Recent research finding
Effect of Complexation of R(+)-α lipoic acid with Cyclodextrin on the Aqueous Solubility and Radical Scavenging Activity.

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Introduction

Alpha-lipoic acid as Antioxidant
In 1959, Rosenberg, et al reported that the administration of alpha-lipoic acid to the guinea-pig which had ascorbic acid deficiency improved its symptom of scurvy and they also reported that the dosage of alpha-lipoic acid to the rat which was given the diet without Vitamin E prevented Vitamin E Deficiency1).
Later, it was reported that alpha-lipoic acid had effect on the diabete neuropathy2) or some other neuropathy3,4) and the association between free radicals and these neuropathy focused on the antioxidant activity of alpha-lipoic acid.

1) Rosenberg, H. R., Culik, R. : 「Effect of α-lipoic acid on vitamin C and vitamin E deficiencies」 Arch. Biochem. Biophys., 80, 86-93 (1959)

2) Jorg, J. Metz, F., Scharafinsky, H. : 「Drug treatment of diabetic polyneuropathy with alpha-lipoic acid or vitamin B preparations. A clinical and neurophysiologic study」 Nervenartz., 59, 36-44 (1988)

3) Altenkirch, H., Stoltenburg-Didnger, H., Wagner, M., Herrman, J., Walter, G. : 「Effects of lipoic acid in hexaxarbon-induced neuropathy」 Neurotoxicol. Treat., 12, 619-622 (1990)

4) Prehn, J. H. M., Karkoutly, C., Nuglisch, J., Peruche, B., Krieglstein, J. : 「Dihydrolipoate reduces neuronal injury after cerebral ischemia」 J. Cer. Blood Flow Metabol., 12, 78-87 (1992)

Defense against oxidative stress

What is alpha-lipoic acid

Alpha-Lipoic acid (ALA) is one of the 5 important coenzymes, which exists in the mitochondrion linked with protein and takes part in the metabolic pathway. ALA has chiral atom on C6(*) (Fig.1) and exists as two enantiomers; R(+)-alpha-lipoic acid (RALA) and S(-)-alpha-lipoic acid (SALA). RALA is the only naturally occurring lipoic acid and works as coenzyme.
RALA and its reduced form, R(+)-Dihydrolipoic acid(R-DHLA) are coupling in vivo and make expression of their antioxidant activity.

Fig.1 Structure of R(+)-α lipoic acid and R(+)-Dihydrolipoic acid.
Thiol/disulfide exchange by lipoic acid is the basis for its modulation of the cell's energy and redox status.

Antioxidant activity of lipoic acid

OVERVIEW OF REACTIVE OXYGEN SPECIES SCAVENGED
BY LIPOIC ACID AND DIHYDROLIPOIC ACID
ALA及びDHLAによって消去される活性酸素種
活性酸素種 ALA DHLA
Hydrogen peroxide
過酸化水素
Yes Yes
Singlet oxygen
一重項酸素
Yes No
Hydroxyl radical
ヒドロキシルラジカル
Yes Yes
Nitric oxide radical
一酸化窒素ラジカル
Yes Yes
Superoxide radical
スーパーオキサイドラジカル
No Yes
Hypochlorous acid
次亜塩素酸
Yes Yes
Peroxynitrite
ペルオキシ亜硝酸
Yes Yes
Peroxyl radical
ペルオキシルラジカル
No Yes

Table1. Overview of reactive oxygen species(ROS) scavenged by lipoic acid and dihydrolipoic acid.5)

5) Packer L., Kraemer L., Rimbach G.:「Molecular aspects of lipoic acid in the prevention of diabetes complications」 Nutrition , 17(10), 888-895 (2001)

Antioxidant network

小西徹也、小久保晋、松郷誠一:「機能性生理活性物質リポ酸の化学と新素材としての可能性」日本食品新素材研究会誌、8(2)51-64 (2005)

<Oxidation reduction : ORP>
Oxidized glutathione GSSG + 2e- → 2GSH: −0.24V
Ubiquinone (CoQ10) (Eo') :+0.10V
Lipoic acid ALA +2e- → DHLA : −0.32V

RALA itself has antioxidant activity and, furthermore, plays a important role to restore other antioxidants by coupling with DHLA. In this way, it keeps balance between oxidative stress and scavenging activity.

Stabilized R(+)-α lipoic acid

RALA is the only naturally occurring lipoic acid, but it is very unstable to form polymeric compound by external stimuli such as heat, photolysis and so on.
For this reason, it has been difficult to use RALA as a neutraceutical fields. Now in the market, racemic mixture, which contains R(+) form and S(-) form at the ratio 1:1, is used for supplement or health food.

Melting point
・Racemic mixture: 60-62℃
・R(+) form: 46-49℃

→ Therefore, stabilization of R(+)-α lipoic acid is needed.

We succeeded in developing stabilized RALA by using Cyclodextrin against heat and acidic condition.

Purpose

RALA scavenges ROS and free radicals, which cause oxidative stress.
The radical scavenging activity of ALA is well known, but the physico-chemical properties of RALA-CD complex has been scarcely reported so far.

In this study, aiming to evaluate physico-chemical properties of stabilized RALA, which is inclusion complex of RALA with Cyclodextrin(CD), its solubility into water and DPPH radical scavenging activity were investigated.

Experiment-Solubility into water

(Procedure)
1. Each amount of RALA-CD; 25mg, 50mg, 75mg, 100mg, is added to 5ml of non-ion water.
2. Mixed for 30min in the bath at temp.=70℃ and left until it is cooled to RT.
3. Filter the sample suspension using 0.2μm membrane.
4. Assay %RALA in the filtered sample solution by HPLC.

(Result)
There was the range in which RALA concentration increased linearly (Fig.3).

Fig.3 Solubility of RALA-CD

Experiment-Binding constant

RALA:CD=1:1 αCD βCD γCD
結合定数
K11(M-1)
UV-vis 135 2330 110

Table2. Binding constant of R(+)-α lipoate anion with CDs.

NaTCA:CD=1:1 αCD βCD γCD
K11(M-1) ST 0 1900 3100
NMR 0 2200 4800

Table3. Complexation of bile salts(NaTCA) by CD. Results obtained by NMR and Surface tension measurements. 7)

7) P.R. Cabrer et al. : 「Complexation of Bile Salts with α-, β- and γ-Cyclodextrins. Effects of the Cavity Size in the Complexation of Steroids.」

Fig.4 Plot of change of UV-vis spectra at λ=245nm of RALA solution at constant concentration (6mM), 25oC and pH7 in different concentrations of CDs.

Experiment-radical scavenging activity

(Procedure)
Sample solution, in which CD or RALA-CD is solved into MilliQ, is mixed with DPPH ethanol solution and soon after the reaction occurred, DPPH radical’s spectrum is measured by ESR.

(Result)
In this experimental system, CD showed no DPPH radical scavenging activity (Fig.5).

Fig.5 DPPH radical scavenging activity of CDs

Experiment-radical scavenging activity

(Result)
RALA-CD showed higher DPPH radical scavenging activity than R(+)-α lipoic acid sodium salt (NaRALA)(Fig.6).

  NaRALA RALA-αCD RALA-βCD RALA-γCD
IC30(mM) 24 7 (11) 3

Fig.6 DPPH radical scavenging activity of RALA-CD and IC30.

Summary

DPPH radical scavenging activity of R(+)-α lipoic acid-CD complex in this experimental system,

1. RALA-CD showed higher DPPH radical scavenging activity comparing to NaRALA . And IC30 order was
RALA-γCD > RALA-αCD > RALA-βCD >NaRALA
The smaller the binding constant of R(+)-lipoate anion with CD is, the higher the DPPH radical scavenging activity is.
Compared to binding constant of NaTCA with CD, the binding constant of lipoate anion with αCD or γCD was smaller. This result indicates that RALA- αCD or RALA- γCD can release RALA molecule in the intestines.
2. CD itself showed no DPPH radical scavenging activity.
This result indicates that the enhancement of RALA-CD complex radical scavenging activity compared to NaRALA is owing to CD complexation.