MSc Daisy Volmer
email: daisy.volmer@ut.ee

John Lilja

David Hamilton

J Simon Bell

Peep Veski

Self-reported competence of Estonian community pharmacists in relation to herbal products: findings from a health-system in transition

Keywords: herbal products, extended community pharmacy services, Estonia

Healthcare reforms in Estonia

. Under these changes the operation of community pharmacies was transferred from the State to private ownership (Volmer et al., 2008).

Estonia joined the European Union (EU) in 2004. Pharmacies in Estonia continue to have a monopoly on the sale of prescription and non-prescription medications, including herbal products. Consumers perceive pharmacists as trustworthy sources of information about prescription and non-prescription drugs (Volmer et al., 2009). However, pharmacy practice and policy in Estonia is increasing shaped by reforms at the European level.

The healthcare reforms described above have created two approaches to the use of herbal products in Estonia. The first approach is based on traditions and practice experience.

The second approach is based on evidence-based medicine and scientific research. These approaches are not necessarily mutually exclusive.

INTRODUCTION

Complementary and alternative medicine (CAM) has been defined as a group of diverse medical and health care systems, practices, and products that are not generally considered part of conventional medicine (Barnes, 2003). Herbal products are one component of CAM, and include both herbal supplements and herbal medications. Herbal supplements, used by some individuals to supplement traditional medical therapies, are not considered as medications and in general are not regulated by drug agencies (Definition of herbal supplement, 2009). Herbal medications have active constituents with known pharmacological effects and they are able to cause side effects and interactions. Herbal medications are usually dispensed by retail pharmacies without prescription and are often regulated as medicinal products (Holst et al., 2009).

The 2007 National Health Interview survey conducted in the US revealed that 74% of adults and 53% of children had used herbal supplements in the past 30 days (National Health Interview Survey, 2007). A similar increase in the use of CAM has been reported in European countries [START_REF] Thomas | Use and expenditure on complementary medicine in England: a population based survey[END_REF].

In Europe legislation concerning herbal medications was not harmonized until 2001.

Prior to European harmonization each country was responsible for its own regulations concerning herbal medicines. In Germany, the therapeutic guide to herbal medicine issued in 1998 contained descriptions of 380 medicinal plants (The Complete German Commission E Monographs, 1999). Requirements for obtaining a marketing authorisation for traditional herbal medicinal products were first presented in the EU

Regulation and use of herbal medications and herbal supplements in Estonia

Legislation related to herbal medicinal products in Estonia follows EU directives. The State Agency of Medicines (SAM) is the institution responsible for categorization of herbal products as medicines or supplements and for licensing of herbal medicinal products. The categorization is made on the basis of the safety of the active components (Medicinal Products Act, 2005). The Estonian Health Care Board is responsible for registration of herbal supplements. However, detailed requirements for the quality, efficacy and safety of herbal supplements have not been developed in Estonia. Currently, the importer of herbal supplements must obtain a quality certificate for each herbal product. Although both herbal medicinal products and supplements are sold in community pharmacies, it is often challenging for pharmacists to access and provide evidence-based information in relation to the quality, efficacy and safety of the herbal supplements.

The use of herbal products has been traditional and popular in Estonia. In 2002 the most popular herbal products were teas for the treatment of minor urinary and upper respiratory tract complaints, followed by stress-relieving remedies and teas to provide a "good night's sleep" [START_REF] Püüa | Marketing of some essential oil herbal drugs in Estonia[END_REF]. Estonians often use herbal products as the first remedy to treat minor illnesses. Information concerning the mode of action and indication 4 is typically obtained from previous experience or provided by a health care professional, usually a pharmacist (Volmer et al., 2007).

The only formal education about medicinal plants and herbal products provided to pharmacists in Estonia is during the five-year Master of Pharmacy degree program at the

University of Tartu (Evaluation of pharmacy training, 2001). Unlike in many other

European countries there are no herbalists in Estonia. In addition, there are very few "health shops" in Estonia from where it is possible to purchase herbal supplements, natural and wellness products, cosmetics and other products.

Attitudes of pharmacists towards herbal products

A systematic review of 19 studies of US and Canadian pharmacists reported both positive and negative attitudes toward the safety and efficacy of dietary supplements (Kwan, et al., 2006). Despite the fact a large proportion of pharmacists received questions about dietary supplements from patients and other health professionals, pharmacists perceived their knowledge was inadequate and recognized the need for additional training. The need for more training in relation to CAM has also been reported among Australian pharmacy students [START_REF] Tiralongo | Attitudes and perceptions of Australian pharmacy students towards complementary and alternative medicine -a pilot study[END_REF]. This need may be reinforced by the expectations of patients who consider pharmacists to be knowledgeable about natural health products (Kwan, et al., 2008). Most published studies investigating the attitudes of pharmacists and pharmacy students toward CAM have been conducted in North America, Asia, Australia and England (Barnes, et al., 2007;Olatunde, et al., in press). No previous The objectives of the study were to (1) explore the self-reported competency of pharmacists in relation to herbal medicines and herbal supplements, and (2) investigate the self-reported provision of community pharmacy services in relation to herbal medicines and herbal supplements. In the context of the current article the herbal medicines and herbal supplements are referred to together as herbal products.

METHODS

Sample and data collection

A random sample of 50% (n=154) of community pharmacies in Estonia was extracted from the Register of pharmacies maintained by the Estonian State Agency of Medicines in February 2005. Each pharmacy in the sample was mailed one copy of the survey instrument and a cover letter. The cover letter requested that the survey instrument should be completed by the pharmacist or assistant pharmacist responsible for managing the sale of non-prescription medications. All pharmacies were provided with a pre-paid addressed envelope to return the survey instrument. A reminder was sent by e-mail to all pharmacies after 2 weeks. All survey instruments were completed anonymously.

Survey instrument

The survey instrument used in the study was adapted from an instrument previously used to survey a stratified random sample of community pharmacists in the US (Bouldin, et

Statistical analyses

All data analyses were performed using the Statistical Package for the Social Sciences (SPSS,Version 11.0,Chicago,IL). Differences between groups of pharmacists were compared using chi square tests for categorical variables and independent samples t-tests for continuous variables. Younger pharmacists were defined as the respondents aged 25-45 years, whereas older pharmacists were those aged 46-65 years. Urban pharmacies were defined as pharmacies located in the official cities, whereas rural pharmacies were defined as pharmacies located in the villages. Differences in pharmacists' attitudes were examined according to age and location of the pharmacy. The level of statistical significance was set at p≤0.05.

RESULTS

Demographic characteristics of the respondents

Of the 154 questionnaires distributed 120 (77.9%) were returned, 89 (74.1%) from city pharmacies and 31 (25.9%) from rural pharmacies. Of the respondents, 114 (95.0%) were pharmacists and 6 (5.0%) were assistant pharmacists. All of the respondents were women. The mean age ± SD of the pharmacists was 42.5 ± 17.8 years, and the mean age of the assistant pharmacists was 39.1 ± 15.4 years. The pharmacists had a mean of 17.5 ± 14.6 years working experience while the assistant pharmacists had a mean of 14.5 ± 13.8 years working experience.

Professional competency of pharmacists

Seventy-seven (64.1%) respondents self-reported their professional knowledge regarding herbal products as good or excellent. Continuing education regarding herbal products was rated as important by 35.8% of the pharmacists (n=43). Pharmacists stated that the best ways of obtaining professional knowledge were written information (n=75) 62.5% and lectures and seminars (n=75) 62.5%. Pharmacists were less likely to view visits from drug representatives (n=47) 39.1% and electronic databases (n=43) 35.8% as important sources of professional knowledge. Pharmacists would like to receive more information about unknown medicinal plants (n=83) 69.1%, safety (n=69) 57.5% and the mode of action (n=65) 54.2% of herbal products. Self-reported professional knowledge was higher among older rather than younger pharmacists (p=0.043). Younger pharmacists were more likely to prefer electronic databases (p=0.027) and visits from drug representatives (p=0.032) as sources of information about herbal products. Among the respondents, 15.8% (n=19) had experienced problems counselling about herbal products, 35.0% (n=42) reported no problems counselling about herbal products and 49.2% (n=59) reported they occasionally encountered problems counselling about herbal products. Of the 65.0% (n=78) of pharmacists who indicated they encountered or occasionally encountered problems, 29.5% (n=23) said this was due to a lack of professional knowledge, 28.2% (n=22) commented on the need for more detailed patient information leaflets and 25.6% (n=20) said they encountered problems because the public misunderstood herbal products. Urban pharmacists were more critical towards the quality of patient information leaflets (p=0.027), and the difficulty encountered when pharmacy customers lacked knowledge of herbal products (p=0.035), than were rural pharmacists.

Customer of herbal products

According to pharmacists, customers routinely came to the pharmacy for information only 45.8% (n=55) or to ask for information and purchase herbal products 60.8% (n=73).

According to the pharmacists, the typical customers were a 41-60 year old woman and a 61 years or older man, both from the city. Respondents from urban pharmacies perceived a higher customer interest in herbal products than respondents from rural pharmacies (p=0.032). More than half of the pharmacists surveyed considered that herbal product ----------------------------------------------------------------------------------------------------------Insert Figure 1 here.

- ---------------------------------------------------------------------------------------------------------Information about herbal products mode of action, administration, side effects and interactions were considered most important by the survey respondents (Figure 2). Younger pharmacists were more likely than older pharmacists to consider information about safety as important (p=0.039).

- ---------------------------------------------------------------------------------------------------------Insert Figure 2 here.

- ---------------------------------------------------------------------------------------------------------

DISCUSSION

This was the first study in Estonia to investigate the attitudes and knowledge of the pharmacists towards herbal products and their consumption. The results of the study provide an insight into the role of pharmacists in relation to the use of herbal products in this health-system in transition.

Sixty four percent of the respondents self-reported that they possessed good knowledge in relation to herbal products. This self-reported level of knowledge was higher than in surveys of pharmacists conducted in other countries. A Finnish study reported that 38% F o r P e e r R e v i e w 10 and 17% of pharmacy owners believed they knew sufficient information about herbal medicines and herbal supplements respectively (Fock, et al., 2004). A survey of pharmacists and attendees at series of international conferences conducted in Singapore reported that 81% pharmacists felt they had inadequate skills and knowledge to counsel patients on herbal medications (Koh, et al., 2003). A survey of Australian pharmacists reported that just 15% reporting they were "very confident" in answering queries about safety, interactions or benefits of CAMs [START_REF] Semple | Community pharmacists in Australia: barriers to information provision on complementary and alternative medicines[END_REF]. This was despite an earlier study finding that 91% of Australian pharmacists believe it is important to have knowledge of both CAM and conventional medicine to be able to inform patients about their treatment options (Naidu, et al., 2005).

Despite high number of pharmacists self-reporting good knowledge, only 35% of Estonian pharmacists and assistant pharmacists had not experienced any problems in relation to counselling about herbal products. It is likely, therefore, that the problems experienced by the pharmacists did not relate to their perceived lack of knowledge.

Estonian pharmacists may have had difficulty accessing evidence-based information about herbal products. Of 200 herbal products available at Estonian pharmacies in 2001, only 50 (25%) had written patient information leaflets (PILs) that contained no errors (Arjakse, 2002). The most common error related to ascribing medical claims to products classified as food supplements. Obtaining evidence-based information about CAMs is also a problem reported internationally. Sources of information about CAMs among pharmacists surveyed in Singapore included books/magazines (64%), friends/family (35.7%), and the Internet (31.4%) (Koh, et al., 2003). The most common sources of 11 information among Australian pharmacists were books and journal articles. This led the authors of several studies to conclude that there is a need for greater access to CAM resources and education [START_REF] Semple | Community pharmacists in Australia: barriers to information provision on complementary and alternative medicines[END_REF]Olatunde, et al., in press).

It is possible that Estonian pharmacists overestimated their knowledge in relation to CAM and particularly concerning herbal products. While there has been a long tradition of herbal product use in Estonia, recognition of the potential for clinically significant herb-drug interactions is more recent (Volmer, et al., 2006). However, as in previous studies (Cuzzolin, et al., 2009), issues concerning the safety of herbal products were rarely discussed with pharmacy customers (Volmer, et al., 2006). Scientific evidence concerning the safety and efficacy of CAMs is typically derived from only small number of randomized controlled trials (RCTs) (Hu, et al., 2005). Herb-drug interactions may have not been covered in the undergraduate pharmacy curricula when many of the respondents undertook their university-based training. Pharmacy education about herbal products in Estonia is primarily orientated to botany and the chemical constituents of plants rather than the pharmacological activity of the active substances (Evaluation of pharmacy training, 2001). Tools such as the 22-item Consolidated Standards of Reporting Trials checklist may assist pharmacists interpreting the results of RCTs (Gagnier, et al., 2006). However, further research is required to determine how Estonian pharmacists communicate the risks and benefits of CAM and particularly herbal products to Estonian pharmacy customers. The majority of Estonian pharmacists self-evaluated their competency in relation to herbal products as good or excellent. However, there was as mismatch between pharmacists' self-evaluated competency and their self-reported provision of advice about herbal products. Health system reforms in Estonia may need to be accompanied by enhanced training for pharmacists to provide advice about the safe and appropriate use of herbal products.

Study strengths and limitations

An important strength of the study was that the survey instrument was mailed to a random sample of half of all Estonian community pharmacies, and a good response rate was achieved. Estonian community pharmacists and assistant pharmacists are almost exclusively female. However, a limitation of the study was that it did not capture the attitudes of male pharmacists. In addition only 6 assistant pharmacists participated in the survey. Unlike in many other countries in Europe, the number of pharmacists working at community pharmacies is higher than the number of assistant pharmacists. Another reason for the low participation of assistant pharmacists in the survey could be incorrect following of the instructions. The envelop with the survey instrument could be opened and completed by pharmacy manager. As with other attitudinal surveys, it is not clear whether pharmacists' self-reported attitudes correspond with their actual behaviour in the pharmacy workplace. Further research is also needed to determine consumers' attitudes toward pharmacists as providers of information about CAM.

INTRODUCTION

Complementary and alternative medicine (CAM) has been defined as a group of diverse medical and health care systems, practices, and products that are not generally considered as part of conventional medicine. Herbal products are one component of CAM, and include both herbal supplements sold as food supplements and herbal medications (Barnes, 2003).

Herbal medications contain active constituents with pharmacological effects and have the potential to cause adverse reactions and drug interactions. Herbal medications are usually dispensed by retail pharmacies without prescription and are often regulated as medicinal products (Holst et al., 2009).

The 2007 National Health Interview survey conducted in the US revealed that 74% of adults and 53% of children had used herbal supplements in the past 30 days ( 2007National Health Interview Survey). A similar increase in the use of CAM has been reported in European countries [START_REF] Thomas | Use and expenditure on complementary medicine in England: a population based survey[END_REF].

In Europe legislation concerning herbal medications was not harmonized until 2001.

Prior to European harmonization each country was responsible for its own regulations concerning herbal medications. In Germany, the therapeutic guide to herbal medication

Healthcare reforms in Estonia

Estonia is a small northern European country (population 1.3 million) located to the south of Finland. Unlike in many European and North American countries there is a long tradition of herbal product use in Estonia. However, the traditions associated with herbal product use are evolving in line with the recent healthcare reforms. Since independence from Soviet Union was restored in 1991 the Estonian health care system has undergone a process of rapid transition. This transition has involved the shift from a centralized, specialist-orientated system to a system based on primary health care and evidence-based medicine [START_REF] Põlluste | Public health reforms in Estonia: impact on the health of the population[END_REF]. Under these changes the operation of community pharmacies was transferred from the State to private ownership (Volmer et al., 2008). Estonia joined the European Union (EU) in 2004. Pharmacies in Estonia continue to have a monopoly on the sale of prescription and non-prescription medications, including herbal products. Consumers perceive pharmacists as trustworthy sources of information about prescription and non-prescription drugs (Volmer et al., 2009). However, pharmacy practice and policy in Estonia is increasing shaped by reforms at the European level.

The healthcare reforms described above have created two approaches to the use of herbal products in Estonia. The first approach is based on traditions and practice experience. The second approach is based on evidence-based medicine and scientific research. These approaches are not necessarily mutually exclusive. Estonian

Regulation and use of herbal medications and herbal supplements in Estonia

Legislation The use of herbal products has been traditional and popular in Estonia. In 2002 the most popular herbal products were teas for the treatment of minor urinary and upper respiratory tract complaints, followed by stress-relieving remedies and teas to provide a "good night's sleep" [START_REF] Püüa | Marketing of some essential oil herbal drugs in Estonia[END_REF]. Estonians often use herbal products as the first remedy to treat minor illnesses. Information concerning the mode of action and indication is typically obtained from previous experience or provided by a health care professional, usually a pharmacist (Volmer et al., 2007). European countries there are no herbalists in Estonia. In addition, there are very few "health shops" in Estonia from where it is possible to purchase herbal supplements, natural and wellness products, cosmetics and other products.

Attitudes of pharmacists towards herbal products

A systematic review of 19 studies of US and Canadian pharmacists reported both positive and negative attitudes toward the safety and efficacy of dietary supplements (Kwan, et al., 2006). Despite the fact a large proportion of pharmacists received questions about dietary supplements from patients and other health professionals, pharmacists perceived their knowledge was inadequate and recognized the need for additional training. The need for more training in relation to CAM has also been reported among Australian pharmacy students [START_REF] Tiralongo | Attitudes and perceptions of Australian pharmacy students towards complementary and alternative medicine -a pilot study[END_REF]. This need may be reinforced by the expectations of patients who consider pharmacists to be knowledgeable about natural health products (Kwan, et al., 2008). Most published studies investigating the attitudes of pharmacists and pharmacy students toward CAM have been conducted in North America, Asia, Australia and England (Barnes and Abbot, 2007;[START_REF] Olatunde | Roles and responsibilities of pharmacists with respect to natural health products: Key informant interviews[END_REF]. No previous studies have investigated the attitudes of pharmacists toward use of herbal medications and herbal supplements in this health system in transition.

METHODS

Sample and data collection

A random sample of 50% (n=154) of community pharmacies in Estonia was extracted from the Register of pharmacies maintained by the Estonian State Agency of Medicines in February 2005. Each pharmacy in the sample was mailed one copy of the survey instrument and a cover letter. The cover letter requested that the survey instrument should be completed by the pharmacist or assistant pharmacist responsible for managing the sale of non-prescription medications. All pharmacies were provided with a pre-paid addressed envelope to return the survey instrument. A reminder was sent by e-mail to all pharmacies after 2 weeks. All survey instruments were completed anonymously.

Survey instrument

The survey instrument used in the study was adapted from an instrument previously used to survey a stratified random sample of community pharmacists in the US (Bouldin, et al., 1999). The survey instrument included items related to (1) the respondent's demographic characteristics, (2) the frequency of consumer requests for information about herbal products, (3) the perceived importance of different

Statistical analyses

All data analyses were performed using the Statistical Package for the Social Sciences (SPSS, Version 11.0, Chicago, IL). Differences between groups of pharmacists were compared using chi square tests for categorical variables and independent samples ttests for continuous variables. Younger pharmacists were defined as the respondents aged 25-45 years, whereas older pharmacists were those aged 46-65 years. Urban pharmacies were defined as pharmacies located in the official cities, whereas rural pharmacies were defined as pharmacies located in the villages. Differences in pharmacists' attitudes were examined according to age and location of the pharmacy.

The level of statistical significance was set at p≤0.05. Of the 154 questionnaires distributed 120 (77.9%) were returned, 89 (74.1%) from city pharmacies and 31 (25.9%) from rural pharmacies. Of the respondents, 114 (95.0%)

RESULTS

Demographic characteristics of the respondents

were pharmacists and 6 (5.0%) were assistant pharmacists. All of the respondents were women. The mean age ± SD of the pharmacists was 42.5 ± 17.8 years, and the mean age of the assistant pharmacists was 39.1 ± 15.4 years. The pharmacists had a mean of 17.5 ± 14.6 years working experience while the assistant pharmacists had a mean of 14.5 ± 13.8 years working experience.

Professional competency of pharmacists

Seventy-seven (64.1%) respondents self-reported their professional knowledge regarding herbal products as good or excellent. Continuing education regarding herbal products was rated as important by 35.8% of the pharmacists (n=43). Pharmacists stated that the best ways of obtaining professional knowledge were written information (n=75) 62.5% and lectures and seminars (n=75) 62.5%. Pharmacists were less likely to view visits from drug representatives (n=47) 39.1% and electronic databases (n=43) 35.8% as important sources of professional knowledge. Pharmacists would like to receive more information about unknown medicinal plants (n=83) 69.1%, safety (n=69) 57.5% and the mode of action (n=65) 54.2% of herbal products.

Self-reported professional knowledge was higher among older rather than younger pharmacists (p=0.043). Younger pharmacists were more likely to prefer electronic databases (p=0.027) and visits from drug representatives (p=0.032) as sources of information about herbal products. Among the respondents, 15 (n=22) commented on the need for more detailed patient information leaflets and 25.6%

(n=20) said they encountered problems because the public misunderstood herbal products. Urban pharmacists were more critical towards the quality of patient information leaflets (p=0.027), and the difficulty encountered when pharmacy customers lacked knowledge of herbal products (p=0.035), than were rural pharmacists.

Customers of herbal products

According to pharmacists, customers routinely came to the pharmacy for information only 45.8% (n=55) or to ask for information and purchase herbal products 60.8% (n=73). According to the pharmacists, the typical customers were a 41-60 year old woman and a 61 years or older man, both from the city. Respondents from urban pharmacies perceived a higher customer interest in herbal products than respondents from rural pharmacies (p=0.032). More than half of the pharmacists surveyed considered that herbal product advertisements in the mass media was the most common sources of information about herbal products used by pharmacy customers (Figure 1).

----------------------------------------------------------------------------------------------------------Insert Figure 1 here.

---------------------------------------------------------------------------------------------------------- Younger pharmacists were more likely than older pharmacists to consider information about safety as important (p=0.039).

----------------------------------------------------------------------------------------------------------Insert Figure 2 here.

- ---------------------------------------------------------------------------------------------------------

DISCUSSION

This was the first study in Estonia to investigate the attitudes and knowledge of the pharmacists towards herbal products and their consumption. The results of the study provide an insight into the role of pharmacists in relation to the use of herbal products in this health-system in transition.

Sixty four percent of the respondents self-reported that they possessed good knowledge in relation to herbal products. This self-reported level of knowledge was higher than in surveys of pharmacists conducted in other countries. A Finnish study reported that 38% and 17% of pharmacy owners believed they knew sufficient information about herbal medications and herbal supplements respectively (Fock and Pietilä, 2004). A survey of pharmacists and attendees at series of international conferences conducted in Singapore reported that 81% pharmacists felt they had inadequate skills and knowledge to counsel patients on herbal medications (Koh, et al., 2003). A survey of Australian pharmacists reported that just 15% reporting they were "very confident" in answering queries about 12 safety, interactions or benefits of CAMs [START_REF] Semple | Community pharmacists in Australia: barriers to information provision on complementary and alternative medicines[END_REF]. This was despite an earlier study finding that 91% of Australian pharmacists believe it is important to have knowledge of both CAM and conventional medicine to be able to inform patients about their treatment options (Naidu, et al., 2005). (Koh, et al., 2003). The most common sources of information among Australian pharmacists were books and journal articles. This led the authors of several studies to conclude that there is a need for greater access to CAM resources and education [START_REF] Semple | Community pharmacists in Australia: barriers to information provision on complementary and alternative medicines[END_REF][START_REF] Olatunde | Roles and responsibilities of pharmacists with respect to natural health products: Key informant interviews[END_REF].

It is possible that Estonian pharmacists overestimated their knowledge in relation to 13 herb-drug interactions is more recent (Volmer, et al., 2006). However, as in previous studies (Cuzzolin and Benoni, 2009), issues concerning the safety of herbal products were rarely discussed with pharmacy customers (Volmer, et al., 2006). Scientific evidence concerning the safety and efficacy of CAMs is typically derived from only small number of randomized controlled trials (RCTs) (Hu, et al., 2006). Herb-drug interactions may have not been covered in the undergraduate pharmacy curricula when Standards of Reporting Trials checklist may assist pharmacists interpreting the results of RCTs (Gagnier, et al., 2006). However, further research is required to determine how Estonian pharmacists communicate the risks and benefits of CAM and particularly herbal products to Estonian pharmacy customers.

Our study was conducted within the context of the Estonian health-system. This means our findings may not be directly generalizable to other countries. However, pharmacists in other countries with long traditions of herbal product use face similar challenges. The health-systems in other Baltic and post-Soviet states have been undergoing rapid transformation. There is a long-tradition of herbal product use in Germany. However, unlike in Estonia, herbal products in Germany are also sold by non-pharmacist government regulated practitioners including "Heilpraktiker" [START_REF] Joos | Use of complementary and alternative medicine in Germany -a survey of patients with inflammatory bowel disease[END_REF]. The challenge associated with combining traditional and western approaches has also been described in Asian countries [START_REF] Connett | Treating childhood asthma in Singapore: when West meets East[END_REF]. Pharmacognosy is widely 14 covered within the undergraduate pharmacy degree program in Estonia. This may be one reason why Estonian pharmacists self-evaluated their competency as good or excellent, while the perceived lack of knowledge has been cited as a barrier to providing advice about herbal products in other countries (Fock and Pietilä, 2004;Koh, et al., 2003;[START_REF] Semple | Community pharmacists in Australia: barriers to information provision on complementary and alternative medicines[END_REF]Naidu, et al., 2005). Nevertheless, health system reforms in Estonia may need to be accompanied by enhanced training for pharmacists to provide advice about the safe and appropriate use of herbal products.

Study strengths and limitations

An important strength of the study was that the survey instrument was mailed to a random sample of half of all Estonian community pharmacies, and a good response rate was achieved. Estonian community pharmacists and assistant pharmacists are almost exclusively female. However, a limitation of the study was that it did not capture the attitudes of male pharmacists. In addition only 6 assistant pharmacists participated in the survey. Unlike in many other countries in Europe, the number of pharmacists working at community pharmacies is higher than the number of assistant pharmacists.

Another reason for the low participation of assistant pharmacists in the survey could be incorrect following of the instructions. The envelope with the survey instrument could be opened and completed by pharmacy manager. As with other attitudinal surveys, it is not clear whether pharmacists' self-reported attitudes correspond with their actual behaviour in the pharmacy workplace. Further research is also needed to determine consumers' attitudes toward pharmacists as providers of information about CAM.

F

  these approaches to meet the healthcare needs and expectations of their customers.

  the attitudes of pharmacists toward use of herbal medications and herbal supplements in this health system in transition.

  1999). The survey instrument included items related to (1) the respondent's demographic characteristics, (2) the frequency of consumer requests for information about herbal products, (3) the perceived importance of different determinants of herbal product use, (4) the perceived importance of different aspects of herbal product information, (5) the respondent's self-reported competency in relation to herbal products, and (6) problems encountered by the respondents in relation to proving advice about herbal products. The content validity of the survey instrument was assessed by a panel of 8 researchers and practitioners with an interest in CAM. The survey instrument was pilot tested for face validity among a convenience sample of five pharmacists. Minor changes to the wording of the items were made based on the feedback received. A copy of the survey instrument can be obtained by contacting the corresponding author.

  mass media was the most common sources of information about herbal products used by pharmacy customers (Figure1).

  Männik G, Axelsson R. 2005. Public health reforms in Estonia: impact on the health of the population. BMJ 331:210-213 Püüa M. Marketing of some essential oil herbal drugs in Estonia [in Estonian]. Master's thesis, University of Tartu, Tartu, Estonia; 2004, 1-52. Semple SJ, Hotham E, Rao D, Martin K, Smith CA, Bloustien GF. 2006. Community pharmacists in Australia: barriers to information provision on complementary and alternative medicines. Pharm World Sci 28(6): 366-373. The Complete German Commission E Monographs, Therapeutic Guide to Herbal Medicines,1st ed. 1998. Integrative Medicine Communications, pub; Bk&CD-Rom edition, 1999. Thomas KJ, Nicholl JP, Coleman P. 2001. Use and expenditure on complementary medicine in England: a population based survey. Complemet Ther Med 9:2-11.

  required to marry these approaches to meet the healthcare needs and expectations of their customers.

  education about medicinal plants and herbal products provided to pharmacists in Estonia is during the five-year Master of Pharmacy degree program at the University of Tartu (Evaluation of pharmacy training, 2001). Unlike in many other

  the study were to (1) explore the self-reported competency of pharmacists in relation to herbal medications and herbal supplements, and (2) investigate the self-reported provision of community pharmacy services in relation to herbal medications and herbal supplements. In the context of the current article the herbal medications and herbal supplements are referred to together as herbal products.

  product use, (4) the perceived importance of different aspects of herbal product information, (5) the respondent's self-reported competency in relation to herbal products, and (6) problems encountered by the respondents in relation to proving advice about herbal products. The content validity of the survey instrument was assessed by a panel of 8 researchers and practitioners with an interest in CAM. The survey instrument was pilot tested for face validity among a convenience sample of five pharmacists. Minor changes to the wording of the items were made based on the feedback received. A copy of the survey instrument can be obtained by contacting the corresponding author.

  products mode of action, administration, side effects and interactions were considered most important by the survey respondents (Figure2).

  CAM and particularly concerning herbal products. While there has been a long tradition of herbal product use in Estonia, recognition of the potential for clinically significant

  many of the respondents undertook their university-based training. Pharmacy education about herbal products in Estonia is primarily orientated to botany and the chemical constituents of plants rather than the pharmacological activity of the active substances (Evaluation of pharmacy training, 2001). Tools such as the 22-item Consolidated

Page 18 of 40 http://mc.manuscriptcentral.com/ptr

  Tiralongo E, Wallis M. 2008. Attitudes and perceptions of Australian pharmacy students towards complementary and alternative medicine -a pilot study. BMC Complement Figure 1. Information sources about herbal products used by Estonian pharmacy customers as reported by Estonian pharmacists.Figure 2. Importance of different types of information concerning herbal products among Estonian pharmacy customers according to the Estonian pharmacists.

		Phytotherapy Research
	Advertisement Side-effects and interactions	36%					54%
	General practitioner Making herbal preparations at home	11%			69%
	Indication						93%
	Package information leaflet			35%
	Administration					81%
	Pharmacist					46%
	Mode of action						93%
	F o F o 0% 10% o F 0% 20%	20% 40%	30%	60%	40%	50% 80%	60% 100%
	r r r
	P P P
		e e r e e r e e r
		R e v i e w R e v i e w R e v i e w
	Altern Med 8:2.

  related to herbal medications in Estonia follows EU directives. The State Agency of Medicines (SAM) is the institution responsible for categorization of herbal products as medications or supplements and for licecing of herbal medications. The categorization is made on the basis of the safety of the active components (Medicinal

Products

Act, 2005)

. The Estonian Health Care Board is responsible for registration of herbal supplements. However, detailed requirements for the quality, efficacy and safety of herbal supplements have not been developed in Estonia. Currently, the importer of herbal supplements must obtain a quality certificate for each herbal product. Although both herbal medicinal products and supplements are sold in community pharmacies, it is often challenging for pharmacists to access and provide evidence-based information in relation to the quality, efficacy and safety of the herbal supplements.

  Despite high number of pharmacists self-reporting good knowledge, only 35% of Estonian pharmacists and assistant pharmacists had not experienced any problems in relation to counselling about herbal products. It is likely, therefore, that the problems experienced by the pharmacists did not relate to their perceived lack of knowledge.

	Estonian pharmacists may have had difficulty accessing evidence-based information
	about herbal products. Of 200 herbal products available at Estonian pharmacies in 2001,
	only 50 (25%) had written patient information leaflets (PILs) that contained no errors
	(Arjakse, 2002). The most common error related to ascribing medical claims to products
	classified as food supplements. Obtaining evidence-based information about CAMs is
	also a problem reported internationally. Sources of information about CAMs among
	pharmacists surveyed in Singapore included books/magazines (64%), friends/family
	(35.7%), and the Internet (31.4%)

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  Figure 1. Information sources about herbal products used by Estonian pharmacy customers as reported by Estonian pharmacists.Figure 2. Importance of different types of information concerning herbal products among Estonian pharmacy customers according to the Estonian pharmacists.

		Phytotherapy Research		Page 40 of 40
	Advertisement Side-effects and interactions	36%		54%
	General practitioner Making herbal preparations at home	11%	69%
	Indication			93%
	Package information leaflet	35%
	Administration		81%
	Pharmacist		46%
	Mode of action			93%
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		w w w w w

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This directive was later replaced with Directive 2003/63/EC (Commission Directive 2003/63/EC amending Directive 2001/83/EC, 2003).


Suresh Kumar

Christopher John Seal

Melanie-Jayne Howes

Geoffrey C Kite

Edward Jonathan Okello

Okello

Seal Cj

M-Jr Howes

Kite Gc

Okello Ej
email: e.j.okello@ncl.ac.uk

induced cytotoxicity in differentiated PC12 cells

Keywords: Withania somnifera, neuroprotection, hydrogen peroxide, amyloid, cytotoxicity, Alzheimer's disease Withania somnifera, neuroprotection, hydrogen peroxide, amyloid, cytotoxicity, Alzheimer's disease

published or not. The documents may come

In vitro protective effects of Withania somnifera (L.)

Dunal root extract against hydrogen peroxide and β-amyloid(1-42) induced cytotoxicity in differentiated PC12 cells

INTRODUCTION

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disease marked by a decline in cognitive functions, primarily memory loss and language deficit which are often accompanied by behavioural and psychological symptoms such as depression, stress, anxiety and mood disturbances.

The pathophysiology of AD is complex and is characterized by neuronal degeneration (cholinergic neurons in particular), abnormal neurofibrillary tangles, toxic β-amyloid (Aβ) plaques and deficiencies of neurochemicals which are essential for neuronal transmission (Ros and Poirier, 2004). Aβ cytotoxicity to neuronal cells has been identified as one of the major features in AD pathology, but the exact mechanisms of the cascade of events leading to neurotoxicity still remain unclear (Fukuyama et al., 1994;Fagarasan and Efthimiopoulos, 1996;Yan et al., 1997;Canevari et al., 2004).

Some studies suggest that one pathway of Aβ induced cytotoxicity could be mediated by free radicals and oxidative stress (Hensley et al., 1994;Behl, 1994Behl, , 1997;;Opazo et al., 2000;Canevari et al., 2004). Evidence obtained using cultured cells, such as sympathetic neurons (Greenlund et al., 1995), has demonstrated that reactive oxygen species are produced during the early, but not the late phase of neuronal cell death, suggesting that their production serves as an early signal, rather than a toxic agent, to mediate apoptosis. It has also been demonstrated that Aβ increases concentrations of hydrogen peroxide (H 2 O 2 ) in cells (Behl et al., 1994). Hydrogen peroxide is one of the (Whittemore et al., 1994;Satoh et al., 1996).

Withania somnifera (L.) Dunal (Solanaceae) root (also known as 'ashwagandha' in Sanskrit and as 'Indian ginseng') is widely used in Ayurvedic medicine [START_REF] Mishra | Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): A review[END_REF]Houghton and Howes, 2005) as a nerve tonic and memory enhancer, with antiaging, anti-stress, immunomodulatory and antioxidant properties (Ziauddin et al., 1996;Archana andNamasivayan, 1999, Bhattacharya et al., 2001;[START_REF] Jayaprakasam | Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves[END_REF]. Some studies have investigated a pharmacological basis to explain the reputed effects of W. somnifera on cognition, and to assess the therapeutic potential for the steroidal derivatives from this species, particularly the withanolides. Studies in vitro and in vivo have investigated the potential of W. somnifera to modulate cholinergic function and extracts of the root, and withanolides isolated from this species (withaferin A, 2,3dihydrowithaferin A and 5β,6β-epoxy-4β-hydroxy-1-oxowitha-2,14,24-trienolide), inhibit acetylcholinesterase (Schliebs et al., 1997;[START_REF] Choudhary | Cholinesterase inhibiting withanolides from Withania somnifera[END_REF][START_REF] Choudhary | Withanolides, a new class of natural cholinesterase inhibitors with calcium antagonistic properties[END_REF][START_REF] Houghton | Uses and abuses of in vitro tests in ethnopharmacology: Visualizing an elephant[END_REF][START_REF] Vinutha | Screening of selected Indian medicinal plants for acetylcholinesterase inhibitory activity[END_REF].

Other studies show that extracts of W. somnifera root, and some of the withanolide derivatives in particular, promote dendrite formation in human neuroblastoma cells in vitro in a dose-dependent manner [START_REF] Tohda | Dendrite extension by methanol extract of Ashwagandha (roots of Withania somnifera) in SK-N-SH cells[END_REF][START_REF] Zhao | Withanolide derivatives from the roots of Withania somnifera and their neurite outgrowth activities[END_REF]; withanolide A and withanosides IV and VI extend axons and dendrites, respectively, in vitro [START_REF] Kuboyama | Axon-or dendrite-predominant outgrowth induced by constituents from Ashwagandha[END_REF], and withanolide A is considered to reconstruct neuronal networks in vivo [START_REF] Kuboyama | Neuritic regeneration and synaptic reconstruction induced by withanolide A[END_REF]. In an animal model of dementia and spinal cord injury, withanolide A, withanoside IV, and withanoside VI restored presynapses and postsynapses [START_REF] Tohda | Dendrite extension by methanol extract of Ashwagandha (roots of Withania somnifera) in SK-N-SH cells[END_REF][START_REF] Kuboyama | Neuritic regeneration and synaptic reconstruction induced by withanolide A[END_REF]. Other studies have investigated various W. somnifera extracts in vivo to explain the putative effects of W. somnifera, including [START_REF] Parihar | Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity[END_REF][START_REF] Sankar | The neuroprotective effect of Withania somnifera root extract in MPTP-intoxicated mice: An analysis of behavioural and biochemical variables[END_REF][START_REF] Bhatnagar | Neuroprotective effects of Withania somnifera Dunal.: A possible mechanism[END_REF]. Root extracts from this species have also been shown to significantly reduce the number of hippocampal degenerating cells in the brains of stressed rodents [START_REF] Jain | Neuroprotective effects of Withania somnifera Dunn. in hippocampal sub-regions of female albino rat[END_REF] and were neuroprotective in animal models of Parkinson's disease [START_REF] Ahmad | Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats[END_REF][START_REF] Sankar | The neuroprotective effect of Withania somnifera root extract in MPTP-intoxicated mice: An analysis of behavioural and biochemical variables[END_REF] and Huntington disease [START_REF] Kumar | Possible neuroprotective effect of Withania somnifera root extract against 3-nitropropionic acid-induced behavioral, biochemical, and mitochondrial dysfunction in an animal model of Huntington's disease[END_REF] . Although some in vivo studies using various animal models suggest W. somnifera extracts can improve antioxidant status and provide neuroprotection [START_REF] Jain | Neuroprotective effects of Withania somnifera Dunn. in hippocampal sub-regions of female albino rat[END_REF][START_REF] Parihar | Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity[END_REF][START_REF] Sankar | The neuroprotective effect of Withania somnifera root extract in MPTP-intoxicated mice: An analysis of behavioural and biochemical variables[END_REF][START_REF] Bhatnagar | Neuroprotective effects of Withania somnifera Dunal.: A possible mechanism[END_REF], there is a paucity of data on the potential protective effects of W. somnifera root, as traditionally used, against H 2 O 2and Aβ (1-42) -induced cytotoxicity which are current targets for novel approaches for the treatment of dementia, especially dementia of the Alzheimer's type (AD). The aim of this study was to investigate the potential neuroprotective effects of an aqueous extract of W. somnifera root against H 2 O 2 -and Aβ (1-42) -induced toxicity under in vitro conditions using a differentiated PC12 cell line as a model of neuronal cells. Extract preparation. The air dried roots were ground to powder consistency using an electric grinder. 1 g of powdered root was infused in freshly boiled de-ionised water (1:50 w/v) for 25 min. The infusion was left to cool to room temperature and centrifuged (12000 rpm, 15 min). The supernatant was re-centrifuged (12000 rpm, 10 min) and then freeze dried. The freeze dried aliquots were reconstituted in de-ionised water prior to assay.

MATERIALS AND METHODS

Plant

Cell line and cell culture. Rat pheochromocytoma (PC12) cell line was a generous gift from the Medical School, Newcastle University. RPMI-1640, penicillin-streptomycin, foetal calf serum, glutamine and nerve growth factor (NGF) were purchased from Invitrogen (UK). Aβ (1-42) and trypan blue were purchased from Sigma (UK).

Cells were maintained in RPMI media supplemented with 10 % heat-inactivated foetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin-streptomycin in humidified 5 % CO 2 and 95 % air at 37 °C. All cells were cultured in culture flasks pre-coated with poly-D-lysine (0.1 mg/mL). Cells were differentiated for 2-3 days using 50 ng/mL NGF. Prior to experimental treatments, cells were microscopically examined to assess differentiation. Eighty percent or more of cells with neurite outgrowth extensions over 2-3-fold cell body size were considered to be differentiated PC12 cells (dPC12). The medium was changed every alternate day. Prior to confluence, cells were dislodged by mechanical scraping and split in 1:3 ratio. Before each experiment, cells were checked for viability using a trypan blue (0.5 %) dye exclusion method (Freshney, 2000). Cells were counted using a haemocytometer and the density was adjusted to 1x10 5 cells/mL prior to plating in 96-well plates; cells in exponential growth phase were used. Assay of H 2 O 2 -induced toxicity. Before treatment, cells were plated at an appropriate density (1x10 4 cells/100µL) in a 96-well plate and incubated for 24 h at 37 o C, so that the cells were acclimatized to the new environment. Cells were pre-incubated with W. somnifera root extract (concentration range: 0.097 -50 µg/mL), prior to exposure to H 2 O 2 (concentration range: 12.5 µM -400 µM) from a freshly prepared 1000 µM stock solution. After 24 h the cell viability was determined by an MTT assay, as described below.

β-Amyloid (1-42) fibrils preparation. Aβ (1-42) peptide was stored at -20 o C until use at room temperature; lyophilized peptide was dissolved in distilled water at a concentration of 0.1 mg/mL, with thorough pipetting over a period of 2 min to clarify the solution. It was then incubated overnight at 37 o C with constant oscillation to form the fibrils used to induce toxicity in dPC12 cells. 8 incubated with MTT (0.5 mg/mL) for 3 h. The dark blue formazan crystals formed in live cells were solubilised with dimethylsulfoxide and ethanol (1:1) and the absorbance was measured at 570 nm using a spectrophotometer (Molecular Device Spectramax Plus 384, equipped with Softmax Pro V5 software) (Mosmann, 1983).

Assay of β-amyloid

Liquid chromatography-serial mass spectrometry. The freeze dried aqueous extract of W. somnifera root was analysed by liquid chromatography-serial mass spectrometry (LC-MS n ) using an HPLC system (Thermo Scientific 'Surveyor' autosampler, pumps and diode array detector) coupled to a 3D ion-trap mass spectrometer (Thermo Scientific 'LCQ Classic') via an electrospray source. Chromatography was achieved on a 150 mm x 4.6 mm, 5 µm C18 column (Phenomenex 'Luna C18(2)') using a 1 ml/min mobile phase gradient of 10 % to 100 % aqueous acetonitrile containing 0.1 % formic acid over 40 min. The flow to the ESI source was reduced to 200 µl/min by a splitter and the source was operated using a needle voltage of ±4.2 kV and desolvation nitrogen gas flows of 80 (sheath) and 20 (auxillary) units. The heated capillary temperature was 220 ºC and standard tuning voltages (obtained from rutin) were used. The instrument was operated using Xcalibur 2.0 software and components in the LC-MS n analyses were detected using MassFrontier 4.0 (both Thermo Scientific). Withaferin A (LGC Standards, UK) was also subjected to LC-MS n analysis using this method.

Statistical analyses.

Results are expressed as the mean ± SEM. Student t-test was used to compare differences between test groups and the negative control (H 2 O 2 or Aβ only) using Graphpad statistical software; p<0.05 were considered significant. When dPC12 cells were incubated with different H 2 O 2 concentrations (12.5 µM to 400 µM) at 24, 48, 72 h time intervals, a concentration-dependent cytotoxicity was observed (Fig. 1). The cell viability was decreased to 50 % at 200 µM H 2 O 2 during 24 incubation, but less than 50% for 48 and 72 h incubation at 200 and 400 µM H 2 O 2 (p<0.001). Thus, 200 µM H 2 O 2 was selected as an appropriate concentration to induce toxicity, with an incubation time of 24 h, to assess the effects of the W. somnifera extract in further experiments.

Effect of W. somnifera extract on viability of dPC12 cells against H 2 O 2 -induced cytotoxicity

As shown in Fig. 2, the aqueous extract of W. somnifera significantly protected dPC12 cells from H 2 O 2 -induced toxicity when cells were pre-incubated with W. somnifera extract for 24 h prior to H 2 O 2 (200µM) exposure. After H 2 O 2 exposure for 24 h, 50 -80 % cell viability was observed at extract concentrations from 6.11 µg/mL to 100 µg/mL, compared with the negative control (H 2 O 2 alone), with 50 and 100 µg/mL extract concentrations showing the greatest improvement compared with the negative control (p<0.001). At the highest extract concentration (200 µg/mL) the cytoprotective effects were abolished, due, perhaps, to direct cytotoxic and antiproliferative properties of W. somnifera being expressed at high concentrations (Sabbaraju et al., 2006;[START_REF] Stan | Ayurvedic medicine constituent withaferin A causes G2 and M phase cell cycle arrest in human breast cancer cells[END_REF]. (1-42) over the concentration range of 0.007 to 2.0 µg/mL (Fig. 3). The cell survival was expressed as a percentage of the control measured in the absence of Aβ peptide. The cell viability was decreased to approximately 50 % at 0.5 µg/mL and no further decrease was observed by increasing the Aβ concentration above 0.5 µg/mL.

Consequently, this concentration of Aβ was selected to induce cytotoxicity in further experiments to investigate the neuroprotective effect of W. somnifera extract against Aβ-induced toxicity.

Effect of W. somnifera extract on viability of dPC12 cells against β-amyloid (1-42)induced toxicity

The pre-treatment of W. somnifera extract (0.75 µg/mL -100 µg/mL) for 24 h prior to Aβ incubation produced dose-dependent attenuation of Aβ-induced toxicity in dPC12 cells. Maximum viability (80 % of positive control) was observed at the highest concentration (100 µg/mL) tested (Fig. 4).

LC-MS n analysis

Positive ion LC-MS n analysis of the W. somnifera extract revealed numerous components (Fig. 5). Component 36 was identified as withaferin A against a standard and together with two other isomeric withanolides (38 & 40), these constituted the main withanolide aglycones in the extract. The majority of the other components, eluting earlier in the analysis, were assigned as derivatives of withanolides (Table 1). Among these were compounds clearly assignable as glycosides of withanolides ( 10 More exact assignment of withanolides from W. somnifera was not determined due to the numerous isomeric forms of withanolides reported in the literature (CCD, 2010).

Other components were also assigned as withanolide derivatives. Following MS2 of the protonated molecule they generated a complex array of product ions, but among these were those with expected m/z values for fragments corresponding to protonated withanolide aglycones. Furthermore, for components such as 14 that generated a product ion m/z 471 (the value expected for a fragment equivalent to protonated withaferin A and isomers), other product ions at m/z 299 and 281 were also observed; Aβ [START_REF] Jayaprakasam | Withanamides in Withania somnifera fruit protect PC-12 cells from beta-amyloid responsible for Alzheimer's disease[END_REF]) have been reported in the fruits of W. somnifera, none have the same molecular mass as 12 and do not display a potential aglycone product ion at m/z 471 [START_REF] Jayaprakasam | Potent lipid peroxidation inhibitors from Withania somnifera fruits[END_REF]). An isomer of 12, component 16, also generated a major product ion at m/z 649, but serial MS analysis of this ion produced a different spectrum to that of 12, in particular the spectrum suggested that the molecular mass of the aglycone was 452. Components 13 and 17 were likely hexosides of 12 (or its isomers) and a likely hexoside of 14 (or isomer) was also present (component 15). Four components (39, 41, 42 & 44) in the LC-MS n analysis were assigned as withanolide sulphates as their molecular masses showed an 80 Da increment over that of known withanolides. Other noted components in the extract were low molecular mass nitrogen containing compounds (1-3).

DISCUSSION

There is growing interest in naturally-derived bioactive compounds with potential neuroprotective properties against a range of neurodegenerative diseases. One area of current pharmacological focus is neuroprotective therapeutic strategies aimed at counteracting H 2 O 2 -and Aβ-induced neurotoxicity associated with AD pathophysiology. In this study, PC12 cells were used as an in vitro model to study the neuroprotective potential of W. somnifera root aqueous extract against H 2 O 2 -and Aβinduced toxicity. PC12 cells were chosen as they acquire neuronal like projections when induced to differentiate with nerve growth factor. The differentiated PC12 cells are both morphologically and physiologically very similar to living neurons in the brain; 13 therefore the results obtained by neuroprotective studies with experimental test plant extracts will be more likely to represent the response when using differentiated, rather than with non-differentiated cells (Datki et al., 2003). To induce Aβ peptide toxicity, the peptide fragment with a length of 1-42 amino acid residues was used, as this fragment aggregates very rapidly and is responsible for inducing toxicity as compared with nonaggregated peptides. The toxic effects of Aβ (1-42) fragments have been well-documented in a number of studies (Behl et al., 1992;Michaelis et al., 1998;Limpeanchob et al., 2008).

The present study demonstrated that pre-treatments of differentiated PC12 cells with an aqueous extract of W. somnifera root significantly protected dPC12 cells against both H 2 O 2 -and Aβ-induced cytotoxicity, in a concentration dependent manner. As Aβ is known to increase free radical production and lipid peroxidation in PC12 cells, leading to apoptosis and cell death (Jung et al., 2007), the cytoprotective effects observed could be attributed to the presence of free radical scavenging compounds in the aqueous extract of W. somnifera. Evidence to support this mode of action has been shown previously. Inhibition of lipid peroxidation due to an antioxidant action has been observed both in vitro and in vivo with extracts of W. somnifera root and some of the component withanolides, including withaferin A [START_REF] Dhuley | Effect of ashwagandha on lipid peroxidation in stress-induced animals[END_REF][START_REF] Panda | Protective role of ashwagandha in cadmiuminduced hepatotoxicity and nephrotoxicity in male mouse[END_REF][START_REF] Bhattacharya | Effect of Withania somnifera glycowithanolides on iron-induced hepatotoxicity in rats[END_REF][START_REF] Ccd ; | Withania somnifera root extract in the regulation of lead-induced oxidative damage in male mouse[END_REF], a compound detected in the W. somnifera extract investigated in the present study. In addition to the withanolides having antioxidant capacity and decreasing lipid peroxidation in rodent brains, withanolides also enhanced catalase and glutathione peroxidase activities in rat frontal cortex and striatum [START_REF] Ccd ; | Withania somnifera root extract in the regulation of lead-induced oxidative damage in male mouse[END_REF][START_REF] Mishra | Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): A review[END_REF][START_REF] Scartezzini | Review on some plants of Indian traditional medicine with antioxidant activity[END_REF][START_REF] Bhattacharya | Antioxidant activity of glycowithanolides from Withania somnifera[END_REF]2001;[START_REF] Subbaraju | Ashwagandhanolide, a bioactive dimeric thiowithanolide isolated from the roots of Withania somnifera[END_REF]. In another 14 study, an extract of W. somnifera was associated with attenuation of memory loss induced by oxidative stress mediated by free radicals in an animal model (Parihar et al., 2004). W. somnifera root extract was also shown to significantly reduce the level of lipid peroxidation and improved the antioxidant status in an animal model of Parkinson's disease [START_REF] Sankar | The neuroprotective effect of Withania somnifera root extract in MPTP-intoxicated mice: An analysis of behavioural and biochemical variables[END_REF]. Other studies showed that W. somnifera root extract also imparted neuroprotective effects, observed in a 6-hydroxydopamineinduced model in rats, and a lithium-pilocarpine induced seizures model of status epilepticus in rats (Kulkarni et al., 1998) and various other animal models of neurological disorders, including Parkinsonism and epilepsy (Sanker et al., 2007;Kulkarni and Dhir, 2008). There is also evidence of neuroprotection by withanolide A, withanoside IV, and withanoside VI, steroidal derivatives isolated from a root extract of W. somnifera, that attenuated Aβ (25-35)-induced axonal, dendritic and synaptic losses and memory deficits in mice [START_REF] Kuboyama | Neuritic regeneration and synaptic reconstruction induced by withanolide A[END_REF][START_REF] Kuboyama | Neuritic regeneration and synaptic reconstruction induced by withanolide A[END_REF][START_REF] Kuboyama | Withanoside IV and its active metabolite, sominone, attenuate Abeta (25-35)-induced neurodegeneration[END_REF]. A study by [START_REF] Jayaprakasam | Withanamides in Withania somnifera fruit protect PC-12 cells from beta-amyloid responsible for Alzheimer's disease[END_REF] demonstrated the ability of withanamides A and C present in W. somnifera fruit extracts to protect undifferentiated PC-12 cells from Aβ cytotoxicity. Thus, the neuroprotective effects of the W. somnifera aqueous root extract against H 2 O 2 -and Aβ-induced cytotoxicity shown in this study, are consistent with other studies that have suggested W. somnifera may be neuroprotective in vivo by mediating antioxidant effects.

One in vitro study showed an interesting finding in which W. somnifera root extract significantly and dose-dependently increased the percentage of cells with neurites in human neuroblastoma SK-N-SH cells [START_REF] Tohda | Dendrite extension by methanol extract of Ashwagandha (roots of Withania somnifera) in SK-N-SH cells[END_REF]. However, one observation in our study was that at high concentrations (> 100 µg/mL), the aqueous extract exhibited cytotoxic and perhaps anti-proliferative properties against the dPC12 cell line, [START_REF] Subbaraju | Ashwagandhanolide, a bioactive dimeric thiowithanolide isolated from the roots of Withania somnifera[END_REF][START_REF] Stan | Ayurvedic medicine constituent withaferin A causes G2 and M phase cell cycle arrest in human breast cancer cells[END_REF]. Indeed, component 32, detected in the W. somnifera extract by LC-MS n analysis (Table 1, Fig. 5), showed mass spectral data consistent with the dimeric thiowithanolide, ashwagandhanolide, a compound reported to inhibit cell proliferation in various tumor cell lines [START_REF] Subbaraju | Ashwagandhanolide, a bioactive dimeric thiowithanolide isolated from the roots of Withania somnifera[END_REF].

In conclusion, this study demonstrated that an aqueous extract of W. somnifera root, in which numerous withanolide derivatives were detected, was neuroprotective against H 2 O 2 -and Aβ-induced cytotoxicity, providing further evidence to explain the use of W. somnifera root in some cognitive disorders. However, further investigations are required to confirm the identity and potency of the specific compounds responsible for the reported neuroprotective effects of W. somnifera against H 2 O 2 -and Aβ-induced cytotoxicity, and to determine whether the observed neuroprotective effects are due to potential polyvalent activities [START_REF] Williamson | Synergy and other interactions in phytomedicines[END_REF] amongst the constituents of the withanolide-rich aqueous extract.

F o r P e e r R e v i e w

21 Table 1. Compounds observed by LC-MS analysis of W. somnifera root aqueous extract. Data presented are the retention time (min), the major ion species found in positive and negative modes (and the assignment of these ions), the deduced molecular mass (M r ) and the suggested molecular mass of the aglycone (A) determined from the positive and / or negative ion MS2 spectra.

  apoptosis in neuronal cells

  various models of oxidative stress and cytotoxicity, and disease mechanisms have been explored, with varying results

  material. The roots of W. somnifera were purchased from a local herbalist in India and were verified and authenticated by Dr George Wake, School of Biology, Newcastle University, UK. A voucher specimen (SK-WS-01) is deposited in the Herbarium of the Medicinal Plant Research Group, Newcastle University, UK.

  (1-42) -induced cytotoxicity. The in vitro toxicity of Aβ (1-42) to dPC12 cells was measured by incubating the cells for 24 h at 37 o C, with increasing concentrations (0.007 -2 µg/mL) of aggregated Aβ (1-42) peptide in a 96-well plate. Cell viability was then assessed by measuring cellular redox activity with the MTT assay described below.MTT assay. PC12 cell viability was determined by the MTT reduction assay. In brief, MTT, a tetrazolium salt, is cleaved to formazan by succinate dehydrogenase, an enzyme in the mitochondrial respiratory chain, by live cells. After pre-incubation of the dPC12 cells with the test extracts and toxicity inducers (H 2 O 2 or Aβ) for 24 h, cells were

  dPC12 cell viability after treatment for 24, 48 and 72 h

  β-amyloid (1-42) -peptide aggregates on dPC12 cells. A concentration-dependent decrease of cell survival was observed after exposure to Aβ

  since, following MS2 of the protonated or ammoniated molecules, they generated fragment ions resulting from cleavage of glycosidic bonds, including protonated aglycone ions at m/z values expected for reported withanolides. Furthermore, upon MS3 analysis, these protonated aglycone ions showed patterns of neutral losses similar to those observed following MS2 analysis of the free withanolides in the extract.

  withaferin A also generated abundant product ions at these m/z values following MS/MS of the protonated molecule. Several of the components assigned as withanolide derivatives appeared to have odd-number molecular masses (i.e. suggesting they contained an odd number of nitrogen atoms), including component 12 that produced the largest chromatographic peak in the analysis. The major ion species generated by positive ESI of 12 (m/z 778) was assigned as [M+H] + on the basis of minor confirmatory ions at m/z 795 [M + NH 4 ] + and m/z 800 [M + Na] + , and the generation of a major ion species at m/z 776 [M -H] -in a negative ion ESI analysis. The MS2 spectrum of protonated 12 showed minor product ions at m/z 471, 299 and 281,suggesting that the compound contained a withanolide moiety. The major product ion was at m/z 649 and MS3 analysis of this ion produced a spectrum that was almost product ion spectrum of protonated 6 ([M+H] + = m/z 649). Although nitrogen-containing compounds (including withanamides that protect PC12 cells from

  reputed anticancer properties of W. somnifera

  Figures legends

Fig. 2

 2 Fig. 2 Protective effect of an aqueous extract of W. somnifera against H 2 O 2 -induced

Fig. 3

 3 Fig. 3 Dose-dependent neurotoxicity of Aβ (1-42) aggregates in differentiated PC12 cells

Fig. 4

 4 Fig. 4 Protective effect of W. somnifera extract against Aβ (1-42) -induced toxicity in

Fig. 5

 5 Fig. 5 MS1 base ion chromatogram from LC-MS n (positive ESI) analysis of W. somnifera root aqueous extract. Peaks are assigned to withanolides, as presented in

Table 1 ;

 1 compound 36 is assigned as withaferin A by comparison with a reference standard of withaferin A.

  The dimeric thiowithanolide, ashwagandhanolide, with the same M r as 32 has been reported by[START_REF] Subbaraju | Ashwagandhanolide, a bioactive dimeric thiowithanolide isolated from the roots of Withania somnifera[END_REF]. **Peak assigned as withaferin A by comparison with a reference standard for withaferin A

						Phytotherapy Research
	23	14.1	934	[M + H]+	932	[M -H]-	933	-	-	916 (20) 726 (53) 579 (40) 562 (100) 544 (30) 518 (22) 356
										(11)
	24	14.2	715	[M + H]+	-	-	714 470 withanolide derivative	679 (33) 622 (53) 471 (100) 299 (33) 281 (15)
						[M +
	25	14.4	816	[M + NH 4 ]+	843	HCOO]-	798 456 withanolide dihexoside	799 (6) 619 (30) 457 (100) 439 (64) 421 (35) 403 (6)
	26	14.5	489	[M + H]+	-	-	488	-	withanolide	317 (100) 299 (37) 281 (16)
						[M +
	Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 27 28 29 30 31 *32 33 34 35 **36 37 38 39 40 15 41 16 17 18 19 20 21 22 42 43 44	Retention time (min) 7.4 7.8 8.9 9.1 9.9 10.1 10.2 10.6 11.0 11.1 11.3 11.5 11.6 11.8 14.7 15.0 15.1 15.2 15.4 15.5 15.8 16.3 17.0 17.4 F Positive ion (m/z) 224 226 226 794 574 649 1112 832 794 962 592 778 940 721 800 762 650 638 762 976 654 705 784 471 o r Assignment [M + H]+ [M + H]+ [M + H]+ [M + H]+ [M + H]+ [M + H]+ -[M + NH 4 ]+ [M + H]+ [M + NH 4 ]+ [M + H]+ [M + H]+ [M + H]+ [M + H]+ [M + NH 4 ]+ [M + H]+ [M + NH 4 ]+ [M + NH 4 ]+ [M + H]+ [M + H]+ [M + NH 4 ]+ [M + H]+ [M + NH 4 ]+ [M + H]+ F o r F o r F o r F o r F o r P e e r R e v i e w Negative ion (m/z) Assignment M r A Assignment --223 NA ---225 NA ---225 NA -792 [M -H]-793 --572 [M -H]-573 --647 [M -H]-648 470 withanolide derivative ----859 [M + HCOO]-814 472 withanolide glycoside 792 [M -H]-793 520 withanolide derivative 943 [M -H]-944 458 withanolide trihexoside 590 [M -H]-591 470 withanolide derivative 776 [M -H]-777 470 withanolide derivative 938 [M -H]-939 470 withanolide derivative 719 [M -H]-720 470 withanolide derivate 827 HCOO]-782 458 withanolide dihexoside 760 [M -H]-761 454 withanolide derivative --632 470 withanolide monohexoside --620 458 withanolide monohexoside 760 [M -H]-761 454 withanolide derivative 974 [M -H]-975 --681 [M + HCOO]-636 458 withanolide monohexuronide 703 [M -H]-704 454 withanolide derivative 811 [M + HCOO]-766 442 withanolide dihexoside (indicative of withanoside V) --470 -withaferin A 18.3 730 [M + H]+ 728 [M -H]-729 --18.6 471 [M + H]+ --470 -withanolide P P P P P 19.4 569 [M + H]+ 567 [M -H]-568 488 withanolide sulphate 19.8 488 [M + NH 4 ]+ --470 -withanolide e e r e e r e e r e e r e e r 11.9 883 [M + H]+ 881 [M -H]-882 -withanolide derivative 20.3 569 [M + H]+ 567 [M -H]-568 488 withanolide sulphate 12.1 778 [M + H]+ 776 [M -H]-777 452 withanolide derivative 12.2 940 [M + H]+ 938 [M -H]-939 -withanolide derivative 12.4 721 [M + H]+ 719 [M -H]-720 452 withanolide derivative 12.6 778 [M + H]+ 776 [M -H]-777 452 withanolide derivative 12.9 897 [M + H]+ 895 [M -H]-896 502? withanolide derivative 13.0 800 [M + NH 4 ]+ 827 [M + HCOO]-782 458 withanolide dihexoside 14.0 800 [M + NH 4 ]+ 827 [M + HCOO]-782 458 withanolide dihexoside 22.8 586 [M + NH 4 ]+ 567 [M -H]-568 488 withanolide sulphate 23.4 280 [M + H]+ --279 --24.1 569 [M + H]+ 567 [M -H]-568 488 withanolide sulphate R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w	Product ions following MS2 of positive ion species listed (m/z; %) 124 (100) 93 (17) 124 (100) 93 (20) 126 (100) 84 (43) 776 (10) 655 (100) 648 (55) 556 (100) 538 (85) 472 (90) 632 (100) 556 (40) 471 (57) 299 (17) 281 (7) unstable ion 815 (5) 635 (7) 473 (100) 665 (100) 521 (80) 945 (916) 983 (30) 621 (100) 459 (95) 575 (80) 574 (100) 471 (35) 435 (20) 299 (30) 281 (20) 760 (10) 649 (100) 632 (55) 556 (20) 471 (22) 299 (9) 281 (4) 922 (70) 886 (68) 865 (86) 649 (100) 471 (16) 783 (6) 603 (44) 459 (8) 441 (100) 423 (8) 405 (9) 744 (7) 633 (100) 616 (61) 455 (24) 471 (100) 621 (7) 603 (14) 459 (100) 441 (72) 423 (27) 405 (12) 744 (7) 633 (76) 615 (100) 455 (34) 772 (23) 604 (100) 417 (25) 457 (100) 439 (53) 421 (19) 403 (3) 687 (20) 455 (26) 437 (94) 419 (100) 401 (67) 767 (20) 605 (19) 443 (100) (425 (24) 407 (7) 435 (10) 299 (74) 281 (100) 175 (6) 712 (20) 601 (41) 583 (100) 423 (77) 405 (48) 453 (15) 435 (100) 417 (94) 399 (63) 371 (17) 263 (49) 211 (22) 197 (13) 533 (17) 515 (28) 497 (32) 453 (73) 435 (91) 417 (78) 397 (49) 361 (100) 283 (20) 471 (65) 453 (100) 435 (32) 417 (71) 399 (54) 289 (23) 263 (36) 703 (16) 592 (100) 574 (41) 471 (17) 299 (12) 281 910) 489 (5) 453 (5) 397 (100) 317 (11) 299 (6) 281 (6) 865 (91) 847 (71) 829 (100) 799 (47) 592 (20) 760 (7) 649 (100) 632 (64) 556 (18) 453 (15) 922 (55) 886 (63) 856 (62) 649 (100) 703 (44) 592 (65) 574 (100) 556 (30) 453 (8) 760 (8) 703 (28) 649 (100) 631 (81) 453 (11) 879 (100) 689 (38) 503 (8) 486 (11) 468 (7) 783 (8) 621 (97) 459 (100) 441 (19) 423 (10) 783 (71) 621 (25) 459 (100) 441 (76) 423 (41) 405 (25) 569 (44) 551 (100) 533 (15) 515 (15) 453 (17) 417 (40) 399 (15) 389 (18) 361 (11) 263 (100) ionisation in positive mode too low
							22
				209x297mm (300 x 300 DPI) 209x297mm (300 x 300 DPI) 209x297mm (300 x 300 DPI)
				209x297mm (300 x 300 DPI) 209x297mm (300 x 300 DPI)

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http://mc.manuscriptcentral.com/ptr Phytotherapy Research

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Sinead Ryan

Denise M O'gorman

Yvonne M Nolan
email: y.nolan@ucc.ie

Evidence that the marine-derived multi-mineral Aquamin has anti-inflammatory effects on cortical glial-enriched cultures

Keywords: inflammation, Aquamin, IL-1beta, TNFalpha, glia, brain Anti-inflammatory properties of Aquamin in cortical glia Inflammation; Aquamin, glia, TNFα, IL-1β, brain

It is well established that neuroinflammation contributes to brain aging, and that cortical cells are particularly vulnerable. Lipopolysaccharide stimulates the release of the proinflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta from glial cells which consequently induces an impairment in neuronal cell function. The food supplement Aquamin is a natural, multi-mineral derived from the red algae Lithothamnion corallioides, rich in calcium, magnesium and 72 other trace minerals.

The aim of this study was to evaluate the anti-inflammatory potential of Aquamin in lipopolysaccharide-stimulated, glial-enriched primary cultures of rat cortex. We report that Aquamin prevented lipopolysaccharide-induced secretion of tumor necrosis factoralpha and interleukin-1beta from cortical glia. These data suggest that nutritional supplements such as Aquamin may play an important role in impeding the detrimental effects of excessive inflammation in the brain.

Evidence that the marine-derived multi-mineral Aquamin has anti-inflammatory effects on cortical glial-enriched cultures.

Introduction

Minerals such as magnesium copper, zinc, manganese and selenium are now recognized as important regulators of inflammation and growing evidence suggests that mineralrich seaweed extracts may play an important role in the regulation of inflammation [START_REF] Granert | Effects of polysaccharide fucoidin on cerebrospinal fluid interleukin-1 and tumor necrosis factor alpha in pneumococcal meningitis in the rabbit[END_REF][START_REF] Jung | Suppressive effect by Hizikia fusiforme on the production of tumor necrosis factor in BV2 murine microglial cells[END_REF]. The food supplement, Aquamin is a natural seaweed-derived multi-mineral from the red algae Lithothamnion corallioides which is rich in calcium, magnesium and trace amounts of other minerals (Table 1). It has recently been shown to provide relief from the symptoms of osteoarthritis ( , 2009) and to be of benefit in digestive and bone health (Aslam et al., 2010a;Aslam et al., 2010b).

It is well established that inflammation contributes to cortical neuronal dysfunction in age-related neurological diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis and Parkinson's disease [START_REF] Minghetti | Role of inflammation in neurodegenerative diseases[END_REF]. Microglia are considered the resident immune cells of the brain and along with astrocytes, constitute the major glial cell types. Under conditions such as neurodegenerative disease, injury, exposure to environmental toxins, infection or age, microglia become activated to release large numbers of mediators such as cytokines and free radicals. Microglialsecreted pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNFα) can influence neurons and their ability to process information and can ultimately contribute to neuronal cell death. Astrocytes provide structural and metabolic support to neurons and also play a role in brain inflammation through antigen presentation and cytokine secretion. It has previously been reported that exposure to the endotoxin lipopolysaccharide (LPS) induces release of IL-1β and TNFα from cortical glia to contribute to neuronal degeneration [START_REF] Cm | Interleukin-1β contributes to dopaminergic neuronal death induced by lipopolysaccharide-stimulated rat glia in vitro[END_REF]. The present study was designed to investigate the anti-inflammatory potential of Aquamin in LPS-stimulated, glial-enriched rat cortical cultures to determine if nutritional supplements, such as Aquamin, may play a role in impeding the detrimental effects of excessive inflammation in the brain.

Materials and Methods

F

Results and Discussion

The data presented demonstrate that LPS induced a significant increase in TNFα (Figure 1A) and IL-1β (Figure 1B) secretion from glial-enriched cortical cultures 24 hours after LPS treatment (p<0.001). Treatment with all doses of Aquamin significantly attenuated the LPS-induced increase in TNFα and IL-1β secretion (p<0.01). Analysis of extracellular concentrations of TNFα revealed that Aquamin prevented its release from LPS-stimulated cortical glial cells in a dose-dependent manner and that all doses of Aquamin significantly attenuated the LPS-induced release of IL-1β. These data suggest that the anti-inflammatory effects of Aquamin may be reliant on blockade of the proinflammatory effects of both TNFα and IL-1β.

Seaweed extracts have previously been reported to inhibit pro-inflammatory cytokine release. Fucoidans from brown algae have been shown to inhibit TNF-α and IL-1 production in cerebrospinal fluid in a rabbit meningitis model [START_REF] Granert | Effects of polysaccharide fucoidin on cerebrospinal fluid interleukin-1 and tumor necrosis factor alpha in pneumococcal meningitis in the rabbit[END_REF] and an extract from the seaweed Hizikia fusiform reduced TNFα production in LPSstimulated murine microglial cells [START_REF] Jung | Suppressive effect by Hizikia fusiforme on the production of tumor necrosis factor in BV2 murine microglial cells[END_REF]. Aquamin is a seaweed-derived multi-mineral rich in calcium and magnesium. It has been shown that IL-1β reduced Ca2+ channel activity and influx in rat cortical neurons [START_REF] Macmanus | Enhancement of (45)Ca(2+) influx and voltage-dependent Ca(2+) channel activity by beta-amyloid-(1-40) in rat cortical synaptosomes and cultured cortical neurons. Modulation by the proinflammatory cytokine interleukin-1beta[END_REF] suggesting that Ca2+ supplementation may be beneficial in situations of excessive IL- et al., 1999). Accumulation of reactive oxygen species in the rat cortex and hippocampus has been demonstrated after LPS treatment [START_REF] Nolan | Evidence that lipopolysaccharideinduced cell death is mediated by accumulation of reactive oxygen species and activation of p38 in rat cortex and hippocampus[END_REF] while an antioxidant-enriched diet has been shown to reverse age-related and inflammatoryinduced neuronal deficits [START_REF] Mcgahon | Age-related changes in oxidative mechanisms and LTP are reversed by dietary manipulation[END_REF]. Consequently, many of the antioxidant-related minerals that compose Aquamin may be anti-inflammatory and may directly or indirectly have neuroprotective properties.

The current study provides clear evidence that Aquamin exerts anti-inflammatory effects by preventing LPS-induced TNFα and IL-1β release from rat cortical glia. This evidence for an anti-neuroinflammatory activity of the food supplement Aquamin may have significant implications for brain health

  and Drug Administration (FDA) GRAS 000028) was prepared from the mineral-rich red marine algae, Lithothamnion corallioides harvested off the Atlantic coasts of Ireland and Iceland under approved licenses. The calcified seaweed was separated from extraneous materials, sterilized, dried and milled under ISO and HACCP certification. Approximately 0.5mg/ml of Aquamin is equivalent to a physiological level of extracellular Ca 2+ . The concentrations of Aquamin were determined from previous experiments using low passage human dermal fibroblasts, calcium sensitive and calcium resistant colon carcinoma cell lines (CBS, Moser, Fet, HCT-116 and SW480)[START_REF] Aslam | Growth-inhibitory effects of a mineralised extract from the red marine algae, Lithothamnion calcareum, on Ca2+,-sensitive and Ca2+-resistant human colon carcinoma cells[END_REF], the murine macrophage cell line RAW 264.7, and the preosteoblastic cell line MC3T3-E1 (unpublished data). Aquamin demonstrated no signs of toxicity at any of the concentrations used in the current study. Glial-enriched cultures were prepared from cortical tissue isolated from seven separate postnatal day 2 Sprague-Dawley rat pups (Biological Services Unit, University College Cork) as previously described[START_REF] Cm | Interleukin-1β contributes to dopaminergic neuronal death induced by lipopolysaccharide-stimulated rat glia in vitro[END_REF]. All scientific procedures were performed under a license issued by the Department of Health and Children (Ireland) and in accordance with the European Communities Council Directive (86/609/EEC). After 7-10 days in vitro (DIV), glia were incubated in the presence or absence of LPS (50 ng/ml) and Aquamin (0.05, 0.1, 0.5, 1 and 2 mg/ml). Twenty-four hours later, supernatant was removed for analysis of TNFα and IL-1β by ELISA (R&D Systems, UK). Untreated culture media was used as a control. The cells were assessed for morphological signs of apoptosis and necrosis (loss of cell membrane asymmetry and attachment, cell shrinkage, cell blebbing, nuclear fragmentation and chromatin condensation) prior to, during and after 24 hours incubation with Aquamin. All cells were considered viable throughout the experiment. ANOVA with post hoc Student Newman-Keuls was used to were significantly different from each other. Results were expressed as means with standard error of the mean (SEM) and deemed significant when p<0.05.

  the brain such as occurs with aging. Increased magnesium in the diet may influence inflammation through reducing the serum level of the proinflammatory C-reactive protein. Copper, zinc and manganese are essential cofactors of the antioxidant enzyme superoxide dismutase and selenium is a vital constituent of the antioxidant glutathione peroxidase. A compromised antioxidant defense has been observed in the cortex of aged rats in parallel with increased IL-1β concentration (McGahon

Figure 1 :

 1 Figure 1: Aquamin prevents LPS-induced release of TNFα and IL-1β. TNFα (A) and

Figure 1 :

 1 Figure 1: Aquamin prevents LPS-induced release of TNFα and IL-1β. TNFα (A) and IL-1β (B) levels from LPS-stimulated glial-enriched cortical cultures. Data are expressed means ± SEM of seven independent experiments, each performed in triplicate. ***p<0.001 vs. control; ++p<0.01; +++p<0.001 vs. LPS (ANOVA). 109x141mm (600 x 600 DPI)

Table 1 :

 1 Typical Mineral Composition of Aquamin.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

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This work was funded by Marigot Ltd. There are no conflicts of interest.