Effects of Sea Buckthorn Berries on Infections and Inflammation: a Double-Blind, Randomized, Placebo-Controlled Trial

ORIGINAL ARTICLE Effects of sea buckthorn berries on infections and inflammation: a double-blind, randomized, placebo-controlled trial

P Larmo1,2, J Alin3, E Salminen4, H Kallio2 and R Tahvonen1,5

1Functional Foods Forum, University of Turku, Turku, Finland; 2Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland; 3Department of Biostatistics, University of Turku, Turku, Finland; 4Department of Radiotherapy and Oncology, Turku University Hospital, Turku, Finland and 5Functional Foods Forum/EPANET, University of Turku, Seina¨joki, Finland

Objective: To study the effect of sea buckthorn berries on the number and duration of common cold (CC) infections. As secondary objectives the effects on digestive and urinary tract infections (DTI, UTI), and serum C-reactive protein (CRP) concentrations were also investigated. Subjects: A total of 254 healthy volunteers were randomly assigned to receive sea buckthorn or placebo product during the study, which 233 of them completed. Results: There were no significant differences in the number or duration of CC or DTI between groups (CC: relative risks (sea buckthorn vs placebo) for the number and duration were 1.15 (95% CI 0.90–1.48) and 1.05 (95% CI 0.87–1.27), respectively). In the sea buckthorn group, as compared to the placebo, the serum CRP concentrations decreased significantly (difference in median change À0.059 mg/l, P ¼ 0.039). The number of UTI was too small to draw solid conclusions, but the results indicate the subject merits further investigation. Conclusion: Sea buckthorn berries did not prevent CC or DTI. However, a reductive effect on CRP, a marker of inflammation, and a risk factor for cardiovascular diseases, was detected. European Journal of Clinical Nutrition (2008) 62, 1123–1130; doi:10.1038/sj.ejcn.1602831; published online 27 June 2007

Keywords: Hippophae¨; common cold; C-reactive protein; infection

Introduction rhamnoides L.) berries have traditionally been used in Eastern medicine and studies indicate that sea buckthorn has The common cold (CC) is a mild upper respiratory illness immunomodulatory properties and positive effects on health caused by numerous viruses. Although the CC is usually a self- (Yang et al., 1999; Johansson et al., 2000; Eccleston et al., 2002; limited illness of short duration, it causes considerable costs in Geetha et al., 2002; Yang and Kallio, 2002; Dorhoi et al., 2006). terms of absence from work, visits to health care providers To our knowledge no clinical trials have been conducted to and the amounts of drugs prescribed (Puhakka et al., 2000; study the efficacy of sea buckthorn in prevention and Heikkinen and Ja¨rvinen, 2003). Sea buckthorn (Hippophae¨ treatment of the CC. The primary objective of this trial was to determine whether regular consumption of sea buckthorn berries affects the number of CC infections or the duration of Correspondence: P Larmo, Department of Biochemistry and Food Chemistry, symptoms in healthy adults. The effects on digestive tract University of Turku, Vatselankatu 2, Turku, FIN-20014, Finland. E-mail: [email protected] infections (DTI), urinary tract infections (UTI) and serum Guarantor: R Tahvonen. concentrations of C-reactive protein (CRP), an important Contributors: RT was responsible for the study concept, supervision and inflammatory and infection marker, were also studied. design. PL contributed to study design and was responsible for recruitment of the participants, data collection and entry, writing the manuscript and with RT and JA for analysis of data and interpretation of results. JA was responsible for the sample size estimation, randomization of the participants, statistical Participants and methods analysis of the data, and contributed to writing the paper. ES and HK contributed to the design of the study. RT, ES and HK revised the manuscript Participants critically for important intellectual content. Received 7 December 2006; revised 3 May 2007; accepted 20 May 2007; Participants of the study were healthy 19- to 50-year-old men published online 27 June 2007 and women, recruited for the study by announcements on Effects of sea buckthorn on infections and inflammation P Larmo et al 1124 the Internet pages and campus of the University of Turku Study design and outcome measures (exclusion criteria: see Supplementary Information at the The study was carried out in the Functional Foods Forum, an European Journal of Clinical Nutrition website). Baseline independent research unit of the University of Turku, information about the participants was collected using a Finland, as a randomized, double-blind, placebo-controlled questionnaire. Participants were informed about the study trial. During the 90-day observation period, the participants protocol. They gave written informed consent to the study were asked to assess daily their symptoms of (1) CC, (2) DTI, procedures, which were approved by the ethics committee of (3) UTI or (4) other infections in a logbook form. In the the Hospital District of Southwest Finland. The study context of each infection type typical symptoms were listed. statistician carried out the randomization procedure in a Participants were asked to report the severity of symptoms double-blind manner (Figure 1). Stratified randomization using a four-point scale with possible responses ranging from was used, the stratification factors being the age of 0 ¼ ‘none’ to 3 ¼ ‘severe’. The upper respiratory infection participant, body mass index, gender, smoking habits symptoms listed were fever, runny nose, nasal congestion, and use of vitamin or mineral supplements. The rando- sore throat, fatigue, headache, earache, nausea, muscular/ mization code was developed using a computer random joint pain or ‘other’ for which option the participant was number generator to select random permuted blocks (block asked to specify the symptom. They were asked to report a length 10). symptom only if, in their assessment, it was caused by an

Assessed for eligibility (n=321)

Randomized (n=254)

Assigned to receive sea Assigned to receive buckthorn (n=127) placebo (n=127)

Withdrew before the Withdrew before the observation period (n=7) observation period (n=5)

Began the observation Began the observation period (n=120) period (n=122)

Withdrew during the Withdrew during the observation period (n=6) observation period (n=4) -symptoms caused by the -symptoms caused by the product (n=1) product (n=2) -other reason/reason not -other reason/reason not known (n=5) known (n=2) One of the withdrawals returned logbooks

Returned logbooks and attended the final meeting Returned logbooks and of the study attended the final meeting (n=116) of the study (n=116)

116 included in ITT 117 included in ITT analysis analysis

Figure 1 Flow of participants enrolled in the study.

European Journal of Clinical Nutrition Effects of sea buckthorn on infections and inflammation P Larmo et al 1125 infection. The daily logbook assessment also included a daily The main outcome measures of the logbook analyses were estimation of overall health (‘do you feel healthy’: yes/no). the number of CC infections and the duration of symptoms. To detect potential differences in the causative viruses Also the number and duration of DTI and UTI were analyzed. between groups, and to confirm the CC diagnoses, the A participant was considered to have a case of CC if he/she participants were asked to give a nasal swab and serum reported at least one symptom at a severity of 1X on the sample on the first to third day they had symptoms of CC severity scale for 1 day or longer. For the duration of the (‘the CC samples’). A second serum sample was taken after 2 symptoms, every day the participant had recorded having weeks. In addition, blood samples were obtained after a 12 h one or more symptoms was calculated. The CC cases were fast, at the beginning and end of the observation period for treated as separate if there were at least 2 consecutive days the analysis of serum CRP concentrations. between them where the participant had not recorded any During the observation period, the participants daily took symptoms. The other infection types were analyzed accord- 28 g of frozen sea buckthorn puree or placebo product ingly. In an effort to explore the sensitivity of the conclu- similar in appearance, taste and smell to the active product. sions for the definitions of infections used, we made The sea buckthorn berries used were of the H. rhamnoides complementary analyses based on the logbook question spp. mongolica cv. Prozcharachnaya origin harvested in ‘do you feel healthy’. The first 7 days of the observation Ostrobothnia, Finland, in autumn 2004 and stored frozen. period were excluded from the analyses to take account of For the puree, berries were mashed in a process where also the incubation period of the CC (Puhakka et al., 2000; the seeds were ground fine. To facilitate the making of Heikkinen and Ja¨rvinen, 2003). Complementary analyses similar placebo product, additives were used (recipes of the concerning only the last month of the observation period sea buckthorn and placebo products, see Supplementary were done in case the prerequisite for effects of sea Information). During the manufacturing process, the buckthorn was reaching certain minimum levels in the temperature of the berries increased to about þ 51C. The active compounds of the body. participants were advised to store the product in the freezer andtakeitfrozenorthawitinsuchawayitdidnotget warm. They were allowed to take the product as such or Statistical analysis with other foodstuffs. Daily consumption was reported in The statistical analyses of the logbook data were carried out the logbooks. using generalized linear models (SAS software, version 9.1.3 To assess the success of blinding, each participant was, at SP2, GENMOD procedure; SAS Institute Inc., NC, USA). The the end of the observation period, asked to guess which CC is a contagious disease, and the number and duration of product he/she had been given. The participants were also it and DTI was assumed to follow negative binomial asked if they thought the study product had been beneficial distribution. In both cases Poisson distribution was first to them. Only after the preliminary statistical analysis was fitted to the data, but the observed overdispersion suggested the treatment code opened for the researchers and the the use of more flexible negative binomial distribution. Due statistician. to the small number of UTI their number and duration was The participants were allowed to use normal medication assumed to follow Poisson distribution. In all statistical during the study. During the observation period and the 1 models, a logarithmic link function was used to convert the month wash-out period the participants were asked not to estimates of the parameters into relative risks (RR; unad- use products containing sea buckthorn. Vitamin and mineral justed, no covariates were used). The estimated RR for supplements were allowed if no megadoses (defined as 10 infections or symptoms were calculated always comparing times the daily intake recommended by the Finnish National the sea buckthorn group (SBG) to the placebo group (PG), Nutrition Council, 2005) were used. Fish oil supplements that is RR significantly less than 1 would denote a desirable were also allowed but the use of all other nutrient sea buckthorn effect. CRP concentrations were analyzed supplements was restricted. Lactic acid bacteria-containing using rank analysis of covariance with the baseline measure- pills and certain probiotic product brands (Gefilus, Rela, ment as a covariate (same software as above). Due to the Yosa), commonly used in Finland, were not allowed. The use abnormal distribution of values, data on the CRP measure- of nutrient supplements and medication was also recorded ments and duration of infections are expressed as median in the logbooks. (range). Fisher’s exact test was used to compare the number Most CC cases are detected in winter. Typically, in Finland of CC samples taken in treatment groups (same software as the CC season begins with a rhinovirus epidemic in above). Two-sided significance tests and significance levels of September–October and ends with a second rhinovirus 0.05 were used throughout. epidemic in April–May. Corona and influenza viruses cause All randomized subjects who returned logbooks were infections typically during the winter (Puhakka et al., 2000). included in the primary analysis (intention to treat (ITT) During a period from mid-January to mid-February 2005 data). In addition, analyses including only participants who about 10–15 participants per day began the 90-day observa- were compliant with the protocol (per protocol, PP, data) were tion period. The study for the participants ended during a conducted (criteria for excluding participants from the PP period from mid-April to mid-May, respectively. analysis, see Supplementary Information). CRP concentrations

European Journal of Clinical Nutrition Effects of sea buckthorn on infections and inflammation P Larmo et al 1126 410 mg/l are considered as a marker of acute infection or the total daily amount of flavonols was about 8.4 mg. inflammation, which could obscure the use of CRP concen- The consumption of flavonols has been estimated to be tration as a risk marker of cardiovascular diseases (Pearson about 20–25 mg/day in the United States, Denmark and et al., 2003). Using both ITT and PP datasets complementary Holland and somewhat higher in Italy, the mean value analyses excluding values 410 mg/l were also made. being 35 mg/day (Manach et al., 2004). The variability of Prestudy sample size estimation was based on simplifying intake even in the same country can be considerable, assumptions made about the number of CC infections and however (Manach et al., 2004). The vitamin C content of duration of symptoms. It was assumed 50% of the partici- the product was 15.6 mg/day, which is about 21% of the pants in the PG would have one case of CC during the recommended daily intake for adults in Finland (Finnish 3-month observation period (Heikkinen and Ja¨rvinen, 2003). National Nutrition Council, 2005). The vitamin E and We considered a reduction of CC incidence to 30% of the carotenoid content of the product was small compared with participants in the SBG having one infection clinically the recommended intakes (Finnish National Nutrition significant. With a sample size of 120 participants/group Council, 2005) and the daily dose contained oil and the trial would have a power of approximately 85% to detect unsaturated fatty acids less than used in previous studies this difference between treatment groups (two-sided tests, reporting positive effects of sea buckthorn oil (Yang et al., 0.05 significance level, 10% assumed dropout rate) if w2-test 1999; Johansson et al., 2000). was used. The number of symptom days was assumed to be 5–7 on average in the PG (Barrett et al., 2002). A 2 days reduction in the duration of CC in the SBG was considered Participants clinically significant (Barrett et al., 2002). If the standard Participants randomized in the sea buckthorn and PGs had deviation was 5 days, 130 persons in both groups would be similar characteristics and there were no major differences in needed to detect a difference of 2 days in treatment groups at their eating habits and lifestyles (Table 1; more character- a power of approximately 85% if the Mann–Whitney U-test istics of the ITT population, see Supplementary Informa- was used (two-sided tests, 0.05 significance level, 10% tion). In both groups 12 participants reported having asthma dropout). or allergy. Based on the information gained from the logbooks the use of medication, vitamins, minerals and nutrient supplements during the observation period did not Laboratory methods differ substantially between treatment groups. There were no Serum CRP concentrations were measured by TYKSLAB major differences in the characteristics of the groups in the (Turku, Finland) with high-sensitivity particle-enhanced PP data either (data not shown). immunoturbidometric assay using Roche tina-quant Overall 9% of the randomized participants dropped out of reagents (Roche Diagnostics, GmbH, Mannheim, Germany) the study and the number of withdrawals was identical and a fully automated analyzer Roche Modular P800 (Roche between groups (Figure 1). Two participants in the SBG and Diagnostics). The flavonolglycosides and vitamin C content one in the PG gave the reason for withdrawal as being of the sea buckthorn puree were analyzed using high- symptoms caused by the study product (PG: rash; SBG: performance liquid chromatograph (HPLC)-UV methods nausea, ulcers in the mouth). The number of noncompliant (Kallio et al., 2005; Tiitinen et al., 2005). For the analysis of participants excluded from PP data was similar in both vitamin E content an HPLC method with fluorescence groups (logbook analyses: SBG 13, PG 14; CRP analyses: SBG detection was used (Kallio et al., 2002b). The oil content of 26, PG 27). the product was analyzed gravimetrically using a chloro- At the end of the observation period, 70% of the form–methanol extraction method (Tiitinen et al., 2005). participants in the SBG guessed their treatment group The fatty acids of the oil were esterified using a boron correctly. In the SBG, 57% of the participants felt the trifluoride method (A˚ gren et al., 1992) and the fatty acid product had positive effects during the observation period methyl esters were analyzed by gas chromatography (Yang (for example, refreshing or energizing). In the PG, 50% of the et al., 1999). The carotenoids were analyzed by the MTT participants guessed they had been receiving the sea buck- Agrifood Research Finland (Jokioinen, Finland) chemistry thorn product and 47% felt the product had had positive laboratory using an in-house HPLC-UV/VIS method. effects.

Results Number and duration of infections Sea buckthorn did not have an effect on CC or DTI risk or Sea buckthorn product duration in the primary ITT data (Table 2). Analyses of the PP The daily dose of sea buckthorn product contained 16.7 mg data yielded the same result (number of infections; CC: RR of flavonol glycosides, the glycosides of isorhamnetin being 1.12, 95% CI 0.87–1.45, DTI: RR 0.99, 95% CI 0.60–1.63; the most abundant (detailed composition of the product, duration of infections; CC: RR 1.05, 95% CI 0.86–1.28, DTI: see Supplementary Information). Calculated as aglycones RR 1.12, 95% CI 0.85–1.46; RR values unadjusted, assumed

European Journal of Clinical Nutrition Effects of sea buckthorn on infections and inflammation P Larmo et al 1127 distributions as described in ‘Statistical analysis’) as did the The participants had only a few UTI during the observa- analyses restricted to the last month of the observation tion period (Table 2). Thus, the results can be considered as period (data not shown). indicative only. The secondary PP analysis suggested a significant positive effect of sea buckthorn on the number, but not the duration, of infections (number of infections: RR 0.43, 95% CI 0.20–0.94; duration of infections: RR 0.60, 95% Table 1 Characteristics of participantsa CI 0.28–1.29; RR values unadjusted, assumed distributions as Characteristics SBG, n (%) PG, n (%) described in ‘Statistical analysis’; in PP data 10 UTI cases in 7 participants). Analyses concerning the last month of the Women 90 (77.6) 90 (76.9) study yielded significant results for both the number and BMI (kg/m2) duration of UTI (data not shown). o25 85 (73.3) 88 (75.2) X25 31 (26.7) 29 (24.8)

Age (years) Number of common cold samples 19–29 60 (51.7) 62 (53.0) The number of CC samples confirms the conclusion of the 30–50 56 (48.3) 55 (47.0) groups not differing in both ITT and PP data (in ITT data the number of participants giving 1 sample: 37 in SBG, 40 in PG; Smoking Yes 8 (6.9) 9 (7.7) 2 samples: 8 in SBG, 4 in PG; 3 samples: 2 in SBG, 0 in PG; P ¼ 0.37; PP data not shown). Thus the samples were not Regular use of vitamin/mineral/nutrient supplements further analyzed. Yes 44 (37.9) 50 (42.7)

Exercise No or occasionally 12 (10.3) 12 (10.3) Subjective sensation of health 1–3 times/week 74 (63.8) 64 (54.7) 43 times/week 30 (25.9) 41 (35.0) Reports of participants’ subjective sensation of health (logbook question ‘do you feel healthy’) varied greatly. Some Consumption of alcohol reported feeling healthy even if they had symptoms of an No or occasionally 36 (31.0) 44 (37.6) infection while others always reported feeling sick if they 1–7 servings/week 72 (62.1) 64 (54.7) 8–14 servings/week 7 (6.0) 8 (6.8) had any symptoms. The median number of sick days during 414 servings/week 1 (0.86) 1 (0.85) the observation period in ITT data was 4 (range 0–45 days) and 6 days (range 0–37 days) in the sea buckthorn and PGs, Consumption of vegetables respectively. The groups did not differ significantly (RR 1.06, Daily 81 (69.8) 87 (74.4) 4once/week 33 (28.5) 30 (25.6) 95% CI 0.76–1.49). PP analysis and analysis using only the oonce/week 2 (1.7) 0 (0) last month of the observation period led to the same conclusion (PP: RR 1.02, 95% CI 0.72–1.46). Consumption of fruits and berries Daily 53 (45.7) 64 (54.7) 4once/week 56 (48.3) 49 (41.9) oonce/week 7 (6.0) 4 (3.4) C-reactive protein

Abbreviations: BMI, body mass index; PG, placebo group; SBG, sea buckthorn Compared to the placebo, there was a small but significant group. reduction in serum concentrations of CRP in the SBG during aValues are numbers of participants (%) in ITT data. SBG n ¼ 116, PG n ¼ 117. the observation period (Table 3). This reduction was evident

Table 2 Effect of consuming sea buckthorn or placebo product on infections in healthy adultsa

CC DTI UTI

SBG PG SBG PG SBG PG

Participants having X1 infections, n (%) 84 (72.4) 84 (71.8) 49 (42.2) 41 (35.0) 4 (3.4) 5 (4.3) Total number of infections 185 161 94 90 6 7 Number of infections, RRb (95% CI) 1.15 (0.90–1.48) 1.06 (0.67–1.68) 0.87 (0.44–1.70) Median duration of symptoms, days (range) 4 (1–87)c 3 (1–28) 1 (1–87)c 1 (1–23) 1.5 (1–4) 3 (1–5) Duration of symptoms, RRb (95% CI) 1.05 (0.87–1.27) 1.08 (0.82–1.43) 0.63 (0.37–1.08)

Abbreviations: CC, common cold; DTI, digestive tract infection; PG, placebo group; SBG, sea buckthorn group; UTI, urinary tract infections. aValues calculated including data from all logbooks received (ITT data). SBG n ¼ 116, PG n ¼ 117. bUnadjusted RR, no covariates were used. For CC and DTI negative binomial distribution was assumed, for UTI Poisson distribution was assumed. cInfections lasting longer than 80 days not included in the estimation model of the relative risk (n ¼ 1).

European Journal of Clinical Nutrition Effects of sea buckthorn on infections and inflammation P Larmo et al 1128 in data containing all measurements and when highly

-value elevated values (410 mg/l) were excluded. P

Discussion

Due to its high acid content the sea buckthorn berry taste 0.0590.0540.0850.090 0.039* 0.049* 0.025* 0.022*

À À À À does not always attract consumers (Tiitinen et al., 2006). We chose a moderate daily amount of the product to have a realistic dose for the normal consumer’s everyday life. As Finns consume on average only about 10 g fresh berries daily (Ma¨nnisto¨ et al., 2003) this was still a substantial addition to the berry intake. a

86. It was expected that the compounds may have synergetic ¼

n effects (Middleton et al., 2000; Puupponen-Pimia¨ et al., 2001) 3.841; 26.540) 3.841; 7.116) 3.841; 26.540) 3.841; 7.116)

À À À À as the study product was the whole berry. The amounts of bioactive compounds in sea buckthorn berry vary depending 86, PG

¼ on the subspecies, area and year of cultivation, and the n maturity of the berries (Gao et al., 2000; Kallio et al., 2002a). It is possible that loss of bioactive compounds took place during the manufacture and storage of the product (de Ancos et al., 2000; Ha¨kkinen et al., 2000). The amounts of vitamins C and E especially were far from the highest amounts 111; PP data: SBG

¼ detected in sea buckthorn berries (Yang and Kallio, 2002; n Tiitinen et al., 2005). CRP (mg/l) When compared to common daily intakes and recom-

112, PG mendations, the intake of flavonols was, by estimation, ¼

n affected most by the sea buckthorn puree. Flavonols along with other flavonoids and polyphenolic compounds influence the immune system and inflammatory cells (Middleton et al., 2000) and have antimicrobial properties (Conti et al., 1998; Middleton et al., 2000; Puupponen-Pimia¨ et al., 2001). 7.371; 16.029)7.371; 3.933)7.371; 0.754 16.029) (0.149; 10.646)7.371; 3.933) 0.724 (0.149; 0.724 8.976) (0.149; 0.049 10.646) ( 0.712 (0.149; 8.976) 0.033 0.031 ( ( 0.028 (

À À À À The most convincing evidence on the efficacy of vitamins in CC treatment or prevention concerns people whose immune

0.010 ( 0.021 ( 0.054 ( 0.062 ( system may not function optimally or who are under À À À À exceptional stress (Hemila¨ et al., 2002, 2004; Meydani SBG PG Difference in change et al., 2004). The participants in the current study were 90.

¼ healthy adults who exercised moderately and very few n smoked. On the grounds of the eating habit information, it is likely that they obtained reasonable intakes of flavonoids BL Change 3 months – BL BL Change 3 months–- BL 90, PG and vitamins in their normal diet. ¼

n Berry phenolics possess antimicrobial activity against bacteria causing DTI and UTI (Puupponen-Pimia¨ et al., 0.688 (0.157; 13.257) 0.654 (0.157; 1.736) 0.730 (0.159; 13.257) 0.688 (0.159; 9.297) 2001, 2005) and cranberry and other berry juices rich in phenolic compounds have been associated with a lowered c c risk of UTI (Kontiokari et al., 2001, 2003). In this study, the

116; PP data: SBG number of UTI and the median duration of infections were ¼

n smaller in the sea buckthorn than in the PG. The comple- 0.05) between the sea buckthorn and placebo groups.

o mentary statistical analyses even suggested significant P efficacy of sea buckthorn. However, the number of UTI was 116, PG 10 mg/l excluded 10 mg/l excluded

¼ too small for reliable conclusions. 4 4 n The definitions of infections were based on self-assessment Serum concentrations of CRP in healthy adults at baseline and after consumption of sea buckthorn or placebo product for 3 months

10 mg/l indicating acute infection or inflammation excluded from analysis. ITT data: SBG of symptoms by the participants, a method frequently used b b 4 in CC studies (Brinkworth and Buckley, 2003; Langkamp- Henken et al., 2004; Turner et al., 2004). The clinical ITT data: SBG Values are medians (range) due to abnormal distribution of the data. Values ITT data Table 3 Abbreviations: BL, baseline;a ITT, intention to treat;b PG, placebo group;c PP, per protocol; SBG, sea buckthorn group. *Significant difference ( ITT data, values PP data PP data, values diagnosis of CC is in most cases simple and can reliably be

European Journal of Clinical Nutrition Effects of sea buckthorn on infections and inflammation P Larmo et al 1129 made by adult patients themselves (Heikkinen and Ja¨rvinen, In conclusion, consumption of sea buckthorn berries 2003). Though allergic and vasomotor rhinitis can mimic the reduced serum concentrations of CRP. Although the change CC, these conditions usually can be easily differentiated was small the results encourage further investigation con- (Heikkinen and Ja¨rvinen, 2003) and in this study, the cerning the effects of sea buckthorn on inflammation and number of participants having allergies was similar in both cardiovascular disease risk factors. The number of UTI was groups. Even with modern diagnostic methods, about too small for solid conclusions, but indicative results suggest 20–30% of cold cases remain without a proven viral cause the subject merits further research. Future studies should also (Heikkinen and Ja¨rvinen, 2003) which makes the CC evaluate the effects of sea buckthorn berries on infections in syndrome in large part subjective (Gwaltney et al.,1996). trials with a longer study period and different dosage, and in However, relying only on self-assessments as the primary populations other than healthy adults. outcomes in our trial is a limitation. Randomization was used to distribute the bias and noise potentially introduced by self- reporting equally between groups, but they still interfere with the detection of true signal (Gwaltney et al., 1996). Acknowledgements The number of CC samples taken was smaller than the number of infections reported in logbooks. That is likely We thank the volunteers who participated in the study. We because samples could only be taken during office hours, also thank Terhi Pohjanheimo and Katja Tiitinen for which made it difficult for participants. Some participants development of the treatment products; Nina Kainulainen, also told they were too sick to leave home to get the samples Raija Nurmi, and Anja Pirinen for excellent assistance; Jukka- taken. Another limitation of the study was the short Pekka Suomela for the flavonol glycoside analysis of the duration. In the case of a natural product it is possible the study product; Saija Hurme for the statistical analyses of CRP effects need more time to develop. data; and Marjo Ma¨kinen-Aakula and Ilkka Liukas for their On the assessment of blinding, more than 50% of the contribution. We acknowledge the Finnish Agency for participants in the SBG guessed they had been receiving the Technology and Innovation, Pakkasmarja Inc., Riitan Herk- active product during the study, raising the question of a ku Inc., Valioravinto Inc., Vinkkila¨ Organic Product, Turku possible placebo effect. However, the logbook analyses University Foundation and ABS Graduate School for finan- showed no significant beneficial effects of sea buckthorn. cial support. Despite the lack of positive results from the logbook analyses, more participants in the sea buckthorn than in the PG reported having benefited from the product when References asked about it at the end of the study. Participants can only be expected to guess 50% right in both groups at the end of A˚ gren JJ, Julkunen A, Penttila¨ I (1992). Rapid separation of serum the study if the active product has no treatment effect and lipids for fatty acid analysis by a single aminopropyl column. J Lipid Res neither of the products have side effects in addition to the 33, 1871–1876. Barrett BP, Brown RL, Locken K, Maberry R, Bobula JA, D’Alessio D products being identical in physical characteristics (Des- (2002). Treatment of the common cold with unrefined echinacea. biens, 2002). 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