Hippophae rhamnoides L. leaf and twig extracts as rich sources of nutrients and bioactive compounds with antioxidant activity

Plants have served for centuries as sources of compounds useful for human health such as antioxidant, anti-diabetic and antitumor agents. They are also rich in nutrients that improve the human diet. Growing demands for these compounds make it important to seek new sources for them. Hippophae rhamnoides L. is known as a plant with health-promoting properties. In this study we investigated the chemical composition and biological properties of bioactive components of ethanol extracts from leaves and twigs of H. rhamnoides L. Chemical components such as the total content of phenolic compounds, vitamins and amino acids and the antioxidant activities of these compounds in cellular and cell-free systems were assessed. The results suggest that the studied extracts are rich in bioactive compounds with potent antioxidant properties. Cytotoxicity and hemotoxicity assays showed that the extracts had low toxicity on human cells over the range of concentrations tested. Interaction with human serum albumin was investigated and conformational changes were observed. Our results indicate that leaf and twig extracts of H. rhamnoides L. should be considered as a non-toxic source of bioactive compounds which may be of interest to the food, pharmaceutical and cosmetic industries.


Results
HPLC analysis. HPLC analysis identified 40 phenolic compounds in the extracts of H. rhamnoides leaves and twigs, respectively (Table 1). Monomeric and polymeric catechins and gallic acid esters such as epigallocatechin were found in both the leaf and twig extracts.
There was a high content of dihydroxybenzoic acids (α-resorcylic and protocatechuic acid) in the twig extracts, whereas the leaf extracts had higher contents of rosmarinic (8-fold), genistic (4-fold), chlorogenic (2.5-fold) and ellagic (12-fold) acids than twig extratcs. The amounts of flavonoids such as rutin, luteolin and hesperidin were higher in the leaf than the twig extracts by about 12-fold, 3.5-fold and 8-fold, respectively. In  www.nature.com/scientificreports/ general, the twig extracts showed lower levels of phenolic compounds than in the leaf extracts except for gallic acid and catechin (1.3-fold and 7-fold higher in twigs than leaves, respectively).

Content of phenolic compounds.
The results indicate similar contents of total phenolic compounds in the H. rhamnoides leaf and twig extracts (Fig. 2): 269.49 mg/g dry matter for leaf extract and 239.41 mg/g dry matter for twig extract. The levels of flavonoids were higher in leaf than in twig extracts (23.7 mg/g and 11.8 mg/g for dry matter of extracts, respectively). Flavonoids accounted for about 9% and 5% of the phenolic compounds in the extracts of leaves and twigs, respectively. However, because AlCl 3 reacts mainly with flavones, flavonols, flavanones and flavanols, the results obtained do not correspond exactly to the total flavonoid contents of the tested extracts.
In contrast to the flavonoids, the catechin concentration was higher in the twig than the leaf extracts (9.3 mg/g and 85 mg/g dry matter of extracts, respectively). The total content of catechins (flavan-3-ols) in the twig extracts was over nine times higher than in the leaf extracts.
Determination of amino acid content. Table 2 shows the total content of amino acids in leaves and twigs from H. rhamnoides. More amino acids were found in leaves (0.62%) than twigs (0.45%). Among all the amino acids, arginine, histidine and proline were most abundant in the twigs (0.087%, 0.058% and 0.067%, respectively), while valine, proline, phenylalanine and arginine were most abundant in the leaves (0.084%, 0.081%, 0.070% and 0.066%, respectively). Due to the method limitation, the content of glutamic and aspartic acid was difficult to evaluate.

Determination of water-soluble vitamins and vitamin E isoforms content. The qualitative
and quantitative contents of water-soluble vitamins in the leaves and twigs of H. rhamnoides are presented in    (Table 3B). H. rhamnoides twigs contained only the α isoform at 1.21 mg/100 g, which was lower than that in the leaves, in which α, ß and γ isoforms were found at 38.2 μg/g, 10.6 μg/g and 8.3 μg/g, respectively.
Antioxidant activity of extracts. Free radical scavenging activity of the H. rhamnoides leaf and twig extracts ( Fig. 3-1) was evaluated using 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical. The antiradical activity of the extracts was dose-dependent in the range of 0-50 µg/ml and at 50 µg/ml amounted to about 75% for both extracts. The H. rhamnoides leaf and twig extracts affected and significantly decreased H 2 O 2 /Fe-induced plasma protein carbonylation. Twig extract was active in the highest concentration (50 μg/ml). Leaf extract exhibited protective activity when 10 μg/ml was used. (Fig. 3-2).
The ability of the H. rhamnoides leaf and twig extracts to decrease ROS production in human fibroblasts was tested with the H 2 DCFDA probe ( Fig. 3-3). Both extracts protected the cells against oxidative stress induced by H 2 O 2 . The leaf extract inhibited ROS content in the cells more effectively than twig extract, but both decreased ROS production significantly ( Fig. 3-4).
Hemolysis and cytotoxicity. The extracts of H. rhamnoides leaves and twigs were practically non-hemolytic at concentrations ranging from 0.5 to 50 µg/ml (Fig. 4A). The twig extract showed slight hematoxicity at concentration 50 µg/ml, but the level of hemolysis did not exceed 5% after treatment with either extract.
The toxicity of extracts was tested on normal human fibroblast (BJ) cells (Fig. 4B). The H. rhamnoides twig extract had a stronger effect on cell viability than the leaf extract but the difference was not statistically significant. The effects did not exceed 20% even after incubation with extracts at the highest concentration (50 µg/ml).
Interaction with human serum albumin (circular dichroism). Human serum albumin exhibits two characteristic negative bands at 202 and 220 nm. Both extracts changed the intensity of these bands that indicated on their interaction with the protein and the effect on its structure. The leaf extract changed the protein secondary structure, (Fig. 5A) more than the twig extract (α-helix content changed from 59.6 to 47.3% and from 64 to 61.4% for the leaf and twig extracts, respectively). The amounts of β-sheet and random coil structures increased from 12.9% to 14.8% and 16.9% to 20.2% respectively for the leaf extract, and from 12.6% and 16.6% to 13% and 17.6%, respectively for the twig extract (Fig. 5B).

Discussion
The aim of our study was to explore phytochemical content, antioxidant properties and potentially toxic effect on normal human cells of ethanol extracts from H. rhamnoides leaves and twigs. H. rhamnoides has been known for centuries in folk medicine. It was believed that different parts of this plant have beneficial properties, strongly connected with their chemical composition 28 . The best-known parts of H. rhamnoides are the berries Table 3. Content of water-soluble vitamins (A) and tocopherol isomers (B) in dry matter of extracts of twigs and leaves from Hippophae rhamnoides L. Values are means ± SD (n = 3). www.nature.com/scientificreports/  www.nature.com/scientificreports/ and leaves. However, the currently-available literature provides also information about twig extracts from H. rhamnoides 5,34,35 .
In order to detect phenolic compounds in leaf and twig extracts from H. rhamnoides, HPLC analysis was conducted. The results revealed that the most abundant compounds in the leaf extract were epigallocatechin, procyanidin, epicatechin, luteolin, rutin, ellagic acid and rosmarinic acid. The twig extract showed the highest contents of α-resorcylic acid, protocatechuic acid, epigallocatechin, catechin, procyanidin B2 and epicatechin. According to other studies, the most abundant phenolics in H. rhamnoides leaf extract prepared as tea-type infusions are isorhamnetin glucosides and kaempferol 5,33,36,37 . We also detected these compounds, though some of them in lower concentrations. The total phenolic contents of both types of extract were similar but there were differences in the flavonoids and catechins pool. The flavonoid content in the leaf extract was twice that in the twig extract; the total content of catechins (flavan-3-ols) in the twig extracts was over nine times higher than in the leaf extracts. When butanol extracts from H. rhamnoides leaves and twigs were investigated, the total phenolics content was higher than we found in ethanol ones (341.5 mg/g and 621.2 mg/g in leaf and twig extracts, respectively) 5,38 . In our study the twig extract contained mainly protocatechuic acid and B-type procyanidin. Similarly, when butanol extracts were tested, catechins and B-type procyanidins were detected in twig extract in the highest concentration 5,38 . Other studies revealed that the butanol extract from leaves was rich in tannins, whereas the butanol extract from twigs was rich in proantocyanidins 5,36 .
Our study showed that rutin and chlorogenic acid are present in both types of extracts but both these compounds are at higher levels in leaves than twigs.
We revealed also three tocopherol isomers (α, β and γ) in the leaf extract, whereas only α-tocopherol was found in the twig extract. In both organs, α isomer was detected in the highest concentration. In another study Kallio et al. described tocopherol contents in berries and seeds, and similarly, Madawala et al. found α-tocopherols in the highest concentration in berry extracts 33,39 . Jaroszewska and Biel estimated the amount of tocopherol in leaves and found 40.98 mg/kg in leaf samples from plants growing in Poland 40 . Vitamins B are a group of water-soluble vitamins that can be found in plant extracts. Plant sources containing vitamins B could be important in the human diet. One of our goals was to measure the total content of vitamins B in leaf and twig extracts from H. rhamnoides. Vitamin B6 was the most abundant in both extracts. Vitamins B were detected in low concentrations, so the extracts could serve rather as alternative source of vitamins.
Some parts of plants are discarded as trash, though they could be considered valuable sources of carbohydrates, proteins, fatty acids and other nutrients. Leaves and twigs from most plants are wasted in large amounts by the agro-food industry 41 . We checked the amino acid contents of ethanol extracts from H. rhamnoides leaves and twigs. We found eight essential amino acids in twig extracts and seven in leaf extracts. The ratio between essential and total amino acid contents was very similar in both extracts: 63% and 66% in leaf and twig extract, respectively. . Interestingly, the percentage of essential amino acids in both extracts was over 60% in our study. The most abundant essential amino acid in twig extract was arginine (0.8719 g/100 g of dry raw material). In leaf extract, valine (0.8369 g/100 g of dry raw material) was the most representative essential amino acid. Lysine and arginine contents are limited in plant materials, so extracts from different parts of H. rhamnoides should be considered attractive sources of amino acids.
It is known that H. rhamnoides is particularly interesting owing to its antioxidant potential. Berries, seeds, leaves and twigs could serve as a valuable sources of antioxidant agents 6,42 . Our analysis of the content of bioactive www.nature.com/scientificreports/ compounds in ethanol extracts of leaves and twigs showed that, in addition to phenolic compounds, they contain several groups of phytochemical that, to varying degrees, exhibit antioxidant properties, namely tocopherols 43 , vitamins [44][45][46] and amino acids 47,48 . Tocopherols were found to reduce lipid peroxidation level and to trap the nitric oxide derivatives. Vitamin B6 easily crosses the membranes and is phosphorylated to a coenzyme form. As a coenzyme pyridoxine it is engaged in many chemical reactions of amino acids chains. Ultimately, it is related to the metabolism of cysteine, which is necessary for glutathione formation. The role of glutathione as one of the most potent antioxidant is well established. In the study regarding thiamine the antioxidant capacity was checked. It was demonstrated that vitamin B1 exhibits ·O 2 − and ·OH scavenging activity. Thiamine is also a precursor of cofactor engaged in the oxidative decarboxylation. Product of such reactions-NADPH can directly scavenge radicals and maintain antioxidant power. Folic acid derivatives also exhibited potency to peroxynitrite and inhibit of lipid peroxidation. The role amino acids in antioxidant defense was also studied. The correlation between peptide concentration and improved antioxidant defense was confirmed. It was found that the presence of tyrosine, phenylalanine, histidine, lysine, and arginine correlated with a higher scavenging activity. Moreover, the presence of hydrophobic amino acids and histidine, proline, methionine and cysteine improved antioxidant activity.
Therefore in our study, we checked antiradical scavenging potential of extracts using the DPPH method. Our study revealed that scavenging activity increased with concentration of extracts (0.5-50 μg/ml) and at 50 μg/ml both extracts reduced DPPH to 70%. In Radekov's study 49 , ethanolic extracts from H. rhamnoides leaves/shoots exhibited stronger antiradical activity than extracts from press cake. Shivapriya et al. showed that extracts from leaves/fruits of H. rhamnoides exhibited antiradical activity in the concentration range 1-300 μg/ml. The IC 50 value for antiradical scavenging activity was 70.91 μg/ml 50 .
In order to investigate the antoxidative activity of extracts at cell level , H2DCFDA was used to measure intracellular ROS content induced by hydrogen peroxide in human fibroblasts. The fluorescence level of oxidized H2DCFDA to dichlorofluorescein (DCF) is equal to the amount of ROS generated inside the cells. Extracts used in the highest concentration of 50 μg/ml strongly protected the cells from ROS production. The leaves extract exhibited better protection (p < 0.001) than the twig extract (p < 0.01) compared with the sample treated with H 2 O 2 only. In the Shivapriya et al. studies, leaf/fruit extract protected neuronal cells from ROS species however when 100 μg/ml was used and the level of ROS content was decreased by 60-70% (p < 0.01) 50 . In our study twig and leaf extract decreased the level of ROS by 62 and 97% respectively comparing to H 2 O 2 -treated cells at 50 μg/ ml.
The high level of phenolic compounds in the alcohol extracts of the leaves and twigs of sea buckthorn should provide those extracts with anti-oxidative properties in protecting lipids and proteins. In the present work, the addition of H 2 O 2 /Fe to human plasma leads to increased oxidation of proteins which, in a concentration-dependent manner was inhibited by both extracts. However, the extracts of from leaves inhibited protein carbonylation even in lower concentrations. Phenolic compounds and tocopherols are mainly responsible for the antioxidant activity of the extracts. Their content in the extract from the leaves is higher, therefore, the protective effect of this extract in the case of oxidation of plasma proteins or oxidative stress in fibroblasts is higher than in the case of the extract from the twigs. Early it was shown that butanol extracts from sea buckthorn leaves and twigs inhibited protein carbonylation in vitro 5,38 .
It is known that bioavailability of phenolic compounds is not high. Nevertheless, it has been shown that in vivo application of, for example, flavonoids, they demonstrated a positive effect on human and animal health. And this group of compounds prevents a variety of diseases, including neurodegenerative disorders. The application of phenolic compounds in high concentrations makes possible to achieve micromolar concentrations in plasma. Such concentration is sufficient enough for the realization of their biological effects 51,52 . Absorption of phenolic compounds dependends on many parameters as molecular weight and lipophilicity and stereoisomerization. Low molecular weight compounds as for example gallic acid and lipophilic are more easily absorbed. There are different transport mechanisms of phenolic compounds. They can be absorbed by either passive diffusion or transporters, such as P-glycoprotein and sodium-glucose cotransporters (SGLT), present in the membrane of enterocytes 53 . Both considered extracts from leaves and twigs of H. rhamnoides L. contain a large amount of low molecular weight phenolic compounds. Therefore, it can be assumed that these components will be sufficiently absorbed.
Albumins are engaged in transport of both endogenous (fatty acids, hormones) and exogenous compounds in the blood 54 . Checking the interactions between albumins and absorbed chemical compounds is important for understanding and explaining the possibility their transportation and realizing biological activity in the body. In our study we examined the conformational changes in human serum albumin (HSA) upon titration with ethanol extracts using circular dichroism method. We observed slight changes in protein secondary structure: decreased α helix structure and increased β helix and random coil. The results obtained suggested that compounds from the extracts can interact with albumin and cause minor conformational changes. The twig extract interacted little stronger with albumin than the leaves extract. Das et al. also reported that extracts containing flavonoids (quercetin, myricetin, kaempferol) could interact with HSA 54 . This indicates that the plant extracts can easily interact with HSA in the bloodstream without significant altering its structure and transport function and can probably be transported in the body.
Extracts from plants are considered as potential sources of biologically active compounds. Therefore, their safety also should be studied. It is known that plant extracts can be toxic to human cells, so checking their cytotoxicity is very important. Guo et al. 55   HPLC analysis. HPLC analysis were performed as previously described 57 . The HPLC system (Summit × 2 Dual-Gradient System, Dionex, Sunnyvale, CA, USA) was equipped with a photodiode-array detector (PDA100 DAD) and fluorescence detector (RF-2000). The phenolic compounds present in the extracts were separated on a RP-C18 column (aQ Hypersil GOLD, 250 × 4.6 mm, 5 µm) joined with a guard column (GOLD aQ Drop-In guards, 10 × 4 mm, 5 µm, Polygen, Gliwice, Poland) at 25 °C. The injection volume of the analyzed samples was 20 µl. A mobile phase composed of water (A) and methanol (POCH, Gliwice, Poland) (B), both with 0.1% formic acid (Sigma-Aldrich, Saint Louis, MO, USA) was used. The injection was started after 2 min of isocratic elution with 5% B, increasing slowly over 30 min to 55% B, followed by 5 min of isocratic elution. Between 37 and 47 min the concentration of phase B increased to 70% followed by 5 min isocratic elution. Then between 52 and 54 min the gradient was returned to the initial 5% B and the column was recalibrated for the next 3 min. The flow rate was 1 cm 3 /min. The absorbance was measured at 235, 280, 325 and 375 nm, and the fluorescence at 420 nm (excitation 270 nm). Phenolic compounds in the H. rhamnoides extracts were identified by comparing the retention times and on-line UV absorption spectra of the analysed samples with the respective data obtained from reference standards (Sigma-Aldrich, Saint Louis, MO, USA, Fluka, Buchs, Switzerland). For all 40 compounds external calibration was performed. Quantification was based on a calibration curves for standards of phenolic compounds covering the range 5-200 µg cm -3 ; the linearity of the calibration curve was verified by the correlation coefficient (r 2 ≥ 0.9994). The optimal wavelengths used for the preparation of calibration curve and quantification of individual metabolites are given in Table 1.
Content of phenolic compounds. The total phenolic content was determined using Folin-Ciocalteu reagent according to Singleton and Rossi 58 . The absorbance of the reaction product was measured at 725 nm www.nature.com/scientificreports/ and the phenolic content was expressed as milligrams per gram of dried extract based on the calibration curve (r 2 = 0.9991) prepared for chlorogenic acid (0-200 g cm -3 ) (Sigma-Aldrich, Saint Louis, MO, USA). The results are given as means ± SD (n = 3). The flavonoid content was determined by the aluminium chloride colorimetric method according to Chang et al. 59 . Reagents AlCl 3 × 6H 2 O and CH 3 COONa were purchased from Chempur, Piekary Śląskie, Poland. The absorbance of the reaction mixture was measured at 415 nm and the flavonoid content was expressed as milligrams per gram of dried extract based on a calibration curve (r 2 = 0.9995) prepared for quercetin (0-100 g cm −3 ) (Sigma-Aldrich, Saint Louis, MO, USA). The results are given as means ± SD (n = 3). The total catechin (flavan-3-ol) content was determined by the vanillin assay method according to Bakkalbasi et al. 60 . Reagents were purchased from Chempur (Piekary Śląskie, Poland, (methanol, H 2 SO 4 )) and Sigma-Aldrich (Saint Louis, MO, USA, (vanillin)). The absorbance of the reaction mixture was measured at 500 nm and the total flavan-3-ol content was calculated from a calibration curve (r 2 = 0.9988) prepared using (+)-catechin (0-100 mg cm -3 ) (Sigma-Aldrich, Saint Louis, MO, USA) and expressed as milligrams per gram of dried extract. The results are given as means ± SD (n = 3). Determination of tocopherol isoforms. The total content of vitamin E isoforms in leaf and twig extracts of H. rhamnoides was determined by HPLC method using a high-performance Agilent 1200 chromatograph (USA) with a four-channel thermostat pump, fluorometric detector and specialized software. Briefly, 5 g of extracts were subjected to alkaline hydrolysis with 5 ml 50% КOH in the presence 50 ml ethanol 0.25 g vitamin C. The resulting mixture was heated in a water bath under reflux at 80 °C for 40 min. Next, 50 ml of water was added to the analyzed sample and transferred to a separatory funnel. Then the mixture was extracted 3 times with 50 ml of isopropyl alcohol. The combined isopropyl extracts were washed with water four times with a volume of 50 ml. The solvent was removed from the isopropyl extract by distillation using a rotary evaporator. Traces of water were removed by drying with anhydrous sodium sulfate. The dry residue was dissolved in 1 ml of the mobile phase (isopropyl alcohol) and applied to 250 × 4.6 mm Zorbax 300SB-C18 column. Antiradical capacity of extracts measured by reduction of DPPH radical. The antioxidant activity of the extracts was estimated using the modified Brand-Williams method 62 . Synthetic free radical DPPH (2,2′-diphenyl-1-picrylhydrazyl, Sigma Aldrich) was used. The DPPH was dissolved in ethanol to a final concentration 8.3 × 10 -5 M. The effect of the ethanol solution of extracts from leaves and twigs of H. rhamnoides was tested at concentrations in the range 0.5-50 μg/ml after 5, 10, 15, 30 and 45 min. The absorbance was measured at λ = 517 nm. Three independent measurements were done. The level of DPPH reduction was calculated as follows:

Determination
where A av = the averaged absorbance of samples containing extracts, A 0 = the absorbance of DPPH solution.
Haemolysis test. Blood

Reactive oxygen species (ROS) in human fibroblast (BJ) cell lines. BJ cells were seeded in an
8-well LabTek chamber (Thermo Fisher Scientific) at density 2 × 10 5 and left overnight to adhere. They were then treated with 50 µg/ml H. rhamnoides leaf and twig extracts for 24 h. After incubation with the extracts the cells were incubated with 80 µM H 2 O 2 for 30 min, then washed with PBS (pH 7.4). Subsequently, 5 µM non-fluorescence probe 2′,7-dichlorodihydrofluorescein diacetate (H 2 DCFDA) was added for 20 min in the dark, and after rinsing with PBS intensity of fluorescent dichlorofluorescein (DCF) was examined in a Leica TCS SP8 confocal microscope with a supercontinuum laser at 485 nm excitation and detection emission at 590 nm. Pictures were analysed and quantitate analysis of ROS level (%) was performed using Leica software.  14.01.2020. To conduct current study the samples were obtained randomly. The blood was collected from healthy volunteers and carefully tested before using in the laboratory. All the experiments published in this manuscript comply with the current laws of the country in which they were performed. The study was approved by the Ethics Committee of the University of Lodz, Poland (NR19/KBBN-UŁ/III/2019). All methods were performed in accordance with the relevant guidelines and regulations.
Ethics approval and consent to participate. All experimental procedures were approved by the Ethics Committee of the University of Lodz, Poland (NR19/KBBN-UŁ/III/2019).

Conclusion
The aim of the present work was to determine the chemical composition and biological properties of leaf and twig extracts from H. rhamnoides L. Both extracts contained similar compositions of phenolics, but in different proportions. Generally, twig extracts contained more catechins than leaf extracts. Essential and non-essential amino acids were found, along with members of the B vitamins family. We also found tocopherol's isoforms in both extracts. However, leaf extract contained all three isoforms, whereas only α-tocopherol was detected in twig extract. Antioxidant properties of both extracts were confirmed by the DPPH assay, the H 2 DCFDA method and protein carbonylation assay. Circular dichroism revealed that bioactive compounds from leaf and twig extracts interacted with albumin slightly modifying the secondary structure. Additionally, the lack of toxic effect of H. rhamnoides L. extracts on human erythrocytes and normal human fibroblast was demonstrated. In view of the results obtained it can be suggested that extracts from H. rhamnoides L. could serve as a non-toxic source of beneficial compounds for a food supplement and cosmetics.

Data availability
All data generated or analyzed during this study are included in this published article. Raw data are available on request from the authors.