The Phytochemical Study of the Populus nigra L. Leaves

 

VA Kurkin*, EA Kupriyanova

Department of Pharmacognosy, Botany and Phytotherapy, Samara State Medical University,

Ulitsa Chapaevskaya 89, Samara, 443099, Russian Federation.

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

 

ABSTRACT:

From the leaves of the black poplar (Populus nigra L., Salicaceae family) there were for the first time isolated rutin (quercetin-3-O-rutinoside), calendoflavobioside (quercetin-3-O-neohesperidoside) (flavonoids) and ferulic acid (phenylpropanoid) by using of liquid column chromatography. The chemical structures of the isolated compounds there were elucidated by means of 1H-NMR-, 13C-NMR-, UV-spectroscopy, mass spectrometry and results of chemical transformations. The isolated flavonoids are the dominant phenolic components of the raw material of the investigated plant. It was determined, that the isolated flavonoids cause the spectral characteristics of the UV spectra of the water-ethanolic extract from the Populus nigra leaves. It was substantiated the expediency of the estimation of the total flavonoids in the leaves of Populus nigra calculated on rutin. As results there was development the method of the quantitative determination of the total flavonoids in the leaves of Populus nigra by the using of the differential spectrophotometry at an analytical wavelength of 414 nm. It was determined that the content of the total flavonoids in the raw materials of the studied plant varies from 2.04±0.09% to 2.99±0.11%. The relative degree of the determination of the total flavonoids in the leaves of black poplar in developed method with confidence probability 0.95 is no more than ± 6.26%.

 

KEYWORDS: Poplar black, Populus nigra L., Leaves, Flavonoids, Rutin, Сalendoflavobioside, Phenylpropanoids, Ferulic acid, Spectrophotometry, Standardization.

 

 


INTRODUCTION:

The species of the genus Populus L. (Salicaceae family) are a valuable plant sources of the phenolic compounds with several pharmacological activity.1,2 Currently, the pharmacopoeial monograph "Populus buds"  was included in the State Pharmacopoeia of the Russian Federation.3  The developed phytopharmaceutical based on the buds of Populus nigra L. and other species of the genus Populus L. have the antimicrobial and anti-inflammatory activities.1-5 It is known about the use of the leaves of this plant in folk medicine as the remedies that have an antibacterial, analgesic and soothing effect.2 In our opinion, the leaves of black poplar are a prospective source of biologically active compounds and medicines, along with the buds.

 

According to the literature data the leaves of black poplar contain the simple phenols (salicin, salicortin, nigracin, populin), phenylpropanoids (3-O-β-D-glucopyranoside of caffeic acid, lignans), tannins, vitamins (C, E), carotenoids (neoxanthin, violaxanthin, lutein), organic acids (formic, oxalic, malonic, glyceric, succinic, fumaric, malic  acids), tartaric, α-ketoglutaric, citric and quinic acids.1,2 Thus there is no information in the literature about the flavonoid composition of the black poplar leaves and about the standardization of this type of the plant raw material.

 

The aim of the research was to conduct a phytochemical study of the leaves of black poplar (Populus nigra L.).

 

MATERIAL AND METHODS:

Plant material:

The leaves of Populus nigra L. were collected on the territory of the Samara region in the month of June 2018. The plant was identified by Director of Botanical Garden of Samara University PhD Svetlana Rozno. The voucher specimen (2018/50) was deposited at the Herbarium of Department of Pharmacognosy, Botany and Phytotherapy of Samara State Medical University (Ulitsa Gagarina 18, Samara, 443079, Russian Federation).

 

Extraction of Plant Materials:

The extraction of the leaves of Populus nigra L. (100g) was carried out with the using of 70% EtOH in ratio 1:10 twice at room temperature and then heating in a boiling water bath during 30 minutes. The obtained water-alcohol extracts were filtered and then evaporated by using a rotary evaporator at low temperature (40-50°C) and reduced pressure to thick residue (about 50ml).

 

Isolation of Compounds from Plant Materials:

The isolation of the compounds from the obtained thick extract of the Populus nigra L. leaves 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 (97:3, 95:5, 93:7, 90:10, 85:15, 80:20, 75:25, 70:30, 60:40). As results, we isolated compounds 1-3. The additional purification of the isolated compounds was carried out by recrystallization from the mixture of alcohol and water. The elution of the compounds was monitored by TLC analysis on “Sorbfil PTLC-AF-A-UV” plates in a system of chloroform-ethanol-water (26: 16: 3). The spots on the plates were detected by luminescence in UV light at a wavelength of 254 and 366 nm and by color after processing the chromatograms with an alkaline solution of diazobenzenesulfonic acid.

 

Methods of Structural Elucidation of Compounds:

UV spectra were recorded using a spectrophotometer “Specord 40” (Analytik Jena, Germany) in cuvettes with a layer thickness of 10mm in the wavelength range from 190nm to 700nm. 1H-NMR spectra were obtained on a  spectrometer “Bruker AM 300” (Bruker, Germany) at a frequency 300 MHz, 13C-NMR spectra were obtained on a spectrometer “Bruker DRX 500” (Bruker, Germany) at a frequency 126.76 MHz, mass spectra were recorded on a mass spectrometer “Kratos MS-30” (Kratos, United Kingdom. The acidic hydrolysis of flavonoid glycosides was carried out with the using of 2% HCl at heating on the boiling water bath during of 2 h.

 

Spectrophotometry Analysis of Plant Materials:

The quantitative estimation of total flavonoids in the Populus nigra L. leaves there was carried out by the using of differential spectrophotometry at an analytical wavelength of 414nm calculated on rutin. The plant raw material was crushed so that its particles passed through a sieve with holes 1mm in diameter. An accurate weighed sample of ground material (about 1g) was placed in a 100mL flask and 30mL 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 60 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. 1mL of the resulting extract was transferred into a 50mL volumetric flask, 2 mL of a 3% alcohol solution of aluminum chloride was added, then the solution volume was adjusted to the mark with 96% ethyl alcohol (test solution). The optical density of the test solution was determined 40 min after preparation on a spectrophotometer at a wavelength of 414nm (to calculate the content of flavonoids). The comparison solution was a solution containing 1mL of an aqueous-alcoholic extract (1:30) and 96% ethyl alcohol, which was adjusted to the mark in a 50mL flask.

The content of total flavonoids in percent (X) in terms of rutin and absolutely dry plant raw materials was calculated by the formula:

            A x 30 x 50 x 100

X = ------------------------------

             m x 240 x (100-W)

Where:

A – optical density of the test solution;

m – mass of plant raw materials, g;

240 – specific absorbance (E1%1cm) of rutin at 414 nm;

W – mass loss on drying, %.

 

RESULTS AND DISCUSSION:

As results of the phytochemical study from the leaves of black poplar (Populus nigra L.) flavonoids 1 and 2 (Fig. 1 and 2) and phenylpropanoid (3) (Fig. 3) there were isolated. With the using of mass spectrometry, UV-, 1H-NMR-, 13C-NMR-spectroscopy and results of chemical transformations (acidic hydrolysis of flavonoid  glycosides) the compounds 1, 2 and 3 there were  identified as rutin (quercetin-3-O-rutinoside), calendoflavobioside (quercetin-3-O-neohesperidoside) and ferulic acid respectively.

 

Figure 1: Chemical structure of rutin (1).

 

Figure 2: Chemical structure of сalendoflavobioside (2).

 

Figure 3: Chemical structure of ferulic acid (3).

 

In the 1H-NMR spectrum of compounds 1 and 2 detected signals of aromatic protons, characteristic for substitution of the flavonoid molecule at C-5 and C-7 (ring A) and also at C-3’ and C-4’ (ring B). The presence of a free hydroxyl group at C-5 in compounds 1 and 2 is confirmed by data from 1H-NMR spectra: single-proton singlet signals at 12.59ppm. The free 7-OH group is confirmed by the reaction with sodium acetate (a bathochromic shift in the UV spectrum of the short-wave band by more than 10nm).6 The presence of a free ortho-dihydroxy group at C-3’ and C-4’ is indicated by a bathochromic shift in the UV spectrum of the long-wave band by 22nm when adding a boric acid solution.6

 

As a result of acid hydrolysis of compounds 1 and 2, is formed an aglycone, identified as quercetin (3,5,7,3’,4’-penthydroxyflavone), which, in contrast to the initial glycosides, in the presence of an aluminum chloride solution gives a greater bathochromic shift in the UV spectrum of the long-wave band – from 372nm to 435 nm.

 

Therefore, compounds 1 and 2 are glycosylated by the 3-OH group. In the acid hydrolysate of both compounds there were found monosaccharides, namely glucose and rhamnose. The principal difference between compounds 1 and 2 is the fact that in their 13C-NMR spectra there are different values of the chemical shifts of C-6" of glucose: 61.07ppm and 66.89 ppm, which indicates the addition of rhamnose, respectively, to 2’-OH of glucose in compound 2 (biose–neohesperidose), and in compound 1 – to CH2OH of glucose (biose – rutinose). Therefore, compound 1 has the structure of quercetin-3-O-(6"-α-L-rhamnopyranosyl)-β-D-glucopyranoside (rutin), and compound 2 has the structure of quercetin-3-O - (2" - α-L-rhamnopyranosyl)-β-D - glucopyranoside (calendoflavobioside).

 

Thus, the compounds 1, 2 and 3 identified on the basis of UV, 1H-NMR-, 13C-NMR and mass spectra, as well as results of acid hydrolysis as rutin (1)7, calendoflavobioside (2)8 and ferulic acid (3)1, were for the first time isolated from the leaves of black poplar. Calendoflavobioside (2) was previously isolated from the inflorescences of Calendula officinalis.8 Ferulic acid (3) was described for black poplar buds.1

 

3-О-Rutinoside of quercetin (rutin) (1). Crystalline substance of yellow color with molecular formula С27Н30О16, m.p. 192-194 0С (water alcohol).  lmax EtOH 258, 266 sh., 362 nm; +NaOAc 270, 303, 370nm;  +NaOAc + H3BO3 270, 303, 384 nm; +AlCl3 273, 304, 360, 418 nm; +AlCl3 + HCl 273, 304, 360, 403 nm; + NaOMe 275, 419 nm. 

 

1H-NMR spectrum (300 MHz, DMSO-d6, δ, ppm, J/Hz): 12.59 (1H, s, 5-ОН-group),  10.80 (1Н, s, 7-ОН), 9.65  (1Н, s, 4’-ОН-group), 9.15 (1Н, s, 3’-ОН-group), 7.52 (1Н, dd, 2.5 and 9 Hz, Н-6’),7.50 (1Н, d, 9 Hz, Н-2’), 6.82 (d, 9 Hz, Н-5’), 6.38 (d, 2.5 Hz, Н-8), 6,18 (d, 2.5 Hz, Н-6), 5,35 (1H, d, 7 Hz, Н-1’’ of glucopyranose), 4,35 (1H,  br. s, Н-1’’’ of rhamnopyranose), 5.2-3.0 (10H, m, 6Н of glucose + 4Н of rhamnose), 0.98 (3H, d, 6 Hz, СН3 of rhamnose).

 

13C-NMR spectrum (126.76  MHz, DMSO-d6, δС, ppm, J/Hz): С-2 (156.60), С-3 (133.30), С-4 (177.36), С-5 (161.21), С-6 (98.67), С-7 (164.06),  С-8 (93.58), С-9 (156.41), С-10 (103.96), С-1’ (116.26),  С-2’ (121.18), С-3’ (144.77),  С-4’ (148.40), С-5’ (115.22),  С-6’ (121.58),  С-1’’ of glucose (100.72), С-2’’ (71.25), С-3’’ (75.90),   С-4’’ (70.56),  С-5’’ (76.45), С-6’’ (66.89), С-1’’’ of rhamnose (101.16), С-2’’’ (70.56),  С-3’’’ (70.37),  С-4’’’ (74.07),  С-5’’’ (68.23),  С-6’’’ (СН3 of rhamnose)  (17.72). Mass-spectrum (ESI-MS, 180 оС, m/z):   М+ 611 (610 + Н), М+ 633 (610 + Nа), М 649 (610 + K).

 

3-О-Neohesperidose of quercetin (calendoflavobioside) (2). Crystalline substance of yellow color with molecular formula С27Н30О16, m.p. 194-196 0С (water alcohol).  257, 266 sh., 363 nm; +NaOAc 270, 303, 372nm;  +NaOAc + H3BO3 271, 304, 385 nm; +AlCl3 273, 304, 360, 416 nm; +AlCl3 + HCl 273, 304, 360, 404 nm; + NaOMe 275, 420 nm. 

 

1H-NMR spectrum (300 MHz, DMSO-d6, δ, ppm, J/Hz): 12.59 (1H, s, 5-ОН-group),  10.75 (1Н, s, 7-ОН), 9.60  (1Н, s, 4’-ОН-group), 9.17 (1Н, s, 3’-ОН-group), 7.52 (1Н, дд, 2.5 and 9 Hz, Н-6’),7.50 (1Н, d, 9 Hz, Н-2’), 6.83 (d, 9 Hz, Н-5’), 6.38 (d 2.5 Hz, Н-8), 6,19 (d, 2.5 Hz, Н-6), 5.32 (1H, d, 7 Hz, Н-1’’ of glucopyranose), 4,37 (1H, br. s,  Н-1’’’ of rhamnopyranose), 4.3-3.0 (10H, m, 6Н of glucose + 4Н of rhamnose), 0.98 (1H, d, 6 Hz, 3Н, СН3 of rhamnose).

 

13C-NMR spectrum (126.76  MHz, DMSO-d6, δС, ppm, J/Hz): С-2 (156.57), С-3 (133.28), С-4 (177.34), С-5 (161.19), С-6 (98.65), С-7 (164.06),  С-8 (93.55), С-9 (156.40), С-10 (103.64), С-1’ (116.24),  С-2’ (121.15), С-3’ (144.72),  С-4’ (148.39), С-5’ (115.29),  С-6’ (121.56),  С-1’’ of glucose (100.72), С-2’’ (72.36), С-3’’ (77.89),   С-4’’ (71.83),  С-5’’ (76.83), С-6’’ (61.07), С-1’’’ of rhamnose (101.16), С-2’’’ (70.75),  С-3’’’ (71.94),  С-4’’’ (74.05),  С-5’’’ (68.21),  С-6’’’ (СН3 of rhamnose)  (17.70). Mass-spectrum (ESI-MS, 180оС, m/z):   М+ 611 (610 + Н), М+ 633 (610 + Nа), М 649 (610 + K).

 

Ferulic acid (3). Crystalline substance of white color molecular formula С10Н10О4, m.p. 168-170о (water alcohol). lmax EtOH 217, 242, 290 sh., 322 nm. 1H-NMR spectrum (300 MHz, DMSO-d6, δ, ppm, J/Hz): 9.18 (1Н, s 4-ОН-group), 7.45 (d, 16 Hz, Н-7), 7.08 (1Н, dd, 2.5 and 9 Hz, Н-6), 7.03 (d, 2.5 Hz, Н-2), 6.93 (d, 9 Hz, Н-5), 6.24 (1Н, d, 16 Hz, Н-8), 3.80 (3Н, s, СН3О-group). Mass-spectrum (ESI-MS, 180оС, m/z): М+ 195 (194 + Н).

 

The compounds 1-3 there were for the first time isolated from the leaves of Populus nigra. The isolated flavonoids 1 and 2 are the dominant phenolic components of the raw material of the investigated plant. It was determined, that the isolated flavonoids cause the spectral characteristics of the UV spectra of the water-ethanolic extract from the Populus nigra leaves.

 

It was shown, that the UV spectrum of extraction from black poplar leaves has two absorption maxima at a wavelength of 270±2nm and 356±2nm (Fig. 4). In addition, in the electronic spectrum of water-alcohol extraction from the leaves of black poplar in the presence of aluminum (III) chloride a bathochromic shift of the long-wave band by 40-50nm was detected (Fig. 5), characteristic for flavonoids. Thus, it is established that the main contribution to the absorption curve is made by flavonoids, including rutin isolated from the leaves of black poplar. The maximum absorption of the solution of rutin and water-alcohol extraction from the leaves of black poplar are in the region of 414±2 nm.

It was substantiated the expediency of the estimation of the total flavonoids in the leaves of Populus nigra calculated on rutin.

 

When developing a method for the quantitative determination of the total flavonoids in black poplar leaves, the extraction ability of alcohols of various concentrations, the ratio of «raw material-extractant» and the extraction time were compared. It is determined that the highest yield of the total of flavonoids calculated on rutin from the leaves of black poplar is observed when using 70% ethyl alcohol as an extractant, the ratio «raw material-extractant» is 1:30, the extraction time is 60 minutes in a boiling water bath.

 

As results there was development the method of the quantitative determination of the total flavonoids in the in the leaves of Populus nigra by the using of the differential spectrophotometry at an analytical wavelength of 414nm. It was determined that the content of the total of flavonoids in the raw materials of the studied plant varies from 2.04±0.09% to 2.99±0.11%. The relative degree of the determination of the total flavonoids in the leaves of black poplar in developed method with confidence probability 0.95 is no more than ± 6.26%.

 

Figure 4: The UV spectra of water-alcohol extraction from black poplar leaves.

 

Designations: 1 – initial solution; 2 - solution with the addition of AlCl3.

 

Figure 5: The differential UV spectrum of extraction from black poplar leaves.

 

CONCLUSION:

As result of the phytochemical study of the leaves of black poplar (Populus nigra L.) from the leaves there were for the first time isolated rutin (quercetin-3-O-rutinoside), calendoflavobioside (quercetin-3-O-neohesperidoside) and ferulic acid, the chemical structures of which there were elucidated by means of 1H-NMR-, 13C-NMR-, UV-spectroscopy, mass spectrometry and results of chemical transformations. It was substantiated the expediency of the estimation of the total flavonoids in the leaves of Populus nigra calculated on rutin. It was development the method of the quantitative determination of the total flavonoids in the in the leaves of Populus nigra by the using of the differential spectrophotometry at an analytical wavelength of 414 nm.

 

It was determined that the content of the total of flavonoids in the raw materials of the studied plant varies from 2.04±0.09% to 2.99±0.11%.

 

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

 

REFERENCES:

1.      Braslavskii VB. Willow, Poplar, and Propolis in Medicine and Pharmacy: Monograph [in Russian]; Samara: Oxford Publ.; 2012; pp. 116.

2.      Plant Resources of the USSR: Flowering Plants, Their Chemical Composition and Use: Families Paeoniaceae – Thymelaeaceae; Leningrad: Nauka; 1986; pp. 336.

3.      The State Pharmacopoeia of the Russian Federation [in Russian], XIV edition, Vol. 4, Moscow; 2018; pp. 1832 [Electronic resource] / URL: http://resource.rucml.ru/feml/pharmacopia/14_4/HTML/index.html - 26.04.2020.

4.      Turetskova VF, Lobanova IYu, Rassypnova SS, Talykova NM. Populus tremula as a promising source of anti-ulcer and anti-inflammatory drugs [in Russian]; Bulletin of Siberian Medicine; 2011; 5: 106-111.

5.      Kurkin VA. Pharmacognosy: A Textbook for Students of Pharmaceutical Universities; Ed. 4th, rev. and add. [in Russian]; Samara:  Oxford Publ.; 2019; pp. 1278.

6.      Mabry TJ, Markham KR, Thomas MB. The Systematic Identification of Flavonoids; Berlin-Heidelberg-New York: Springer Verlag, 1970; pp. 354.

7.      Akkola EK, Sόntara I, Keleşb H, Sezika E, Gόrlerc G. Bioassay-guided isolation and characterization of wound healer compounds from Morus nigra L. (Moraceae); Rec. Nat. Prod.; 2015; 9(4): 484–495.

8.      Komissarenko NF, Chernobai VT, Derkach AI. Flavonoids of inflorescences of Calendula officinalis; Chemistry of Natural Compounds; 1988; 24(6): 675-680.

 

 

 

 

Received on 27.08.2020           Modified on 14.10.2020

Accepted on 18.11.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(8):4192-4196.

DOI: 10.52711/0974-360X.2021.00726