Identification of Saponins, Flavonoids and Other Phenolic Compounds in Polysaccharide-containing Mixed Herbal Product (Pectorales species No 2) by Ultra-performance Liquid Chromatography with Photodiode arrays and Tandem Quadrupole Mass-selective Detectors

Chevidaev V.V.¹*, Bokov D.O.^1,2, Bobkova N.V.¹, Sergunova E.V.¹, Kovaleva T.Yu.¹,

Nicheporchuk P.A.¹, Klyukina E.S.¹, Bondar A.A.¹, Evgrafov A. A.¹, Yakubovich L. M.¹,

Samylina I.A.¹, Luferov A.N.¹, Gildeeva G.N.¹, Smolyarchuk E.A.¹, Bessonov V.V.^2,3

¹Sechenov First Moscow State Medical University, 8 Trubetskaya St.,

Bldg. 2, Moscow, 119991, Russian Federation.

²Federal Research Center of Nutrition, Biotechnology and Food Safety,

2/14 Ustyinsky pr., Moscow, 109240, Russian Federation.

³Рeoples’ Friendship University of Russia (RUDN University),

6 Miklukho-Maklaya Street, Moscow, 117198, Russian Federation.

*Corresponding Author E-mail: chevidaev_v_v@staff.sechenov.ru

ABSTRACT:

In Russian pharmaceutical practice, pectorales species No. 2 (phytopectol No.2) is used as part of complex therapy for upper respiratory tract diseases. Pectorales species No. 2 (PS No.2) includes in its composition polysaccharide-containing medicinal plant raw materials, leaves of plantain (Plantaginis majoris folia), leaves of coltsfoot (Tussilaginis farfarae folia), and licorice roots (Glycyrrhiza radices). In addition to polysaccharides, the listed medicinal plant raw materials contain saponins, flavonoids, hydroxycinnamic acids. These groups of compounds can exhibit antibacterial, antiviral, and anti-inflammatory effects. The aim of this study is to determine the profile of saponins, flavonoids and other phenolic compounds in PS No. 2. To achieve this aim, the UPLC-PDA-MS/MS method was used. Results: Chemical analysis of herbal tea PS No. 2 identified a variety of flavonoids, including isoquercitrin, kaempferol and its derivatives, and liquiritigenin glycosides. Hydroxycinnamic acids, including chlorogenic and isochlorogenic acids, a number of dicaffeoylquinic acids (1,3-O-, 3,5-O-, 4,5-O-), 3,5-Di-O-caffeoyl-4-O-muconolactonylquinic acid, and 3,4,5-tricaffeoylquinic acid, were also found in the extract. Detected compounds from the group of saponins are represented by glycyrrhizic acid, glycyrrhizic acid, isoglycyrrhizic acid, licoricesaponin G2, isolicoricesaponin G2. Conclusions: PS No. 2 is rich in saponins, flavonoids and hydroxycinnamic acids. These compounds may be responsible for the anti-inflammatory effect of the herbal tea. To standardize PS No. 2, it is recommended to carry out a quantitative determination of glycyrrhizic acid as a marker compound.

KEYWORDS: Pectorales species No 2, Plantago major L., Tussilago farfara L., Glycyrrhiza glabra L, UPLC-PDA-MS/MS.

INTRODUCTION: 

In the treatment of bronchopulmonary diseases, both synthetic drugs and herbal medicines are used. The latter are used as monocomponent or polyherbal formulations. Medicines of polyherbal formulations are currently of interest to medicine and science^1-6. Pectorales species No. 2 (PS No.2) is the mixed herbal products (MHP) or herbal teas (HP), being polyherbal formulations for the treatment of bronchopulmonary diseases.

PS No.2 is composed of 40% coltsfoot leaves (dried leaves from Tussilago farfara L., Fam Asteraceae), 30% greater plantain leaves (dried leaves from Plantago major L., Fam Plantaginaceae) and 30% licorice roots (dried roots of Glycyrrhiza glabra L. and Glycyrrhiza uralensis Fisch. ex DC. Fam. Lamiaceae). PS No.2 refers to expectorants of herbal origin. The final dosage form for use is an infusion - an aqueous extract that exhibits expectorant and anti-inflammatory effects. The crushed PS No.2 is a mixture of heterogeneous particles of plant origin up to 7mm in size. The color of the particles is yellow, brownish-yellow, brown, grayish-green, greenish-white. Purple blotches are sometimes observed (Figure 1). The smell is rather weak or absent, the taste of the aqueous extract is sickly sweet, with a feeling of mucous.

Figure 1. External signs of PS No. 2

The main group of biologically active compounds (BAC) of PS No.2 are polysaccharides. However, insufficient attention is paid to the study of the component composition of PS No.2. Regulatory documentation governing the quality of PS No.2 requires revision. Regulatory documentation of medicinal plant materials in the composition of PS No.2 is presented by pharmacopeial monographs included in the State Pharmacopoeia of the Russian Federation, 15th edition. To improve the regulatory documentation on PS No.2, it is necessary to know the composition of all groups of BAC in PS No.2 that have a pharmacological effect, including phenolic compounds and saponins.

Table 1 summarizes the literature data on saponins, flavonoids and other polyphenolic compounds contained in the components of PS No. 2.

Table 1. Chemical composition of flavonoid, phenolic compounds and saponins in PS No. 2

BAS group	Chemical compounds
Coltsfoot leaves (Folia Tussilaginis farfarae)
Flavonoids	Kaempferol, astralagin, nicotiflorin, guayaverine, isoquercetin, rutin, quercetin, luteolin, apigenin,hesperidin ^7-10.
Polyphenolic compounds	4,5-dicaffeoylquinic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid, ferulic acid, caffeic acid, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, syringic acid, p-coumaric acid, rosmarinic acid, cinnamic acid ^8-12.
Greater plantain leaves (Folia Plantaginis majoris)
Flavonoids	Luteolin, quercetin, rutin, naringenin, hyperoside, chrysin, kaempferol, baicalein, baicalin, isorhamnetin^13-15.
Polyphenolic compounds	Gallic acid, catechol, catechin, caffeic acid, vanillic acid, syringic acid, ellagic acid, coumarin, ferulic acid^13-14.
Licorice roots (radices Glycirrhizae)
Flavonoids	Liquritigenin, hispoglabridin A, glabridin, 4-O-methylglabridin, isoviolantin, genistein, neoliquiroside, luteon, uralenol, pratensin, homobutein, kaempferol, licoisoflavanone, abiochanin A, licflavone A, lycoisoflavone B^16-20.
Polyphenolic compounds	5”-dimethyl-[2, 1-b] furano-coumestane, 1,2-bezenediol, 3-methoxy-trans-isoeugenol, liconolignan, glycoricone, lycofuranone^16,17.
Saponins	Glycyrrhizic acid, glycyrrhetinic acid, uraloside, glycyrrhizin, glycyrrhizol, uralenoside, glycyrrhizasaponin, glyeurysaponin,licoricesaponin A3, licoricesaponin B2 licoricesaponin G^21,22.

In addition to the expectorant and anti-inflammatory action, caused mainly by polysaccharides, the components of PS No.2 also have other activities. Flavonoids and polyphenolic compounds of coltsfoot leaves have high antioxidant and antibacterial activity²³. Alcoholic extracts of plantain leaves have antibacterial and antiviral effects²⁴. Licorice root flavonoids have antitumor and antiviral activity²⁵.

In this paper, the authors used UPLC-PDA-MS/MS methods to determine the composition of polyphenolic compounds and flavonoids in PS No.2. UPLC is advanced chromatographic techniques that are widely used in the physicochemical analysis of chemical compounds^26-29.

The aim of the present work was the determination of the main saponin, flavonoid and other phenolic compounds in PS No.2.

MATERIALS AND METHODS:

Plant material:

The PS No.2 (JSC “Krasnogorskleksredstva”, Russia) samples were purchased in Moscow pharmacy network. The samples corresponded to the requirements of State Pharmacopoeia of Russian Federation (SPRF) XV edition.

Sample preparation:

The extraction methodology involved accurately weighing a sample (1.5g). Extraction was then performed under reflux with 25ml of 70% methanol. The extraction lasted for 1hour. This process was then repeated twice more, using 10ml of solvent each time. All extracts were combined in a rotary evaporator. The dry residue was dissolved in 10ml of methanol of the same concentration. An aliquot of 2ml of the resulting solution was centrifuged (15,000rpm, 5min), after which the supernatant was collected for subsequent chromatography.

Determination of phenolic compounds and saponins:

The PS No 2 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% (33-36min).

The mobile phase flow rate was 0.3ml/min.

The separation was achieved on Acquity UPLC BEH (Bridged Ethylene Hybrid) C18 (150×2.1mm, particle size 1.7µm) chromatography column. The column temperature was 35⁰C. The sample volume was 5 µl. Conditions for UV detection: 220-550nm. 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:

The molecular masses of the compounds were determined using mass spectrometry data recorded under positive and negative ionization conditions, with detection of [M+H]⁺, [M+Na]⁺, and [M-H]⁻ ions. The structure of the aglycone was established based on analysis of fragmentation patterns and UV spectra.. Some compounds declared according to literature data were not detected. This may be explained by their low content for obtaining high-quality spectra, the inability to ionize under ESI conditions, high lipophilicity and, as a consequence, indelibility from the column under given conditions, or the unavailability of their structural formula in the literature. Especially many compounds with an unknown structure are declared in the chemical composition of licorice root. In the leaves of the common coltsfoot were identified 3-O-β-D-glucopyranoside (astralagin) (Mm=594g/mol), 3-O-a-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside (nicotiflorin) (434g/mol), 3-O-β-L-arabinopyranoside (guayaverine) (Mm=594g/mol), 3-O-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside (rutin) (Mm=594g/mol), 3,4-hydroxybenzoic acid (Mm=154 g/mol), ferulic acid (Mm=194g/mol), caffeic acid (Mm=180 g/mol), gallic acid (Mm=170g/mol), p-hydroxybenzoic acid (Mm=138g/mol), syringic acid (Mm=198g/mol), vanillin (Mm=152g/mol), p-coumaric acid (Mm=164g/mol), benzoic acid (Mm=122g/mol), hesperidin (Mm=610g/mol), rosmarinic acid (Mm=360 g/mol), cinnamic acid (Mm=148g/mol), quercetin (Mm=302g/mol), luteolin (Mm=286g/mol), apigenin (Mm=270g/mol), sinapinic acid (Mm=224g/mol). In the leaves of the greater plantain were identified gallic acid (Mm=170g/mol), catechol (Mm=110g/mol), catechin (Mm=290g/mol), caffeic acid (Mm=180 g/mol), vanillic acid (Mm=168g/mol), syringic acid (Mm=198g/mol), ellagic acid (Mm=302g/mol), coumarin  (Mm=146 g/mol), ferulic acid (Mm=194g/mol), luteolin  (Mm=286 g/mol), rutin (Mm=610g/mol), naringenin  (Mm=272 g/mol), hyperoside  (Mm=464g/mol), chrysin  (Mm=254 g/mol), baikalein (Mm=270g/mol), baikalin (Mm=446 g/mol), p-coumaric acid (Mm=164g/mol), isorhamnetin (Mm=316g/mol), glabrene (Mm=324g/mol). In the licorice roots were identified glabridin (Mm=324 g/mol), naringenin (Mm=566g/mol), formononetin (Mm=562g/mol), glycyrrhetinic acid (Mm=470g/mol), 4-hydroxyglycyrrhetic acid (Mm=486g/mol).

The criterion for data collection was the peak area, which had to be at least 1% of the total signal at 260 nm, resulting in a higher classification of the sample composition (Table 2).

Table 2 . PS No. 2 compounds with a peak area of at least 1% of all areas at 260 nm.

Name	Retention time (RT)	Molecular weight	UV absorbance wavelength	Potential structure
Chlorogenic Acid	4.53	353	326
Isochlorogenic Acid	4.86	353	325
Kaempferol-3-dimethylglucosylglucoside	6.58	337	638
Liquiritigenin 7-Apiofuranoside-4'-Glucoside	8.59	550	272
Liquiritigenin 4'-O-Apiosylglucoside	8.88	550	275
Isoquercitrin	9.09	464	355
Kaempferol-3-methylglucoside	9.43	462	350
3,5-Di-O-caffeoyl-4-O-Muconolactonyl-quinic acid	9.98	640	338
4,5-O-Dicaffeoylquinic acid	10.24	516	326
3,5-O-Dicaffeoylquinic acid	10.53	516	326
Quercetin-X(370 nm)	11.3	366	460
1,3-O-Dicaffeoylquinic acid	11.56	516	326
Kaempferol-3-malonylglucoside	11.71	534	344
256-Apiosylglucoside(370nm)	12.58	550	363	-
268-Apiosylglucoside (250nm)	12.94	562	250	-
iso-256-Apiosylglucoside(370nm)	13.07	550	372	-
268-Х (250nm)	13.53	500	252	-
Kaempferol	14.94	348	286
3,4,5-Tricaffeoylquinic acid	15.61	678	328
268-malonylglucoside(250nm)	15.97	516	250
Licoricesaponin A3	18.18	985	252
Licoricesaponin G2	19.62	838	252
Isolicoricesaponin G2	21.56	838	252
Glycyrrhizic Acid	23.12	822	252
Isoglycirrhizic Acid	24.99	822	250
Unknown	26.78	940	262

The PS No.2 chromatogram is shown in the figure 2.

Figure 2. Chromatogram of flavonoids, saponins and other phenolic compounds of PS No.2

Figure 3. UV and mass spectra of chlorogenic acid and isochlorogenic acid, m/z = 353 in the negative ion mode, wavelength=330 nm

Peaks with retention times of 4.53, 4.86, 9.98, 10.24, 10.53, 11.56 and 15.61min contain the same fragment with m/z=353 in the positive ion mode and a similar UV spectrum with a maximum at about 330nm. Based on this, it can be assumed that they are all derivatives of caffeic acid. The peak with a retention time of 4.53min coincides in spectra and retention time with chlorogenic acid. The peak with a retention time of 4.86 min has the same spectra as chlorogenic acid and can presumably have the structure of isochlorogenic acid. (Figure 3).

Peaks with retention times of 10.24, 10.53 and 11.56 min have the same spectrum with m/z=516 in the negative ion mode and a UV spectrum with a maximum around 326 nm, presumably are isomers of dicaffeoylquinic acid with different positions of addition of caffeic acid to quinic acid (Figure 4).

The peak with a RT=9.98 min, a wavelength of 338 nm and m/z=640 in the negative ion mode can have the structure 3,5-di-O-caffeoyl-4-O-muconolactonyl-quinic acid with accuracy up to the sites of attachment. The peak with a RT=15.61 min, a wavelength of about 328 nm and m/z=678 in the negative ion mode can have the structure of 3,4,5-tricaffeoylquinic acid with accuracy up to the sites of attachment (Figure 5).

Figure 4. UV and mass spectra of 4,5-O-dicaffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 3,5-O- dicaffeoylquinic acid, m/z = 516 in the negative ion mode, wavelength=326 nm

Figure 5. UV and mass spectra of 3,5-Di-O-caffeoyl-4-O-Muconolactonyl-quinic acid, m/z=640 in the negative ion mode, wavelength=338 nm and 3,4,5-tricaffeoylquinic acid, m/z=678 in the negative ion mode, wavelength=328 nm

Peaks with RT=6.58, 9.43, 11.17 and 14.94 min contain the same fragment with m/z=286 and a similar UV spectrum with a maximum at about 350 nm. Based on this, we can assume that all of them are derivatives of kaempferol.

The peak with a retention time of 6.58 min has m/z=638 in the negative ion mode and a possible structure of kaempferol-3-dimethylglucosylglucoside accurate to the attachment sites. The peak with a retention time of 9.43 min has a m/z= 462 in the negative ion mode and a possible structure of kaempferol-3-methylglucoside with accuracy up to the attachment sites. The peak with a retention time of 11.71 min has a m/z= 534 in the negative ion mode and a possible structure of kaempferol-3-malonylglucoside with accuracy up to the attachment sites (Figure 6).

The peak with a RT=14.94 min has m/z=286 in the negative ion mode and is presumed to be free kaempferol (Figure 10). The peaks with RT= 9.09 and 11.3 min contain the same fragment with m/z=302 in the negative ion mode and a similar UV spectrum with a maximum at about 360 nm. Based on this, it can be assumed that all of them are quercetin derivatives. The peak with RT= 9.09 min has m/z=464 in the negative ion mode can be belong to isoquercitrin. The peak with RT=11.3 min has m/z=460 in the negative ion mode and it was not possible to find a suitable structure for it (Figure 7).

Figure 6. UV and mass spectra of kaempferol-3-dimethylglucosylglucoside, m/z = 638 in the positive ion mode, wavelength=337 nm, kaempferol-3-methylglucoside, m/z=462 in the negative ion mode, wavelength=350 nm and kaempferol-3-malonylglucoside, m/z = 534 in the negative ion mode, wavelength=344 nm

Figure 7. UV and mass spectra of kaempferol, m/z = 286 in the positive ion mode, wavelength=348 nm, isoquercitrin, m/z = 464 in the negative ion mode, wavelength=355 nm and unknown compound, m/z = 460 in the negative ion mode, wavelength=366 nm

Peaks with RT=8.59 and 8.88 min contain the same fragment with m/z=257 in the positive ion mode and m/z=550 in the negative ion mode and a similar UV spectrum with a maximum around 272-275nm. Based on this, it can be assumed that they are all isomers of liquiritgenin apiosyl-glucoside. The peak with a RT=26.78 min has m/z=940 in the negative ion mode, contains a fragment with m/z= 352 in the positive ion mode and a similar UV spectrum with a maximum of about 262 nm. Unfortunately, it was not possible to find a suitable structure for the chromophore part (Figure 8).

Peaks with RT= 18.18, 23.12 and 24.99 min contain the same fragment with m/z 452 in the negative ion mode and similar UV spectrum with maximum around 252 nm. Based on this it can be assumed that they are derivatives of glycyrrhetic acid.

The peak with RT=18.18 min has m/z 985, which can be assumed as licoricesaponin A3. The peaks with RT=19.62 and 21.56 min have m/z=838 in the negative ion mode and contain the same fragment with m/z=468 in the positive ion mode and a similar UV spectrum with a maximum at about 252 nm. Based on this, it can be assumed that they are isomers of the derivative 24-Hydroxyglycyrrhetic acid (Figure 9).

Figure 8. UV and mass spectra of liquiritigenin 7-apiofuranoside-4'-glucoside, liquiritigenin 4-O-apiosylglucoside, m/z=257 in the positive ion mode and m/z = 550 in the negative ion mode, wavelength=272-275 nm and unidentified compound, m/z=940 in the negative ion mode, m/z=352 in the positive ion mode, wavelength=262 nm

Figure 9. UV and mass spectra of licoricesaponin A3, m/z=985 in the negative ion mode, wavelength=252 nm, licoricesaponin G2 and isolicoricesaponin G2, m/z=838 in the negative ion mode, m/z=468 in the positive ion mode, wavelength=252 nm

Figure 10. UV and mass spectra of glycyrrhizinic acid and isoglycyrrhizic acid, m/z=822 in the negative ion mode, wavelength=250-252 nm

The two peaks with RT=23.12 and 24.99 min have the same mass of 822 in the positive ion mode. Based on this, it can be assumed that they are isomers of glycyrrhizic acid (Figure 10).

Peaks with RT=12.58 and 13.07min have the same m/z=550 in the negative ion mode, contain the same fragment with m/z=256 in the positive ion mode and a similar UV spectrum with a maximum of about 370 nm. Unfortunately, it was not possible to find a suitable structure for the chromophore part of these components, but it is clear that they contain a carbohydrate part and belong to apiosylglucosides (Figure 11).

Figure 11. UV and mass spectra of unidentified apiosylglucosides, m/z=550 in the negative ion mode, m/z=256 in the positive ion mode, wavelength=370 nm

Peaks with RT=12.92, 13.53 and 15.97min have m/z=562, 500, 516 in the negative ion mode and contain the same fragment with m/z=268 in the positive ion mode and a similar UV spectrum with a maximum of about 250nm. Unfortunately, it was not possible to find a suitable structure for the chromophore part of these components, but it is clear that the first contains a carbohydrate part - apiosylglucoside, and the last – malonylglucoside (Figure 12).

Thus, the UPLC-UV-MS/MS analysis of PS No. 2 extract allowed us to identify a number of phenolic compounds and saponins. Among the phenolic compounds, derivatives of hydroxycinnamic acids were detected, in particular chlorogenic and isochlorogenic acids, as well as a number of di- and tricaffeoylquinic acids (1,3-O-, 3,5-O-, 4,5-O-dicaffeoylquinic, 3,4,5-tricaffeoylquinic) and their structural analogue, 3,5-Di-O-caffeoyl-4-O-muconolactonylquinic acid. Among the flavonoids, various kaempferol glycosides (kaempferol-3-dimethylglucosyl glucoside, kaempferol-3-methylglucoside, kaempferol-3-malonylglucoside), its aglycone, isoquercitrin, and two liquiritigenin glycosides were identified. Furthermore, the extract contained saponins such as glycyrrhizic and isoglycyrrhizic acids, as well as licoricesaponins G2 and isolicoricesaponin G2.. The literature data of PS No. 2 determined flavonoids, phenolic compounds and saponins pharmacological activity is shown in Table 6.

Figure 12. UV and mass spectra of unidentified apiosylglucosides and malonylglucoside, m/z=562, 500, 516 in the negative ion mode, m/z=268 in the positive ion mode, wavelength=250 nm

Table 6. PS No. 2 determined compounds pharmacological activity

Compound’s name	Pharmacological effects according to literature data
Chlorogenic acid	Antioxidant, anti-inflammatory, antidiabetic, cardioprotective, neuroprotective, antimicrobial, liver protective, antitumor^30,31
Isochlorogenic acid	Antioxidant, anti-inflammatory, hypoglycemic, neuroprotective, cardiovascular protective, liver protective^32
4,5-O-dicaffeoylquinic acid	Anti-inflammatory^3^3
1,3-O-dicaffeoylquinic acid	Anti-inflammatory^3^3
3,5-O- dicaffeoylquinic acid	Anti-inflammatory^3^3
3,5-Di-O-caffeoyl-4-O-muconolactonyl-quinic acid	Antiviral^3^4
Kaempferol-3-malonylglucoside	Antioxidant^3^5
Kaempferol	Anti-inflammatory, anti-cancer, cardiovascular protective, liver protective, neuroprotective, cardioprotective^36
Isoquercitrin	Antioxidant, anti-cancer, cardiovascular protective^37
Liquiritigenin 7-apiofuranoside-4'-glucoside	No data available
Liquiritigenin 4-O-apiosylglucoside	No data available
Glycyrrhizinic acid	Anti-inflammatory, anti-diabetic, antioxidant, anti-tumor, antimicrobial, anti-viral^38
Isoglycyrrhizic acid	Anti-inflammatory, anti-diabetic, antioxidant, anti-tumor, antimicrobial, anti-viral^38
Licoricesaponin G2	Antibacterial, anti-inflammatory, antioxidant properties, antitumor, hepatoprotective^39,40
Isolicoricesaponin G2	Antibacterial, anti-inflammatory, antioxidant properties, antitumor, hepatoprotective^39,40

CONCLUSIONS:

The expectorant and anti-inflammatory effects of PS No. 2 can be caused by both polysaccharides and phenolic compounds and saponins. For some of the compounds of these groups, for example glycyrrhizic acid, it is possible to standardize PS No. 2.

In this study, hydroxycinnamic acids and their derivatives (chlorogenic acid, isochlorogenic acid, 4,5-O-dicaffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 3,5-O- dicaffeoylquinic acid, 3,5-Di-O-caffeoyl-4-O-muconolactonyl-quinic acid, 3,4,5-tricaffeoylquinic acid), flavonoids (kaempferol-3-malonylglucoside, kaempferol, isoquercitrin, liquiritigenin 7-apiofuranoside-4'-glucoside, liquiritigenin 4-O-apiosylglucoside) and saponins (glycyrrhizinic acid, isoglycyrrhizic acid, licoricesaponin G2, isolicoricesaponin G2) were found in PS No. 2.

All of PS No 2 components contain kaempferol. Hydroxycinnamic acids are mostly in greater plantain leaves and coltsfoot leaves. There is no data about hydroxycinnamic acids presence in licorice roots. Saponins are present only in licorice roots.

ACKNOWLEDGEMENT:

The authors would like to express special gratitude to Nedialkov P.T. for his assistance in preparing this manuscript and valuable advice.

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Received on 20.03.2025      Revised on 13.08.2025

Accepted on 17.11.2025   Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):201-214.

DOI: 10.52711/0974-360X.2026.00030

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