Identification of flavonoids and hydroxycinnamic acids in polysaccharide-containing mixed herbal product (Pectorales species No 1) by Ultra-Performance Liquid Chromatography with Photodiode arrays and Tandem quadrupole Mass-selective detectors

Kakhramanova S.D.^1,2*, Bokov D.O.^1,3, Luferov A.N.¹, Samylina I.A.¹, Rendyuk T. D.¹,

Sergunova E.V.¹, Bondar A.A.¹, Fedorova L. V.¹, Klyukina E.S.¹, Malysheva M.O.¹,

Tikhomirova E.A.¹, Baeva V.M.¹, Stepanova O.I.¹, Yakubovich L.M.¹, Selifanov A.V.³,

Bessonov V.V.³

¹Sechenov First Moscow State Medical University, Institute of Pharmacy,

Department of Pharmaceutical Natural Science. Russian Federation.

²Federal State Budgetary Institution “Scientific Centre for Expert Evaluation of Medicinal Products”.

Russian Federation.

³Federal Research Center of Nutrition, Biotechnology and Food Safety, Russian Federation.

*Corresponding Author E-mail: ksd113@yandex.ru

ABSTRACT:

The pectoral species No 1 (phytopectol No 1) is widely used in Russian medicinal practice. Pectoral species No 1 (PS No 1) contains coltsfoot leaves (Tussilaginisfarfarae folia), marshmallow roots (Althaeae radices), oregano herb (Origani vulgaris herba). This research aims to determine the flavonoid profile of PS No 1. These biologically active compounds (BAC) can have pharmacological effects such as antioxidant, antibacterial. The UPLC/PDA/MS/MS method was used for flavonoids’ determination. Chromatographic system: ultra-performance liquid chromatograph Waters Acquity (Waters Corporation, USA). Column: 2.1×150 mm Acquity UPLC BEH (Bridged Ethylene Hybrid) C18 (particle size – 1.7 µm). The column temperature and the injection volume were 35 °C and 5 µL, respectively. Mobile phase A – a mixture of water, acetonitrile, and formic acid (95:5:0.1). Mobile phase B – a mixture of acetonitrile and formic acid (100:0.1). A gradient program was used for elution. Results: The pectoral species No 1 was established to contain various flavonoids as apigenin 7-O-β-glucuronide, luteolin 7,4'-diglucuronide-3'-glycoside, luteolin 7-O-glucuronide, luteolin 7-O-[β-D-glucuronosyl-(1→2)-β-D-glucuronide]-4'-O-β-D-glucuronide, rutin. Some derivatives of hydroxycinnamic acid such as caffeic acid, chlorogenic and isochlorogenic acids, isochlorogenic acid C (4,5-dicaffoylquinic acid), isochlorogenic acid A (3,5-dicaffoylquinic acid), 3,4,5-tricaffoylquinic acid were also identified in the herbal tea. Conclusions: The polyphenolic complex of pectoral species No. 1 was described; it was rich in flavonoids (flavonols and flavones) and hydroxycinnamic acid derivatives. The reported anti-inflammatory effect of the mixture herbal product (herbal tea) might be due to high flavonoid content. Thus, we recommend the flavonoids to be chosen as an active marker for the standardization of pectoral species No 1.

KEYWORDS: Flavonoids, hydroxycinnamic acids, polysaccharides, Origanum vulgare L., Tussilagofarfara L., Althaea officinalis L., pectoral species No 1, apigenin, luteolin, kaempferol, quercetin.

INTRODUCTION:

The mixed herbal products (MHP) or herbal teas (HP), being polyherbal formulations1^-6, have been widely used for the complex treatment of bronchopulmonary system diseases. The pectoral species No 1 (Phytopectol No. 1 (Pectorales species No. 1 (PS No. 1) is one of these herbal products and according to the project of the pharmacopoeial monograph (PM) it is composed of 40% marshmallow roots (dried roots from Althaea officinalis L. or A. armeniaca Ten., Fam. Malvaceae), 40% coltsfoot leaves (dried leaves from Tussilagofarfara L., Fam Asteraceae) and 20% oregano herb (dried aerial parts of Origanum vulgare L., Fam. Lamiaceae). According to the instructions for use, PS No 1 refers to the expectorants of plant origin pharmacological groups; its infusion has an expectorant as well as an anti-inflammatory effect. PS No. 1 is a mixture of heterogeneous particles of plant material; it has a greenish-gray color with white, grayish-white, greenish-brown, brown, and violet inclusions (Figure 1). The smell is weak, fragrant; the taste of the aqueous extract is spicy, with a sensation of mucus.

Figure 1. Phytopectol No. 1 (Pectorales species No. 1)

It is known that the main group of PS No 1 biologically active compounds (BAC) is polysaccharides. Insufficient attention was paid to the study of the PS No 1 component composition despite its fame and popularity. Currently, there are pharmacopoeial monographs (PM) on the individual components of the MHP; normative documentation (ND) regulating the quality of the PS No 1 itself requires revision. For the normative documentation improvement, it is necessary to know the composition of all PS No 1 BAС groups that have a pharmacological effect, including flavonoids.

There is information for the crude herbal drugs flavonoid profile in PS No 1. So, the main flavonoids of Origanum vulgare L. herb have been reported to be apigenin, luteolin, and their glycosides7^-¹⁰. Also, some hydroxycinnamic acid derivatives have been identified in the oregano herb9, as well. The literature data have shown that the roots of Althaea officinalis L. contain caffeic acid and isoscutellarin flavone¹², while coltsfoot leaves contain kaempferol and quercetin glycosides¹³. There are no data about flavonoids in Althaea armeniaca L. roots.

Researchers from different countries studied the pharmacological activity of coltsfoot leaves, marshmallow roots, and oregano herb. Research data are presented in Table 1.

The aim of the present work was the determination of the main polyphenol complex compounds, particularly flavonoids, in pectoral species No 1.

Table 1. PS No 1 components’ pharmacological activity

Extract	The main BAS group, compound		Biological/ pharmacological effect
Coltsfoot leaves (Folia tussilaginisfarfarae)
Fractionated extract, Fermented extract		kaempferol 3-O-[3,4-O-(isopropylidene)-α-L-arabinopyranoside]	Hypoglycemic effect¹⁴
Ethanol extract		Flavonoids: rutin, quercetinpentoside, quercetin glycoside, kaempferol glycoside, tricaffeoylquinic acid, quinic acid, chlorogenic acid	Antioxidant activity; Antimicrobial activity²⁹
Marshmallow roots (Radices althaeae)
Aqueous extracts		The total amount of phenolic compounds in terms of gallic acid	Antimicrobial activity¹⁶
Aqueous extracts		Hydrophilic compounds	Antiproliferative effect; Decreased cisplatin-induced aberration¹⁷
polysaccharide-depleted methanol-water (1:1) extract		Flavons: hypolaetin-8-O-β-D-glucuronopyranosyl-1''',4''-β-O-D-glucopyranoside, hypolaetin-4'-methlyether-8-O-β-D-glucopyranoside-2″-O-sulfate, hypolaetin-8-O-β-D-glucopyranoside-2″-O-sulfate, hypolaetin-4'-methylether-8-O-β-D-glucuronopyransoide-3″-O-sulfate, isoscutellarein-4'-methlyether-8-O-β-D-glucuronopyransoide-3″-O-sulfate; Cumarin: scopoletin-O-β-D-glucopyranosyl-L-rhamnoside	Anti-inflammatory effect¹⁸
Ethanol extract		quercetin, rutin, apigenin, isorhamnetin, scopoletin, coumarins, kaempferol	antioxidant, antitumor, antibacterial, cardioprotective, anti-inflammatory immunomodulatory effects¹⁹
Aqueous extract, methanol extract		rhamnogalacturonan	Bronchodilator effect, β-adrenergic effect²⁵
Alcoholic extract		The total amount of phenolic compounds	Hypolipidemic effect²⁶
Oregano herb (Herbaorigani vulgaris)
Alcoholic extract, essential oils		polyphenolic compounds. terpenes: thymol, carvacrol	Antioxidant effect (suppression of the destructive effect of diazinon on the body)²⁰
Essential oils		Terpenes: α-thujene, myrcene, α-terpinene, o-cymen, γ-terpinene, thymol, carvacrol	Antioxidant activity²¹
Essential oils		Terpenes: β-caryophyllene epoxide ‑ 13.3%; β-caryophyllene ‑ 8.2%; cymen‑ 5.2% Flavonoids: The sum of flavonoids in terms of catechin	Antioxidant activity
Essential oils		Terpenes: α-pinene, α-thujene, camphene, α-terpinene, γ-terpinene, p-cymene, trans-linalol oxide, 1-octen-3-ol, cis-linalol oxide, linalool, hotrienol, β-caryophyllene, α-humulene, α-terpineol, borneol, β-bisabolene, p-cymene-8-ol, caryophyllene oxide, elemol, thymol, carvacrol	Chelating ability
Fractionated extract		Hydroxycinnamic acids: rosmarinic acid, oleanolic acid, ursolic acid	Tyrosinase inhibition;
Aqueous extract, ethanol extract		Sum of biologically active compounds	Antimicrobial activity²²
Ethanol extract		Hydroxycinnamic acids: rosmarinic acid, chlorogenic acid, p-coumaric acid, gentisic acid (2,5-dihydroxybenzoic acid); Flavonoids: hyperoside, isoquercetin, rutin, quercetin, quercetrin, luteolin	Antioxidant activity

MATERIALS AND METHODS

Plant material:

The pectoral species No 1 (Phytopectol No 1, Pectorales species No 1; JSC “Krasnogorskleksredstva”) samples were purchased in Moscow Pharmacy Network. The samples corresponded to the requirements of State Pharmacopoeia of Russian Federation (SPRF) XIV edition.

Sample preparation:

About 2.0 g (accurately weighed) of crushed PS No 1 (particle size passing through a 0.5 mm sieve) is placed in a 250 ml conical flask with a ground stopper, 50 ml of hexane is added. Flask is combined with a reflux condenser, heated in a water bath, maintaining a weak boil for 1 h. Then the flask is cooled to room temperature, the contents of the flask are filtered through a folded paper filter, discarding the hexane extract. The filter is placed back into the flask, the operation is repeated one more time with hexane, then 2 times with chloroform. Then 50 ml of 70% methanol is added the extraction procedure is repeated, the extract is collected in a 250 ml flask. The extraction is repeated twice more. The combined alcohol extract is evaporated on a rotary vacuum evaporator, dissolved in 5 ml of 70% methanol, filtered through a membrane filter (0,45 μm, Agilent Technologies, USA), and used for UPLC analysis.

Determination of flavonoids and hydroxycinnamic acids:

The PS No 1 extract was studied using an Acquity UPLC TQC system (Waters Corporation, USA). The system consists of ultra-performance liquid chromatography (UPLC) system and sequential quadrupole mass-spectrometry (MS/MS) with photodiode array (PDA) detectors. The UPLC chromatographic system contains a binary gradient pump, sample delivery systems (including a column heater), a photodiode array detector (Waters PDA, USA) and a triple quadrupole mass-spectrometric detector (Waters ACQUITY Triple Quadrupole Detector (TQD), USA). The MassLynx 4.1 program was used to process the experimental chromatographic data and to control the instruments.

Chromatography was run on Waters Acquity ultra-perfomance liquid chromatograph (Waters, USA). Mobile phase A was a mixture of water, acetonitrile, and formic acid (95:5:0.1). Mobile phase B was a mixture of acetonitrile and formic acid (100:0.1). The gradient elution was formed by mixing the mobile phase A and mobile phase B according to the gradient program: MPhА: - 95-50% (0-30 min), 50-0% (30-32 min), 0-95% (32-33 min), 95% (33-36 min); MPhВ: 5-50% (0-30 min), 50-100% (30-32 min), 100-5% (32-33 min), 5% (33-36 min). The mobile phase flow rate was 0.3 ml/min.

The separation was achieved on Acquity UPLC BEH (Bridged Ethylene Hybrid) C18 (150×2.1 mm, particle size 1.7 µm) chromatography column. The column temperature was 35 ⁰C. The sample volume was 5 µl. Conditions for UV detection: 220-550 nm. Conditions for MS/MS detection: TQD (Waters Corporation, USA) mass spectrometer operated in alternating positive and negative electrospray ionization (MS-ES+/-) regimes. Detector parameters in the positive and negative ion modes: Voltage on capillary - +3 kV (ES+), -3kV (ES–); Voltage on the nozzle - +50 V (ES+), -30 V (ES–); Capillary temperature +450 ⁰С (ES+), +350 ⁰С (ES–); Source temperature - +120 (ES+), +120 (ES–); Drying gas flow rate - 50 liters/h (ES+), 50 liters/h (ES–); Mass scanning range - from 100 to 1500 U (ES+), from 100 to 1500 U (ES–).

RESULTS AND DISCUSSION:

Chromatograms obtained during the pectoral species No 1 analysis are presented in Figure 2-14. In the positive ion mode, the protonated molecule with m/z = 446 was seen in the one peak with RT of 10.49 min (Figure 2).

Figure 2. Chromatogram and mass-spectrum of apigenin (m/z = 271) derivative (flavone) in the positive and negative ion mode

This component may be apigenin derivative, it may be apigenin 7-O-β-glucuronide. In the positive ion mode, the presence of the protonated molecule at m/z = 287 due to flavonoid aglycone was established in nine peaks at 5.87, 5.94, 7.99, 8.54, 9.17, 9.67, 10.27, 10.87 and 11.18 min. These components may be luteoline, kaempferol, and/or isoscutellarein derivatives (Figure 3).

Figure 3. Chromatogram of apigenin 7-O-β-glucuronide (retention time – 10.49 min) in the positive and NIM, wavelength 350 nm, m/z = 446 and flavonoids (luteolin, kaempferol, isoscutellarein derivatives; RT‑ 5.87, 5.94, 7.99, 8.54, 9.17, 9.67, 10.27, 10.87 and 11.18 min) in the positive ion mode, wavelength 350-370 nm, m/z = 287

The molecular ion m/z = 638 was seen in the two peaks with retention times of 5.87 and 5.94 min (Figure 4). These components may be luteolin derivatives.

Figure 4. Chromatogram and mass-spectra of flavonoids (luteolin derivatives; RT ‑ 5.87 and 5.94 min) in the positive and negative ion mode, wavelength 350-370 nm, m/z = 638

A protonated molecule at m/z = 800 appeared in a peak with RT of 7.99 min. This component may be luteolin 7,4’-diglucuronide-3’-glucoside (Figure 5).

Figure 5. Chromatogram and mass-spectra luteolin 7,4’-diglucuronide-3’-glycoside (RT‑ 7.99 min) in the positive and negative ion mode, wavelength 350-370 nm, m/z = 800

A protonated molecule at m/z = 462 was appeared in a peak with RT of 8.54 min. The component may be luteolin 7-O-glucuronide (Figure 6).

Figure 6. Chromatogram and mass-spectra of luteolin 7-O-glucuronide (RT‑ 8.54 min) in the positive and negative ion mode, wavelength 350-370 nm, m/z = 462

The molecular ion m/z = 814 was seen in the one peak with a retention time of 9.16 min. This component may be luteolin 7-O-[β-D-glucuronosil-(1→2)-β-D-glucuronide]-4’-О-β-D-glucuronide (Figure 7).

Figure 7. Chromatogram and mass-spectrum of luteolin 7-O-[β-D-glucuronosyl-(1→2)-β-D-glucuronide]-4'-O-β-D-glucuronide (RT 9.16 min) in the positive and NIM, wavelength 350-370 nm, m/z = 814

In the positive ion mode, the molecular ion m/z = 303 was seen in the four intensive peaks with RT of 7.75, 8.01, 8.22, and 9.16 min. These components may be quercetin derivatives.

The molecular ion m/z = 610 peak is seen in the one peak with a retention time 7.75 min. This component may be rutin (Figure 8).

Figure 8. Chromatogram and mass-spectrum of rutin (quercetin derivative; RT – 7.75 min) in the positive and negative ion mode, wavelength 350-360 nm, m/z = 610

In the positive ion mode, the molecular ion m/z = 354 was seen in the two peaks with RT of 3.68 and 3.78 min. These components may be chlorogenic and isochlorogenic acids (Figure 9).

Figure 9. Chromatograms and mass-spectra of chlorogenic and isochlorogenic acid (RT – 3.68 and 3.78 min) in the positive and negative ion mode, wavelength 240-330 nm, m/z = 354

In the positive ion mode, the molecular ion m/z = 516 was seen in the one peak with a retention time of 9.38 min. This component may be isochlorogenic acid C or 4,5-dicaffoylquinic acid (Figure 10).

Figure 10. Chromatograms and mass-spectra of isochlorogenic acid C and 4,5-dicaffoylquinic acid (retention time – 9.38 min) in the positive and negative ion mode, wavelength 240-350 nm, m/z = 516

The molecular ion [M + H] m/z = 516 was seen in the one peak with a retention time of 10.76 min (Fig. 11). This component may be isochlorogenic acid A or 3,5-dicaffoylquinic acid too. These chemical structures are isomers, so their output sequence can be reversed.

Figure 11.Chromatograms and mass-spectra of isochlorogenic acid A and 3,5-dicaffoylquinic acid (retention time – 10.76 min) in the positive and negative ion mode, wavelength 240-330 nm, m/z = 516

The molecular ion [M+H] m/z = 678, [M+H] m/z = 677 was seen in the one peak with a retention time of 14.81 min. This component may be 3,4,5-tricafeoylquinic acid (Figure 12).

Table 4. PS No 1 determined flavonoids’ pharmacological activity

Compound/component	Pharmacological effects according to literature data
Apigenin 7-O-β-glucuronide	Antioxidant, anti-inflammatory, inhibition of ACHE, antitumor effects³⁸
Luteolin 7,4’-diglucuronide-3’-glycoside	Antioxidant, anti-inflammatory, antimicrobial, antitumor effects³⁸
Luteolin 7-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronide]-4’-O-β-D-glucuronide	Antioxidant, anti-inflammatory, antimicrobial, antitumor effects³⁸
Rutoside	antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective, cardioprotective, antidiabetic (hypoglycemic) effects³⁰^,³¹
Caffeic acid	Antimicrobial, immunomodulatory effects³²^,³³
Chlorogenic acid	Antioxidant activity, antibacterial, hepatoprotective, cardioprotective, anti-inflammatory, antipyretic, neuroprotective, hypolipidemic, antiviral, antimicrobial, antihypertensive effects³⁴
Isochlorogenic acid	Anti-inflammatory, antioxidant effects³⁷
4,5-Dicaffeoylquinic acid (Isochlorogenicacid C)	Antiviral activity³⁷
3,5-Dicaffeoylquinic acid (Isochlorogenicacid А)	Anti-inflammatory, Antimicrobial, Antioxidant effects³⁸
3,4,5-Tricaffeoylquinic acid	Antiviral activity³⁵