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Laboratory of Cellular Biochemistry, Graduate School of Human Health Sciences, Siebold University of Nagasaki, Nagayo, Nagasaki 851-2195, Japan
Published, JLR Papers in Press, April 1, 2004. DOI 10.1194/jlr.M300502-JLR200
1 To whom correspondence should be addressed. e-mail: shidoji{at}sun.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GGA and phytanic acid were detected in 24 out of 25 herbs tested. The present study is the first report of natural GGA in medicinal herbs.
Abbreviations: Chl, chlorophyll; CHL-P, geranylgeranyl reductase; CRABP, cellular retinoic acid binding protein; GGA, geranylgeranoic acid; GGOH, geranylgeraniol; GGPP, geranylgeranyl diphosphate; IPP, isopentenyl diphosphate; LC/MS, liquid chromatography/mass spectrometry; phytylPP, phytyl diphosphate; PPAR
, peroxisome proliferator-activated receptor ; RT, retention time; SIR, selected ion recording
Supplementary key words apoptosis • cancer chemoprevention • hepatoma
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although acyclic retinoid shares characteristics of natural retinoids in vitro and in vivo (6), acyclic retinoid apparently differs from natural retinoids such as all-trans and 9-cis retinoic acids in the following respects: 1) acyclic retinoid upregulated the cellular level of albumin mRNA in human hepatoma-derived cell lines, HuH-7 and PLC/PRF-5, while all-trans retinoic acid downregulated the expression (5); 2) acyclic retinoid induced apoptosis in these cell lines, whereas neither all-trans nor 9-cis retinoic acid did (9); and 3) acyclic retinoid showed no growth-promoting activity in vitamin A-deficient animals (unpublished observation). Therefore, we have speculated that acyclic retinoid may have a different metabolism from retinoic acid in cells and animals and may mimic other biological components such as acyclic diterpenoids, for example, geranylgeranoic acid (GGA), from which it differs for the additional double bond at a position of C4 and C5.
From a chemical structural point of view, GGA belongs to isoprenoids or terpenoids, which are the most functionally and structurally varied group of plant metabolites. Plants have two distinct biosynthetic routes for the formation of isopentenyl diphosphate (IPP; C5), either in the mevalonic acid pathway or in the methylerythritol phosphate pathway (10) (Scheme 1)
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To our knowledge, GGA (C20) has so far been recognized as only a synthetic substance, and there has been no report of the existence of natural GGA in plants or animals. Although a variety of cyclic diterpenoids have been discovered in nature, there is limited information available on acyclic diterpenoids, except for phytanic acid (3,7,11,15-tetramethyl hexadecanoic acid). Phytanic acid (C20) is an isoprenoid-derived 3-methyl branched-chain fatty acid, originating from the phytol (C20) side chain of plant Chl. Although phytanic acid is easily found in normal human plasma, there has been no report of the existence of phytanic acid in plants except for some dry lichen species from Lecanorales (13) and walnuts (14). Microorganisms, which are present in the rumen of ruminants, are thought to release phytol from Chl, after which phytol is converted into phytanic acid (15). Because humans are not capable of releasing phytol from Chl (16), all phytanic acid is considered to enter the human body via the diet. In particular, ruminant fats, fish, and dairy products are rich sources of phytanic acid (17).
Based upon the chemical structural similarity of GGA to phytanic acid, it seems reasonable for us to assume that GGA can be enzymatically derived from geranylgeraniol (GGOH; C20), which is a common precursor of all natural diterpenoids hydrolyzed from GGPP by a phosphatase enzyme (Scheme 1). If this metabolic pathway exists in plant cells, we should be able to find GGA as a natural component of traditional medicinal herbs, especially liver disease-preventive herbs.
In the first part of the present study, we show that exogenous GGA is a micromolar inducer of apoptosis in human hepatoma-derived cells. In the second and main part, we searched for naturally occurring acyclic diterpenoid acids, including GGA in plants. Liquid chromatography/mass spectrometry (LC/MS) analysis was performed with a variety of traditional medicinal plants mainly used as liver tonics to clarify whether these plants contain GGA as natural substances. We carried out a screening of 25 dry herbs and some fresh herbs. The identification of GGA was performed by chemical modification through both methylation and hydrogenation. Here we show a representative LC/MS analysis of GGA in herbal extracts from Schisandra chinensis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Human hepatoma-derived cell lines, PLC/PRF-5 and HuH-7, human hepatoblastoma-derived cell line, HepG-2, and mouse c-H-ras transformed hepatocyte-derived cell line, MLE-10, which was kindly supplied by Dr. T. Kitagawa (Cancer Institute, Tokyo) (18), were all maintained with Dulbecco's-MEM containing 10% fetal bovine serum (FBS). Hepatocytes were prepared from livers of 8-week-old male Balb/c mice after digestion with collagenase perfusion. The isolated hepatocytes were cultured with Williams' medium E supplemented with 50 ng/ml epidermal growth factor, 10–7 M insulin, 10–6 M dexamethasone, and 10% FBS.
Treatment of cells with isoprenoids
Twenty four hours before the treatment of cells in 6-well plates with isoprenoids, the media were replaced by FBS-free Dulbecco's-MEM. After addition of the isoprenoids at the indicated concentrations, viable cells were counted by the trypan blue dye-exclusion method at the indicated time points.
DNA fragmentation assay
After the treatment with isoprenoids, the detached and the loosely attached cells were collected by flushing with phosphate buffered saline, and the cell suspension was centrifuged at 400 g for 10 min. The pelleted cells were lysed with 0.5% SDS in 10 mM Tris-HCl buffer, pH 8.0, containing 25 mM EDTA and 0.1 M NaCl, and digested with 0.1 mg/ml proteinase K at 50°C overnight. The DNA was extracted twice with a solvent mixture of phenol:chloroform:isoamyl alcohol (25:24:1; v/v/v). After precipitation with ethanol and digestion with 10 µg/ml RNase A, the washed DNA was loaded onto a 1.5% agarose gel in 89 mM Tris, 80 mM boric acid, and 0.2 mM EDTA, pH 8.0. The fragmented DNA was stained with ethidium bromide after electrophoresis (100 V, 1.5 h) with a 100-bp DNA ladder maker (Invitrogen, CA).
Preparation of plant extracts
The dried fruits of S. chinensis (Schisandra; China) were homogenized in methanol with Polytron-MR3100 (probe type: 3012/2, KINEMATIKA, Switzerland) and extracted with methanol/chloroform (1:1; v/v) at 4°C overnight. The resultant monophasic extracts were evaporated to dryness and the residues were dissolved in ethanol and then partitioned with H2O/ethanol/n-hexane (1:2:4; v/v/v). The upper organic phase was evaporated to dryness, and the residues were dissolved in ethanol. The ethanolic solution was analyzed by LC/MS. The other dried herbs were also extracted in the same procedure. Curcuma longa (turmeric, rhizome; China), Glycyrrhiza uralensis (licorice, root; China), Rheum palmatum (rhubarb, root; China), Saussurea lappa (saussurea, root; China), Gentiana scabra (gentian, root; China), Bupleurum falcatum (bupleurum, root; China), Astragalus membranaceus (astragalus, root; China), Trichosanthes kirilowii (snake gourd, root; China), Coptis chinensis (coptis, root; China), Scutellaria baicalensis (skullcap, root; China), Cornus officinalis (cornelian cherry, fruit; China), Cinnamomum cassia (cinnamon, bark; China), Magnolia obovata (magnolia, bark; Japan), Commiphora abyssinica (myrrh, resin; China), and Ganoderma lucidum (reishi, whole plant; China) were purchased from a local pharmacy. Rosa canina (rosehip, fruit; Chile), Cynara scolymus (artichoke, stem and leaves; Germany), Echinacea augustifolia (echinacea, root; Egypt), Taraxacum officinale (dandelion, root; Hungary), and Citrus aurantium (orange peel, peel; Egypt) were from a local herb shop. Picrorrhiza kurroa (hellebore, rhizome; India), Bauhinia variegata (mountain ebony, bark; India), and purified Commiphora mukul (Indian bdellium tree, resin; India) were generously provided by Dr. S. P. Sardeshmukh (Ayurveda Hospital and Research Center of Baratiya Sanskriti Darshan Trust, India). Emblica officinalis was from the Institute of Traditional Oriental Medicine (Tokyo, Japan). Other plants analyzed in additional experiments are the fresh herbs Aloe barbadensis (aloe, gel) and Vinca rosea (rose periwinkle, whole plant), which were obtained from a local private garden, and marine algae, Nori (sun-dried laver) and Kajime (dried seaweed), which were purchased from a local seafood store.
HPLC/MS analysis of herbal extracts
HPLC was performed using a Waters 2690 separations module (Waters, MA) equipped with a semimicro Capcell-Pak UG80 reverse-phase C18 column (2.0 mm x 50 mm, 3 µm; Shiseido Fine Chemicals, Tokyo). The mobile phase consisted of methanol 50 mM ammonium acetate aqueous solution (90:10; v/v) at a flow rate of 0.1 ml/min. One microliter of a sample was injected into the column by the automatic injector. Inline MS was performed by using a Waters ZMD 2000 (Micromass, UK) with an electrospray interface in the negative or positive ion mode, specifically with a capillary voltage of 3 kV and a cone voltage of 30 V. Dehydrated nitrogen was used for both cone gas (56 l/h) and desolvation gas (496 l/h), with the source and desolvation temperatures being held at 130°C and 350°C, respectively. The data acquisition program (MassLynx NT, version 3.5, Micromass) was set to scan in the total ion current at m/z 200 to m/z 350 negative ions, or in the selected ion mode at m/z 303.4, m/z 311.4 of negative ion, and m/z 319.5 of positive ion.
Methylation with diazomethane
The methylation with diazomethane was conducted with authentic GGA and herbal extracts in the same procedure. A 100 µl aliquot of samples was dried under nitrogen, and the residues were treated with diazomethane. To this end, diazomethane was released from 130 mg of N-methyl-N-nitro-N-nitrosoguanidine by addition of 0.5 ml of water and 0.6 ml of 5 M NaOH, and the generated gas was trapped in 3.0 ml of ice-cold ether in a diazomethane generator (millimole size, GL Sciences, Tokyo). A sufficient amount (0.5 ml) of the diazomethane-ether solution was added to the dry residues, and the mixture was left for 30 min at room temperature. After the reaction, the solvent was removed under nitrogen, and the methylated residues were redissolved in 0.25 ml of ethanol. The methylated herbal extracts were compared with the behavior of a methylated GGA standard on LC/MS.
Catalytic hydrogenation
The catalytic hydrogenation was conducted with authentic GGA and herbal extracts in the same procedure. A 0.1 ml aliquot of samples was diluted in 5 ml of ethanol and stirred under H2 gas in the presence of 10 mg of Pt2O catalyst for 3 h. After the reaction, the spent Pt2O was removed by centrifugation and the supernatant was evaporated to dryness. The hydrogenated residues were redissolved in 0.1 ml of ethanol. The perhydrogenated GGA standard and herbal extracts were compared with the behavior of authentic phytanic acid on LC/MS.
Quantitative measurement of GGA by selected ion recording
The standard curve for quantification of GGA was made from mass peak areas of ion plots in the selected ion recording (SIR) at m/z 303.4 of the negative ion using MassLynx NT software, version 3.5. GGA standard solution (0.2 µl) was injected into the reverse-phase column ranging in amounts from 50 pg to 0.6 ng and detected by LC/MS. The concentrations of GGA in herbal extracts were calculated from the mean mass area by SIR-LC/MS chromatogram with triplicate injection based upon the standard curve.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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expression (9). Here, we report that the exogenously added all-trans GGA induced cell death in HuH-7, PLC/PRF-5, and MLE-10 cells at micromolar concentrations (Fig. 1A)
and in 16 h (Fig. 1B) but not in HepG-2 cells and mouse primary-cultured hepatocytes. Cell death was due to apoptosis as revealed by DNA stepladder formation (Fig. 2)
. GGA-induced apoptosis was also morphologically evidenced by chromatin condensation stained with Hoechst 33258 (data not shown).
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- or 
-saturation slightly decreased the activity, but neither 4,5,8,9-tetradehydration nor 4,5-didehydro-10,11,14,15-tetrahydration of GGA kept the activity. The reduction of a terminal carboxylic group to an alcohol group abolished the cell killing activity and so did the reduction in the number of isoprene units to farnesoic acid.
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In total negative ion mode, no peaks were found around at a retention time (RT) of 7.65 min corresponding to authentic GGA. However, as shown in Fig. 5, a GGA-like component (Fig. 5B, peak f) was unveiled as a prominent peak with the same RT as authentic GGA (Fig. 5A) in SIR for m/z 303.4 [M-H]–, which is a base ion of GGA (one mass unit lower than the molecular mass). Because arachidonic acid has the same molecular mass as GGA, it was possible to mistake this popular fatty acid for GGA in a chromatogram of m/z 303.4 [M-H]–, although the acid was eluted at an earlier RT of 6.5 min (Fig. 5B, peak e) than that of GGA. In a further analysis, peak f was precisely cochromatographed with authentic GGA added internally (Fig. 5C). These results indicate the compound in peak f has the identical RT with GGA in the C18 column and may have the same molecular weight as GGA.
Structural characterization of compound in peak f
To confirm the existence of a terminal carboxyl group in the structure of a compound in peak f of Fig. 5B, a further analysis by LC/MS was performed after methylation with diazomethane. LC/MS of authentic GGA after methylation (Fig. 6A)
revealed a conversion of m/z –303.4 [M-H]– (7.65 min) to m/z +319.5 [M+H]+ (22 min). Reduction of GGA (peak I) at 7.65 min by methylation gave a product (peak II) at 22 min in an appreciable amount, suggesting that the carboxyl-end of GGA was esterified with diazomethane in diethyl ether to give methylated GGA (Fig. 6A). The small peak I at 7.65 min after methylation contained GGA that remained underivatized (3%).
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To obtain inside information about the chemical structure of the compound in peak f of Fig. 5, catalytic hydrogenation of putative isoprene units was carried out, followed by an analysis by reverse-phase LC/MS. As shown in Fig. 7A , LC/MS of authentic GGA exposed to catalytic hydrogenation revealed a conversion of m/z 303.4 [M-H]– (7.65 min) to 311.4 m/z [M-H]– (13.9 min). Reduction of GGA (peak I) at 7.65 min by hydrogenation gave rise to a product (peak III) at 13.9 min in an appreciable amount at the same RT as authentic phytanic acid, suggesting that four double-bonds in GGA were fully saturated through incorporation of eight hydrogen atoms. No partially hydrogenated GGA was detected. The small peak I at 7.65 min after hydrogenation contained GGA that remained underivatized (1.8%). Further, an additional experiment of arachidonic acid with catalytic hydrogenation confirmed that perhydro-arachidonic acid eluted approximately 5 min later than perhydro GGA at the same RT as arachidic acid (20:0n-6) at m/z 311.4 [M-H]– (results not shown).
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However, the calculated amount of the 13.9-min peak (phytanic acid) produced after hydrogenation was more than the expected area of the reduced peak f. To explore the presence of possible sources for phytanic acid other than GGA or peak f, GGA derivatives saturated to the different extents, such as di- (m/z 305.4), tetra- (m/z 307.4), and hexa- (m/z 309.4) hydrogenation, were surveyed on LC/MS before and after hydrogenation. As shown in Fig. 8
, the prominent components eluted at m/z 303.4, 305.4, 307.4, and 309.4 prior to hydrogenation decreased or disappeared after being subjected to hydrogenation, whereas peak j corresponding to phytanic acid significantly rose. These components were demonstrated to elute at clearly different RTs from common straight-chain fatty acids with the same molecular weight, such as 20:4n-6 (arachidonic acid, m/z 303.4), 20:3n-6 (dihomo-
-linolenic acid, m/z 305.4), 20:3n-9 (mead acid, m/z 305.4), 20:2n-6 (11,14-eicosadienoic acid, m/z 307.4), and 20:0 (arachidic acid, m/z 311.4), respectively, except that an authentic standard corresponding to 20:1 at m/z 309.4 was not available (Fig. 8). Therefore, it is strongly suggested that peaks g, h, and i might correspond to di-, tetra- and hexa-hydro GGAs, respectively. These results indicate that the significant amount of peak j was produced during the hydrogenation, possibly arising from partially saturated compounds in peaks f, g, h, and i. The similar elution profile of putative GGA derivatives, including phytanic acid, was observed in all herbal extracts as described in Materials and Methods.
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GGA was detected for almost all dry herbs in this screening. GGA concentration in herbs ranged from 0.1 µg/g to 2.9 µg/g (Table 1), in decreasing order (>1 µg/g): turmeric (root), schisandra (fruit), licorice (root), Indian gooseberry (fruit), rhubarb root (root), hellebore (root), saussurea (root), cornelian cherry (fruit), and rosehip (fruit). There was no significant difference in GGA content according to the plant part used.
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Aside from dry herbs, the same LC/MS analysis was performed with fresh herbs, including aloe (A. barbadensis; gel) and rose periwinkle (V. rosea; whole plant), and marine algae, including Nori (sun-dried laver) and Kajime (dried seaweed), which had no significant amount of GGA (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies from our group showed that GGA and 4,5-didehydro GGA were both potent ligands for CRABP (3) and nuclear retinoid receptors (4), so we called these acids "acyclic retinoids." Consistent with our findings and definition of acyclic retinoids, some other acyclic compounds have been reported, such as methoprene acid (19) and phytanic acid (20), as ligands for retinoid-X receptor. At the initial point of our study, we thought that apoptosis-inducing activity of 4,5-didehydro GGA was attributable to the retinoidal function; however, natural retinoids such as all-trans and 9-cis retinoic acids were unexpectedly unable to induce apoptosis in HuH-7 cells (9). Henceforth, 4,5-didehydro GGA was expected to mimic other natural isoprenoids such as GGA rather than retinoids in terms of apoptosis induction in hepatoma cells. In fact, GGA itself actively induced apoptosis in human hepatoma- (21) and mouse hepatocyte-derived cell lines (Figs. 1, 2). Furthermore, 2,3-dihydro GGA possessed this activity (Fig. 3), even though the -saturation of GGA totally destroyed the ligand activity for retinoid receptors (4), suggesting that the induction of apoptosis by GGA may not be mediated through retinoid receptors. Recently, it was reported that phytanic acid transactivated peroxisome proliferator-activated receptor (PPAR), as well as polyunsaturated fatty acids (22). GGA may well be a potent ligand for PPAR and for some other orphan receptors. We cannot even exclude the possibility that GGA may induce apoptosis through a hypothetical GGA-specific receptor, which is now under investigation in our laboratory by using a technique of RNAi with a whole genomic siRNA expression library.
Inasmuch as GGA and its derivatives were shown to induce apoptosis in human hepatoma cell lines (Fig. 3), our next question was whether GGA is present in nature. Isoprenoids are synthesized in all organisms but are especially abundant and diverse in plants, with tens of thousands of compounds reported to date (23). To our knowledge, however, there has been no report of the existence of the acyclic diterpenoid GGA in the isoprenoid biosynthetic pathway in plants or animals.
The present characterization of a GGA-like compound in herbal extracts revealed its structure by LC/MS analysis. The compound, coeluted with authentic GGA, was converted to a product corresponding to GGA methylester after its methylation with diazomethane and was converted to a product incorporated with eight hydrogen atoms corresponding to phytanic acid by catalytic hydrogenation. These results strongly support a conclusion that the GGA-like compound is structurally identical with GGA, which has four isoprenyl double-bonds and a carboxyl group in terminal with a molecular mass of 304, although further isolation analysis is required to warrant the existence of natural GGA.
The amounts of GGA in herbs were in the range of micrograms/grams (Table 1), suggesting that GGA may be relevant to micronutrients such as vitamins. GGA and its derivatives are active to induce apoptosis in hepatoma-derived HuH-7 cells at concentrations of 1
10 µM (Fig. 1). On the supposition that all of the ingested GGA will appear in blood, several hundred grams of herbs will be required to reach the effective concentrations. It is also worthwhile to mention that the ingested GGA may be selectively taken up and accumulated in tumor cells, because tumor cells show the enhanced expression of CRABP, to which GGA may be bound (3,4). Ratios of GGA to phytanic acid contents varied by 60-fold in our samples of herbs. What does determine a proportion of GGA to phytanic acid in the dry herbs? Chl, the main constituent of the photosynthetic apparatus in plants, consists of two moieties, chlorophyllide and phytol. The hydrogenation of GGPP is catalyzed to phytyl diphosphate (phytylPP) by a geranylgeranyl reductase (CHL-P), as indicated in Scheme 1. It is reported that CHL-P uses GGPP and geranylgeranylated chlorophyllide as substrates and directs phytylPP to the tocopherol- and Chl-synthesizing pathway (24). CHL-P catalyzes a stepwise reduction of geranylgeranyl derivatives to phytyl derivatives. This reductase broad specific for geranylgeranyl moiety could catalyze GGA to phytanic acid conversion in a stepwise mode. Therefore, once GGA is produced from GGOH in plants, a proportion of phytanic acid to GGA contents would be attributable to CHL-P activity.
Furthermore, the finding of other GGA derivatives with only subtle differences on numbers of double bonds in herbal extracts is informative for supporting the existence of GGA and CHL-P activity. We could demonstrate that these peaks did not represent the well-known straight-chain fatty acids, and that these were all converted to phytanic acid by hydrogenation. In the present study, the exact positions of the double bonds for m/z 305.4, 307.4, and 309.4 [H-M]– were not assigned because authentic standards were not available. However, based upon a mode of CHL-P enzyme action (25), it is reasonable to speculate that these may be 6,7-dihydro (for m/z 305.4), 6,7,10,11-tetrahydro (for m/z 307.4), and 6,7,10,11,14,15-hexahydro (for m/z 309.4) GGAs. Obviously, further structural analysis is required of each isolated compound and additional examination of biological activity for these GGA derivatives found in herbs.
In terms of its distribution, interestingly, GGA and phytanic acid were observed in almost all dry herbs examined in this screening. In additional experiments of other plants, however, fresh herbs, including aloe (A. barbadensis; gel) and rose periwinkle (V. rosea; whole plant), used as liver tonics had no detectable GGA and its derivatives. Marine algae, including Nori (sun-dried laver) and Kajime (dried seaweed), contained only GGA derivatives, including phytanic acid, with no significant amount of GGA. Although little is known about the presence of phytanic acid in higher plants (14), most of the medicinal dry herbs used in the present study contained both GGA and phytanic acid as a characteristic common to all, independent of their own specific effects, such as liver tonics. The accumulation of these compounds is supposed to be involved with a certain type of plants, such as "dried" plants, because it has been reported that phytanic acid was not found in any food consisting of nondried vegetable (26).
The information that dry herbs, not fresh plants, contain GGA and its derivatives could provide a new vista on the biological significance of GGA in plants. A large variety of products are derived from isoprenoids in plants for their growth and response to environmental stress, which is closely associated with plant defense mechanisms (11). For example, Nah, Song, and Back (27) have reported that farnesyl disphosphatase and geranylgeranyl disphosphatase (Scheme 1) were induced in rice seedlings when they were exposed to UVC. Unlike mammals, the isoprenoid metabolism in plants is a cell autonomous process (28). It can, therefore, be presumed that the reactions to environmental stress, such as drying or UV rays, occur in the plant body as plant defense mechanisms even after collecting herbs off the soil. In this context, the secondary metabolites in the isoprenoid pathway, such as GGOH-derived diterpenoids in response to environmental stress, can be synthesized and accumulated during preparation of dry herbs.
The drying process in plants may be a sort of "dying" process that results in cell death. Though it can be a broad interpretation, it is intriguing for us to speculate that GGA may be involved in apoptosis as a defense mechanism in plants. Apoptosis has now been recognized as an indispensable facet of development, defense responses, and tissue sculpturing in both animal and plant, though little is known about the molecular mechanisms of apoptosis in plant cells (29). As the present study demonstrates the possibility that GGA is a natural component of dry herbs, it may also be one of the putative intracrine regulators for apoptosis in plant cells. Traditional medicine has utilized dry herbs for thousands of years, which is not only because of preservation, but may be because of naturally occurring substances induced by a defense mechanism for drying, which may be taken into account empirically in order to obtain the optimal efficacy.
It is interesting that we found the same substance in nature as the one that has been chemically synthesized for cancer chemoprevention of hepatoma. GGA and its 4,5-didehydro derivative were already reported to both inhibit experimental hepato-carcinogenesis and induce differentiation and apoptosis in human hepatoma-derived cell lines. Further, a one-year intake of 4,5-didehydro GGA already has been proven effective for prevention of second primary hepatoma and increased the 5-year survival rate by a phase II double-blinded, placebo-controlled clinical trial with relatively low toxicity (1,2).
Our research suggests that cancer-preventive GGA and its derivatives could be synthesized in the isoprenoid biosynthetic pathway in plants. With regard to the practical use of GGA contained in herbs in order to prevent second primary hepatoma, we have not obtained yet a clear interpretation of the relation between the amount of natural GGA and its effect from the perspective of cancer prevention. However, the presence of natural GGA in herbs is informative for supporting the therapeutic efficacy of 4,5-didehydro GGA in the phase II clinical trial (1) and also will provide concrete information on whether natural GGA and its derivatives can be expected to play an important role in improving primary health care besides cancer prevention (30).
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ACKNOWLEDGMENTS |
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Manuscript received December 8, 2003 and in revised form February 24, 2004.
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REFERENCES |
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