Effect of Matricaria chamomilla hydro-alcoholic and flavonoids rich extracts on rat isolated uterus

Introduction Chamomile belongs to Asteraceae family and grows naturally in many places (1). Several species of chamomile are spread over the world, however, the most popular of all is known as German chamomile (Matricaria chamomilla L.) (2,3). M. chamomilla is cultivated in many parts of the world for its historical use in the food and cosmetic products as well as herb infusions for its medicinal benefits (4-7). In Iran, M. chamomilla is known as Babuna and traditionally is used for the treatment of a number of ailments such as gastrointestinal problems including indigestion, carminative, flatulence, colic and diarrhea (8). Other common traditional uses of M. chamomilla are for treatment of inflammatory diseases such as bronchitis, eczema and other skin problems (9). M. chamomilla is also traditionally used for treatment of common cold and fever (5). It is believed that M. chamomilla has analgesic effect and therefore it is used for treatment of painful conditions such as toothache, dysmenorrheal and rheumatism (4). Phytochemical studies identified presence of essential oils, flavonoids and coumarins constituents in the M. chamomilla (10-12). Antispasmodic and antihttp://www.herbmedpharmacol.com doi: 10.15171/jhp.2020.05


Introduction
Chamomile belongs to Asteraceae family and grows naturally in many places (1). Several species of chamomile are spread over the world, however, the most popular of all is known as German chamomile (Matricaria chamomilla L.) (2,3). M. chamomilla is cultivated in many parts of the world for its historical use in the food and cosmetic products as well as herb infusions for its medicinal benefits (4)(5)(6)(7). In Iran, M. chamomilla is known as Babuna and traditionally is used for the treatment of a number of ailments such as gastrointestinal problems including indigestion, carminative, flatulence, colic and diarrhea (8). Other common traditional uses of M. chamomilla are for treatment of inflammatory diseases such as bronchitis, eczema and other skin problems (9). M. chamomilla is also traditionally used for treatment of common cold and fever (5). It is believed that M. chamomilla has analgesic effect and therefore it is used for treatment of painful conditions such as toothache, dysmenorrheal and rheumatism (4). Phytochemical studies identified presence of essential oils, flavonoids and coumarins constituents in the M. chamomilla (10)(11)(12). Antispasmodic and anti-inflammatory effects of M. chamomilla is attributed to the constituents of the essential oil and the flavonoids (13,14). Most abundant flavonoids which have been identified in M. chamomilla are apigenin, luteolin, quercetin, and isorhamnetin which often are found in the glycoside forms (15,16). As mentioned above M. chamomilla is famous for its antispasmodic activity especially on the gastrointestinal tract and pharmacological studies have shown that both aqueous and hydro-alcoholic extracts of M. chamomilla inhibit guinea pig ileum contractions induced by acetylcholine (ACh) and histamine (17)(18)(19). As a gastrointestinal smooth muscle relaxant, it is expected that M. chamomilla can also relax uterus smooth muscle. Nevertheless, so for there is no report on relaxant effect of M. chamomilla extract on isolated uterus. On the other hand, there are suggestions that M. chamomilla consumption is associated with increase in uterine tonicity (20). However, so far there is no pharmacological evidence which could confirm or reject this assumption. Therefore, the objective of this research was to investigate both excitatory and inhibitory effects of M. chamomilla hydro-alcoholic and its flavonoids rich extracts on uterus contractions.

Methods and Materials
Extract preparation Chamomile was collected during flowering time and the flowers were separated and dried in shade. A voucher specimen was identified and deposited in the herbarium of School of Pharmacy and Pharmaceutical Sciences (NO:1391). Dried flowers were ground into fine powder using an electronic grinder (Keep, Korea). Hydroalcoholic extract was prepared by maceration method (21). Four hundred grams of dried powder was placed in a large container and 2 liters of ethanol (70%) was added, well mixed and top of the container was sealed. After 24 hours, solute was separated by help of a Buchner funnel. The maceration process repeated twice by adding fresh ethanol and all the collected solutes were added together and concentrated by rotary apparatus. Following drying a sample of extract, the percentage of remaining liquid in the extract was determined and extract yield was calculated.
For preparation of flavonoids rich extract, solvent in solvent partitioning technique was employed (22). For this purpose, 50 g of concentrated hydro-alcoholic extract was added into decanter filled with one liter of chloroform and water (1:1). This mixture was shaken for 20 minutes and then left until two liquid phases were completely separated. The lower chloroform phase was decanted and 500 mL fresh chloroform was added into decanter and the process was repeated. The remaining chloroform in the aqueous phase was removed by rotary evacuation at 30°C. Then hydrochloric acid was drip into the aqueous solution and pH of the liquid was adjusted (pH=2). After that, 500 mL ethyl acetate was added into the aqueous solution and well shaken. Fifteen minutes later, the lower aqueous phase was collected and by adding equal volume of ethyl acetate the process was repeated thrice. Final ethyl acetate phase was collected and concentrated as flavonoids extract. All the fractions were concentrated on the rotary evaporator at 60˚C for pharmacological studies. Flavonoids contents of separated fractions were assessed by aluminum chloride colorimetric method as described before (22). Ethyl acetate fraction possessed highest contents of flavonoids and therefore considered as flavonoids rich fraction.
Tissue preparation Pharmacological studies were performed on isolated rat uterus. The animals were handled in accordance to international principles for laboratory animal use and care (23). For this purpose a day before experiment, Wistar non-pregnant female rats (180-220 g) were given a subcutaneous injection of estradiol (100 µg/kg)). Estrogen pretreated rats were killed and uterine horns were dissected and placed in oxygenated physiological solution (Tyrode's solution). In the pharmacology laboratory, uterine horns were separated and two ends were tied up with separate pieces of cotton thread. One end of the tissue was attached to special hook and secured into Harvard organ both filled with Tyrode's solution. Other thread was tied up into lever of an isotonic Harvard transducer. The tissue was hold under 1 g tension and continuously gassed with oxygen through the experiment. Following calibration of Oscillograph, the tissue was washed several times and allowed to relax into a stable baseline. Uterine contractions were amplified and recorded on Harvard Universal Oscillograph. Effect of the extract or drug was examined on uterine spontaneous contraction, electrical field stimulation (EFS), ACh, oxytocin and KCl induced contractions. Initially a series of pilot experiments were carried out to establish effective concentration of the extracts and then full concentration-response ranges of the extract were constructed. Relaxant effect of the extract was compared with vehicle treated time-matched controls using other uterine horn. Nifedipine was used as positive standard drug for comparison.

Drug and solutions
Tyrode's solution was composed of following chemicals: NaCl=136.9 mM; CaCl 2 =1.8 mM; NaHCO 3 =11.9 mM; MgCl 2 =1.05 mM; KCl=2.68 mM; glucose=5.55 mM; NaH 2 PO 4 =0.42 mM. Tyrode's solution was prepared daily in distilled water and saturated with oxygen. KCl was prepared as 2 M stock solution in distilled water. Acetylcholine (ACh, Sigma) was made up as 100 mM solution (acidified with 0.1 mL acetic acid) and diluted to 0.25 mM stock solution. Oxytocin (Aburaihan Pharm., Iran) stock solution was prepared from 10 IU/mL ampoule in distilled water. Estradiol valerate (10 mg/mL ampoule, Aburaihan Pharm., Iran) was diluted in vegetable liquid oil as 100 µg/mL stock solution for subcutaneous injection. Nifedipine (Sigma) was initially prepared as 10 mM stock solution in dimethyl sulfoxide (DMSO), further dilution was made up in DMSO or distilled water. All the extracts were made up as 40 mg/mL stock solution in 50% DMSO. Further dilution was prepared in distilled water. Unless stated, all the chemicals were from Merck (Germany).
Measurements and data analysis Uterine tonic contraction was measured as maximum amplitude of contraction from recorded tissue baseline. Uterine rhythmic contraction was measured as mean amplitude. The relaxation of isolated tissue preparations was expressed as percent of the control response mediated by added spasmogen. Drug concentration causing 50% of maximum inhibitory response (IC 50 ) was calculated by plotting full concentration-response curve for each tissue. All the results are presented as mean ± standard error of mean (ESM) for each group of results (n=6). Each test group was compared with its corresponding timematched control group treated with equivalent amount of the vehicle. One-way analysis of variance (ANOVA) or Student's t test was used for statistical analysis. P values less than 0.05 was considered statistically significant. SigmaPlot program was used for statistical analysis and plotting the graphs.

Results
Isolated rat uterus primed with estrogen, exhibited irregular spontaneous periodic activities. These spontaneous contractions were not sustainable and gradually subsided down over the time. Repeated washing with fresh Tyrode's solution, facilitated uterine smooth muscle basal tension settlement. Addition of hydro-alcoholic extract of M. chamomilla into the organ bath had no effect the basal tension but significantly potentiated spontaneous uterus contractions in comparison to the control group. On the contrary, flavonoids rich extract of M. chamomilla attenuated uterine spontaneous activities ( Figure 1).
Addition of KCl (2.5, 5, 10 and 20 mM) caused rhythmic contractile response imposed on a small tonic contraction. KCl at 40 mM bath contraction only induced tonic contraction in rat isolated uterus. Increasing KCl concentration to 80 mM produced a similar sustained tonic contraction with slightly higher amplitude. Effect of chloroform, flavonoids and aqueous fractions were initially screened on KCl induced tonic contraction. The aqueous fraction had no effect of KCl (80 mM) responses, while chloroform and flavonoids fractions concentration dependently inhibited KCl induced contraction ( Figure  2). As the flavonoids rich fraction was four times more potent than the chloroform fraction, the flavonoids rich fraction was tested in the subsequent experiments.
Flavonoids rich fraction in a concentration manner inhibited uterus responses to above mentioned KCl concentrations. Inhibitory effect of flavonoids rich fraction was compared with the hydro-alcoholic extract of M. chamomilla in Figure 3. After washing the tissue with fresh Tyrode's solution, the response to KCl was restored. The inhibitory concentration causing 50% of maximum response (IC 50 value) is presented in Table 1 for comparison. Time-matched control tissue treated with equivalent volume of vehicle (DMSO) showed no significant changes in the contraction induced by KCl (ANOVA) (Figure 3).
Addition of oxytocin (0.001 IU/mL) into the bath produced strong and more regular rhythmic uterine contractions. Both hydro-alcoholic and flavonoids rich extract of M. chamomilla in a concentration dependent   (Figure 4). The inhibitory effect of the extract was reversible following washing the tissue with fresh Tyrode's solution. No significant changes were observed in time-matched control tissue treated with vehicle (ANOVA). However, the flavonoids extract was at least 10 times more potent than the hydro-alcoholic extract. The IC 50 values are compared in the Table 1.
Addition of ACh (1µM) into the organ bath solution induced a rapid phasic response in rat uterus smooth muscle within 30 seconds contact time. Uterus response to addition of ACh, concentration dependently was attenuated by addition of flavonoids rich fraction. Inhibition of uterus response was started with 40 µg/ mL and complete inhibition was achieved with 400 µg/ mL flavonoids extract in the bath ( Figure 5). The hydroalcoholic extract of M. chamomilla reversibly inhibited uterine smooth muscle contraction induced by ACh but at bath concentration above 400 µg/mL ( Figure 5). For comparison of IC 50 see Table 1. Equivalent volume of vehicle had no significant inhibitory effect on ACh responses (ANOVA).
EFS applied via a parallel platinum weirs caused a single sharp contraction in rat isolated smooth muscle suspended in organ bath. Hydro-alcoholic extract of M. chamomilla at similar ranges of concentration which inhibited oxytocin and ACh contractions, reduced uterine response to EFS. Inhibition of tissue response was reversible. Flavonoids rich extract also attenuated tissue response to EFS but at much lower concentrations ( Figure 6). Concentrationresponse curve are shown in Figure 6 and IC 50 values are compared in Table 1. Although at higher concentration, DMSO slightly affected the uterine response to EFS, but these changes were not statistically significant (ANOVA).
Nifedipine in a concentration dependent manner inhibited uterine contraction produced by KCl, ACh, oxytocin and EFS (Figure 7).

Discussion
Myometrium is an excitable tissue which implies that its contraction is accompanied by cell membrane excitation (24). Smooth muscle excitation can arise from membrane depolarization or receptor activation (25). Current understanding of the cellular basis of uterine contractility    is that, increase in intracellular calcium ions is essential for normal uterine contractility. Calcium ions either come from extracellular fluid following arising of action potential or released from intracellular stores (26)(27)(28)(29)(30)(31)(32)(33). Following depolarization of the myometrial cell membrane, Ca 2+ influx occurs via voltage-gated Ca 2+ channels (29). Rise in Ca 2+ ions stimulate myosin light chain kinase via Ca 2+calmodulin and force rises within the myometrium (34). Addition of KCl into the organ bath, causes myometrial cell membrane depolarization and opening of voltage-   gated Ca 2+ channels (27)(28)(29)(30). Calcium ion entry in the rat uterus mostly occurs via L-type Ca 2+ channels (33). In the presence of nifedipine, a blocker of L-type Ca 2+ channels, Ca 2+ ion influx is reduced and therefore contraction is inhibited.
Oxytocin is a powerful modulator of uterus contractions. It acts on its own specific receptors on the myometrium and increases activity of phospholipase-C on the cell membrane and in turn production of inositol triphosphate (IP 3 ) is increased (35). Rise in cytosolic Ca 2+ ions results from interaction of IP 3 with specific receptors on the sarcoplasmic reticulum (SR). ACh interacts with the muscarinic M 3 receptors which exist on the myometrium and in a similar way increases uterine contraction by raising intracellular IP 3 production (32). It should be mentioned that some drugs might have more than one mechanism of action. Oxytocin, for example, also impairs Ca 2+ efflux from the cell (27). As in the case of other smooth muscles, rat uterus is innervated by autonomic nerves system manipulating both excitation and inhibition responses (36). Application of EFS produced a rapid response indicating that excitatory neurotransmitters response is prominent. Inhibition of EFS by nifedipine indicates that L-type Ca 2+ channels are involved in the process of excitation of uterus smooth muscle cells (37).
Physiological responses of uterus muscle vary at different stages of menstrual cycle (38). Therefore, to synchronize cycle time, all the rats used in these experiments were treated with estradiol. Rat uterus horns primed with estrogen exhibited spontaneous periodic contraction in vitro. This is because uterus is known as a myogenic organ, meaning that uterine smooth muscles contracts in absence of nervous or stimulating agents (27). It has been proposed that spontaneous contraction of rat uterus is associated with prostaglandin production (39).
Hydro-alcoholic extract of M. chamomilla did not affect uterus basal tension but potentiated both amplitude and frequency of spontaneous rhythmic contractions of rat isolated uterus. However, hydro-alcoholic extract at similar concentration ranges only inhibited uterine contractile responses to oxytocin, ACh, KCl and EFS. These results show that hydro-alcoholic extract M. chamomilla mainly affects myogenic induced contractions in rat uterus. This does not imply that M. chamomilla has no effect on uterus contractility induced by above spasmogens because first of all, both spasmogenic and spasmolytic effects of the extract are seen at similar concentration ranges. Therefore, it is likely that spasmogenic activity simply was not revealed. Secondly, M. chamomilla extract exhibited a relatively weak spasmogenic activity and in presence of a strong spasmogen such as oxytocin it would not make any significant difference to oxytocin response or other strong spasmogen.
Bioactivity assessment of fractions separated from hydro-alcoholic extract of M. chamomilla revealed that aqueous fraction had no effect on uterus contraction while both chloroform and flavonoids rich fractions in a concentration dependent manner inhibited uterus contraction. This indicates that the main bioactive compounds responsible for inhibitory action of M. chamomilla possessed non-polar properties. As the flavonoids rich fraction was 4 times more potent than the chloroform fraction, it is likely that the active substances may belong to flavonoids group. Apigenin and luteolin are two bioactive flavonoids with spasmolytic activities on smooth muscles (40). Both of these compounds have been identified in M. chamomilla hydro-alcoholic extract (15,16). Unlike hydro-alcoholic extract, flavonoids rich fraction did not potentiate spontaneous activity of rat isolated uterus. Therefore, it is likely that compounds responsible for stimulatory effect of M. chamomilla were not present in any significant amount in the flavonoids rich extract.

Conclusion
These finding indicate that M. chamomilla hydro-alcoholic extract contains mixtures of pharmacological active constituents. Some components possessed spasmodic activity on rat isolated uterus while others possessed spasmolytic activities. Active antispasmodic components are concentrated in the flavonoids rich fraction, suggesting that it is likely that they are a form of flavonoids. These results provide pharmacological evidence that crude extract of M. chamomilla has strong potential to enhance uterus spontaneous contractile activity and thus should be avoided in pregnancy.