In Russia, 1500 ha of greenhouse complexes have been put into operation in the past 5 years; the total area of such enterprises increased by 10% in 2021 [1]. The increasing number of such enterprises leads to a rapid increase in the mass of plant waste, i.e., non-fruit secondary raw materials (stems, leaves, and roots) as a result of plant vegetation that are not used commercially. The main way to utilize green waste is composting, although plant biomass can be a source of economically important products with added commercial value [2].

One major agricultural crop in the greenhouse crop rotation is the cucumber (Cucumis sativus L., the gourd family, or the Cucurbitaceae) [3]. The biological peculiarity of this culture is formation of elongated shoots (stems and leaves) with the mass reaching 50–60% of fruit mass, i.e., according to preliminary estimates, can be up to 1 million tons per year [4]. In spite of the broad involvement of C. sativus fruits in human economic activity as a food product, the studies of green shoots of this species are limited only by the general data on chemical composition [5], in the absence of information about the detailed composition of metabolites and potential biological activity [6]. Previously, it has been established that extracts of C. sativus leaves and some phenolic compounds in their composition are able to inhibit the activity of human pancreatic lipase [7]. This digestive enzyme is involved in the hydrolysis of triglyceride ester bonds and the formation of free fatty acids [8]; excessive intake of the latter leads to hyperlipidemia resulting in atherosclerosis and cardiovascular diseases [9]. Lipase inhibitors normalize lipid metabolism and mitigate the negative consequences of hyperlipidemia. Various compounds are effective lipase inhibitors [10], including those of natural origin; among the latter, special attention should be focused on flavonoids, which inactivate lipase and lower lipid concentration [11]. Taking the volume of production of C. sativus into account, this crop can be a promising raw material for the pharmaceutical industry, in particular, for obtaining phytodrugs with hypolipidemic activity.

The aim of the present work was to study the flavonoid composition of C. sativus leaves, to isolate the major compounds, and to determine their inhibitory effects on the activity of human pancreatic lipase.

MATERIALS AND METHODS

Experimental conditions. The leaves of C. sativus (variety Alphabet F1, Amur F1, Dachny F1, Dynamite F1, Courage F1, Nezhinsky, Siberian garland F1, Siberian express F1, Strelets F1, Tornado F1) were collected in the experimental greenhouse of the Institute of General and Experimental Biology, Siberian Branch of the Russian Academy of Sciences (Republic of Buryatia, Russia) and dried at 45°С to a moisture content of 5% or less in a PRO ShSP-U 35/150-120 convection desiccant drying oven (Novye Tekhnologii, Russia).

Column chromatography was performed using polyamide, normal (SiO2) and reversed-phase silica gel (RP-SiO2), and Sephadex LH-20 (Sigma-Aldrich, United States). Spectrophotometry was performed with a SF-2000 spectrophotometer (OKB Spektr, Russia). Mass spectra were recorded with an LCMS-8050 TQ mass spectrometer (Shimadzu, Japan) [12]; NMR spectra were recorded with a VXR 500S spectrometer (Varian, United States). Preparative HPLC was performed with a LC-20 Prominence liquid chromatography system (Shimadzu) equipped with a Shim-pak PREP-ODS column (20 mm × 250 mm × 15 µm) and a SPD-M30A photodiode detector (Shimadzu) at a rate of 1.0 mL/min and a column temperature of 20°С.

Extraction and isolation of compounds from C. sativus leaves. Crushed raw material (Siberian Express F1 variety; 5 kg) was extracted twice with 70% isopropanol (1 : 25, 70°С); the extract was filtered through a cellulose filter and vacuum dried (1.4 kg). Then, the extract was partitioned by solid-phase extraction (SPE) on polyamide (10 kg), which was washed with water (SPE-1, 420 g), 70% ethanol (SPE-2, 150 g) and 0.5% ammonia solution in 90% ethanol (SPE-3, 170 g).

The ability to inhibit the activity of human pancreatic lipase was determined by spectrophotometry as described [13]; it has been shown that the SPE-3 fraction is more active (IC50 63.22 µg/mL) than SPE-1 (IC50 > 500 µg/mL) and SPE-2 (IC50 158.63 µg/mL); therefore, further studies were performed with SPE-3. For isolating individual compounds, flash chromatography of SPE-3 (160 g) was performed on Sephadex LH-20 (a column of 2 × 90 cm, ethanol–water eluent 80 : 20 for fraction A, 70 : 30 for fraction B, and 40 : 60 for fraction C); then fractions B and C were separated in RP-SiO2 (a column of 2 × 40 cm, water–acetonitrile eluent 95 : 5, 90 : 10, 80 : 20, 70 : 30, 60 : 40, 50 : 50) and SiO2 (3 × 40 cm, ethylacetate–ethanol 100 : 0, 90 : 10, 80 : 20, 70 : 30, 60 : 40).

Closely eluting compounds were separated by preparative HPLC with water (I) and acetonitrile (II) as eluents. The elution program was as follows (elution time, min; % of acetonitrile in water): 0–50 min, 5–27%; 50–100 min, 27–40%; 100–150 min, 40–52%. As a result, the following compounds were isolated: isovitexin-2"-O-glucoside-6"-O-ferulate (630 mg), isovitexin-2"-O-glucoside-6"-O-p-coumarate (160 mg), isovitexin-2"-O-(6"'-O-feruloyl)-glucoside-6"-O-ferulate (340 mg), and the known compounds isovitexin-4'-O-glucoside-2"-O-(6""-O-feruloyl)-glucoside (1.6 g), isovitexin-4'-)-glucoside-2"-O-(6""-O-p-coumaroyl)-glucoside (8.3 g), isovitexin-2"-O-(6""-O-feruloyl)-glucoside (810 mg) and isovitexin-2"-O-(6""-O-p-coumaroyl)-glucoside (430 mg) identified by the data of UV, NMR spectroscopy and mass spectrometry [14, 15].

Isovitexin-2"-O-glucoside-6"-O-ferulate (1). The general formula is C37H38O18. The UV spectrum (MeOH, λmax, nm): 273, 292, 322. HR-ESI-MS, m/z: 769.5211 [M–H]– (calc. 769.6776 for C37H37O18 [M–Н]–). ESI-MS, m/z (%): 769 [M–H]– (18), 607 [(M–H)–C6H10O5]– (31), 593 [(M–H)–C10H8O3]– (63), 431 [(M–H)–C6H10O5–C10H8O3]– (100), 341 [(M–H)–C6H10O5–C10H8O3–90]– (15), 311 [(M–H)–C6H10O5–C10H8O3–120]– (8). The NMR1H spectrum (500 MHz, DMSO-d6, 330 K, δH, ppm): Table 1. The NMR 13C spectrum (125 MHz, DMSO-d6, 330 K, δС, ppm): Table 1.

Table 1. The signals of NMR 1H (500 MHz, DMSO-d6, 330 K, δH, ppm, J/Hz) and 13C (125 MHz, DMSO-d6, 330 K, δС, ppm) spectra of compounds 1–3
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Isovitexin-2"-O-glucoside-6"-O-p-coumarate (2). The general formulas is C36H36O17. The UV spectrum (MeOH, λmax, nm): 270, 290, 319. HR-ESI-MS, m/z: 739.3731 [M–Н]– (calc. 739.6520 for C36H35O17 [M–Н]–). ESI-MS, m/z (%): 739 [M–H]– (12), 593 [(M–H)–C9H6O2]– (58), 577 [(M–H)–C6H10O5]– (17), 431 [(M–H)–C6H10O5–C9H6O2]– (100), 341 [(M–H)–C6H10O5–C9H6O2–90]– (19), 311 [(M–H)–C6H10O5–C9H6O2–120]– (15). The NMR 1H spectrum (500 MHz, DMSO-d6, 330 K, δH, ppm): Table 1. The NMR 13C spectrum (125 MHz, DMSO-d6, 330 K, δС, ppm): Table 1.

Izovitexin-2"-O-(6"'-O-feruloyl)-glucoside-6"-O-ferulate (3). The general formula is C47H46O21. The UV spectrum (MeOH, λmax, nm): 272, 292, 321. HR-ESI-MS, m/z: 945.7209 [M–H]– (calc. 945.8470 for C47H45O21 [M–Н]–). ESI-MS, m/z (%): 945 [M–H]– (28), 679 [(M–H)–C10H8O3]– (21), 607 [(M–H)–C10H8O3–C6H10O5]– (11), 593 [(M–H)–2C10H8O3]– (42), 431 [(M–H)–2C10H8O3–C6H10O5]– (100), 341 [(M–H)–2C10H8O3–C6H10O5–90]– (9), 311 [(M–H)–2C10H8O3–C6H10O5–120]– (6). The NMR 1H spectrum (500 MHz, DMSO-d6, 330 K, δH, ppm): Table 1. The NMR 13C spectrum (125 MHz, DMSO-d6, 330 K, δС, ppm): Table 1.

Hydrolysis. Acid hydrolysis was performed in 2 M TFA (5 mL per 5 mg of the compound) at 100°С for 2 h; then the acid was removed under vacuum in the presence of methanol and the dry residue was separated on polyamide (3 g, Capital Analytical cartridges, Great Britain), which was washed with water (100 mL, eluate I), methanol (150 mL, eluate II) and 0.5% ammonia in methanol (150 mL, eluate III). In eluate I, monosaccharides were detected by HPLC after the reaction with 3-methyl-1-phenyl-2-pyrazoline-5-one, as well as after reductive amination with L-tryptophane to determine whether they belong to the D/L series [16]. The incubation with 0.3% NaOH was performed as described previously; enzymatic hydrolysis was performed with β-glucosidase (3.2.1.21, 30 U/mg, Sigma-Aldrich; 2 U in 500 µL of 100 mM phosphate buffered saline, pH 5.0), which was used for incubation of compounds by the method described in [17].

Biological activity. The effects of fractions and individual compounds on lipase activity were studied by spectrophotometry [13] using human pancreatic lipase (3.1.1.3, 50 U/mg; Lee Biosolutions, United States) with p-nitrophenyl-butyrate, p-nitrophenyl-laurate and p-nitrophenyl-stearate (all from Sigma-Aldrich) as substrates. Orlistate (tetrahydrolipstatin, Sigma-Aldrich) was used as a positive control. The inhibitory activity was expressed as IC50 (the concentration causing a 50% inhibition of enzyme activity) in µg/mL, which was determined graphically after plotting the dependence of inhibitory activity on concentration.

Lethality test. The test was carried out with the cysts of Artemia salina (Arsal, Russia), which were incubated in artificial seawater (Sigma-Aldrich) with the tested substance (20–2000 µg/mL) or 0.9% NaCl solution (control) as described [18].

Statistical analysis. Statistical processing was performed by one-way analysis of variance (ANOVA). The significance of differences between the mean values was determined using Duncan’s multiple range test. The differences at p < 0.05 were considered statistically significant.

RESULTS AND DISCUSSION

The extract of C. sativus leaves was fractionated by solid-phase extraction (SPE) on polyamide to obtain SPE-1–SPE-3 fractions; SPE-3 had the maximum inhibitory effect on human pancreatic lipase. SPE-3 was then separated by flash chromatography with Sephadex LH-20, reverse and normal phase silica gel and preparative HPLC, which resulted in isolation of compounds 1–7; their structure was studied using the data of UV, NMR spectroscopy, mass spectrometry and chemical transformations (Fig. 1). Four compounds were the known natural flavonoids and C-glycoside isovitexin (apigenin-C-glucoside) derivatives, including isovitexin-4'-O-glucoside-2"-O-(6""-O-feruloyl)-glucoside (4), isovitexin-4'-O-glucoside-2"-O-(6""-O-p-coumaroyl)-glucoside (5), isovitexin-2"-O-(6""-O-feruloyl)-glucoside (6), and isovitexin-2"-O-(6""-O-p-coumaroyl)-glucoside (7) (Fig. 2). These compounds were found previously in the leaves of C. sativus infected by Podosphaera fuliginea, which is the pathogen causing powdery mildew in Cucurbitaceae species [15] and have been described in the tissues of healthy plants for the first time.

Fig. 1.
figure 1

A chromatogram (HPLC–mass spectrometry, negative ionization, the mode of recording isolated ions with m/z 739, 769, 901, 931, 945) of the SPE-3 fraction of Cucumis sativus leaves (a) and mass spectra of compounds 1 (b), 2 (c), 3 (d), 4 (e), 5 (f), 6 (g) and 7 (h). The numerals above the peaks indicate the number of compound and the value m/z of deprotonated ion.

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Fig. 2.
figure 2

The structure of compounds 1–7 isolated from the leaves of C. sativus.

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Compound 1, after the hydrolysis with 2 M TFA, broke up into isovitexin, ferulic acid, and D-glucose and had a UV spectrum typical of C,O-glycosyl-apigenins acylated with hydroxycinnamic acids [14]. The incubation with 0.3% NaOH resulted in the formation of isovitexin-2′′-O-glucoside, which is possible in case of acylation of the carbohydrate part of the molecule. Fragmentation in the mass spectrum of negative ionization in 1 was similar to that of isovitexin-2"-O-(6""-O-feruloyl)-glucoside (6) [15]; however, 1 demonstrated formation of a fragment with m/z 607 caused by removal from the deprotonated ion of hexose, which is possible in case of terminal localization of the latter. This fact was confirmed by the results of enzymatic hydrolysis of 1 with β-glucosidase, resulting in the formation of isovitexin-6"-O-ferulate [15], while the treatment of 6 with this enzyme did not lead to structural changes. The comparative analysis of NMR spectra of 1 and isovitexin-2"-O-glucoside showed their similarity, except for the presence of additional signals belonging to the trans-ferulic acid fragment (Table 1). Localization of the feruloyl fragment at atom C-6" of 6-C-bound glucose has been confirmed by the data of the HMBC spectrum, with correlations between the signals of H-6" protons (δH 4.10, 4.38) and the carbon of the carbonyl group C-9"" (δС 166.0). Thus, the structure of compound 1 was defined as apigenin-6-C-(2"-O-β-D-glucopyranosyl-6"-O-feruloyl)-β-D-glucopyranoside (isovitexin-2"-O-glucoside-6"-O-ferulate), one isomer of which is isovitexin-2"-O-(6′′′′-O-feruloyl)-glucoside isolated previously from C. sativus [15]. Compound 2 was structurally similar to 1 but contained the fragment of n-coumaric acid, as was demonstrated by the results of acid and alkaline hydrolysis, UV and NMR spectroscopy, and mass spectrometry. Hence, it was possible to establish that 2 is apigenin-6-C-(2"-O-β-D-glucopyranosyl-6"-O-p-coumaroyl)-β-D-glucopyranoside (isovitexin-2"-O-glucoside-6"-O-p-coumarate). The compound structurally similar to 2 is isovitexin-2"-O-(6""-O-p-coumaroyl)-glucoside, which is also found in C. sativus [15].

The data of mass spectrometry indicated that the molecular weight of compound 3 was higher by 176 AMU (C47H46O21) than that of 1 (C37H38O18). As in the case of 1, acid hydrolysis of 3 was accompanied by the formation of isovitexin, ferulic acid and D-glucose; however, the mass spectrum of the compound was shown to contain ions with m/z 769 and 593, indicating the removal of two feruloyl fragments. In the NMR 13C spectrum, there are downfield shifts of C-6" (δС 61.2 → 63.9) and C-6"' (δС 60.9 → 64.7) signals of glucose fragments relative to those in isovitexin-2"-O-glycoside [19], while the HMBC spectra demonstrated correlations between the signals of protons H-6" (δH 4.12, 4.32) and H-6"' (δH 4.10, 4.38) and the carbons of carbonyl groups C-9"" (δС 165.9) and C-9""' (δС 166.1), respectively. The revealed peculiarities indicated that compound 3 was isovitexin-2"-O-(6"'-O-feruloyl)-glucoside-6"-O-ferulate or apigenin-6-C-{2"-O-(6"'-O-p-coumaroyl)-β-D-glucopyranosyl-6"-O-n-coumaroyl}-β-D-glucopyranoside.

The results of quantitative analysis of the leaves of ten Russian varieties of C. sativus at the age of 14–16 weeks have shown that the total content of compounds 1–7 may vary from 3.78 mg/g (Amur F1) to 7.44 mg/g (Dachny F1) (Table 2). Different varieties demonstrated different abilities to accumulate individual compounds, while concentration of the dominant compounds was 0.30–5.34 mg/g for 3, 0.75–2.53 mg/g for 4 and 1.73–4.19 mg/g for 5. There are no literature data on the content of individual compounds in the leaves of C. sativus, but it is known that the total concentration of this group of compounds in varieties grown in other countries is lower: up to 1 mg/g in China [20], up to 1.46 mg/g in Indonesia [21], and up to 2 mg/g in the United States [22].

Table 2. The contents of compounds 1–7 and their sums (Σ1–7) in the leaves of Russian varieties of C. sativus (mg/g of dry plant)*
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The study of biological activities of isolated compounds has shown that compounds 1–7 had an inhibitory effect on the activity of human pancreatic lipase (Table 3). p-Nitrobenzene esters with different fatty chain lengths, including butyrate, laurate, and stearate, were used as substrates. The reference compound, orlistate, caused the maximum inhibition of hydrolysis of the short-chain substrate p-nitrophenyl butyrate (IC50 = 0.012 µg/mL). Under these conditions, the maximum activity was demonstrated by flavonoid 3 (IC50 = 5.69 µg/mL), while other compounds were less effective (IC50 11.28–90.83 µg/mL). With long-chain p-nitrophenyl stearate used as a substrate, the inhibitory activity of Orlistat decreased (IC50 = 3.14 µg/mL) and the activity of flavonoids increased; the efficiency of 3 (IC50 = 1.62 µg/mL), 6 (IC50 = 2.72 µg/mL) and 1 (IC50 = 2.94 µg/mL) exceeded that of the reference compound. In the case of substrate with medium-chain fatty acids, flavonoids 1–3, 6, 7 inhibited lipase activity less efficiently (IC50 2.83–7.52 µg/mL) than Orlistat (IC50 = 0.53 µg/mL). Food triglycerides in most cases are glycerol and fatty acid esters with a chain length of 14 to 22 carbon atoms, i.e., medium- to long-chain [23]. In this context, the fact that the flavonoids of C. sativus could have an inhibitory effect on hydrolysis of this group of glycerides is of great importance.

Table 3. Inhibition of the activity of human pancreatic lipase by compounds 1–7 and orlistat in the presence of three substrates, IC50, µg/mL ± S.D.
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The experiments with Artemia salina showed that Orlistat caused 50% death of organisms at a concentration of 125 µg/mL, while the concentration of flavonoids 1–7 causing the same percentage of death was higher: 1000 µg/mL, which indicates their greater safety compared to the reference compound.

Previously, the efficiency of flavonoids as pancreatic lipase inhibitors has been shown for some apigenin glycosides that are structurally similar to the studied compounds [24]; however, this kind of activity has been established for the first time for acylated derivatives of isovitexin. When considering the interrelationship between the chemical structure and biological activity of flavonoids from C. sativus, it can be noted that the most active compound 3 contained two feruloyl fragments and a free hydroxyl group at C-4'. The activity of mono-acylated glycosides 1, 2, 6, and 7 was slightly lower; if a glucose residue was substituted for the hydroxyl at C-4', compounds 4 and 5 lost the ability to inhibit lipase activity. Thus, the presence of fragments of ferulic or n-coumaric acids in the structure of flavonoids, as well as the free hydroxyl at C-4' in ring B influenced the inhibitory effect on pancreatic lipase, as has been shown previously for other flavonoids detected in Camellia sinensis, Eremochloa ophiuroides, and Litchi chinensis [25]. The known lipase inhibitors from the group of natural apigenins, isovitexin and saponarin (isovitexin-7-O-glucoside), demonstrated the lower efficiency (IC50 > 100 µg/mL) [24] compared to the most active flavonoid 3, which allowed this compound to be considered a promising bioactive agent.

These studies have shown that the green waste formed during the cultivation of C. sativus is a plant source for obtaining acylated flavonoids capable of inhibiting the activity of human pancreatic lipase. These compounds can be used in the development of new drugs for the treatment and prevention of lipid metabolism disorders.