INTRODUCTION:

Tagetes patula L. (spreading marigold) is a well-known decorative plant that is used in folk medicine.^1,2 In our opinion, the flowers of this plant are interesting as potential medicinal plant raw material. The plants of the genus Tagetes are known to be potential sources of biologically active compounds such as simple phenols, essential oil, vitamins (carotenoids), and flavonoids (patulitrin, patuletin, quercetin, isoquercitrin, quercimeritrin, isorhamnetin and its glycosides).^1-6 The antimicrobial properties and wound-healing and antioxidant activity were reported for T. patula flowers.^7-9 In our opinion, this pharmacological activity is due to the flavonoids in the raw material of this plant. Therefore, standardization of T. patula flowers for flavonoid content is a actual problem.

The main and diagnostically significant flavonoids for plant raw material of the genus Tagetes are patulitri (7-O-β-D-glucopyranoside of 3,5,7,3’,4’-pentahydroxy-6-methoxyflavone) and patuletin (3,5,7,3’,4’-pentahydroxy-6-methoxyflavone).

The aim of this study was to develop a method for the quantitative determination of the content of patulitrin and patuletin in the flowers of Tagetes patula by HPLC.

MATERIAL AND METHODS:

Plant material:

The flowers of the Tagetes patula L. (variety "Tangerine") was collected on the territory of the Samara region, in the Botanical Garden of Samara University (August-September 2018 and 2019).

Extraction of Plant Materials:

The extraction of the flowers of the Tagetes patula L. (150g) was carried out with the using of 70% EtOH in ratio 1:5, combining the maceration method with subsequent thermal extraction in a boiling water bath. The obtained water-alcohol extract was filtered and then evaporated by using a rotary evaporator at low temperature (40-50°C) and reducing pressure to thick residue (about 200ml).

Isolation of Compounds from Plant Materials:

The isolation of flavonoids 1 and 2 from the obtained thick extract of the Tagetes patula L. flowers was carried out with the using of the method of adsorption column chromatography. For this the obtained concentrated extract was dried on L 40/100 silica gel and the resulting powder was applied to a silica gel layer formed in chloroform. The chromatographic column was eluted with chloroform and a mixture of chloroform-ethanol in various ratios (99:1, 98:2, 97:3, 90:10, 85:15, 80:20, 70:30, and 60:40). The eluates were divided into fractions of approximately the same volume (200ml each), then evaporated under vacuum. The elution of the compounds was monitored by TLC analysis on “Sorbfil PTLC-AF-A-UV” plates in a system of chloroform-ethanol-water (25:18:2). The spots on the plate were detected by luminescence in UV light at a wavelength of 254 and 366nm and by color after processing the chromatograms with an alcoholic solution of aluminum chloride. The fractions obtained by elution with 60:40 CHCl₃–EtOH afforded the dominant compound 1 with R_f 0.60. The fractions eluted by 93:7 CHCl₃–EtOH afforded a compound 2 with R_f 0.80.

As results, we isolated flavonoids 1 and 2. The additional purification of the isolated flavonoids was carried out by recrystallization from the mixture of alcohol and water.

Methods of Structural Elucidation of Flavonoids:

UV spectra were recorded using a spectrophotometer “Specord 40” (Analytik Jena, Germany) in cuvettes with a layer thickness of 10 mm in the wavelength range from 190 nm to 700 nm. ¹H-NMR spectra were obtained on a spectrometer “JNM-ECX 400” (JEOL Ltd., Japan) at a frequency 399.78 MHz, ¹³C-NMR spectra were obtained on a spectrometer “JNM-ECX 400” (JEOL Ltd., Japan) at a frequency 100.52 MHz.

HPLC of Plant Materials:

The raw material was crushed so that its particles passed through a sieve with holes 3 mm in diameter. An accurate weighed sample of ground material (about 1 g) was placed in a 100mL flask and 50mL of 70% ethyl alcohol was added. The flask was closed with a stopper and weighed on a balance accurate to 0.01g. The flask was attached to a reflux condenser and heated in a boiling water bath for 45 min. After boiling, the flask was cooled for 30 min, closed with the same stopper, weighed again, and the extractant was added to its original weight. The resulting aqueous-alcoholic extract was filtered through a paper filter.

HPLC analysis was carried out using a Milichrom-6 chromatograph (NPAO Nauchpribor) under the following conditions of reverse-phase chromatography in an isocratic mode: steel column KAH-6-80-4 (2 mm x 80 mm; Separon-C18, 7μm), mobile phase - acetonitrile: 1% solution of acetic acid in water in a ratio of 3:7, elution rate - 100μL/min, eluent volume - 2500 μL. The compounds were detected at a wavelength of 360 nm. Volumes of injected samples: 4 μl (reference solutions of patulitrin and patuletin, the aqueous EtOH extract from T. patula flowers).

The compounds were detected at wavelengths of 360 nm. At least 3 concurrent definitions are carried out.

In parallel, 4μl of solutions of reference samples of patulitrin and patuletin were injected into the chromatograph and chromatographed as described above. The peak areas of patulitrin and patuletin were determined. The average peak area was calculated based on the results of three parallel determinations.

The retention time was determined and the peaks of patulitrin and patuletin were identified on the chromatogram of the test solution. The peak areas of patulitrin and patuletin were calculated on the chromatogram and the average peak area was calculated using three parallel determinations.

The content of patulitrin (X) in the flowers of the Tagetes patula L. in terms of the dry raw materials was calculated by the formula:

where:

S is the area of the patulitrin peak on the test solution chromatogram;

S₀ is the area of the patulitrin peak in the reference solution chromatogram;

m is the exact weight of plant raw material, g;

m₀ is the exact weight of patulitrin reference sample, g;

V is the volume of a volumetric flask in which an aliquot of aqueous-alcoholic extract was diluted, ml;

V₀ is the volume of a volumetric flask in which an aliquot of reference solution of patulitrin was diluted, ml;

V₁ is the volume of the injected test solution, μl;

V₂ is the volume of the injected sample of the reference solution of patulitrin, μl;

W is mass loss on drying, %.

The content of patuletin in the flowers of the Tagetes patula L. in terms of the dry raw materials as a percentage (X) was calculated using a similar formula.

Note: The preparation of patulitrin reference solution. About 0.05 g (accurately weighed) of the reference sample of patulitrin (content of the main substance ≥98%) is placed in a volumetric flask with a capacity of 50 ml, dissolved in a small amount of 70% ethanol, diluted to the mark with 70% ethanol, mixed.

The preparation of patuletin reference solution. About 0.05 g (accurately weighed) of the reference sample of patuletin (content of the main substance ≥98%) is placed in a volumetric flask with a capacity of 50 ml, dissolved in a small amount of 70% ethanol, diluted to the mark with 70% ethanol, mixed.

RESULTS AND DISCUSSION:

In the course of our research were isolated from Tagetes patula L. patulitrin (1) and patuletin (2) (Fig. 1), identified with using of UV, ¹H-NMR, ¹³C-NMR spectroscopy and chemical transformations.

Figure 1: The chemical structures of flavonoids isolated from Tagetes patula L. flowers:

patulitrin (1) and patuletin (2).

Patulitrin (7-O-β-D-glucopyranoside of 3,5,7,3’,4’-pentahydroxy-6-methoxyflavone) (1). The crystalline substance is a bright-yellow color composition C₂₂H₂₂O₁₄; m.p. 250–252°C (water alcohol). l_mаx ^EtOH 266, 382nm;+NaOAc 266, 384nm;+NaOAc+ H₃BO₃272, 400nm;+АlCl₃ 276, 382sh, 443nm; + АlCl₃ + HCl 275, 382sh, 438 nm; + NaOMe 303, 373, 445 (sh) nm.

¹H-NMR spectrum (399.78 MHz, DMSO-d₆, δ, ppm, J/Hz): 12.47 (1H, s, 5-ОН- group), 9.48 (3Н, br. s, 3-ОН- group, 7-ОН- group и 4’-ОН-group), 7.70 (1Н, d, 2.5 Hz, Н-2’), 7.52 (1Н, dd, 2.5 и 8.5 Hz, Н-6’), 6.92 (1Н, s, Н-8), 6.88 (1Н, d, 8.5 Hz, Н-5’), 5,11 (1Н, d, J = 7,12, Н-1’’of glucopyranose), 3,75 (3H, s, ОСН₃ at С-6), 3,3-4,6 (6Н of glucopyranose).

¹³C-NMR spectrum (100.52 MHz, DMSO-d₆, δ_C, ppm): 176.66 (С-4), 156.89 (С-7 ), 151.94 ( С-5), 151.58 (С-9), 148.43 (С-2), 148.22 (С-4’), 145.49 (C-3’ ), 135.31 (C-3), 132.32 (С-6), 122.39 (С-1’), 120.56 (С-6’), 116.08 (С-2’), 115.93 (С-5’), 104.12 (С-10), 100,64 (C-1’’ of glucose), 94.36 (С-8), 77,75 (C-5’’ of glucose), 77.20 (C-3’’ of glucose), 73.72 (C-2’’ of glucose), 70,08 (C-4’’ of glucose), 61.15 (C-6’’ of glucose), 56.56 (CH₃O at С-6).

Patuletin (3,5,7,3’,4’-pentahydroxy-6-methoxyflavone) (2). Crystalline substance of a bright-yellow color composition C₁₆H₁₂O₈; m.p. 265-267°C (dec.) (water alcohol). l_mаx ^EtOH 264, 296 sh, 378 nm; + NaOAc 268, 382 nm; + NaOAc + H₃BO₃270, 396 nm; + АlCl₃274, 381sh, 438 nm; + АlCl₃ + HCl 275, 381sh, 436 nm; + NaOMe 328, 368sh, 428 (sh) nm.

¹H-NMR spectrum (399.78 MHz, DMSO-d₆, δ, ppm, J/Hz): 12.54 (1H, s, 5-ОН-group), 10.65 (1Н, s, 7-ОН-group), 9.56 (1Н, s, 4’-ОН-group), 9.32 (1Н, s, 3-ОН-group), 7.64 (1Н, d, 2.5 Гц, Н-2’), 7.50 (1Н, dd, 2.5 and 8.5 Hz, Н-6’), 6.85 (1Н, d, 8.5 Hz, Н-5’), 6.48 (1Н, s, Н-8), 3,73 (3H, s, ОСН₃at С-6).

¹³C-NMR spectrum (100.52 MHz, DMSO-d₆, δ_C, ppm): 176.56 (С-4), 157.50 (С-7), 152.27 (С-5), 151.84 (С-9), 148.24 (С-2), 147.46 (С-4’), 145.49 (C-3’), 135.31 (C-3), 132.32 (С-6), 122.39 (С-1’), 120.56 (С-6’), 116.08 (С-2’), 115.93 (С-5’), 104.12 (С-10), 94.15 (С-8), 56.55 (CH₃O at С-6).

UV spectra of solutions of the water-alcohol extracts from T. patula flowers and solutions of the isolated flavonoids (1 and 2) were studied (Fig. 2).

Figure 2: The UV spectra of water-alcohol extraction from Tagetes patula L. flowers and flavonoids.

Designations: 1 – extraction; 2 – patulitrin; 3 – patuletin.

Patulitrin (1) and patuletin (2) were found to be mainly responsible for the absorption curve of the water-alcohol extract from T. patula flowers (Fig. 2). In our opinion, these flavonoids are important for the diagnostics of this plant raw material. As both flavonoids have an absorption maximum in the long-wave region of the electronic spectrum at 360±2 nm, we selected this wavelength for detecting of the analyzed substances by means of HPLC analysis.

It was determined that under the specified chromatography conditions, when using the acetonitrile – water system in a ratio of 3:7, it is possible to identify the analyzed components – patulitrin and patuletin (Fig. 3-6).

Figure 3: HPLC chromatogram of water-alcohol extraction from Tagetes patula L. flowers and flavonoids.

Designations: 1 – extraction; 2 – patulitrin; 3 – patuletin.

Figure 4: HPLC chromatogram of water-alcohol extraction from Tagetes patula L. flowers.

Designations: 1 –patulitrin; 2– patuletin.

Figure 5: HPLC chromatogram of patulitrin.

Figure 6: HPLC chromatogram of patuletin.

The retention times of peaks of substances on chromatograms of reference sample, as well as in the extraction from the flowers of Tagetes patula L. are presented in Table 1.

Table 1 – The retention times of peaks of flavonoid in flowers of Tagetes patula L.

Flavonoid	The retention times of peaks, min
Flavonoid	Reference sample	In the extraction
Patulitrin	3,188	3,009
Patuletin	10,770	11,385

Table 2 – The content of patulitrin and patuletin in water-alcohol extraction from Tagetes patula L. flowers (variety "Tangerine").

No.	Plant raw material sample	Patulitrin content, %	Patuletin content, %
1.	The flowers of the T. patula L. (Samara, the Botanical Garden of Samara University (August 2018).	5,28 ±0,17	0,0063
2.	The flowers of the T. patula L. (Samara, the Botanical Garden of Samara University (September 2018).	5,11 ±0,18	0,0023
3.	The flowers of the T. patula L. (Samara, the Botanical Garden of Samara University (August 2019).	5,64 ±0,17	0,0143

Since the content of patuletin in the flowers of T. patula is much lower than the content of patulitrin (Table 2), it is advisable to standardize this raw material only on the content of patulitrin.

The metrological characteristics of the proposed HPLC procedure indicate that the error in determining the average result of the patulitrin content in the flowers of the T. patula L. with a confidence level of 95% is ± 3.32% (Table 3).

Table 3 - Metrological characteristics of the method for the quantitative determination of patulitrin in the flowers of the T. patula L.

*Sample*	f	𝑋̅, %	S	*P, %*	*t (P,f)*	ΔX	𝜀*̅, %*
Tagetes patula L. flowers	10	5,12	0,3808	95	2,23	±0,17	±3,32

Designations: f – degrees of freedom; 𝑋̅ – average; S – standard deviation; P – confidential probability, t - Student's t-test, ΔX– half-width of the confidence interval of the mean result; 𝜀̅ – mean relative error.

CONCLUSION:

The results of the studies carried out indicate the advisability of the standardization of the flowers of Tagetes patula by determination of the content of the main and diagnostically significant flavonoid, patulitrin, using the HPLC method and detection on a UV detector at a wavelength of 360 nm.

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

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Received on 31.07.2021 Modified on 07.10.2021

Research J. Pharm. and Tech. 2022; 15(5):2216-2220.

DOI: 10.52711/0974-360X.2022.00368