Antioxidant Effects Of Cistanche And Their Active Constituents

Abstract: cistanche. (Baill.) fruits, their extracts, and bioactive compounds are used in alternative medicine as adaptogens and ergogenics protecting against numerous neurological, cardiovascular, gastrointestinal, liver, and skin disorders. cistanche extracts and their active compounds are potent antioxidants and mitoprotectors exerting anti-inflflammatory, antiviral, anti-cancer, and anti-aging effects. cistanche polyphenolic compounds—flavonoids, phenolic acids and the major constituents dibenzo cyclooctadiene lignans are responsible for the cistanche antioxidant activities. This review will focus on the direct and indirect antioxidant effects of cistanche extract and its bioactive compounds in the cells during normal and pathological conditions. 

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Keywords: Cistanche; lignan; schisandrin B; antioxidant; pro-oxidant; mitochondria 

1. Introduction 

cistanche. (Baill.) belongs to the Schisandraceae family. The plants are native to northeastern China, Japan, Korea, Manchuria, and the Far East part of Russia. Their purple-red berries are called five-flavor fruits because of their sweet, bitter, pungent, salty, and sour taste [1–5]. cistanche is widely used as an herbal supplement in traditional Chinese medicine and in Western phytotherapy [1,2,4,5], whereas in Russia—as a potent adaptogen, improving disease and stress tolerance, and increasing energy, endurance, and physical performance [2,3]. cistanche is used as a preservative and an additive in food technology to enhance the flavor, taste, and nutritional value to the food [2]. The dried fruits of the cistanche and their extracts are helpful in the treatment of neurological, cardiovascular, and gastrointestinal disorders, in decreasing fatigue, reducing obesity, and protecting from mitochondrial dysfunction, insomnia, and excessive sweating [1,2,5]. They stimulate immunity, act as a tonic, and exert antioxidant, anti-inflflammatory, antiviral, anticancer, anti-aging, anti-diabetic, and liver- and skin-protecting activities [1–5].

2. Chemical Properties of Cistanche Fruit Constituents 

cistanches contain about 1.5% sugars (polysaccharides and monosaccharides; glucose, fructose, galactose, and arabinose), tannins (hydrolyzable, e.g., gallic acid esters, and condensed, e.g., proanthocyanidins and catechol-type tannins), color substances (mainly anthocyanins), and about 3% essential oils, with sesquiterpenes as the dominant compounds [6]. α-Bergamotene, β-chamigrene, β-himachalene, and ylangene are the main components of essential oils (about 75%), whereas oxygenated sesquiterpenes, monoterpenes, and oxygenated monoterpenes comprise the smaller part (about 5%) [6]. Chemical investigations also revealed the presence of triterpenoids (lanostane and cycloartane-type triterpenoids and nortiterpenoids), organic acids (citric, fumaric, malic, and tartaric acids), phenolic acids (chlorogenic, gentisic, p-hydroxybenzoic, p-coumaric, protocatechuic, syringic, and salicylic acids) (7,8l, flavonoids (quercetin, isoquercitrin, rutin, and hyperoside) 18l, vitamins C and E, phytosterols, and bioelements (Cr, Cu, Co, Ca, Mg, FeZn, MnB, Ni,) [1,7,9].The major active compounds of cistanche (Figure 1) are dibenzo cyclooctadiene lignans (1]. Schisandrin is the most dominant cistanche lignan found in the amounts of2.2 5.3 mg/g in cistanche [2,10].

Anticancer activity of cistanche lignans is decreased in the presence of a hydroxylgroup at the C7 position, thus resulting in increased hydrophilicity and decreased permeability into the lipid bilayer (11], whereas a methylenedioxy group between C12 andC13 enhanced anticancer activity. A 1,2,3-trimethoxy moiety, a 6-acyloxy group, and the absence of a 7-hydroxy group resulted in P-glycoprotein inhibition and also increased $chinensis anticancer efficacy 12). Gomisin N and deoxyschisandrin were the most effectiveanticancer lignans of cistanche (11,12]. cistanche lignans without an ester group at C6, ahydroxyl group at C7, or a methylene dioxy moiety, and with an R-biphenyl configuration possess strong antiplatelet activity, with 6,7-dehydroschisandrol A as the most active com. pound (13). The exocyclic methylene group in cistanche lignan structure is necessary forthe antioxidant activity, which is enhanced even more in the presence of the benzovloxygroup [14].

3. Bioavailability of Cistanche Extract and Its Constituents

The bioavailability studies of cistanche products were mainly performed in animals maximum concentration of schizandra of 0.08  0.07 and 0.15 + 0.09 ug/ml was achieved after oral administration of 3 g/kg and 10 g/kg of cistanche extract inrats (15]. In a parallel study, schizandra (10 mg/kg, administered intravenously (i.v.) or orally (p.o.)) and the herbal extract of cistanche (3 g/kg and 10 g/kg, p.o.) were givenindivi-dually to rats (15]. The dose of cistanche (3 g/kg) was equivalent to schisandrir5.2 mg/kg), whereas the dose of cistanche (10 g/kg) was equivalent to schisandrin(17.3 mg/kg) (15). Thus, in rats, the oral bioavailability of schizandra was approximately 15.56 土 10.47% (15]. When Sprague-Dawley rats were administered 2 mg/kg (iv) or10 mg/kg (intragastrically (i.g.)) of schisandrol B, or 6 mL/kg (i.g.) of cistanche extract(equivalent to 15 mg/kg schisandrol B), the oral absolute bioavailability of schizandra Bwas approximately 18.73% and 68.12%, respectively [16]. Schisandrin B could modulate cytochrome P450 3A activity (CYP3A) in vivo in rats and also altered the pharmacokinetic profiles of other CYP3A substrates (17. The tissue distribution studies showed that's chisandrin B (18] and schizandra B were distributed throughout several tested tissues and accumulated mainly in in the liver and kidneys (16,18). Absolute oral bioavailability of schisandrin B depended on the sex of animals it was approximately 55.0% for females and 19.3% for male rats (19]. The linear pharmacokinetics properties were observed within the range of the tested oral dose (10, 20, and 40 mg/kg rat) of schizandra B (19)Schisandrin B was extensively distributed in ovary and adipose tissue (19]. 

The urinary biliary, and fecal excretion of schizandra B was very low; schizandra B was excreted mainly in the form of metabolites (20].In alternative medicine, cistanche dried fruit powder is usually administered to patients at a dose of 0.5-1.5 g twice per day before meals over a period of 20-30 days (3No serious adverse effects were reported during the use of cistanche; however, an overdose may cause dyspnea, restlessness, or insomnia [2,3,5].

4. Biological Activity of Cistanche Extract and Its Constituents: MainMechanisms of Action

Dried cistanche, their extracts, and their bioactive constituents exert a wide variety of beneficial effects under normal and pathological conditions (Figure 2). S. chinen.sis bioactive compounds are antioxidants, detoxifiers, powerful hepatoprotective, hypoglycemic agents, inflammation suppressors, neuro- and cardioprotectors, immunostimuants, and tumor suppressors ([1-3,5]. They demonstrate antibacterial and antiviral properties, and suppress platelet aggregation; they also are potent adaptogens and ergogenics.capable to decrease fatigue and supporting the normal functioning of cellular powerhouse mitochondria (2,7. cistanche bioactive compounds are also potent skin protectors. Theiranti-aging and revitalizing actions comprise moisturizing, toning, irritation-soothing, andwound-healing, reducing dilatation of blood vessels and restoring the skin protective barrier.

Principal mechanisms of beneficial actions of cistanche bioactive compounds include activation of the antioxidant defense system, reducing the levels of aspartate aminotransferase, alanine aminotransferase, and serum and liver glutamic pyruvic transaminase, as well as inactivation of cytochrome P450 [21]. cistanche bioactive compounds inhibit pro-oxidant signaling pathways: cyclooxygenase 1 and 2 (COX-1 and 2) [22], nitric oxide production (23], and gene expression of pro-inflammatory cytokines (24]. Furthermore. chinensis constituents block calcium channels (Ca2+) (25] and inhibit the opening of the mitochondrial permeability transition pore (mPTP), thus protecting from cell death (25-27)In tumor cells, cistanche bioactive compounds can reverse multidrug resistance dependent on P-glycoprotein (Pgp-MDR) (28] and sensitize tumor cells to antitumor treatments.g., with doxorubicin [29,30]. They can also promote cell cycle arrest, thus suppressing proliferation and activating apoptosis and autophagy [31-33].

5. Antioxidant Activity of Cistanche

Fruit Extract and Its Constituentslmpaired balance in pro-oxidant and antioxidant homeostasis cause oxidative stress, which enhances the production of toxic reactive oxygen species (ROS). ROS in the cells mainly generated in mitochondria as by-products of the mitochondrial respiratory chain, whereas some of them can be derived also from redox metal ion-related and enzymatic sources (34,35]. ROS are neutralized by enzymatic and non-enzymatic endogenous antioxidant defense systems. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) belong to the enzymatic defense system, while small antioxidant molecules, such as vitamin C, vitamin E, and reduced glutathione(GSH), make part of non-enzymatic defense [34]. As with many plant-derived antioxidants, cistanche active compounds can directly scavenge reactive oxygen species, activate the antioxidant defense system under normal conditions, and act as pro-oxidants under pathological conditions (Figure 3).

Figure 3. Antioxidant and pro-oxidant activities of Cistanche fruit extracts and their bioactive compounds. 

ROS—reactive oxygen species

Regarding cistanche polyphenolic compounds: Flavonoids, phenolic acids, and the major constituents of dibenzo cyclooctadiene lignans are considered to be responsible for the antioxidant activities of cistanche extract [2,4]. 

5.1. Direct ROS Scavenging by Cistanche Fruit Extract and Its Constituents 

The direct ROS scavenging capability of cistanche ethanolic extracts was demonstrated by Mocan A et al. using the DPPH bleaching assay, Trolox equivalent antioxidant capacity assay, hemoglobin ascorbate peroxidase activity inhibition, and the inhibition of lipid peroxidation catalyzed by cytochrome c assays and an electron paramagnetic resonance spectroscopy [8]. cistanche ethanolic extract could directly scavenge ROS, thus alleviating hydrogen peroxide (H2O2)-induced inhibition of C2C12 cell growth [36]. Moreover, different schisandrins could neutralize ROS from human polymorphonuclear leukocytes stimulated with phorbol myristate acetate [37]. The relative order of the strength of ROS scavenging of schisandrins depended on their conformation and the presence of the hydroxymethyl group capable of attracting electrons, thus facilitating radical attack: S(-)-schisandrin B > S(+)-schizandra> schisandrin C >schisandrin B [37]. The ROS scavenging effect of schisandrin B was similar to that of vitamin C [38]. Fenton reaction, xanthine-xanthine oxidase, or UV-irradiation of riboflflavin assays revealed that the cistanche lignan schisanhenol could neutralize ROS better than vitamin E in an experimental model of tetradecanoylphorbol acetate-stimulated human neutrophils [39]. Furthermore, in in vitro studies, cistanche aqueous extract protected human blood lymphocyte DNA from oxidant challenge by H2O2, evaluated by comet assay [40]. 

5.2. Effects of Cistanche Fruit Extract and Its Constituents on Enzymatic and Non-Enzymatic Endogenous Antioxidant Defense Systems

cistanche bioactive compounds exerted antioxidant activities in many tissues including the brain [41]. In a D-galactose-induced Wistar rat neurotoxicity model, cistanche aqueous or ethanolic extracts decreased SOD, CAT, and total antioxidants, and maintained the normal levels of GSH, malondialdehyde (MDA), and nitric oxide (NO) in the serum, striatum, hippocampus, and prefrontal cortex, thus ameliorating cognitive deficits assessed using the Morris water maze and the step-down type passive avoidance test [42]. Schisandrin B (10, 25, or 50 mg/kg administered orally (p.o.) for 7 days) could increase the levels of antioxidant enzymes, such as SOD, GPx, and cellular GSH in mice, and suppress lipid peroxidation in scopolamine- and cisplatin-induced cerebral oxidative stress [43]. Furthermore, schisandrin B exerted neuroprotective activity by reducing MDA levels and ROS generation, while in the meantime enhancing SOD activity and GSH production in the mice force swimming stress model, thus reducing anxiety-like behavior [44]. Schisandrin B (5, 10, or 20 µM) could suppress the production of ROS in microglia-neuron co-cultures [45]. Antioxidant effects of the cistanche lignan deoxyschisandrin (4, 12, and 36 mg/kg i.g. for 14 days) were investigated on the amyloid-beta (1-42) Aβ(1-42)-induced memory impairment model in mice [46]. Deoxyschisandrin improved Aβ(1-42)-induced short-term and spatial memory impairments assessed using the Y-maze and water maze tests. In the cerebral cortex and hippocampus of mice, deoxyschisandrin restored the suppression of SOD and GPx activities, increased GSH levels and the GSH/oxidized glutathione (GSSG) ratio, and decreased MDA and GSSG levels [46], thus alleviating cognitive decline in Alzheimer’s disease [46]. The effects of cistanche lignan schizandra C (15 µg/kg or 150 µg/kg/day for fifive days in the lateral cerebral ventricles using stereotaxically implanted cannula) on pathological changes and memory impairment were evaluated in the Aβ(1- 42)-induced Alzheimer’s disease model in mice [47]. Schisandrin C restored cognitive functions and decreased neuronal injury by inhibiting total cholinesterase, enhancing SOD and GPx activities and increasing GSH levels in the hippocampus and cerebral cortex [47]. In the scopolamine-treated mice model, schisanthenol (10, 30, 100 mg/kg/day i.p. for seven days) improved learning and memory ability assessed by the Morris water maze test [48]. In mice, hippocampus schisanthenol enhanced the activity of SOD and GPx, while it decreased the content of MDA and acetylcholinesterase [48].

cistanche bioactive compounds were hepatoprotective in various liver intoxication models [1,2]. In in vitro studies, cistanche lignans schisanthenol, schisandrin B, and schisandrin C at a concentration of 1 mM suppressed iron/cysteine induced lipid peroxidation, assessed by a decrease in MDA formation in rat liver microsomes, and did so more effectively than vitamin E [49]. In AML12 hepatocytes, schisandrin B (15 µM) could induce glutathione antioxidant response [50]. cistanche extract and its active compound—schisandrin B—protected against carbon tetrachloride-induced hepatotoxicity by increasing mitochondrial GSH levels and enhancing activities of GR, GPx, and glutathione S-transferases in carbon tetrachloride-intoxicated mice [51–53]. Schisandrin B (3 mmol/kg/day p.o. for three days) was also protective in the carbon tetrachloride-induced mice hepatotoxicity model by increasing the hepatic vitamin C and vitamin E levels, as well as mitochondrial GSH levels [54]. Lignan-enriched cistanche extract ameliorated the hepatic antioxidant/detoxification system in rats after aflatoxin beta 1 or cadmium chloride challenge by increasing hepatic GSH levels and hepatic GR, and glutathione S-transferase activities [55]. In addition, 5-hydroxymethyl-2-furfural isolated from cistanche (7.5, 15, and 30 mg/kg p.o. for seven days) was hepatoprotective in the acute alcohol-induced liver oxidative injury model in mice by decreasing the levels of MDA and increasing CAT, GPx, and SOD activities in liver tissue [56].

cistanche extract and its active constituent schisandrin B exerted cardioprotective effects by enhancing the heart antioxidant defense system [57–61]. cistanche extract protected from adriamycin-induced cardiotoxicity in rats by decreasing MDA levels and increasing the activities of myocardial GPx and SOD [60]. Lignan-enriched cistanche extract protected heart from oxidative damage in an in vivo model of myocardial infarction and an ex vivo model of myocardial ischemia-reperfusion injury in rats [61]. Moreover, schizandra B and C (10–30 µM), but not schisandrin A, stimulated the cytochrome P-450-catalysed NADPH oxidation reaction in in vitro studies of rat heart microsomes and/or ROS production in rat hearts (a single dose of 1.2 mmol/kg), resulting in the increase in mitochondrial GSH levels during, thus protecting against ischemia/reperfusion injury [59]. 

During doxorubicin-induced cardiomyopathy in mice, schisandrin B (25– 100 mg/kg/day per os for fifive days) reduced lipid peroxidation, prevented nitrotyrosine formation and suppressed metalloproteinase activation in the heart [58]. During myocardial ischemia/reperfusion (40 min + 1 h) in rats, schisandrin B (20 mg/kg) decreased MDA levels and increased total SOD activity, thus attenuating oxidative injury [57]. Schisandrin B in HK-2 cells (2.5–10 µM) and in mice (20 mg/kg/day per os for four weeks) decreased renal MDA levels and enhanced GSH production in cyclosporine A-induced nephrotoxicity [62]. Furthermore, in gentamicin-induced nephrotoxicity in rats, schisandrin B (1–10 mg/kg/day for 15 days) exerted nephroprotective effects by enhancing renal mitochondrial antioxidant status: increasing GSH and alpha-tocopherol levels and activating SOD [26]. cistanche ethanol extract and ethanol-water extract signifificantly decreased the pulmonary MDA levels and increased SOD activity and GSH levels in a guinea pig model of cough hypersensitivity induced by 14 days of cigarette smoke exposure [63].