Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Bitter tastants alter gastric-phase postprandial haemodynamics Michael K. McMullen a,n,1, Julie M. Whitehouse a, Peter A. Whitton b, Anthony Towell c a b c

Division of Complementary Medicine, University of Westminster, London, UK Naturally Scientific, Nottingham, UK Department of Psychology, University of Westminster, Westminster, UK

art ic l e i nf o

a b s t r a c t

Article history: Received 2 October 2013 Received in revised form 20 April 2014 Accepted 27 April 2014

Ethnopharmacological relevance: Since Greco-Roman times bitter tastants have been used in Europe to treat digestive disorders, yet no pharmacological mechanism has been identified which can account for this practice. This study investigates whether the bitter tastants, gentian root (Gentian lutea L.) and wormwood herb (Artemisia absinthium L.), stimulate cephalic and/or gut receptors to alter postprandial haemodynamics during the gastric-phase of digestion. Materials and methods: Normal participants ingested (1) 100 mL water plus capsules containing either cellulose (placebo-control) or 1000 mg of each tastant (n ¼14); or (2) 100 mL of water flavoured with 500 or 1500 mg of each tastant (a) gentian (n ¼ 12) and (b) wormwood (n ¼ 12). A single beat-to-beat cardiovascular recording was obtained for the entire session. Pre/post-ingestion contrasts with the control were analysed for (1) the encapsulated tastants, in the “10 to 15” minute post-ingestion period, and (2) the flavoured water in the “5 to 10” minute post-ingestion period. Results: Water, the placebo-control, increased cardiac contraction force and blood pressure notwithstanding heart rate decreases. Encapsulated tastants did not further alter postprandial haemodynamics. In contrast gentian (500 and 1500 mg) and wormwood (1500 mg) flavoured water elicited increased peripheral vascular resistance and decreased cardiac output, primarily by reducing stroke volume rather than heart rate. Conclusions: Drinking 100 mL water elicits a pressor effect during the gastric-phase of digestion due to increased cardiac contraction force. The addition of bitter tastants to water elicits an additional and parallel pressor effect due to increased peripheral vascular resistance; yet the extent of the post-prandial blood pressure increases are unchanged, presumably due to baroreflex buffering. The vascular response elicited by bitter tastants can be categorised as a sympathetically-mediated cephalic-phase response. A possible mechanism by which bitter tastants could positively influence digestion is altering gastricphase postprandial haemodynamics and supporting postprandial hyperaemia. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Artemisia absinthium Bitter tastants Cephalic response Gentiana lutea Postprandial hyperaemia Postprandial haemodynamics

1. Introduction Prior to the modern era of medicine, fluid preparations derived from bitter tasting plants were regularly “given to promote appetite and thus to aid digestion” (Douthwaite, 1963). Today, the treatment of digestive disorders with bitter tastants continues in both traditional medicine systems of eastern and southern Asia (Chang-Liao et al., 2011; Williamson, 2002) and modern European phytotherapy (Knö ss and Stolte, 2009b; Koch, 2009b; Schulz et al., 2004). However, despite their widespread usage, the pharmacological activity of bitter tastants has as yet to be either

n Corresponding author. Current address: Life Force Research, Backegardsv. 4, 45930 Ljungskile, Sweden. Tel.: þ 46 706227384; fax: þ46 522 20559. E-mail address: [email protected] (M.K. McMullen). 1 Current address: Life Force Research, Ljungskile, Sweden.

scientifically investigated (Laurence et al., 1997) or clinically studied (Heinrich et al., 2012).

1.1. Bitter tastant theories Although the pharmacological mechanism by which bitter tastants impact digestion is unknown, pharmacologists have acknowledged that the mechanism is likely to be chemosensory. There are two principal and one less well known hypothesis: (1) The cephalic-response model (i.e. responses originating from the head) proposes that with intake of bitter tastants “the appetite is sharpened because the gustatory nerves are stimulated; this reflexively leads to dilation of the gastric vessels and to an increase in the gastric and salivary secretions” (Hale White, 1892). This hypothesis drew support from Pavlov’s

http://dx.doi.org/10.1016/j.jep.2014.04.041 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: McMullen, M.K., et al., Bitter tastants alter gastric-phase postprandial haemodynamics. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.041i

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research on the autonomic nerve system (Hale-White, 1920). A more modern account of this hypothesis is that “bitter stimuli pass primarily by way of the glossopharyngeal nerve to a special group of cells in the cerebral cortex. The taste is interpreted there as bitter, and causes stimuli to be forwarded through the vagus nerve to both the salivary gland and the stomach …. This stimulation of the digestive processes enhances the appetite” (Robbers and Tyler, 1999). The cephalic-response model is supported in the European Medical Agency’s Assessment Report on gentian which states: “it is fact that the bitter constituents stimulate the gustatory nerves in the mouth and give rise to an increase in the secretion of gastric fluid and bile” (Knö ss and Stolte, 2009a). Notably the modern view (Mills and Bone, 2000; Sandberg and Corrigan, 2001; van Wyk and Wink, 2004) focuses entirely on the vagal stimulation of gastric secretions and excludes the circulatory component that was suggested a century before by Hale White. Some pharmacologists have suggested that earlier work (Glatzel, 1968) indicates that “bitter principles act reflexively on the cardiovascular system causing a decrease of in heart rate and cardiac stroke volume” (Schulz et al., 2004). However, the statistical analysis upon which this concept is based has been shown to be faulty, and there is no evidence from cardiovascular research indicating that bitter tastants increase vagal tone (McMullen, 2013). (2) The local-response model proposes that bitter tastants “act directly on the mucosa of the upper part of the gastrointestinal tract and especially on the bitter receptors of the tongue stimulating the release of saliva and gastric juices” (Heinrich et al., 2012). This hypothesis is supported by recent observations that bitter, sour, sweet and umami taste receptor cells are present in the stomach, duodenum, jejunum, ileum and colon of rats (Horn, 2008). Some proponents of the cephalicresponse model accept that local stimulation augments cephalic-elicited vagal stimulation (Hale White, 1892; Knö ss and Stolte, 2009a; Robbers and Tyler, 1999). In contrast, other proponents of the cephalic-response model maintain that there is insufficient evidence that local stimulation produces effects and that if bitter tastants are to be effective “they must be tasted” (Mills and Bone, 2000). (3) Weiss hypothesised that bitter tastants also elicit sympathetic stimulation. Similarly to Hale White (1892) he proposed that “the appetite-inducing action of bitters is probably due to improved circulation in the abdominal organs” (Weiss, 1988). Furthermore he proposed that the general tonic action of bitter tastants was due to repeated stimulation of the sympathetic nervous system by bitter tastants. A key reason for the lack of research on the mechanisms of bitter tastants is the lack of investigative tools for assessing digestive activity. While it is well accepted that cephalic-phase responses modulate the production of digestive secretions via the efferent branch of the vagus nerve (Zafra et al., 2006), techniques to measure digestive secretions are not routinely available for researchers (Furness, 2006).

and continues for 10 to 20 min after intake has ceased. During this phase increases of celiac blood flow and velocity are accompanied by a decrease of celiac vascular resistance. Systemically these changes are accompanied by increases of heart rate, cardiac output and systemic blood pressure. During the second phase of digestion, the intestinal-phase, similar changes occur in the superior mesenteric arteries as occurred for celiac artery during the gastricphase. Systemically, there are smaller heart rate increases, decreases in mean and diastolic blood pressure and reductions in skeletal muscle blood flow (Someya et al., 2008). The increased splanchnic blood circulation during digestion is referred to as postprandial hyperaemia. Postprandial hyperaemia facilitates gastrointestinal motor and secretory activity as well as the absorption and removal of digested substances (Hall, 2011). Crucially, postprandial hyperaemia requires compensatory postprandial sympathetic activation, namely increased cardiac activity, to preserve systemic blood pressure levels (Sidery and Macdonald, 1994; van Baak, 2008). Inadequate postprandial hyperaemia is associated with digestive problems (Mensink et al., 2011) and systemically with postprandial hypotension (Brignole et al., 2001). Postprandial hypotension is an independent predictor of mortality and may trigger syncope, falls, strokes, transient ischaemic attacks, angina and myocardial infarctions (Luciano et al., 2010). This study investigates whether bitter tastants stimulate cephalic and/or gut receptors to alter haemodynamics (i.e. cardiovascular activity) during the gastric-phase of digestion. Gentiana lutea radix L. Fam. Gentianaceae (gentian) and Artemisia absinthium herba L. Fam. Asteraceae (wormwood) were chosen for this study because both plants 1) are well-known for their bitter taste (Sweetman, 2002); 2) serve as standards in research on bitter tastants (Olivier and van Wyk, 2013); 3) are used in Mediterranean cultures to flavour alcoholic beverages, known as aperitifs, which are traditionally taken to stimulate the appetite (Bruneton, 1999) i.e. they are part of the “Mediterranean diet”; 4) contain compounds known to stimulate multiple cephalic bitter taste receptor cells. Gentian contains the secoiridoid glycoside amarogentin, which stimulates the human taste receptors (hTAS2Rs) 1, 4, 39, 43, 46, 47 and 50, while wormwood contains the sesquiterpene absinthin, which stimulates the hTAS2Rs 10, 14, 46 and 47 and thujone, a compound found in the essential oil of wormwood, which stimulates hTAS2Rs 9 and 14 (Meyerhof et al., 2010). Gentian also contains numerous other secoiridoid glycosides which are known to taste bitter, as well as bitter carbohydrates (Knö ss and Stolte, 2009a). Similarly, wormwood contains numerous compounds regarded as bitter tasting, particularly the sesquiterpenes (Koch, 2009a), although some of these sesquiterpenes appear to be hTAS2R46 partial agonists or antagonists (Brockhoff et al., 2011); 5) have similar bitterness indexes: gentian, 10,000 to 30,000 and wormwood, 10,000 to 25,000 (Wagner and Bladt, 2001).

1.2. Postprandial hyperaemia 2. Methods The development of precise cardiac stroke volume measurement in the 1980s stimulated studies on the cardiovascular response to eating in dogs. The findings indicated that the cardiovascular system responded to the challenge of food intake in two distinct phases (Chou and Coatney, 1994). Similar phases have subsequently been reported for humans (Harthoorn and DransWeld, 2008; Someya et al., 2008). The first phase of digestion, the gastric-phase, begins with the intake of food and/or drink

This investigation was approved by the University of Westminster Ethics Committee (03/04–08) and conforms to the principles outlined by the Declaration of Helsinki. Written informed consent was obtained from the participants, who were volunteers recruited from the staff, students and associates of the University of Westminster, London. Hypertensive individuals (systolic pressure 4140 mmHg or diastolic pressure 4 90 mmHg), smokers,

Please cite this article as: McMullen, M.K., et al., Bitter tastants alter gastric-phase postprandial haemodynamics. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.041i

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pregnant women and those on prescribed medication were excluded from the study. In Part 1, the impact of 1000 mg encapsulated gentian and wormwood on cardiovascular parameters was compared to a placebo-control. In Part 2, the impacts of water flavoured with either, 500 and 1500 mg of gentian (Part 2a), or 500 and 1500 mg wormwood (Part 2b), were compared to the placebo-control measures from Part 1. 2.1. Plant material and extract preparation. The samples of gentian and wormwood were purchased from the Organic Herb Trading Co., UK with a Certificate of Conformance (date of issue 01/02/11). The certifier was the Soil Association, licence number P938. The batch number for the gentian root was 5317FR and the country of origin was France. The batch number for the wormwood herb was 5481BG and the country of origin was Bulgaria. Both samples were classified as organic, nonirradiated and pesticide free. Specimens of both samples have been retained. Gentian and wormwood fluid extracts were prepared using a Soxhlet extractor and 96% ethanol (European Pharmacopeial standard). Then ethanol was removed under vacuum and the extracts were suspended in a solution of lecithin and water. The gentian and wormwood solutions were then reconstituted with alcohol at the two sample strengths 500 or 1500 mg mL  1 and the final alcohol level was circa 40% for all extracts. During the production of the fluid extracts some chemical changes may have occurred. The identity of the tastants was confirmed by analysis of the 1500 mg mL  1 samples (volume 6 μL) using HPLC. HPLC was performed with diode array and infrared detection using a Genesis C18 column (15 cm  0.46 cm). Separation was by a gradient system with solvent A (800 g water, 154.6 g acetonitrile containing 1 mMol phosphoric acid) and solvent B (200 g water, 625.6 g acetonitrile containing 1 mMol phosphoric acid). The gradient was 80% solvent A and 20% solvent B at the start, increasing to 100% solvent B after 28 min, held for 8 min before decreasing to 20% solvent B over the next 4 min. Reference samples from Sigma-Aldrich (USA) were used to confirm the identity and quantity of the secoiridoid glycosides, amarogentin (9.082 min) and gentiopicrine (9.774 min), present in the gentian extract at 17.6 and 20.5 g L  1, respectively. The sesquiterpenes, absinthin (9. 707 min) and anabsinthin (9.082 min) were present in the wormwood extract at 5.7 and 1.6 g L  1, respectively. 2.2. Test substances 2.2.1. Part 1 Capsules containing either gentian, wormwood or cellulose (placebo-control) were prepared. The gentian and wormwood capsules each contained circa 333 mg powdered plant material without fillers. Each intervention was comprised of three capsules (size 0) and was administered with 100 mL room-temperature water. Capsules were opaque (blue) and identical to ensure double-blinding. The presentation order of the capsules was randomised. The gelatine capsules conformed to British Pharmacopeia standards and the estimated disintegration time was 10 min. 2.2.2. Part 2 Both gentian and wormwood fluid extracts were administered at two strengths: 500 and 1500 mg mL  1. These are referred to as G500, G1500, W500 and W1500, respectively. Each flavoured water intervention was comprised of 1 mL extract administered in 100 mL room-temperature water. The alcohol content of these

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interventions was 0.4%. Interventions were administered in a randomised manner using a dark opaque cup to minimise visual cues. Consequently, neither the participants, nor the administrator, were aware of the amount of bitter tastant administered on each test occasion. 2.3. Procedure Prior to an experimental session, participants were required to abstain from food and drink for 2 h, excepting water. This requirement was verbally confirmed at the start of each session. Testing was conducted in silence in a quiet room at a temperature of circa 22 1C. A haemodynamic monitoring system (Finometer PRO, Finapres Medical Systems, Amsterdam, The Netherlands) was attached to their left hand and a single beat-to-beat recording was produced for the entire test session. The test session started in the supine posture to calibrate the Finometer (Return-to-Flow) and allow the participants’ cardiovascular system to stabilise (Pickering et al., 2005). Participants then moved to a sitting position and a 120 s pre-ingestion period was followed by the gastric-phase period which started with the presentation and ingestion of the interventions. The ingestion process was allowed to proceed at a pace determined individually by participants, both to ensure their personal comfort and to reduce confounding autonomic noise, and varied in length from 30 to 60 s. The gastric-phase period lasted for 15 min. 2.4. Cardiovascular measurements The Finometer records the finger-pulse contour and provides continuous beat-to-beat readings of a number of cardiovascular parameters. An infrared plethysmograph in a finger cuff records (200 Hz) the pulsation of the arterial diameter. Cuff pressure clamps the artery’s unstretched diameter and is attuned so that finger arterial pressure is reflected in the cuff pressure. The Finometer provides the measured heart rate, cardiac contraction force (dP/dt) and brachial systolic and diastolic pressure. Additionally, Modelflow software, which is integrated into the Finometer system, automatically computes for healthy individuals: stroke volume, cardiac output and peripheral vascular resistance; as well as the body-surface area adjusted values (Dubois and Dubois formula): “indexed stroke volume,” “indexed cardiac output” and “indexed peripheral vascular resistance” (Guelen et al., 2003, 2008; Imholz et al., 1998; Jansen et al., 2001; van Lieshout et al., 2003; van Lieshout and Wesseling, 2001). The indexed values are reported rather than the non-indexed values. 2.5. Participants We applied the data from a previous study (McMullen et al., 2011) when calculating the number of participants required to change heart rate with the program Power and Precision (v4, Biostat, New Jersey, USA). The standard deviation of the pre–post differences was calculated as 2.4 beats per minute (bpm), alpha was set at 0.05, power required was 80% and required heart rate increase set at 3.0 bpm. The number of participants required was calculated as n ¼ 12 (two-tailed). The participant characteristics in the groups were: Part 1 capsules: The 14 participants (11 female) with a mean (standard deviation and range) age of 43 (7 10.0, 23 to 64) years, a mean weight of 70 ( 717.3, 47 to 104) kg, a mean height of 1.70 (70.12, 1.53 to 1.93) m and a mean BMI of 24 (74.0, 19 to 31) kg m  2. Part 2a gentian group: The 12 participants (10 female) had a mean (standard deviation and range) age of 46 (713.3, 22 to

Please cite this article as: McMullen, M.K., et al., Bitter tastants alter gastric-phase postprandial haemodynamics. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.041i

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63) years, a mean weight of 70 (7 9.8, 59 to 86) kg, a mean height of 1.70 (70.10, 1.58 to 1.93) m and a mean BMI of 24 (72.6, 21 to 30) kg m  2. Part 2b wormwood group: The 12 participants (10 female) had a mean (standard deviation and range) age of 42 (713.0, 20 to 62) years, a mean weight of 67 (712.4, 50 to 92) kg, a mean height of 1.70 (70.11, 1.55 to 1.93) m and a mean BMI of 23 (7 2.9, 19 to 30) kg m  2. None of the individuals who participated in Part 1 participated in Part 2 whereas; some individuals participated in both Parts 2a and 2b. There were no differences between the pre-ingestion values of the groups in Part 1 and Part 2. 2.6. Analysis Mean parameter values for the pre-ingestion and gastric-phase periods were extracted from the recording. No analysis of the initial 5 min post-ingestion period was undertaken because in this period cardiovascular measures were subject to the influences of ingesting and swallowing and neither of the activities were standardised. Three different periods were analysed. First, to investigate the effect of drinking 100 mL water on post-prandial haemodynamics, pre–post comparisons were undertaken for the placebo-control capsules using the post-ingestion period 5 to 15 min. For the capsules of Part 1, the post-ingestion period 10 to 15 min was selected for pre–post comparisons. This interval corresponds to the period immediately after the capsule disintegration. A previous study, using identical capsules to those used in this study, reported that encapsulated caffeine elicited increases in diastolic pressure in the period 10 to 15 min after ingestion (McMullen et al., 2011). For Part 2, the post-ingestion period 5 to 10 min was selected for pre–post comparisons. This interval can be expected to exclude haemodynamic changes associated with the drinking process such as body movement and swallowing. As the age of the participants varied widely, from 20 to 64 years, significant pre–post comparisons were tested to determine if the magnitude of changes were age dependent. For this Pearson

correlation analysis was utilised with the significance level set at po 0.05 (two-tailed). For Part 1 within-participant repeated measures ANOVA analyses were applied while for Part 2 between-participant repeated measures ANOVA analyses were applied. The significance level was set at p o0.05 (two-tailed) and the number of planned contrasts were equal to the number of degrees of freedom (NCSS 9. NCSS, LLC. Kaysville, Utah, USA). Changes where p o0.10 (Bland, 2000) are also noted. 2.7. Measurement sensitivity A number of strategies were employed to increase both the accuracy of measurements and analytical sensitivity. First, we utilised beat-to-beat recordings rather than less sensitive intermittent measures (Langewouters et al., 1998). Second, the postingestion measurements were derived from either 5 or 10 min segments. The use of longer segments than used in previous research, reduces the loss of statistical power due to fluctuations of neural tone (McMullen et al., 2012a) and stroke volume oscillations due to the impact of breathing (McMullen et al., 2010). Third, the use a single of summary measure derived from a series of readings safeguards statistical validity (Matthews et al., 1990).

3. Results and discussion 3.1. Overview This study reports two distinct novel findings. First, in the 5 to 15 min following the intake of 100 mL water and the placebo capsules, healthy adults experienced small increases of systolic pressure circa 4 mmHg. The pressor effect resulted from increases of cardiac contraction force, i.e. dP/dt, rather than changes of either heart rate or vascular tone. The magnitude of the pressor effect was not age correlated. The post-ingestion responses following the ingestion of encapsulated gentian and wormwood were not different to the post-ingestion responses for the placebo-

Table 1 Parameter means and standard deviations for pre-ingestion and post-ingestion periods. Substance

Period (min)

HR (bpm)

dP/dt (mmHg s  1)

iSV (mL m  2)

iCO (L m  2)

iPR (MU m  2)

SP (mmHg)

DP (mmHg)

PC

Pre 5–10 10–15 5–15 Pre 5–10 Pre 5–10 Pre 5–10 Pre 5–10 Pre 10–15 Pre 10–15

70.37 10.9 69.0 7 11.2 69.17 11.5 69.17 11.3∧ 67.6 7 7.2 66.47 6.2 69.8 7 5.6 68.37 6.5 69.4 7 6.6 68.77 6.7 72.4 7 7.4 71.0 7 7.3 69.3 7 10.8 68.47 10.4 69.6 7 9.7 69.0 7 10.4

7797 174 8577 147 840 7 150 8497 146∧∧ 8017 207 8517 229 788 7 214 839 7 209 878 7 176 9127 228 939 7 251 960 7 229 803 7 205 8727 219 8167 168 862 7 165

41.8 7 10.3 43.8 7 9.9 43.2 7 9.7 43.5 7 9.8† 45.17 8.1 44.9 7 8.2n 42.17 5.4 41.6 7 4.7n 37.8 7 6.7 38.0 7 6.8 39.2 7 9.9 37.8 7 8.5nn 42.5 7 9.2 43.17 9.6 43.6 7 13.3 44.3 7 12.3

2.89 7 0.69 2.99 7 0.69 2.95 7 0.70 2.977 0.69 3.02 7 0.55 2.977 0.57n 2.93 7 0.42 2.82 7 0.47nn 2.58 7 0.38 2.58 7 0.40 2.79 7 0.61 2.647 0.51nn 2.917 0.65 2.92 7 0.60 2.977 0.76 3.007 0.71

0.6770.20 0.66 70.20 0.6770.19 0.66 70.19 0.59 70.16 0.62 70.16# 0.59 70.10 0.65 70.17nn 0.74 70.14 0.76 70.16 0.69 70.18 0.75 70.19nn 0.63 7 0.18 0.64 70.17 0.63 70.21 0.65 70.22

121.6 7 12.4 125.7 7 12.3 125.0 7 10.2 125.3 7 11.1∧∧ 121.0 7 11.8 125.17 10.2 118.0 7 11.5 123.8 7 14.0 125.17 7.7 127.3 7 8.9 123.17 9.0 126.7 7 8.0 121.0 7 12.7 125.3 7 12.9 122.5 7 11.9 126.6 7 12.5

77.0 7 7.5 78.3 7 8.6 78.3 7 7.9 78.3 7 8.2∧∧ 72.8 7 5.3 75.2 7 5.4 72.3 7 7.7 75.6 7 8.9 78.6 7 4.5 79.5 7 4.2 75.7 7 8.1 78.3 7 7.4 76.7 7 9.7 78.6 7 9.4 77.17 8.6 79.17 9.3

G500 G1500 W500 W1500 GC WC

HR ¼heart rate, dP/dt ¼cardiac contraction force, iSV¼ indexed stroke volume, iCO¼indexed cardiac output, iPR ¼ indexed peripheral vascular resistance, SP¼ systolic pressure, DP¼ diastolic pressure, bpm ¼beats per minute, PC¼ placebo control, G500¼ 500 mg gentian extract, G1500¼ 1500 mg gentian extract, W500¼ 500 mg wormwood extract, W1500 ¼1500 mg wormwood extract, GC ¼ gentian capsules containing 1000 mg, WC ¼ wormwood capsules containing 1000 mg. ∧

¼p o 0.05. ¼ p o0.01. † ¼ 0.05 rp o 0.10, Pre–post ingestion comparisons for the control condition. n ¼p o 0.05. nn ¼ po 0.01. # ¼ 0.05 r po 0.10, Placebo-controlled pre–post ingestion comparisons. ∧∧

Please cite this article as: McMullen, M.K., et al., Bitter tastants alter gastric-phase postprandial haemodynamics. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.041i

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3.2.1. Placebo-control results Changes were observed between the pre-ingestion and the 5 to 15 min post-ingestion period for the placebo-control condition which was composed of 100 mL water and three cellulose capsules (Table 1 and Fig. 1). The mean differences, with statistical values and 95% confidence intervals (CI), were as follows: heart rate decreased by 1.2 (t¼2.39, p ¼0.033; 95% CI:  2.3 to  0.1) bpm, dP/dt increased by 69 (t¼ 3.989, p¼ 0.002; 95% CI: 32 to 107) mmHg s  1, systolic pressure increased by 3.8 (t ¼3.795, p ¼0.002; 95% CI: 1.6 to 5.9) mmHg, diastolic pressure increased by 1.3 (t ¼2.532, p ¼0.025; 95% CI: 0.2 to 2.3). Additionally, the placebocontrol elevated indexed stroke volume by 1.7 (t ¼2.061, p ¼0.060; 95% CI:  0.1 to 3.5) mL m  2. These various changes can be attributed largely, if not completely, to increases in dP/dt. The decrease in heart rate is likely due to baroreflex activity resulting from the increased blood pressure. The baroreflex is a negative feedback loop which acts acutely, i.e. over periods of seconds and minutes (Levick, 2010). The baroreceptor system is part of the autonomic nerve system and buffers blood pressure levels, opposing both increases and decreases in arterial pressure (Hall, 2011).

2013). Second changes in dP/dt have not previously been reported as contributing to haemodynamic changes in postprandial hyperaemia. The difference between our findings and previous studies may be attributed to both design differences and the enhanced measurement sensitivity utilised in this study. The bulk of the research on the postprandial haemodynamic responses to water, typically 500 mL, has focused on the responses of the elderly or those with autonomic disorders. For these groups water’s pressor effect peaks in the intestinal-phase of digestion about 30 min after ingestion (May and Jordan, 2010; Young and Mathias, 2013). Whereas this study utilised continuous beat-to-beat measures previous research with healthy individuals have measured blood pressure at either 5, 10 or 15 min intervals (Ahuja et al., 2009; Jordan et al., 2000; Routledge et al., 2002; Scott et al., 2001). The blood pressure increases of 3.8/1.3 mmHg were due to increased cardiac activity, specifically dP/dt, even though the heart rate decreased. Following ingestion of 100 mL water, the increased cardiac activity acted to increase blood pressure which acts to support postprandial hyperaemia and maintain systemic blood pressure. The increase of dP/dt can be categorised as a sympathetic response (Klabunde, 2005) and is likely elicited by receptors in the gut (May and Jordan, 2010). In related research it has been reported that both gastric distension (van Orshoven et al., 2004) and water ingestion (Scott et al., 2001) increases peripheral sympathetic neural discharge though neither study reported increased blood pressure. Other studies have also observed decreased heart rate, albeit without a change in blood pressure, in the gastric-phase following the ingestion of either 350 mL (Ahuja et al., 2009) or 500 mL (Brown et al., 2005) water. In the current study, the reduction of heart rate, although small, 1.2 bpm, indicates that the increases of blood pressure are being buffered by the baroreflex system. Consequently individuals with an impaired baroreflex, or otherwise compromised autonomic system, may be more susceptible to large blood pressure changes in the gastric-phase than previously appreciated (Young and Mathias, 2013).

3.2.2. Postprandial haemodynamic response to water The finding, that the ingestion of 100 mL water elicits increases of blood pressure in the gastric-phase of digestion, is novel on two counts. First, the consensus view is that even the ingestion of relatively large amounts of water (500 mL) by healthy adults elicits no pressor response (May and Jordan, 2010; Young and Mathias,

3.2.3. Gentian and wormwood capsules There was no difference between the pre–post differences of the placebo-control capsules and the pre–post differences of either the gentian or wormwood capsules. This finding contrasts with a previous report which demonstrated that encapsulated caffeine, also a bitter tastant, elicited increases in diastolic pressure

control capsule. Second, the addition of the larger amount of bitter tastants to the water altered gastric-phase postprandial haemodynamics. Compared to placebo-control, bitter-flavoured water elicited an additional and parallel pressor effect which resulted from increased vascular tone. Again, the magnitude of these changes were not age correlated. The combined impact of the two pressor effects did not lead to increased blood pressure. Rather, blood pressure increases were equivalent for bitterflavoured water and the control condition because increases of peripheral vascular resistance resulted in decreased cardiac activity. As encapsulated gentian and wormwood elicited no haemodynamic changes, the increases in peripheral vascular resistance elicited by bitter-flavoured water were likely due to cephalic stimulation.

1.5

0.8

1.3

0.6

1.1

0.4

0.9

0.2 z scores

z scores

3.2. Part 1: Ingestion of capsules

0.7 0.5 0.3

-0.2 -0.4

0.1

-0.6

-0.1

-0.8

-0.3 -0.5

0

0

3

6

9

12

15

-1

0

3

Minutes after intake HR

dP/dt

SV

6

9

12

15

Minutes after intake CO

SP

DP

PR

Fig. 1. Pre–post ingestion cardiovascular parameter changes following the ingestion of 100 mL water over a 15 min period presented as average readings of 1 min intervals. Parameter values are expressed as Δ z scores (mean¼ 0, standard deviation ¼1) based on changes from pre-ingestion measures. (a) Cardiac parameters, (b) Vascular parameters. HR ¼heart rate, dP/dt¼ cardiac contraction force, SV¼ indexed stroke volume, CO¼indexed cardiac output, SP ¼ systolic pressure, DP ¼ diastolic pressure, PR ¼ indexed peripheral vascular resistance.

Please cite this article as: McMullen, M.K., et al., Bitter tastants alter gastric-phase postprandial haemodynamics. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.041i

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Fig. 2. Pre–post ingestion cardiovascular parameter changes following the ingestion of 100 mL either the placebo condition, water plus 3 capsules, or bitter flavoured water over a 10 min period presented as average readings of 1 min intervals. PC¼ placebo control, G500¼ 500 mg gentian extract, G1500¼1500 mg gentian extract, W500 ¼500 mg wormwood extract, W1500 ¼1500 mg wormwood extract. (bpm¼ beats per minute, MU ¼medical unit ¼mmHg s mL  1). (a) Heart rate, (b) dP/dt—cardiac contraction force, (c) indexed stroke volume, (d) indexed cardiac output, (e) indexed peripheral vascular resistance, (f) systolic pressure, (g) diastolic pressure.

(McMullen et al., 2011) during the 10 to 15 min post-ingestion period. Both studies were conducted under identical conditions and with the same batch of capsules. The contrasting results indicate that, although the molecular structures of bitter receptors are present throughout the gastrointestinal tract (Horn, 2008), the distribution of individual bitter receptors may differ. 3.3. Part 2: Gentian and wormwood flavoured water Changes in the cardiovascular system were observed following the ingestion of water flavoured with both gentian and wormwood (see Table 1 and Fig. 2). 3.3.1. Gentian flavoured water results Both quantities of gentian altered the haemodynamic response to water with the larger quantity, G1500, producing greater increases than the smaller quantity, G500 (see Table 1 and

Fig. 2). The principal impact of G1500 was an increase of the vascular tonus, with indexed peripheral vascular resistance increasing by 0.07 (t¼3.184, p ¼ 0.002; 95% CI: 0.03 to 0.11) MU m  2. G500 elevated indexed peripheral vascular resistance by 0.04 (t ¼1.818, p ¼0.074; 95% CI: 0.00 to 0.08). Both G500 and G1500 decreased indexed stroke volume by 2.2 (t¼ 2.128, p¼ 0.038; 95% CI:  4.3 to  0.1) and by 2.5 (t¼2.465, p ¼0.017; 95% CI: 4.6 to 0.5) mL m  2, respectively, as well as decreasing indexed cardiac output by 0.15 (t¼2.226, p ¼0.030; 95% CI:  0.29 to 0.02) and 0.20 (t ¼2.946, p¼ 0.005; 95% CI:  0.34 to  0.06) L m  2, respectively.

3.3.2. Wormwood flavoured water results Only the larger quantity of wormwood, W1500, altered the cardiovascular response to water (see Table 1 and Fig. 2). Similarly to gentian, the principal impact on the cardiovascular system was increased vascular tonus. W1500 increased indexed peripheral

Please cite this article as: McMullen, M.K., et al., Bitter tastants alter gastric-phase postprandial haemodynamics. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.041i

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vascular resistance by 0.07 (t ¼3.105, p ¼0.003; 95% CI: 0.02 to 0.11) MU m  2 as well as decreasing both indexed stroke volume by 3.4 (t¼ 3.294, p ¼0.001; 95% CI:  5.5 to  1.3) mL m  2 and indexed cardiac output by 0.25 (t ¼3.631, po 0.001; 95% CI:  0.38 to  0.11) L m  2. 3.3.3. Bitter tastants alter postprandial haemodynamics The responses elicited by the larger doses of gentian and wormwood were similar: increases of peripheral vascular resistance, decreases of stroke volume and cardiac output without alterations of heart rate, dP/dt or blood pressure. The increases of peripheral vascular resistance did not lead to increases of blood pressure larger than those occurring in the control condition, presumably because blood pressure increases were buffered by the baroreflex. The increased peripheral vascular resistance acted to reduce the cardiac workload as is indicated by the decreases of the cardiac activity parameters. Thus the increased peripheral vascular resistance is acting in parallel with the increased dP/dt to support post-prandial hyperaemia. It is unlikely that these increases of peripheral vascular resistance involved gastrointestinal receptors, as the encapsulated tastants elicited no changes (Section 3.2.3). Consequently the increase in vascular tonus can be attributed to the stimulation of the cephalic receptors. As both gentian and wormwood contain a number of compounds, of which only some are bitter, the pharmacological activity cannot be attributed to any particular compound. 3.4. Implications for bitter tastant theories Previous investigation of taste’s effect on haemodynamics is limited. Increases of heart rate and skin resistance were reported following the tasting of quinine (bitter), citric acid (sour) and salt (Rousmans et al., 2000) however it is unclear whether these shortlived haemodynamic changes are startle reactions (Bradley and Lang, 2007) or indicators of substantial haemodynamic changes. The intake of a 0.9% saline solution may have increased heart rate 10 min after intake, however these results are unclear as statistical comparisons were made with pre-ingestion values and not between the saline and the control condition (Brown et al., 2005). Chewing has been reported to increase heart rate while chewing with chocolate-taste increased heart rate to a greater extent. These increases were limited to the chewing period with heart rate returning to normal within 5 min. Neither chewing, nor chewing with taste, impacted on mean blood pressure (Someya and Hayashi, 2008). Black coffee tastes bitter (Breslin and Huang, 2006) due to the presence of quinides rather than caffeine (Frank et al., 2006) even though caffeine stimulates the hTAS2Rs 5,10,14, 43 and 46 (Meyerhof et al., 2010). Drinking black coffee (67 mL espresso from 16.5 g beans containing circa 130 mg caffeine) elicits increases of heart rate extending for 15 to 30 min without affecting blood pressure, yet the response is due to the presence of caffeine rather than the bitter quinides (McMullen et al., 2011, 2012b). The response can be attributed to the impact of caffeine on the cephalic taste receptors and likely results from vagal withdrawal rather than sympathetic activation. The current findings indicate that some bitter tastants, such as gentian and wormwood, can elicit cephalic-phase responses that alter postprandial haemodynamics during the gastric-phase of digestion. As vascular tonus is under the control of the sympathetic nerve system, the increases in peripheral vascular resistance are attributable to increased sympathetic activity. The observed increase of systemic blood pressure can be expected to reduce the extent of inadequate postprandial hyperaemia and so is consistent with suggestions of both Hall White and Weiss that bitter tastants improve appetite by increasing the gastric circulation. Changes of

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the circulatory system were observed after only 5 min indicating that bitter tastants likely act more quickly than appreciated. Many texts, including the European Medical Agency Community Herbal Monograph for gentian (Knö ss and Stolte, 2009b), suggest that to be effective bitter tastants should be consumed 15 to 30 min prior to eating (Mills and Bone, 2000; Sandberg and Corrigan, 2001; Schulz et al., 2004). The finding that increased blood pressure due to increased peripheral vascular resistance is buffered by the baroreflex can explain the frequently reported observation (Mills and Bone, 2000; Schulz et al., 2004) that those with poor digestion are benefited by the intake of bitter tastants whereas those with normal digestion experience no effect. When inadequate postprandial hyperaemia is the cause of poor digestion, the intake of bitter tastants will increase peripheral vascular resistance and so act to increase systemic blood pressure. This will support postprandial hyperaemia and consequently improve the digestive process. Whereas, in cases of normal postprandial hyperaemia the increases in blood pressure, due to increased peripheral vascular resistance, will be buffered by the baroreflex. Additionally, the increased peripheral vascular resistance may act to reduce cardiac workload via the baroreflex and reduce the extent of cardiac problems, associated with cardiac insufficiency, which may occur following eating (Luciano et al., 2010). The findings support the hypothesis that bitter tastants elicit cephalic-phase responses involving the sympathetic nervous system. The findings do not necessarily clash with the hypothesis that bitter tastants elicit “stimulation of gastric juices via the nervus vagus” (Olivier and van Wyk, 2013) as the actions of the sympathetic and parasympathetic systems are largely integrated rather than opposing (Jänig, 2006). However, cephalic elicited vagal activity may not be required to explain the action of bitter tastants if sympathetic mediated cephalic-phase responses are sufficient to promote normal digestive function. Although gentian and wormwood did not locally stimulate gut receptors to elicit haemodynamic changes in the gastric-phase of digestion, it remains to be assessed whether bitter tastants stimulate gut receptors during the intestinal-phase of digestion. 3.5. Hypothesis testing and potential application: Postprandial hypotension This research provides a rationale for testing the response to intake of bitter tastants elicited by individuals with postprandial hypotension. Postprandial hypotension is a particular problem amongst the aged and for one group of low-level-care elderly it was shown to be an independent predictor of all-cause mortality, with no predictive value explained by the other blood pressure indices: orthostatic hypertension, hypertension, pulse pressure and mean arterial pressure (Fisher et al., 2005). As a blunted sympathetic response is thought to cause postprandial hypotension (Luciano et al., 2010), it is unclear whether bitter tastants will be able to elicit sympathetically mediated vascular responses in these individuals. The testing of such individuals may aid in establishing whether bitter tastants affect vagal tone. It also is possible that bitter tastants may be of therapeutic use in reducing postprandial hypotension. For the treatment of a similar condition, fainting, the two English herbalists, John Gerard and Nicholas Culpeper, both recommended gentian during the 16th and 17th century, respectively (Brendler et al., 2003). 3.6. Limitations In Part 1 it is possible that the cellulose in the placebo capsules may have influenced the findings. To avoid this potential confounding in future studies, test substances could be delivered in

Please cite this article as: McMullen, M.K., et al., Bitter tastants alter gastric-phase postprandial haemodynamics. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.041i

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capsules containing the same amount of cellulose, or some other base, as the placebo and impregnated with the fluid preparations. It is also possible, that there may be some differences between the chemical composition of the bitter tastants in the flavoured waters and the capsules, resulting from the extraction procedures, and that these differences may have responsible for dissimilarity of responses elicited by the two groups of test substances (Section 2.1).

4. Conclusions The current findings have implications across a wide range of disciplines including pharmacology, cardiology, neuroscience, perceptual psychology, gastroenterology and dietetics. Normal individuals experience increases of blood pressure in the gastric phase of digestion following the ingestion of 100 mL water. This hypertensive activity results from increased dP/dt and is likely a reflex response originating from gut receptors. The magnitude of the blood pressure increase is sufficient to trigger baroreflex buffering, as evidenced by reductions of heart rate. The addition of the bitter tastants gentian and wormwood to water elicits cephalic-phase responses, which alter post-prandial haemodynamics. An increased vascular tone, acts in parallel with the elevated dP/dt, to increase blood pressure yet the magnitude of the blood pressure increases is unchanged as cardiac activity decreases presumably due to baroreflex activity. This cephalicphase response elicited by the bitter tastants is likely a reflex response involving the sympathetic nerve system. The findings indicate the existence of a chemosensory pharmacological mechanism that is consistent with the traditional use of these bitter tastants to treat digestive disorders.

Sources of funding None.

Acknowledgements None.

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Please cite this article as: McMullen, M.K., et al., Bitter tastants alter gastric-phase postprandial haemodynamics. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.04.041i