Bioactivity of Phytosterols and Their Production in Plant in Vitro Cultures

Review pubs.acs.org/JAFC

Bioactivity of Phytosterols and Their Production in Plant in Vitro Cultures Begoña Miras-Moreno, Ana Belén Sabater-Jara, M. A. Pedreño, and Lorena Almagro* Department of Plant Biology, Faculty of Biology, University of Murcia, Campus de Espinardo, E-30100 Murcia, Spain ABSTRACT: Phytosterols are a kind of plant metabolite belonging to the triterpene family. These compounds are essential biomolecules for human health, and so they must be taken from foods. β-Sitosterol, campesterol, and stigmasterol are the main phytosterols found in plants. Phytosterols have beneficial effects on human health since they are able to reduce plasma cholesterol levels and have antiinflammatory, antidiabetic, and anticancer activities. However, there are many difficulties in obtaining them, since the levels of these compounds produced from plant raw materials are low and their chemical synthesis is not economically profitable for commercial exploitation. A biotechnological alternative for their production is the use of plant cell and hairy root cultures. This review is focused on the biosynthesis of phytosterols and their function in both plants and humans as well as the different biotechnological strategies to increase phytosterol biosynthesis. Special attention is given to describing new methodologies based on the use of recombinant DNA technology to increase the levels of phytosterols. KEYWORDS: biosynthethic pathway, in vitro cultures, phytosterols

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INTRODUCTION Phytosterols are thoroughly widespread in plants and are similar to cholesterol in terms of physiological functions and structure. Phytosterols differ from cholesterol (Figure 1) by

beneficial effects on human health since they have hypocholesterolemic capacity, and they can also act as antidiabetic, anticancer, and antiinflammatory agents.4 Due to the high value of these compounds, new strategies have been designed for increasing the production of these bioactive compounds since phytosterols are accumulated in relatively low amounts in plants. In this way, plant in vitro cultures under elicitation could be a valuable alternative source for the production of phytosterols. In this review, we describe the biosynthesis and bioactivity of phytosterols and the different strategies to increase their accumulation in plant in vitro cultures including selection of in vitro plant cultures, the stage of cell differentiation of these cultures, optimization of the culture medium, elicitation, and the use of recombinant DNA technology on different plant tissues.

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THE BIOSYNTHETIC PATHWAY OF PHYTOSTEROLS A pair of isomeric C5 diphosphates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), constitutes the two initial precursors of isoprenoid biosynthetic pathway (Figure 2). These C5 units are mainly generated through the cytosolic mevalonate (MVA) pathway, which is the main route for the production of farnesyl diphosphate (FPP, C-15), which, in turn, is transformed into squalene (C-30), the central precursor for all phytosterols.5 These C5 units might also be provided from methylerythritol 4-phosphate under specific circumstances as it was described by Opitz et al.6 in feeding experiments with stable isotope-labeled precursors.

Figure 1. Chemical structures of cholesterol (A), campesterol (B), βsitosterol (C), and stigmasterol (D).

having a methyl or ethyl group at C-24. Phytosterols are found in plant tissues as free alcohols and glycosides as well. Also, they form esters when they are combined with ferulic acid, fatty acids, or p-coumaric acid. The most important phytosterols, βsitosterol (C-29 carbon skeleton), campesterol (C-28), and stigmasterol (C-29), contribute up to 98% of all the phytosterols found in plants (Figure 1). As phytosterols are not synthesized in the human body, they must be consumed from foods. The main food sources of these compounds are vegetable oils, nuts, cereals, and other vegetables.1 Phytosterols participate in essential cellular processes since they modulate permeability and fluidity of membranes. In addition, they are precursors of steroid hormones and are involved in plant defense.2,3 Besides, phytosterols have © 2016 American Chemical Society

Received: Revised: Accepted: Published: 7049

May 24, 2016 September 6, 2016 September 11, 2016 September 12, 2016 DOI: 10.1021/acs.jafc.6b02345 J. Agric. Food Chem. 2016, 64, 7049−7058

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Journal of Agricultural and Food Chemistry

Figure 2. Simplified metabolic pathway of phytosterol in plants. HMGR: 3-hydroxy-3-methylglutaryl-coenzyme A reductase. MVA: mevalonic acid. IPP: isopentenyl pyrophosphate. DMAPP: dimethylallyl diphosphate. GPP: geranyl diphosphate. FPP: farnesyl diphosphate. FPPS: farnesyl diphosphate synthase. SS: squalene synthase. SO: squalene epoxidase. CAS: cycloartenol synthase.

function and overexpressors) showed that the lanosterol pathway also contributes about 1.5% of total sterol biosynthesis in plants.9

Cytoplasmic MVA pathway involves the binding of acetylCoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutarylCoA,5 which is the precursor of MVA formed by the enzyme 3hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR).7 Then, MVA is transformed to mevalonate 5-diphosphate (MVAPP), which is converted into IPP by the action of diphosphomevalonate decarboxylase.8 Three C5 units are condensed to produce FPP (C-15) by the enzyme FPP synthase (FPPS).9 Studies have been devoted to find out the specific biosynthetic pathway of phytosterols, but it is still difficult because some enzymes of their biosynthetic pathway remain unknown. The two first steps in the phytosterol biosynthesis involves the transformation of FPP to squalene and the formation of 2,3-oxidosqualene in two reactions catalyzed by squalene synthase (SS) and epoxidase (SO), respectively. Then, cycloartenol synthase (CAS) produces the cyclization of 2,3oxidosqualene to form cycloartenol (Figure 2). Cycloartenol is then methylated, resulting in 24-methylene cycloartanol, which is converted into 24-methylene lophenol through several enzymatic reactions. The next step is the transformation of 24-methylene lophenol into 24-ethylene lophenol (Figure 2), which is the precursor of avenasterol, giving rise to fucosterol, β-sitosterol, and stigmasterol, consecutively. The other branch of this pathway consists of the conversion of 24-methylene lophenol to campesterol through four enzymatic steps (Figure 2). Although plants mainly use the cycloartenol pathway to biosynthesize sterols,10 studies employing Arabidopsis lines with mutations in lanosterol synthase encoding gene (loss-of-

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FUNCTIONS OF PHYTOSTEROLS IN PLANTS Phytosterols Modulate the Physicochemical Properties of Plant Membranes. Phytosterols are essential membrane compounds that alter the biophysical properties of membranes like, for example, fluidity and permeability.11 Phytosterols are necessary for proper vesicle trafficking and interacting with integral proteins which regulate the physical state of the lipid bilayer and phospholipids.10 Phytosterols are also able to regulate the activity of the plasma membrane H+ATPase in maize roots.12 Additionally, phytosterols can also change the lipid content in the bilayer by inducing alterations in the activity of some proteins associated with the plasma membrane.13 In this way, some studies have suggested the existence of membrane microdomains enriched in sphingolipids and phytosterols that are key compounds in the recruitment of molecular components involved in the intracellular signaling pathways related to the defense.14 On the other hand, campesterol is a precursor of brassinosteroid biosynthesis, which modulates many physiological activities involved in plant development and defense.15−17 Plant Defense. Phytosterols have been related to plant defense responses against various types of stress both biotic and abiotic.18,19 In fact, Wang et al.20 showed that the overexpression of the enzyme HMGR, which is responsible of the formation of the precursors for the biosynthesis of phytosterols, could be involved in increasing resistance to Botrytis cinerea in 7050

DOI: 10.1021/acs.jafc.6b02345 J. Agric. Food Chem. 2016, 64, 7049−7058

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Antidiabetic Effects of Phytosterols. Although phytosterols have been reported as compounds with antidiabetic effect, further clinical evidence is necessary to assess this function. In this way, Misawa et al.31 analyzed the effect of lophenol and cycloartanol, obtained from Aloe vera, in obese animal models of type II diabetes, Zucker diabetic fatty rats. Male rats were fed with lophenol and cycloartanol at 25 g/(kg day) for 44 days. The presence of these sterols in male rats decreased the hyperglycemia, and glucose levels in blood were 39.6 and 37.2% lower than those in the control, in the presence of lophenol and cycloartanol, respectively. Misawa et al.32 showed that the administration of lophenol and cycloartenol in Zucker diabetic fatty rats decreased the levels of the random and fasting blood glucose at 0 (start), 3, and 5 weeks after treatments, and reduced significantly visceral fat weight. In addition, the expression levels of hepatic genes which encode gluconeogenic enzymes (glucose-6-phosphatase and phosphopyruvate carboxykinase) and lipogenic enzymes (acetyl-CoA carboxylase and fatty acid synthase) decreased significantly after the administration of lophenol and cycloartenol. Anticancer Effects of Phytosterols. Phytosterols have been associated as compounds able to inhibit the angiogenesis involved in the progression of tumoral cells.23,33 In fact, Choi et al.34 studied the effect of campesterol obtained from Chrysanthemum coronarium on human umbilical vein endothelial cells, and these authors found a reduction in the proliferation of these cells after treatment with campesterol. Also, β-sitosterol and campesterol decreased the production of prostaglandin E and I, factors which stimulated angiogenesis.35 On the other hand, reactive oxygen species play a key role in carcinogenesis since they provoke DNA damage. However, βsitosterol enhanced the activity of antioxidant enzymes such as glutathione peroxidase and superoxide dismutase in macrophage cultures induced with oxidative stress, suggesting that βsitosterol could prevent carcinogenesis caused by high levels of reactive oxygen species.36 The addition of β-sitosterol also resulted in a reduction of cell growth of prostate cancer lines increasing the levels of prostaglandins and arresting the cell cycle of prostate cancer cells at the G2/M phase.37 Moreover, βsitosterol triggered cytotoxicity and apoptosis in human monoblastic leukemia in a dose-dependent manner. The enhancement in apoptosis triggered by β-sitosterol was related to a downregulation of Bcl-2, the activation of caspase-3, and the degradation of poly(ADP-ribose) polymerase.38 Bioavailability of Phytosterols in Humans. Depending on dietary habits, a person who ingests between 160 and 500 mg/day of phytosterols absorbs less than 20% of campesterol and about 7% of β-sitosterol in the small intestine.39 Like cholesterol, phytosterols are absorbed from the diet in the proximal part of the small intestine, and they are incorporated into micelles.40 The absorption of phytosterols is lower than that of cholesterol due to the selectivity and the return into the intestinal lumen mediated by ABC transporters. Also, there is a close relationship between the low rate of absorption of different phytosterols and their chemical structure as regards the length of lateral chain. In fact, the absorption levels of campesterol, which has a methyl group, are higher than those of β-sitosterol, which has an ethyl group.39 Once absorbed, the phytosterols are esterified with fatty acids by the action of acyl CoA-cholesterol acyl transferase and combined with apolipoproteins, triglycerides, and cholesterol in order to form chylomicrons. Then, these chylomicrons are released to the lymph, and subsequently to the liver, where they will be used

Arabidopsis plants. Likewise, these authors showed that the silencing of SS gene increased the growth of Pseudomonas syringae by enhancing nutrient efflux into the apoplast of Nicotiana benthamiana.20 In Arabidopsis plants, Griebel et al.19 also observed that a cytochrome P450, which encodes C22sterol desaturase transforming β-sitosterol into stigmasterol, was significantly induced in the presence of non-host pathogens.

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FUNCTIONS OF PHYTOSTEROLS IN HUMANS Over the past 20 years, phytosterols have had special interest because of their cholesterol-lowering activities and their effect protective against cardiovascular diseases.21,22 Besides, phytosterols have other important pharmacological properties like antiinflammatory and antidiabetic activities4 and antitumoral effects.23 Phytosterols Reduce Plasma Cholesterol Levels. The cholesterol-lowering activity of phytosterols is mainly due to their structural similarities to cholesterol because they are able to reduce cholesterol absorption in the small intestine.24,25 In fact, phytosterols reduce plasma cholesterol levels since they are more lipophilic than cholesterol itself, and therefore, phytosterols displace to cholesterol from the phospholipid micelles. These micelles are an important vehicle for both transport and absorption of cholesterol in the human body.4,25 Rozner and Garti24 indicated that phytosterols are able to inhibit the enzyme acetyl-CoA acetyltransferase, which catalyzes the conversion of intracellular free cholesterol into cholesteryl esters, by preventing cholesterol esterification and their incorporation into chylomicrons. The unesterified cholesterol is excreted into the intestinal lumen from the intestinal cell through an ABC transporter. Some studies have demonstrated that phytosterols increase the expression of the genes which encode these ABC transporters and trigger cholesterol efflux from cells into the small intestinal lumen.22 Rozner and Garti24 also indicated that cholesterol-esterase can use phytosterol fatty acid esters as substrates due to their structural similarity with cholesterol-esters. Thus, due to the rapid hydrolysis of phytosterol fatty acid esters, phytosterols could displace cholesterol from the micelle, allowing cholesterol and its esters to be accumulated in the oil phase. The absorption of cholesterol is decreased (30−40%) after ingestion of 1.5−2.0 phytosterol g/day. Moreover, a dose of 2.0 g/day provoked a reduction of 10% in plasma LDL-cholesterol, and therefore this has been proposed as optimal dose.4,26 However, doses higher than 2.0 g/day are generally not recommended because phytosterols do not provoke any additional decrease in cholesterol levels and may trigger a reduction of levels of carotenoid and α-tocopherol in plasma.4 Antiinflammatory Effects of Phytosterols. The regulation of the immune system and decrease of inflammatory disorders is another beneficial effect of phytosterols.4 In fact, Bouic and Lamprecht27 suggested that phytosterols could regulate the immune system by increasing the activity of T lymphocytes and natural killer cells which are phenotypically lymphocytes that contribute to innate immunity, adaptive immunity, and placental reproduction. In addition, β-sitosterol was able to increase the effect of vitamin D on the immune function of macrophages,28 and repress mitogen-induced IL-2 production in cells of human Jurkat T in vitro.29 In the same way, Devaraj et al.30 showed that phytosterols decreased the levels of proinflammatory cytokines. 7051


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keratinocyte proliferation and differentiation, and stimulation of hyaluronic synthesis increased the epidermal thickness.51 Moreover, phytosterols both improved skin elasticity and decreased skin roughness in in vivo studies; therefore they improved the structure and function of mature skin. In addition, phytosterols could also be applied for hair care due to their hair softening and conditioning properties.52 For these reasons, skin care products rich in phytosterols could provide moisture, softening, and elasticity to the skin. However, the benefits of phytosterols on skin and hair have not been studied in depth, and they are now emerging. Phytosterol Extraction. Currently, the most frequent process used for the extraction of phytosterols at industrial scale is mostly based on the extraction from two major raw materials, vegetable oils and resins oils (also known as tall oil, ref 53 and references therein). The most common source of phytosterols is a mixture of crude edible oils. The extraction of vegetable oils is performed by methods similar to those used for the extraction of fat using nonpolar solvents such as hexane. Prior to use, the crude vegetable oils must be refined to remove impurities. In this chemical or physical refining process, high temperatures and pressures which increase the cost of the final product are required, although the extraction process is very effective, and allows recovery of up to 90% of total phytosterols contained in the oils. The alternative source of phytosterols is tall oil obtained as byproducts in the manufacture of paper pulp from conifers, ref 53 and references therein. In this case, tall oil is subjected to a refining process and crystallization, which allows the concentration of phytosterols. However, as in the above process, chemicals and high temperatures that provoke the degradation of nonesterified sterols are required, and more than half of the β-sitosterol present in the crude resin oil is destroyed,54 so that the total content of sterols can range between 8 and 20% by weight.53 In addition, these methods of extraction described above produce a large quantity of waste organic solvents that are toxic to both human health and the environment. Therefore, it is desirable to develop new methods and technologies that allow us to obtain phytosterols without generating environmental problems. In this way, supercritical fluid extraction has been used as an efficient technology to extract phytosterols from different plant raw materials.55 This technology has significant advantages over the use of conventional liquid organic solvents for extraction processes, and it is faster and environmentally friendly. More specifically, supercritical CO2 is the most effective method for selective extraction of phytosterols.55 However, the use of supercritical CO2 has the disadvantage of the high cost of high pressure equipment, and also, it implies the use of organic solvents, which sometimes makes the process economically unfeasible, if the compound obtained is not of high added value. Another way to produce these compounds is the use of plant in vitro cultures, which have emerged as a new alternative with great potential. Biotechnological Approaches To Improve Phytosterol Production Using in Vitro Cultures. As we described previously, the strategy commonly used to obtain these bioactive compounds is their extraction from plant raw material. However, the production of phytosterols from plants could lead to the extinction of endangered plant species. Moreover, the biosynthesis of these compounds could be increased in plants when they are treated with stressor agents.56 For these reasons, a large number of strategies to increase phytosterol production using plant in vitro cultures, in

for the synthesis of bile salts or will be incorporated into LDL to transport them to peripheral tissues.23 Those phytosterols that are not used usually are secreted by cells into the blood, released to the liver, and excreted in the bile.40 Safety in the Use and Consumption of Phytosterols. As phytosterols are high value compounds, great effort is being made in order to analyze their possible adverse effects on human health. In this way, Lees et al.41 showed that hypercholesterolemic patients treated with a dose of 3 g of phytosterols/day had high levels of campesterol in serum (4− 21 mg/dL). These results suggested that a high accumulation of phytosterols could induce the occurrence of atherosclerosis. In addition, phytosterols can also be accumulated in the brain. In fact, a high intake of phytosterols in wild-type mice provoked their accumulation in the brain.42 Vanmierlo et al.43 also observed that a phytosterol-enriched diet displayed an increase in phytosterol levels in brain, serum, and liver in mice. Some studies have described a close relationship between high doses of phytosterols (greater than or equal to 2.2 g of free sterols) and a decrease in plasma of carotenoid and αtocopherol levels,44 which have a key role in protecting LDLcholesterol in plasma.25 In fact, Kritchevsky et al.45 showed a dose-dependent decrease of total cholesterol, carotenoid, and LDL-cholesterol in plasma with a high intake of phytosterols. Moreover, a reduction on carotenoid levels in plasma could provoke changes in the activity of antioxidant enzymes like glutathione-S-transferases (GST) which are associated with the prevention of cancer. At the moment, only a small reduction of GST activity after phytosterol intake has been observed.46 These negative effects of phytosterols could be solved by enhancing the intake of foods rich in α-tocopherol and βcarotene to prevent the risk of these side effects as described by Noakes et al.47 in some clinical studies. Functional Foods and Biocosmetics Containing Phytosterols. The beneficial effects of phytosterols on human health have increased the intake of phytosterol-enriched foods. In fact, there are more than 40 patents on functional foods containing phytosterols and more than 15 European marketed products including margarine which has a hypocholesterolemic effect.48 Currently, there is a new social culture about the consumption of functional foods enriched in bioactive compounds like phytosterols, due to an increase of blood cholesterol levels of the western population.49 On the other hand, the use of cosmetics has taken a new course in recent years since consumers are much more interested in the origin, safety, and sustainability of the products they purchase. A new class of personal care products called biocosmetics have arisen through new technologies, considering also the concept of sustainable development.50 In this way, phytosterols are added to various cosmetic products for their properties of moisturizing and regenerating the skin.49 In fact, when they are applied to the human skin, they keep the structural integrity of cell membranes, so that it could improve moisture retention. However, the most striking biological effect of phytosterols is based on their antiinflammatory properties, which could be used in antiaging cosmetic products. Some in vitro clinical studies have shown that phytosterols and polyphenols improved sun-damaged skin, so that these compounds could be used as ingredients in skin care formulations in order to improve skin health. In fact, when apple seed phytosterols were applied in a cutaneous reconstruction, a differential gene expression, related to 7052


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Journal of Agricultural and Food Chemistry Table 1. Strategies To Increase the Production of Phytosterols in Plant in Vitro Cultures material Catharanthus roseus cell cultures Tabernaemontana divaricata cell cultures Apium graveolens cell cultures Morinda citrifolia cell cultures Nicotiana tabacum cell cultures Solanum tuberosum cell cultures nonorganogenic calli from flax hypocotyl shoot organogenic calli from flax hypocotyl embriogenic calli from flax hypocotyl somatic embryos from flax hypocotyl somatic shoot from flax hypocotyl Chenopodium rubrum cell cultures Cissus quadrangularis callus

flax calli Euphorbia characias calli Centella asiatica calli Centella asiatica cell cultures Uncaria tomentosa cell cultures Spartina patens callus flax cell cultures Solanum malacoxylon cell cultures

Solanum lycopersicum cell cultures Capsicum annum cell cultures Daucus carota cell cultures Lemna paucicostata vitroplants flax cell cultures

phytosterol production

refs

B5 medium for 21 days B5 medium for 14 days MS medium/stationary phase

strategy

604.9 μg campesterol/g DW 311.02 μg β-sitosterol/g DW 3.170 μg total phytosterols/g DW

50 50 51

MS MS MS MS MS

2.650 2.320 2.300 1.190 1.320

medium/stationary phase medium/stationary phase medium/stationary phase medium/stationary phase medium with 0.6 mg/L 2,4-D and 1.6 mg/L ZEA for 8 weeks

MS medium with 0.6 mg/L 2,4-D and 1.6 mg/L zeatin for 8 weeks

μg μg μg μg μg

total total total total total

phytosterols/g phytosterols/g phytosterols/g phytosterols/g phytosterols/g

DW DW DW DW DW

51 51 51 51 52

1.480 μg total phytosterols/g DW

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1.450 μg total phytosterols/g DW 2.130 μg total phytosterols/g DW 2.100 μg total phytosterols/g DW 402.5 or 260.1 μg total phytosterols/g DW 272 μg β-sitosterol/g DW 167 μg stigmasterol/g DW 2 weeks of cultivation 257 μg β-sitosterol/g DW 150 μg stigmasterol/g DW 4 weeks of cultivation 262 μg β-sitosterol/g DW 157 μg stigmasterol/g DW MS medium with indole-3-acetic acid (5 mg/L) for 6 weeks 10−12-fold higher than control whithout hormone MS medium with 2,4-D (5 mg/L) for 6 weeks 6−8-fold higher than control whithout hormone MS medium with 2,4-D (0.5 mg/L) + zeatin (0.5 mg/L) for 26 days 400 μg β-sitosterol/g DW MS medium with 2,4-D production of β-sitosterol improved MS medium with 3 mg/L N-(2-chloro-4-pyridyl)-N-phenylurea for 190 μg total phytosterols/g DW 4 weeks elicitation with 100 and 200 μM methyl jasmonate for 30 days 220 and 170 μg total phytosterols/g DW elicitation with pectin for 20 days 120 μg total phytosterols/g DW elicitation with 170 or 340 mmol/L NaCl for 10 weeks decrease of total sterol production 1 mg/L β-glucan for 144 h 192.58 μg total phytosterols/g DW 40 μM (Z)-3-hexenol for 144 h 231.61 μg total phytosterols/g DW benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester for 48 h 130 μg β-sitosterol/g DW 17 μg campesterol/g DW 20 μg stigmasterol/g DW 50 mM CDM for 96 h 30 μg extracellular isofucosterol/g DW 50 mM CDM for 96 h 850 μg extracellular β-sitosterol/g DW 50 mM CDM for 144 h 10.8 mg extracellular total phytosterols/g DW 50 mM CDH for 144 h 4 mg extracellular total phytosterols/g DW 10 μM silver nitrate +100 μM methyl jasmonate for 28 days 1.52 mg total phytosterols/g DW 50 mM CDH + 1 mg/L β-glucan for 144 h 503.88 μg extracellular total phytosterols/g DW 50 mM CDH + 40 μM (Z)-3-hexenol for 144 h 530.81 μg extracellular total phytosterols/g DW 50 mM CDM for 144 h 1.325 μg extracellular total phytosterols/g DW 50 mM CDH for 144 h 259.47 μg extracellular total phytosterols/g DW MS medium with 0.6 mg/L 2,4-D and 1.6 mg/L zeatin for 8 weeks MS medium with 0.6 mg/L 2,4-D and 1.6 mg/L zeatin for 8 weeks MS medium with 0.6 mg/L 2,4-D and 1.6 mg/L zeatin for 8 weeks 11 days or 8 weeks cultivation 6 weeks of cultivation

52 52 52 53 54 54 54 54 54 52 55 56 57 58 62 59 59 63

60 65 61 61 67 59 59 59 59

higher levels of phytosterols.57 Saiman et al.57 analyzed nine Catharanthus roseus cell lines grown in Murashige and Skoog or Gamborg liquid medium. They observed that the maximal production of campesterol (604.90 μg/g dry weight (DW)) was detected in one C. roseus cell line grown for 21 days in B5 medium enriched with 30 g/L sucrose and 1.86 mg/L 1naphthylacetic acid, whereas the maximal production of βsitosterol (approximately 311.02 μg/g DW) was detected in C. roseus cell lines grown for 14 days in B5 medium enriched with 1.86 mg/L 1-naphthylacetic acid and 30 g/L sucrose or 20 g/L glucose.

particular plant cell cultures, represent an alternative system to the extraction from plant raw material. One approach consists of the empirical testing of plant cell material and in vitro cultivation conditions. The selection of highly productive plant cell lines and the elicitation have resulted in an enhancement of phytosterol production. Another strategy is the rational approach in which information about the biological system is used to engineer the phytosterol biosynthetic pathways.56 Selection of Highly Productive Plant Cell Lines. Screening of high-yielding cell cultures has allowed the selection of plant cell cultures which are able to produce 7053


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chloro-4-pyridyl)-N-phenylurea (190 μg/g DW) while the lowest levels were found in those calli grown with 1 mg/L N(2-chloro-4-pyridyl)-N-phenylurea (approximately 150 μg/g DW). Elicitation Strategies To Improve the Accumulation of Phytosterols in Plant in Vitro Cultures. Some strategies have been designed to increase the amount of phytosterols in plant in vitro cultures. In this way, the use of elicitors like methyl jasmonate, pectins, NaCl, β-glucan, (Z)-3-hexenol, benzothiadiazole, cyclodextrins (CDs), and silver nitrate changes the levels of phytosterols.64−68 Plant cell cultures produce plant metabolites in the presence of jasmonates. However, the addition of 100 or 200 μM methyl jasmonate to C. asiatica cell cultures provoked a decrease in total content of phytosterols (approximately 220 and 170 μg/g DW, respectively) compared to control cells (approximately 240 μg/g DW)64 after 30 days of elicitation. In a similar way, the treatment with pectin (obtained from cultures of Trichoderma sp, Pestalotia sp, Epicoccum nigrum, and Alternaria tenuis) did not alter the production of sterols in Uncaria tomentosa cell cultures (approximately 120 μg/g DW).65 Besides, a decrease in the total phytosterol production in callus of Spartina patens was detected by using two different concentrations of NaCl (170 and 340 mmol/L) for 10 weeks.69 However, there are some elicitors which increase the biosynthesis of phytosterols. Thus, the addition of 1 mg/L βglucan, which are oligosaccharides derived from the fungal cell walls, or 40 μM (Z)-3-hexenol, which is important in plant signaling, provoked an important enhancement of intracellular phytosterols in flax cell cultures,66 192.58 ± 61.03 or 231.61 ± 31.64 μg/g DW in the presence of β-glucan or (Z)-3-hexenol, respectively, being 13- and 16-fold higher than control cell cultures (14.53 ± 0.02 μg/g DW), respectively. In addition, βglucan or (Z)-3-hexenol did not provoke any significant differences in cell growth when they were added to flax cell cultures.66 Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester, an effective inducer of systemic acquired resistance, increased the production of phytosterols in Solanum malacoxylon cell cultures.70 In fact, the cellular levels of β-sitosterol, campesterol, and stigmasterol increased at the end of the second day (approximately 130, 17, and 20 μg/g DW, respectively) with regard to the controls (approximately 40, 6, and 5 μg/g DW). On the other hand, CDs, which are well-defined cyclic oligosaccharides, can increase the extracellular phytosterol production in plant cell cultures.71Specifically, β-CDs, which contain 7 glucopyranose residues, have been used for enhancing phytosterol accumulation in plant cell cultures.66−68,72 In fact, the elicitation with β-CDs produces a high extracellular accumulation of phytosterols in plant cell cultures. Therefore, β-CDs act as elicitors of the biosynthesis of phytosterols and as promoters of the secretion of them into the culture medium, allowing both their accumulation and their recovery from the culture medium.73 In fact, the addition of β-CDs to Solanum lycopersicum cv Micro-Tom cell cultures induced a substantial enhancement in the production of isofucosterol after 96 h of treatment (4.6-fold, approximately 30 μg/g DW) while a decrease in cell growth (10%) was observed.67 Capsicum annum cell cultures also accumulated high amounts of β-sitosterol (approximately 850 μg/g DW) after 96 h of treatment with methylated-β-cyclodextrins (CDM).72 In a similar way, a high level of phytosterols was produced at 144 h in Daucus carota cell cultures when they were elicited with CDM (10.8 mg/g

Moreover, Dyas et al.58 observed that the levels of phytosterols obtained depend on plant species. In fact, they observed that the maximal levels of these compounds were detected in cell cultures of Tabernaemontana divaricata (3.170 μg/g DW) > Apium graveolens (2.650 μg/g DW) > Morinda citrifolia (2.320 μg/g DW) > Nicotiana tabacum (2.300 μg/g DW) > Solanum tuberosum (1.190 μg/g DW). The Production of Phytosterols Depends on Both Cell Differentiation Stage and Cell Age of Culture. The content of phytosterols in flax is dependent on the level of cell differentiation.59 Thus, nonorganogenic calli (1.32 mg/g DW), shoot organogenic calli (1.48 mg/g DW), and embryogenic calli (1.45 mg/g DW) contained phytosterol levels higher than those found in dry seeds (0.94 mg/g DW). However, the production of phytosterols was significantly lower than those observed in their respectively derived somatic embryos and shoots (2.13 and 2.1 mg/g DW, respectively) (Table 1).59 On the other hand, the production of phytosterols is also closely related to the cell age of cultures used. In fact, the ratio of the main phytosterols (β-sitosterol, stigmasterol, and campesterol) remained approximately constant in Chenopodium rubrum cell cultures, but their overall amount (402 μg/g DW) was reduced as cell cultures got older (260 μg/g DW).60 In contrast, Sharma et al.61 observed that the total amount of βsitosterol (272 μg/g DW) and stigmasterol (167 μg/g DW) was higher in 6 week old callus of Cissus quadrangularis than those found in their respective callus cultivated for 2 (βsitosterol, 257 μg/g DW, and stigmasterol, 150 μg/g DW) or 4 (β-sitosterol, 262 μg/g DW, and stigmasterol, 157 μg/g DW) weeks.61 Therefore, the production of phytosterols in these plant in vitro cultures is strongly associated with the cell age of culture. Empirical Testing of in Vitro Cultivation Conditions To Increase the Production of Phytosterols. Plant growth regulators alter both the culture growth and production of phytosterols in C. quadrangularis cultures.61 Thus, the best conditions for obtaining callus induction was the use of Murashige and Skoog medium which contained 3% sucrose, 1naphthaleneacetic acid (2.0 mg/L), and 6-benzylaminopurine (0.5 mg/L). However, the maximal levels of β-sitosterol and stigmasterol were identified in the presence of indole-3-acetic acid (5 mg/L) or 2,4-dichlorophenoxyacetic acid (2,4-D, 5 mg/ L) being 10−12-fold and 6−8-fold higher than those detected in control conditions without hormones, respectively. Moreover, Cunha and Ferreira et al.59 analyzed the effects of both 2,4-D (0.5 mg/L) + zeatin (0.5 mg/L) and indole-3-butyric acid (0.6 mg/L) + zeatin (0.5 mg/L) on phytosterol production in flax calli. In both cases, a significant increase in β-sitosterol levels was observed in the presence of 2,4-D (0.5 mg/L) + zeatin (0.5 mg/L) during the first half of the growth period, reaching the maximal value after 26 days of cultivation (400 μg/g DW). In the same way, Fernandes-Ferreira et al.62 also observed an increase in β-sitosterol levels in the presence of 2,4-D in Euphorbia characias calli. The highest efficiency of 2,4-D could be due to the effect of this plant hormone by promoting higher callus growth rates than other auxins. In a similar way, Mangas et al.63 analyzed the effect of different concentration of N-(2-chloro-4-pyridyl)-N-phenylurea on the total content of sterols in nonorganogenic calli of Centella asiatica observing a direct relationship between the levels of this kind of cytokinin and the concentration of phytosterols inside the cells. In fact, the total content of phytosterols was higher in calli grown with 3 mg/L N-(27054


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Journal of Agricultural and Food Chemistry Table 2. Metabolic Engineering To Increase the Production of Phytosterols in Plant in Vitro Cultures material hairy root cultures of Centella asiatica hairy root cultures of Platycodon grandif lorum hairy root cultures of Panax ginseng

adventitious roots of Panax ginseng adventitious roots of Bupleurum falcatum L. somatic embryos of Eleutherococcus senticosus Rupr.

strategy

phytosterol production

overexpression of decarboxylase overexpression of overexpression of overexpression of overexpression of overexpression of

refs 69 70

mevalonate-5-diphosphate

740 μg total phytosterols/g DW 1.6-fold higher in transgenic hairy root lines than in control lines 4.436 μg β-sitosterol/g DW

FPPS SS SS SS + methyl jasmonate SS

3.854 μg β-sitosterol/g DW 600 μg total phytosterols/g DW 270 μg total phytosterols/g DW 285 μg total phytosterols/g DW 3.750 μg total phytosterols/g DW

71 72 73 73 74

overexpression of FPPS overexpression of HMGR

71

metabolites than undifferentiated cell cultures. Thus, Kim et al.76 obtained C. asiatica hairy roots overexpressing FPPS. These C. asiatica transgenic hairy roots produced high levels of squalene, which had a positive effect on the production of phytosterol. In fact, the phytosterol levels in transgenic hairy root lines were approximately 2-fold higher (approximately 740 μg/g DW) than those found in control lines (approximately 335 μg/g DW), indicating that FPPS had a regulatory function in phytosterol biosynthetic pathway (Table 2). The overexpression of HMGR in hairy root cultures of Platycodon grandif lorum also induced an accumulation of phytosterols which was 1.6 times higher in transgenic hairy root lines than in control lines.77 In order to elucidate the role of mevalonate-5-diphosphate decarboxylase and FPPS in triterpene biosynthesis, the genes encoding these enzymes were introduced in P. ginseng hairy roots.78 The transgenic lines overexpressing mevalonate-5diphosphate decarboxylase produced the greatest levels of βsitosterol (4.436 μg/g DW), while the transgenic lines overexpressing FPPS produced lower levels of β-sitosterol (3.854 μg/g DW) compared to nontransformed control roots (Table 2). All these results suggest that hairy root cultures could be a powerful material for increasing the bioproduction of phytosterols. On the other hand, adventitious transgenic roots of Panax ginseng which overexpress the SS gene showed an increased production of phytosterols (600 μg/g DW) compared to those found in the wild type (200 μg/g DW), indicating that SS gene plays an important regulatory role in the biosynthesis of phytosterols. In addition, the production of squalene was higher in actively dividing embryogenic callus of P. ginseng while its production decreased in adventitious roots and plants.79 In a similar way, transgenic lines of Bupleurum falcatum L. adventitious roots overexpressing the SS gene in the sense orientation produced high levels of phytosterols (approximately 270 μg/g DW) but this production decreased in the antisense orientation (approximately 180 μg/g DW). Interestingly, the addition of methyl jasmonate to transgenic lines in the sense orientation induced an increase in the total phytosterol levels (approximately 285 μg/g DW).80 Finally, the production of phytosterols was also studied in transgenic somatic embryos of Eleutherococcus senticosus Rupr. overexpressing an SS gene from P. ginseng using Agrobacteriummediated transformation.81 In this case, the levels of β-sitosterol and stigmasterol (approximately 1.750 and 2.000 μg/g DW, respectively) were significantly enhanced, compared to wild type (approximately 700 and 1.000 μg/g DW, respectively, Table 2).

DW), being approximately 2.7-fold higher than when carrot cells were elicited with hydroxypropylated β-cyclodextrins (CDH) (approximately 4 mg/g DW, Table 1). In addition, the intracellular phytosterol production was substantially lower than those observed in the extracellular medium at 144 h of cultivation (1.08 mg/g DW).68 The joint action of elicitors is a strategy commonly used to enhance the biosynthesis of phytosterols. Indeed, 10 μM silver nitrate and/or 100 μM methyl jasmonate is an efficient strategy to enhance phytosterol biosynthesis in Lemna paucicostata vitroplants. The joint action of both elicitors retarded the growth of L. paucicostata vitroplants, and they increased the levels of phytosterols (1.52 mg/g DW) compared to the control (1.03 mg/g DW).74 Moreover, the combined treatment of methyl jasmonate and CDs was also evaluated on phytosterol production in carrot cell cultures.68 The levels of phytosterols in the presence of both elicitors were 1.5-fold lower than those detected with CDM alone, observing a greater decrease as the concentration of methyl jasmonate increased. Likewise, Almagro et al.66 also showed that CDM-treated flax cells produced high extracellular levels of phytosterols in a dose-dependent manner. In fact, the maximum phytosterol production (1.325 μg/g DW) was obtained with 50 mM CDM after 144 h of treatment (Table 1). In this case, 25 or 50 mM CDM did not alter the cell growth in flax cell cultures. Almagro et al.66 also described that the treatment of flax cell cultures with β-glucan or (Z)-3-hexenol resulted in an increased intracellular phytosterol production (192.58 μg/g DW and 231.61 μg/g DW, respectively). In this case, no significant differences on extracellular phytosterol production were found between CDM alone (1.325 μg/g DW) or in combination with β-glucan (1.278 μg/g DW) or (Z)-3-hexenol (1.507 μg/g DW). However, these authors showed that the amounts of phytosterols were greater when flax cell cultures were treated with 50 mM CDH and β-glucan (503.88 μg/g DW) or (Z)-3hexenol (530.81 μg/g DW) compared to CDH alone (259.47 μg/g DW) (Table 1). Taking into account all results, β-CDs are the best elicitors to obtain phytosterols from plant cell cultures. In this process, βCDs provoke the secretion of phytosterols into the culture medium, allowing both their accumulation and their recovery from the culture medium.75 Metabolic Engineering as an Alternative Strategy To Increase the Phytosterol Production. The culture of hairy roots is also used in order to produce plant metabolites, since these tissues present a high rate of growth in the absence of hormones in the culture medium, and these cultures provide differentiated cells genetically more stable than undifferentiated cells. Also, in some cases, they are able to produce more plant 7055


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(8) Henrikson, C. V.; Smith, P. F. Conversion of mevalonic acid to γ, γ-dimethylallyl pyrophosphate by Mycoplasma. J. Bacteriol. 1966, 92, 701−706. (9) Ohyama, K.; Suzuki, M.; Kikuchi, J.; Saito, K.; Muranaka, T. Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 725−730. (10) Boutté, Y.; Grebe, M. Cellular processes relying on sterol function in plants. Curr. Opin. Plant Biol. 2009, 12, 705−713. (11) Neelakandan, A. K.; Nguyen, H. T.; Kumar, R.; Tran, L. S. P.; Guttikonda, S. K.; Quach, T. N.; Aldrich, D. L.; Nes, W. D.; Nguyen, H. T. Molecular characterization and functional analysis of Glycine max sterol methyl transferase 2 genes involved in plant membrane sterol biosynthesis. Plant Mol. Biol. 2010, 74, 503−518. (12) Grandmougin-Ferjani, A.; Schuler-Muller, I.; Hartmann, M. A. Sterol modulation of the plasma membrane H+-ATPase activity from corn roots reconstituted into soybean lipids. Plant Physiol. 1997, 113, 163−174. (13) Roche, Y.; Gerbeau-Pissot, P.; Buhot, B.; Thomas, D.; Bonneau, L.; Gresti, J.; Mongrand, S.; Perrier-Cornet, J. M.; Simon-Plas, F. Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts. FASEB J. 2008, 22, 3980−3991. (14) Mongrand, S.; Stanislas, T.; Bayer, E. M.; Lherminier, J.; SimonPlas, F. Membrane rafts in plant cells. Trends Plant Sci. 2010, 15, 656− 663. (15) Asami, T.; Nakano, T.; Fujioka, S. Plant brassinosteroid hormones. Vitam. Horm. 2005, 72, 479−504. (16) Gudesblat, G. E.; Russinova, E. Plants grow on brassinosteroids. Curr. Opin. Plant Biol. 2011, 14, 530−537. (17) Fridman, Y.; Savaldi-Goldstein, S. Brassinosteroids in growth control: how, when and where. Plant Sci. 2013, 209, 24−31. (18) Devarenne, T. P.; Ghosh, A.; Chappell, J. Regulation of squalene synthase, a key enzyme of sterol biosynthesis, in tobacco. Plant Physiol. 2002, 129, 1095−1106. (19) Griebel, T.; Zeier, J. A role for β-sitosterol to stigmasterol conversion in plant−pathogen interactions. Plant J. 2010, 63, 254− 268. (20) Wang, K.; Senthil-Kumar, M.; Ryu, C. M.; Kang, L.; Mysore, K. S. Phytosterols play a key role in plant innate immunity against bacterial pathogens by regulating nutrient efflux into the apoplast. Plant Physiol. 2012, 158, 1789−1802. (21) Moreau, R. A.; Whitaker, B. D.; Hicks, K. B. Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses. Prog. Lipid Res. 2002, 41, 457−500. (22) García-Llatas, G.; Rodríguez-Estrada, M. T. Current and new insights on phytosterol oxides in plant sterol-enriched food. Chem. Phys. Lipids 2011, 164, 607−624. (23) Woyengo, T. A.; Ramprasath, V. R.; Jones, P. J. H. Anticancer effects of phytosterols. Eur. J. Clin. Nutr. 2009, 63, 813−820. (24) Rozner, S.; Garti, N. The activity and absorption relationship of cholesterol and phytosterols. Colloids Surf., A 2006, 282, 435−456. (25) Jones, P. J.; AbuMweis, S. S. Phytosterols as functional food ingredients: linkages to cardiovascular disease and cancer. Curr. Opin. Clin. Nutr. Metab. Care 2009, 12, 147−151. (26) Rocha, M.; Bañuls, C.; Bellod, L.; Jover, A.; Victor, V. M.; Hernandez-Mijares, A. A review on the role of phytosterols: new insights into cardiovascular risk. Curr. Pharm. Des. 2011, 17, 4061− 4075. (27) Bouic, P. J.; Lamprecht, J. H. Plant sterols and sterolins: a review of their immune-modulating properties. Altern. Med. Rev. 1999, 4, 170−177. (28) Alappat, L.; Valerio, M.; Awad, A. B. Effect of vitamin D and βsitosterol on immune function of macrophages. Int. Immunopharmacol. 2010, 10, 1390−1396. (29) Aherne, S. A.; O’Brien, N. M. Modulation of cytokine production by plant sterols in stimulated human Jurkat T cells. Mol. Nutr. Food Res. 2008, 52, 664−673.

In conclusion, phytosterols are biologically active compounds with a high cholesterol-lowering activity in humans. In fact, phytosterols can also reduce plasma cholesterol levels due to the fact that they are more lipophilic than cholesterol itself, and therefore phytosterols can displace to cholesterol from the phospholipid micelles. Moreover, phytosterols also have antiinflammatory, antidiabetic, and anticancer effects. As a result of their beneficial properties, the demand for these compounds has enhanced, and therefore, new alternative strategies to produce them from natural sources have been developed. Through elicitation, plant cell cultures might provide adequate amounts of phytosterols without manipulating them with genetic engineering. However, metabolic engineering also appears to increase the production of these compounds in hairy and adventitious roots in in vitro cultures. In the future, scale-up to industrial processes will have to be achieved and optimized, in order to develop a sustainable and environmentally friendly production of phytosterols.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +34868884904. Fax: +34868883963. Funding

This work has been supported by the Ministerio de Economiá y Competitividad (BIO2014-51861-R) and the Fundación Seneca-Agencia de Ciencia y Tecnologiá de la Región de Murcia (No. 19876/GERM/15). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED 2,4-D, 2,4-dichlorophenoxyacetic acid; CAS, cycloartenol synthase; DMAPP, dimethylallyl diphosphate; DW, dry weight; FPP, farnesyl diphosphate; FPPS, FPP synthase; GPP, geranyl diphosphate; GST, glutathione-S-transferases; CDH, hydroxypropylated β-cyclodextrins; IPP, isopentenyl diphosphate; CDM, methylated-β-cyclodextrins; MVAPP, mevalonate 5diphosphate; MVA, mevalonic acid pathway; SO, squalene epoxidase; SS, squalene synthase

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REFERENCES

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