Rhamnus pallasii subsp. sintenisii fruit, leaf, bark and root: Phytochemical profiles and biological activities

2.1 Plant material

R. pallasii subsp. sintenisii fruits, leaves, barks, and roots were collected in August 2020 from the Chakhmaqlu mountains of North Khorasan province, Iran (37°29′34′′N 56°56′52′′E) (Fig. 1). A voucher specimen (803893) has been deposited in the Gonbad Kavous University herbarium. The individual portions were dried at 30 °C in a well-ventilated room and stored in the dark until use.

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Fig. 1

Rhamnus pallasii subsp. sintenisii and map showing the location of the sampling (Chakhmaqlu altitudes, North Khorasan, Iran).

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2.2 Chemicals and reagents

Caffeic acid, gallic acid, quercetin, cyanidin-3-glucoside, butylated hydroxytoluene (BHT), sodium hydroxide, hydrochloric acid, sodium molybdate, sodium carbonate, sodium acetate, aluminum chloride, potassium acetate, potassium chloride, Folin-Ciocalteu reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), ether petroleum, methanol and formic acid were purchased from Sigma Aldrich (USA). The other compounds that were employed were of analytical grade. Aqueous solutions were also prepared using deionized water. Microorganism cultures were obtained from Iranian microbial collections, Pasteur Institute of Iran. The cultures of Gram-positive Staphylococcus aureus (ATCC 9144) and Gram-negative Escherichia coli (ATCC 25922) were used for the study.

2.3 Preparation of the extracts

The dried powder of species' fruits, leaves, barks, and roots (2 g) were extracted separately with 20 ml of EP at room temperature for 24 h (three times), and residues were extracted with water-methanol under the same conditions (80%). All of these extracts were filtered using a vacuum pump, and the organic solvents were extracted using a rotary evaporator at 40 °C under decreased pressure. Finally, concentrated extracts were lyophilized to dryness in a freeze dryer and stored in darkness at +4 °C for further analysis. The extraction yields (w/w) for EP extracts ranged from 1.2% to 3.5% and 2.9% to 6.3% for HM extracts.

2.4 Quantification of total phenolic content

The total phenolic content (TPC) of HM extracts from species' fruits, leaves, bark, and roots was evaluated using the Folin-Ciocalteu spectrophotometric method described by Singleton et al. (1999), with minor modifications (Singleton et al., 1999). In brief, 200 µL of diluted extracts were combined with 0.25 M Folin–Ciocalteu reagent, 600 µL of H₂O, and 1000 µL of 1.0 M Na₂CO₃. The absorbance of the solutions was measured at 760 nm after 1 h of incubation at room temperature in the dark. The findings were reported in milligrams of gallic acid equivalents (GAE) per gram of dried extract (mg GAE/g DE).

2.5 Quantification of total flavonoid content

The total flavonoid content (TFC) of all HM extracts was measured using the aluminum chloride colorimetric method described previously (Zhishen et al., 1999), with quercetin standard. In brief, diluted extracts or quercetin standard solutions were combined with 720 µL of distilled water, 90 µL of 5% NaNO₂, 600 µL of NaOH, and 90 µL of AlCl₃. The absorbance of reaction mixtures was measured at 510 nm after incubation at room temperature, and the TFC was reported as milligram of quercetin equivalents per gram of dry extract (mg QE/g E).

2.6 Quantification of total phenolic acid content

The total phenolic acid content (including hydroxycinnamic acid derivatives) was assessed using the Matkowski et al. (2008) method for determining the interaction of phenolic acids with sodium nitrite-sodium molybdate. Each extract (1 ml) was combined with 2 ml HCl (0.5 M), 2 ml Arnow reagent (10 g sodium molybdate and 10 g sodium nitrite diluted to 100 ml with deionized water), 2 ml NaOH (8.5 % w/v), and 3 ml water. The solutions were compared to a control mixture that did not contain Arnow reagent. The absorbance at 490 nm was measured, and the total hydroxycinnamic acid concentration was estimated using a caffeic acid calibration curve and represented as mg caffeic acid equivalent (CAE) per gram of dried extract (mg CAE/g DE).

2.7 Quantification of anthocyanin content

The anthocyanin content of all HM extracts was measured using differential pH methods (Camelo-Méndez et al., 2013), two diluted solutions were prepared, one in 0.4 M sodium acetate buffer with a pH of 4.5; and the other in 0.025 M potassium chloride buffer with a pH of 1.0. At 510 and 700 nm, the absorbance was determined using a spectrophotometer. The absorbance was calculated as follows: A = (A₅₁₀ − A₇₀₀) pH₁ − (A₅₁₀ − A₇₀₀) pH_4.5.

The content of anthocyanins was determined using the absorbance of (A) and the molar absorptivity of cyanidin 3-glucoside (29,600). The TAC values were calculated as mg cyanidin-3-glucoside per gram dry extract. TAC = (A/e × L) × (449.2) × D/G × V × 100.

Where A is absorbance; e (26,900) is the molar extinction coefficient, of cyanidin 3-glucoside (Giusti and Jing, 2008); L (1 cm) is the cell length; 449.2 is anthocyanins molecular weight; D is dilution factor; V (ml) is final volume and G (mg) is the dry weight (dw) of samples.

2.8 DPPH radical-scavenging activity

The DPPH radical scavenging activities of HM extracts was monitored according to the method of Cavin et al. (1998). Five different concentrations of each extract were added to 915 μL methanol, then 200 μL DPPH solution in methanol (0.022%) were added. After 30 min incubation at room temperature in the dark, the reaction mixture's absorbance was measured at 517 nm. The absorbance of extracts was compared to that of methanol without DPPH as a blank.

DPPH radical-scavenging activity was determined by: % Inhibition rate = (A _control − A _sample)/A _control × 100.

The effective concentration necessary to inhibit 50% of the DPPH radicals was expressed by IC₅₀ value (half maximal inhibitory concentration).

2.9 Antibacterial activity

The antibacterial activity of R. pallasii extracts against Gram-positive Staphylococcus aureus (ATCC 9144) and Gram-negative Escherichia coli (ATCC 25922) was tested using the microdilution broth technique as described by Suffredini et al. (2006) with certain modifications. Strains were obtained from Iranian microbial collections, Pasteur Institute of Iran. Two-fold serial dilutions of the extracts of fruit, leaf, bark and root were prepared in Mueller Hinton Broth ranging from 25 to 0.195 mg/ml in 96-wells plates. Additionally, gentamicin discs and each fraction's solvents were employed as positive and negative controls, respectively. After 24 h of incubation, the plates inhibitory effect on bacteria growth was determined visually by examining the growth in each well. After incubation, the MIC value was determined as the lowest concentration of plates at which microorganisms displayed no observable growth. Additionally, the MBC value was calculated using the lowest concentration of plates that exhibited no bacterial growth. Each microorganism was subjected to three independent analyses.

2.10 HPLC-ESI-MS analysis

The HPLC-ESI-MS analysis was implemented by using the Waters Alliance 2695 HPLC system, which was connected with micro mass quattro micro API mass spectrometer with electrospray ion source (ESI). A standard solution containing uracil, 4-hydroxymethyl benzoate, 4-hydroxy ethyl benzoate and benzophenone was injected into the device in order to validate the reliability of the system.

2.11 GC–MS analysis

The GC–MS analyses were performed using an Agilent 6890 gas chromatograph linked to a 5973 MSD mass spectrometer and an HP-5 ms column with a 30 m 0.25 mm i.d. and 0.25 m film thickness. On the basis of Wiley 7n.L and NIST libraries, the chemical profiles of fruit, leaf, bark, and root were identified. Separation of the compounds occurred at a rate of 3°Celsius per minute along a temperature gradient extending from 50 to 280 °C. The instrumentation used a 250 °C analyzer and ion source, a split ratio of 1:20, a 1 μL injection volume, a 70 eV ionization potential, a helium carrier gas flowing at 1.0 ml/min, and a mass range of 50–550 m/z. The components were identified by comparing their mass spectra to those in the Wiley 7.0 mass spectral library and the literature (Adams, 2007). This procedure is similar to that described by Faizi et al., but with minor variations (Faizi et al., 2014).

2.12 Statistical analysis

Each test was conducted in triplicate. Results were expressed as a mean ± standard deviation. SPSS statistics version 20 software and ANOVA procedures were used for statistical analysis. A significance level of 0.05 was considered.

3.1 Total phenolics (TPC), total flavonoids (TFC), total phenolic acids (TFAC) and total anthocyanin contents (TAC)

Fig. 2a illustrates the phytochemical analysis of R. pallasii fruit, leaf, bark, and root extracts. The extracts contained a total of 69.8 1.4 to 232.8 2.5 mg GAE/g DE. The fruit extract had the greatest TPC concentration (232.8 1.5 mg GAE/g), followed by the leaf (208.6 2.3 mg GAE/g), the bark (124.3 1.8 mg GAE/g), and the root (69.8 1.4 mg GAE/g) extracts, respectively. The TPC of a methanolic extract of R. alaternus examined in the literature (Moussi et al., 2015). The leaves contained 77.8 mg GAE/g TPC, which was lower than the value observed in our investigation. Another study determined the TPC of a 60% ethanol extract of R. prinoides stems to be 228.21 ± 13 mg of GAE/g (Chen et al., 2020). Additionally, the TPC of R. lycioides leaves was 259.33 ± 4.95 mg of GAE/g, which was greater than the value found in our study (Benamar et al., 2019). A similar result was achieved when species total flavonoids content was determined. However, the fruit extract (187.03 ± 2.09 mg QE/g DE) had a higher TFC value than the leaf (98.6 ± 2.5 mg QE/g), bark (83.3 ± 1.2 mg QE/g), and root (46.8 ± 2.4 mg QE/g) extracts. The TFC values calculated in this study for the leaves extract were higher than those previously reported for R. alaternus collected in Algeria (30.11 ± 5.76 mg QE/g) (Moussi et al., 2015), but were lower than those previously reported for R. alaternus collected in Tunisia (283 ± 11 mg QE/g) (Ammar et al., 2007). In previous research, the TFC of methanol extracts of R. kurdica and R. lycioides leaves was determined to be 86.32 ± 2.98 mg catechin equivalent per mg plant and 74.08 ± 2.10 mg catechin equivalent per g dry extract, respectively (Gholivand and Piryaei, 2014; Benamar et al., 2019). Additionally, we determined the total anthocyanin concentration of fruit, leaf, bark, and root extracts (Fig. 2a). The fruit (75.14 ± 0.03 mg cyanidin 3-glucoside/g DE) and bark (51.76 ± 0.02 mg/g) extracts had the highest TAC values, followed by the leaf and root extracts at 42.21 ± 1.02 and 12.41 ± 2.3 mg/g, respectively. Gholivand discovered that R. kurdica flowers and leaves have a significant concentration of anthocyanin (21.53 ± 0.57 and 12.36 ± 0.84 g/100 mg fw, respectively) (Gholivand and Piryaei, 2014). The anthocyanin content of extracts varied considerably. These distinctions are related to the diversity of chemicals that make up plant pigments. Finally, the extracts total phenolic acid content (TPAC) was determined (25.01 ± 1.3 to 63.14 ± 2.2 mg CAE/g DE). The fruit extract (63.14 ± 2.2 mg/g) had the highest TPAC value, followed by the leaf (52.4 ± 1.5 mg/g), bark (41.3 ± 2.2 mg/g), and root (25.01 ± 1.3 mg/g) extracts, respectively.

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Fig. 2

Total phenolics, total flavonoids, total anthocyanins and total phenolic acids contents (a) and antioxidant activities (b) of the HM extracts of R. pallasii.

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3.2 Antioxidant activity

For the first time, the antioxidant activities of several parts of R. pallasii were determined using the DPPH radical scavenging assay, and the results were reported as IC₅₀ values (Fig. 2b). Among the R. pallasii extracts, the fruit and leaf extracts displayed the highest scavenging activity, with IC₅₀ values of 7.52 ± 2.1 and 11.81 ± 1.06 g/ml, respectively, which are significantly more active than the positive control butylated hydroxytoluene (BHT) (IC₅₀ = 19.3 ± 1.06 g/ml). The bark and root extracts had the lowest IC₅₀ values, at 20.01 ± 2.5 and 22.39 ± 0.10 g/ml, respectively. The extracts free radical scavenging activity may be a result of their high TPC and TFC content, which have hydrogen-donating capabilities (Rice-Evans et al., 1997). Only a few publications in the literature have discussed the DPPH assay in relation to other species. Our values are lower than those previously reported for other species, including methanolic leaf extract of R. kurdica (IC₅₀ of 21.04 ± 1.35 g/ml), 60 % ethanolic stem extract of R. prinoides (IC₅₀ of 51.21% 0.046 g/ml), and methanolic leaf extract of R. lycioides (IC₅₀ of 29.69 ± 0.33 g/ml) (Gholivand and Piryaei, 2014; Chen et al., 2020; Benamar et al., 2019).

3.3 Antibacterial activity

Table 1 summarizes the antimicrobial activity of EP and HM extracts of R. pallasii fruit, leaf, bark, and root. The extracts were antimicrobial against both microorganisms that cause food poisoning (Staphylococcus aureus and Escherichia coli). The results indicated that the extracts had significantly more antibacterial action against S. aureus than against E. coli, owing to the bacterial strains different cell wall structures. Gram-negative bacteria have an outer membrane composed of lipids and a polysaccharide component that serves as a barrier to antimicrobial drug penetration (Lambert, 2002). When compared to gentamicin, the antibacterial effects of the HM extract of fruit and EP extract of root were much stronger than those of other extracts (positive control). The extracts had MIC and MBC values of 0.39 to 3.12 and 0.78 to 6.25 mg/ml respectively. No investigations on the antibacterial activity of R. pallasii have been conducted to date, however various studies have reported on the antimicrobial activity of other species. Molla demonstrated that methanol and chloroform leaf extracts of R. prinoides were bactericidal against four bacterial strains with MIC values ranging from 8.13 to 32.5 mg/ml (Molla et al., 2016). EP and methanolic extracts of R. alaternus had no detectable inhibitory action on gram-negative and gram-positive bacteria until 6 mg/ml (Ben Ammar et al., 2007). Another study found that the MIC and MBC values of R. prinoides fruit and leaf extracts against S. aureus and E. coli were 1.3 to 5.23 and 2.08 to 8.33 g/ml, respectively (Kibret, 2019). Carranza et al. discovered that methanolic extracts of R. californica leaves had MIC values of 5.0 to 6.0 mg/ml against six bacterial species (Carranza et al., 2015). The results of this study suggest that the significant antibacterial activity of the EP and HM extracts of R. pallasii may be attributed to the presence of terpenes, phenols, and flavonoids, which act as antimicrobial agents via a variety of different mechanisms (Guimarães et al., 2019).

3.4 GC–MS analysis

GC–MS analysis was used to determine the chemical composition of an EP extract of R. pallasii (fruit, leaf, bark, and root). The extract yields were 3.5 % (fruit), 1.2 % (leaf), 3.1 % (bark), and 2.8 % (stem) based on the plant's dry weight. The chemicals that have been identified are listed in Table 2. A total of 68 chemicals were isolated from EP extracts of various plant sections. Our findings indicate that fruit extract contains 26 compounds that account for 93.62 % of its composition; leaf extract contains 29 compounds that account for 92.97 % of its composition; bark extract contains 38 compounds that account for 96.97 % of its composition; and root extract contains 28 compounds that account for 89.92 % of its composition. Each extract contained the following major compounds (in percentages): fruit (oleic acid 45.93 %, stigmastan-3,5-diene 9.76 %, -tocopherol 6.60 %); leaf (-tocopherol 44.28 %, hentriacontane 8.93 %, clionasterol 7.56 %); bark (clionasterol 12.99 %, -tocopherol 11.89 %, tridecane 6.43 %); and root (-tocop Generally, the extracts are a good source of biological components. Terpenes, phenolics, fatty acids, fatty esters, steroids, and hydrocarbons are all significant types of chemicals found in R. pallasii extracts (Table 2). A review of the literature indicated that no data on GC–MS studies of R. pallasii extracts were given, while data on the volatile components of other species were reported (Chouitah et al., 2012; Mekala et al., 2017).

3.5 LC-ESI-MS analysis

The profile of bioactive chemicals in HM extracts of R. pallasii fruits, leaves, barks, and roots was published in this work for the first time using an LC-ESI-MS method in the negative ion mode. All extracts included 59 chemicals, including 24 flavonols, 6 flavones, 4 flavanones, 3 flavanonols, 6 phenolic acids, 10 anthraquinones, 3 naphthaenic lactone derivatives, 2 naphthalene derivatives, and 1 coumarin derivative (Table 3). Fig. 3A-H illustrates the total ion chromatogram (TIC) of extracts and instances of extracted ion chromatograms (EIC). Peaks were identified using molecular weights, retention times (Rt), complete ESI-MS, and matching mass adducts ([M−H]^-, [2 M], [2 M−H], [M−2H], and [M−2H + Na]), as well as comparisons to published data. Only ten of the 59 compounds had been identified previously in R. pallasii, and they were all kaempferol, quercetin, isorhamnetin, mearnsetin, aromadendrin, taxifolin, eriodictyol, pallasiin, -sorinin, and physclon-8-O- β-primeveroside from the barks of Turkish species and leaves of Georgian species (Sakushima et al., 1983; Coşkun et al., 1984). There are no data on the phytochemical profiles of other components of this plant to our knowledge.

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Fig. 3

Chromatograms and corresponding mass adducts in the HM extracts of R. pallasii. (A) Total ion chromatogram (TIC) of fruit; (B) TIC of leaf; (C) TIC of bark; (D) TIC of root; (E) Emodin chromatogram (XIC) and mass adducts, m/z 269.837; (F) Kaempferol-4′-O-rhamninoside XIC and mass adducts, m/z 739.469; (G) Quercetin-3-O-robinobios XIC and mass adducts, m/z 609.991; (H) Emodin-1-glucoside XIC and mass adducts, m/z 431.823.

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