Effect of Alpinia officinarum Hance alcohol extracts on primary dysmenorrheal

Ling Huang， Jun-Qing Zhang， You-Bin Li， Mi Liu， Hui-Ming Deng， Yu-Chao Luo， Yin-Feng Tan， Jie Hou， Gui-Wei Yao， Wei-Wei Guan

School of Pharmaceutical Sciences， Hainan Medical University， Haikou 571101， Hainan， China

Effect of Alpinia officinarum Hance alcohol extracts on primary dysmenorrheal

Ling Huang， Jun-Qing Zhang， You-Bin Li， Mi Liu， Hui-Ming Deng， Yu-Chao Luo， Yin-Feng Tan， Jie Hou， Gui-Wei Yao， Wei-Wei Guan✉

School of Pharmaceutical Sciences， Hainan Medical University， Haikou 571101， Hainan， China

ARTICLE INFO

Article history:

Received 15 May 2016

Received in revised form 16 June 2016

Accepted 1 July 2016

Available online 20 September 2016

Alpinia officinarum Hance alcohol

Objective: To study the effect of Alpinia officinarum Hance （A. officinarum） 80% alcohol extract on the primary dysmenorrhea. Methods: A. officinarum 80% alcohol extract were enriched by macroporous adsorption resins. Female mice of primary dysmenorrhea model were established by oxytocin induction； the ef f ects of A. officinarum 80% alcohol extract on primary dysmenorrhea were observed by body twist method； and the homogenate level of prostaglandin F2α（PGF2α）， prostaglandin E2（PGE2） and Ca2+in the uterus were observed in oxytocin-induced female mice. Results: The writhing frequency of primary dysmenorrhea mice was signif i cantly decreased after treatment of A. officinarum 80% alcohol extract and the level of PGF2α， PGE2and Ca2+in mice uterus was signif i cantly decreased （P＜0.05， P＜0.01）in groups of mice treated with middle and high dosage of A. officinarum 80% alcohol extract compared with that of model group. Conclusions: These fi ndings suggest that A. Officinarum 80% alcohol extract can signif i cantly relieve primary dysmenorrhea.

1. Introduction

Primary dysmenorrheal， which is a common gynecological problem， is defined as cramping pain in the lower abdomen occurring just before or during menstruation， and it occurs in up to 50% of menstruating females and causes signif i cant disruption in quality of life［1-4］. Principal pharmacological therapies for primary dysmenorrheal include nonsteroidal anti-inflammatory drugs （NSAIDs）［4-7］ or oral contraceptive pills （OCPs）［8，9］. NSAIDs reduce myometrial activity by inhibiting prostaglandin（PG） synthesis and reducing vasopressin secretion. However， the failure rate of NSAIDs is often 20% to 25%. Besides， NSAIDs havelong-term adverse ef f ects involving disorders of the liver， kidney，and digestive systems［10］. OCPs suppress ovulation and thin the endometrial lining which reduces menstrual fl uid volume along with the amount of PG produced， thus reducing the pain associated with uterine contractions［11］. However， OCPs can cause side ef f ects including nausea and water retention and may not be suitable for all women， especially those pursuing pregnancy［12，13］.

Because of these limitations of conventional treatments， herbal medicines are considered as feasible alternatives for the treatment of pain or primary dysmenorrhea［14，15］. Herbal medicines used for treating pain or primary dysmenorrhea have been used for long time in China， and currently these therapies are increasingly being used worldwide［16，17］. Herbal medicines are relatively well tolerated by patients because of fewer adverse ef f ects and lower recurrence rates associated with them. During the last few decades， an increasing number of preclinical studies investigating the effi cacy of herbal medicines in cell and animal models for primary dysmenorrhea have been published.

Alpinia officinarum Hance （A. officinarum） is a perennial medicinalplant that is mainly distributed in the tropical and subtropical regions of Southeast Asia. It is commonly used as a food additive，and a kind of Chinese medicinal material， and has been listed as Affinal Drug and Diet by National Health and Family Planning Commision of the People's Republic of China due to its high safety. The most medicinally active part of the plant is the rhizome，which has important medical value. The rhizomes of A. officinarum are widely used in China for relieving stomach aches， treating colds， invigorating the circulatory system， reducing swelling and invigorating blood circulation and antiemetic［18-21］.

Nevertheless， there are little studies that have addressed the pharmacological ef f ects of A. officinarum on impairment in primary dysmenorrhea. Taking the above into consideration， this study aims to clarify the pharmacology ef f ects of A. officinarum ef f ective parts on impairment in pain perception and dysmenorrhea， and provide theoretical basis for the industrialization development of A. officinarum rhizome.

2. Materials and methods

2.1. Experimental animals

Female BALB/c mice （25±5 g， 8-10 week old） were purchased from Medical Experimental Animal Center of Hunan Province and kept in a room at 22-24 ℃ with alight/dark cycle of 12/12 h and 55%-60% relative humidity in Experimental Animal Center of Hainan Medical University. They had free access to standard rodent chow and clean tap water. All procedures of animal experiments in this study were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People’s Republic of China. The design of animal experiment was approved by Experimental Animal ethics review committee of Hainan Medical University.

2.2. Main instrument

AB-8 macroporous absorptive resin was purchased from Bohong Resin Technology Co. Ltd （Shanghai， China）； Waters alliance 2695 high-performance liquid chromatography was sourced from Waters Science and Technology Co. Ltd （Milford， USA）； YLS-6B hot plate gauge was purchased from Jinan Yiyan Technology Development Co. Ltd （Shandong， China）.

2.3. Chemicals and reagents

The dried root of A. officinarum was purchased from Xuwen A. officinarum planting base （Xuwen， China）； aspirin was purchased from Bayer AG （Leverkusen， Germany）； diethylstilbestrol and oxytocin was purchased from Dalian Meilun Technology Co. Ltd，（Shenyang， China）.

2.4. A. officinarum 80% alcohol extracts preparation

A total of 10 kg dried root of A. officinarum was identified by associate professor Jianping Tian from School of Pharmacy， Hainan Medical University （Haikou， China）. After crushing， A. officinarum powder was ref l uxing extracted by 12 times volume of water for 2 h，which was repeated twice. The 2 times extraction were fi ltrated and combined， then A. officinarum aqueous extract were obtained， the aqueous extract was subjected to macroporous resin AB-8 column chromatography， then were eluted with 80% alcohol successively，the 80% eluent were collected and concentrated by vacuum drying to obtain the 80% alcohol extracts. A fl avonoids and diarylheptanoids enriched extract （280 g） were collected after the 80% alcohol eluent were concentrated by vacuum drying， the total content of fl avonoids and diarylheptanoids were more than 65% measured by HPLC.

2.5. Establishment of mice dysmenorrhea model

A total of 50 Balb/c mice were intragastric administrated with diethylstilbestrol （0.8 mg/mL， 0.1 mL/ 10 g wt） for seven days to establish primary dysmenorrhea models， six hours after medication of diethylstilbestrol from the seventh day， 50 Balb/c mice were randomly divided into 5 groups with 10 mice in each group and administrated with aspirin 0.5 g/kg （positive control group）； normal saline （model group）； A. officinarum 80% alcohol extracts low dose group （crude drugs 0.75 g/kg）， medium dose group （crude drugs 3.00 g/kg）， high dose group （crude drugs 12.00 g/kg） for another seven days. Other 10 Balb/c mice which were intragastric administrated with normal saline for 13 d （0.1 mL/ 10g wt） were considered as normal control group， each group of fi nal volume of administration was 0.1 mL/10 g wt. On the 13th day， each group of mice were treated with intraperitoneal injection of oxytocin 33U/ kg to induce the writhing reaction after administration with A. officinarum extracts［22，23］.

2.6. Writhing test of A. officinarum 80% alcohol extracts on primary dysmenorrhea

Writhing test were used to observe the ef f ects of A. officinarum extracts on primary dysmenorrhea mice. On the 13th day， each group of mice were treated with intraperitoneal injection of oxytocin 33U/ kg to induce writhing reaction 30 min after administration with A. officinarum 80% alcohol extract， then the number of writhing was observed. Mice were placed in a box， and the number of writhingwas counted for the next 30 min. The writhing was characterized by abdominal contraction， stretching or bending of the body， trunk and/ or pelvis ending with limbs extension［24］.

Analgesia percentage = （writhing number of model group -writhing number of medicated groups/writhing number of model group） ××100%.

2.7. Preparation of uterine smooth muscle tissue homogenate

Injection of oxytocin in mice after 1 h， intraperitoneal injection of chloral hydrate anesthesia， open the abdominal cavity， and the uterine tissues were then dissected for further investigation. The uterine tissues were homogenized in 9 volumes of normal saline. Following centrifugation at 3 000 rpm for 15 min at 4 ℃， the supernatant was used to measure the levels of PGF2α， PGE2and Ca2+.

2.8. PGF2α， PGE2and Ca2+enzymelinked immunosorbent assay

The supernatant abtained from 2.7 was used to measure the levels of PGF2α， PGE2and Ca2+in uterine smooth muscle tissue of mice by enzyme linked immunosorbent assay （ELISA） in accordance with manufactures instructions［25-27］.

2.9. Statistical analysis

The results were expressed as mean±SD Statistical comparisons were made using Student’s t-test and the chosen level of signif i cance was P＜0.05.

3. Results

3.1. Writhing test of A. officinarum extracts on primary dysmenorrhea

Mice models of primary dysmenorrhea which were induced by oxytocin were established to investigate the ef f ects of A. Officinarum 80% alcohol extract for relieving dysmenorrhea symptoms. As shown in Table 1， the model group mice all showed body writhing reaction compared with normal control group indicating that the primary dysmenorrhea model was successfully established. Compared with model group， the writhing number of positive control group were signif i cantly reduced， and the analgesia percentage was 84.00% （P＜0.01）. The mice writhing numbers were significantly decreased by A. officinarum 80% alcohol extract low， medium and high dose group， and their analgesia percentage were 59.22%，61.87% and 84.35% respectively （P＜0.05， P＜0.01）.

3.2. PGF2αlevels of oxytocin-induced writhing mice

The synthesis and release of PGs increase is one of the important reasons of cause primary dysmenorrhea， and this study tested the ef f ect of A. officinarum 80% alcohol extract on the PG level of the dysmenorrhea model mice uterus. As shown in table 1， compared with the normal control group， the level of uterus tissue PGF2αwas signif i cantly increased in model group； compared with model group， the PGF2αlevel of uterus tissue was signif i cantly decreased in positive control group （P＜0.05）； PGF2ααlevel of uterine tissue was decreased in some degree after administrated with A. officinarum 80% alcohol extract all dose group （P＜0.05）.

3.3. PGE2levels of oxytocin-induced writhing mice

As shown in table 1， compared with the normal control group， the level of uterus tissue PGE2in model group mice was signif i cantly increased； compared with model group， the PGE2level was significantly decreased in positive control group uterus tissue（P＜0.05）， the uterine tissues PGE2level was signif i cantly reduced by A. officinarum 80% alcohol extract high dose group （P＜0.05）.

3.4. Ca2+levels of oxytocin-induced writhing mice

As shown in table 1， compared with the normal control group，the level of Ca2+in uterus tissue was significantly increased in model group （P＜0.01）； compared with model group， the Ca2+levelwas signif i cantly decreased in positive control group uterus tissue（P＜0.01）， and the uterine tissues Ca2+level were significantly reduced by A. officinarum 80% alcohol extract high dose group（P＜0.01）.

Table 1Ef f ects of A. officinarum 80% alcohol extract on writhing reaction， analgesia of primary dysmenorrheal model mice， and levels of PGF2αα， PGE2and Ca2+in their uterine tissue （n=10）.

4. Discussion

Primary dysmenorrhea is def i ned as a cramp-like pain in the lower abdomen before or during menstruation without any identif i able pelvic pathology， it may be accompanied by lower back pain，nausea， vomiting， and diarrhea and it is frequently found in young nullipara［28］. Modern medical science holds that the emergence of primary dysmenorrhea is related with many factors. Among them，temporary ischemia of myometrium and endometrium of the uterus plays an important role. The ischemia may result from pressured intermuscular blood vessels induced by forceful contraction of uterine arteries and paroxysmal contraction of uterine smooth muscles［29］. Traditional Chinese Medicine thinks that primary dysmenorrhea is often caused by blood stasis blocking the uterus. In some previous studies， A. officinarum showed the ef f ects of regulating the contractive amplitude， frequency， and mobility of gastrointestinal smooth muscle and improving microcirculation. Therefore， we hypothesized that A. officinarum 80% alcohol extracts perhaps could alleviate dysmenorrhea. We established the dysmenorrhea mice models by administation of diethylstilbestrol and oxytocin to further study the ef f ects of A. officinarum on dysmenorrhea.

After administrated with diethylstilbestrol and oxytocin， the abdominal contraction， stretching or bending of the body， trunk and pelvis ending with limbs extension were observed in all model mice suggesting that mouse dysmenorrhea model was established successfully. The dysmenorrhea latency was remarkably increased by A.offi cinarum 80% alcohol extracts， while after administration of A. officinarum in each group， the body writhing reaction caused by oxytocin were decreased. The analgesia ef f ect of A. officinarum 80% alcohol extract high dose group was even better than aspirin.

COX enzymes are involved in inflammatory pathways and responsible for formation of pro-inf l ammatory PGs. PGs which can cause constriction in uterine smooth muscle and sensitize spinal neurons to pain are regarded as the most pain factor to primary dysmenorrohea［30］. PGF2αand PGE2， two naturally occurring PG， are used to induce labor in medicine. PGF2ααand PGE2can combined with their receptor on the spiral arterioles increased uterine contractility， resulting in ischemia pain［31］. In the present study， it was found that the PGF2αand PGE2levels in uterus were all signif i cantly decreased after treatment with A. officinarum 80% alcohol extract medium and high groups.

The levels of Ca2+in the dysmenorrheal model mice uterus were also detected. Overload of intracellular Ca2+can cause uterine smooth muscle contraction and reduce the endometrial blood supply，leading to the occurrence of dysmenorrhoea. Therefore， calcium channel blocking agents decrease myometrial contractility and beneficial in cases of dysmenorrhea［32，33］. The treatment of A. officinarum 80% alcohol extract， the medium and high doses groups，all resulted in the signif i cant decrease of Ca2+levels. This indicated that one mechanism of A. officinarum 80% alcohol extracts treating dysmenorrhea may act on Ca2+channel to decrease intracellular Ca2+concentration. The above results provide theoretical evidence that medicines extracted from A. officinarum 80% alcohol extract could be produced to treat dysmenorrhea in clinic.

In conclusion， the results of the current study provide evidence that A. officinarum might be effective for treating primary dysmenorrhea. Considering the advantages of better tolerability，shorter contraindication list and lower incidence of side effects，A. officinarum 80% alcohol extract might be considered as an alternative agent of NSAIDs and analgesic in treatment of primary dysmenorrhea.

Conflict of interest statement

We declare that we have no conf l ict of interest.

Acknowledgments

This study was supported by the key project supported of Hainan Province（Grant no. ZDZX2013008） and Natural Science Foundation of China （Grant No. 2014 81403006）.

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10.1016/j.apjtm.2016.07.012

Ling Huang， Ph.D， Associate Professor， School of Pharmaceutical Sciences， Hainan Medical University， Haikou 571101， Hainan， China.

E-mail: jqzhang2011@163.com

Tel: 0898-66891789

✉Corresponding author: Wei-Wei Guan， School of Pharmaceutical Sciences， Hainan Medical University， Haikou 571101， Hainan， China.

E-mail: puer6@163.com

Tel: 86-898-66893460

Foundation Project: This study was supported by the key project supported of Hainan Province （Grant No. ZDZX2013008） and Natural Science Foundation of China （Grant No. 2014 81403006）.

extracts

Relieve

Primary dysmenorrhea