INTRODUCTION:

The flax seeds (Fig. 1) are pharmacopeial medicinal plant raw material; Pharmacopoeial monograph (PM) on the Lini usitatissimi semina (PM.2.5.0026.15) is included into the State Pharmacopoeia of the Russian Federation XV edition¹, the medicinal plant raw materials are also represented by the State Pharmacopoeias of other countries (Belarus and Great Britain).

The European Scientific Cooperative on Phytotherapy (ESCOP) has published the monograph on flax seeds. The “Flax seeds” herbal drug and medicinal plant raw material drugs, containing fatty oil flax seeds, are registered in the State Register of Medicinal Remedies of the Russian Federation. Linum usitatissimum L. is an annual plant, which belongs to the Linaceae family. Fiber flax (used to produce flax fiber), common flax (used to produce flax oil), and intereum flax (combined using) are flax subspecies.²^,³ Chemical composition of flax seeds is various. At the average, flax seeds contain 40% of lipids, 23% of proteins, and 29% of carbohydrates mainly represented by soluble and insoluble dietary fibers. Flax seeds also contain a lot of biologically active substances (BAS), they are phenol acid (0.8-1%,p-, o-coumaric acid, etc.), lignans (0.9-3% of diglucosidesecoisolariciresinol, etc.), flavonoids (0.3-0.7% of herbacetin), vitamins (up to 3%), and mineral substances (1-2%), mucilages.⁴^-¹³

Figure 1: Flax seeds

Flax seeds are of various pharmacological properties. The fatty acids (especially, α-linoleic acid) and the polysaccharides containing in the flax and lignan slime are mainly active components. Application of seeds is considered safe despite of existence of anti-nutrients (cyan-glycosides, phytic acid, and linatin). Flax seeds and drugs have the antioxidant, anti-inflammatory, antiatherogenic, hormonal, and laxative effects. Seeds also have vitamin properties: 9 g of flax seeds contain the daily value of δ-aminolevulinic acid and 21-50 g of flax seeds contain the daily value of vitamin E. Flax seeds reduce risk of cardiovascular, oncological, autoimmune, kidney, and neurological diseases.¹⁴^-²⁰

Currently, the regulatory documentation, which regulates quality of flax seeds, needs to be completed with new sections and additional quality ratings.

This research aims to study the possibilities of improving the methods for standardization of flax seeds and multicomponent drugs on their basis.

MATERIALS AND METHODS:

Research objects:

The Linum usitatissimum (the whole seeds produced by A/S “Krasnogorckleksredstva”), the “Dicloran ® Plus” drug (Unique Pharmaceutical Laboratories) containing 30 mg/g of fatty flax seeds oil, and BAA “Linseed oil in capsules” (RUSKAPS) containing fatty flax seeds oils in the extra virgin gelatin capsules are the subjects of this research.

The flax seeds infusion has been prepared in accordance with prescribing information for the drug. 1.5 teaspoon of the seeds is covered by 200 ml (1 glass) of hot boiled water, than covered by the plug, infused in water bath for 15 minutes, and strained; remaining raw materials are squeezed.

Methods of determination of polysaccharides:

Determination of polysaccharides is performed by gravimetry in accordance with PM.2.5.0026.15. SPRF XV ed.

Analytical raw material samples are pounded to be the particles passing through the sieve, which holes are of 2 mm size. About 10.0 g (accurately weighed sample) of granular raw materials are placed into a 500 ml flask covered with ground glass joint, then, they are mixed with 200 ml of water. The flask is joined with backflow condenser and boiled while mixing on the electric oven for 30 minutes. Water extraction is reproduced two more times. The first time, 200 ml of water are extracted, the second time 100 ml of water are extracted. Aqueous extractions are mixed, cooled to room temperature, and filtered into a 500 ml flask through 5 gauze layers put into the glass funnel of 66 mm diameter and previously moistened with water. The filters are moistened with water, volume of solution is increased with up to the note, and then it is mixed (solution A). 25.0 ml of the solution A are placed into a 200 ml glass, combined with 100 ml of 96 % alcohol, mixed, and warmed through water bath at 60 °C temperature during 5 minutes. In an hour, contents of the glass are managed into the centrifuge tubes and centrifuged at a speed of 5000 r/min during 30 minutes. The supernatant fluid is filtered under the vacuum at a residual pressure of 13-16 kPa through a glass filter “POR-16” with pores of 40 mm diameter and dried to constant mass at a temperature of 100-105 °С. Then, sediment is quantitatively transferred on the same filter and filtered with 20 ml of ethyl acetate. The filter with sediment is dried in air, then, it is dried at a temperature of 100-105 °С to constant mass. Contents of summarized polysaccharides containing in the sample of completely dried raw material (taking into consideration moisture) is calculated in percent of difference of mass of the filter with sediment less then mass of the filter.¹

Methods of determination of fatty acid composition of fatty oil:

The following standard samples and reagents are used to determine fatty acid composition: stabilized chloroform (qualified by CP, “Chimmed”, Russia), methanol (qualified by CP, “Aquametria”, Russia), commercial mixture FAME 37 Component mix in dichloromethane (Sigma Aldrich, Bellefone, USA), the methyl ether of undecanoic acid standard (C11-methyl ether, Sigma Aldrich, Bellefone, USA), the triglyceride tridecenoic acid standard (TAGS11, Toronto Research Chemicals, Canada), acetyl chloride (Acrosorganics, Belgiom), hexane for chromatography (GOST (the RF standards and regulations) 31665-2012 “Chimmed”, Russia), and water cleared with a MilliQ system.

To analyze fatty acid composition of flax seeds and the “Dicloran ® Plus” drug, quantitate extraction of fatty oil is performed. For this purpose, 2-3 g of drug or of granular flax seeds (2 mm) are placed into a 50 ml test tube. 30 ml of mixed chloroform and methanol (2:1 volume) are added to each sample, then, the test tubes are closed with plugs and mixed in rotation shaker (BiosanOS-10, Latvia) during 1.5 hours. Then, 10 ml of water cleared with a MilliQ system are added, mixed, and centrifuged at a speed of 3000 r/min during 5 minutes. Lower chloroform solution of lipophilic fraction of each sample is bottled into previously weighed 50 ml round-bottom flasks. Chloroform is recovered from the flasks with the vacuum rotation evaporator (Heidolph “Hei-VapAdvantage”, Germany). Remained lipophilic fraction is dried at a temperature of 100-105 °С (drying oven Binder FED 53, Germany) to constant mass during 5-10 min, the flask is frozen and weighed in analytical balance (“Ohaus” AR 3130) to constant mass. To analyze fatty acid composition of fatty oil in capsules, extraction is not performed.

To analyze fatty acid composition, about 10 mg of lipophilic fraction of each sample are put into the test tubes with the screw plug with the layer to note mass. Then, 800 µl of mixed solution of inner methanol standard (methyl ether of undecanoic acid, C11:0-methyl ether C=0.593 mg/ml, glyceryltritridecanoate, TAGS13:0, C=0.513 mg/ml), and buthylhydroxytoluene (C=0.012 mg/ml) are put into the test tubes as antioxidant. 1 ml of methanol, 20 µl of hexane, 20 µl of acetyl chlorideare added to samples. The test tubes are closed with plugs and placed into the oven at 80 °С temperature for an hour methylation after short mixing. After samples cooling to room temperature, 2.5 ml of hexane and 100 µlof water cleared with a MilliQ system are added and mixed with the rotation shaker for about 10 seconds. After layering of 1 ml of upper hexane methyl ether layer, 2.5 ml of hexane, and 100 µl of water cleared with a MilliQ system are transferred into vials to be GC-analyzed.

Chromatographic separation is performed with a gas chromatograph 7890А (Agilent Technologies, USA) with an auto sampler 7683B (Agilent Technologies, USA), flame ionization detector, and 100 m column Agilent J&W GC ColumsSelect FAME, 0.25 mm×0.25 µm (Agilent Technologies, Netherlands). The conditions of the GC-FID analysisare the following: volume of the introduced sample is 1 µl; flow separation mode is 30:1; the carrier gas is nitrogen; flow rate is of 0.9 ml/min; the sample injector temperature is of 260°С; the detector temperature is of 240°С; chromatography separating conditions are the following: the set temperature is of 140°С (5 min isothermal line); then, set temperature increases up to 220 °С with a speed of 4 °С/min during 25 min. Collection of data and processing of chromatograms and accumulation spectrum are performed with the Agilent Chem Station Rev.B.04.03 and “Microsoft Excel 2007” software accordingly.

Determination of contents of fatty acids as individual compounds is calculated in terms of undecanoic acid methyl ether as inner standard, the level of complete interesterification and methyl ether extraction containing in hexane is controlled with TAGS13 standard. For calculation, the conversion factor of methyl ether contents of fatty acids into free fatty acids contents (according to the standard procedure)²¹, as well as previously determined detector response coefficients for each fatty acid (calculated using the FAME 37 Component mix) were used.

Methods of determination of vitamins:

The following standard samples and reagents are used to determine vitamins: vitamin В₂ (R4500 98%, Sigma Aldrich, USA), vitamin C (A92902 99%, Sigma Aldrich, USA), vitamin E (T3251 (±)-α-tocopherol 96%, T3376 DL-α-tocopherolacetate96%, Sigma Aldrich, USA), carotenoids (74283 (±)-α-Carotene 95%, C4582 β-carotene 95%, Sigma Aldrich, USA) of potassium phosphate monobasic (pure, Panreac, Spain), ortho-phosphoric acid (85%, Panreac, Spain), trichloracetic acid (99.5%, Merck, Germany), amylorisin (fungal α-Amylase, (fungal α-Amylase, trading house “Biopreparat”, Russia), and non-aqueous sodium acetate (S2889 99% Sigma Aldrich, USA). The vitamins are determined by the methods described in works.²²^-²⁵

To determine the level of water-soluble vitamins (vitamin C and riboflavin) containing in flax seeds infusion, the infusion is prepared in accordance with the production recipe. Aliquote, containing in 2 ml of prepared infusion, is selected into the Eppendorf test tubes, than, 100 µl of trifluoroacetic acid is added to settle proteins, and centrifuged in the 5424 Eppendorf centrifuge (maximal rotation is up to 20000 r/sec, Eppendorf, Federal Republic of Germany) at a speed of 14500 r/min during 10 minutes.

To determine the level of vitamin C in ground flax seeds, vitamin C is extracted with distilled water by intensive mixing in the IKA Vortex 2 rotation shaker (IKA, Germany) at less than 60 °C temperature; the samples are infused on the UV bath (RK 31, Bandelin electronic (sn: 329.00072514.029), Germany). Then, volume of distilled water is increased up to the unit of flask and mixed. Aliquote, containing in 2 ml of prepared infusion, is selected into the Eppendorf test tubes, than, 100 µl of trifluoroacetic acid are added to settle proteins, and centrifuged in the Eppendorf centrifuge at a speed of 14500 r/min during 10 minutes.

To determine the level of riboflavin in granular seeds, consecutive acid and enzyme hydrolysis are performed. For this purpose, the sample of granular flax seeds is placed into a 100 ml conical flask, then, 50 ml of 0.1 M hydrochloric acid solution are added, than, the flask is opened and shaken on the rotation shaker IKA Vortex 2 (IKA, Germany) during 1 minute. Then, flask with sample is placed on the water bath STEGLER WB-2 (STEGLER, China) at 95 °C temperature (acid hydrolysis of the sample) for 40 minutes. After cooling, sample is mixed with the necessary amount of 2.5 M sodium acetate solution up to рН=5. The pH-solution of sample is controlled with the pH meter РВ-11 (Sartorius AG, Germany). Then, 0.2 g of amylorisin (fungal α-amylase, trading house “Biopreparat”, Russia) are added to sample, shaken on the rotation shaker IKA Vortex 2 (IKA, Germany) during 1 minute, then, opened flasks are placed on the water bath STEGLER WB-2 (STEGLER, China) at 50 °C temperature (enzyme hydrolysis) for 3 hours. After hydrolysis, samples are cooled, volume is increased up to 100 ml, and samples are mixed. Aliquote, containing in 2 ml of prepared infusion, is selected into the Eppendorf test tubes, than, 100 µl of trifluoroacetic acid is added to settle proteins and centrifuged in the 5424 Eppendorf centrifuge (Eppendorf, Federal Republic of Germany) at a speed of 14500 r/min during 10 minutes.

To determine the level of fat-soluble vitamins (vitamin Е and carotenoids), sequential extraction of chloroform and methanol in 1:3 proportion is performed. At first, 5 ml of chloroform are added to the sample and mixed with the shaker (BiosanOS-10, Latvia) for 10 minutes. Then, 15 ml of methanol are added to the sample and mixed with the shaker (BiosanOS-10, Latvia) for 10 minutes. The sample is settled for 1 minute, than, supernatant layer is selected into the Eppendorf test tubes and centrifuged into the 5424 Eppendorf centrifuge (Eppendorf, Federal Republic of Germany) at a speed of 14500 r/min for 10 minutes.

Chromatographic separation of vitamins is performed with the liquid chromatograph Aglient 1100 (Agilent Technologies, USA) as well as with the delivery and degassing system into two solvents, with the diode array, the column oven, and the autoinjector (autosampler). Collection of data and processing of chromatograms and accumulation spectrum are performed with the Agilent Chem Station software.

Conditions of chromatography of water-soluble vitamins (C and B₂).

Column: Atlantis C₁₈is of 4.6×250 mm and 5 mum (Waters, USA); flow rate is of 0.9 ml/minutes. Total time of analyze is 25 minutes. Measurements are performed at wave long of 254 (ascorbic acid) and 450 Nm (riboflavin). Mobile phase is gradient elution with mixed acetonitrile (A) 0% and phosphate buffer (7.4 mm of potassium phosphate monobasic + phosphoric acid) рН=2.5 (B) 100%. Gradient: 0 minutes. – 0% A, 5 minutes. – 0% A, 22 minutes. – 40% A, 23 minutes. – 0% A, 25 minutes. – 0% A.

Conditions of chromatography fat-soluble vitamins (E and carotenoid). Column: Thermo C₁₈is of 2.1× 100 mm and 5 mum (Thermo, USA); flow rate is of 1.0 ml/minutes. Total time of analyze is 24 minutes. Measurements are performed at wave long of 224 and 290 Nm (vitamin E). Mobile phase is gradient elution with mixed 60% of acetonitrile (A) and 40% of distilled water (B). Gradient: 0 minutes – 60% A, 5 minutes – 60% A, 15 minutes – 100% A, 19 minutes. – 100% A, 20 minutes – 60% A, 24 minutes – 60% A.

RESULTS AND DISCUSSION:

Contents of polysaccharides:

Contents of polysaccharides in flax seeds and in flax seeds infusion are determined by gravimetry, it is of 8.23±0.38% and 0.62±0.04% consequently (Table 1). The contents index of polysaccharides in flax seeds is regulated by SPRF XV ed. (not less than 7 %). The contents index of polysaccharides in the infusion is determined for the first time and may be recommended to be included into the regulatory documentation.

Table 1: Contents of polysaccharides

Name of sample	Contents of polysaccharides, %
Flax seeds	8.23±0.38
Flax seeds infusion	0.62±0.04

Contents of fatty oil and its fatty acid composition:

Results of analysis to determine contents of fatty oils and its fatty acid composition are shown in Table 2. α-linoleic acid, oleic acid, and linoleic acid are the main fatty acids for marking all subjects. The characteristic ratio of acids (about 4:1:1) may be used to confirm identity of fatty flax seeds oil drugs and DS in food. The fatty acid profile may be used to identify and to differentiate the unsaturated fatty acids subjects.²⁶ The GC-FID chromatograms are illustrated in figure 2.

Figure 2: GC-FID chromatograms; 1 - DS “Flax seeds oil in capsules”; 2 - flax seeds oil; “Dicloran ® Plus” drug. Indications: C11 - internal standard No. 1, C13 - internal standard No. 2

Fatty acid composition is important characteristics to be controlled during the pharmacopeial analysis of medicinal plant raw materials.²⁷

Contents of vitamins:

Results of determining analysis of contents of lipid- and water-soluble vitamin (Vitamin B2, C, E, and carotenoid) are shown in Table 3, the HPLC-UV/VIS chromatogram are illustrated in figures 3, 4. The vitamin B₂, the vitamin E, and carotenoid contents indexes are recommended for quality assurance of flax seeds; vitamin B₂index is recommended for quality assurance of infusion; carotenoid contents indexes are recommended for quality assurance of the DS “Flax seeds oil in capsules”; the vitamin E contents index is recommended for quality assurance of the “Dicloran ® Plus” drug. There is no purpose to regulate contents of vitamin C because of its high lability as well as because these raw materials and drugs are not the vitamin C sources, the main pharmacological effect is caused by other BAS group.

Table 2: Contents of fatty acids

Name of fatty acid	Index of fatty acids	Flax seeds oil in capsules	Flax seed soil	“Dicloran ® Plus” drug
Name of fatty acid	Index of fatty acids	mg/g of sample
Myristic acid	14:0	1±0	<1	-
Palmitic acid	16:0	41±8	20±4	2±0
Palmitoleic acid	16:1 9-cis	1±0	2±0	-
Margaric acid	17:0	1±0	2±0	-
Heptadecenoic acid	17:1	1±0	2±0	-
Stearic acid	18:0	24±5	17±3	3±1
Elaidic acid	18:1 9-cis	1±0	-	-
Oleic acid*	18:1 9-cis	128±26	60±12	6±1
Vaccenic acid	18:1 11-trans	8±0	3±1	<1
Linoleic acid*	18:2	154±31	61±12	5±1
γ-Linoleic acid	18:3 ω-6 6 cis,9 cis,12cis	2±0	1±0	-
α-Linoleic acid*	18:3 ω-3 9 cis,12 cis,15cis	541±108	234±47	20±4
Arachidic acid	20:0	2±0	1±0	-
Eicosatrienoic acid n6	20:3 8,11,14-cis	4±1	<1	-
Behenic acid	22:0	1±0	1±0	-
Lignoceric acid	24:0	<1	<1	-
In terms of contents of lipids, mg/g of sample		1000**	438±53	277±33

* major fatty acids containing in fatty oils of flax seeds

** mass of accurately weighed sample of fatty oil extracted from capsule.

At the moment, many issues related to the use of flax seeds remain under development and discussion. It is important to develop up-to-date regulatory documentation that would allow assessing the quality of both medicinal plant raw materials and preparations based on it.^28-30

CONCLUSION:

Flax seeds are valuable medicinal and food raw material because of its various complex of biologically active substances (BAS). They have a great potential for pharmaceutical practice and food industry. There is a purpose to standardize the “Flax seeds” medicinal plant raw material and the drugs based on medicinal plant raw material in accordance with the prescribing information for applying of the raw materials. The development/improvement requirements may be set out as follows. To prepare the medicinal plant raw material infusion, it is necessary to determine summarized polysaccharides (pharmacopeial method: gravimetry/ spectrophotometric) and qualitative composition of polysaccharide complex (modern physicochemical methods: HPLC-RD, etc.). To prepare medicinal plant raw material fatty oil and the drugs containing fatty oil, it is necessary to determine contents of fatty oils, fatty acid composition of the medicinal plant raw material, and contents of the fat-soluble vitamins.

In summary, it is important to note that it is necessary to standardize flax seeds in the following BAS groups: polysaccharides, fatty oil, and vitamins. So, the regulatory documentation of PM.2.5.0026.15 “Flax seeds” is to be completed. There is a purpose to include such sections as “Determination of the main groups of biologically active substances (fatty oil: histochemical reaction with Sudan III)” and “Quantitation (“amount of fatty oil is not less than...”)” into the PM “Fatty flax seeds oil”, as well as to determine the PM “Fatty flax seeds oil”.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.02.2024 Modified on 30.05.2024

Research J. Pharm. and Tech 2024; 17(10):4840-4846.

DOI: 10.52711/0974-360X.2024.00744

Name of vitamin	Subjects of research
Name of vitamin	Flax seeds	Flax seeds infusion	DS “Flax seeds oil in capsules”	“Dicloran ® Plus” drug
Vitamin B₂	31.00±4.65 µg/100 g	2.85±0.43 µg/100 ml	–	–
Vitamin C	330.00±49.50 µg/100 g	45.00±6.75 µg/100 ml	–	–
Vitamin E (summarized tocopherols in terms of acetate)	19.60±2.94 µg/100 g	–	27.00±4.05 µg/100 g	1.10±0.17 µg/100 g
Carotenoids	10,30±1,55µg/100 g	–	14.90±2.24 µg/100 g	–