X-Message-Number: 30412
Date: Mon, 4 Feb 2008 20:57:07 -0800 (PST)
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Subject: Green tea polyphenols induce freeze tolerance

  [Some cryoprotectants, such as dimethylsulfoxide have shown strong
reduction in freezing damage at concentrations too low to significantly
reduce ice formation. Much of the effect of this cryoprotectant thus must be
due to other effects, which I will term "freeze tolerance". It is commonly
believed that only vitrification or complete ice crystal formation
suppression can completely preserve organ functionality. However the only
existing animal model of survival at dry ice temperatures is Canada's
woollybear caterpillar. This caterpillar is known not to vitrify, so its
ability to survive at temperatures below the glass transition must be due to
freeze tolerance. The result of billions of years of evolution in the
form of this hardy Arctic animal implies that vitrification is not
neccessary for full recovery from below glass transition temperatures.
  Green tea polyphenols are an example of an additive that can induce
freeze tolerance. Below, 500 mug of green tea polyphenol (GTP) offered
protection against freeze-thaw damage that was slightly superior to that
offered by 10% (v/v) dimethylsulfoxide. Moreover, NO morphological
alterations were noted when both GTP and dimethylsulfoxide were added. The
woolybear caterpillar cryopreservation secret thus may no longer be a
complete mystery.
  Green tea polyphenols possess the additional property of reducing ethylene
glycol and glycerol toxicity, as well as probably reducing the toxicity of
other cryoprotectants as well. GTP may aid current vitrification solutions,
both by reducing cryoprotectant toxicity, as well as by lowering the
absolute amount of  cryoprotectants needed as well, since complete
vitrification would hopefully no longer be required.
  The major limitation in the use of this inexpensive additive is its
relatively poor permeation into tissue. This can be partially corrected by
the use of liposomes, and/or terpenes. However, until this permeation issue
is fully resolved duplicating the woolybear caterpillar's amazing feat of
survival at ultra-low temperatures without vitrification will remain -just-
out of reach.]

Biotechnol Lett. 2005 May;27(9):655-60.
Protection of osteoblastic cells from freeze/thaw cycle-induced oxidative
stress by green tea polyphenol.
  Han DW, Kim HH, Lee MH, Baek HS, Lee KY, Hyon SH, Park JC. Department of
Medical Engineering, Yonsei University College of Medicine, 134
Shinchon-dong, Seodaemun-gu, Seoul, 120-752, Korea.
  Green tea polyphenol (GTP) together with dimethylsulphoxide (DMSO) were
added to a freezing solution of osteoblastic cells (rat calvarial
osteoblasts and human osteosarcoma cells) exposed to repeated freeze/thaw
cycles (FTC) to induce oxidative stress. When cells were subjected to 3
FTCs, freezing medium containing 10% (v/v) DMSO and 500 mug GTP ml(-1)
significantly (p<0.05) suppressed cell detachment and growth inhibition by
over 63% and protected cell morphology. Furthermore, the alkaline
phosphatase activity of osteoblastic cells was appreciably maintained after
2 and 3 FTCs in this mixture. Polyphenols may thus be of use as a cell
cryopreservant and be advantageous in such fields as cell transplantation
and tissue engineering.
PMID: 15977073

[Catechin, a constituent of green tea,  completely blocked all morphological
alterations caused by a 50% glycerol solution.]

Pharmacol Res. 2003 Nov;48(5):503-9.
Catechin, a natural antioxidant protects against rhabdomyolysis-induced
myoglobinuric acute renal failure.
  Chander V, Singh D, Chopra K. Pharmacology Division, University Institute
of Pharmaceutical Sciences (U.I.P.S.), Punjab University, Chandigarh 160014,
India.
  Rhabdomyolysis-induced myoglobinuric acute renal failure accounts for
about 10-40% of all cases of acute renal failure (ARF). Reactive oxygen
intermediates have been demonstrated to play an etiological role in
myoglobinuric renal failure. This study was performed to explore the
protective effect of catechin-a natural antioxidant in an experimental model
of myoglobinuric ARF in rats. Four groups of rats were employed in this
study, group 1 served as control, group 2 was given 50% glycerol (8 ml
kg(-1), i.m.), group 3, glycerol plus catechin (40 mg kg(-1), p.o. for 4
days, twice a day) and group 4 was given only catechin (40 mg kg(-1), p.o.),
respectively. Renal injury was assessed by measuring serum creatinine, blood
urea nitrogen (BUN), creatinine, and urea clearance. The oxidative stress
was measured by renal malondialdehyde (MDA) levels, reduced glutathione
levels and by enzymatic activity of catalase, glutathione reductase (GR) and
superoxide dismutase (SOD). Glycerol administration resulted in a marked
renal oxidative stress, significantly deranged the renal functions as well
as renal morphology. All these factors were significantly improved by
catechin treatment. Catechin, due to its antioxidative activity, reduced the
toxicity of myoglobin in the renal tissues, and thus exerted a
renoprotective effect in this rhabdomyolysis mimicking model.
PMID: 12967597

[Green tea reduces ethylene glycol toxicity.]

J Urol. 2005 Jan;173(1):271-5.
Preventive effects of green tea on renal stone formation and the role of
oxidative stress in nephrolithiasis.
  Itoh Y, Yasui T, Okada A, Tozawa K, Hayashi Y, Kohri K. Department of
Surgical Medicine, Nephro-urology, Nagoya City University Graduate School of
Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya City, Aichi
467-8601, Japan.
  PURPOSE: Urinary stones are similar to arteriosclerosis in epidemiology,
mechanism, calcification composition and age at frequent occurrence. The
calcification that occurs in arteriosclerosis is inhibited by antioxidants.
Green tea leaves contain approximately 13% catechins, which have been shown
to have antioxidant effects. We investigated the inhibitory, antioxidative
effects of green tea on calcium urinary stone formation. MATERIALS AND
METHODS: A total of 120 Wistar rats were divided into 4 groups, namely group
1-control rats receiving saline, group 2-stone group rats administered
ethylene glycol (EG) and vitamin D3, group 3-drink group rats administered
EG, vitamin D3 and green tea given as drinking water, and group 4-powder
group rats administered EG, vitamin D3 and 2.5% powdered green tea leaves
mixed in a powder diet. Pooled 24-hour urine samples and blood samples were
collected and the 2 kidneys were excised 7, 14 and 21 days after
administration, respectively. One kidney was used for immunohistological
examination of osteopontin, superoxide dismutase (SOD), p65, p53 and bcl-2
expression, in situ hybridization of osteopontin and detection of apoptosis,
while the other was used for quantitative analysis of SOD activity. RESULTS:
Green tea treatment decreased urinary oxalate excretion and calcium oxalate
deposit formation. Green tea treatment increased SOD activity compared with
the stone group. The degree of apoptosis in the stone group was
significantly increased compared with the drink and powder groups.
CONCLUSIONS: The inhibitory effect of green tea on calcium oxalate
urolithiasis is most likely due to antioxidative effects.
PMID: 15592095

[The major limitation in the use of green tea polyphenols is their poor
permeation into tissue.]

J Neurosci Methods. 2005 Jun 30;145(1-2):255-66.
Optimal conditions for peripheral nerve storage in green tea polyphenol: an
experimental study in animals.
  Matsumoto T, Kakinoki R, Ikeguchi R, Hyon SH, Nakamura T. Department of
Orthopedic Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku,
Kyoto 606-8507, Japan.
  Our previous study demonstrated successful peripheral nerve storage for 1
month using polyphenol solution. We here report two studies to solve
residual problems in using polyphenols as a storage solution for peripheral
nerves. Study 1 was designed to determine the optimal concentration of the
polyphenol solution and the optimal immersion period for nerve storage. Rat
sciatic nerve segments were immersed in polyphenol solution at three
different concentrations (2.5, 1.0, and 0.5 mg/ml) for three different
periods (1, 7, and 26 days). Electrophysiological and morphological studies
demonstrated that nerve regeneration from nerve segments that had been
immersed in 1mg/ml polyphenol solution for 1 week and in Dulbecco's modified
Eagle's medium (DMEM) for the subsequent 3 weeks was superior to the
regeneration in other treatment groups. In study 2, the permeability of
nerve tissue to polyphenol solution was investigated using canine sciatic
nerve segments stored in 1.0mg/ml polyphenol solution for 1 week and in DMEM
for the subsequent 3 weeks. Electron microscopy revealed that the Schwann
cell structure within 500-700 microm of the perineurium was preserved, but
cells deeper than 500-700 microm were badly damaged or had disappeared. The
infiltration limit for polyphenol solution into neural tissue is inferred to
be 500-700 microm.
PMID: 15922041

Int J Pharm. 2006 Mar 9;310(1-2):131-8. Epub 2006 Jan 18.
Enhancement of the transdermal delivery of catechins by liposomes
incorporating anionic surfactants and ethanol.
  Fang JY, Hwang TL, Huang YL, Fang CL. Pharmaceutics Laboratory, Graduate
Institute of Natural Products, Chang Gung University, 259 Wen-Hua 1st Road,
Kweishan, Taoyuan 333, Taiwan.
  The aim of this study was to develop and evaluate liposomal formulations
encapsulating tea catechins, which possess antioxidant and chemopreventive
activities. Liposomes were characterized for size, zeta potential, and
entrapment efficiency. Both in vitro and in vivo skin permeation were
examined using nude mouse skin as a model. The results suggested that the
liposomal composition plays an important role in affecting the efficiency of
transdermal catechin delivery. Incorporation of anionic surfactants such as
deoxycholic acid (DA) and dicetyl phosphate (DP) in the liposomes in the
presence of 15% ethanol increased the (+)-catechin permeation by five to
seven-fold as compared to the control. The flexibility of bilayers is
suggested as an important factor governing the enhancing effect of
liposomes. Intercellular spaces within the stratum corneum but not shunt
routes are the major pathways for catechin delivery from liposomes.
(+)-Catechin and (-)-epicatechin are isomers which showed similar
encapsulation efficiencies and skin permeation in liposomes.
(-)-Epigallocatechin-3-gallate showed the highest encapsulation rate and in
vivo skin deposition level in liposomes among all catechins tested. The
stability and in vitro tranepidermal water loss test indicated the safety of
the practical use of liposomes developed in this study.
PMID: 16413711

Biol Pharm Bull. 2007 Feb;30(2):343-9.
Transdermal delivery of tea catechins and theophylline enhanced by terpenes:
a mechanistic study.
  Fang JY, Tsai TH, Lin YY, Wong WW, Wang MN, Huang JF. Pharmaceutics
Laboratory, Graduate Institute of Natural Products, Chang Gung University,
Taoyuan, Taiwan.
  Using in vitro and in vivo techniques, terpenes were evaluated as
enhancers to improve the skin permeation of therapeutically active agents
derived from tea, including tea catechins and theophylline. The in vitro
permeation was determined by Franz cells. The skin deposition and
subcutaneous amounts of drugs sampled by microdialysis were evaluated in
vivo. Terpenes varied in their activities of enhancing drug permeation. The
oxygen-containing terpenes were effective enhancers of drug permeation,
whereas the hydrocarbon terpenes were much less efficient. Oxygen-containing
terpenes with a bicyclic structure had reduced enhancing activity. Terpenes
enhanced tea catechin permeation to a much greater degree than they did
theophylline. The isomers of (+)-catechin and (-)-epicatechin showed
different permeation behaviors when incorporated with terpenes. In the in
vivo status, terpenes promoted the skin uptake but not the subsequent
subcutaneous concentration of (-)-epigallocatechin gallate (EGCG). Both
increased skin/vehicle partitioning and lipid bilayer disruption of the
stratum corneum (SC) contributed the enhancing mechanisms of terpenes for
topically applied tea catechins and theophylline based on the experimental
results from the partition coefficient and transepidermal water loss (TEWL).
alpha-Terpineol was found to be the best enhancer for catechins and
theophylline. The high enhancement by alpha-terpineol was due to macroscopic
perturbation of the SC and the biological reaction in viable skin as
evaluated by TEWL and colorimetry.
PMID: 17268077

[The nature of glycerol induced toxicity has never been convincingly
explained by cryobiologists. However the main cause of glycerol toxicity has
been known for over a decade according to claims by toxicologists, who
apparently never talk to cryobiologists! Glycerol induced toxicity is
reputed to be largely mediated by iron, since iron chelators like catechin,
naringin, etc can completely block glycerol induced morphological
alterations.]

Drug Metab Rev. 1999 Nov;31(4):971-97.
Oxidant mechanisms in toxic acute renal failure.
  Baliga R, Ueda N, Walker PD, Shah SV. University of Mississippi Medical
Center, Jackson 39216, USA.
  Over the last decade, there is accumulating evidence for a role of
reactive
oxygen metabolites in the pathogenesis of a variety of renal diseases,
including gentamicin, glycerol, cisplatin, and cyclosporine A models of
toxic acute renal failure. Gentamicin has been shown both in in vitro and in
vivo studies to enhance the generation of reactive oxygen metabolites. Iron
is important in models of tissue injury, presumably because it is capable of
catalyzing free-radical formation. Gentamicin has been shown to cause
release of iron from renal cortical mitochondria. Scavengers of reactive
oxygen metabolites as well as iron chelators provide protection in
gentamicin-induced nephrotoxicity. In glycerol-induced acute renal failure,
an animal model of rhabdomyolysis, there is enhanced generation of hydrogen
peroxide, and scavengers of reactive oxygen metabolites and iron chelators
provide protection. Although the dogma is that the myoglobin is the source
of iron, recent studies suggest that cytochrome P450 may be an important
source of iron in this model. In addition, there are marked alterations in
antioxidant defenses, such as glutathione, as well as changes in heme
oxygenase. Several recent in vitro and in vivo studies indicate an important
role of reactive oxygen metabolites in cisplatin-induced nephrotoxicity.
Thus, catalytic iron is increased both in vitro and in vivo by cisplatin,
and iron chelators as well as hydroxyl radical scavengers have been shown to
be protective. Recent studies indicate that cytochrome P450 may also be an
important source of the catalytic iron in cisplatin nephrotoxicity.
Cyclosporine A has been shown to enhance generation of hydrogen peroxide in
vitro and enhance lipid peroxidation in vitro and in vivo. Antioxidants have
been shown to be protective in cyclosporine A nephrotoxicity. This
collective body of evidence suggests an important role for reactive oxygen
metabolites in toxic acute renal failure and may provide therapeutic
opportunities of preventing or treating acute renal failure in humans.
PMID: 10575556

[Tea polyphenols may reduce cryoprotectant toxicity by, for example
chelating calcium to reduce ethylene glycol toxicity, and chelating iron to
reduce glycerol toxicity. Propylene glycol and dimethysulfoxide release even
more intracellular calcium than ethylene glycol does, so calcium chelation
probably would reduce their toxicity as well.]

Biol Trace Elem Res. 1998 Oct;65(1):75-86.
Effect of green tea and black tea on the metabolisms of mineral elements in
old rats.
  Zeyuan D, Bingying T, Xiaolin L, Jinming H, Yifeng C. Institute of Food
Nutrition, Nanchang University, China.
  A 2-mo experiment with the white Sprague-Dawley (SD) rats was conducted to
investigate the effect of the water extracts of black tea (BTWE) and green
tea (GTWE) and the black tea leaves (BTF) and the green tea leaves (GTF) on
the metabolism of mineral elements. One hundred eight 12-mo-old white SD
rats were randomly divided into 13 groups; 6 of these drank the BTWE or GTWE
in which the water extracts concentrations of black tea or green tea were,
respectively, 0.6%, 1.2%, and 2.4%, and 6 of these had black tea leaves
(BTF) and green tea leaves (GTF) added in which the contents of BTF or GTF
were, respectively, 0.5%, 1.0%, and 2.0%, one of these was control. The teas
and their water extracts could promote the absorption of manganese and
copper. In GTF, BTF, GTWE, and BTWE, the apparent absorption rates of
manganese were significantly increased. The manganese contents in the tibia
were also elevated, and the differences between GTWE and GTF were
significant. The apparent absorption rates of copper and the copper contents
in the tibia were increased, but not significantly. The teas and their water
extracts inhibited the absorption of calcium (p > 0.05) and iron (p < 0.05).
The cerebrum calcium contents were significantly decreased, but the contents
of calcium and iron in tibia were not significantly changed. Compared with
the control, although the apparent absorption rates of aluminum in all
experimental groups were not observed to be different, the aluminum contents
in the tibia (p > 0.05) and cerebrum (p < 0.05) were increased. GTF and GTWE
decreased the apparent absorption rates of zinc, but BTF and BTWE increased
them; the zinc contents in tibia were a little improved, whereas its
contents in the cerebrum were gradually decreased with the increase of tea
leaves dose and tea concentration.
PMID: 9877538

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