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Testing of Perilla frutescens extract and Vicenin 2 for their antispasmodic effect.



Perilla frutescens

Vicenin 2

Antispasmodic effect


Gastrointestinal discomfort is frequently observed. The effects of Perilla frutescens extract and Vicenin 2 (a compound in this extract) were assayed in rat ileum with or without stimulation with acetylcholine or [Ba.sup.2+]. Both had no direct spasmolytic effect, but both decreased acetylcholine- or [Ba.sup.2+]-induced contraction of rat ileum indicating an antispasmodic effect. This is valuable because effects were only observed when spasms were induced and may disturb the patient. The extract and the compound may be used to maintain and improve gut health.

[c] 2012 Elsevier GmbH. All rights reserved.


Maintaining optimal digestive health is particularly important for vitality and well being throughout all stages of life. The efficiency of each person's digestive tract can make all the difference in daily energy and overall health. Nearly everybody experiences gastrointestinal discomfort, which affects quality of life.

Symptoms can be bloating, altered intestinal mobility and transit as well as abdominal pain. Many times these symptoms are combined with poor mood, lack of concentration and energy and may consequently prevent people from sleeping, working, exercising and socializing with friends.

Gastrointestinal discomfort is mainly triggered by ileum contractions, which can be caused by several factors. For example, daily stress, food sensitivity and allergies, infections, genetic preposition, altered gut flora or deregulation of brain-gut cross-talk may lead to development of gastrointestinal disorders, dyspepsia or irritations like irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD).

Patients with freshly diagnosed gastrointestinal disorders may have dyspepsia (35%), bile disturbance (21%), obstipation (22%), intestinal bowel syndrome (IBS) (16%) and others (6%) (Fintelmann and MenBen 1996). Promoting digestive comfort includes regulation of transit through the gastrointestinal tract and easing pain associated with digestion as well as to support the reduction of causes leading to gastrointestinal discomfort.

The cholinergic system may be involved in the development of intestinal discomfort as well as in many gastrointestinal diseases. Serotonin (95% of the neurotransmitters found in GI tract) can stimulate cholinergic neurons to release acetylcholine which results in smooth muscle contraction. Serotonin participates in mucosal sensory transduction and peristaltic reflex. When intraluminal pressure increases, enterochromaffin cells release serotonin, which stimulates vagal and intrinsic (enteric) afferent nerve fibers which then initiates the peristaltic reflex (Bulbring and Crema 1958; Bulbring and Lin 1958; Bulbring and Crema 1959) and peristaltic reflex. GI symptoms including IBS may result from dysregulation of a brain-gut cross talk. In addition to serotonin cholecystokinin. substance P. calcitonin gene-related polypeptide, and even more compounds including toxic compounds such as barium ions ([Ba.sup.2+]) may be involved in a complex, yet unknown manner leading to ileum contraction and disturbances.

For many plant extracts (Angelica roots, chamomile flowers, Caraway fruits, milk thistle fruits, peppermint leaves, Lemon balm leaves) spasmolytic properties have been shown (Izzo et al. 1996; Reiter and Brandt 1985; Achterrath-Tuckermann et al. 1980; Forster et al. 1980; Forster 1983; Liersch et al. 2003). Therefore, it was interesting to investigate the effect of the nutraceutical Perilla frutescens extract and of the isolated compound Vicenin 2 with respect to their beneficial physiological effects to prevent gastrointestinal discomfort and to maintain a healthy gut.

Perilla extract is obtained from Perilla frutescens (L) leaves, an annual eatable herbaceous plant native to Asia. Perilla frutescens is a member of the family Lamiaceae. Perilla leaves are used as food and in traditional medicine. In foods the leaves are used fresh or fried as spice, as garnish, salad and in wrappings for sushi. Traditional medical applications are all linked to infections of the respiratory tract and the immune system.

During our phytochemical investigation of Perilla leaves, we discovered the compound Vicenin 2 which has not been described as constituent for Perilla leaves before. Our intention was to develop a special Perilla leaf extract, which included the known constituents, like Apigenin or Luteolin as well as the new discovered compound Vicenin 2. Our Perilla leaf extract was, therefore, standardized on a special flavonoid fractions, as given in the paper by the fingerprint.

Our goal was to investigate the anti-spasmodic effects of the standardized PeriIla leaf extract and to show that also the newly identified molecule Vicenin 2 has anti-spasmodic effects. Therefore we tested the newly identified molecule in comparison to the standardized extract, which includes all the known flavonoids, di-glycosides, etc. Perilla frutescens extract and the compound Vicenin 2 (called AUC-P and AUC-V, respectively) were compared for their spasmolytic and antispasmodic effect with respect to both the cholinergic system and nonspecific contraction mediated by [Ba.sup.2+]. Therefore, rat ileum was precontracted with increasing concentrations of either acetylcholine or [Ba.sup.2+]. The shift of either concentration response curve by both the above mentioned extract and compound should be determined. As positive controls (validation of the experiments) either atropine or papaverine were used depending on the type of experiment.

Materials and methods

Extracts and calculation of their concentrations to be used in the experiments

Two samples were investigated called AUC-P (Perilla frutescens water extract) and AUC-V (isolated Vicenin 2) were supplied by Vital Solutions GmbH, Germany (Dr. Sybille Buchwald-Werner) and the Company Amino Up Chemical Co., Ltd. (Dr. Hajime Fujiil), Sapporo, Japan. They are hydrophilic and could easily be dissolved.

Reverse-phase HPLC (RP-HPLC) was performed using a Hitachi apparatus equipped with an L-7420 detector (at 320 nm) and a 250 mm x 4.6 mm I.D., 5 [micro]m, ODS, Capsule Pack UG-120 (Shiseido, Japan). Separation was achieved with an increasing amount of 0.1% acetic acid, 5% methanol and 10% water in acetonitrile (B) in 0.1% aqueous acetic acid (A): 0-10 min, 12.5% B, isocratic; 10-25 min, 12.5-90% B, linear gradient; 25-30 min, 90% B, isocratic; 30-31 min, 90-12.5% B, linear gradient; and 31-40 min, 12.5% B, isocratic at a flow rate of 0.8 ml/mm.

From the company it is expected that the dose of the extract (AUC-P) for humans will be between 5 and 100 mg/70 kg b.w. which will correspond to 0.071 and 1.43 mg/kg b.w. Due to the increased metabolism in rodents compared to humans the doses were calculated as approx. between 0.5 and 10 mg/kg b.w. The central (blood) compartment (hydrophilic extracts!) is approx. 7% of whole body volume, i.e. between 0.5/70g and 10 mg/70 g blood. Since blood is in direct contact with ileum, a concentration of 7-140 mg/kg (0.007-0.140 mg/g) is expected for ileum. Note: Calculations were not corrected for specific gravity (density) of liquids (possibly differing from exactly 1.0) and for absorption (assumed to be 100%; no data available). Therefore, it was reasonable to translate these in vivo calculations into the in vitro experiments and in these experiments 10 and 104.1 of a stock solution (0.05 g/m1= 5 x [10.sup.-2]g/ml with respect to AUC-P) per 10 [micro]l incubation bath were used. Therefore, the final concentrations were 0.05 and 0.5 mg/ml bath volume with respect to AUC-P.

AUC-V calculation is slightly different. Since the isolated compound may be 10 times more concentrated compared to the whole extract AUC-P, the stock solution was only 0.005 g/m1=5 x [10.sup.-3]g/ml). Therefore, the final concentrations of AUC-V were 0.005 or 0.05 mg/m1 bath volume.

Acetylcholine sulfate and barium chloride were from Roth, Karlsruhe, Germany, and Sigma-Aldrich Chemie, Deisenhofen, Germany. All other reagents and compounds used for incubations were obtained from Roth, Karlsruhe, Germany.

Rat ileum

Under a protocol approved by the Animal Care and Use Committee of Munster, Germany, male and female rats (Charles River, Sulzfeld, Germany) weighing 200-320g were used. They received a standard diet (Altromin, Lage/Lippe, Germany) and tap water ad libitum. The rats were anesthetized (C[O.sup.2]). The proximal ileum was removed, washed and placed in Krebs-Henseleit solution. 1 cm segments were placed in 10 ml organ baths with a resting tension of 1.0g (preload). One end was attached to a force displacement transducer (lever transducer B40, Typ 373) combined with a two-channel amplifier (type 301, Hugo Sachs Elektronik, March, Germany); for the recording of tension changes a multi-pen recorder was used (Rikadenki Kogyo, Tokyo, Japan). The composition of Krebs-Henseleit solution was (mM): NaCI 118.1, KCI 4.7, Ca[Cl.sub.2] 2.5, MgS[O.sub.4] 1.2, NaHC[O.sub.3] 25 and glucose 5.6. The solution was kept at 37 [degrees]C. pH 7.4 and gassed with carbogen (a 95% 02/5% C[O.sub.2] mixture). Equilibration time for the ileum before starting the experiment was at least 30 min under a resting tension of 1.0 g. The longitudinal contraction was measured according to the method of Magnus (1904), modified, using an isotonic contraction transducer and amplifier, and recorded with a pen recorder (Okpanyi et al. 1993). After the tissues had been pretreated with the extracts for 3 min cumulative concentration response curves using either acetylcholine or barium ion were recorded isotonically in the organ bath, and the effect was allowed to reach a steady state at each concentration.

The spasmodic agent for the induction of intestinal contraction was acetylcholine or [Ba.sup.2+] used in various concentrations up to 10 [micro]g/m1 and 4 [micro]g/ml, respectively. The results were normalized to 100% (maximum effect of either acetylcholine or barium chloride alone).

Statistical analysis

Differences between entire curves were obtained by comparing F-values of the best fitted curves by using GraphPad-Prism (version 3.00. GraphPad Software, Inc., 1999). p <0.05 was regarded as statistically significant. EC50 values were calculated. E[C.sub.50] and SEM are given as effect of acetylcholine or barium concentration in [mu]g/l without and in the presence of test substances. Data were calculated from individual curves.


The Perilla frutescens extract was characterized; a fingerprint obtained by using RP-HPLC method is shown in Fig. 1. It comprises, e.g. 10 different flavonoids, caffeic acid and rosmarinic acid.


As shown in Fig. 2 AUC-P and AUC-V induced a slight (not significant) relaxation from their own. The data are shown as % of maximum acetylcholine effect. This means that they lack a direct spasmolytic effect.


Next the effect of various acetylcholine concentrations and the interaction with both AUC-P and AUC-V are shown (Fig. 3). Low concentrations of extract AUC-P (0.05 mg/ml final bath concentration) and compound AUC-V (0.005 mg/ml) shifted the acetylcholine curve to the right in a competitive manner; compound AUC-V also shifts the curve downward possibly indicating an additional non-competitive antispasmodic effect. The data indicate a neurotropic antispasmodic effect. Atrophic (positive control) shows the expected competitive neurotropic antispasmodic effect indicating that the setup of experiment is working (validation). The use of atropine is mimicked by the marketed N-butylscopolamine (Buscopan[R]). All curve data of Fig. 3 were recalculated for their E[C.sub.50] values shown in Table 1.
Table 1 Half maximal effects (E[C.sub.50]) of extracts and
compounds as derived from data shown in Figs. 3 and 5.

Compound           E[C.sub.50] + S.E. of inhibitory effect
                 [[mu]g/ml](acetylcholine stimulation) (a)

None                                    0.62 [+ or -] 0.03

plus Extract                          0.99 [+ or -] 0.05 *

plus Compound                          133 [+ or -] 0.03 *

plus Atropine                         1.67 [+ or -] 0.02 *

Compound           E[C.sub.50] + S.E. of inhibitory effect
                 [[mu]g/ml](acetylcholine stimulation) (b)

None                                    1.62 [+ or -] 0.02

plus Extract                            1.98 [+ or -] 0.06

plus Compound                         2.04 [+ or -] 0.05 *

plus Papaverine                       2.13 [+ or -] 0.09 *

* p<0.05 compared to no addition (none).

(a.) Note: Data are derived from Fig. 3.

(b.) Note: Data are derived from Fig. 5.


These experiments were repeated while the concentration of extracts was increased 10 fold: AUC-P from 0.05 to 0.5 mg/m1 and AUC-V from 0.005 to 0.05 mg/ml (final bath concentration). The data are shown in Fig. 4. Increasing the concentrations of AUC-P and AUC-V results in a stronger rightward and downward shift of the acetylcholine curve compared to data of Fig. 3 which indicates a concentration-dependent effect of both AUC-P and AUC-V.


Next the effect of various [Ba.sub.2+] concentrations and the interaction with both AUC-P and AUC-V are shown (Fig. 5). Low concentrations of extract AUC-P (0.05 mg/ml final bath concentration) and compound AUC-V (0.005 mg/ml) shifted the [Ba.sub.2+] curve downwards indicating a non-competitive antispasmodic effect. The data indicate a musculotropic antispasmodic effect. Papaverine (positive control) shows the expected non-competitive musculotropic antispasmodic effect indicating that the setup of experiment is correct and working (validation). All curve data of Fig. 5 were recalculated for their E[C.sub.50] values shown in Table 1.

These experiments were repeated while the concentration of extract and compound was increased 10 fold: AUC-P from 0.05 to 0.5 mg/ml and AUC-V from 0.005 to 0.05 mg/m1 (final bath concentration). The data are shown in Fig. 6. AUC-P and AUC-V addition results in a rightward and downward shift of the [Ba.sub.2+] curve which indicates a non-competitive inhibition (antispasmodic effect) of the [Ba.sub.2+] effect. The effects were as strong as compared to these at the lower concentrations shown in Fig. 5.




A spasmolytic effect in intestinal smooth muscle in vitro has been observed in several herbal drugs used in the therapy of motility-related functional gastro-intestinal diseases or discomfort. Extracts and drugs have to be tested in two ways: first whether they have an effect from their own (so-called spasmolytic effect), and second whether they have an antispasmodic effect with respect to compounds inducing smooth muscle contraction.

Both extracts have a weak (not significant) effect From their own; this means they have no spasmolytic activity.

An antispasmodic effect was observed. The antispasmodic effect on smooth muscles leading to a relief from gastrointestinal symptoms including gastrointestinal discomfort and bowel disease symptoms can be achieved by two types of effective compounds: they should either have neurotropic or musculotropic antispasmodic effects or even both. Physiologically a neurotropic effect is mediated via acetylcholine; a musculotropic effect is mediated via e.g. toxic compounds. Both the extract AUC-P and the compound AUC-V have an antispasmodic activity, inhibiting neurotropic and musculotropic activity. What is important, their effects show a concentration dependency with respect to their neurotropic antispasmodic activity. As known from many other plant extracts their effects are not as strong as typical lead compounds (positive controls): in this case the effects were minor compared to either atropine (acetylcholine experiments) or papaverine ([Ba.sub.2+] experiments).

The impact of these experiments has to be defined: [Ba.sub.2+] -induced spasm is an overall effect involving intracellular [Ca.sup.2+] and other mechanisms, e.g. inhibiting K+ channels. Regarding neurotropic effects, in addition to that of acetylcholine (neurotropic effect; as was performed in this study), histamine (antiallergic effect) and possibly serotonin as neurotransmitter should be included.

In conclusion: both the extract and the compound (AUC-P and AUC-V, respectively) are not (or only hardly) effective as spasmolytics (no precontractions induced). This makes them ineffective if there is no major disease with respect to contraction. However, they have antispasmodic effects using two different mechanisms of action probably helping when there is an irregular contraction leading to Cl symptoms. Thus the Perilla frutescens extract (AUC-P) and the compound Vicenin 2 (AUC-V) do not appear to be a remedy, but can be used prophylactically to maintain and improve gut health.

* Corresponding author at: Department of Pharmacology. Institute of Medicinal Chemistry. University of Munster. Hittorfstr. 58-61 48149 Munster. Germany.

E-mail address: (E.J. Verspohl).

0944-7113/$--see front matter [c] 2012 Elsevier GmbH. All rights reserved.


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Eugen J. Verspohl (a), *, Hajime Fujii (b), Kohei Homma (b), Sybille Buchwald-Werner (c)

(a.) Department of Pharmacology, institute of Medicinal Chemistry, University of Minister, Minister, Germany

(b.) AMMO Up Chemicals, Saporo, Japan

(c.) Vital Solutions GmbH, Langenfeld, Germany
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Article Details
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Author:Verspohl, Eugen J.; Fujii, Hajime; Homma, Kohei; Buchwald-Werner, Sybille
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:May 15, 2013
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