Corresponding author: Alona Savych ( alonasavych@gmail.com ) Academic editor: Maya Georgieva
© 2021 Alona Savych, Svetlana Marchyshyn, Halyna Kozyr, Nadiya Yarema.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Savych AO, Marchyshyn S, Kozyr H, Yarema N (2021) Determination of inulin in the herbal mixtures by GC-MS method. Pharmacia 68(1): 181-187. https://doi.org/10.3897/pharmacia.68.e55051
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The herbal mixtures due to the wide range of biologically active substances can influence on various links of the pathogenetic mechanism of development of diabetes mellitus and its complications. The carbohydrates, especially inulin, deserve the particular attention through their hypoglycemic, hypolipidemic, anticholesterolemic and detoxifying activities. The aim of the study was to investigate the content of inulin in the herbal mixtures No. 3, No. 4, No. 7, No. 13 and No. 19, which are used in folk medicine for the prevention and treatment of diabetes mellitus type 2 in Ukraine. The quantity content of inulin was defined by the difference between fructose as a product of enzymatic hydrolysis and fructose, a constituent of sucrose and free fructose, taking into account the empirical factor for the conversion of fructose from inulin. The carbohydrates were separated by gas chromatography-mass spectrometry after conversion into volatile derivatives as aldononitrile acetate. According to the results, the herbal mixture No. 3 contains 458.97 mg/g of inulin, the herbal mixture No. 4 – 99.21 mg/g, the herbal mixture No. 7 – 139.93 mg/g, the herbal mixture No. 13 – 203.84 mg/g, the herbal mixture No. 19 – 359.65 mg/g. The availability of inulin and its high content in the investigated herbal mixtures due to the presence of inulin-containing medicinal plants, such as Cichorium intubus roots (mixtures No. 3 and No. 13), Taraxacum officinale roots (mixtures No. 3, No. 7 and No. 19), Arctium lappa roots (mixture No. 4), Inula helenium rhizome with roots (mixture No. 7).
diabetes mellitus, herbal mixture, inulin, GC-MS
Diabetes mellitus is a major problem of World Health Organization, as the epidemiological situation is becoming alarming – the number of patients with diabetes is increasing every year, and with it the number of deaths and disabilities through the development of micro- and macroangiopathies (
One such area is phytotherapy, as it has a number of advantages over traditional therapy with using oral synthetic agents, namely, it is low-toxic, has a mild pharmacological effect and can be used for long periods without significant side effects, is well combined with synthetic drugs (
Thus, for this purpose, it is advisable to study the phytochemical compounds, namely the inulin from group of carbohydrates in the investigated herbal mixtures, which are used in folk medicine for the prevention and treatment of diabetes mellitus type 2 in Ukraine (
Polysaccharide complexes, including inulin, are very important active substances for the prevention and treatment of diabetes mellitus and diabetic angiopathies (
The aim of study was to determine the quantitative content of inulin in the herbal mixtures No. 3, No. 4, No. 7, No. 13 and No. 19 with reliable hypoglycemic activity established during the screening testing (
It was used the herbal raw materials harvested in June – August 2019 in Ternopil region and Charpathians (Vaccinium myrtillus leaf) (Ukraine) during the study. After harvesting, the raw materials were dried, crushed and brought back to standard according to the general GACP requirements (
For the study were used the five different herbal mixtures with reliable hypoglycemic activity established during the screening testing (
The herbal mixtures | The herbals | Quantity of herbals in mixtures, g |
---|---|---|
No. 3 | Urtica dioica leaf | 26.32 |
Cichorium intubus roots | 26.32 | |
Rosa majalis fruits | 21.05 | |
Elymys repens rhizome | 15.79 | |
Taraxacum officinale roots | 10.52 | |
Total: 100.0 | ||
No. 4 | Arctium lappa roots | 26.32 |
Elymys repens rhizome | 26.32 | |
Zea mays columns with stigmas | 21.05 | |
Helichrysum arenarium flowers | 15.79 | |
Rosa majalis fruits | 10.52 | |
Total: 100.0 | ||
No. 7 | Inula helenium rhizome with roots | 10.0 |
Helichrysi arenarium flowers | 20.0 | |
Zea mays columns with stigmas | 20.0 | |
Origanum vulgari herb | 20.0 | |
Rosa majalis fruits | 20.0 | |
Taraxacum officinale roots | 10.0 | |
Total: 100.0 | ||
No. 13 | Cichorium intubus roots | 26.32 |
Elymys repens rhizome | 26.32 | |
Helichrysum arenarium flowers | 21.05 | |
Rosa majalis fruits | 15.79 | |
Zea mays columns with stigmas | 10.52 | |
Total: 100.0 | ||
No. 19 | Urtica dioica leaf | 20.0 |
Taraxacum officinale roots | 20.0 | |
Vaccinium myrtillus leaf | 20.0 | |
Rosa majalis fruits | 20.0 | |
Mentha piperita herb | 20.0 | |
Total: 100.0 |
All applied reagents were of analytical grade (≥ 95% purity). Standard reagents including D-arabinose, D-glucose, D-fructose, saccharose were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The water used in the studies was produced by MilliQ Gradient water deionizaton system (Millipore, Bedford, MA, USA). Inulinase, acetate buffer, methanol, hydroxylamine hydrochloride, pyridine, dichloroethane, hydrochloride acid, heptanes, ethyl acetate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
The quantity content of inulin in the herbal mixtures was studied by GC-MS method. Chromatographic separation was performed on a gas chromato-mass spectrometric system model 6890N/5973inert (Agilent Technologies, USA) using a capillary column HP-5ms (30 m×0.25 mm×0.25 mkm, Agilent Technologies, USA). The evaporator temperature was 250 °C, the interface temperature – 280 °C. The separation was performed in the mode of temperature programming – the oven temperature was initially at 160 °C, held for 8 min, then ramped at the rate of 5 °C/min to 240 °C and finally held at this temperature for 6 min. The samples 1 μL were administered in a 1:50 flow divider mode. The detection was held in the SCAN mode in the range of (38–400 m/z). The carrier gas flow rate through the column was 1.2 mL/min.
The samples of herbal raw materials were grinded into a powder by laboratory mill, then about 50–80 mg (accurately mass) was placed in a glass vial and 4 mL of 0.1 M acetate buffer (pH 4.5) was added. Extraction of inulin was performed in the ultrasonic bath at 80 °C for 3 hours. The resulting extracts were centrifuged at 3000 rpm and the supernatants were evaporated to dryness on a rotary evaporator. One part of the extract was used for enzymatic hydrolysis of inulin with 100 μL of inulinase at 60 °C for 30 min (
To obtain the aldonitrile monosaccharide derivatives, an aliquots 0.6 mL of the extracts were taken and 0.3 mL of a derivatizing reagent (32 mg/mL of hydroxylamine hydrochloride in the mixture of pyridine/ methanol (4:1, v/v)) was added. Samples were incubated in a preheated water bath shaker at 75 °C for 25 min. After incubation, 1.0 mL of acetic anhydride was subsequently added to the samples and incubated at 75 °C for 15 min. 2 mL of dichloromethane was added to the mixture, the excess of the derivatization reagents was removed by the double extraction with 1 M hydrochloride acid solutions and water. Dichloromethane layer was dried and dissolved into 300 μL of the mixture of heptane/ethyl acetate (1:1, v/v) (
Identification of enzymatic hydrolysis products, free monosaccharides and disaccharide – sucrose was performed by comparing of the retention time of the mixture of standard and using the NIST 02 mass spectrum library. Quantitative analysis was performed by adding a solution of internal standard – arabinose 0.25 mg in the test samples. Under normal conditions of derivatization, the ketone carbohydrate (fructose) is converted into an aldo carbohydrate (glucose) (
The concentration of total fructose (C1, mg/mL), free fructose (C2, mg/mL) and sucrose (Csucr, mg/mL) was determined by the method of internal standards according to the formula:
where Sx – peak area of the studied substance;
mst – mass of the internal standard injected into the sample, mg;
Sst – peak area of the internal standard;
mx – mass of sample of raw materials, mg;
Vsol – volume of solvent for extraction, mL;
Vextr – volume of extract for derivatization, mL.
The concentration (C3, mg/mL) of fructose released from sucrose was calculated by the formula:
where sucr –concentration of sucrose, mg/mL;
B – empirical factor for the conversion of fructose from sucrose (2.13).
Quantitative content (X, mg/g) of inulin was determined as the subtraction from total content of fructose after enzymatic hydrolysis, free fructose and fructose released by decomposition of sucrose according to the formula:
where C1 – concentration of total fructose, mg/mL;
C2 – concentration of free fructose, mg/mL;
C3 – concentration of fructose released from sucrose, mg/mL,
A – empirical factor for the conversion of fructose from inulin (1.03);
m1 – mass of raw materials on which was calculated, g.
The empirical factor for the conversion of fructose from inulin and sucrose (the factor of conversion of inulin to fructose and sucrose to fructose) was determined by sequential processing of samples with different amounts of inulinase using arabinose as the internal standard and determining the amount of fructose released (
During the study it was detected the inulin in all investigated herbal mixtures No. 3, No. 4, No. 7, No. 13 and No. 19 by the products of its enzymatic hydrolysis after conversion into volatile derivatives as aldononitrile acetate (Figs
The results of the GC-MC analysis of carbohydrates in the herbal mixtures.
No. of peak on chromatograms | Retention time, min | Identified substance | Derivatization products | Content in the herbal mixtures, mg/g | ||||
---|---|---|---|---|---|---|---|---|
No. 3 | No. 4 | No. 7 | No. 13 | No. 19 | ||||
FREE CARBOHYDRATES | ||||||||
1. | 5.56 | arabinose | 2,3,4,5-tetra-O-acetyl-D-arabinononitrile | internal standard | ||||
2. | 12.41 | glucose | 2,3,4,5,6-penta-O-acetyl-D-gluconitrile | 23.01 | 37.80 | 17.67 | 11.09 | 26.99 |
3. | 18.80 | fructose | naphthalene-1-carboxylic acid, 4-butylamino-6,7-dimethoxy-2-methyl-ethyl ester | 51.56 | 87.08 | 17.70 | 16.54 | 38.29 |
4. | 19.06 | fructose | 1-nitro-4-phenoxyanthraquinone | 49.88 | 82.48 | 16.66 | 15.98 | 37.55 |
5. | 32.75 | sucrose | sucrose octaacetate | 58.74 | 5.84 | 9.25 | 24.27 | 17.14 |
CARBOHYDRATES AFTER HYDROLISIS | ||||||||
1. | 5.56 | arabinose | 2,3,4,5-tetra- O-acetyl- D-arabinononitrile | internal standard | ||||
2. | 12.41 | glucose | 2,3,4,5,6-penta-O-acetyl-D-gluconitrile | 60.29 | 44.98 | 32.94 | 33.08 | 48.23 |
3. | 18.80 | fructose | naphthalene-1-carboxylic acid, 4-butylamino-6,7-dimethoxy-2-methyl-ethyl ester | 297.68 | 140.75 | 90.79 | 128.62 | 224.46 |
4. | 19.06 | fructose | 1-nitro-4-phenoxyanthraquinone | 276.95 | 127.87 | 83.77 | 113.19 | 208.61 |
During the chromatographic analysis of the herbal mixtures extracts to which inulinase was added, the total content of D-fructose as a product of enzymatic hydrolysis and free D-fructose was determined (Figs
The results of the quantitative study showed that the herbal mixture No. 3 contains 458.97 mg/g of inulin, the herbal mixture No. 4 – 99.21 mg/g, the herbal mixture No. 7 – 139.93 mg/g, the herbal mixture No. 13 – 203.84 mg/g, the herbal mixture No. 19 – 359.65 mg/g (Fig.
The detection of inulin and establishment of its high content in the investigated herbal mixtures is a predictable result,because these phytomixtures include the medicinal inulin-containing plants: Cichorium intubus roots (herbal mixtures No. 3 and No. 13), Taraxacum officinale roots (herbal mixtures No. 3, No. 7 and No. 19), Arctium lappa roots (herbal mixture No. 4), Inula helenium rhizome with roots (herbal mixture No. 7) (
The chromatographic analysis of inulin as an important biologically active substance with hypoglycemic, hypolipidemic, anticholesterolemic and detoxifying activities in the herbal mixtures indicate the advisability of the further pharmacological and phytochemical research of these phytomixtures as promising herbal medicines for the prevention and treatment of diabetes mellitus and its complications.
The authors have no funding to report.
The authors have declared that no competing interests exist.
The authors have no support to report.
We established for the first time, the quantity content of inulin in herbal mixtures No. 3, No. 4, No. 7, No. 13 and No. 19 after enzymatic hydrolysis by GC-MS method. The obtained results make these phytomixtures perspective for the future medical application against diabetes, its complications and metabolic disorders. However, in the future studies phytochemical and pharmacological investigations should be undertaken to better assess their potential.