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Phytomedicine 18 (2011) 836– 842

Contents lists available at ScienceDirect

Phytomedicine

jou rn al hom epage: www.elsev ier .de /phymed

ffect of Bacopasides on acquisition and expression of morphine tolerance

halid Rauf ∗, Fazal Subhan, Muzaffar Abbas, Amir Badshah, Ihsan Ullah, Sami Ullahepartment of Pharmacy, University of Peshawar, Peshawar, Pakistan

r t i c l e i n f o

eywords:acopa monnieriacopasidesoleranceorphineigh performance liquid chromatorgraphy

HPLC)

a b s t r a c t

Opioids are extensively used for the management of both chronic malignant and non malignant pains.One major serious limitation associated with chronic use of opioids is the development of tolerance toits analgesic effect. The effect of Bacopa monnieri, a renowned ayurvedic medicine for acquisition andexpression of morphine tolerance in mice, was investigated. Bacopa monnieri, n-Butanol fraction wasanalyzed on High performance liquid chromatography (HPLC), for Bacopaside A major components i.e.Bacoside A3, Bacopaside ll and Bacosaponin C. Antinociceptive effect of n-Butanol extract of Bacopa mon-nieri (n Bt-ext BM) (5, 10 and 15 mg/kg) was assessed on hot plate. Effect of different doses of n Bt-extBM on morphine antinociception was also assessed. n Bt-ext BM was also screened for development oftolerance to antinociceptive effect of Bacopa monnieri by administering 15 mg/kg n Bt-ext BM for seven

days. Tolerance to morphine analgesia was induced in mice by administering intraperitoneally (I.P.)20 mg/kg morphine twice daily for five days. Acute and Chronic administration of 5, 10 and 15 mg/kg nBt-ext BM significantly reduced both expression and development of tolerance to morphine analgesia inmice. Additionally, Bacopa monnieri was found to enhance antinociceptive effect of morphine in intol-erant animals. However, no tolerance to Bacopa monnieri antinociceptive effect was observed in sevendays treatment schedule. These findings indicate effectiveness of Bacopa monnieri for management ofmorphine tolerance.

ntroduction

Clinical management of chronic malignant and non malignantains is still a major challenge to the medical communities acrosshe globe, with an economic impact of around 100 billion annuallyRenfrey et al. 2003). Opioids are still the drugs of choice for the

anagement of chronic refractory malignant and non malignantains. One major limitation that narrows the therapeutic value ofpioids for the management of chronic pains is development ofolerance that needs escalating doses to produce same analgesicffects consequently causing cumbersome side effects, includingoor quality of life, sedation, constipation, and respiratory depres-ion. There is growing concern among clinicians regarding clinicalse of opioids, their tolerance, subsequent addiction and abusemong patients taking opioids. As there are meager chances ofeplacement of opioid in near future by some new magical panacea

or chronic pains, the medical communities are confronting withhallenges of opioid tolerance, addiction and non medical useWalwyn et al. 2010).

∗ Corresponding author. Tel.: +92 091 9216750; mobile: +92 3459824468.E-mail address: khalidrauf@upesh.edu.pk (K. Rauf).

944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.phymed.2011.01.023

© 2011 Elsevier GmbH. All rights reserved.

As plants provide a prolific source of chemically diverse bioac-tive compounds, there is renewed interest in phytomedicines withcentral effects for the management of opioid tolerance and otherdrug of abuse management (Abenavoli et al. 2009; Almeida et al.2001; Muscoli et al. 2010) and many plants have been prelimi-narily screened for this purpose and a lot of work is underway,with some encouraging and significant results (Ernst 2006; Liuet al. 2009). Bacopa monnieri (L.) Wettst. (Brahmi) is perennial herbfrom Scrophulariaceae family, found in marshy places across Asiaand Europe including Pakistan (Qureshi and Raza Bhatti 2008).Bacopa monnieri having reputed nootropic effects has been exten-sively used for various diseases in ayurvedic system of medicinesince time immemorial (Russo and Borrelli 2005). The plant hasmany active moieties but its major bioactive putative component isBacoside A mainly responsible for its neuropharmacological effects(Deepak et al. 2005). Bacoside A is a mixture of major compo-nents, Bacoside A3 (C47H76O18) Fig. 1, Bacopaside II (C47H76O18)Fig. 2 and Bacosaponin C (C46H74O17) Fig. 3 and isomer of Bacos-aponin C (Deepak et al. 2005). The plant has been reported to haveanalgesic (Vohora et al. 1997) anxiolytic (Calabrese et al. 2008),

antidepressant (Sairam et al. 2002), anti-inflammatory (Channaet al. 2006) hepatoprotective (Sumathi and Nongbri 2008) cura-tive effect in ulcer (Sairam et al. 2001) and memory enhancingproperties (Uabundit et al. 2010). The plant has been found to be

K. Rauf et al. / Phytomedicine 18 (2011) 836– 842 837

ure of

sceti2

twmgo

Fig. 1. Struct

afe and well tolerated in humans as herbal products are commer-ially available for enhancing memory in old age patients (Pravinat al. 2007). Bacopa monnieri has been reported to have a pro-ective effect against morphine toxicity at various organs level,ncluding, liver, kidneys and brain (Sumathi and Niranjali Devaraj009).

The aim of this study was, firstly to quantify Bacoside A concen-ration in locally available Bacopa monnieri, secondly to investigate

hether tolerance develops to the antinociceptive effects of Bacopaonnieri with its repeated administration, and thirdly to investi-

ate the effects of Bacopa monnieri on acquisition and expressionf morphine tolerance in mice.

Fig. 2. Structure of B

Bacoside A3.

Materials and methods

Animals

Balb-C mice (25–30 g) of either sex, bred in the department ofPharmacy, University of Peshawar animal house were used in allexperiments. Mice were housed at 22 ± 2 ◦C under a 12 h light /12h dark cycle, having free access to food and water. All experiments

were performed between 08.00 h and 05.00 h of the day. The ani-mals were acclimatized to the laboratory conditions for at least2 h before the start of the experimental procedure. All experimentswere performed in accordance with rules, guidelines and ethics laid

acosaponin C.

838 K. Rauf et al. / Phytomedicine 18 (2011) 836– 842

re of

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D

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P

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C

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Fig. 3. Structu

own by the Ethical Committee, Department of Pharmacy Univer-ity of Peshawar.

rugs

Morphine sulphate was gifted by PDH Laboratories Lahoreakistan through proper channel, while, naloxone hydrochloridend acetonitrile (HPLC Grade) were purchased from Sigma localistributor. Bacoside A3, Bacopaside ll and Bacosaponin C wereifted by Prof. Dr. Ikhlas Khan, the National Center for Natural Prod-cts Research, University of Mississippi USA. For extraction purpose

ocal commercial grade n-hexane, acetone methanol and n-Butanolere obtained from Haq chemicals Peshawar. All drugs were dis-

olved in saline and were injected I.P. Control animals receivedormal saline.

lant material

Plant was collected from Ramali Stream near Quaide Azamniversity Islamabad in April and was authenticated by Prof. Dr.uhammad Ibrar, Department of Botany, University of Peshawar.

he plant aerial parts were washed, shade dried and coarsely pow-ered. During this method, 500 g plant material was first defattedith n-Hexane, and then with acetone. The plant material was

hen extracted with methanol in soxhlet apparatus. The methanolraction (yield 14 g) was further sub fractionated to get n-Butanolraction (Yield 824 mg) which is reported to be richest in Bacosideshe major active components of Bacopa monnieri (Kahol et al. 2004).his fraction of Bacopa monnieri was used for the assessment ofntinociceptive effect and its impact on acquisition and expressionf morphine tolerance.

hromatographic analysis of Bacopa monnieri n-Butanol fraction

n Bt-ext BM was analyzed on HPLC/UV for quantification of Baco-ide A3, Bacopaside II, and Bacosaponin C using Phrompitayyaratethod (Phrompittayarat et al. 2007) with slight modification.

riefly 5 mg of n-Butanol fraction was dissolved in 5 ml of methanoly sonication for 5 min. The solution was then centrifuged at 3000

Bacopaside II.

revolution per minutes (rpm) for 10 min and filtered through 0.45u filter. As this fraction is very rich in bacosides, 200 �l of the cen-trifuged solution was further diluted with HPLC grade methanolto make up a volume of 10 ml to avoid column over loading andsubsequent deformed peaks. HPLC system equipped with LC-20ATdouble pump (Shimadzu, Japan), a Rheodyne injector with 20 �lloop and SPD-20A UV Visible detector (PDA) and ShimPack C18

column (150 mm × 4.6 mm, 5 �m particle size) was used in theexperiment. The mobile phase consisted of 0.2% phosphoric acidand acetonitrile (60:40, v/v). The Mobile phase pH was adjustedto 3.0 with 3 M NaOH. The total run time was 22 min at flow rateof 0.6 ml/min using wavelength of 205 nm. The peaks and reten-tion timings were first confirmed by standards of the respectiveBacosides Fig. 4.

Determination of antinociceptive activity (hot plate method)

Antinociceptive response of n Bt-ext BM was assessed by HotPlate method using Harvard Hot Plate (Harvard apparatus U.K.) at54 ± 0.1 ◦C. Animals were acclimatized to the laboratory for at least2 h before the start of the experiment. All animals were screenedfor pre test latency before drug administration. Only those animalshaving a pre test latency of <15 s were included in the experiment.A cut off time of 30 s was set to avoid any injury to the animal. After30 min of pre-testing, animals were administered with standarddrug-morphine sulphate, plant extract or saline and were tested forlatency on hot plate maintained at 54 ± 0.1 ◦C at 30 and 60 min afterdrug administration (Eddy and Leimbach 1953). Percent Analgesiawas calculated with the help of following formula:

Percent analgesia = test latency − control latencycut − off time − control latency

× 100

Induction and evaluation of morphine tolerance

Tolerance to morphine analgesia was produced in animals usinga five day schedule of 20 mg/kg morphine sulphate twice dailyinjection. Antinociceptive response of all groups was assessed at30 min and 60 min, on day one and day six by hot plate method

K. Rauf et al. / Phytomedicine 18 (2011) 836– 842 839

ion, di

um

Am

t1t1ea

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Effect of n Bt-ext of Bacopa Monnieri on development of morphinetolerance

As shown in Fig. 7, n Bt-ext BM exhibited significant(***P < 0.001) antinociceptive response in all three (5, 10, and

0

20

40

60

80

100Morphine group

saline group

30 60

Perc

en

t A

nalg

esia

Fig. 4. HPLC Chromatogram of Bacopa monnieri n-Butanol fract

sing 10 mg/kg morphine sulphate I.P. Control group received nor-al saline.

ssessment of effect of Bacopa monnieri on development oforphine tolerance

To assess the effect of Bacopa monnieri on induction of morphineolerance, animals (n = 8) received single injection of 5, 10, and5 mg/kg n Bt-ext BM daily along with 20 mg/kg twice daily injec-ion of morphine sulphate for five days. Antinociceptive response to0 mg/kg of morphine sulphate was tested on hot plate as describedarlier on day one and day six. Control group received normal salinelong morphine tolerance induction schedule.

ssessment of effect of Bacopa monnieri on expression oforphine tolerance

Animals (n = 8) received 20 mg/kg twice daily morphine sul-hate for five days and on day six separate groups received a single

njection of n Bt-ext BM, in dose of 5, 10 and 15 mg/kg 30 min beforeose of 10 mg/kg morphine. Control group received normal saline0 min before morphine dose 10 mg/kg on day six. Antinociceptiveesponse was tested at 30 min and 60 min interval.

ntinociceptive effect of Bacopa monnieri and its combinationith morphine

Antinociceptive effect of Bacopa monnieri n-Butanol fraction, 5,0 and 15 mg/kg alone or in combination with 10 mg/kg morphineas also tested on hot plate at 30 min and 60 min time post injection

s described earlier.

valuation of tolerance to the antinociceptive effect of Bacopaonnieri

To assess the antinociceptive effect after repeated administra-ion of Bacopa monnieri, animals (n = 8) were administered singlenjection of 15 mg/kg n Bt-ext BM (maximum tolerable dose) for

aximum period of time, i.e. seven consecutive days. Antinocicep-ive effect of the 15 mg/kg n Bt-ext BM was assessed on hot platen day one and day seven at 30 min and 60 min interval. Controlroup received 15 mg/kg n Bt-ext BM on day one and day six, andormal saline for five days.

tatistics

The results obtained were expressed as mean ± SEM of percentrotection. The means of all percent protection were subjected totudent’s t-test, one way ANOVA/two way ANOVA as needed fol-owed by appropriate post hoc tests. P-value <0.05 were consideredo be statistically significant.

splaying 1. Bacoside A3; 2. Bacopaside II; and 3. Bacosaponin C.

Results

Chromatographic analysis of Bacoside A3, Bacopaside II, andBacosaponin C

The results revealed that n Bt-ext BM is bacoside A rich as itcontains Bacoside A3 (3.49 �g/mg), Bacosaponin C (2.4 �g/mg) andBacopaside ll as (3.6 �g/mg), likewise the total quantity of BacosideA three major components is 9.5 �g/mg of n-Butanol fraction or15.67 �g/g of dry powder.

Induction and evaluation of morphine tolerance

Antinociceptive effect of morphine sulphate in hot plate test onday one

Antinociceptive response of a single dose 10 mg/kg morphinesulphate was almost similar in both pre treatment groups (n = 8)at both 30 min and 60 min time on day one, as depicted inFig. 5

Antinociceptive effect of morphine sulphate in hot plate test onday six

As shown in Fig. 6, animals that received 20 mg/kg twice dailyschedule elicited a decreased antinociceptive response both at30 min and 60 min time, on day, six, exhibiting development oftolerance to the antinociceptive effect of morphine Fig. 6. Twoway ANOVA clearly indicate that there is significant decrease inan antinociceptive response (***P < 0.001).

Time (minutes)

Fig. 5. Day 1 analgesia of morphine group and saline group in mice. Animals receivedmorphine sulphate (10 mg/kg I.P.) or saline on day one and were tested for antinoci-ceptive response at 30 min and 60 min of administration. Each point representsmean ± SEM of percent protection (n = 8) (Student’s t-test).

840 K. Rauf et al. / Phytomedicine 18 (2011) 836– 842

0

20

40

60

80

100saline group

Morphine group

6030

******

Time (minutes)

Pe

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nt

An

alg

es

ia

Fig. 6. Effect of 10 mg/kg morphine sulphate on day six in morphine treated groupand saline group in mice. Animals received morphine sulphate (10 mg/kg I.P.) orsaline on day six and were tested for antinociceptive response at 30 min and 60 minof administration. Each point represents mean ± SEM of percent protection (n = 8)(***P < 0.001, values significantly different as compared to saline (two way ANOVA)).

0

20

40

60

80

100Morphine + SAL

Morphine + 5mg BM

Morphine + 10mg BM

Morphine + 15mg BM

30

***

******

*** ***

***

60

Time (minutes)

Pe

rce

nt

An

alg

es

ia

Fig. 7. Effect of chronic administration of n Bt-ext BM 5, 10, and 15 mg/kg on devel-opment of morphine tolerance as compared to morphine group. Animals received5, 10 and 15 mg/kg n-Butanol single injection daily along with morphine toleranceinduction schedule of 20 mg/kg morphine sulphate twice daily and were tested forantinociceptive response at 30 min and 60 min of administration in hot plate test.Ew

1s

Et

1a

Fe(1aAt

0

20

40

60

80

100SAL

5mg BM

10mg BM

30 60

Time(minutes)

15mg BM

******* ****** ***

Perc

en

t A

nalg

esia

Fig. 9. Antinociceptive effect of n Bt-ext of Bacopa monnieri in mice. Animals were

Bacoside A in doses above 50 �g/kg has been found to equally effec-tive in acute pain models in mice, this might have important future

ach point represents mean ± SEM of percent protection (n = 8) (***P < 0.001, twoay ANOVA followed by Bonferroni post tests).

5 mg/kg) treated groups as compared to morphine groups on dayix (ANOVA followed by Bonferroni post tests).

ffect of n Bt-ext of Bacopa monnieri on expression of morphineolerance

As depicted in Fig. 8, single injection of n Bt-ext BM (5, 10, and5 mg/kg) significantly attenuated expression of morphine toler-

nce (***P < 0.001, ANOVA followed by Bonferroni post tests).

0

20

40

60

80

100Morphine + SAL

Morphine + 5mg BM

Morphine + 10mg BM

Morphine + 15mg BM

30 60

time (minutes)

*

*** ** *

*

***

** *

Perc

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ig. 8. Effect of Acute administration of Bacopa monnieri 5, 10, and 15 mg/kg n Bt-xt on expression of morphine tolerance as compared to morphine group. Animalspost morphine induced tolerance) received single injection of n Bt-ext BM 5, 10 and5 mg/kg on day six prior to 10 mg/kg morphine challenge dose and were tested forntinociceptive test. Each point represents mean ± SEM of percent protection (n = 8).NOVA followed by Bonferroni post tests revealed significant difference between

reatment groups and control group (***P < 0.001).

administered n-Butanol fraction (10 and 15 mg/kg) and were screened on hot platefor percent protection at 30 and 60 min. Each point represents mean ± SEM of per-cent protection (n = 8) (*P < 0.05 and ***P < 0.001, values significantly different ascompared to saline (two way ANOVA followed by Bonferroni post tests)).

Antinociceptive effect of n Bt-ext of Bacopa monnieri in hot platetest

As shown in Fig. 9, n Bt-ext BM exhibited antinociceptiveresponse in hot plate test. Two way ANOVA followed by Bonferronipost tests revealed significant antinociceptive activity (***P < 0.001)at all doses tested.

Antinociceptive effect of n Bt-ext of Bacopa monnieri incombination with morphine

As depicted in Fig. 10, n Bt-ext exhibited antinociceptiveresponse in hot plate test. Two way ANOVA followed by Bonferronipost tests revealed significant antinociceptive activity (*P < 0.05,***P < 0.001) at all doses tested (5, 10 and 15 mg/kg).

Development of tolerance to the antinociceptive effect of n Bt-extBacopa monnieri

As depicted in Fig. 11, n Bt-ext BM exhibited no tolerance toantinociceptive response in hot plate test both on day one and dayseven.

Discussion

HPLC analysis of locally available Bacopa monnieri indicates thepresence of around 15.67 �g of Bacoside A (Bacopaside A3, Baco-paside II and Bacopaside C)/gram of dry powder. Administering

clinical implications in management of both chronic malignant andnon malignant pains. The above results reveal that Bacopa monnierihas an antinociceptive effect comparable to morphine in acute pain

0

20

40

60

80

100Morphine + SAL

Morphine + 5mg BM

Morphine + 10mg BM

Morphine + 15mg BM

6030

Time (minutes)

*

******

*******

Perc

en

t A

nalg

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Fig. 10. Antinociceptive effect of n Bt-ext of Bacopa monnieri in combination withmorphine sulphate (10 mg/kg) in mice. Animals received 5, 10 and 15 mg/kg of n Bt-ext BM 30 min before morphine administration and were tested for antinociceptiveeffect at 30 min and 60 min. Each point represents mean ± SEM of percent protec-tion (n = 8) (*P < 0.05 and ***P < 0.001, values significantly different as compared tocontrol (two way ANOVA followed by Bonferroni post tests)).

K. Rauf et al. / Phytomedicin

30 600

20

40

60

80

100BM n-butanol 15mg/kg

saline group

Time (minutes)

Perc

en

t A

nalg

esia

Fig. 11. Antinociceptive effect of n Bt-ext of Bacopa monnieri on day one and dayseven. Animals were administered n Bt-ext for seven days (15 mg/kg) twice dailyand were screened for antinociceptive effect on hot plate at 30 min and 60 min. Eachptsa

mcob1tcoowcBsdbB

(ctnss(pi2oprehmm(

Si((bvDded

oint represents mean ± SEM of percent protection (n = 8). Student’s t test revealedhat tolerance does not develop to maximum tolerable dose of Bacopa monnieri ineven days and the comparison of n Bt-ext BM on day seven and control groupnalgesia was found to be statistically non significant.

odels, with an added benefit that it enhances morphine antinoci-eptive effect also. This effect highlights a newer facet of researchn the role of Bacopa monnieri in clinical cases where opioids are toe used. Animals were administered a maximum tolerable dose of5 mg/kg Bacopa monnieri (BM) n-Butanol fraction, for seven days,o assess development of tolerance to its antinociceptive effect andoncluded that no tolerance develops to the antinociceptive effectf Bacopa monnieri. In mice administration of more than 15 mg/kgf n-Butanol fraction of Bacopa monnieri (BM) led to death of animalithin three to four days. As Bacopa monnieri has got both antinoci-

eptive and anti-inflammatory effects, it’s high time to evaluateacopa monnieri role in management of pain models either as sub-titute or adjuvant to opioids with an objective to minimize opioidsose, tolerance and cumbersome side effects. Bacopa monnieri haseen found to be effective in neuropathic pain models (Sahoo andehera 2010).

Bacopa monnieri has got calcium channel blocking effectChanna et al. 2003) also, this might be one contributing factor, asalcium channels blockers have capability to diminish morphineolerance (Smith et al. 1999). Additionally sustained Ca2+ chan-el blockade results in modulation in the adenyl cyclase effectorsystem activated by �-opioid receptor activation, leading to theuppression of opioid tolerance and restoration of super-sensitivityHurlé et al. 2000). Nitric oxide modulates the actions of mor-hine and related analgesics and tolerance to morphine analgesia

s likely prevented by nitric oxide synthase inhibition (Zafar et al.002) and tolerance to morphine analgesia is potentiated by nitricxide (Dambisya and Lee 1996; Toda et al. 2009). Nitric oxide anderoxynitrite (ONOO−) are reported as the key players that cur-ent literature suggests have a major role in both acquisition andxpression of morphine tolerance (Bryant et al. 2009). Peroxynitriteas been the center of all current research in the pursuit of newerolecules from both natural and synthetic origin, for the manage-ent of chronic pains, neuropathic pains, and morphine tolerance

Salvemini 2009; Salvemini and Neumann 2009).Bacopa monnieri has been reported (Russo and Borrelli 2005;

hinomol and Muralidhara 2011) that it protects against NOnduced oxidative stress and rotenone induced oxidative stressHosamani and Muralidhara 2009) in dose dependent mannerBhattacharya et al. 2000; Kapoor et al. 2009). Bacopa monnieri haseen found to be an effective and well tolerated phytomedicine in

arious clinical trials in old age patients also (Calabrese et al. 2008;eb et al. 2008). Currently Bacopa monnieri is available as stan-ardized herbal product alone or in combination with other plantxtracts for various cognitive disorders and preliminary clinicalata of safety and tolerability in various age groups has promis-

e 18 (2011) 836– 842 841

ing results (Pravina et al. 2007; Qureshi et al. 2010). Additionally,Bacopa monnieri not only suppresses acquisition and developmentof morphine tolerance but also protects liver, kidneys and brainfrom toxic effects of chronic morphine use (Sumathi and NiranjaliDevaraj 2009; Sumathy et al. 2001, 2002). Addition of Bacopa mon-nieri as an adjuvant therapy for the management of opioid tolerancehas many added benefits like it is effective both as single dose orrepeated dose and has the capacity to enhance opioids analgesia.

Conflict of interest

No conflict to disclose.

Acknowledgements

We sincerely thank Higher Education Commission of Pakistanfor the grant of PhD Scholarship for the author.

We sincerely thank and acknowledge Prof. Dr. Ikhlas A. Khan,the National Center for Natural Products Research, Mississippi, MS,USA for a gift of HPLC standards of Bacoside A3, Bacopaside II, andBacosaponin C.

We gratefully acknowledge Ministry of Health, Pakistan andMinistry of Narcotics control, Pakistan, for their support in acqui-sition of Morphine sulphate. We sincerely thank M/S Punjab DrugHouse labs, for the gift of Morphine sulphate through proper chan-nel.

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