternative drugs or medicinal agents to treat or manage diabetes. Medicinal plants offer an interesting subject for further research into the identification of their bioactive constituents, their mechanism of action and their development into phytomedicinal products to help alleviate the disease.
The use of α-glucosidase inhibitors is a therapeutic approach used to reduce postprandial hyperglycaemia in diabetic patients. It works by retarding the absorption of carbohydrates and glucose [
17]. The α-glucosidase enzyme is located in the brush border membrane of the small intestine, and its function is to breakdown carbohydrates to form the more absorbable monosaccharides. α-Glucosidase inhibitors delay and reduce postprandial glucose and insulin levels [
18]. Thus, the inhibition of the carbohydrate-hydrolysing enzyme using α-glucosidase inhibitors is a plausible pathway to combat diabetes [
19‐
21]. In recent years, numerous investigations have been carried out on the α-glucosidase inhibitory activity of various plant extracts in the hope of discovering new or more potent α-glucosidase inhibitors [
22‐
26].
The quality of plant material produced from herb and medicinal plant plantations is greatly influenced by a number of factors, including the environment, cultivation conditions, and management practises, such as temperature, irradiance, fertilizer supply, irrigation and harvesting time [
27,
28]. The cultivated plant material should be harvested at the optimum growth stage when the parameters associated with the raw material quality are at their peak [
27,
29‐
32]. This is because different metabolites are biosynthesized and may be present at different stages of plant growth. The age of the crop at harvesting time is known to significantly affect the level of bioactive compounds in herbs and medicinal plants [
31,
33,
34]. Thus, it is necessary to investigate the alterations in the production of the bioactive constituents in relation to the plant’s age to ascertain the optimum time for crop harvest that would assure the potency of the biological or pharmacological effect of the final plant product. For the herbal industry in particular, these aspects are very important in the standardization and quality control of the herbal produce, either in the upstream or the downstream stages.
Presently, study on variations in the levels of bioactive constituents with respect to the maturation of
C. caudatus, specifically the α-glucosidase inhibitory properties, is still underexplored. Previously, Mediani et al. [
29,
30] evaluated the antioxidant activity of
C. caudatus at different growth stages along with the effect of various drying methods on the harvested sample. However, the influence of the growth stages on the α-glucosidase inhibitory activity of the harvested samples was not examined. Moreover, Javadi et al. [
35] investigated the α-glucosidase inhibitory activity of
C. caudatus samples, but this study specifically examined the effect of different post-harvest storage times (0–12 h). They reported that the biological activity decreased after 10 h of storage. In a continued study, Javadi et al. [
36] further reported that some compounds contributing to the α-glucosidase activity were catechin, α-linolenic acid, α-D-glucopyranoside and vitamin E. Thus, more information is needed with regards to the best practices in the cultivation of this medicinal plant to provide proper guidelines and a standard operating procedure for farmers to produce quality plant material for a specific biological use.
The present study reports the evaluation of the metabolite profiles of
C. caudatus at different stages of its growth and its correlation to the α-glucosidase inhibitory properties. The experiment was carried out with a specific cultivar and strictly followed agronomic conditions. The study was divided into two parts. First, to ensure good representation of the α-glucosidase inhibitory constituents, the best solvent for the optimum extraction of the bioactive compounds was determined. The optimum solvent system was then employed in the plant age-metabolite correlation study. An NMR-based metabolomics approach [
37] was used to analyse the solvent-metabolite and plant age-metabolite correlations. Identification of the bioactive metabolites was further supported by 1D and 2D NMR spectroscopy and dereplication via LC-MS/MS metabolite profiling.
Methods
Chemicals
Absolute ethanol (EtOH) for the extraction of plant samples was purchased from HmBg Chemicals Inc. (Germany). For the α-glucosidase inhibitory assay, quercetin, glycine, 4-nitrophenyl-α-D-glucopyranoside and α-glucosidase from Saccharomyces cerevisiae were purchased from Sigma Aldrich (St. Louis, USA). Folin-Ciocalteu’s phenol reagent, gallic acid, and sodium carbonate (Na2CO3) were also supplied by Sigma Aldrich. For NMR measurements, deuterated methanol (CD3OD), non-deuterated KH2PO4, sodium deuterium oxide (NaOD), trimethylsilyl propionic acid-d4 sodium salt (TSP) and deuterium oxide (D2O) were purchased from Merck (Darmstadt, Germany). For liquid chromatography mass spectrometry (LC-MS) analysis, HPLC grade methanol, acetonitrile, acetic acid and hydrochloric acid were purchased from Merck (Darmstadt, Germany). Liquid nitrogen was supplied by MOX Company (Petaling Jaya, Malaysia).
Plant materials
Plant material used for the study was obtained from the germplasm collection of the Agriculture Technology Park, Universiti Putra Malaysia (UPM). Plant identity was confirmed by Dr. Shamsul Khamis, the botanist at the Institute of Bioscience, UPM, and a voucher specimen (No. SK 2511/14) was deposited at the Herbarium of the Institute. An experimental plot was established at the park in November 2013. Prior to planting, the soil was first treated, fertilized, turned and covered with black plastic to prevent weeds from growing in the soil. Four equidistant cut-out holes numbered 1 to 4 (thus groups of four per biological replicate) were made on the plastic covering, and 5 to 7 seeds were sown in the centre of each hole. As the plants grew larger, the number of plants was reduced to one per hole. Organic fertilizer was applied at the beginning of planting and every 2 weeks thereafter. Pesticide treatment was avoided during the growth period. Plants in the same plot were used for both solvent-metabolite and plant age-metabolite correlation metabolomics studies. For the solvent-metabolite correlation study, C. caudatus leaves were randomly collected from the plants growing in the plot and thoroughly mixed. Additionally, for the plant age-metabolite correlation study, leaf samples (comprising the top eight young leaves but excluding the shoot tip) were collected at different growth stages of the plant, i.e., at 6, 8, 10, 12 and 14 weeks. Each age group consisted of six individual plants (six biological replicates).
Plant sample preparation
Leaf samples of
C. caudatus were quenched directly by immersion in liquid nitrogen during field collection prior to transport to the laboratory for analysis. This was to ensure that the degradation of metabolites due to enzymatic reactions was minimized [
38]. The samples were further freeze-dried before being ground into a fine powder. For the solvent-metabolite correlation study, the powdered leaf material was divided into 6 × 6100 g portions, for extraction with EtOH:water (E:W) solvent systems consisting of ratios of 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100. To facilitate extraction, each replicate was sonicated for 1 h at room temperature. The extraction procedure for each replicate was repeated three times, and the filtrates were pooled, filtered (Whatman filter paper no 1), evaporated to dryness under vacuum, and stored at − 80 °C prior to metabolomics analysis. For the plant age-metabolite correlation study, the freeze-dried samples were similarly extracted using an 80:20 E:W solvent system. All samples were weighed, labelled and stored at − 80 °C prior to metabolomics analysis.
For
1H-NMR measurements, the sample preparation was carried out according to Kim et al. [
39,
40] with slight modifications. A 10 mg sample of each extract was dissolved in 0.375 mL of deuterated methanol (CD
3OD) and 0.375 mL of phosphate buffer (pH 6.0) prepared in D
2O containing 0.1% TSP (w/w). The sample mixture was sonicated for 15 min, vortexed for 2–3 min and centrifuged at 13000 rpm for 10 min. The supernatant (0.6 mL) was collected, transferred to a 5 mm NMR tube and subjected to
1H-NMR measurement.
For LC-MS/MS analysis, each test sample was prepared by dissolving 1 mg of the extract in 1 mL of methanol and subjecting the solution to ultrasonication for 30 min at room temperature. The test sample was then filtered and kept at 4 °C prior to the analysis.
Measurement of total phenolic content
The Folin-Ciocalteu method as described by Zhang et al. [
41] was adopted for the measurement of total phenolic content (TPC), with minor modifications. Aliquots of 20 μL of each serial dilution (6.25, 12.5, 25, 50, 75, 100, 125, 250, 500 ppm) prepared from a stock solution (0.5 mg mL
− 1) of the respective test extract were loaded onto a 96-well microplate, alongside the same series of serial dilutions of quercetin as a positive standard. Folin-Ciocalteu’s reagent (100 μL) was added to each well, mixed thoroughly using a vortex mixer, and the mixture was allowed to rest for 5 min at room temperature. This was followed by the addition of 80 μL of 7.5% (w/v) sodium carbonate solution and diluted to a final volume of 200 μL with distilled water. After thoroughly mixing, the plate was covered and left in the dark at room temperature. After 30 min, the absorbance of the reaction mixtures was measured at 765 nm against a blank (solvent used for extraction) using a microplate reader. The analysis was performed in triplicate. A standard calibration curve was constructed using gallic acid solutions of different concentrations (12.5, 25, 50, 75, 100, 125, 250, 500 and 1000 ppm). The TPC results are expressed as g gallic acid equivalents (GAE) per g dry weight of the fresh sample (g GAE g DW
− 1).
Measurement of α-glucosidase inhibitory activity
The α-glucosidase inhibitory activity was assayed in a 96-well plate following the method described by Collins et al. [
42] with slight modifications. Briefly, 4 mg of plant extract was dissolved in 1 mL of ethanol to prepare a stock solution, and 5 mg mL
− 1 quercetin in ethanol was used as a positive control [
43]. Subsequently, 10 μL of sample solutions of different concentrations (0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1 and 0.2 mg mL
− 1 for the test extract and 0.004, 0.008, 0.0156, 0.0313, 0.0625, 0.125, 0.25 mg mL
− 1 for quercetin) were added to 100 μL of α-glucosidase type 1 from
S. cerevisiae (Sigma G5003) solution (0.02 U well
− 1) in 30 mM phosphate buffer (pH 6.5). The sample mixture was then incubated for 5 min at room temperature [
44]. In the meantime, 60 mg of 4-nitrophenyl-α-D-glucopyranoside (PNPG) was dissolved in 20 mL of 50 mM phosphate buffer (pH 6.5). The PNPG solution (75 μL) was added to each well, and the reaction mixtures were incubated for an additional 15 min at room temperature. The reaction was terminated by adding 50 μL of 2 M glycine (pH 10) to each well. The optical densities (ODs) were then immediately read at 405 nm using a microplate reader [
44]. The results are described as IC
50 inhibition values. The percent inhibition was calculated using the following formula:
$$ \%\mathrm{inhibition}=\left[\right({\mathrm{A}}_{\mathrm{c}}-{\mathrm{A}}_{\mathrm{e}}/{\mathrm{A}}_{\mathrm{c}}\Big]\ \mathrm{x}\ 100\% $$
where Ac is the difference in absorbance between the control (with enzyme) and the blank control (without enzyme);and
Ae is the difference in absorbance between a sample (with enzyme) and the blank sample (without enzyme).
To calculate the IC50 value, a plot of the assay concentration (serial dilutions) of each test extract versus the % inhibition was constructed. The IC50 value was then estimated using the fitted line, y = mx + c (where m and c are numbers), and IC50 = (50-c)/m, where m is the slope of the line and c is the y-intercept.
Measurement of NMR spectra
1H-NMR spectra were measured on a 500 MHz Varian UNITY INOVA NMR spectrometer (Varian Inc., California, USA) functioning at a frequency of 499.91 MHz and temperature of 26 °C. For data acquisition, a single pulse proton experiment with Presat was used with the following set of parameters (3.53 s acquisition time, 64 scans with 1.5 s presaturation delay, spectral width: − 2 to 14 ppm). For structural elucidation, both 1D and 2D NMR experiments were used. The J-resolved spectrum was acquired in 50 min 18 s, and the relaxation delay was 1.5 s. The heteronuclear multiple bond coherence (HMBC) spectra were obtained using 64 scans, which were achieved in 6 h, 9 min and 9 s.
Multivariate data analysis
All the 1H-NMR spectra were manually phased and baseline corrected using Chenomx software (v. 5.1, Alberta, Canada). The 1H-NMR spectrum of each sample was processed and binned (0.04 ppm bin width) over the spectral region of δ 0.50 to δ 10.00 ppm. All spectra were then automatically converted to ASCII files and transferred into a Microsoft Excel (version 1997–2003) worksheet. Residual water (δ 4.70–4.90 ppm) and methanol (δ 3.27–3.31 ppm) regions were excluded from the spectral data to retain only signals from endogenous metabolites. The resulting dataset was saved as an Excel file and further subjected to multivariate analysis using SIMCA-P+ version 13.0 (Umetrics, Umea°, Sweden).
LC-MS/MS analysis
For the LC-MS/MS analysis, a Dionex C18 reversed-phase column (Dionex, Sunnyvale, USA) with dimensions of 250 (l) × 2.0 mm (i.d.) and 2.5 μm particle size was used. Analysis was performed on a Dionex Ultimate 3000 HPLC equipped with a photodiode-array detector (PDA-3000) at 26.9 °C (thermostatted column compartment). The mobile phase used was double distilled water containing 0.1% acetic acid (solvent A) and HPLC grade acetonitrile containing 0.1% acetic acid (solvent B). The addition of acetic acid to the mobile phase enhanced compound peak sharpness by inducing the ionization of metabolites [
45]. Sample elution was performed in a gradient manner with 10 to 100 mL for solvent A and 90 to 0 mL solvent B. The injection volume was 15 μL with a constant flow rate of 1.00 mL min
− 1. The flow was split to allow 200 μL min
− 1 of eluent into the mass spectrometer. The total LC run time was 35 min. The mass spectrometry (MS) measurement of the sample was performed on a MicroTOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). The source conditions were: nebulizer gas nitrogen (N
2) at 0.2 bar and dry gas (N
2) at 3.0 L min
− 1, dry temperature at 180 °C, capillary voltage at 4500 V and end plate offset at − 500 V. Data acquisition was performed by HyStar Application version 3.2, while data processing was carried out with DataAnalysis Version 3.4 by Bruker Daltonik GmbH.
Statistical analysis
The results are expressed as the mean ± SD. The statistical significance of the results was evaluated using one-way ANOVA with Duncan’s post hoc test. Significant differences were based on p values where p < 0.05 was considered significantly different. Correlations among the different activities were performed by Pearson’s correlation test, where the IC50 values were converted to 1/IC50 to inverse the relationship between absorbance and activity.
Conclusions
In the present study, α-glucosidase inhibitory activity data of C. caudatus leaves at different growth stages extracted with different solvent combinations were correlated with NMR results. Among the six EtOH:water solvent combinations, C. caudatus leaves extracted with EtOH:water (80:20) showed the highest α-glucosidase inhibitory activity. Among the harvesting ages, 10-week-old C. caudatus leaf samples were found to be the best in terms of α-glucosidase inhibitory activity. Rutin, quercetin 3-O-glucoside, quercetin 3-O-xyloside, quercetin 3-O-arabinofuranoside and quercetin 3-O-rhamnoside were identified as the major flavonoid glycosides responsible for the plant’s activity. Moreover, through 1H-NMR metabolomics along with multivariate data analysis, a strong correlation was found between TPC and C. caudatus leaf samples harvested at the 10th week and extracted with EtOH:water (80:20). Our results indicated the best age and solvent combination to harvest and extract C. caudatus leaves, respectively, to retrieve the bioactive constituents with maximum α-glucosidase inhibitory activity.
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