Penchom Peungvicha et al/JAASP 2014; 3:299‐305

RESEARCH PAPER

In vitro uterine relaxant activity of Fire Ha Kong Recipe in rats

Penchom Peungvicha1,*, Yuvadee Wongkrajang1, Boontium Kongsaktrakoon1, Kanjana Jittiporn2 and Somsak Nualkaew3 1Department of Physiology, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand, 2Department of Cardio-thoracic Technology, Faculty of Allied Health Sciences, Naresuan University, Thailand, and 3Pharmaceutical Chemistry and Natural Product Research Unit, Faculty of Pharmacy, Mahasarakham University, Kantarawichai, Mahasarakham, 44150, Thailand

Key words: Abstract Fire Ha Kong recipe uterine contractions Fire Ha Kong recipe in a concentration range of 10- in vitro test 40 µl/ml was used to test in an in vitro uterine rat contraction inhibition. Contraction frequencies and amplitudes of the normal uterine of estrogen-primed non-pregnant rats were monitored. A decline in the Correspondence: Penchom Peungvicha frequencies of uterine contractions of 43.8 - 93.6% Department of Physiology was detected. Addition of Fire Ha Kong at the final Faculty of Pharmacy concentrations of 20-40 µl/ml inhibited the Mahidol University amplitudes of contraction by 57.1% and 447 Sri-Ayuthaya Road 85.7%, respectively. The reductions in area under Rajathevi the curve (AUC) of contractions were 57.1% and Bangkok 10400 85.7% for the added concentrations. Fire Ha Kong Thailand recipe revealed inhibitory effects on the uterine -11 E-mail: response to oxytocin (10 M) 31.9% and 29.3% [email protected] only in frequency but not in amplitude and AUC in comparison to oxytocin alone. These results indicate that the tocolytic potential of Fire Ha Kong recipe may involve oxytocin receptor-dependent pathway or reduce the intracellular availability of calcium ions.

Introduction The use of herbal medicines during relate to the notion of restoring and is energy after the child delivery common among the Asian cultures. (Kaewsarn et al, 2003). Heating is It has gained a popularity among the most simple way of restoring young highly-educated Chinese energy, and to do so, there have women (Chuang et al, 2009). Thai been various postpartum practices, traditional postpartum practices including lying by fire, taking hot

299 Pharmacology of a traditional recipe baths and drinks, taking foods with The effect of Fire Ha Kong on the ‘hot taste’ spices, etc. uterine contraction has not been well explained, though its use has Fire Ha Kong, a Thai name of one of been clinically accepted. This its traditional recipes meaning 5 explanation should provide sub- piles of fire, is composed of an equal stantial information for sustainably amount each of 5 ‘hot’ spices or hot- improving and promoting its optimal tasted herbs, i.e. dried fruits of use. Acacia concinna D.C., young seeds of black pepper (Piper nigrum Linn.), Therefore, the aim of this study was dried roots of red leadwort to investigate the effect of Fire Ha (Plumbago indica Linn.), rhizomes of Kong recipe on the uterine ginger (Zingiber officinale Roscoe.) contraction of non-pregnant rats and and alum. Fire Ha Kong has been oxytocin-induced uterine contraction on the National List of Essential in vitro. Medicines of Thailand since 2011 (Thai FDA, 2014) with an indication Materials and methods to be a herbal remedy for gynaecologic treatment. It is Animals : Female rats weighted believed to strengthen postnatal 150-160 g, were obtained from mothers, potentially to warm up the Salaya Animal Center, Mahidol bodies, clear uterine discharge and University, Nakornprathom, regain normal sizes of the uteri. Thailand. Animals were housed, in a o Pepper seeds have been shown to maintained condition at 25 ± 1 C, possess spasmolytic activity in 60% RH, a 12 h light-dark cycle, in uterus of rats (Naseri and Yahyavi, groups of 5 per cage, and 2007), aqueous extract of ginger acclimatized for with free access to rhizome could inhibit uterine smooth food and water about 1 week in the in rats (Ghayur laboratory animal room before and Gilani, 2007) but Plumbago starting the experiments. All indica showed no effect on uterine experiments were carried out in contraction and no reports about accordance to the guidelines for care Acacia concinna and Alum. and use of experimental animals by the approval of Institutional Animal Uterine contraction is one of the Care and Use Committee, Faculty of substantial indicators for parturition Pharmacy, Mahidol University or delivery. After parturition, these (PYR012/2553). contractions help the uterine muscle to continue to constrict the uterine Plant material: The plants were blood vessels, and bring about a collected as follows: dried fruits of decrease in the amount of vaginal Acacia concinna and black pepper bleeding after delivery and cause from Chanthaburi province, dried pains in post-partum periods. roots of red leadwort from Uterine contraction is also used to Suphanburi province, rhizomes of evaluate the effect of oxytocin ginger from Ratchaburi province and (Challis et al 2000), and thus could alum from herbal drug store in be used to screen the effect of a Mahasarakham province. They were herbal remedy on uterus which identified by comparison with the should be an immediate action specimens at Forest Herbarium, required for postpartum treatment. Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and

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Environments, Bangkok. The strip of the uterus was cut, tied both voucher specimens (A. concinna: ends by silk threads and bathed in O MSU.PH-AC1, P. nigrum: MSU.PH- Kreb’s solution (37 C, 95%O2/5% PN1, P. rosea: MSU.PH-PR1, Z. CO2). The bottom end of the uterus officinale: MSU.PH-ZO1) were strip was attached to an aeration deposited at the Department of tube fixed in the organ bath and other end was connected to the Pharmacognosy, Faculty of Phar- gauge coupler of the oscillograph macy, Mahasarakham University, (Gould, USA). The uterine strip was Mahasarakham, Thailand. stretched to the basal tension at 1 g. After a 30-min equilibration period, Preparation and extraction of Fire Ha Kong which was prepared in Fire Ha Kong: Dried crude samples dilutions was added and incubated of A. concinna, P. nigrum, P. rosea, for 5 min followed by 5 min resting. Z. officinale and alum, 20 g of each, During the 5-min resting period, were pulverized and mixed in a Krebs solution was used to rinse the roller mill and the pulverized mix uterus strip 3 times. Then, oxytocin was sieved through a sieve no. 80 to (10-11M) was added and incubated obtain about 100 g of fine powder of for 5 min. The frequency (number of Fire Ha Kong. A sample of Fire Ha contraction cycles in 5 min), Kong was extracted in 70% ethanol amplitude (average peak height in for 7 days, followed by condensing cm) and area under the curve (AUC and freeze-drying to obtain dried in cm2) were recorded. brown powder (a yield of about 22%). Statistical analysis: Statistical analysis of the data was performed Preparation of drugs and Fire Ha using paired Student’s t test as well Kong for in vitro testing: Oxytocin as one way ANOVA followed by (Syntocinon ampule 10 I.U/ml= 20 Tukey-Kramer multiple comparison μg/ml, Sandoz Company, test. P<0.05 was chosen as a Switzerland) was diluted to 10-11 M criterion for significance. by Kreb’s solution. Estradiol benzoate injection (China Chemical Result and discussion & Pharmaceutical, Taiwan) was used as received. Figure 1 and Table 1 revealed that addition of Fire Ha Kong to the Fire Ha Kong was dissolved in 4% organ bath at concentrations of 20- dimethyl sulphoxide (BDH, England) 40 g/ml produced significant in Kreb’s solution to obtain a stock reductions in the uterine contraction solution of Fire Ha Kong of 2 mg/ml, of estrogen-primed non-pregnant which was further diluted to 10, 20 rats in vitro. The decreases in the and 40 µg/ml by Kreb’s solution for normal frequency of contraction use. were 74.9% and 93.6%, while the reductions were 56.9% and 85.7% In vitro uterine contraction: Each for amplitude. The inhibitions in the rat was intramuscularly injected by normal AUC of uterine contraction estradiol benzoate at a dose of 1 were amounted to 57.1% and mg/kg for 24 h to induce the estrous 85.7% for the added concentrations respectively. stage, followed by sacrifice by CO2 inhalation and removal of the uterus (avoiding its stretching). A 3-cm

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Figure 1 Representative tracing record of contractions of rat uterus strips after control and treated with Fire Ha Kong at 3 final concentrations 1g

control Fire Ha Kong Fire Ha Kong Fire Ha Kong 10 g/ml 20 g/ml 40 g/ml

5min

Table 1 Mean (± SEM) of the effect of Fire Ha Kong (FHK) on rat oxytocin-induced uterine contractions (n = 5-6) Amplitude(cm) AUC (cm2) Frequency (cycles per 5 min) Control 2.67±0.51 5.39±1.54 2.67±0.52 Vehicle 2.65±0.45 5.26±1.83 2.33±0.82 (-0.7%) (-2.4%) (-2.7%) FHK 2.31±0.43 4.27±1.28 1.50±0.84* 10 g/ml (-13.5%) (-20.8%) (-43.8%) 20 g/ml 1.15±1.28**,+ 2.31±2.56*, + 0.67±0.82***,+ (-56.9%) (-57.1%) (-74.9%) 40 g/ml 0.38±0.94***,++ 0.77±1.88**, ++ 0.17±0.41***,++ (-85.7%) (-85.7%) (-93.6%) * p<0.05, ** p< 0.005, *** p< 0.001 compared to control + p<0.05, ++ p<0.005, +++ p< 0.001 compared to 10 g/ml

Figure2 and Table 2 showed that frequency of contraction as 37.9% -11 oxytocin (10 M)- induced and 29.3% as compared to the contraction of rat uterus incubated values obtained after addition of with Fire Ha Kong recipe in the oxytocin alone but no significant above added concentrations was change both in amplitude of markedly declined only in the contraction and AUC.

Figure 2 Representative tracing showed the effect of Fire Ha Kong on oxytocin-induced uterine contractions 1g

Oxytocin Oxytocin Oxytocin Oxytocin + Fire Ha Kong +Fire Ha Kong +Fire Ha Kong 10 g/ml 20 g/ml 40 g/ml

5min

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Table 2 Mean (± SEM) of the effect of Fire Ha Kong (FHK) on rat oxytocin-induced uterine contractions (n = 5-6)

Amplitude(cm) AUC (cm2) Frequency (cycles per 5 min) Oxytocin 3.04±0.37 7.62±1.20 19.33±1.97 oxytocin + FHK 3.26±0.41 7.52±1.39 16.50±4.18 (10 g/ml) (7.2%) (-1.31%) (-14.6%) oxytocin + FHK 3.15±0.52 6.85±1.27 13.17±3.87* (20 g/ml) (3.6%) (-10.1%) (-31.9%) oxytocin + FHK 3.01±0.31 6.47±1.20 13.67±3.01* (40 g/ml) (-0.99%) (-15.1%) (-29.3%) * p < 0.05 compared to oxytocin alone.

The in vitro uterine contractions availability of calcium ions depress were recorded for 3 min immediately the contractile activity of isolated after addition of oxytocin10-11M human uterine muscle (Poli, 1990). incubated with vehicle or Fire Ha Thus, Fire Ha Kong recipe might Kong in the organ bath followed by reduce the intracellular availability of three times washing with Krebs calcium ions in . solution during 5 min rest period. Oxytocin, a hormone involved in It is well-known that estrogen numerous physiologic processes, stimulates proliferation in the plays a central role in the myometrium, accomplishing the mechanisms of parturition and considerable myometrium growth lactation. It acts through its that is necessary for the forceful receptor, which belongs to the G- contractions of labor. Thus, the protein-coupled receptor estrogen-primed uterus is a good superfamily, while Gq/phospholipase model for the research about uterine C (PLC)/inositol 1,4,5-triphosphate contractility in the non-pregnancy (IP3) is the main pathway via which condition. Data of the present study it exerts its action in the showed that Fire Ha Kong recipe myometrium (Vrachnis et al, 2011). produced marked inhibition in the It is fundamentally known that spontaneous uterine contraction of oxytocin-induced uterine contraction estrogen-primed non-pregnant rats is mediated through a typical class in frequency, amplitude and area of G protein-coupled receptor which under the curve which support the is primarily coupled via Gq proteins medicinal use of the formula. The to phospholipase c-beta (Kiss and results are in agreement with those Mikkelsen, 2005). Moreover obtained by PSU report 2010 that oxytocin-induced uterine contraction Fire Ha Kong recipe could reduce is attributed to an increase of frequency of uterine contraction at cytosolic free calcium originating at doses of 0.1-10 g/ml but not from inositol triphosphate (IP3)- amplitude of contraction. In this sensitive intracellular stores and study the dose range was increased calcium influx via receptor-operated to 40 g/ml and showed more effect channels (Phillippe and Chien, 1995; in amplitude of contraction. Trujillo et al., 2000). Fire Ha Kong Spontaneous uterine contraction is recipe also revealed the inhibitory closely dependent on the effects on the uterine response to cytoplasmic concentration of free oxytocin (10-11M) only in frequency calcium ions (Bolton, 1979) and but not in amplitude and AUC in drugs that reduce the intracellular comparison to oxytocin alone.

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