Water-Based Extraction of Bioactive Principles from Blackcurrant Leaves and Chrysanthellum americanum: A Comparative Study

Abstract

The water-based extraction of bioactive components from flavonoid-rich medicinal plants is a key step that should be better investigated. This is especially true when dealing with easy-to-use home-made conditions of extractions, which are known to be a bottleneck in the course for a better control and optimization of the daily uptake of active components from medicinal plants. In this work, the water-based extraction of Blackcurrant (Ribes nigrum) leaves (BC) and Chrysanthellum americanum (CA), known to have complementary pharmacological properties, was studied and compared with a previous work performed on the extraction of Hawthorn (Crataegus, HAW). Various extraction modes in water (infusion, percolation, maceration, ultrasounds, microwaves) were compared for the extraction of bioactive principles contained in BC and CA in terms of extraction yield, of amount of flavonoids, phenolic compounds, and proanthocyanidin oligomers, and of UHPLC profiles of the extracted compounds. The qualitative and quantitative aspects of the extraction, in addition to the kinetic of extraction, were studied. The optimized easy-to-use-at-home extraction protocol developed for HAW was found very efficient to easily extract bioactive components from BC and CA plants. UHPLC-ESI-MS and high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were also implemented to get more qualitative information on the specific and common chemical compositions of the three plants (including HAW). Their antihyaluronidase, antioxidant, and antihypertensive activities were also determined and compared, demonstrating similar activities as the reference compound for some of these plants.

Keywords: Chrysanthellum americanum; blackcurrant; enzymatic activity; flavonoid; granulometry; hawthorn; infusion; polyphenol; procyanidin; water-based extraction.

Figure 1

Kinetics of extraction of Chrysanthellum americanum (CA, lot no. 559980) and blackcurrant (BC, lot no. 55870) monitored by UV absorbance at 198 nm. (A) Infusion mode at 500 rpm stirring speed with the temperature profile (in black). (B) Maceration mode at 500 rpm stirring speed and at 60 °C. A total of 2.5 g of plant material in 250 mL water was systematically used. In total, 100 μL of the solution was taken and added to 4 mL water before each UV measurement. If the absorbance values were above 1.7, dilution in 8 mL water (instead of 4 mL) was performed, but the experimental values were multiplied by 2 to allow for a comparison with dilutions in 4 mL. Error bars are ± 1 SD on n = 3 repetitions of independent extractions. For ground materials, 1 mm grinder was used (see Section 2.2).

Figure 2

Relative size distributions of the raw and ground CA (A) and BC (B) plant materials obtained by laser granulometry in dry mode and variation of the density of CA (C) and BC (D) plant materials as a function of the particle diameter. D₁₀ (□), D₅₀ (∆), and D₉₀ (◯) with D_x being the tenth decile of the distribution. See Section 2.2 for more details on the experimental conditions. Lot number for CA: 559980. Lot number for BC: 55870.

Figure 2

Relative size distributions of the raw and ground CA (A) and BC (B) plant materials obtained by laser granulometry in dry mode and variation of the density of CA (C) and BC (D) plant materials as a function of the particle diameter. D₁₀ (□), D₅₀ (∆), and D₉₀ (◯) with D_x being the tenth decile of the distribution. See Section 2.2 for more details on the experimental conditions. Lot number for CA: 559980. Lot number for BC: 55870.

Figure 3

UHPLC profiles of BC extracts issued from infusion Bodum^® extraction mode for ‘fine’ ground BC material monitored at 280, 320, and 360 nm. Typical UV spectra of the main compounds are given on the right. UV spectrum of Chlorogenic acid represents hydrocinnamic acid derivatives, UV spectrum of Quercetin 3- rutinoside (2) represents Quervetin 3-O-galactoside (3^a), Quercetin 3-O-glucoside (3^b), and Quercetin-3-6-malonyl-glucoside (4^a), UV spectrum of Kaempferol-3-O-hexoside (5) represents Kaempferol-3-O-rutinoside (4^b), Kaempferol-malonylglucoside (6), and Kaempferol-malonylglucoside isomer (7). See Table 4 for peak ID. Lot number: 55870. See Figure S4 for UHPLC profiles of other lots.

Figure 4

UHPLC profiles of CA extracts issued from infusion Bodum^® extraction mode for ‘fine’ ground CA material monitored at 273 nm. Typical UV spectra of main compounds are given on the right. UV spectrum of Chlorogenic acid (1) represents hydrocinnamic acid derivatives (12, 13, 15), UV spectrum of Isookanin-7-O-glucoside (9) represents Eriodicyol-7-O-glucoside (8^a), UV spectrum of Luteolin-7-O-glucuronide (11) represents 6,8-C,C-diglucosyl-apigenin (8^b) and Apigenin-7-glucuronide (14). See Table 5 for peak ID. Lot number: 559980. See Figure S5 for UHPLC profiles of other lots.

Figure 5

PCA score plot of all mass features measured by ESI(-) FT-ICR MS issued from HAW (green, lot numbers: CB58120 and APC27031904), BC (purple, lot number: 55870), and CA (orange, lot numbers: 559980, CP44120 and NH558088) extracts. The annotations #1 and #2 refer to the replicate number.

Figure 6

Venn diagram achieved from BC, CA, and HAW samples analyzed in ESI(-) FT-ICR MS. Heteroatom class distribution and van Krevelen diagram concern features specifically and commonly observed in all samples. Lot numbers: see Figure 5.

Figure 7

Percentage of inhibition of tested enzymes: hyaluronidase (in red) and ACE (in green) and ABTS antioxidant assay (in blue) in the presence of different extracts obtained from HAW, BC, and CA. For the enzyme inhibition assays, the plant extracts were screened at 1 mg mL⁻¹ and the inhibition percentages of hyaluronidase and ACE were calculated according to Equations (2) and (3), respectively. The antioxidant capacities of the plant extracts were determined at 0.01 mg mL⁻¹ and calculated according to Equation (4). The absorbance of the multiwell plates was read twice for both ACE inhibition and ABTS antioxidant capacity assays and the average of obtained results were plotted. All assays were carried out in triplicates (n = 3). Plant extracts were obtained from HAW (#1/#2, ground 1 mm, lot n°20335 and #1/#2, ground ‘fine’, lot n°CB58120), BC (#1/#2, ground ‘fine’, lot n°55870) and CA (#1/#2, ground ‘fine’, lot n°559980). EGCG, hyaluronidase referenced inhibitor, showed 98% hyaluronidase inhibition at 1 mg mL⁻¹, and Trolox, an antioxidant reference, demonstrated 64% antioxidant capacity at 0.01 mg mL⁻¹. Both references were used to validate the methods (more details in Table S2).