GABA_A Receptor Modulators from the Chinese Herbal Drug Junci Medulla – The Pith of Juncus effusus

• Abstract
• Material and Methods
• Supporting information
• Acknowledgements
• References

Abstract

The gamma-amino butyric acid (GABA) type A (GABA_A) receptor represents a crucial target for clinical agents in the treatment of anxiety and insomnia. Using the two-microelectrode voltage clamp technique on recombinant α ₁ β ₂ γ _2S GABA_A receptors, effusol (1) and dehydroeffusol (2) were isolated in a bioactivity-guided approach from the pith of Juncus effusus L. Both compounds concentration-dependently enhanced GABA induced chloride currents (I_GABA) by a maximum 188 ± 20 (1) and 239 ± 18 % (2), independent of the benzodiazepine (BZ) binding site. This activity on the GABA_A receptor may explain the traditional use of J. effusus as a sedative and anxiolytic agent in Chinese medicine.

Anxiety and insomnia are central nervous system disorders with a high prevalence, especially in developed countries. It is estimated that over 16 % of the world's population suffer from some form of anxiety disorder [1], and more than 30 % frequently experience sleep disturbances [2].

A variety of drugs such as the commonly prescribed benzodiazepines (BZs) are available for the treatment of both disorders [3]. Although their use is safe compared to the now mostly obsolete barbiturates, they can generate a range of adverse effects such as lethargy, daytime sedation, amnesia, or involuntary muscle relaxation. Furthermore, long-term use of BZs, as would be necessary for the treatment of chronic insomnia or anxiety, leads to tolerance against the anxiolytic and sedative effects. Moreover, dependence and withdrawal symptoms can occur, which further limits their use [4]. This high risk of adverse effects combined with the limited prescription time of BZs and other conventional drugs [5] might be one of the reasons why an increasing percentage of insomnia and, particularly, anxiety patients rely on herbal preparations [6]. Although the use of herbal drugs is not free of unwanted side effects, they are still considered safe, well tolerated, and effective due to the experience gained from long-term usage. Nonetheless, scientific evaluation of their activity and underlying molecular mechanisms are rare [7], [8].

A Chinese herbal medicine frequently prescribed against childhood insomnia and night terrors is Junci Medulla – the pith of Juncus effusus L. [9]. Several compound classes were detected in J. effuses, such as phenanthrene derivatives, cyclo-artane type triterpenes, flavonoids, phenolic acid derivatives, as well as essential oil components, β-sitosterol and α-tocopherol, and could already be isolated from J. effusus and related species [9], [10]. Studies regarding their pharmacological activity mainly focus on the phenanthrene derivatives, which displayed anti-algal, antimicrobial, phototoxic, and cytotoxic activities, whereby similar compounds derived from other plant families were studied for their anticancer, spasmolytic, and anticoagulative activities, as reviewed by Kovacs et al. [11]. The anxiolytic and sedative activities of one compound from J. effusus, dehydroeffusol, have recently been established in vivo [12], whereas the underlying mechanisms are still unknown.

A key target for sedation and anxiolysis is the γ-amino butyric (GABA) type A (GABA_A) receptor, which is the major inhibitory neurotransmitter receptor in the mammalian brain. The GABA_A receptor is a ligand-gated ion channel that transmits its inhibitory signals due to the opening of a chloride channel and subsequent hyperpolarization of the neuronal membrane. The heteropentameric GABA_A receptor can be assembled from a range of 19 different subunits (α _1–6, β _1–3, γ _1–3, δ, ε, π, θ, and ρ _1–3). From a large number of possible receptor assemblies, only some have been found in vivo in the mammalian brain until now, with the α ₁ β ₂ γ ₂ receptor being the most abundant [13]. Interestingly, the GABA_A receptor seems to be a valid target for natural products, since many plant derived compounds are able to influence the GABA_A receptor [14].

In the present study, we investigated the effects of J. effusus extracts on recombinant α ₁ β ₂ γ _2S GABA_A receptors expressed in Xenopus laevis oocytes in a bioassay-guided manner. Potentiation of GABA-induced chloride currents (I_GABA) on the GABA_A receptor was determined using the two-electrode voltage clamp technique by means of an automated fast perfusion system.

In a preliminary extract screening of 4 J. effusus extracts of different polarity, the EtOAc extract displayed the highest ability to potentiate I_GABA (170 ± 24 %, n = 3, [Fig. 1 A]) and therefore was selected for further bioactivity-guided isolation. The EtOAc extract was subjected to vacuum liquid chromatography (VLC) on silica gel, using different mixtures of petroleum ether and EtOAc as eluents. The resulting fractions were screened for their activity on the GABA_A receptor, revealing that only VLC fractions 4 and 5 could significantly enhance I_GABA to an almost similar extent (153 ± 48 % and 127 ± 23, [Fig. 1 B]). VLC fractions 4 and 5 were subfractionated by column chromatography on Sephadex LH-20 using EtOAc or EtOAc : MeOH (95 : 5) as the mobile phase. Five subfractions could be gained from VLC fraction 5 for further activity screening, with subfraction 5–3 displaying the highest activity (266 ± 58, [Fig. 1 C]). TLC screening revealed one major blue fluorescent zone. The same compound was isolated from VLC fraction 4 on Sephadex LH-20 using EtOAc : MeOH (95 : 5) as the mobile phase. The compound was identified by MS, 1D- and 2D-NMR experiments as effusol (1, ≥ 96 % purity) and spectral data was in good accordance with the data published in the literature [15]. Additionally, another predominant constituent was isolated from cumulative fraction 5–5, and its structure was determined as dehydroeffusol (2, ≥ 98 % purity) by MS and NMR experiments and compared to existing spectral data ([Fig. 2]) [16].

To gain further insight into the molecular mechanism of action, both compounds were investigated for their ability to potentiate I_GABA. Compounds 1 and 2 displayed a concentration-dependent effect with a maximal potentiation of 188 ± 20 % (EC₅₀ = 31 ± 8 µM, n _H = 2.5 ± 0.6, n = 7) for 1 and 239 ± 18 % (EC₅₀ = 27 ± 6 µM, n _H = 1.4 ± 0.2, n = 4) for 2 ([Fig. 3]). Furthermore, the interaction of 1 and 2 with the benzodiazepine (BZ) binding site was determined, since BZs are the most commonly prescribed drugs used in the treatment of sleep and anxiety disorders. I_GABA enhancement induced by 2 (50 µM) could not be blocked by the co-application of the BZ antagonist flumazenil (1 µM, [Fig. 4 A] and [B]). Moreover, the co-application of 2 (50 µM) with diazepam (0.3 µM) leads to an additive increase in I_GABA enhancement ([Fig. 4 C] and [D]). Similar results were obtained for 1 (Fig. 1S in Supporting Information), indicating that both components do not mediate their I_GABA potentiating effects via the BZ binding site. Both compounds were not as active as the positive control diazepam. These findings are in good accordance with the work of Bhattacharyya [15], where dehydroeffusol was found to have anxiolytic and sedative effects in mice, albeit lower than those of diazepam. However, the compound did not exhibit the typical muscle relaxant effects of diazepam suggesting a binding site distinct from the diazepam binding site. This again supports our results from the in vitro test, where we could not block the activity of the phenanthrene derivatives with flumazenil.

To summarize, we found scientific evidence for the traditional use of J. effusus on the molecular level which is at least partly due to 1 and 2. Moreover, the ability of 2 to modulate I_GABA may explain the sedative and anxiolytic effects of this compound in vivo.

Material and Methods

Junci medulla, the pith of J. effusus L., was purchased from Plantasia (lot.: 660 558). A voucher specimen (No. JS-07–11-JE) is deposited in the Department of Pharmacognosy, University of Vienna.

For a preliminary activity screening, 50 g of ground plant material were extracted successively with solvents of increasing polarity (petroleum ether, EtOAc, methanol, and water), with the EtOAc extract showing the highest activity. Thus, 2.0 kg of ground pith were extracted exhaustively with EtOAc to yield 13.4 g of a dark brown residue. Ten g of this residue were subjected to VLC on silica gel (65 × 3.5 cm, i. d.) using petroleum ether : EtOAc mixtures (1 L each) in ratios of 9 : 1 (pre-run), 8.5 : 1.5, 8 : 2, 7.5 : 2.5, 6.5 : 3.5, 6 : 4, 5 : 5, 3 : 7, 1.5 : 8.5, 0 : 10, from which the latter 9 were used for activity testing. Fraction 5 (petroleum ether : EtOAc 6.5 : 3.5, 0.96 g) was further fractionated on a Sephadex LH-20 column (65 × 1.5 cm, i. d.) using EtOAc as eluent (5 mL fractions, 10 mL/h flow) resulting in six subfractions. Subfraction 5–3 displayed one prominent blue fluorescent zone in the TLC screening, which was identified as 1 by MS and NMR experiments and data comparison with the literature. The crystalline residue of cumulative fraction 5–5, which was gained from recrystallization in EtOAc, was purified by semipreparative HPLC on RP-18. Using a water (solvent A) – methanol (solvent B) gradient from 60 to 80 % B in 15 minutes with a flow rate of 26.7 mL/min, compound 2 (9 mg, purity ≥ 98 %) eluted at 9.9 min. VLC fraction 4 (petroleum ether : EtOAc 6 : 4, 0.68 g), which also contained 1 and 2, was fractionated on Sephadex LH-20 (70 × 1 cm, i. d.) using EtOAc : MeOH (95 : 5) as eluent to yield 220 fractions (5 mL fractions, ∼ 10 mL/h flow). Compound 1 was eluted as a pure compound in subfraction 4–150, to yield 14 mg (purity ≥ 96 %).

NMR spectra were recorded in CD₃Cl or d ₆-DMSO on a Bruker Avance 500 spectrometer at 500 MHz (¹H NMR) and 125 MHz (¹³C NMR). ESIMSⁿ spectra were obtained on a 3D-ion trap mass spectrometer (HCT; Bruker Daltonics) and recorded in positive and negative ion modes. Semipreparative HPLC of compound 2 was performed using a Shimadzu system consisting of two LC-8A pumps, a SPD-M20A diode array detector, a FRC-10A fraction collector, and a CBM-20A interface, engaging a Nucleosil C-100 RP-18 column (250 × 21 mm, i. d., 5 µm; Machery Nagel). TLC was carried out on precoated silica gel plates KG60 F₂₅₄ (Merck) with toluene : EtOAc : MeOH : acetic acid in a ratio of 35 : 10 : 1 : 2.5 as the mobile phase. Zones were detected in visible light and under UV₃₆₆ after spraying with anisaldehyde sulphuric acid reagent and heating for 5 to 10 min at 105 °C.

Preparation of stage V–VI oocytes from Xenopus laevis (NASCO) and injection of cRNA was performed as previously described [17]. The animal experiments were approved by the Austrian Federal Ministry for Science and Research. One to 3 days after cRNA injection, GABA_A receptor expressing oocytes were screened for GABA-evoked currents as previously described [18]. More details are given in Supporting Information.

Potentiation of the GABA-induced chloride current (I_GABA) in percent was defined according to the formula:

I_GABA (%) = [I_{(GABA + Comp)}/I_GABA − 1] × 100

Where I_(GABA+Comp) is the current response in the presence of a given compound, and I_GABA is the control GABA-induced chloride current. Origin Software (OriginLab Corporation) was used to generate concentration-response curves. Data were fitted by nonlinear regression analysis to the equation 1/(1+(EC₅₀/[compound]n _H), where EC₅₀ is the concentration of the compound that increases the amplitude of the GABA-evoked current by 50 % of the compound-induced maximum response, and n _H is the Hill coefficient. Responses were graphed as mean ± standard error (S. E. M.) from at least three oocytes out of ≥ two different batches. Statistical significance was calculated using one-way ANOVA followed by Tukey's test with a confidence interval of p < 0.05.

Supporting information

Detailed information on the pharmacological assays, the benzodiazepine binding site experiment of 1 (1S), and ¹H- and ¹³C-NMR data of 1 (2S and 3S) and 2 (4S and 5S) are available as Supporting Information.

Acknowledgements

This work was supported by the University of Vienna (Initiative Group “Molecular Drug Targets”) and P 19614-B11 (S. H.); this project was also supported in part by the Sino-Austria-Project, supported by the Austrian Federal Ministry of Science and Research and Federal Ministry of Health.

Conflict of Interest

All authors declare no conflict of interest.

References

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Univ. Prof. Mag. Dr. Brigitte Kopp

Department of Pharmacognosy
University of Vienna

Althanstraße 14

1090 Vienna

Austria

Phone: +43 14 27 75 52 55

Fax: +43 14 27 75 52 56

Email: brigitte.kopp@univie.ac.at

References

• 1 Somers J M, Goldner E M, Waraich P, Hsu L. Prevalence and incidence studies of anxiety disorders: a systematic review of the literature.  Can J Psychiatry. 2006;  51 100-113
  Search in Google Scholar
• 2 Roth T. Sleep and society.  Sleep Med. 2009;  10 S1-S2
  Search in Google Scholar
• 3 Stevens J C, Pollack M H. Benzodiazepines in clinical practice: consideration of their long-term use and alternative agents.  J Clin Psychiatry. 2005;  66 21-27
  Search in Google Scholar
• 4 Lader M, Tylee A, Donoghue J. Withdrawing benzodiazepines in primary care.  CNS Drugs. 2009;  23 19-34
  Search in Google Scholar
• 5 Sullivan S S. Insomnia pharmacology.  Med Clin North Am. 2010;  94 563-580
  Search in Google Scholar
• 6 Roy-Byrne P P, Bystritsky A, Russo J, Craske M G, Sherbourne C D, Stein M B. Use of herbal medicine in primary care patients with mood and anxiety disorders.  Psychosomatics. 2005;  46 117-122
  Search in Google Scholar
• 7 Kinrys G, Coleman E, Rothstein E. Natural remedies for anxiety disorders: potential use and clinical applications.  Depress Anxiety. 2009;  26 259-265
  Search in Google Scholar
• 8 Salter S, Brownie S. Treating primary insomnia – the efficacy of valerian and hops.  Aust Fam Physician. 2010;  39 433-437
  Search in Google Scholar
• 9 Bensky D, Clavey S, Stoeger E. Chinese herbal medicine: Materia Medica. 3rd edition. Seattle: Eastland Press; 2006: 1311
  Search in Google Scholar
• 10 Della Greca M, Fiorentino A, Monaco P, Previtera L. Cycloartane triterpenes from Juncus effusus.  Phytochemistry. 1994;  35 1017-1022
  Search in Google Scholar
• 11 Kovacs A, Vasas A, Hohmann J. Natural phenanthrenes and their biological activity.  Phytochemistry. 2008;  69 1084-1110
  Search in Google Scholar
• 12 Liao Y J, Zhai H F, Zhang B, Duan T X, Huang J M. Anxiolytic and sedative effects of dehydroeffusol from Juncus effusus in mice.  Planta Med. 2011;  77 416-420
  Search in Google Scholar
• 13 Olsen R W, Sieghart W. GABA(A) receptors: subtypes provide diversity of function and pharmacology.  Neuropharmacology. 2009;  56 141-148
  Search in Google Scholar
• 14 Johnston G A, Hanrahan J R, Chebib M, Duke R K, Mewett K N. Modulation of ionotropic GABA receptors by natural products of plant origin.  Adv Pharmacol. 2006;  54 285-316
  Search in Google Scholar
• 15 Bhattacharyya J. Structure of effusol: A new phenolic constituent from Juncus effusus.  Experientia. 1980;  36 27-28
  Search in Google Scholar
• 16 Shima K, Toyota M, Asakawa Y. Phenanthrene derivatives from the medullae of Juncus effusus.  Phytochemistry. 1991;  30 3149-3151
  Search in Google Scholar
• 17 Khom S, Baburin I, Timin E N, Hohaus A, Sieghart W, Hering S. Pharmacological properties of GABA(A) receptors containing gamma1 subunits.  Mol Pharmacol. 2006;  69 640-649
  Search in Google Scholar
• 18 Baburin I, Beyl S, Hering S. Automated fast perfusion of Xenopus oocytes for drug screening.  Pflugers Arch. 2006;  453 117-123
  Search in Google Scholar

Univ. Prof. Mag. Dr. Brigitte Kopp

Department of Pharmacognosy
University of Vienna

Althanstraße 14

1090 Vienna

Austria

Phone: +43 14 27 75 52 55

Fax: +43 14 27 75 52 56

Email: brigitte.kopp@univie.ac.at

• Supplementary Material