JP7217134B2 - How to identify saw palmetto oil - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Publication: Proceedings of the 65th Annual Meeting of the Japan Society for Food Science and Technology Publication date: August 22, 2018 Name of the meeting: The 65th Annual Meeting of the Japan Society for Food Science and Technology Publication date: August 23, 2018 Day

The present invention relates to a method for discriminating saw palmetto fruit-derived oil from fake oil.

Saw palmetto is a small plant of the palm family that is distributed in the southeastern part of North America, and has been used as a medicinal herb by the indigenous people of America since ancient times. The main components of saw palmetto fruit are fatty acids such as lauric acid and oleic acid, and trace amounts of higher alcohols and plant sterols are also included. Saw palmetto oil is expected to be effective in preventing and improving frequent urination and in hair care. At present, about 10 types of saw palmetto oil are distributed in the Japanese market, but many cheap counterfeit oils such as artificial saw palmetto oil whose fatty acid composition is adjusted using animal and vegetable oils are distributed.

The fake oils are often saw palmetto oil blends that have been diluted using cheaper undeclared vegetable oils (palm, canola, coconut, etc.). If the imitation oil has a similar fatty acid composition to that of the saw palmetto fruit oil, it is difficult to distinguish with conventional analytical methods.

Methods for determining whether a product distributed as saw palmetto oil is a counterfeit oil have so far been used to analyze fatty acid composition, viscosity, color, and β-sitosterol, which is a standard component. However, the fatty acid composition is determined by adding animal and plant-derived fatty acids, the viscosity is determined by the addition of emulsifiers, the color is determined by the addition of colorants, and β-sitosterol is determined by the addition of other plant-derived products. It was adjusted to resemble the oil, and lacked a decisive factor to determine whether it was fake oil.

As an improvement measure, saw palmetto oil manufacturers and others have proposed a method of discriminating fake oil (Non-Patent Documents 1 and 2), but the method is not yet highly accurate.

Anthony B. et al. “A phytochemical comparison of saw palmetto products using gas chromatography and 1H nuclear magnetic resonance spectroscopy metabolomic profiling” Journal of Pharmacy and Pharmacology, 66 (2014) pp. 811-822 Matteo P. et al. “Combined use of isotopic fingerprint and metabolomics analysis for the authentication of saw palmetto (Serenoa repens) extracts”Fitoterapia 127 (2018) pp.15-19

An object of the present invention is to provide a method for identifying saw palmetto oil with simple steps.

As a result of intensive research by the present inventors to solve the above problems, by extracting and analyzing the highly polar fraction of the sample, trace components peculiar to saw palmetto oil raw materials were detected, and saw palmetto oil was identified as fake oil. The present invention was completed by discovering that it can be distinguished.

That is, the present invention is as follows.
[1] First step of extracting a highly polar fraction of the test sample, second step of analyzing the highly polar fraction of the test sample obtained in the first step, and analysis of the test sample obtained in the second step A method for identifying saw palmetto oil comprising a third step of comparing the results to a standard result for saw palmetto oil.
[2] A first step of extracting a highly polar fraction of the test sample, a second step of analyzing the highly polar fraction of the test sample obtained in the first step, and analysis of the test sample obtained in the second step A method for identifying saw palmetto oil, comprising a third step of determining that the test sample is equivalent to saw palmetto oil if, in the results, linolenic acid or cinnamic acid is present in the highly polar fraction of the test sample.
[3] The analysis of the second step is high-performance liquid chromatography, and the analysis result of the third step is a chromatogram obtained by a measurement wavelength of 212 nm or 280 nm using an ultraviolet-visible detector or a PDA detector. 1] or [2].
[4] A first step of extracting a highly polar fraction of the test sample, a second step of analyzing the highly polar fraction of the test sample obtained in the first step, and analysis of the test sample obtained in the second step Saw palmetto oil comprising a third step of determining that the test sample is equivalent to saw palmetto oil if, in the results, a substance of molecular weight 270 and molecular formula C ₁₈ H ₂₂ O ₂ is present in the highly polar fraction of the test sample. method of determination.
[5] The analysis in the second step is high-performance liquid chromatography, and the analytical result in the third step is a chromatogram obtained at a measurement wavelength of 412 nm using an ultraviolet-visible detector or a PDA detector, [4] The method described in .

According to the method of the present invention, saw palmetto oil can be discriminated from fake oil by a simple process.

It is a chromatogram of each sample obtained at a measurement wavelength of 212 nm. It is a chromatogram of each sample obtained at a measurement wavelength of 280 nm. It is a chromatogram of each sample obtained at a measurement wavelength of 412 nm. It is the figure which compared the peak of saw palmetto oil obtained at the measurement wavelength of 212 nm with the peak of linolenic acid. It is the figure which compared the peak of saw palmetto oil obtained at the measurement wavelength of 280 nm with the peak of cinnamic acid. It is a peak mass spectrum of saw palmetto oil obtained at a measurement wavelength of 412 nm. It is a peak mass spectrum of saw palmetto oil obtained at a measurement wavelength of 412 nm.

The present invention is a method for distinguishing between saw palmetto oil and fake oil by extracting and analyzing a highly polar fraction from what is present as saw palmetto oil.

<First step of extracting a highly polar fraction of the test sample>
In the present invention, the test sample used is one present as saw palmetto oil. Ground saw palmetto fruit (positive control), vegetable oils such as palm oil, canola oil, and coconut oil (negative control) can also be used for comparison.

A highly polar fraction of a test sample can be extracted using known methods. A highly polar fraction of the test sample can be obtained with a highly polar solvent. Snyder's polarity parameter is used herein for the polarity value of the solvent. A highly polar solvent herein has a Snyder polarity parameter of 3.9 or higher. A highly polar fraction can also be obtained by liquid-liquid extraction, in which case the solvent mixture ratio is preferably 1:2 to 2:1, more preferably 1:1.

As used herein, "Snyder's polarity parameter" means a parameter for quantitatively expressing the polarity of an organic solvent defined by Snyder (LR Snyder, Journal
of Chromatography A, Vol. 92, p.223-230, 1974). Snyder's polarity parameter values for specific organic solvents can be obtained, for example, by referring to values described in published literature (e.g., Wako Analytical Circle No. 11, "Chromatography Q &A"), or by calculating equilibrium constants according to said literature. It can be calculated by measuring.

Highly polar solvents include, for example, methanol, ethanol, propanol, butanol, acetone, pyridine, dimethylsulfoxide and dimethylformamide. May contain water. For example, hexane, cyclohexane, benzene, chloroform, dichloromethane, tetrahydrofuran may be used to degrease the test sample.
The amount of solvent used for extraction is not particularly limited, but it is usually 200 to 10,000 parts by volume, preferably 500 to 2,000 parts by volume, per 100 parts by volume of the test sample.

Extraction of the highly polar fraction of the test sample is carried out at room temperature to heating (for example, 20 to 100° C.) while standing or stirring. In addition, the extraction time is preferably 30 minutes or more in order to sufficiently transfer the highly polar fraction of the test sample. A test sample is separated into a high-polarity fraction and a low-polarity fraction by extraction, and the high-polarity fraction is fractionated.

The highly polar fraction is then concentrated and dried as required. Known vacuum concentration, membrane concentration, freeze concentration, vacuum drying, spray drying, or freeze drying can be used to concentrate and dry the highly polar fraction of the test sample.
Since the highly polar fraction is subjected to analysis in the next step, it is dissolved in an organic solvent if necessary. Dimethylformamide and methanol, for example, can be used as this organic solvent.

<Second step of analyzing the highly polar fraction of the test sample>
A well-known analysis method can be used for the analysis of the highly polar fraction of the test sample. Since the component contained in the highly polar fraction is in a trace amount, a method capable of detecting the trace component is preferable. Specifically, High Performance Liquid Chromatography (HPLC for short), Gas Chromatography (GC for short), Nuclear Magnetic Resonance (NMR for short) analysis, Mass Spectrometry abbreviated MS) and the like, and two or more of these may be used in combination. For the purpose of analyzing trace components, it is more preferable to use ultrahigh-performance high-separation liquid chromatography (common name: UPLC) as one type of high-performance liquid chromatography.

When analyzing the highly polar fraction of the test sample by high performance liquid chromatography (hereinafter sometimes referred to as HPLC), HPLC is performed by reverse phase or normal phase chromatography. As the column, silica gel, alumina, styrene-divinylbenzene copolymer, polymer gel such as polymethacrylate as a carrier, and a column having an octadecylsilyl group (C18) are preferred, but octyl group (C8), butyl group (C4), A column having groups such as trimethyl group (C3) and aminopropyl group (NH2) can also be used. Among these, an ODS column (C18 column) having an octadecyl group and using silica gel as a carrier is preferable in that analysis can be performed with higher precision. When the C18 column is used, the peaks of each component contained in the highly polar fraction of the test sample are separated well, and the confidence intervals in the analysis are widened both at the upper and lower limits. A commercially available apparatus may be used for HPLC, and a commercially available column may be used. The carrier packed in the column can be spherical or crushed, and its particle size is usually 1.5 to 3 μm. It is required to have a high number of theoretical plates from the viewpoint of high-precision analysis, and the spherical carrier is 2 μm or less. is preferred, and 1.8 µm is more preferred. A guard column can also be used for the purpose of preventing clogging due to contaminants in the sample. The size of the column to be used depends on the volume of the sample to be injected, but for example, a column with an inner diameter of 1 to 5 mm and a length of 30 to 200 mm may be used. Preferably, one with an inner diameter of 2.1 mm and a length of 50 mm is used. The volume of the sample injected into the HPLC is not particularly limited as long as it can be injected into the column and does not overflow, but is preferably 1 μL to 10 μL, more preferably 2 μL to 5 μL.

A mobile phase with little or no UV absorption is used for HPLC analysis. Acetonitrile, methanol, tetrahydrofuran, and the like can be mentioned as such, but acetonitrile is preferred because it does not absorb UV. Elution may be performed by a gradient elution method in which the composition and concentration of the mobile phase are gradually changed. The flow rate of the mobile phase during analysis is 0.05 mL/min to 2 mL/min, preferably 0.1 mL/min to 1 mL/min.

<Third step of comparing the analysis results of the test sample with the standard results of saw palmetto oil>
When the analysis method is HPLC, the detector can be an ultraviolet-visible detector, an absorbance detector such as a PDA detector, a mass spectrometry detector, or the like.
The inventors of the present invention have found that characteristic peaks are present in the chromatograms of saw palmetto oil at measurement wavelengths of 212 nm, 280 nm and 412 nm in HPLC analysis using an ultraviolet-visible detector. The chromatogram of saw palmetto oil at a measurement wavelength of 212 nm has a peak at the same retention time as linolenic acid (C ₁₈ H ₃₀ O ₂ ). In the chromatogram of saw palmetto oil at a measurement wavelength of 280 nm, there is a peak at the same retention time as cinnamic _{acid ( C9H8O2} ). In the chromatogram of saw palmetto oil at a measuring wavelength of 412 nm, there is a peak for a substance with a molecular weight of 270 and a molecular formula of C ₁₈ H ₂₂ O ₂ .

In the present invention, obtaining a chromatogram of the test sample at measurement wavelengths of 212 nm, 280 nm and 412 nm, and comparing the chromatogram of the test sample with the chromatogram of saw palmetto oil, which is the standard result, is useful for distinguishing saw palmetto oil. is. A test sample is linolenic acid (C ₁₈ H ₃₀ O ₂ ), cinnamic acid (C ₉ H ₈ O ₂ ) or saw palmetto if at least one peak of a substance with molecular weight 270 and molecular formula C ₁₈ H ₂₂ O ₂ is present. It can be determined to be equivalent to oil. Also, if even one of these peaks does not exist, it can be determined that the oil is a counterfeit saw palmetto oil.

In addition, when saw palmetto oil is diluted with animal or vegetable oil, the peak shape of the chromatogram changes and the area of the peak peculiar to saw palmetto oil decreases compared to the chromatogram of saw palmetto oil. can identify something.

Results obtained using linolenic acid, cinnamic acid, or substances of molecular weight ₂₇₀ and molecular formula _C18H22O2 as standard results for saw _palmetto oil and analyzing the highly polar fractions of the test samples when the analytical method is not HPLC. is useful in distinguishing saw palmetto oil. If linolenic acid, cinnamic acid, or a substance with a molecular weight of 270 and a molecular formula of C ₁₈ H ₂₂ O ₂ can be detected by analyzing the highly polar fraction of the test sample, it can be determined to be equivalent to saw palmetto oil. . Also, if the presence of any one of linolenic acid, cinnamic acid, and a substance with a molecular weight of 270 and a molecular formula of C ₁₈ H ₂₂ O ₂ cannot be detected, it can be determined to be a counterfeit saw palmetto oil.

The standard results of saw palmetto oil used for comparison may be obtained at the same time as the analysis results of the test sample, or the past results obtained under the same conditions as the analysis conditions of the test sample may be used as the standard results.

The present invention will now be described in more detail with specific examples, but the present invention is not limited to the following specific examples.

[Examples 1 to 3] Comparison of chromatograms of ultrahigh-performance high-separation liquid chromatography Saw palmetto oil product 1, product 2, product 3, product 4, and product 5 were used as test samples, and saw palmetto dried fruit samples were exceeded for comparison. It was subjected to high-performance high-separation liquid chromatography (hereinafter referred to as UPLC). Product 1 is real saw palmetto oil.

(Preparation of test sample)
About 1 g of each of the saw palmetto oil products 1 to 5 was taken, 5 mL of 90% methanol was added, and the degreasing operation using 5 mL of hexane was repeated three times. 3.5 mL of the 90% methanol layer was collected and dried. After that, it was dissolved in 150 μL of methanol and subjected to analysis.

(Preparation of fruit sample)
After freeze pulverization, about 1 g was taken, and chloroform/methanol (1:1) was added and stirred. After filtering the suspension, the filtrate was dried. 5 mL of 90% methanol was added to the dried product, and degreasing operation with 5 mL of hexane was performed three times. After that, 3.5 mL of the 90% methanol layer was collected and dried again. The resulting dried product was dissolved in 150 μL of dimethylformamide and subjected to analysis.
As for product 4, a 5-fold dilution was used because there was an excessively detected peak.

(UPLC analysis)
Each sample was supplied to UPLC under the following conditions. The resulting chromatograms are shown in Figures 1-3. The arrows in each figure indicate peaks characteristic of saw palmetto oil at the respective measurement wavelengths. 1 to 3, it can be determined that products 2 and 3 among the test samples are saw palmetto oil equivalents, and products 4 and 5 are counterfeit oils.

(Analysis conditions) (UPLC)
Equipment: Waters ACQUITY UPLC
Column: Waters ACQUITY UPLC HSS C18 (100×2.1 mm id)
Mobile phase: A = 5% MeCN/ _H2O + 0.05% HCOOH, B = MeCN + 0.05% HCOOH
Gradient: %B = 10 (0 min) → 50 (5-7.5 min) → 100 (9-15 min)
Column temperature: 40℃
Flow rate: 0.40 mL/min
Injection volume: 0.5 μL
Detector: PDA
Measurement wavelength: 212 nm (Example 1), 280 nm (Example 2), 412 nm (Example 3)

[Test Example 1] Analysis of specific components of saw palmetto oil by HPLC (preparation of high-concentration sample)
1 mL of saw palmetto oil was liquid-liquid partitioned with 5 mL of 90% methanol and 5 mL of hexane. 3.5 mL of the 90% methanol layer was collected and dried. After that, it was dissolved in 150 μL of methanol.

(HPLC analysis)
It was expected that the characteristic peak at the measurement wavelength of 212 nm (hereinafter referred to as peak A) was linolenic acid and the characteristic peak at the measurement wavelength of 280 nm (hereinafter referred to as peak B) was cinnamic acid. In order to confirm this, the retention times were compared with a linolenic acid standard and a cinnamic acid standard under the following conditions. The results are shown in FIGS. Since both peak A and peak B had the same retention time as the standard, peak A was presumed to be linolenic acid and peak B was cinnamic acid.

(Analysis conditions) (HPLC)
Instrument: JASCO LC2000 Plus Column: Waters XSelect ^TM HSS C18 (150 x 3.0 mm id)
Mobile phase: A = 5% MeCN/ _H2O + 0.05% HCOOH, B = MeCN + 0.05% HCOOH
Gradient: %B = 0 (0 min) → 50 (10-20 min) → 100 (23-38 min)
Column temperature: 40℃
Flow rate: 0.40 mL/min
Injection volume: 5 μL
Detector: UV
Measurement wavelength: 212 nm, 280 nm

[Test Example 2] Analysis of specific components of saw palmetto oil by liquid chromatography/high-resolution mass spectrometry (LC/MS analysis) (preparation of high-concentration sample)
Samples were prepared in the same manner as in Test Example 1. 1 mL of saw palmetto oil was liquid-liquid partitioned with 5 mL of 90% methanol and 5 mL of hexane. 3.5 mL of the 90% methanol layer was collected and dried. After that, it was dissolved in 150 μL of methanol.

(LC/MS analysis)
LC/MS analysis was performed to identify the molecular weight and molecular formula of the characteristic peak (hereafter referred to as peak C) at a measurement wavelength of 412 nm. The results are shown in FIG. 6 and Tables 1 and 2. A mass spectrum of peak C was obtained and the molecular weight of peak C was estimated to be 270. The _putative molecular _formula was _assumed to be _{C18H22O2 or C16H20N30} , but could not be identified.

(Analysis conditions) (LC/MS)
Instrument (LC): Waters ACQUITY Instrument (mass spectrometer): Waters SynaptG2-S Column: ACQUITY UPLC HSS C18 (100 x 2.1 mm id)
Mobile phase: A = _H2O + 10 mM _HCOONH4 , B = MeCN
Gradient: %B = 10 (0 min) → 50 (5-7.5 min) → 100 (9-15 min)
Column temperature: 40℃
Flow rate: 0.40 mL/min
Injection volume: 2 μL
Detector: UV (212, 280, 412 nm), MS (APCI)

(LC/MS/MS analysis)
To identify the molecular formula of Peak C, LC/MS/MS analysis was performed. Sample preparation and analysis conditions were the same as for LC/MS. The results are shown in FIG. Two fragment ions thought to be derived from dehydration were detected, and peak C was thought to have two or more oxygen atoms, so the molecular formula of peak C was presumed to be C ₁₈ H ₂₂ O ₂ .

Claims (4)

a first step of extracting a highly polar fraction of the test sample;
In the second step of analyzing the highly polar fraction of the test sample obtained in the first step, and the analysis results of the test sample obtained in the second step, cinnamic acid is present in the highly polar fraction of the test sample In some cases, a method for identifying saw palmetto oil, comprising a third step of determining that the test sample is equivalent to saw palmetto oil. The analysis of the second step is high performance liquid chromatography,
The method according to claim 1 , wherein the analytical result of the third step is a chromatogram obtained with a measurement wavelength of 280 nm using a UV-visible detector or a PDA detector. a first step of extracting a highly polar fraction of the test sample;
In the second step of analyzing the highly polar fraction of the test sample obtained in the first step, and in the analysis results of the test sample obtained in the second step, the highly polar fraction of the test sample has a molecular weight of 27
0, A method for identifying saw palmetto oil, comprising a third step of determining that the test sample is equivalent to saw palmetto oil if a substance of the molecular formula C ₁₈ H ₂₂ O ₂ is present. The analysis of the second step is high performance liquid chromatography,
4. The method according to claim 3 , wherein the analytical result of the third step is a chromatogram obtained with a measurement wavelength of 412 nm using a UV-visible detector or a PDA detector.

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