J Neural Transm (2008) 115: 35–42 DOI 10.1007/s00702-007-0821-6 Printed in The Netherlands

Euphorbia hirta reverses chronic stress-induced anxiety and mediates its action through the GABAA receptor benzodiazepine receptor-Cl2 channel complex H. Anuradha1 , B. N. Srikumar2, B. S. Shankaranarayana Rao2 , M. Lakshmana1 1 2

Department of Pharmacology, Government College of Pharmacy, Bangalore, India Department of Neurophysiology, National Institute of Mental Health and Neurosciences, Bangalore, India

Received 13 July 2007; Accepted 7 September 2007; Published online 18 December 2007 # Springer-Verlag 2007

Summary. Chronic stress is known to result in impairment of learning and memory and precipitate several affective disorders including depression and anxiety. Drugs of natural origin are known to possess several effects on the central nervous system and are emerging as promising alternative therapies. In this context, the hydroalcoholic extract of Euphorbia hirta (Eh) was evaluated for anxiolytic property in chronically stressed rats subjected to elevated plus maze (EPM) and open field test (OFT). Eh treatment (200 mg=kg, p.o.; seven days) showed marked anti-anxiety activity in chronic immobilization stress. In contrast, the forced swim stress-induced anxiety was only partially decreased by Eh. Co-treatment of rats with flumazenil (0.5 mg=kg, i.p.), bicuculline (1 mg=kg, i.p.) or picrotoxin (1 mg=kg, i.p.) resulted in a significant reduction of anxiolytic effect of Eh indicating that its actions are mediated through GABAA receptor-benzodiazepine receptorCl
channel complex. Thus, our studies indicate that Eh is a potential anxiolytic drug, which might be beneficial in the treatment of stress-induced anxiety disorders. Keywords: Anxiety; stress; herbal drugs; Euphorbia hirta; elevated plus maze; open field test

Introduction Stress is any perturbation of either physiological or psychological homeostasis. While appropriate stress responses help in resisting the diseases and are beneficial, severe and prolonged stress can be harmful (McEwen 2002). There is a wealth of evidence indicating that stress impairs learning and memory and precipitates several affective disorders including depression and anxiety (McEwen 2000). Earlier, we have demonstrated that 21 days of restraint stress impairs learning in T-maze (Sunanda et al. Correspondence: B. S. Shankaranarayana Rao, Department of Neurophysiology, National Institute of Mental Health and Neurosciences (NIMHANS), Hosur Road, PB 2900, Bangalore 560 029, India e-mail: [email protected], [email protected]

2000) and partially baited radial arm maze (Srikumar et al. 2006, 2007) tasks and several investigators have demonstrated that exposure of animals to stress produces anxiety (Adamec et al. 1999; Vyas et al. 2002; Govindarajan et al. 2006). Drugs obtained from natural plant sources are emerging as alternative=adjuvant therapies in the treatment of psychiatric disorders (Kienzle-Horn 2002; Carlini 2003). Extracts of Kava-kava (Piper methysticum) and St. John’s wort (Hypericum perforatum) are known to have clinical effectiveness in double blind, randomized placebo controlled trials in the treatment of anxiety and depression, respectively (Ernst 2002). Extracts of valerian, hops, lemon balm, passion flower are few other self-medicated herbal preparations employed for the prevention and treatment of neuropsychiatric disorders such as anxiety, sleep disorders, convulsions, cognitive impairment and depression (Beaubrun and Gray 2000). Whole plant or leaf extracts obtained from Euphorbia hirta (Eh) is known to have a multitude of actions in the biological system. Traditional preparations of this drug have shown antidiarrhoeal, antiamoebic and spasmolytic activities in vitro (Galvez et al. 1993; Tona et al. 2000). This plant has been used locally in many parts of Africa and Australia for the treatment of numerous diseases like hypertension, edema and malaria (Johnson et al. 1999; Tona et al. 1999b). Extracts of Euphorbia hirta Linn have been found to possess central analgesic, antipyretic, anti-inflammatory properties in addition to its central antidepressant, sedative and anxiolytic effects (Lanhers et al. 1990, 1991; Johnson et al. 1999). Although the active ingredients of this drug still remain to

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be determined (Galvez et al. 1993; Vijaya et al. 1995), the anxiolytic activity of this drug has been established in mice subjected to two-compartment, staircase and light=dark choice situation tests (Lanhers et al. 1990). Thus, it is known that stress induces anxiety and Eh demonstrates anxiolytic activity. However, it is not known whether Eh would produce anxiolytic activity in chronically stressed rats. Our present study addresses this issue. We administered Eh to rats subjected to two different stressors, chronic immobilization stress (CIS) and forced swim stress (FSS) and assessed the anxiety in the elevated plus maze (EPM) and the open field test (OFT). Further, in order to understand the mechanism underlying the anxiolytic action of this drug, we co-administered antagonists of the GABAA receptor-benzodiazepine receptor-Cl
channel complex with Eh and evaluated anxiety in the EPM. Materials and methods Experimental animals Male Wistar rats weighing 200–225 g (2–2.5 months old) obtained from Central Animal Research Facility at National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore was used for the experiments. The rats were housed and maintained in standard laboratory conditions. Food and water was provided ad libitum, unless specified otherwise in stress protocols. The experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996 and the institutional animal ethics committee approved the experimental protocols. All efforts were made to minimize both the suffering and the number of animals used.

Induction of stress Two types of stressors were applied to separate groups of animals namely, chronic immobilization (CIS), 2 h daily for 10 days (Vyas et al. 2002) and forced swim stress (FSS) (Roche et al. 2003). All the stress protocols were followed between 10 h and 12 h. CIS consisted of complete immobilization in rodent immobilization bags as described elsewhere (Vyas et al. 2002). For the FSS, the swimming apparatus measuring 60 cm in height and 45 cm in diameter was used. Water was filled to a level of 30 cm. The 30 cm depth allowed rats to swim or float without allowing their tails to touch the bottom of the tank. Temperature was maintained at 23  1 C. On the first day of stress, each animal was forced to swim for a period of 15 min. Later, individual animals were forced to swim for a period of 5 min regularly for 10 days. Immediately after the swim session, rats were removed from the tank, dried and put in a warming cage that was covered with towels for 10 min. Rats were then returned to their respective home cages (Roche et al. 2003).

Drug treatment Hydroalcoholic extract of the whole plant of Euphorbia hirta was supplied by the Himalaya Drug Company, Bangalore, India. Eh was dissolved in distilled water and was administered at a dose of 200 mg=kg per orally, for seven days, i.e. from the fifth through tenth day of the stress, one hour prior to stress and on the eleventh day. This dosage and duration of treatment was based on a preliminary dose response study with a poly-herbal formulation containing the extract of Eh, GS-02 (unpublished observations). Flumazenil, bicuculline and picrotoxin were obtained from Sigma Chemical Co.,

H. Anuradha et al. St. Louis, MO. Flumazenil (a benzodiazepine receptor antagonist; 0.5 mg= kg, i.p.) in 2% DMSO–water mixture, bicuculline (GABAA receptor antagonist; 1 mg=kg, i.p.) in 0.1 N HCl and picrotoxin (GABAA-gated chloride channel blocker; 1 mg=kg, i.p.) in 2% ethanol–water mixture was administered daily for seven days. Vehicle treated groups received only the respective vehicles through the respective routes. The data from different vehicle controls was pooled, since there was no significant difference between groups. For the mechanism of action studies, the antagonists were administered one hour prior to Eh treatment for seven days.

Behavioral testing The behavior of these rats in the EPM or OFT was recorded on the eleventh day in case of stress experiments or on the seventh day in case of mechanism of action studies. The behavioral experiments were conducted in a sound attenuated room designed for psychopharmacological studies. Rats were subjected to EPM test as described elsewhere (Vyas et al. 2002). Briefly, the plus maze was made up of wood and had two open arms and two closed arms, crossing each other in the form of a plus. The dimensions of closed arm were 50  10  50 cm and that of open arm were 50  10 cm with a central platform of 10  10 cm area. The whole apparatus was situated at a height of 50 cm above the ground (Vyas et al. 2002). Two hours after the last drug administration, rats were placed in the center square at the beginning of the test facing one of the closed arms. Behavior of animals in the EPM was video tracked using a web camera (Logitech quickcam 640  480 VGA, 24-bit) and stored for offline analysis. The whole apparatus was thoroughly cleaned using 5% ethanol before placing each rat in the maze. To encourage exploration into the open arms, the room was dimly lit. Animals were monitored in an adjacent room so that presence of the observer would not affect their behavior. The following parameters were observed for evaluation of behavior, percentage open arm entries, percent time spent in the open arms and number of head dips, reflecting anti-anxiety behavior. Closed arm entries and vertical rearing was measured as indication of exploratory activity of the rat. The total duration of the experiment for each rat was 5 min. Four hours after EPM testing, these animals were subjected to the OFT. For the OFT, the apparatus was made up of wood and had dimensions of 90  90  30 cm. It was divided into sixteen equal squares-four in the centre and twelve in the periphery. Rat was placed in the centre of the apparatus facing one of the sides at the start of the experiment and its locomotor activity was video tracked using a web camera (Logitech quickcam 640  480 VGA, 24-bit) and stored for offline analysis. Each rat was observed for a period of 5 min. The apparatus was thoroughly cleaned using 5% ethanol before the start of each trial. Criteria for evaluation were the crossings made in the central and peripheral squares, vertical rearing and defecation. Increased central to peripheral crossings and reduction of defecation was an indication of anti-anxiety effect of drug. Vertical rearing and total locomotion was used as an indication of exploratory activity. Statistical analysis The behavioral data was analyzed offline in a blind fashion by coding the files and the codes were broken only after the analysis was completed. Data was statistically analyzed by one-way analysis of variance (ANOVA) followed by a Tukey’s post-hoc test. Values are expressed as Mean  SEM, p < 0.05 was considered statistically significant.

Results Eh treatment reverses CIS-induced anxiety The effect of CIS and Eh treatment on performance in the EPM is presented in Fig. 1. Rats subjected to CIS exhibited

Reversal of stress-induced anxiety by Euphorbia hirta

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Fig. 1. Effect of chronic treatment of Euphorbia hirta (200 mg=kg, p.o.) on CIS-induced anxiety in the elevated plus maze. (A) % Open arm entries, (B) % time spent in the open arms, (C) number of head dips, (D) number of vertical rearings, (E) number of closed arm entries and (F) time spent in the closed arm. NC Normal control; CIS Rats subjected to 10 days of chronic immobilization stress; CIS þ Veh, CIS þ Eh Rats subjected to CIS followed by vehicle or Euphorbia hirta 200 mg=kg, p.o. treatment, respectively. Values represent Mean  SEM (n ¼ 6); ###p < 0.001 vs. normal control;  p < 0.001 vs. CIS group; one-way ANOVA followed by a Tukey’s post-hoc test

very poor percent open arm entries [F3,20 ¼ 174.9; p< 0.001] (Fig. 1A), percent time spend in the open arm [F3,20 ¼ 36.02; p < 0.001] (Fig. 1B), and number of head dips [F3,20 ¼ 22.16; p< 0.001] (Fig. 1C). Eh treatment to stressed

rats restored the stress-induced decrease in percent open arm entries, percent time spent in the open arms and the number of head dips (Fig. 1A–C). Further, CIS rats also demonstrated a decreased number of closed arm entries

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Fig. 2. Effect of chronic treatment of Euphorbia hirta (200 mg=kg, p.o.) on CIS-induced anxiety in the open field test. (A) Number of crossings in the center, (B) number of crossings in the periphery, (C) total number of crossings, (D) number of vertical rearings and (E) defecation. NC Normal control; CIS Rats subjected to 10 days of chronic immobilization stress; CIS þ Veh, CIS þ Eh Rats subjected to CIS followed by vehicle or Euphorbia hirta 200 mg=kg, p.o. treatment respectively. Values represent Mean  SEM (n ¼ 6); ##p < 0.01; ### p < 0.001 vs. normal control;  p < 0.01;  p < 0.001 vs. CIS group; one-way ANOVA followed by a Tukey’s post-hoc test

[F3,20 ¼ 11.86; p< 0.001] (Fig. 1E) and increased time spent in the closed arm [F3,20 ¼ 12.46; p< 0.001] (Fig. 1F). However, the number of vertical rearings was unaltered by CIS [F3,20 ¼ 2.08; p> 0.05] (Fig. 1D). In CIS rats subjected to Eh treatment, there was an increase in the closed arm entries and decrease in the time spent in the closed arms with no change in the number of vertical rearings (Fig. 1D–F). Figure 2 shows the effect of CIS and Eh on performance in the OFT. CIS rats showed decreased number of crossings in center [F3,20 ¼ 12.38; p < 0.001] (Fig. 2A), decreased

peripheral crossings [F3,20 ¼ 17.03; p< 0.001] (Fig. 2B) and decreased total crossings [F3,20 ¼ 19.49; p < 0.001] (Fig. 2C). Further CIS also decreased the number of vertical rearings [F3,20 ¼ 21.79; p < 0.001] (Fig. 2D) and increased defecation [F3,20 ¼ 26.93; p< 0.001] (Fig. 2E). Eh treatment significantly restored the CIS induced decrease in the number of central, peripheral and total crossings, and vertical rearings (Fig. 2A–D). CIS-induced increase in defecation was also reversed by Eh treatment (Fig. 2E). To assess any adverse effect of Eh treatment on

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Reversal of stress-induced anxiety by Euphorbia hirta Table 1. Effect of Eh treatment to rats subjected to forced swim stress (FSS) on EPM and OFT behavior Test

Behavior

EPM

1. 2. 3. 4. 5. 6.

% of open arm entries % of time spent in the open arms No. of head dips No. of vertical rearings No. of closed arm entries Time spent in the closed arms (sec)

OFT

1. 2. 3. 4. 5.

No. of central crossings No. of peripheral crossings Total No. of crossings No. of vertical Rearings Defecation

NC

FSS þ Veh

FSS ##

FSS þ Eh ##

16.36  1.26 5.88  0.79 5.67  0.33 11.50  2.26 6.67  0.76 208.33  7.83

4.58  2.27 1.02  0.68### 6.00  1.86 14.83  2.20 4.83  0.95 271.67  9.40###

5.15  2.38 1.01  0.69### 6.33  1.43 16.67  1.48 5.17  0.65 258.00  7.45##

10.08  4.86 5.80  2.99 5.17  0.87 13.00  1.71 4.83  0.48 234.67  5.69

2.50  0.43 38.33  3.24 40.83  3.22 10.50  0.96 0.67  0.21

0.83  0.54 33.50  6.22 34.33  6.24 11.00  0.73 2.83  0.17###

1.67  1.31 40.50  7.82 42.17  8.62 10.33  0.80 2.50  0.22###

0.67  0.42 38.50  7.59 39.17  7.62 12.00  0.58 1.50  0.43

NC Normal control; FSS rats subjected to forced swim stress; FSS þ Veh rats subjected to forced swim stress and vehicle treatment; FSS þ Eh rats subjected to forced swim stress and Euphorbia hirta treatment. Data is represented as mean  SEM. ##p < 0.01; ###p < 0.001 vs. NC group;  p < 0.05 vs. FSS group; one-way ANOVA followed by a Tukey’s post-hoc test.

the health of the CIS animals, body weight was measured. Eh treatment to CIS animals did not have any apparent adverse effect on the health of these rats. Normal control animals had an average of 9.98% increase in body weight over a period of 10 days (during which CIS group of rats underwent CIS). In CIS rats, this was decreased (0.67%), while in CIS þ Eh group of rats, there was a partial restoration (2.85%). However, this was not statistically significant.

toxin (GABAA-gated chloride channel blocker) significantly attenuated the Eh-induced increase in percent open arm entries and percent time spent in the open arms (Fig. 3A and B). However, Eh-induced increase in the number of head dips were not affected by bicuculline or picrotoxin treatment (Fig. 3C). Further, the number of vertical rearings, number of closed arm entries and time spent in the closed arm were unaltered by any of the antagonist cotreatment (Fig. 3D–F).

Effect of Eh treatment on FSS-induced anxiety

Discussion

Rats subjected to 10 days of forced swim stress displayed higher levels of anxiety compared to normal control rats (Table 1). The percent open arm entries [F3,20 ¼ 10.65; p< 0.001] and percent time spent in the open arms [F3,20 ¼ 15.19; p< 0.002] were significantly decreased and time spent in the closed arms was increased [F3,20 ¼ 13.02; p < 0.001] (Table 1) in FSS rats. However, the number of head dips, vertical rearings and closed arm entries were not altered by FSS (Table 1). Unlike CIS, treatment with Eh did not reverse the anxiogenic effect of FSS in the EPM (Table 1). In the OFT, FSS did not demonstrate any anxiety except in defecation [F3,20 ¼ 12.70; p< 0.001] and this increase in defecation was reversed by Eh treatment (Table 1).

The various therapeutic actions of Eh have already been documented through various in vivo, in vitro and cell line studies (Vijaya et al. 1995; Johnson et al. 1999; Tona et al. 2004). Although the exact constituents mediating its benefits are still under debate, extracts of this drug has long been known to be exploited by many Australian and African tribes for its medicinal values (Tona et al. 1999a, b, 2000, 2004). Lanhers et al. (1990, 1991) have reported analgesic, sedative, antidepressant and anxiolytic properties of Eh. Data obtained from our study indicate the powerful anxiolytic action of Eh and is in accordance with earlier reports on anxiolytic action of Eh in mice. However, this is the first study in our knowledge demonstrating the anxiolytic effects of this drug in EPM and OFT paradigms in rats. Numerous studies have been carried out to understand the role of various lifestyle factors contributing to stress, its biological ramification as well as its clinical implications such as in the development of depression and anxiety (McEwen 2000; Vyas et al. 2002). In our earlier studies, we have demonstrated that stress can lead to behavioral abnormalities such as learning and memory impairment in a T-maze (Sunanda et al. 2000), radial arm maze (Srikumar et al. 2006) tasks and anxiety in the EPM (Vyas et al.

GABAA receptor-benzodiazepine receptor-Cl
channel complex mediates the anxiolytic action of Eh The effect of flumazenil, bicuculline and picrotoxin on Eh’s anxiolytic effects is shown in Fig. 3. Co-treatment with flumazenil appreciably blocked the Eh-induced increase in percent open arm entries, percent time spent in the open arms and number of head dips (Fig. 3A–C). Co-treatment with bicuculline (GABAA receptor antagonist) or picro-

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Fig. 3. Anti-anxiety effect of chronic treatment of Euphorbia hirta 200 mg=kg, p.o. in rats subjected to elevated plus maze and the mechanism of antianxiety action of the drug. (A) % Open arm entries, (B) % time spent in the open arms, (C) number of head dips, (D) number of vertical rearings, (E) number of closed arm entries and (F) time spent in the closed arm. NC Normal control; Eh rats subjected to 7 days of treatment with Euphorbia hirta 200 mg=kg, p.o. Eh þ Flu, Eh þ Bic, Eh þ Pic rats treated with Euphorbia hirta 200 mg=kg, p.o. and flumazenil (0.5 mg=kg, i.p.), bicuculline (1 mg=kg i.p.) and picrotoxin (1 mg=kg i.p.), respectively. Values represent Mean  SEM (n ¼ 6); ##p < 0.01; ###p < 0.001 vs. normal control;  p < 0.001 vs. Eh group; one-way ANOVA followed by a Tukey’s post-hoc test

2002). Rats, when subjected to CIS showed anxiogenic responses in the EPM and OFT (Figs. 2 and 3), reiterating earlier findings (Padovan et al. 2000; Vyas et al. 2002).

Antidepressants (selective serotonin reuptake inhibitors, SSRIs) are still the mainstay in the pharmacotherapy of stress disorders and prognosis is far from satisfactory (Hamner

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Reversal of stress-induced anxiety by Euphorbia hirta

et al. 2004). In this study, 7 days treatment of hydroalcoholic extract of Euphorbia hirta reversed the CIS-induced anxiety (Figs. 1 and 2). This is an important finding since herbal drugs are thought to be a good alternative or supplement in the therapy of psychiatric disorders. However, it has to be borne in mind that there are not enough controlled double-blind clinical trials (Ernst 2002). Extracts of Kavakava (Piper methysticum) have been shown to be useful clinically in the treatment of anxiety. In animal models of stress-induced anxiety, very few studies have examined the usefulness of herbal drugs. Kava-kava extract has been shown to be anxiolytic in the chick social separation stress procedure, an anxiolytic bioassay and it was found that the fraction containing highest concentration of dihydrokavain demonstrated anxiolytic properties (Smith et al. 2001; Feltenstein et al. 2003). Our finding that Eh treatment ameliorates CIS-induced anxiety is in accordance with these reports that drugs of natural origin can be useful in stress-induced anxiety. In the EPM, besides decreased open arm exploration, CIS rats exhibited decreased closed arm entries and increased time spent in the closed arm (Fig. 2E and F). The open field test, in addition to being used to study anxiety is a good model for depression (Kulkarni 1977). Decreased crossings in the periphery and total number of crossings are an indication of depression. Eh treatment restored all these functions, indicating an additional antidepressant activity. This is important because anxiety and depression can present together and some of the antidepressants are also good anxiolytics. Although, a direct effect on locomotor activity in mediation of the antidepressant activity of Eh cannot be ruled out, this is less likely because neither Eh per se nor co-treatment of antagonists altered the closed arm entries or time spent in the closed arms (Fig. 3E and F). However, these results are only indicative and additional experiments would be needed to demonstrate unequivocally that Eh is an antidepressant. Further, the effects of acute versus chronic Eh treatment and the effect of Eh on the acute stress brought on by a behavioral test such as EPM or OFT need to be addressed in the future studies. Drugs like diazepam that is used as anxiolytic act through the GABAA receptor-benzodiazepine receptorCl
channel complex. Euphorbia hirta also appears to mediate its anxiolytic action through this complex since all of the three antagonists, flumazenil, bicuculline and picrotoxin inhibited Eh-induced increase in open arm exploration. The effects of these antagonists on EPM behavior are similar to previous studies that have used these antagonists to study the mechanism of action of anti-anxiety drugs (Ferris et al. 2001; Rao et al. 2003; Langen et al. 2005).

Rats subjected to 10 days of FSS demonstrated anxiety in the EPM. This finding is in accordance with earlier reports of swim stress-induced anxiety in the EPM (Heinrichs et al. 2002; Chaki et al. 2004, 2005). Contrary to our findings in CIS, the anxiolytic activity of Eh in the plus maze was found to be much reduced in rats subjected to FSS. We also observed that this drug was not effective in the OFT. Interestingly, we observed that diazepam treatment also did not seem to affect FSS-induced anxiety as much as it did to CIS-induced anxiety (unpublished observations). It has been established that anxiety can result from an alteration in serotonin levels, which is the prime reason for the use of selective serotonin reuptake inhibitors (SSRI) in therapeutic regimens (Roche et al. 2003). FSS is known to result in the alteration of serotonin levels in the brain. These serotonergic pathways innervating structures such as the frontal cortex, amygdala, hypothalamus and hippocampus are activated by anxiogenic stimuli including psychosocial stress (Wright et al. 1992). Although the exact mechanism is still unknown, prolonged stress is believed to preferentially desensitize presynaptic 5HT1A receptors and cause an indirect disturbance of the benzodiazepine-GABA receptors, which may further potentiate anxiety (Millan 2003). Expression of GABAA receptors and their subunits are also known to be modified by stress resulting in altered behavior in various experimental models of anxiety (Pericic et al. 2001) which could contribute to the inefficacy of anxiolytic drugs acting via this receptor. However, it remains to be established whether these mechanisms are responsible for the lack of efficacy of Eh in FSS-induced anxiety. In conclusion, the present study clearly demonstrates the anxiolytic potential of Euphorbia hirta particularly in CISinduced anxiety. And the actions of Eh could be mediated at least in part through the GABAA receptor-benzodiazepine receptor-Cl
channel complex.

Acknowledgements This study was partially funded by The Himalaya Drug Company, Bangalore, India. We acknowledge Salesworth Education Foundation, Bangalore, India for a scholarship to A. H.

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