Raphanus sativus (Radish): Their Chemistry and Biology

Leaves and roots of Raphanus sativus have been used in various parts of the world to treat cancer and as antimicrobial and antiviral agents. The phytochemistry and pharmacology of this radish is reviewed. The structures of the compounds isolated and identified are listed and aspects of their chemistry and pharmacology are discussed. The compounds are grouped according to structural classes.


INTRODUCTION
The plant family of Cruciferae contains many important vegetables of economic importance. Raphanus sativus L. is originally from Europe and Asia. It grows in temperate climates at altitudes between 190 and 1240 m. It is 30-90 cm high and its roots are thick and of various sizes, forms, and colors (see Fig. 1). They are edible with a pungent taste. Salted radish roots (Takuan), which are consumed in the amount of about 500,000 tons/year in Japan, are essentially one of the traditional Japanese foods. The salted radish roots have a characteristic yellow color, which generates during storage.
This specie is used popularly to treat liver and respiratory illnesses [1]. The antibiotic activity of its extracts and its time persistence validates its effectiveness in microbial sickness as reported in traditional medicine. The root's juice showed antimicrobial activity against Bacillus subtilis, Pseudomonas aeruginosa, and Salmonella thyphosa. The ethanolic and aqueous extracts showed activity against Streptococcus mutans and Candida albicans. Aqueous extract of the whole plant presents activity against Sarcinia lutea and Staphylococcus epidermidis [2]. Aqueous extract of the leaves showed antiviral effect against influenza virus. Aqueous extract of the roots showed antimutagenic activity against Salmonella typhimurium TA98 and TA100. In this review, the metabolites produced by R. sativus are presented according to structural classes. (See also Tables 1 through 10 at the end of this paper.)

Enzymes
A number of enzymes are present in both the cytoplasm and the cell wall, and in some cases it has been shown that the cell wall isozymes differ from those of the cytoplasmic [19]. When radish seedlings are grown in the dark, β-fructosidase (βF) first accumulates in the cytoplasm, then slowly increases in the cell wall. Charge heterogeneity of cytoplasmic enzymes resides in the polypeptides, while the formation of the basic cell wall occurs as a result of post-translational modifications that can be inhibited by tunicamycin [20].
Cysteine synthase (EC 4.2.99.8) was purified to near homogeneity (275-fold) in 11.5% yield from mature roots. It was relatively stable, retaining most of its activity in standing for several days at room temperature [21].
Catalase and glutathione reductase activities increased considerably in the root and leaves after 24-h exposure to cadmium, indicating a direct correlation with Cd accumulation. PAGE enzyme activity staining revealed several superoxide dismutase isoenzymes in leaves. The main response may be via activation of ascorbate-glutathione cycle for removal of hydrogen peroxide or to ensure availability of glutathione for synthesis of Cd-binding proteins [26].
A γ-glutamyl transpeptidase was found. It catalyzed the release of CySH-Gly from glutathione, the release of alanine from γ-glu-Ala, and the formation of γ-glutamyl dipeptides. A dipeptide formed from Smethylcysteine and glutathione or γ-glu-Ala was characterized as γ-glutamyl-S-methylcysteine [27].
Two cationic isoperoxidases (C1 and C3) and four anionic isoperoxidases (A1, A2, A3n, and A3) were isolated from Korean R. sativus L. root. All the six isoperoxidases are glycoproteins composed of a single polypeptide chain. The molecular weights of C1, C3, A1, and A2 were ca. 44,000, while anionic isoperoxidase A3n and A3 have molecular weights of 31,000 and 50,000, respectively. N-terminal amino acid sequences were determined for A1, A3n, and C3, while A2 was found to have a blocked terminal residue [28]. Analysis of digested products of the two major N-glycans of C3 suggested that corefucosylated trimannosylchitobiose may contain a different linkage from the typical α-1,6 of native Nlinked oligosaccharide [29].
Thiamin-binding substances were found in the radish. There were two kinds of compounds; one was heat labile and Pronase sensitive, and the other was heat stable and Pronase resistant. It would be inferred that the former is protein and the latter is a nonprotein compound [30].
βF is an isozyme (glycoprotein) found in the cytoplasm and cell walls of the radish. The nonglycosylated cytoplasmic and cell wall βF forms have the same relative molecular mass, but glycosylated forms have different oligosaccharide side chains with respect to size and susceptibility to αmannosidase and endoglycosidase D digestion [31]. 7-Glucoside de zeatin, isolated from radish cotyledons, occurs naturally as glycoside with β-glucose as substituent. A large number of derivatives of purine are glucosylated, but adenine derivatives with alkyl side chains at least three carbon atoms in length at position N6 are preferentially glucosylated [20].

Glucosinolates
Glucosinolates are very stable water-soluble precursors of isothiocyanates. The relatively nonreactive glucosinolates are converted to isothiocyanates on wounding of the radish. The tissue damage releases Myrosinase, purified to homogeneity from daikon, has a specific activity of 280 µMol/min/mg protein with sinigrin as a substrate [33]. Glucosinolate contents of seed of radish cultivar ranged from 37-87 µmol/g seed. The 5-vinyl-2-oxazolidinethione, 3-butenyl, 4-pentenyl, and phenethyl isothiocyanate were found in industrially extracted rapeseed oils. The compounds were hydrolysis products from glucosinolates present in the seed [34].

Oil Seed Components
The seeds of the radish contain a high percentage of oil. Chromatographic analysis of these oils showed clearly their complete similarities to cottonseed oil [36]. The steam volatile constituents of fresh radish of Japanese and Kenyan origin have been studied. The overall pattern of compounds in the two materials was similar. Major components are pentyl hexyl, 4-methylpentyl isothiocyanate, dimethyl disulfide, methyl methanethiolsulfinate, and 1-methylthio-3-pentanone [37]. Oil radish seeds contained 1.21 µmol of total alkenylglucosinolates (AG/g), consisting mostly of progoitrin and gluconapin [38].
Among the anthocyanins, pelargonidine and cyanidine were responsible for red and violet color in corollas and roots in all inbred progenies. The absence of pelargonidine and cyanidine resulted in a white color. The flavonoid, quercetine, was also found in both corolla and root [42]. Anthocyanins extracted from epidermal tissue resulted in juices with fairly low initial ˚Brix (1.3˚), containing 400 mg anthocyanin/100 ml. This compound provided color similar to FD&C Red#40. Radish concentrate extract represents a promising natural alternative to the use of FD&C Red#40 [43].

Pigments
Salted radish roots have a characteristic yellow color, which generates during storage. 4-Methylthio-3butenyl-glucosinolate (4-MTBG) is the substrate of the main pungent principle of radish and is one of the essential factors for the formation of the yellow pigment. The yellow compound 1-(2´-pyrrolidinethion-3´-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxilic acid is presumed to have been the condensation product from the degradation of 4-methylthio-3-butenylisothiocyanate and L-tryptophan, which carboline compound is considered to play an important role in the formation of the yellow pigment in salted radish roots [52].

Polysaccharides
Pectic substances were extracted from the leaves with oxalate buffer of pH 4.25 as weakly acidic pectic polysaccharide (WAP) and pectic acid. WAP was appreciably hydrolyzed by exo-and endopolygalacturonases and the galacturonic acid content (17.3-25.8%) was much lower than the pectic acids, though the neutral sugar components of both pectic substances were almost the same. The arabinose-galactose side chains were very long or highly branched in pectine compared with those in pectic acids. These compounds are probably inherent pectic components of the cell walls of the vegetables [53]. Rhamnose, glucose, and xylose were also isolated. Lipopolysaccharides (LPS) were isolated from radish roots [54].

Proteoglycan
An L-arabino-D-galactan-contained proteoglycan was isolated from hot phosphate-buffered saline extract of radish seeds by ethanol fractionation. The proteoglycan consisted of 86% of a polysaccharide component-contained L-arabinose and D-galactose as major sugar constituents, together with small proportions of D-xylose, D-glucose, and uronic acids, and 9% of a hydroxyproline-contained protein. Arabinogalactan from radish seed had a high content (81%) of L-arabinose and its basic structure seemed to be similar to that of the polysaccharide component of the proteoglycan [55].

Sulfur Compounds
Radish leaves contain only one of the sulfonium diateroisomers of S-adenosylmethionine (AdoMet), which has a remarkable variety of biochemical functions. It is an allosteric enzyme effector and a precursor of spermine biosynthesis, spermidine, and ethylene. It is also the methyl group donor for most biological transmethylation reactions, wherein transfer of its methyl group converts AdoMet to the homocysteine analog (AdoHcy). Much of the chemistry and biochemistry of AdoMet derives from the fact that it is a sulfonium compound [56]. 1-(2´-Pyrrolidinethion-3´-yl)-1,2,3,4-tetrahydro-β-carboline-3carboxilic acid was found in radish root. This carboline compound is considered to play an important role in the formation of the yellow pigment in salted radish roots.

Other Constituents
β-Carotene was isolated from radish. Vitamin C content in fresh hotbed radishes ranged from 17.95-27.86 mg% [57]. Also identified was β-sitosterol from R. sativus seeds [40]. The contents of raphanusol A and B in radish increased at the lighted side and decreased in the shaded side. The differential distribution of raphanusol A and B in the hypocotyls is closed correlated with growth suppression at lighted side [58].

Allergic Contact
In the radish, the allyl isothiocyanate released enzymically from simigrin, a thioglycoside, was identified as a possible sensitizing substance. In some cases, it can produce allergic contact and dermatitis [59]. The leaves of this plant also contained glucoparin that produced allergic contact.

Antimicrobial Activity
Crude juice of the radish inhibited the growth of Escherichia coli, Pseudomonas pyocyaneus, Salmonella typhi, and Bacillus subtilis in vitro. This common plant may be an important source of antimicrobial substances [60]. The cysteine-rich peptides (Rs-AFP1 and Rs-AFP2) isolated from R. sativus showed substantial antifungal activity against several fungal species with minimal inhibitory concentration (MIC) of 30-60 µg/ml. Both Rs-AFPs are among the most potent antifungal proteins characterized. Moreover, their antibiotic activity shows a high degree of specificity to filamentous fungi [16]. The active region of the antifungal protein appears to involve β-strands 2 and 3 in combination with the loop connecting those strands [61]. Rs-AFP1 and Rs-AFP2 are highly basic oligomeric proteins composed of small (5-kDa) polypeptides that are rich in cysteine. These proteins are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs. Moreover, Rs-AFPs are preferentially released during seed germination after disruption of the seed coat [62]. Two purified antifungal proteins RAP-1 and RAP-2 isolated from Korean radish seeds (R. sativus) exhibited growth-inhibitory activities against Candida albicans and Saccharomyces cerevisiae [63]. The protein AFP1 isolated from the radish showed antifungal activity against Fusarium culmorum [17].
Caffeic acid showed antifungal properties in vitro against Helminthosporium maydis. It has antibacterial, antifungical activities. Ferulic acid is active against Sytaphylococcus aureus, Bacillus subtilis, Corynebacterium, diphtheria, Aspergillus niger, and Candida albicans. These acids displayed antibacterial activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and the Gram-negative Escherichia coli and Kliebsiella pneumoniae. The MIC values were 1.56-3.13 µg/ml. These p-hydroxybenzoic acid (hydroxycinnamic, p-hydroxybenzoic) showed marked activity against Gram-positive bacteria.
The inoculation of sliced daikon roots with the bacterium Pseudomonas cichorii induced the formation of several antifungal compounds including brassinin, methoxybrassinin, spirobrassinin, and 3indolecarbaldehydes [64].
The radish released biocidal compounds, mainly isothiocyanates, produced during the enzymic degradation of glucosinolates present in the plant cell. The highest fungicidal activity depended on concentration of isothiocyanates [65].

Antioxidative Activity
The red radish pigment (pelargodinin-3-sophoroside-5-glucoside) had almost the same antioxidative activity as BHT at the same concentration. The inhibition ratio could reach more than 93% by the 0.01% pigment addition [66]. Also, the caffeic acid showed antioxidative activity.

Antiviral Activity
Caffeic acid and pelargonidin are virucidal for several enveloped viruses [41]. The lipopolysaccharides showed antiherpes activity.

Hypotensive
Sinapine was extracted with methanol. It is a hypotensive constituent of laifuzi (Semen raphani) and seed of R. sativus [5].

Platelet Aggregation Inhibitor
The 6-methyl-sulfinylhexyl-isothiocyanate (MS-ITC) was isolated from wasabi horseradish (Japanese domestic) as a potential inhibitor of human platelet aggregation in vitro. It is a potential inducer of GST (glutathione S-transferase). In the mechanism of MS-ITC, the isothiocyanate moiety of MS-ITC plays an important role for antiplatelet and anticancer activities because of its high reactivity with sulfhydryl (-SH) groups in biomols (GSH, cysteine, residue in a certain protein) [69].

Immunological Properties
The AGPs isolated from the radish showed immunological properties. Radish AGPs R-I, R-II, crude fraction R-C, and turnip AGP B-II reacted with eel anti-H serum, indicating that these AGPs shared common antigenic determinants [70]. The root's AGPs were composed mainly of L-arabinose and Dgalactose, but were distinguishable from each other in their contents of L-fucose as well as of protein and hydroxyproline. Structures of AGPs from the root, seeds, and mature leaves were essentially similar [71]. Proteoglycan from radish leaves and seeds appeared to share common antigenic determinant [55].

Phytoalexins
The inoculation of sliced daikon roots with the bacterium Pseudomonas cichorii induced the formation of several antifungal compounds including brassinin, methoxybrassinin, spirobrassinin, and 3indolecarbaldehydes [64].

Pungent Principle
The pungent principle extracted from the radish root is trans-4-methylthio-3-butenyl-isothiocyanate. Also isolated was the cis-isomeride, in a trans-cis ratio of 4:1 [72]. 2-Thioxo-3-pyrrolidinecarbaldehyde (TPC) is a major product generated from the pungent principle of radish. This compound possesses antimicrobial activity with the MIC against fungi and bacteria ranging from 50-400 µg/ml, while yeasts were more resistant. The antifungal and antibacterial actions were due to the sporicidal and bactericidal activities. A dose-dependent inhibition of the uptakes of both oxygen and the precursors for RNA and DNA was observed, suggesting that TPC caused damage to the mitochondrial functions and biosynthetic systems [73].

Serological Activity
AGPs were presumably responsible for expression of the serological activity. In their immunological reactions with rabbit antiradish leaf AGP antibody, the root AGPs were shown to share common antigen determinant with those of seed and leaf AGPs [10]. Arabino-3,6-galactan associated with a hydroxyproline-rich protein portion, which might be responsible for the serological H-like activity of the AGPs [12]. Two L-arabino-D-galactan-contained glycoproteins having potent inhibitory activity against eel anti-H agglutinin were isolated from the saline extract of mature radish leaves [70].

Intestine Motility Stimulation
The effect of radish aqueous extract at doses of 10 µg/ml to 2 mg/ml caused a dose-dependent increase in contractions of the duodenum, jejunum, and ileum. Ileal contraction was remarkably inhibited by pretreatment of atropine (10 -7 M) by 10 min. Oral administration of radish extract (300-500 mg/kg body wt) to mice improved the intestinal transit of charcoal and this was significantly attenuated by coadministration of atropine (50 mg/kg). These results suggest that radish extract stimulates gastrointestinal motility through activation of muscarinic pathways [74]. Scopoletin is an antispasmodic agent.

Cardiovascular Disease Prevention
Radish powder decreased the lipid levels by increasing the fecal excretion of total lipids, triglycerides, and total cholesterol. Catalase and glutathione peroxidase (GSH-Px) activities in red blood cell (RBC) were most remarkably increased by radish. Superoxide dismutase (SOD), catalase, and GSH-Px activities in the liver were increased by radish powder. Xanthine oxidase (XOD) activities in the liver were decreased by radish. Flavonoids and vitamin C in radish may inhibit lipid peroxidation, promote liver and RBC catalase, and inhibit XOD activities in animals tissues. Radish can be recommended for the treatment and prevention of diseases such as cardiovascular disease and cancer and for delaying aging [75].

Other Activities
Lipopolysaccharides (LPS) were isolated from radish having a macrophage activating with ED 50 of 0.4-100 ng/ml. These compounds can be used as antidiabetic agents in pharmaceutical or veterinary fields. Also the LPS showed analgesic activity [54].