Phytochemistry and pharmacology of sea buckthorn (Elaeagnus rhamnoides; syn. Hippophae rhamnoides): progress from 2010 to 2021

Sea buckthorn (Elaeagnus rhamnoides; syn. Hippophae rhamnoides) is a thorny shrub or a small tree belonging to the Elaeagnaceae family, native to Eurasia. Sea buckthorn fruit is rich in vitamins and minerals, oils from the seeds and fruit flesh find use in medicine and the cosmetic industry or as nutraceutical supplements. Fruit, leaves and other parts of buckthorn have been used in traditional medicine, especially in China, Tibet, Mongolia, and Central Asia countries, and are a rich source of many bioactive substances. Due to its health-promoting and medicinal properties, the plant has been extensively investigated for several decades, and its phytochemical composition and pharmacological properties are well characterized. The years 2010–2021 brought significant progress in phytochemical research on sea buckthorn. Dozens of new compounds, mainly phenolics, were isolated from this plant. Numerous pharmacological studies were also performed, investigating diverse aspects of the biological activity of different extracts and natural products from sea buckthorn. This review focuses on the progress in research on sea buckthorn specialized metabolites made in this period. Pharmacological studies on sea buckthorn are also discussed. In addition, biosynthetic pathways of the main groups of these compounds have been shortly described. Graphical abstract


Introduction
Sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson; syn. Hippophae rhamnoides L.) is a dioecious, deciduous, thorny shrub or a small tree (Elaeagnaceae), native to Eurasia. It has elongate-oblanceolate or elongate-spathulate leaves, and small, juicy fruit, yellow, orange, or red in colour, with very short (1.5-2 mm) stalks (Fig. 1). The plant lives in a symbiotic association with root-nodule-forming, nitrogen-fixing Frankia actinomycetes. It is cold resistant, drought and salt tolerant, with low soilrequirements, which makes it well adapted to growth in different habitats and climatic zones. Sea buckthorn (SB) has become a horticultural crop plant, cultivated in many countries of Europe and Asia, as well as in Canada. Its popularity has been growing over last decades, mainly due to the high nutritional value and medicinal properties of its fruit. It has also found use in the ecological restoration of degraded lands, e.g. for the afforestation of mining heaps. SB fruit is very rich in vitamin C, it contains also significant amounts of vitamin E, vitamin K, b-carotene, as well as mineral elements (mainly P, K, Ca and Mg). The fruit is used in the food industry, to produce juices, jams, syrups etc.. Moreover, both seeds and flesh of the fruit contain valuable oils (100-160 g kg -1 seeds, 20-105 g kg -1 fresh soft parts), finding use in medicine, cosmetic industry, or as nutraceutical supplements. Fruit, leaves and other parts of SB have been used in traditional medicine, especially in China, Tibet, Mongolia, and Central Asia countries, and the plant is listed in the Chinese Pharmacopeia (Suryakumar and Gupta 2011;Kalia et al. 2011;Letchamo et al. 2018;Ciesarova et al. 2020). Due to its health-promoting and medicinal properties, the plant has been extensively investigated for several decades, and its phytochemical composition and pharmacological properties are well characterized.
The taxonomy of Hippophae genus has been a source of controversies. In the first monography (1908) of Elaeagnaceae, only one species was recognized, H. rhamnoides, with three subspecies: ssp. rhamnoides, ssp. salicifolia, and ssp. tibetana. Later, Rousi (1971) recognized ssp. salicifolia, and ssp. tibetana as separate species, and distinguished nine subspecies of H. rhamnoides: ssp. carpatica, ssp. caucasica, ssp. fluviatilis, ssp. gyanthensis, ssp. mongolica, ssp. rhamnoides, ssp. sinensis, ssp. turkestanica, and ssp. yunnanensis. This taxonomic division was questioned by some researchers, who raised ssp. gyanthensis to the level of species, and distinguished a few more new species. More recent taxonomic studies (www.theplantlist.org) applied the work of A. Nelson (1935), H. rhamnoides taxon was synonymized, and its updated name is Elaeagnus rhamnoides. Most of the subspecies recognized by Rousi were elevated to species level (i.e. Hippophae carpatica, H. caucasica etc.), but these taxonomic names are described as unresolved (Letchamo et al. 2018). However, both names, Hippophae rhamnoides, and Elaeagnus rhamnoides, have been used in recent publications on SB, and the former seems to be far more popular.
The taxonomic issues are far beyond the scope of this narrative review, as well as beyond the knowledge and competences of its author. Readers interested in SB taxonomy may find more details in publications of Letchamo et al. (2018) and Bartish (2016). The aim of this study is to review the progress in the research on SB (regardless of the Latin name, Hippophae rhamnoides (including all subspecies), or Elaeagnus rhamnoides used in publications) specialized metabolites made from 2010 to 2021. Publications about the biological activity of SB extracts and their constituents have also been described. However, the number of available articles on pharmacological properties of different extracts or natural products from this plant is very large, so that section of the article only highlights some important aspects of research made during the last twelve years. The literature search was performed using Google Scholar, PubMed, and Scopus search engines, with a time limit from 2010 to 2021. Keywords ''sea buckthorn '' or 'rhamnoides' were combined with 'flavonoids','ellagitannins',''phenolic compounds'','phytosterols','triterpenoids','alkaloids',''anticancer activity'',''antiviral activity'',''clinical trial'',etc. Finally,97 original articles about SB from this period were included in this review.

Phytochemistry
The years 2010-2021 brought significant progress in phytochemical research on sea buckthorn; several tenths of new natural products were isolated from fruit, seeds, and leaves of this plant, mainly flavonoids, flavonolignans, and triterpenoid saponins. Apart from the above-mentioned groups, different parts of SB were shown to contain ellagitannins, phenolic acids, lignans, naphthols, naphthoquinones, anthraquinonoids, triterpenoids, phytosterols, carotenoids, volatile compounds, norsesquiterpenoids, and alkaloids. This review is focused on these issues. In the case of ellagitannins and triterpenoids, some older data have been also presented, as these two groups were relatively rarely mentioned in the literature about SB, though they significantly contribute to the bioactivity of this plant. In addition, biosynthetic pathways of the main groups of SB specialized metabolites have been shortly described.
Due to the great scientific interest in flavonoids and anthocyanins, as well as proanthocyanidins, their pathway of biosynthesis has been investigated for a long time, and is well characterized (Fig. 3). The first committed step in biosynthesis of SB flavonols is the formation of naringenin chalcone, the result of the condensation of p-coumaroyl-CoA (originating from L-phenylalanine) with 3 malonyl-CoA units, catalysed by chalcone synthase. In the next step, the chalcone undergoes stereospeciphic cyclization to (2S)-naringenin by chalcone isomerase. Naringenin is subsequently hydroxylated by flavanone 3-hydroxylase to form dihydrokaempferol, and then oxidized to kaempferol by flavonol synthase. Additional hydroxyl groups are added to the B ring of flavonoids by flavonoid 3'-hydroxylase and flavonoid 3 0 5'-hydroxylase, at the stage of naringenin, dihydrokaempferol, or kaempferol. Biosynthesis of isorhamnetin and syringetin additionally involves methylation, catalysed by S-adenosyl methionine-dependent O-methyl transferases. Glycosylation of the aglycones is catalysed by UDP-glycosyltransferases (Alseekh et al. 2020;Dastmalchi 2021;Liu et al. 2021). Acylation is the final step in biosynthesis of some SB flavonoids, conducted usually by BAHD-acyltransferases (BAHD-ATs), employing CoA-activated acids; however, serine carboxypeptidase-like acyl transferases (SCPL-ATs), using 1-O-b-glucose esters as donor molecules, may also be sometimes involved in acylation of flavonoids, as shown for Brasica napus (Bontpart et al. 2015;Wilson et al. 2016;Alseekh et al. 2020).
Gallic acid and glucose are the initial substrates for the biosynthesis of ellagitannins (Fig. 3). Gallic acid derives from the shikimate pathway, and is probably  directly synthesized from 3-dehydroshikimic acid, in the reaction catalysed by the dual activity 3-dehydroquinate dehydratase/shikimate dehydrogenase; however, its biosynthesis via protocatechuic acid is not excluded (Salminen 2014;Bontpart et al. 2016;Rock 2017). Formation of 1-O-galloyl-b-D-glucose (glucogallin) from gallic acid and UDP-glucose, is the first step of ellagitannin biosynthesis. The molecule is subsequently further galloylated, with the use of other glucogallin molecules as galloyl donors, to form 1, 2, 3, 4, 6-penta-O-galloylglucose; these reactions are most probably catalysed by SCPL acyl transferases (Wilson et al. 2016). Further steps of the ellagitannin biosynthesis are only generally known, partly due to a  The galloyl moieties of the pentagalloylglucose are subsequently subjected to reactions of oxidative coupling (which may be catalysed by laccases), and formation of hexahydroxydiphenoyl units (HHDP), which are a characteristic structural feature of ellagitannins. In this way tellimagrandins II and casuarictin are formed. The addition of more galloyl units, combined with the oxidative coupling reactions (giving HHDP and dehydrohexahydroxydiphenoyl (DHHDP) groups), the removal of gallic acid residues, other modifications, as well as oligomerization lead to formation of plethora of compounds (Ascacio-Valdes et al. 2011;Salminen 2014;Yamashita et al. 2021).
Saponins constitute a quite recently discovered group of SB constituents (Fig. 8). Six triterpenoid glycosides were isolated from seeds of H. rhamnoides ssp. sinensis: . Another compound, arjunglucoside I (93) has been recently isolated from SB leaves (Yuca et al. 2022 (Fig. 9). Then, two molecules of farnesyl diphosphate (a common substrate for the synthesis of sesquiterpenoids, triterpenoids and sterols) are joined into squalene, by squalene synthase. Squalene is oxidized by squalene monooxygenase (squalene epoxidase) to 2,3-oxidosqualene, the branch point intermediate in biosynthesis of sterols and triterpenoids, which is subsequently cyclised by different oxidosqualene cyclases (e.g. a-amyrin synthase, bamyrin synthase, lupeol synthase). Synthesis of SB triterpenoid acids involve further oxidation of a-amyrin, b-amyrin, or lupeol by cytochrome P450 enzymes, which catalyse the addition of additional hydroxylic groups, oxidation of OH groups, and transformation of C28 methyl group into the COOH group. The biosynthesis of known SB saponins involves the formation of sugar chains at 3-OH group of the aglycones, and the addition of a single glucose moiety at 28-COOH, catalysed by different UDP-glycosyltransferases (Thimmappa et al. 2014;Rahimi et al. 2019;da Silva Magedans et al. 2021).

Pharmacology
Sea buckthorn is a medicinal plant, used in traditional and conventional medicine, and cultivated for its fruit, rich in health-promoting compounds. For these reasons, the plant has attracted scientific attention for many years, and its pharmacological properties have been studied in many ways. As in the case of many other plants, it seems that antioxidant, anti-inflammatory, anticancer, as well as antimicrobial activity were the most often investigated bioactivities of sea buckthorn. Antidiabetic, anti-hyperlipidemic, hepatoprotective, and neuroprotective properties of the plant were also frequently described. In contrast, new studies on antiviral activity of SB seem to be rare. As it has been mentioned above, the number of publications on the pharmacological activities of SB is very large, and most of them describe properties of SB oils, extracts, or fractions. This section of the article only highlights some important aspects of research published between 2010 and 2021, and is focused (when possible) on activities of compounds isolated from SB. Reports on clinical trials and toxicological studies are also described.

Anticoagulant and antiplatelet activity
Butanol extracts from SB leaves and twigs were tested for their influence on human plasma haemostasis, and were found to prolong activated thromboplastin time (APTT) at 50 lg mL -1 (Skalski et al. 2019a). Another experiment showed that the phenolic fraction of the SB leaf butanol extract prolonged prothrombin time (PT), and the low-polarity fraction (rich in triterpenoids) of the SB twig butanol extract extended APTT, at the concentration range 0.5-50 lg mL -1 (Skalski et al. 2018). In turn, isorhamnetin 3-O-bglucoside-7-O-(3-isovaleryl)-a-rhamnoside prolonged thrombin time (TT) in human plasma. Moreover, 22 and isorhamnetin inhibited the thrombin-induced human platelet aggregation at 10 lg mL -1 , but had no influence on ADP-or collagen-activated platelets (Skalski et al. 2019b). Phenolic and low-polarity fractions of SB leaf and twig butanol extracts inhibited the thrombin-induced adhesion of human platelets to collagen, at 0.5-50 lg mL -1 . The same preparations inhibited the thrombin-and ADP-induced adhesion of the platelets to fibrinogen, while the phenolic and low polarity fractions from twigs also suppressed the ADPinduced aggregation of platelets. In addition, all tested preparations decreased superoxide anion level, and inhibited lipid peroxidation in thrombin-activated platelets, while they did not inhibit lipid peroxidation in the resting platelets . The influence of the total SB fruit flavonoids on vascular endothelial injury was investigated in blood stasis model rats. The flavonoid preparation caused a reduction of whole-blood viscosity, lengthening of PT and APTT at the doses of 0.24 and 0.48 g kg -1 b.w., and decreasing of the level of fibrinogen. The reduction of levels of thromboxane B2, 6-ketoprostaglandin F1a, von Willebrand factor (vWF),and thrombomodulin was also observed (Wei et al. 2017).

Anticancer activity
Anti-proliferative and cytotoxic activity of extracts from fruits of H. rhamnoides spp. yunnanensin, sinensis, mongolica, turkestanica, and their 15 phenolics were tested on human liver cancer cell line HepG2. The median effective doses (ED 50 ) were determined to evaluate their effect for the proliferation of the cells, 0.85, 1.21, 3.03, and 3.31 mg mL -1 for extracts from the fruit of spp. yunnanensin, mongolica, turkestanica, and sinensis, respectively. Isorhamnetin, kaempferol, and quercetin were the most potent proliferation inhibitors (ED 50 29.0-80.0 lM), their 3-O-sophoroside-7-O-rhamnosides of kaempferol and isorhamnetin, as well as isorhamnetin 3-O-glucoside-7-O-rhamnoside were the least active (ED 50-[ 800 lM). The half maximal cytotoxicity concentrations (CC 50 ) of the preparations were usually distinctly higher (e.g. 8.31-16.8 mg mL -1 for the extracts, and 76.2-202 lM for the flavonol aglycones), which indicates that the anticancer activity of the tested preparations may be attributed mainly to their anti-proliferative properties (Guo et al. 2017 Grey et al. (2010) compared the influence of heptane, ethyl acetate, ethanol, water extracts from SB fruit (prepared successively), as well as ethanol-water fruit extracts (in concentrations up to 2%) on proliferation of HepG2 and Caco-2 cells. The ethanol-water extract, and ethyl acetate extract were the most active proliferation inhibitors. Their activity was dosedependent, and the ethyl acetate extract had also proapoptotic properties. The activity of the ethyl acetate extract was attributed to its high ursolic acid content. The effect of the ethanol extract from SB leaves on the growth and differentiation of cells of human acute myeloid leukemia (KG-1a, HL60, U937) was also determined. The extract inhibited proliferation of the tested cell lines in a dose-dependent manner, and it showed some cytotoxic activity against HL60, at 100 lg mL -1 , due to the induction of apoptosis (Zhamanbaeva et al. 2014). Similarly, a hot water extract from SB leaves inhibited proliferation of rat glioma C6 cells in a dose-dependent manner (up to 49.5% inhibition at 62 lg mL -1 , on the 3 rd day). It also reduced viability of the glioma cells (at 0.62-62 lg mL -1 ), while non-cancerous mice NIH/ 3T3 fibroblast cell line was not affected. The treatment was accompanied by a significant decrease in production of reactive oxygen species in the glioma cells, upregulation of expression of the pro-apoptotic Bax protein, and caused some changes in morphology of nuclei. These reactions suggest that the leaf extract's anti-proliferative properties may be caused by the induction of early events of apoptosis (Kim et al. 2017). In another experiment, SB leaves were sequentially extracted with cyclohexane, hexane, diethyl ether, ethyl acetate, methanol, and water. The water extract turned out to cause a dose-dependent (3.12-50 lg mL -1 ) inhibition of proliferation of C4-2 and LNCaP prostate cancer cells. Moreover, it reduced migration of C4-2, and was shown to inhibit the nuclear translocation of GFP-tagged androgen receptor (AR). Expression of AR and AR-responsive genes was also downregulated (Masoodi et al. 2020). Proanthocyanidins from SB seeds decreased viability of human breast cancer MDA-MB-231 cells (IC 50 37.5 lg ml -1 ), inducing apoptosis. The preparation was a potent inhibitor (IC 50 0.087 lg mL -1 ) fatty acid synthase (FAS), active in a dose-dependent manner against intracellular FAS in MDA-MB-231 cells (which overexpress the enzyme); the inhibition of intracellular FAS activity induced apoptosis in the cancer cells (Wang et al. 2014). The cytotoxic activity of low polarity fractions of butanol extracts from SB fruit, leaves and twigs were tested against HT29, HCT116, PC3, AGS, MCF7, HS27, and PBMCs cell lines. The determined IC 50 values were 14.58-74.58 lg ml -1 for the twig fraction, 23.33-68.00 lg ml -1 for the leaf fraction, and 18.00-42.92 lg ml -1 for the fruit fraction, respectively. Further experiments on the most sensitive cell lines (HT29, PC3, AGS) showed that the cytotoxic effect of the tested preparations was caused by induction of apoptosis. Moreover, the fractions had moderate genotoxic activity (the comet assay; up to * 16% DNA in tail) at their IC 50 concentrations. The cytotoxic properties of the low-polarity fractions was attributed to the presence of diverse triterpenoid acids (Marciniak et al. 2021). HRWP-A, a highly methylated homogalacturonan pectin from the fruit of SB significantly inhibited the growth of the Lewis lung carcinoma in mice. It seems HRWP-A did not have any direct cytotoxic properties, but the anticancer effect was due to its immunomodulatory activity. It enhanced the lymphocyte proliferation, augmented the cytotoxicity and phagocytosis of macrophages, increased levels of TNF-a and NO, and stimulated activity of NK cells and cytotoxic lymphocytes in the tumour-bearing mice (Wang et al. 2015).
Rats with type 2 diabetes, induced by streptozotocin and high-fat diet, were fed with a diet supplemented with a water extract from SB seed residue (400 mg kg -1 b.w.), for 6 weeks. The treatment caused a statistically significant reduction of body weight, serum glucose, total cholesterol, and LDL-C levels in the diabetic rats. Their insulin sensitivity index also increased (Zhang et al. 2010). The hypoglycaemic and hypolipidemic effect of the total flavonoids from SB seeds was tested in a high-fat diet fed mouse model. The obese mice were fed with a high-fat chow, supplemented with SB flavonoids (at three doses: 50, 100,150 mg kg -1 b.w.), for 12 weeks. A significant reduction of the total body weight, liver weight and epididymal fat pad weight was observed, accompanied by the decrease of the serum total cholesterol, LDL-C, and glucose levels (Wang et al. 2011). Antidiabetic potential of SB juice, and the juice enriched with L-quebrachitol (5 mL daily) was tested on db/db mice in a 10 week experiment. The treatment caused a significant improvement of glucose tolerance and the integrity of pancreatic tissue. The feed intake, body weight gain, blood glucose level, and the expression of insulin receptor b in the liver were also reduced. It seems the presence of L-quebrachitol contributed to the antidiabetic effect of the juice (Xue et al. 2015).

Antimicrobial activity
Extracts and a phenolic-rich fraction from SB leaves inhibited growth of Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus phecalis, Escherichia coli, Salmonella typhi, Shigella dysenteriae, and Streptococcus pneumoniae in the agar-diffusion assay (up to 0.5-1 mg mL -1 ). However, the activities were lower than those for 10 lg mL -1 ampicilin (Upadhyay et al. 2010;Yogendra Kumar et al. 2013). Jeong et al. (2010) determined Minimum Inhibitory Concentrations (MIC) against Bacillus subtilis, S. aureus, E. coli, Salmonella typhimurium, as well as yeasts Pichia jadinii and Candida albicans, for methanol extracts from SB roots and stem, and their hexane, ethyl acetate, butanol and water fractions. While MIC values against the bacteria were rarely lower than 500-1000 lg mL -1 (except for S. aureus), the preparations were more active against the fungi -125 lg mL -1 for preparations from the roots, 62 lg mL -1 for the crude extract from the stem, and its hexane and ethyl acetate fractions, and 125 lg mL -1 for its butanol and water fractions. Butanol extracts from SB leaves and twigs significantly inhibited growth of Candida glabrata G1 (MIC 15.6 and 3.9 lg mL -1 for the twig and leaf extract, respectively), and C. albicans ATCC 10,231 (MIC 250 and 31.5 lg mL -1 for the twig and leaf extract, respectively). Moreover, the extracts had antivirulence properties, inhibiting the formation of biofilm by C. albicans ATCC 10,231 (80.6% and 15.3% of inhibition at 0.5 MIC, for the twig and leaf extract, respectively) (Sadowska et al. 2017). In contrast, phenolic fractions and low-polarity fractions from butanol SB leaf extract and butanol twig extract did not inhibit growth of diverse bacteria (S. aureus, Streptococcus mutans, B. cereus, Lactobacillus acidophilus, Helicobacter pylori, E. coli, Proteus vulgaris, P. aeruginosa), and yeasts (C. albicans, C. parapsilosis, C. krusei, C. glabrata) in a significant degree (MIC mostly 0.5-1.0 mg mL -1 or higher). However, the twig and leaf preparations, as well as the phenolic and low polarity fractions of SB fruit extract suppressed the adhesion and formation of biofilm by S. aureus and C. albicans at 0.5 MIC; the inhibitory activity was generally stronger against C. albicans. In the case of S. aureus, the investigated low-polarity fractions were more active than the phenolic ones ( Ró _ zalska et al. 2018).

Antiviral activity
Ellagitannins from SB leaves were known to be active against influenza and Herpes viruses, while SB leaf extract was found to be active also against dengue virus (Suryakumar and Gupta 2011). In more recent research, 7 phenolic compounds and quinones from SB fruit peels were tested for their antiviral activity.

Other activities
Several flavonolignans from SB fruit exerted immunosuppressive properties. Compounds 44,47,48,33 (cinchonain Ia), and 34 (cinchonain Ib) inhibited the concavalin A-induced proliferation of BALB/c mouse spleen cells, with IC 50 values of 41.82,19.42,20.19,48.05,and 46.91 lM,respectively. Other compounds,including 43,46,, 35 (mururin A), and 36 (mururin B) showed moderate neuroprotective activity. The compounds increased the survival rate of PC12 cells from 50.30% (negative control) to 61.08-71.63%, at 10 lM (Ma et al. 2020). Watermethanol extracts from the fruit of different cultivars of SB exerted moderate anticholinergic activity, their IC 50 values ranged from 20.16 to 40.60 lg mL -1 ; the samples had also strong inhibitory activity against butylcholinesterase, with IC 50 \ 0.01 lg mL -1 (Tkacz et al. 2020). The results suggest that SB fruit may find use in the prevention and treatment some neurological disorders, including neurodegenerative diseases. Ethyl acetate fraction of SB leaf extract increased the viability of neuronal PC-12 cells treated with H 2 O 2 , their membrane integrity (inhibition of lactate dehydrogenase release), and reduced their intracellular ROS level in a dose-dependent manner. The fraction had also some anti-apoptotic properties (Cho et al. 2017). SB fruit juice, administered to Wistar rats with induced epilepsy (at the dose of 1 mL kg -1 b.w., for 1 month), significantly reduced the epileptiform activity, improved abnormalities of behaviour, and improved histological structure of cortex and hippocampus (Ladol and Sharma 2021).
Kalemba-Dro_ zd_ z et al. (2020) investigated the protective effect of different pasteurized fruit juices (bilberry, chokeberry, cranberry, Goji, Noni, rosehips, raspberry, and SB) against DNA damage. Human lymphocytes were incubated with 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine (PhiP), in presence or absence of 0.1% solutions of the juices. All tested juices efficiently protected lymphocytes against DNA damage induced with 100 lM PhiP. Moreover, the juices inhibited the formation of ROS; it seems the inhibitory activity of the SB juice was higher than those of Noni, cranberry, chokeberry, and raspberry juices. In absence of PhiP, the SB juice did not increase ROS production in lymphocytes, unlike cranberry and Noni juices. Zhang et al. (2018) tested 46 compounds and groups of compounds isolated from SB fruit, including flavonoids, carotenoids, phospholipids, phenolic acids, aliphatic acids, fatty acids, sterols, for their hepatoprotective activity in rats. Some of them, mainly flavonoids and phenolic acids, inhibited the self-activation of hepatic stellate cells (HSCs), which are known to be engaged in the development of liver fibrosis, with IC 50 values ranging from 46.03 (isorhamnetin 3-O-rutinoside) to 186.34 lM (zeaxanthin dipalmitate). The most active compounds (isorhamnetin 3-O-rutinoside, isorhamnetin and protocatechuic acid) were also shown to reduce viability TGF-b-activated HSCs, decreased levels of TNF-a, IL-1, and IL-6 secreted by the them, as well as they induced the HSC cell arrest. Moreover, a vaguely described preparation ''active components of SB berries'' alleviated rat liver fibrosis (caused by the bile duct ligation) during in vivo experiment. Maheshwari et al. (2011) showed that the ethyl acetate fraction of the 70% ethanol SB leaf extract protected rats from CCl 4 -induced oxidative liver damage. The fraction, administered orally at 25-75 mg kg -1 b.w., for 7 days, decreased the serum activity of alanine aminotransferase, aspartate aminotransferase, and bilirubin level. The treatment prevented the depletion of hepatic antioxidant enzymes (SOD, catalase, glutathione peroxidase, glutathione reductase), and glutathione level. It also inhibited lipid peroxidation and protein carbonylation, as well as prevented histopathological changes in rat livers.

Clinical trials
In the years 2010-2021, different preparations from SB were subjected to at least several clinical trial studies. Lehtonen et al. (2010) investigated the influence of a crushed SB fruit, an oil-free SB fruit (after the supercritical CO 2 extraction), and ethanolextracted oil-free fruit on postprandial hyperglycemia and insulin response. Each of the ten healthy male volunteers consumed breakfasts of yogurt ? 50 g glucose, containing the above mentioned supplements and a placebo on consecutive days of the experiment, in a randomized order. The amounts of the fruit preparations corresponded to 200 g of fresh SB fruit. The trial showed that the SB fruit and the oil-free fruit significantly suppressed the postprandial peak insulin response, and stabilized postprandial hyperglycemia and subsequent hypoglycemia. The third preparation was not active. The results suggest that the SB oil, containing carotenoids, phytosterols, and tocopherols had a minor influence on the investigated parameters, and ethanol-soluble compounds, possibly flavonoids and other phenolics, were responsible for the observed antidiabetic effect. In the article of Lehtonen et al. (2011), the dried SB fruit, as well as SB oil (supercritical CO 2 -extracted), an ethanol extract from the oil-free SB fruit (spray-dried in a 1:1 mixture with maltodextrin), and frozen bilberries were tested for their ability to alleviate problems associated with obesity and metabolic syndrome, in an experiment with randomized cross-over study design. These fruit preparations were consumed by 80 female volunteers for 33-35 days (intervention periods), in an independently randomized order; the intervention periods were separated by 30-39-day wash-out periods. Doses of the fruit preparations were supposed to be equivalents of 100 g of fresh berries. The consumption of SB fruit, and SB oil caused a small, but statistically significant reduction of waist circumference. The SB fruit reduced also the serum glucose and TNFa levels; the latter parameter was also decreased by the ethanol extract. Another publication, based on the same or a very similar experiment, provided some metabolomic data about the participants' serum samples. The SB fruit, and (particularly) SB oil tended to improve serum lipid parameters (e.g. lowering serum cholesterol, LDL-C, serum triglycerides), though the observed changes were usually not statistically significant. The positive effects were the best seen in participants who had a higher cardiometabolic risk at the beginning of the trial (Larmo et al. 2013). Vashishtha et al. (2017) investigated the influence of the SB seed oil (supercritical CO 2 -extracted; 0.75 mL per day, for 30 days) on cardiovascular risk factors in people with hypertension in the randomized, controlled, double blind longitudinal study (106 volunteers, three groups: I -the healthy taking the oil, IIhypertensive persons taking the oil, and III -hypertensive persons taking a placebo, sunflower oil). The treatment caused a statistically significant reduction of serum LDL-C, total cholesterol, oxidized LDL, and triglycerides, more visible in people with hypertension. In contrast, the consumption of SB fruit puree (a double-blind, randomized, placebo-controlled trial; 111 volunteers with hypercholesterolemia, two groups; 90 mL of the puree or placebo per day, for 90 days) did not improve the serum lipid parameters. However, diastolic blood pressure was significantly decreased. In addition, the SB treatment tended to have an anti-inflammatory effect (a reduction of levels of hypersensitive C-reactive protein and soluble vascular cell adhesion molecule-1) (Zhou et al. 2020). Rodhe et al. (2013) evaluated the effect of the supercritical CO 2 -extracted SB oil on oral health, inflammation, and DNA damage in haemodialysis patients by a randomized, double-blinded, and placebo-controlled crossover study. The experiment comprised two treatment periods (for the oil and placebo, dosed 2 g per day; 8 weeks) separated by a wash-out period. The oil did not have any significant influence on the investigated parameters. The effect of the consumption of a SB oil on vaginal atrophy in postmenopausal women was tested in a placebocontrolled, randomized, double-blind, study (96 volunteers; two groups; 3 g of SB oil or placebo per day, for 3 months). A significantly better rate of the improvement in the integrity of vaginal epithelium was observed in the SB oil group, as compared to the placebo group. The treatment did not significantly influenced serum lipid parameters (Larmo et al. 2014). A SB emulsion (with its juice and oil) was shown to alleviate symptoms of functional dyspepsia in children. The study comprised 120 children (11 months -10 years), divided into three groups: I -a group treated with the SB emulsion; II -a group treated with domperidone (a dopamine antagonist, used in treatment of nausea); III -a group treated with the SSB emulsion and domperidone. The SB emulsion was a daily doses of 5-15 g, depending on the children's age, for 8 weeks. The application of the SB emulsion significantly improved appetite factors, increased levels of leptine and neuropeptide Y, and improved gastric emptying, gastrointestinal digestive functions, as well as children's growth and development (Xiao et al. 2013). Gao et al. (2014) investigated effectiveness of sea buckthorn capsules (Hebei Shengxing Seabuckthorn Pharmaceuticals, Shijiazhuang City, China) in treatment of nonalcoholic fatty liver disease.
Patients (94) were divided into two groups: a treatment group, taking the SB capsules (daily dose-4.5 g, for 90 days), and a control group, taking placebo capsules. At the end of the experiment, the SB treatment caused a statistically significant reduction of levels of serum alanine aminotransferase, LDL-C, triglycerides, collagen type IV, and hyaluronic acid, as compared to the control group. Liver stiffness measurement, and liver/spleen ratio were also improved. Sea buckthorn (especially the oils) is well known for its wound and burn-healing properties. Abdullahzadeh and Shafiee (2021) compared the effectiveness of a cream containing 40% of ''the active ingredient of sea buckthorn'' fruit in treatment of second-degree burns, with that of a 1% silver sulfadiazine (SSD, a topical medicine commonly used for treatment of burns) cream. A randomized triple-blind clinical trial comprised 55 hospital patients. The sea buckthorn-based preparation significantly shortened the period of burn healing, as compared to the 1% SSD cream.
Toxicity studies Acute and sub-chronic toxicity of SB fruit oil was evaluated on mice and rats, respectively. The oil, obtained from the ground dried fruit by supercritical CO 2 extraction, was shown to contain isorhamnetin (0.12 mg g -1 ). In the acute toxicity test, the oil was administered by gavage, in a single dose (at 20 mL kg -1 b.w.). After 14 days, there was no mortality among the mice, and no sign of toxicity was observed in their organs. In the sub-chronic toxicity test, the oil was given to rats at 2.5, 5, and 10 mL kg -1 b.w., for 90 days. No significant changes in haematology and serum biochemistry were observed. Administration of the oil did not significantly influence food consumption and body weight gains, the weight of organs or their histology. The maximum tolerated dose of SB oil was determined as [ 20 mL kg -1 b.w. for mice, and the no-observedadverse-effect-level (NOAEL) was [ 10 mL kg -1 b.w. for rats (Zhao et al. 2017). Genotoxicity and teratogenicity of the same (or similar) oil was also determined, using mice and rats. Genotoxicity tests comprised the mice sperm abnormality test (the oil doses 2.34, 4.68, and 9.36 g kg -1 b.w., for 5 days), the in vivo mice bone marrow micronucleus test (the same oil doses, for 2 days), as well as the bacterial reverse mutation assay (Salmonella typhimurium; 8-5000 lg of the oil per plate). Teratogenicity of the oil was determined on rats (the oil doses 1.17, 2.34, 4.68 g kg -1 b.w., for 10 days). The tested SB oil was not genotoxic or teratogenic under the applied experimental conditions (Wen et al. 2020). In another experiment, SB seed oil was administered intramuscularly to rabbits (0.5, 1, 1.5 mL kg -1 b.w., once a week, for 7 weeks). A dose-dependent increase in the total serum antioxidant capacity was observed with maximum 48 h after the oil injection. The treatment did not significantly influence haematological parameters and serum biochemistry, no significant histopathological changes were observed in organs (Ali et al. 2011). Tulsawani (2010 investigated the influence of the water extract from SB fruit on rats, in a 90 day experiment. The extract was administered by gavage, at the dose of 100, 250 and 500 mg kg -1 b.w. No toxic effect was observed. However, an increase in serum glucose at 250 and 500 mg kg -1 doses was detected at the end of the experiment, and 100 mg kg -1 b.w per day was determined as the NOAEL for the extract. A herbal antioxidant supplement, containing SB pulp and ''extract of indigenous medicinal plants of high altitudes that tone up the gastric and intestinal functions'' was orally administered to mice at 2, 4, 8, and 10 g kg -1 b.w., to test its acute toxicity. During 14 days, no change in mice behaviour and body weight gains were observed. To determine sub-acute toxicity of the preparation, 2, 4, and 8 g kg -1 b.w. doses were administrated daily, for one month. No change in the animal behaviour, body weight gains, haematological and biochemical parameters of blood, as well as organ histology was observed (Ali et al. 2012). The above results seem to confirm that SB oils and fruit extracts are generally safe food additives.

Conclusion
Sea buckthorn is currently a well-known plant. Its vitamin-rich fruit is more and more frequently consumed, usually in processed form, seed and fruit pulp oils are used in the cosmetic industry, and medicine. The plant is listed in the Chinese Pharmacopoeia, and its different parts are used in the traditional medicine of China, Tibet, Mongolia, and other countries of central and eastern Asia. Healthpromoting and medicinal properties of SB caused a large scientific interest in this plant. As it has been shown in this review, The years 2010-2021 brought significant progress in phytochemical research on SB. At least 24 new flavonoids, 10 flavonolignans, 6 saponins, 3 alkaloids, two sesquiterpenoids, one ellagitannin, and one phenylpropanoid derivative were isolated during this period from SB fruit, seeds, and leaves. In addition, dozens of known specialized metabolites were also isolated, some of them for the first time from this plant. Summarizing, SB is a rich source of natural products, including flavonoids, phenolic acids, ellagitannins, triterpenoids, phytosterols, carotenoids, and volatile compounds. Flavonolignans, lignans, sesquiterpenoids, naphthalenes, anthraquinonoids, triterpenoid saponins, alkaloids or carboxylic acid derivatives were also found. These compounds undoubtedly significantly contribute to medicinal properties of SB. During the period in question, diverse pharmacological effects of SB extracts and natural products were extensively investigated, including antioxidant, anti-inflammatory, anticancer, antidiabetic, antithrombotic, antimicrobial, antiviral, immunosuppressive, neuroprotective activity, and other effects. Casuarinin, one of the main ellagitannins of SB leaves, seems to be most promising compound, a potent a-glucosidase inhibitor and free radical scavenger, potentially useful in treatment of diabetes. Sea buckthorn flavonolignans, reported for the first time from the plant, also constitute an interesting group of phytochemicals. Among them, compounds 47 and 48 had significant immunosuppressive properties, while 43 and 46 showed distinct neuroprotective activity in in vitro tests.
The reviewed publications suggest that preparations made from different parts of SB may be potentially useful in the prevention and treatment of neoplasms, metabolic syndrome, diabetes, and other inflammation-and oxidative stress-related disorders and diseases, or as anti-virulence agents, decreasing the formation of biofilm by pathogenic microbiota. In the author's opinion, future research should pay more attention to SB triterpenoids, triterpenoid saponins, and ellagitannins. Though ellagitannins seem to be the dominant phenolics of SB leaves, considerable amounts of saponins can be found in the seeds and leaves, and triterpenoids occur in all parts of this plant, these compounds were relatively rarely investigated (especially ellagitannins and saponins), as compared to SB flavonoids, phenolic acids, or carotenoids. Triterpenoids, saponins, and ellagitannins are known for their bioactivity, and undoubtedly significantly contribute to the pharmaceutical properties of sea buckthorn.

Supplementary information
A list of sea buckthorn specialized metabolites (with their molecular formulas, exact masses, PubChem CID numbers, CAS numbers, and SMILES strings) is provided as a supplementary file.