Kazakh Ziziphora Species as Sources of Bioactive Substances

Ziziphora species represent the prototypical example of the Lamiaceae family. The phytochemicals present in Ziziphora include monoterpenic essential oils, triterpenes and phenolic substances belonging to the flavonoids. In Kazakh traditional medicine, Ziziphora species possess several medicinal uses. In particular, Z. bungeana Lam. and Z. clinopodioides Lam. are used for the treatment of illnesses related to the cardiovascular system or to combat different infections. Unfortunately, the majority of the information about the complex Ziziphora species is only available in Russian and Chinese language, therefore, we decided gather all available information on Kazakhstan Ziziphora, namely its content compounds, medicinal uses and published patents, to draw the attention of scientists to this very interesting plant with high medicinal potential.


Taxonomy of Ziziphora spp. and Their Typical Habitat
Taxonomy of Ziziphora spp. is complicated, as its world population is represented by more than 30 different species. This genus belongs to a very large Lamiaceae family with very similar taxonomic signs. In the flora of Kazakhstan, this genus can be subdivided into six species: Z. bungeana Lam., Z. clinopodioides Lam., Z. interrupta Juz., Z. pamiroalaica Juz., Z. tenuior L., and Z. vichodceviana Tkatsch. ex Tuylaganova. It is not completely clear if Z. bungeana is not simply a subspecies derived from Z. clinopodioides [1].
Ziziphora plants are annual or perennial and herbaceous or sub-shrubby. Their leaves are short petiolate or sub-sessile; the leaf blade is abaxially glandular. Verticillasters are scattered on the leaf axils or crowded in a terminal capitulum; floral leaves occur as large as stem leaves or can be reduced. Ziziphora species blossom from June to September according to the surrounding conditions. The calyx of Ziziphora plants appears to be narrowly cylindric, straight to slightly curved, 13-veined, villous, annulated at throat, obscurely 2-lipped, with the upper lip 3-toothed and lower lip 2-toothed; the teeth are subequal, close together, rarely divergent after anthesis. The corolla limb of the flower is 2-lipped: upper lip straight, margin entire, apex emarginate; lower lip spreading, 3-lobed, and middle lobe narrower than suborbicular lateral lobes. The anterior stamens are fertile, reaching the upper corolla lip, and posterior stamens are rudimentary, short, or absent; anther cells are linear, with only

Patents
There are several patents registered for the Ziziphora species and their application (or application of their isolated compounds) in the area of medicine. Capsules containing the mixture of dried aerial part extract Z. bungeana with Artemisia rupestris and Arctium lappa extracts are used to treat different viral infections of the upper respiratory tract. The patent applications also include the assays on the antipyretic activity of the extract in rabbits, anti-inflammatory activity in rats and antitussic activity in mice. The antiviral activity of the preparation was also evaluated in vitro [30].
The method for obtaining the flavonoid fraction of the Z. bungeana extract was also patented, combining the extraction of the Z. bungeana aerial part with organic solvent, with the dispersion of the extract into aqueous phase and filtration through macroporous resin, further washed with ethanol to get a flavonoid-rich extract [31]. This flavonoid fraction is believed to be useful in the treatment of cardiovascular diseases. Other patents cover the usage of Z. bungeana polyphenol and flavonoid fraction [32]. A flavonoid preparation from Z. bungeana to treat cardiovascular disorders is also patented [33].
Z. clinopodioides is also a component of Chinese traditional medicinal preparation for the treatment of paroxysmal supraventricular tachycardia [34]. Z. clinopodioides essential oil can be used as an oral spray to improve hygiene of oral cavity, suppress inflammation and suppress the growth of oral pathogenic bacteria [35]. Z. clinopodioides essential oil can be used in agriculture. The method for obtaining this oil and its application as an anti-fungal preparation against plant pathogenic fungus Sclerotinia sclerotiorum was patented [36]. The HPLC fingerprint for Z. clinopodioides compounds has also been developed using reversed-phase chromatography of diosmin (7), linarin (8) and pulegone (53) [37].
The antioxidant activity of flavonoid substances depends on the arrangement of the functionalities on the skeleton. Especially, the substitution and number of hydroxyl groups affects the antioxidant activity mediated by radical scavenging and metal ion chelation. As the substitution of Ziziphora-isolated flavonoids is not entirely favourable for scavenging and chelation, the antioxidant effect may be more related with suppression of ROS formation either by inhibition of enzymes or by upregulation or protection of antioxidant defences. Flavonoids contribute to ROS generation inhibition by the affection of the enzymes involved in their production, like microsomal monooxygenase, glutathione S-transferase, mitochondrial succinoxidase, NADH oxidase, and others. The antioxidant activity of Z. clinopodioides was tested by several methods (DPPH, superoxide, and hydroxyl radical scavenging activity). Given the high polyphenol and flavonoid content, the greatest activity was observed in ethyl acetate extract [5]. Monoterpenic glucoside shizonepetoside A (83) and simple flavonoids apigenin (1), luteolin (3) and diosmetin (6) showed potent inhibitory effects on NO production. The stereochemistry of monoterpenic glucosides is important for this effect according to these results [4]. Vasorelaxant activity was shown by those Z. clinopodioides extracts that had high concentration of polyphenolic substances [135]. The mechanism of its vasorelaxant action was also elucidated. The bioactivity guided separation of CH 2 Cl 2 part of a hydroalcoholic extract of the whole plant, using an in vitro model of rat-isolated thoracic aortic rings led to isolation of several compounds, from which apigenin (1) and chrysin (2) showed the greatest activity [39]. Therefore, some structure-activity relationships can be assumed: the presence of 4 1 -hydroxy group of flavonoid, no methyl substitution at C-4 1 and absence of continual substitution at positions 5, 6 and 7 of the flavonoid skeleton [39]. These results should be interpreted carefully, as these tests were carried out ex vivo on normal rat aortas, and differences can be observed after application of compounds or extracts to hypertonic animals or human.
In general, lipophilic flavonoids (flavonoids aglycons or methoxylated and prenylated flavonoids) are synthesized by plants as a part of defence against microbial infection; therefore, they can be used for antimicrobial therapy in humans. Lipophilic flavonoids isolated from Ziziphora species like chrysin (2), acacetin (5) or thymonin (4) have antimicrobial effects and are components of, for example, propolis, a well-known antimicrobial active material [50,61]. Antibacterial flavonoids probably possess multiple cellular targets rather than one specific site of action. One of their actions at the molecular level is to form a complex with proteins through nonspecific forces such as hydrogen bonding and hydrophobic effects as well as by covalent bond formation. Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins, and others [136]. Lipophilic flavonoids can also kill microbes by causing disruption of the microbial membranes [136]. Therefore, the presence of a number of lipophilic flavonoids can contribute to overall antibacterial effect of the traditional medicinal usage of Ziziphora extracts.
As it is well known, inflammation is a normal biological process in response to tissue injury, microbial pathogen infection, and chemical irritation. Inflammation is initiated by migration of immune cells from the blood vessels and release of mediators at the site of damage. This process is followed by further recruitment of inflammatory cells and release of reactive oxygen and nitrogen species and pro-inflammatory cytokines to combat the cause of inflammation, and later to repair caused damage. Acute inflammatory process is rapid and self-limiting, but prolonged inflammation triggers chronic disorders. Natural products are often used to combat diseases connected with chronic inflammation [137,138]. The effectiveness of methanolic extract obtained from Z. clinopodioides for treating inflammatory bowel disease was tested in dextran sulphate-induced colitis model in mice. The parameters of inflammatory process were observed and it was found that the TNF-α level and NO level were decreased and level of antioxidative defence was restored to almost normal level [23]. Ziziphora is relatively rich in flavonoids, which can be considered responsible for the anti-inflammatory potential of this plant. Flavonoids like apigenin (1) [46], luteolin (3) [53,54], diosmin (7) [82], its aglycone diosmetin (6) [79] and linarin (8) [88,89] are reported to possess anti-inflammatory effects.

Triterpenes and Steroids
There is not much information about triterpenes obtained from Ziziphora species, however, their presence is confirmed and some unpublished results showed their relatively high concentrations. The main triterpenic compounds identified till date in Ziziphora spp. are oleanolic acid (26), ursolic acid (27) and maslinic acid (28), together with daucosterol (29) as a representative of plant steroids. The bioactivity of all these compounds was well reviewed (with the exception of 29) [139,140].
Oleanolic acid (26) and maslinic acid (28) are representatives of β-amyrin type of pentacyclic triterpenes with the carboxyl group at position C-17 of the triterpenic skeleton. Both these compounds are relatively abundant in nature and are active components of many plants with medicinal properties. Oleanolic (26) and maslinic (28) acids and their derivatives are often used as components in medical drugs with effect on the cardiovascular system. These compounds help combat different so-called "civilization" diseases, for example cardiovascular diseases including atherosclerosis and diabetes, and even cancer. This could be because they have anti-inflammatory and antioxidative properties, and both cytoprotective and cytotoxic activity depending on the conditions and type of cells. Albeit, their activity is relatively indistinctive, and the multiple potentials of these triterpenes makes them good candidates for semi-synthesis and synthesis of potent drugs [141]. Ursolic acid (27) is an α-amyrin type of triterpene, again with carboxylic function at C-17. Similarly to oleanolic (26) and maslinic (28) acid, it can be isolated from several plant species with potent medicinal properties [142]. Similar to previously mentioned triterpenic acids, it shows activities beneficial in the treatment of civilization diseases like for example cancer, cardiovascular diseases or chronic inflammations [142].
Daucosterol (29) is a natural phytosterol-a glucoside derived form β-sitosterol. As we did not find relevant information about its bioactivity, we tried to summarise its effects in Table 2. Several activities of daucosterol (29) are again in accordance with therapeutic potential of Ziziphora species observed both in folk medicine and scientific studies. The anti-inflammatory effect of daucosterol (29) was observed both in vitro and in vivo [152,153] and a 5-LOX inhibitory effect was observed [154]. Daucosterol (29) also acts as scavenger of free radicals in vitro [155] and as an antioxidant [156] and it inhibits nitric oxide production in LPS-activated RAW264.7 cells [157]. Furthermore, 29 showed antiproliferative [164,166] and cytotoxic [167] activity against different cancer cell lines; it induces apoptosis [165] and inhibits MDA-MB-231 cancer cell migration [169]. Some antimicrobial activities of 29 were observed, mainly against E. coli, S. aureus and H. pylori. The inhibition of H. pylori growth can be beneficial in the treatment of gastric ulcer lesions, because daucosterol (29)-mediated suppression of HCl/ethanol-induced gastric lesions was observed by Jeong et al. [177].

O H
Ar-turmerone (101) Z. tenuior [94] Significant repellent action against Sitophilus zeamais and toxic effect against Spodoptera frugiperda [413]; insecticidal activities against Nilaparvata lugens and Plutella xylostella [414] Inhibition of platelet aggregation induced by collagen (IC50, 14.4. µM) and arachidonic acid (IC50, 43.6. µM) and no effect on PAF and thrombin-induced platelet aggregation on washed rabbit platelets [415]; anti-inflammatory effects through blocking of NF-κB, JNK and p38 MAPK signalling pathways in amyloid β-stimulated microglia [416] Review on activity [417] O Essential oils are almost always complex mixtures of numerous substances, and therefore their biological effects are often described as the result of a synergism of all molecules or they mirror major activities of molecules present at the highest concentrations [418]. Moreover, the synergistic action is beneficial because the bacteria can undergo adaptation to maintain their membrane functionality in the presence of sub-inhibitory concentrations of antibacterial compounds and the resistance can occur, but the complex action of essential oil can help suppress this resistance [419].
Therefore, only the biological activities of essential oils in their entirety or of their main compounds have usually been evaluated. There are some reports about bioactivities of Ziziphora essential oils, mainly connected with evaluation of antibacterial activity. Moreover, antioxidant properties and anti-inflammatory effect was evaluated using different methods. 5-LOX was inhibited by Z. clinopodioides esstential oil (could be due to the presence of compounds structurally related to fatty acids serving as substrate of LOX) [420].
Generally, the major compounds reflect the biophysical and biological characteristics of the parent essential oils quite well (as visible for example for Origanum oil and carvacrol (73) [421]), and the exhibition of their effects depends on their concentration [422,423]. The very complex mixture of compounds present in essential oil also strongly affects the smell, thickness, texture, colour and cell penetration [424], lipophilic or hydrophilic attraction and fixation on cell walls and membranes, and cellular distribution [418]. Therefore, it is sometimes better to analyse the activity of the entire essential oil and compare it with the activity of pure main components. However, some reports highlight the antagonism of single components of essential oil [281,425], so the information about the activity of pure compounds could be useful. As visible from Table 3, we tried to summarize all information about biological effects of compounds present in Ziziphora essential oils available in recent literature, but for some compounds the information is missing or it is scarce.
Reports on the essential oils of different Ziziphora species often discuss their antibacterial activity. The essential oils obtained from different Kazakh Ziziphora species are generally rich in oxygenated monoterpenes (see Table 3); their antibacterial effect can be attributed to the presence of these compounds; however, this effect is not the consequence of the presence of oxygenated monoterpenes only. Z. clinopodioides essential oils were found to be effective against both Gram-negative and Gram-positive bacterial species [184,426]. The presence of thymol (62) could be responsible for the antibacterial activity. The activity of Z. tenuior essential oil was lower [426]. Similar results have been presented by Salehi et al. [21], showing good activity of Z clinopodioides subsp. rigida essential oil against several bacterial strains (with the exception of insensitive P. aeruginosa). Thymol (62) and pulegone (53) showed at least partial responsibility for the antibacterial effects of these materials. Also, the assays carried out on Z. clinopodioides subsp. bungeana essential oils showed activity against both Gram-positive and negative bacterial species, and pulegone (53) and 1,8-cineol (72) were assigned as compounds responsible for the effect [201]. The high concentration of pulegone (53) is mentioned when the antimicrobial activity of essential oils is analysed: it showed activity especially against C. albicans and S. typhimurium. C. albicans was found to be susceptible to pulegone (53), which was found to be twice as effective as nystatin [427,428].
In other test, the antibacterial activity of essential oil and methanolic extract from Z. clinopodioides was compared using 52 Gram-positive and Gram-negative bacterial species, with disc diffusion method [11]. Both tested materials varied in level of activity, with much higher activity of essential oil. Pulegone (53), limonene (38), and piperitone (45) appeared to be the most active substances; however, further information about antibacterial activity of these compounds is not abundant.
We attempted to summarise compounds found in the literature with the antibacterial effect of Ziziphora essential oils and, if possible, their underlying mechanisms (Table 3). Carvacrol (73) and thymol (62) are placed in the first place, as their antibacterial and antiseptic effect is well known [429]. Their synergic action has been described previously and is well reviewed [369]. Carvacrol (73) acts on B. cereus via depletion of intracellular ATP pool, changes the membrane potential and increase the permeability of membrane for protons and potassium. Carvacrol (73) integrates into the lipidic monolayer of the cell membrane, changes its fluidity and damaging its functions [430]. There are evidences of other mechanisms of antibacterial effect, such as interaction with DNA. Moreover, application of carvacrol (73) has been found to inhibit the formation of bacterial biofilm, which is one of the mechanisms of bacterial resistance [369].
Thymol (62), an aromatic p-menthane type monoterpene phenol, isomeric to carvacrol (73), is established as a good antimicrobial agent, interacting both with outer and inner cytoplasmic cell membranes via incorporation of the polar head group region into the lipid bilayer. This interaction changes the properties of the cell membrane and leads to its increased permeability/ disintegration [431][432][433]. Moreover, thymol (62) can also up-or down-regulate the genes encoding the outer membrane protein synthesis. Beside this, it is able to inhibit the enzymes involved in protection against thermal stress, to affect the synthesis of ATP or to alter citric acid metabolic pathways [434,435].
As mentioned above, carvacrol (73) and thymol (62) exert a synergic effect, similar to many other combinations of components of essential oils against different common human pathogens (carvacrol/thymol (73/62), terpinene-4-ol/myrcene (70/31), carvacrol/p-cymene (73), eugenol/thymol (25/62), eugenol/carvacrol (25/73), cinnamaldehyde/eugenol (106/25), citronellol/geraniol and others) [436][437][438]. The synergic action of p-cymene and carvacrol (73) combination is based on the high affinity of p-cymene to the cytoplasmic membrane and its bonding to the membrane causes its expansion, altering its potential and resulting in its higher sensitivity to the action of carvacrol (73) [358]. Some of these combinations of compounds with synergic activities are also present in Ziziphora essential oils. The mechanism of thymol (62) and carvacrol (73) synergism was also elucidated and reviewed [132,439]; however, the mechanistic studies describing the mechanisms of synergy are relatively scarce. Owing to their hydrophobic nature, 73 and 62 interact with the lipid bilayer of cytoplasmic membranes, causing loss of integrity and leakage of cellular material. This effect can, in general, increase the permeability of the membrane to other antimicrobial compounds by general disintegration of the membrane or by formation of a large number of pores.
Of note, essential oil components of the thymol (61) and carvacrol (72) type can act as antagonists, as several essential oils showed lower activity than their single monoterpenic components [281]. The review of Bassolé and Juliani [439] showed some examples of synergic, additive or even antagonistic activity of well-known components of essential oils in different bacterial species.
Carvacrol (72) and thymol (61) are often mentioned as inhibitors of growth of food-borne pathogens. These pathogens are represented for example by different strains of Salmonella, Shigella, E. coli or Clostridium. The activity of the essential oils of Z. tenuior and Z. clinopodioides against food-borne bacteria has been proven by Aliakbarlu and Shameli [426]. Together with the results of experiments on the antiradical activity of Z. clinopodioides essential oil, which showed better activity than Z. tenuior [426], Z. clinopodioides essential oils can be seen as promising food preservatives. This observation is supported by some other reports that examined the single components identified in Ziziphora essential oils ( Table 3).
Several components of Ziziphora essential oils were tested and found to possess antioxidant activity, proven in both in vitro and in vivo assays. For example, monoterpenic α-terpinene (36), terpinolene (37) [217,218], thymol (62) [293], borneol (67) [324], carvacrol (73) [273,294,297,299], and sesquiterpenic caryophylene (91) [392] were found to possess antioxidant activity. The antioxidant activity of Lamiaceae essential oils is known, so they can be used in their entirety or their single compounds can be used as food preservatives; moreover, their antibacterial activity and relative non-toxicity makes them more beneficial than some synthetic antioxidants.

Potential Toxicity
Monoterpenic pulegone (53) is present in many Lamiaceae plants. It is commonly connected with potential toxic effect of so called pennyroyal oil. In high doses, it can cause hepatic failure, central nervous system toxicity, gastritis, renal and pulmonary toxicity, and, in very serious cases, death [451,452]. Assays carried out on mice showed its hepatotoxicity and pulmonary toxicity [451,453]. The toxic potential of pulegone (53) is connected to its extensive metabolism in liver, which includes its oxidation to menthofuran, p-cresol and other compounds. These compounds can be further metabolized and cause depletion of glutathione; then, they can covalently bind to proteins and modify their function, causing cell injury [454].

Conclusions
The traditional medicine of Kazakhstan uses Ziziphora species (Lamiaceae) to combat several diseases. Especially, Z. bungeana Lam. and Z. clinopodioides Lam. are used for the treatment of illnesses connected with cardiovascular system or to combat different infections. We gathered information about four Kazakh Ziziphora species, their traditional utilization and the compounds identified in extracts obtained from these plants. This review presented information about each compound and their bioactivities. We can conclude that as a typical example of the Lamiaceae family, phytochemicals present in Ziziphora are represented especially by monoterpenic essential oil, phenolic substances belonging to the flavonoids and phenolic acids, and triterpenes. The presence of these particular compounds with confirmed activity can be seen as proof of the traditional use and validation of numerous patent applications. We hope that the review on the compounds isolated from Ziziphora, their medicinal uses and published patents will draw the attention of scientists to this very interesting plant with high medicinal potential.