Galloyl Esters from Rhubarb are Potent Inhibitors of Squalene Epoxidase, a Key Enzyme in Cholesterol Biosynthesis

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Galloyl glucoses and galloyl proanthocyanidins obtained from rhubarb (Rhei Rhizoma, Rheum palmatum L., Polygonaceae); e.g. 1,2,6-tri-O-galloyl-β-D-glucose (IC₅₀ = 0.63 μM), 1,6-di-O-galloyl-2-O-cinnamoyl-β-D-glucose (IC₅₀ = 0.58 μM), procyanidin B-2 3,3′-di-O-gallate (IC₅₀ = 0.54 μM), and procyanidin B-5 3,3′-di-O-gallate (IC₅₀ = 0.55 μM), were found to be potent inhibitors of rat squalene epoxidase (SE). The inhibition at submicromolar level was far more potent than that of chemically synthesized substrate analogs. It was demonstrated for the first time that the cholesterol-lowering effect of rhubarb may be attributed to the potent inhibition activities of SE, a rate-limiting enzyme of cholesterol biogenesis.

Squalene epoxidase (SE) (EC 1.14.99.7) is a non-metallic, flavoprotein monooxygenase that catalyzes the conversion of squalene to (3S)2,3-oxidosqualene, a rate-limiting step of cholesterol biogenesis [1]. Vertebrate SE requires FAD, NADPH, a supernatant protein factor, and NADPH-cytochrome P-450 reductase as co-factors. The unstable, membrane-bound 64 KDa enzyme has been purified, cloned [2], [3], and recombinant proteins functionally expressed in E. coli are now available for further characterization of the enzyme [4]. SE is the only known non-cytochrome P-450 enzyme that epoxidizes an unactivated alkene.

Since regulation of the level of SE enzyme has clinical importance for modulation of cholesterol biosynthesis, enzyme inhibitors for SE have been a potential target for the design of cholesterol-lowering drugs. To date, several potent SE inhibitors such as chemically synthesized squalene analogs and allylamine derivatives have been developed, however, there are as yet no reports of human clinical trials [5], [6].

In our previous paper [7], we reported that out of ca. 300 plant and herbal medicine extracts tested for SE enzyme inhibition activities, rhubarb (Rhei Rhizoma, Rheum palmatum L.) showed significant enzyme inhibition (IC₅₀ = 5 μg/mL) as well as 30 other plant extracts including those from green tea (Camellia sinensis (L.) O. Kuntze), Agrimonia pilosa Ledeb., Aleurites fordii Hemsley, Euphorbia jolkinii Boiss., Myrica rubra Sieb. et Zucc., and Polygonum multiflorum Thunb. Interestingly, these plants have been known to be rich sources of polyphenols, and rhubarb [8] has been reported to efficiently reduce plasma cholesterol levels as in the case of green tea [9], [10] and Polygonum multiflorum [11].

Active principles of green tea were found to be (-)-epigallocatechin-3-O-gallate (EGCG) (IC₅₀ = 0.69 μM), and other flavan-3-ols with a galloyl (3,4,5-trihydroxybenzoyl) group at C-3. In contrast, flavan-3-ols without galloyl substitution such as (-)-epicatechin did not show significant SE enzyme inhibition (IC₅₀ > 1000 μM). The presence of a galloyl moiety was thus suggested to be important for the inhibition activity, although gallic acid itself did not show potent SE inhibition (IC₅₀ = 73 μM) [7]. In this paper, we describe that gallic acid derivatives from rhubarb are also potent inhibitors of SE, the rate-limiting enzyme of the cholesterol biosynthesis.

A hot-water extract of rhubarb showed 70 % enzyme inhibition toward the recombinant rat SE at 50 μg/mL concentration. Like green tea, rhubarb is also rich in galloyl compounds such as galloyl glucoses and galloyl proanthocyanidins. These galloyl compounds were indeed found to be excellent inhibitors of SE enzyme (Table [1]).

First, a simple galloyl ester of glucose; 1,2,6-tri-O-galloyl-β-D-glucose (1) (IC₅₀ = 0.63 μM) showed SE inhibition as potent as the previously reported tea gallocatechin, EGCG (IC₅₀ = 0.69 μM). The inhibition at the submicromolar level was far more potent than that of known vertebrate SE enzyme inhibitors such as chemically synthesized squalene analogs; trisnorsqualene alcohol (IC₅₀ = 4 μM for pig SE; IC₅₀ = 7.9 μM for rat recombinant SE in our assay system) [15], trisnorsqualene cyclopropylamine (IC₅₀ = 2 μM for pig SE) [16], and trisnorsqualene difluoromethylidene (IC₅₀ = 5.4 μM for rat SE) [17], but not so potent as an allylamine derivative, NB-598 (IC₅₀ = 0.75 nM for human HepG2 SE) [18]. In addition, another class of galloyl glucoses containing a phenylpropanoyl moiety; 1-O-galloyl-2-O-cinnamoyl-β-D-glucose (2) (IC₅₀ = 0.71 μM), 1,6-di-O-galloyl-2-O-cinnamoyl-β-D-glucose (3) (IC₅₀ = 0.58 μM), and 1,6-di-O-galloyl-2-O-p-coumaroyl-β-D-glucose (4) (IC₅₀ = 3.3 μM), and a stilbene glucoside, resveratrol 4′-O-β-D-(6″-O-galloyl)-glucose (5) (IC₅₀ = 3.6 μM), also showed good enzyme inhibition. On the other hand, phenylbutanone glucosides; lindleyin (6) (IC₅₀ > 50 μM) and isolindleyin (7) (IC₅₀ = 10 μM), which may be responsible for the analgesic and anti-inflammatory action of rhubarb [13], did not show potent SE inhibition.[]

Dimers of (-)-epicatechin-3-O-gallate (ECG, IC₅₀ = 1.3 μM); procyanidin B-2 3,3′-di-O-gallate (8) (IC₅₀ = 0.54 μM) and procyanidin B-5 3,3′-di-O-gallate (9) (IC₅₀ = 0.55 μM), and a ECG oligomer; rhatannin (IC₅₀ = 1 μg/mL), as a mixture of rhatannin I (10) and II (11), were also found to be potent inhibitors of SE. In contrast, oligomers of (-)-epicatechin, a flavan-3-ol without gallate, did not show significant enzyme inhibition (IC₅₀ > 1000 μM). The occurrence of galloyl proanthocyanidins is limited in the plant kingdom, e.g., in rhubarb, tea, grapes, Polygonum multiflorum, and Myrica rubra, although proanthocyanidins are widely distributed [19]. It should be noted that extracts from most of these plants showed potent SE inhibition as reported in our previous work. Finally, another major component of rhubarb; a dianthrone glucoside, sennoside A (12), also showed good SE inhibition (IC₅₀ = 7.6 μM).

As in the case of green tea [7], we postulate that the cholesterol lowering effect of rhubarb may be explained by the potent inhibition activities of SE, a rate-limiting enzyme of cholesterol biogenesis. The non-metallic flavoprotein-mediated epoxidation has been proposed to proceed via formation of superoxide anion and flavin C(4a)-hydroperoxide intermediate [1]. Presumably, the potent enzyme inhibition by galloyl esters would be caused by scavenging reactive oxygen species required for the enzyme reaction. It has been reported that the reactivity of galloyl esters with reactive oxygen species such as superoxide anion was one of the highest measured rates of reduction of superoxide anion by any chemical antioxidant [20]. Finally, it is likely that the antioxidative galloyl esters also inhibit other oxygenase reactions involved in the cholesterol biogenesis as well as oxidation of LDL cholesterol. Further analysis of the enzyme inhibition mechanism are now in progress.

Materials and Methods

Chemicals: [1,25-¹⁴C]Squalene (57.1 mCi/mmol) and trisnorsqualene alcohol were synthesized in our laboratory. A hot-water extract of rhubarb was a gift from Alps Pharmaceuticals, Co. Ltd. (Gifu, Japan). Isolindleyin (7) and rhatannin (10, 11) were obtained from Tsumura, Co. Ltd. (Tsukuba, Japan). Sennoside A and gallic acid were purchased from Wako Chemicals (Tokyo, Japan) and Tokyo Kasei (Tokyo, Japan), respectively. Other compounds were isolated from rhubarb [12] [13] [14]. The purities of compounds were all over 95 % except for rhatannin (as a mixture).

Enzyme assay: Enzyme assay was carried out as described in the previous paper using a recombinant rat SE [7]. The recombinant enzyme, with an apparent K _M = 3.8 μM and k _cat = 4.1 min^-1 for squalene, showed properties very similar to those of native microsomal enzyme with regard to co-factor requirement, pH dependency, and sensitivity to most of known SE enzyme inhibitors. The assay mixture contained in a total volume of 200 μL of 20 mM Tris-HCl, pH 7.4, the recombinant rat SE (1.5 μg/mL), NADPH-cytochrome P-450 reductase (0.05 U), 1 mM NADPH, 0.1 mM FAD, 0.1 % Triton X-100, and [1,25-¹⁴C]squalene (5 μM, 2 × 10⁴ dpm), and was incubated at 37 °C for 1 hour [7]. Samples were dissolved in 2 μL of 70 % ethanol in water. All experiments were carried out in duplicate. The IC₅₀ values presented as means ± sem were determined by non-linear regression analysis of % control versus semi-log concentration. Dose-inhibition curves were generated with ten sample concentrations. Trisnorsqualene alcohol, a known SE inhibitor [15], was employed as a positive control.

Acknowledgements

We thank Drs. Teruo Ono and Jun Sakakibara (Niigata University, School of Medicine) for the recombinant rat SE clone used in this study. We are also indebted to Alps Pharmaceuticals, Co. Ltd. for providing a hot-water extract of rhubarb, and to Tsumura, Co. Ltd. for providing compounds (7), (10), and (11). This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas (A) from the Ministry of Education, Sciences, Sports and Culture Japan (no. 12045254 to I.A.).

References

• 1 Abe  I,, Prestwich  G D.. Squalene epoxidase and oxidosqualene: Lanosterol cyclase. Comprehensive natural products chemistry, 2.10,. In: Barton DHR, Nakanishi K, editors Vol. 2. Oxford:; Pergamon, 1999: 267-98
  Search in Google Scholar
• 2 Sakakibara  J,, Watanabe  R,, Kanai  Y,, Ono  T.. Molecular cloning and expression of rat squalene epoxidase.  J. Biol. Chem.. 1995;;  270 17-20
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 20 Jovanovic  S V,, Hara  Y,, Steenken  S,, Simic  M G.. Antioxidant potential of gallocatechins. A pulse radiolysis and laser photolysis study.  J. Am. Chem. Soc.. 1995;;  117 9881-8
  CrossrefPubMedSearch in Google Scholar

Dr. Ikuro Abe

University of Shizuoka School of Pharmaceutical Sciences

52-1 Yada

Shizuoka 422-8526

Japan

Email: abei@ys7.u-shizuoka-ken-ac.jp

Phone: +81-54-264-5662

References

• 1 Abe  I,, Prestwich  G D.. Squalene epoxidase and oxidosqualene: Lanosterol cyclase. Comprehensive natural products chemistry, 2.10,. In: Barton DHR, Nakanishi K, editors Vol. 2. Oxford:; Pergamon, 1999: 267-98
  Search in Google Scholar
• 2 Sakakibara  J,, Watanabe  R,, Kanai  Y,, Ono  T.. Molecular cloning and expression of rat squalene epoxidase.  J. Biol. Chem.. 1995;;  270 17-20
  CrossrefPubMedSearch in Google Scholar
• 3 Nakamura  P,, Sakakibara  J,, Izumi  T,, Shibata  A,, Ono  T.. Transcriptional regulation of squalene epoxidase by sterols and inhibitors in Hela cells.  J. Biol. Chem.. 1996;;  271 8053-6
  CrossrefPubMedSearch in Google Scholar
• 4 Nagumo  A,, Kamei  T,, Sakakibara  J,, Ono  T.. Purification and characterization of recombinant squalene epoxidase.  J. Lipid Res.. 1995;;  36 1489-97
  PubMedSearch in Google Scholar
• 5 Abe  I,, Tomesch  J C,, Wattanasin  S,, Prestwich  G D.. Inhibitors of squalene biosynthesis and metabolism.  Nat. Prod. Rep.. 1994;;  11 279-302
  CrossrefPubMedSearch in Google Scholar
• 6 Abe  I,, Prestwich  G D.. Development of new cholesterol-lowering drugs: Inhibitors of squalene biosynthesis and metabolism.  Drug Discovery Today. 1998;;  3 389-90
  CrossrefPubMedSearch in Google Scholar
• 7 Abe  I,, Seki  T,, Umehara  K,, Miyase  T,, Noguchi  H,, Sakakibara  J,, Ono  T.. Green tea polyphenols: Novel and potent inhibitors of squalene epoxidase.  Biochem. Biophys. Res. Commun.. 2000;;  268 767-71
  CrossrefPubMedSearch in Google Scholar
• 8 Aonuma  S,, Mimura  T,, Tarutani  M.. Effects of Coptis, Scutellaria, rhubarb, and Bupleurum on the serum cholesterol and phospholipid of rabbits.  Yakugaku Zasshi. 1957;;  77 1303-7
  PubMedSearch in Google Scholar
• 9 Muramatsu  K,, Fukuyo  M,, Hara  Y.. Effect of green tea catechins on plasma cholesterol level in cholesterol-fed rats.  J. Nutr. Sci. Vitaminol.. 1986;;  32 613-22
  PubMedSearch in Google Scholar
• 10 Fukuyo  M,, Hara  Y,, Muramatsu  K.. Effect of tea leaf catechin, (-)-epigallocatechin gallate, on plasma cholesterol level in rat.  J. Jpn. Soc. Nutr. Food Sci.. 1986;;  39 495-500
  PubMedSearch in Google Scholar
• 11 Yamahara  J,, Sawada  T,, Fujimura  H.. Experimental atherosclerosis in mice fed a butter-rich diet and effect of Kanpo-prescriptions and crude drugs on this model.  Proc. Symp. WAKAN-YAKU. 1983;;  16 167-70
  PubMedSearch in Google Scholar
• 12 Nonaka  G,, Nishioka  I,, Nagasawa  T,, Oura  H.. Tannins and related compounds. I. Rhubarb (1).  Chem. Pharm. Bull.. 1981;;  29 2862-70
  PubMedSearch in Google Scholar
• 13 Kashiwada  Y,, Nonaka  G,, Nishioka  I.. Tannins and related compounds. XLVII. Rhubarb (6). Isolation and characterization of new p-hydroxyphenylbutanones, stilbenes and gallic acid glucosides.  Chem. Pharm. Bull.. 1986;;  34 3237-43
  PubMedSearch in Google Scholar
• 14 Kashiwada  Y,, Nonaka  G,, Nishioka  I,, Nishizawa  M,, Yamagishi  T.. Studies on rhubarb (Rhei Rhizoma). XIV. Isolation and characterization of stilbene glucosides from Chinese rhubarb.  Chem. Pharm. Bull.. 1988;;  36 1545-9
  PubMedSearch in Google Scholar
• 15 Sen  S E,, Prestwich  G D.. Trisnorsqualene alcohol, a potent inhibitor of vertebrate squalene epoxidase.  J. Am. Chem. Soc.. 1989;;  111 1508-10
  CrossrefPubMedSearch in Google Scholar
• 16 Sen  S E,, Prestwich  G D.. Trisnorsqualene cyclopropylamine: a reversible, tight-binding inhibitor of squalene epoxidase.  J. Am. Chem. Soc.. 1989;;  111 8761-2
  CrossrefPubMedSearch in Google Scholar
• 17 Moore  W R,, Schatzman  G L,, Jarvi  E T,, Gross  R S,, McCarthy  J R.. Terminal difluoro olefin analogues of squalene are time-dependent inhibitors of squalene epoxidase.  J. Am. Chem. Soc.. 1992;;  114 360-1
  CrossrefPubMedSearch in Google Scholar
• 18 Horie  M,, Tsuchiya  Y,, Hayashi  M,, Iida  Y,, Iwasawa  Y,, Nagata  Y,, Sawasaki  Y,, Fukuzumi  H,, Kitani  K,, Kamei  T.. NB-598: a potent competitive inhibitor of squalene epoxidase.  J. Biol. Chem.. 1990;;  265 18075-8
  PubMedSearch in Google Scholar
• 19 Nonaka  G,, Muta  M,, Nishioka  I.. Myricatin, a galloyl flavanol sulfate and prodelphinidin gallates from Myrica rubra.  Phytochemistry. 1983;;  22 237-41
  CrossrefPubMedSearch in Google Scholar
• 20 Jovanovic  S V,, Hara  Y,, Steenken  S,, Simic  M G.. Antioxidant potential of gallocatechins. A pulse radiolysis and laser photolysis study.  J. Am. Chem. Soc.. 1995;;  117 9881-8
  CrossrefPubMedSearch in Google Scholar

Dr. Ikuro Abe

University of Shizuoka School of Pharmaceutical Sciences

52-1 Yada

Shizuoka 422-8526

Japan

Email: abei@ys7.u-shizuoka-ken-ac.jp

Phone: +81-54-264-5662