BIOLOGICAL ACTIVITIES OF THAI TRADITIONAL REMEDY CALLED PRASACHANDAENG AND ITS PLANT INGREDIENTS

BY

MISS ALISA SANGPHUM

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN APPLIED THAI TRADITIONAL MEDICINE FACULTY OF MEDICINE THAMMASAT UNIVERSITY ACADEMIC YEAR 2016 COPYRIGHT OF THAMMASAT UNIVERSITY

Ref. code: 25595611031526QZI BIOLOGICAL ACTIVITIES OF THAI TRADITIONAL REMEDY CALLED PRASACHANDAENG AND ITS PLANT INGREDIENTS

BY

MISS ALISA SANGPHUM

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN APPLIED THAI TRADITIONAL MEDICINE FACULTY OF MEDICINE THAMMASAT UNIVERSITY ACADEMIC YEAR 2016 COPYRIGHT OF THAMMASAT UNIVERSITY

Ref. code: 25595611031526QZI

(1)

Thesis Title BIOLOGICAL ACTIVITIVES OF THAI TRADITIONAL REMEDY CALLED PRASACHANDAENG AND ITS PLANT INGREDIENTS Author Miss Alisa Sangphum Degree Master of Science Major/Faculty/University Applied Thai Traditional Medicine Faculty of Medicine Thammasat University Thesis Advisor Associate Professor Arunporn Itharat, Ph.D. Academic Years 2016

ABSTRACT

Prasachangdaeng remedy (PC) has long been used for relief of fever and internal heat. It is composed with 12 plants as Chan Daeng (Dracaena loureiri Gagnep.) stems, Mueng Kon (Helicia terminalis Kurz.) roots, Ma Now (Citrus aurantifolia Swing.) roots, Ma Prang Wan (Bouea macrophylla Griff.) roots, Proh Horm (Kaempferia galanga L.) rhizome, Kot Hua Bua (Ligusticum chuanxiong Hort.) rhizome, Chan Tet (Myristica fragrans Houtt.) stems, Fang Saeng (Caesalpinia sappan L.) stems, Bua Luang (Nelumbo nucifera Gaertn.) pollens, Boon Ngak (Mesua ferrea L.) flowers, Sa Ra Pee (Mammea siamensis Kosterm.) flowers, and Ma Li (Jasminum sambac L.) flowers. It is in the National List of Essential Medicines in Thailand, but there is no report for anti-inflammatory activity of this remedy. Some plant ingredients in this remedy were investigated for antibacterial and anti-inflammatory activities but there is no report of PC and some plant ingredients in PC remedy. Thus, the objectives of this research were to study on antibacterial and anti-inflammatory activities of PC remedy extracts by different extraction method and its plant ingredients. Raw material of its plant ingredients from PC remedy were tested for standardization following Thai Herbal Pharmacopoeia (THP). All plant ingredients of PC remedy were accepted by standard values. PC remedy was also accepted by THP

Ref. code: 25595611031526QZI (2) criteria (the moisture content of PC remedy was 6.74±0.67%, total ash as 5.69±0.08% and acid insoluble ash as 1.78±0.03%). PC remedy was extracted by different methods by maceration in 95% ethanol and decoction. The yields of PC ethanolic extracts (PCE) were higher than water extract (PCW) (17.28% and 3.35%, respectively). Each plant ingredients were also extracted by 95% ethanol. The ethanolic extract of Dracaena loureiri Gagnep. stems exhibited the highest yield and Myristica fragrans Houtt. stems exhibited the lowest yield (22.76% and 1.24%, respectively). For decoction; Ligusticum chuanxiong Hort. Rhizomes water extract exhibited the highest yield and Myristica fragrans Houtt. stems exhibited the lowest yield (27.29% and 0.85%, respectively) Antibacterial activity were evaluated by disc diffusion method to determine the inhibition zone and by using microtitre plate-based antibacterial assay to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values and tested against three of gram positive bacteria; Staphylococcus aureus (ATCC 25923), Staphylococcus aureus MRSA (DMST 2065) and Streptococcus pyogenes (ATCC 19615) and four types of gram negative bacteria; Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Salmonella typhimurium (DMST 22842) and Shigella dysenteriae (DMST 151161). The PCE and PCW showed antibacterial activity against S.aureus MRSA (MIC = 0.312 mg/ml, MBC = 2.5 mg/ml and MIC = 1.25 mg/ml, MBC = 1.25 mg/ml, respectively). The inhibition zones of all plant extracts were in the range of 7 to 26 mm. The ethanolic and water extracts of Caesalpinia sappan L. stems showed the largest inhibition zone against S.aureus MRSA as 26 and 20 mm., respectively. The ethanolic extract of Mammea siamensis Kosterm. flowers exhibited the best gram positive bacteria activity against S.aureus MRSA with MIC and MBC values of 0.004 and 0.019 mg/ml., respectively. The ethanolic extract of Caesalpinia sappan L. stems exhibited the best inhibitory gram negative bacteria activity against S.dysenteriae with MIC and MBC values of 0.156 and 0.156 mg/ml. Most of water extracts of PC remedy and its plant ingredients had no antibacterial activity against all strains. Neither ethanolic nor water extracts of PC remedy and its plant ingredients were able to inhibit gram negative K. pneumonia. except, the ethanolic and water extracts of Caesalpinia sappan L. stems exhibited the inhibitory gram negative bacteria activity against K. pneumonia. with

Ref. code: 25595611031526QZI (3)

MIC value = 1.25 mg/ml, MBC value = 5 mg/ml, and MIC value = 2.5 mg/ml, MBC value = 5 mg/ml., respectively. Anti-inflammatory activity was evaluated by inhibition Nitric Oxide (NO) production which induced by Lipopolysaccharide (LPS) from murine macrophages cell lines (RAW264.7). The results found that the PCW showed better anti-inflammatory activity than PCE (IC50 value of 16.87±2.51 and 39.70±1.48 µg/ml.,respectively). The ethanolic and water extracts of Caesalpinia sappan L. stem showed the highest anti- inflammatory activity with IC50 value 5.42±0.24 µg/ml and 7.60±0.28 µg/ml, respectively. The stability test by accelerated condition (40ºC, 75%RH for 6 months) of Prasachandaeng extract found that the water extract of PC remedy was stable in anti- inflammatory activity by inhibitory NO production from RAW264.7 cell lines for at least 8 months. In conclusion, the PCE showed good antibacterial activities while the PCW showed good anti-inflammatory activity. The ethanolic extract of Mammea siamensis Kosterm. flowers showed the effective antibacterial activities against MRSA and the ethanolic (FSE) and water (FSW) extract of Caesalpinia sappan L. stems showed the effective anti-inflammatory activity. Furthermore, the FSW and FSE also showed good antibacterial activities. Thus, these results support to use the PCE for treat infection fever and MRSA infection and using PCW for treat fever from inflammation. Thus, these results are related on using PC in Thai Traditional Medicine scripture.

Keywords: Prasachandaeng, Thai Traditional remedy, Antibacterial, Anti- inflammatory

Ref. code: 25595611031526QZI (4)

หัวข้อวิทยานิพนธ์ ฤทธิ์ทางชีวภาพของต ารับยาไทยชื่อยาประสะจันทน์แดงและ สมุนไพรในต ารับ ชื่อผู้เขียน นางสาวอลิษา แสงพุ่ม ชื่อปริญญา วิทยาศาสตรมหาบัณฑิต สาขา/คณะ/มหาวิทยาลัย สาขาวิชาการแพทย์แผนไทยประยุกต์ คณะแพทยศาสตร์ มหาวิทยาลัยธรรมศาสตร์ อาจารย์ที่ปรึกษาวิทยานิพนธ์ รองศาสตราจารย์ ดร.อรุณพร อิฐรัตน์ ปีการศึกษา 2559

บทคัดย่อ

ต ารับยาประสะจันทน์แดงเป็นต ารับยาสมุนไพรไทย ซึ่งมีการใช้รักษาอาการไข้ตัว ร้อน และบรรเทาอาการร้อนใน กระหายน ามายาวนาน ประกอบด้วยสมุนไพรทั งหมด 12 ชนิด ได้แก่ แก่นจันทน์แดง (Dracaena loureiri Gagnep.), รากเหมือดคน (Helicia terminalis Kurz.), ราก มะนาว (Citrus aurantifolia Swing.), รากมะปรางหวาน (Bouea macrophylla Griff.), หัว เปราะหอม (Kaempferia galanga L.), โกฐหัวบัว (Ligusticum chuanxiong Hort.), แก่น จันทน์เทศ (Myristica fragrans Houtt.), แก่นฝางเสน (Caesalpinia sappan L.), เกสรบัวหลวง (Nelumbo nucifera Gaertn.), ดอกบุนนาค (Mesua ferrea L.), ดอกสารภี (Mammea siamensis Kosterm.) และดอกมะลิ (Jasminum sambac L.) ต ารับยาประสะจันทน์แดงเป็นต ารับ ที่อยู่ในบัญชียาหลักแห่งชาติ แต่อย่างไรก็ตามต ารับยาประสะจันทน์แดงนั น ยังไม่มีรายงานผลการวิจัย ถึงฤทธิ์ต้านการอักเสบ ในส่วนของสมุนไพรในต ารับบางชนิด มีงานวิจัยถึงฤทธิ์ต้านเชื อแบคทีเรียและ ฤทธิ์ต้านการอักเสบ แต่สมุนไพรบางตัวในต ารับไม่มีรายงานวิจัยถึงฤทธิ์ดังกล่าว ดังนั นวัตถุประสงค์ ของการวิจัยนี เพื่อศึกษาถึงฤทธิ์ต้านเชื อแบคทีเรียและฤทธิ์ต้านการอักเสบของสารสกัดด้วยเอทานอล และสารสกัดด้วยน าของต ารับยาประสะจันทน์แดงและสมุนไพรไทยในต ารับ ต ารับยาประสะจันทน์แดงและสมุนไพรในต ารับแต่ละชนิดผ่านเกณฑ์มาตรฐานการ ควบคุมคุณภาพมาตรฐานยาสมุนไพรไทย โดยต ารับยาประสะจันทน์แดงผ่านเกณฑ์มาตรฐาน ดังต่อไปนี ค่าบ่งชี ปริมาณน า 6.74±0.67% ปริมาณเถ้าทั งหมด 5.69±0.08% และปริมาณเถ้าที่ไม่ ละลายในกรด 1.78±0.03% ต ารับยาประสะจันทน์แดงและสมุนไพรแต่ละชนิด น ามาสกัดด้วยวิธีการหมักด้วยเอ ทานอลความเข้มข้น 95% และสกัดด้วยวิธีการต้มน า สารสกัดด้วยเอทานอลได้ผลผลิตร้อยละมากกว่า

Ref. code: 25595611031526QZI (5)

สารสกัดด้วยน า เท่ากับ 17.28% และ 3.35% ตามล าดับ สมุนไพรแต่ละชนิดที่สกัดด้วยเอทานอล ความเข้นข้น 95% พบว่าแก่นจันทน์แดงได้ผลผลิตร้อยละมากที่สุด และแก่นจันทน์เทศได้ผลผลิตร้อย ละน้อยที่สุดเท่ากับ 22.76% และ 1.24% ตามล าดับ ในส่วนของสมุนไพรที่สกัดด้วยการต้มน า พบว่า โกศหัวบัวได้ผลผลิตร้อยละมากที่สุด และแก่นจันทน์เทศได้ผลผลิตร้อยละน้อยที่สุด เท่ากับ 27.29% และ 0.85% ตามล าดับ การศึกษาฤทธิ์ต้านแบคทีเรียด้วยวิธี Disc diffusion เพื่อหาเส้นผ่าศูนย์กลางของ Inhibition Zone และวิธี Microtitre plate-based antibacterial assay เพื่อหาค่าความเข้มข้น ต่ าสุดที่สามารถยับยั งการเจริญเติบโตของเชื อแบคทีเรีย (MIC) และค่าความเข้มข้นต่ าสุดที่สามารถ ฆ่าเชื อแบคทีเรีย (MBC) โดยทดสอบกับเชื อแบคทีเรียแกรมบวก 3 สายพันธุ์ คือ Staphylococcus aureus (ATCC 25923), Staphylococcus aureus MRSA (DMST 2065) และ Streptococcus pyogenes (ATCC 19615) และเชื อแบคทีเรียแกรมลบ 4 สายพันธุ์ คือ Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 700603), Salmonella typhimurium (DMST 22842) และ Shigella dysenteriae (DMST 151161) พบว่าสารสกัดด้วยเอทานอลและสารสกัดด้วยน าของต ารับประสะจันทน์แดง มีฤทธิ์ในการยับยั งเชื อ แบคทีเรียและฆ่าเชื อแบคทีเรีย S. aureus MRSA โดยมีค่า MIC เท่ากับ 0.312 มิลลิกรัมต่อมิลลิลิตร , MBC เท่ากับ 2.5 มิลลิกรัมต่อมิลลิลิตร และ MIC เท่ากับ 1.25 มิลลิกรัมต่อมิลลิลิตร, MBC เท่ากับ 1.25 มิลลิกรัมต่อมิลลิลิตรตามล าดับ โดยสารสกัดพืชสมุนไพรเดี่ยวทั งหมดมีค่าเส้นผ่าศูนย์กลางของ Inhibition Zone อยู่ในช่วง 7-26 มิลลิมตร สารสกัดด้วยเอทานอลและสารสกัดด้วยน าของแก่นฝาง เสนมีค่าเส้นผ่าศูนย์กลางของ Inhibition Zone ในการยับยั งเชื อแบคทีเรีย S. aureus MRSA กว้าง ที่สุดคือ 26 และ 20 มิลลิเมตร ตามล าดับ สารสกัดด้วยเอทานอลของดอกสารภี พบว่ามีฤทธิ์ต้านเชื อ แบคทีเรียแกรมบวกดีที่สุดต่อเชื อ S. aureus MRSA โดยมีค่า MIC และ MBC เท่ากับ 0.004 และ 0.019 มิลลิกรัมต่อมิลลิลิตรตามล าดับ สารสกัดด้วยเอทานอลของแก่นฝางเสน พบว่ามีฤทธิ์ต้านเชื อ แบคทีเรียแกรมลบดีที่สุดต่อเชื อ S. dysenteriae โดยมีค่า MIC และ MBC เท่ากับ 0.156 และ 0.156 มิลลิกรัมต่อมิลลิลิตรตามล าดับ สารสกัดด้วยน าของต ารับยาประสะจันทน์แดงและสมุนไพร เดี่ยวแต่ละชนิด พบว่าส่วนใหญ่ไม่มีฤทธิ์ในการต้านเชื อแบคทีเรียทั งหมด ทั งสารสกัดด้วยเอทานอล และสารสกัดด้วยน าของต ารับยาประสะจันทน์แดงและสมุนไพรเดี่ยวแต่ละชนิดไม่มีฤทธิ์ต้านเชื อ แบคทีเรีย K. pneumonia ยกเว้นสารสกัดด้วยเอทานอลและสารสกัดด้วยน าของแก่นฝางเสน พบว่า มีฤทธิ์ต้านเชื อแบคทีเรีย K. pneumonia โดยมีค่า MIC เท่ากับ 1.25 มิลลิกรัมต่อมิลลิลิตร, MBC เท่ากับ 5 มิลลิกรัมต่อมิลลิลิตร และ MIC เท่ากับ 2.5 มิลลิกรัมต่อมิลลิลิตร, MBC เท่ากับ 5 มิลลิกรัม ต่อมิลลิลิตรตามล าดับ

Ref. code: 25595611031526QZI (6)

การทดสอบฤทธิ์ต้านการอักเสบโดยการดูการยับยั งการสร้าง Nitric oxide จาก เซลล์ RAW264.7 เมื่อถูกกระตุ้นด้วย LPS พบว่าสารสกัดด้วยน าของต ารับยาประสะจันทน์แดงมี

ฤทธิ์ต้านการอักเสบมากกว่าสารสกัดด้วยเอทานอล โดยมีค่า IC50 เท่ากับ 16.87±2.51 ไมโครกรัมต่อ มิลลิลิตร และ 39.70±1.48 ไมโครกรัมต่อมิลลิลิตรตามล าดับ สารสกัดด้วยเอทานอลและสารสกัดด้วย

น าของแก่นฝางเสน พบว่ามีฤทธิ์ต้านการอักเสบที่ดีที่สุด โดยมีค่า IC50 เท่ากับ 5.42±0.24 ไมโครกรัม ต่อมิลลิลิตร และ 7.60±0.28 ไมโครกรัมต่อมิลลิลิตรตามล าดับ การศึกษาความคงตัวของสารสกัดด้วยน าต ารับยาประสะจันทน์แดงภายใต้สภาวะ เร่งที่อุณหภูมิ 40±2 องศาเซลเซียส ความชื นสัมพันธ์ 75±5 เปอร์เซ็นต์ นาน 6 เดือน เมื่อน าสารสกัด ด้วยน าของต ารับยาประสะจันทน์แดงมาทดสอบฤทธิ์ต้านการอักเสบ พบว่ามีความคงตัวประมาณ 8 เดือน จากผลการทดลอง สรุปว่าสารสกัดด้วยเอทานอลของต ารับยาประสะจันทน์แดงมี ฤทธิ์ต้านเชื อแบคทีเรียได้ดี ในขณะที่สารสกัดด้วยน าของต ารับยาประสะจันทน์แดงมีฤทธิ์ต้านการ อักเสบได้ดี สารสกัดด้วยเอทานอลของดอกสารภี พบว่ามีประสิทธิภาพในการต้านเชื อแบคทีเรีย S. aureus MRSA และสารสกัดด้วยเอทานอลกับสารสกัดด้วยน าของแก่นฝางมีประสิทธิภาพในการ ต้านการอักเสบและยังมีฤทธิ์ต้านเชื อแบคทีเรียได้ดีอีกด้วย จากผลการทดลองที่ได้กล่าวข้างต้นนั น สามารถน ามาสนับสนุนการใช้สารสกัดด้วยเอทานอลของต ารับยาประสะจันทน์แดงในการรักษา อาการไข้ที่เกิดจากการอักเสบและการติดเชื อที่เกิดจากเชื อแบคทีเรีย S. aureus MRSA และการใช้ สารสกัดด้วยน าของต ารับยาประสะจันทน์แดงในการรักษาอาการไข้หวัดที่เกิดจากการอักเสบ ผลการ ทดลองข้างต้นนี มีความสอดคล้องกันกับคัมภีร์แผนไทย

ค าส าคัญ: ประสะจันทน์แดง, ต ารับยาไทย, ฤทธิ์ต้านเชื อแบคทีเรีย, ฤทธิ์ต้านการอักเสบ

Ref. code: 25595611031526QZI (7)

ACKNOWLEDGEMENTS

This study was completed successfully by research fund of faculty of Medicine, Thammasat University. My gratefully thankfulness to my major advisor, Associate Professor Dr.Arunporn Itharat for her good advice, counsel, support and encouragement for this successful research and thesis writing during my all study in faculty of Medicine, Thammasat University. Thank you to Associate Professor Sukanya Jesadanont, Faculty of Pharmacy, Chulalongkorn University, Dr.Srisopa ruangnoo, Faculty of Medicine, Thammasat University and Assistant Professor Rathapon Asasutjarit, Faculty of Pharmacy, Thammasat University for their useful comments. Special thanks to Mr.Norman Mungall for checking and helping the sentences in this thesis. I am grateful my friends and staffs in Faculty of Medicine, Thammasat University for their good help, support and suggestion to me. Finally, the most important is my lovely parent. I love their so much to give opportunity for me to study in Master Degree.

Miss Alisa Sangphum

Ref. code: 25595611031526QZI (8)

TABLE OF CONTENTS

Page ABSTRACT (IN ENGLISH) (1)

ABTRACT (IN THAI) (4)

ACKNOWLEDGEMENTS (7)

LIST OF TABLES (12)

LIST OF FIGURES (13)

LIST OF ABBREVIATIONS (15)

CHAPTER 1 INTRODUCTION 1

1.1 Introduction 1 1.2 Objectives of this study 3 1.2.1 Overall objectives 3 1.2.2 Specific objectives 3

CHAPTER 2 REVIEW OF LITERATURE 4

2.1 Pathogenic bacteria 4 2.1.1 Staphylococcus aureus 4 2.1.2 Staphylococcus aureus MRSA 5 2.1.3 Streptococcus pyogenes 6 2.1.4 Escherichia coli 7 2.1.5 Shigella dysenteriae 8 2.1.6 Salmonella typhimurium 9 2.1.7 Klebsiella pneumonia 10

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2.2 Inflammation 11 2.3 Prasachandaeng remedy and its plant ingredients 13 2.3.1 Bouea macrophylla Griff (ANACARDIACEAE) 13 2.3.2 Caesalpinia sappan Linn. (FABACEAE) 14 2.3.3 Citrus aurantifolia Swing. (RUTACEAE) 15 2.3.4 Dracaena loureiri Gagnep. (AGAVACEAE) 16 2.3.5 Helicia terminalis Kurz. (PROTEACEAE) 17 2.3.6 Jasminum sambac Linn. (OLEACEAE) 18 2.3.7 Kaempferia 9alangal Linn. (ZINGIBERACEAE) 19 2.3.8 Ligusticum chuanxiong Hort. (APIACEAE) 20 2.3.9 Mammea siamensis Kosterm. (CLUSIACEAE) 21 2.3.10 Mesua ferrea Linn. (CALOPHYLLACEAE) 22 2.3.11 Myristica fragrans Houtt. (MYRISTICACEAE) 23 2.3.12 Nelumbo nucifera Gaertn. (NELUMBONACAE) 24 2.4 Biological activities of Prasachandaeng remedy and its plant 25 ingredients

CHAPTER 3 RESEARCH METHODOLOGY 31

3.1 Materials 31 3.1.1 Chemicals and reagents 31 3.1.1.1 Extraction 31 3.1.1.2 Quality control 31 (1) Acid insoluble ash 31 (2) Extractive vale 31 3.1.1.3 Antibacterial activities 32 (1) Disc diffusion method 32 (2) Microtitre plate-based antibacterial assay 32 3.1.1.4 Anti-inflammatory activities 33 (1) Assay for NO inhibitory effects 33 3.1.2 Instruments 34 3.1.3 Plant materials 36

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3.2 Conceptual frame work 38 3.2.1 Preparation of crude extracts 39 3.2.1.1 Maceration 39 3.2.1.2 Decoction 39 3.2.2 Quality control 39 3.2.2.1 Loss on drying 39 3.2.2.2 Total ash 40 3.2.2.3 Acid insoluble ash 40 3.2.2.4 Extractive value 41 3.2.3 Antibacterial activities 41 3.2.3.1 Microbial strains 41 3.2.3.2 Preparation of inoculums 41 3.2.3.3 Preparation of test disc 41 3.2.3.4 Disc diffusion method 42 3.2.3.5 Minimum inhibitory concentration 42 3.2.3.6 Minimum bactericidal concentration 43 3.2.4 Anti-inflammatory activities 43 3.2.4.1 Animal cell lines 43 3.2.4.2 Preparation of sample solution 43 3.2.4.3 Assay for NO inhibitory effects in RAW264.7 cells 44 3.2.4.4 MTT assay 45 3.2.5 The stability test of Prasachandaeng extracts 45 3.2.6 Statistical analysis 45

CHAPTER 4 RESULTS AND DISCUSSION 46

4.1 Preparation of crude extracts 46 4.1.1 Percentage of yield 46

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4.2 Quality control 50 4.2.1 Loss on drying 50 4.2.2 Total ash 50 4.2.3 Acid insoluble ash 51 4.2.4 Extractive values 51 4.3 Antibacterial activities 57 4.3.1 Disc diffusion method 57 4.3.2 Minimum inhibitory concentration (MIC) and minimum 61 bactericidal concentration (MBC) by microtitre plate-based antibacterial assay 4.4 Anti-inflammatory activities 64 4.5 The stability test of Prasachandaeng extract 70 4.5.1 The stability test of Prasachandaeng extract on 70 anti-inflammatory by inhibition Nitric oxide production release from RAW 264.7 cell lines

CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 72

REFERENCES 76

APPENDIX

APPENDIX 81

BIOGRAPHY 83

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LIST OF TABLES

Tables Page 2-1 Biological activities of Prasachandaeng remedy 25 2-2 Biological activitives of Prasachandaeng remedy plant ingredients 26 3-1 List of chemicals and reagents of extraction 31 3-2 List of chemicals and reagents of quality control 31 3-3 List of chemicals and reagents of extractive value 31 3-4 List of chemicals and reagents of disc diffusion method 32 3-5 List of chemicals and reagents of microtitre plate-based antibacterial assay 32 3-6 List of chemicals and reagents of assay for NO inhibition effects 33 3-7 List of instruments, plastic and glass wares 34 3-8 List of plant materials in Prasachandaeng remedy 37 4-1 The percentage yields of the ethanolic and water extracts of 47 Prasachandaeng remedy and its plant ingredients 4-2 Results of quality controls of prasachandaeng remedy and its plant 52 ingredients 4-3 Antibacterial activity of the ethanolic and water extracts of 59 Prasachandaeng remedy and its plant ingredients by disc diffusion method 4-4 Minimum inhibitory concentration (MIC) and minimum 62 bactericidal concentration (MBC) of the ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients by using microtitre plate-based antibacterial assay 4-5 Anti-inflammatory activities by Griess reagent and MTT assay on 65 mouse leukemic macrophage cell lines (RAW 264.7) 4-6 Anti-inflammatory activities by Griess reagent and MTT assay on 71 RAW 264.7 of the stability test of Prasachandaeng extract

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LIST OF FIGURES

Figures Page 2-1 A scanning electron micrograph of S. aureus 4 2-2 A scanning electron micrograph of a human neutrophil ingesting MRSA 5 2-3 A scanning electron micrograph of S. pyogenes 6 2-4 A scanning electron micrograph of E.coli 7 2-5 A scanning electron micrograph of S. dysenteriae 8 2-6 A scanning electron micrograph of S. typhimurium 9 2-7 A scanning electron micrograph of K. pneumonia 10 2-8 Cascade of events and systemic responses of the acute phase response 12 2-9 Bouea macrophylla Griff. 13 2-10 Caesalpinia sappan Linn. 14 2-11 Citrus aurantifolia Swing. 15 2-12 Dracaena loureiri Gagnep. 16 2-13 Helicia terminalis Kurz. 17 2-14 Jasminum sambac Linn. 18 2-15 Kaempferia galanga Linn. 19 2-16 Ligusticum chuanxiong 20 2-17 Mammea siamensis Kosterm. 21 2-18 Mesua ferrea Linn. 22 2-19 Myristica fragrans Houtt. 23 2-20 Nelumbo nucifera Gaertn. 24 3-1 Conceptual framework of thesis 38 4-1 The percentage yield of the ethanolic extracts of Prasachandaeng remedy 48 and its plant ingredients 4-2 The percentage yield of the water extracts of Prasachandaeng remedy 49 and its plant ingredients 4-3 The loss on drying (%) of Prasachandaeng remedy and its plant ingredients53 4-4 The total ash contents of Prasachandaeng remedy and its plant ingredients 54 4-5 The acid insoluble ash contents of Prasachandaeng remedy and its 55 plant ingredients

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LIST OF FIGURES (CONTINUED)

Figures Page 4-6 The extractive values of Prasachandaeng remedy and its plant ingredients 56

4-7 Anti-inflammatory activity (IC50 (µg/ml) ± SEM) by Griess reagent on 68 RAW 264.7 of the ethanolic extracts (N=3)

4-8 Anti-inflammatory activity (IC50 (µg/ml) ± SEM) by Griess reagent on 69 RAW 264.7 of water extracts (N=3) 4-9 Anti-inflammatory activities by Griess reagent and MTT assay on 71 RAW 264.7 of the stability test of Prasachandaeng extract

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LIST OF ABBREVIATIONS

Symbols/Abbreviations Terms

% Percent > More than ≥ More than or equal < Less than ≤ Less than or equal = Equal µg Microgram µg/ml Microgram per milliliter µm Micrometer µl Microliter ºC Degree Celsius ºF Degree Fahrenheit ATCC American type culture collection CFU Colony forming unit CFU/ml Colony forming unit per milliliter

CHCl3 Chloroform cm Centimeter

CO2 Carbon dioxide CPE Cytopathic effect DMSO Dimethyl sulfoxide EMEM Earle’s minimal essential medium et al. Et alii, and other FBS Fetal bovine serum g Gram g/kg Gram per kilogram GEN Gentamicin

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LIST OF ABBREVIATIONS (CONTINUED)

Symbols/Abbreviations Terms

HCl Hydrochloric acid

IC50 Concentration causing 50% inhibition effect iNOS Inducible nitric oxide synthase LPS Lipopolysaccharide m Meter MBC Minimal bactericidal concentration MHA Mueller Hinton Agar MHB Mueller Hinton Broth MIC Minimum inhibition concentration ml Millilitre mm Millimetre mg/kg Millimetre per kilogram mg/ml Milligram per millilitre MRSA Methicillin-resistant Staphylococcus aureus MTT Thiazolyl blue tetrazolium bromide or 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H- tetrazolium bromide NA Nutrient agar

NaHCO3 Sodium bicarbonate NaOH Sodium hydroxide NI No inhibition zone nm Nanometer NO Nitric oxide NT Not tested PBS Phosphate buffer saline PGE2 Prostaglandin E2 PGs Prostaglandins

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LIST OF ABBREVIATIONS (CONTINUED)

Symbols/Abbreviations Terms

P/S Penicillin-Streptomycin RAW264.7 Murine macrophage leukemia RH Relative humidity RNA Ribonucleic acid RPMI 1640 Roswell Park Memorial Institute 1640 SEM Standard error of mean THP Thai Herbal Pharmacopoeia TNF Tumor necrosis factor

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CHAPTER 1 INTRODUCTION

1.1 Introduction

Fever is a cardinal symptom of infectious or inflammatory agents, and is characterized by an increase in body temperature induced by an elevation in thermoregulatory set point (Fraga et al., 2015). Staphylococcus aureus causes superficial, deep-skin, soft-tissue infections, endocarditis and bacteremia with metastatic abscess formation and a variety of toxin-mediated diseases including gastroenteritis, staphylococcal scalded-skin syndrome and toxic shock syndrome (Martineau et al., 1998). Methicillin - resistant Staphylococcus aureus (MRSA) is a major cause of hospital - acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes (Enright et al., 2002). Streptococcus pyogenes is a colonizer of the human oropharynx and skin that can cause very various clinical presentations ranging from common and limited diseases such as pharyngo-tonsillitis, impetigo, erysipelas or cellulitis to life- threatening invasive diseases such as necrotizing fasciitis, pneumonia or streptococcal toxic shock syndrome (Cunningham, 2008). Escherichia coli is one of the most common pathogens in nosocomial and community-acquired infections in humans (Li et al., 2015). Shigella is the etiological agent of shigellosis, a disease responsible for more than 500,000 deaths of children per year, in developing countries (Willer et al., 2004). Salmonella is the pathogenic agent of salmonellosis, a major cause of enteric illness and typhoid fever, leading to many hospitalisations and a few rare deaths if no antibiotics are administered (Hadjinicolaou et al., 2009). and Klebsiella pneumoniae is a common Gram-negative bacterium with worldwide distribution, classically causing nosocomial infections, community-acquired pneumonia, liver abscess, and urinary tract infections (Melot et al., 2015).

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Infectious diseases are disorders caused by organisms such as bacteria, viruses, fungi or parasites. Many organisms live in and on our bodies. They are normally harmless or even helpful, but under certain conditions, some organisms may cause disease. Signs and symptoms vary depending on the organism causing the infection but often include fever and fatigue (World Health Organization, 2011). Inflammation and fever usually occurs after the infections. Inflammation and infection are closely linked in critically ill patients. When adaptive immunity fails to prevent or control infection, an exacerbated or maintained inflammatory response emerges as consequence. It could be considered as an attempt of the immune system to fight the infection, which, on so many occasions, is insufficient. In turn, uncontrolled inflammatory responses impair the development of specific and targeted responses against the infecting microbe, closing a vicious circle. The consequences of unbalanced inflammatory and adaptive responses to infection are microbial escape and tissue damage, contributing to the physiopathogenesis of sepsis, acute respiratory distress syndrome, or multiorganic failure (Bermejo-Martin et al., 2014). Mostly, antibiotics are prescribed for illness caused by bacteria. However, they cannot kill all disease-causing bacteria. Thus, Thai traditional medicine is an alternative treatment for bacterial infection. Prasachandaeng remedy is an antipyretic drug. It is used to relieve fever and internal heat. In addition, it has been used to relieve thirst by mixing with jasmine water. Prasachandaeng remedy has been published in National List of Essential Medicines 2011 (Department for Development of Thai Traditional and Alternative Medicine, 2011). The remedy consists of twelve plants such as: stem of Chan Daeng (Dracaena loureiri Gagnep.), root of Mueng Kon (Helicia terminalis Kurz.), root of Ma Now (Citrus aurantifolia Swing.), root of Ma Prang Wan (Bouea Burmanica Griff.), rhizome of Phro Hom (Kaempferia galanga L.), rhizome of Kot Hua Bua (Ligusticum chuanxiong Hort), stem of Chan Tet (Myristica fragrans L.), stem of Fang Saeng (Caesalpinia sappan L.), pollen of Bua Luang (Nelumbo nucifera Gaertn.), flower of Boon Nag (Mesua ferrea L.), flower of Sa Ra Pee (Mammea siamensis Kosterm.), and flower of Ma Li (Jasminum sambac Lour.)

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There are many reports of antibacterial activity of some plant ingredients in this remedy such as Myristica fragrans L., Citrus aurantifolia Swing., Kaempferia galanga L., Nelumbo nucifera Gaertn., Mesua ferrea L. and Jasminum sambac Lour. Some plant ingredients which exhibited anti-inflammation activities are Dracaena loureiri Gagnep., Ligusticum chuanxiong Hort., Caesalpinia sappan L. and Mammea siamensis Kosterm. However, there is no research report of anti-inflammation activity on Prasachandaeng remedy extract and extracts of some of its plant ingredients. Therefore, the objective of this research were to determine antibacterial and anti- inflammation activitys of Prasachandaeng remedy and its plant ingredient extracts by different extraction methods bacterial infection against three types of gram positive bacteria; Staphylococcus aureus (ATCC 25923), Staphylococcus aureus MRSA (DMST 20651) and Streptococcus pyogenes (ATCC 19615), and four types of gram negative bacteria; Escherichia coli (ATCC 25922), Shigella dysenteriae (DMST 151161), Salmonella typhimurium (DMST 22842) and Klebsiella pneumoniae (ATCC 700603).

1.2 Objectives of this study

1.2.1 Overall objective The overall objectives of this research are to study the antibacterial activity and anti-inflammation activity of the ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients. 1.2.2 Specific objectives To investigate antibacterial activity of ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients. To investigate anti-inflammation activity of ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients by using inhibitory NO production from RAW264.7 cell lines.

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CHAPTER 2 REVIEW OF LITERATURE

2.1 Pathogenic bacteria

2.1.1 Staphylococcus aureus

Figure 2-1 A scanning electron micrograph of S. aureus (Available from https://www.cdc.gov/hai/organisms/staph.html, 2011)

Staphylococcus aureus is a gram-positive, round-shaped bacterium which common bacterium found on the skin and in the noses of up to 30% of healthy people and animals. S. aureus is important because it has the ability to make seven different toxins that are frequently responsible for food poisoning. Staphylococcal toxins are fast acting, sometimes causing illness in as little as 30 minutes. Symptoms usually develop within one to six hours after eating contaminated food. Patients typically experience several of the following: nausea, vomiting, stomach cramps, and diarrhoea. The illness is usually mild and most patients recover after one to three days. In a small minority of patients the illness may be more severe. Anyone can develop a staphylococcal infection, although certain groups of people are at greater risk, including people with chronic conditions such as diabetes, cancer, vascular disease, eczema, and lung disease. In a healthcare setting, the risk of more serious staphylococcal infection is higher because patients often have weakened immune systems or have undergone procedures such as surgery or have intravenous catheters according to Centres for Disease Control and prevention, 2011.

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2.1.2 Methicillin-resistant Staphylococcus aureus (MRSA)

Figure 2-2 A scanning electron micrograph of a human neutrophil ingesting MRSA (Available from https://www.bcm.edu/departments/molecular-virology-and- microbiology/emerging-infections-and-biodefense/mrsa, 2015)

Methicillin-resistant Staphylococcus aureus (MRSA) is a type of staphylococcal bacterium that is resistant to certain antibiotics called beta-lactams. MRSA is any strain of Staphylococcus aureus that has developed, through horizontal gene transfer and natural selection, multi- resistance to beta-lactam antibiotics. These antibiotics include methicillin and other more common antibiotics such as oxacillin, penicillin and amoxicillin. In the community, most MRSA infections are skin infections. More severe or potentially life-threatening MRSA infections occur most frequently among patients in healthcare settings. Anyone can get MRSA on their body from contact with an infected wound or by sharing personal items, such as towels or razors that have touched infected skin. MRSA infection risk can be increased when a person is in activities or places that involve crowding, skin-to-skin contact, and shared equipment or supplies. People including athletes, day-care and school students, military personnel in barracks, and those who recently received inpatient medical care are at higher risk according to Centres for Disease Control and prevention, 2016.

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2.1.3 Streptococcus pyogenes

Figure 2-3 A scanning electron micrograph of S. pyogenes (Available from http://www.bacteriainphotos.com/ streptococcus_pyogenes_3D.html, 2016)

Streptococcus pyogenes is a species of bacteria, spherical, Gram- positive bacterium that is the cause of group A streptococcal infections. S. pyogenes displays streptococcal group A antigen on its cell wall. S. pyogenes typically produces large zones of beta-hemolysis (the complete disruption of erythrocytes and the release of hemoglobin) when cultured on blood agar plates and are therefore also called Group A (beta-hemolytic) Streptococcus (abbreviated GABHS). These bacteria are aerobic and an extracellular bacterium, made up of non-motile and non-sporing cocci. As expected with a streptococci, it is clinically important in human illness that can cause many different infections, including strep throat, scarlet fever, impetigo and others according to Centres for Disease Control and prevention, 2008.

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2.1.4 Escherichia coli

Figure 2-4 A scanning electron micrograph of E.coli (Available from https://en.wikipedia.org/wiki/Escherichia_coli, 2005)

Escherichia coli is a gram-negative, facultatively anaerobic, rod- shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli are harmless and actually are an important part of a healthy human intestinal tract. However, some E. coli are pathogenic, meaning they can cause illness, either diarrhoea or illness outside of the intestinal tract. The types of E. coli that it can cause of diarrhoea which can be transmitted through contaminated water or food, or through contact with animals or persons. The prevention methods for E. coli transmission include hand washing, improved sanitation and drinking water, as transmission occurs through faecal contamination of food and water supplies according to Centres for disease control and prevention, 2015.

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2.1.5 Shigella dysenteriae

Figure 2-5 A scanning electron micrograph of S. dysenteriae (Available from http://www.foodpoisonjournal.com/food-poisoning- information/what-to-know-about-shigella/#.WK1_82_yjIV, 2015)

Shigella dysenteriae is a species of the rod-shaped bacterial genus Shigella. S. dysenteriae, spread by contaminated water and food, causes the most severe dysentery because of its potent and deadly Shiga toxin, but other species may also be dysentery agents. Contamination is often caused by bacteria on unwashed hands during food preparation or soiled hands reaching the mouth. Shigellosis is a diarrheal disease caused by a group of bacteria called Shigella. There are four different species of Shigella: S. sonnei (the most common species in the United States), S. flexneri, S. boydii and S. dysenteriae. S. dysenteriae and S. boydii are rare in the United States, though they continue to be important causes of disease in the developing world. S. dysenteriae type 1 can cause deadly epidemics. Symptoms of shigellosis typically start 1–2 days after exposure and include: diarrhoea (sometimes bloody), fever, abdominal pain, tenesmus according to Centres for Disease Control and prevention, 2015.

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2.1.6 Salmonella typhimurium

Figure 2-6 A scanning electron micrograph of S. typhimurium (Available from https://en.wikipedia.org/wiki/Salmonella, 2005)

Salmonella typhimurium is a pathogenic Gram-negative bacteria predominately found in the intestinal lumen. S. typhimurium is a bacterium that makes people sick. It was discovered by an American scientist named Dr.Salmon and has been known to cause illness for over 125 years. Most people infected with Salmonella develop diarrhoea, fever and abdominal cramps between 12 and 72 hours after infection. The illness usually lasts 4 to 7 days, and most individuals recover without treatment. In some cases, diarrhoea may be so severe that the patient needs to be hospitalized. In these patients, the Salmonella infection may spread from the intestines to the blood stream, and then to other body sites. In these cases, Salmonella can cause death unless the person is treated promptly with antibiotics. The elderly, infants, and those with impaired immune systems are more likely to have a severe illness according to Centres for Disease Control and prevention, 2015.

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2.1.7 Klebsiella pneumonia

Figure 2-7 A scanning electron micrograph of K. pneumonia (Available from http://sevilla.abc.es/andalucia/cordoba/sevi-klebsiella-guerra-infinita- 201604162017_noticia.html, 2016)

Klebsiella is a type of Gram-negative bacterium that can cause different types of healthcare-associated infections, including pneumonia, bloodstream infections, wound or surgical site infections and meningitis. Increasingly, Klebsiella bacteria have developed antimicrobial resistance, most recently to the class of antibiotics known as carbapenems. Klebsiella bacteria are normally found in the human intestines. They are also found in human stool (faeces). In healthcare settings, Klebsiella infections commonly occur among sick patients who are receiving treatment for other conditions. Patients whose care requires devices like ventilators (breathing machines) or intravenous (vein) catheters and patients who are taking long courses of certain antibiotics are most at risk for Klebsiella infections. Healthy people usually do not get Klebsiella infections according to Centres for Disease Control and prevention, 2012.

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2.2 Inflammation

Inflammation comprises a series of cellular changes which ultimately facilitate phagocytosis and either the killing of microorganisms or the digestion of cell debris. This is followed by proliferation of connective tissue cells and repair of the intercellular matrix. Both of these events require the complex control of various cell types at a local level. There is increasing evidence that, while many molecules may mediate effects such as vascular permeability and chemotaxis, cytokines exert control in a very sophisticated way with remarkable duplication of key elements and complex negative and positive interaction with each other and other hormones. The local processes of inflammation are facilitated by systemic metabolic changes that mobilize energy in the form of glucose and fatty acids and amino acid building blocks. Fever up-regulates several enzymatic reactions in inflammatory and tissue cells which, perhaps being potentially hazardous, operate at a suboptimal level at normal body temperature. Leukocytosis provides an increased supply of phagocytic cells. Counter-regulatory effects such as the release of cortisol come into play and curtail the potentially destructive effects of inflammation. Liver production of acute phase proteins provides an increased tissue supply of certain inflammatory mediators and inhibitors. All these events are, in part, controlled by the same pleotropic cytokines which exert key local controls. The cluster of systemic responses which accompany trauma and inflammation, regardless of cause are known as the acute phase response. The response is remarkably stereotyped regardless of the localized or systemic nature of the underlying inflammation. Many threads of evidence support the notion, put forward by several workers over the past 40 years, that this is an adaptive response conditioning the “milieu interieur” so that inflammation and healing may progress optimally.

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A complex array of hormones and cytokines induce and control the components of the response and it is rapidly becoming clear that these two systems and the nervous system are closely integrated (Whicher, 1984).

Figure 2-8 Cascade of events and systemic responses of the acute phase response (Whicher, 1984)

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2.3 Prasachandaeng remedy and its plant ingredients

2.3.1 Bouea macrophylla Griff.

Figure 2-9 Bouea macrophylla Griff. (Original picture)

Common name (Thai): Ma Prang Wan Family: ANACARDIACEAE Botanical characteristics: Bouea macrophylla Griff. is a tropical fruit tree native to Southeast Asia. The tree is related to the mango. The evergreen tree grows to height of 25 meters. Its leaves are lanceolate to elliptic and range from 13 to 45 cm (5 to 17 inches) long and from 5 to 7 cm (2 to 3 inches) wide. The fruit (resembling a mango) are green in colour and mature to orange or yellow. They grow to roughly 2 to 5 cm (0.7 to 1.9 inches) in diameter. The entire fruit, including its skin is edible. The fruit range from sweet to sour in flavour and have a light smell of turpentine. In Thailand, it flowers in November to December and fruit appears from April to May (Food and Agriculture Organization, 2007). Part used: roots Traditional use: relieve common cold, detoxify (Wutthithammawet, 2002)

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2.3.2 Caesalpinia sappan L.

Figure 2-10 Caesalpinia sappan L. (Original picture)

Common name (Thai): Fang Saeng Family: FABACEAE Botanical characteristics: Caesalpinia sappan L. is a species of flowering tree in the legume family that is native to Southeast Asia and the Malay Archipelago. This plant has many uses. It possesses medicinal properties as an antibacterial and anticoagulant. It also produces a valued type of reddish dye called brazilin, used for dyeing fabric as well as making red paints and inks. Slivers of heartwood are used for making herbal drinking water in Kerala. Heartwood also contains juglone (5-hydroxy-1, 4-naphthoquinone). Homoisoflavonoids (sappanol, episappanol, 3'-deoxysappanol, 3'-O-methylsappanol, 3'-O-methylepisappanol and sappanone A) can also be found in C. sappan. The wood is somewhat lighter in color than Brazilwood and its other allies but the same tinctorial principle appears to be common to all. C. sappan was a major trade commodity during the 17th century, when it was exported from Southeast Asian nations (especially Siam) aboard red seal ships to Japan (Badami et al., 2003). Part used: stems Traditional use: blood tonic, promote lymphatic system (Wutthithammawet, 2002)

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2.3.3 Citrus aurantifolia Swing.

Figure 2-11 Citrus aurantifolia Swing. (Original picture)

Common name (Thai): Ma Now Family: RUTACEAE Botanical characteristics: C. aurantifolia Swing is a hybrid citrus fruit, which is typically round, lime green, 3–6 centimetres (1.2–2.4 in) in diameter and containing acidic juice vesicles. There are several species of citrus trees. Limes are an excellent source of vitamin C and are often used to accent the flavours of foods and beverages. They are grown year-round in tropical climates and are usually smaller and less sour than lemons, although varieties may differ in sugar and acidic content. Plants with fruit called "limes" have diverse genetic origins; limes do not form a monophyletic group (Penjor et al., 2014). Part used: root Traditional use: relieve common cold, detoxify, anti-inflammatory (Wutthithammawet, 2002)

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2.3.4 Dracaena loureiri Gagnep.

Figure 2-12 Dracaena loureiri Gagnep. (Original picture)

Common name (Thai): Chan Daeng Family: AGAVACEAE Botanical characteristics: Dracaena loureiri Gagnep is a shrub or slender much-branched tree. When this plant becomes old, it has a red core in the stem and then the stem gradually decays until all cores become red, this core wood is called Chan Daeng. Most D. loureiri grows in the high mountains, but it can be found in many parts of Thailand. D. loureiri has been used as a folk medicine e.g., antipyretic, anti- inflammatory and for pain relief (Ichikawa et al., 1997). Part used: stems Traditional use: relieve common cold, blood tonic, promote hepatic system (Wutthithammawet, 2002)

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2.3.5 Helicia terminalis Kurz.

Figure 2-13 Helicia terminalis Kurz. (Original picture)

Common name (Thai): Mueng Kon Family: PROTEACEAE Botanical characteristics: Helicia terminalis Kurz. is a tree growing 5-15 m tall. Young branchlets rust-colored, tomentose, glabrescent. Leaves dimorphic, simple or tripartite to pinnatipartite; petiole 1-2.5 cm for simple leaves, 4-5 cm for divided leaves; leaf blade (leathery, glabrescent in age), base cuneate to attenuate, margin entire, apex shortly acuminate, acuminate, or sometimes obtusely acute; secondary and reticulate veins conspicuous on both surfaces. Simple leaf blade oblanceolate to oblong, 12-35 × 4-10 cm. Divided leaf blade 25-60 × 15-50 cm, lobes 1-7 pairs. Inflorescences axillary or ramiflorous, rarely terminal on short branchlet, pilose; bracts of flower pairs linear to subulate 0.5-2 mm; floral bracts linear, ca. 0.5 mm. Perianth white or yellowish. Fruit brownish and ellipsoid 3-4.5 × 1.7-3 cm. This species is used medicinally (Flora of China, 1995). Part used: roots Traditional use: relieve common cold, tonic coolant, detoxify (Wutthithammawet, 2002)

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2.3.6 Jasminum sambac L.

Figure 2-14 Jasminum sambac L. (Original picture)

Common name (Thai): Ma Li Family: OLEACEAE Botanical characteristics: Jasminum sambac is an evergreen vine or shrub reaching up to 0.5 to 3 m (1.6 to 9.8 ft.) tall. The species is highly variable, possibly a result of spontaneous mutation, natural hybridization and auto polyploidy. Cultivated Jasminum sambac generally do not bear seeds and the plant is reproduced solely by cuttings, layering, marcotting and other methods of asexual propagation. The leaves are ovate, 4 to 12.5 cm long and 2 to 7.5 cm wide. The phyllotaxy is opposite or in whorls of three, simple (not pinnate, like most other jasmines). They are smoothing (glabrous) except for a few hairs at the venation on the base of the leaf. The flowers bloom all throughout the year and are produced in clusters of 3 to 12 together at the ends of branches. They are strongly scented with a white corolla 2 to 3 cm in diameter with 5 to 9 lobes. The flowers open at night (usually around 6 to 8 in the evening), and close in the morning, a span of 12 to 20 hours. The fruit is a purple to black berry 1 cm in diameter (Li and Zhang, 2015). Part used: flowers Traditional use: relieve common cold, cardio tonic (Wutthithammawet, 2002)

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2.3.7 Kaempferia galanga L.

Figure 2-15 Kaempferia galanga L. (Original picture)

Common name (Thai): Proh Horm Family: ZINGIBERACEAE Botanical characteristics: Kaempferia galanga is a monocotyledonous plant in the ginger family and one of four plants called Galangal. It is found primarily in open areas in Indonesia, southern China, Taiwan, Cambodia and India, but is also widely cultivated throughout Southeast Asia. The plants need high humidity and do well in pots alongside warm growing orchids or in beds beneath greenhouse staging. Leaves are spreading flat on the ground, round-ovate or sub- orbicular, deltoid-acuminate, thin, green; flowers white, 6-12 on a short scape, fugacious, fragrant opening successively; lip biloped with lilac or purple spots (Zhou et al., 1989). Part used: rhizomes Traditional use: urticaria, relieve cough (Wutthithammawet, 2002)

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2.3.8 Ligusticum chuanxiong Hort.

Figure 2-16 Ligusticum chuanxiong (Original picture)

Common name (Thai): Kot Hua Bua Family: APIACEAE Botanical characteristics: Ligusticum chuanxiong is one of the most popular herbs in China and Korea, where it grows wild and has been cultivated for centuries. It is a perennial plant with hermaphrodite flowers that are pollinated by insects. The root and rhizomes are used for herbal remedies. In additional to its medicinal uses. Ligusticum can be combined with certain foods and beverages as a flavor component and to add fragrance to some soaps and cosmetics. In traditional Chinese medicine. Ligusticum has pungent and warm properties and is associated with the liver, gallbladder and pericardium meridians. Its main functions are to promote the flow of blood, dispel wind and relieve pain. Many practitioners prescribe it to treat irregular menstrual periods, dysmenorrhea and headaches. It is also given to patients with inflammation caused by injuries, carbuncles and boils (Xiao et al., 2002). Part used: rhizomes Traditional use: carminative, relieve common cold (Wutthithammawet, 2002)

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2.3.9 Mammea siamensis Kosterm.

Figure 2-17 Mammea siamensis Kosterm. (Original picture)

Common name (Thai): Sa Ra Pee Family: CLUSIACEAE Botanical characteristics: Mammea siamensis is a small, evergreen tree up to 15 m tall and 10-30 cm in diameter. The flowers are polygamous with a globular calyx opening into two valvate sepals. There are 4 to 6 petals. Indehiscent drupes are formed, containing 1 to 4 seeds. The leaves are rigid, coriaceous and often have pellucid dots. Mammea is native to Myanmar, Thailand, Laos, Cambodia and Vietnam. The genus Mammea is reported to contain various types of xanthones (Poobrasert et al., 1998). Part used: flowers Traditional use: tonic, cardio tonic, blood tonic (Wutthithammawet, 2002)

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2.3.10 Mesua ferrea L.

Figure 2-18 Mesua ferrea L. (Original picture)

Common name (Thai): BoonNag Family: CALOPHYLLACEAE Botanical characteristics: Mesua ferrea is a slow-growing tree named after the heaviness and hardness of its timber. It is widely cultivated as an ornamental due to its graceful shape, grayish-green foliage with a beautiful pink to red flush of drooping young leaves and large, fragrant white flowers. It is native to wet, tropical parts of Sri Lanka, India, southern Nepal, Burma, Thailand, Indochina, the Philippines, Malaysia and Sumatra where it grows in evergreen forests, especially in river valleys. In the eastern Himalayas and Western Ghats in India it grows up to altitudes of 1500 meters while in Sri Lanka up to 1000 meters. The tree can grow over 30 meters tall, often buttressed at the base with a trunk up to 2 meters in diameter. The bark of younger trees has an ash grey color with flaky peelings, while in old trees the bark is dark ash-grey with a red-brown blaze. It has simple, opposite, narrow, oblong to lanceolate, blue-grey to dark green leaves that are 7–15 cm long and 1.5–3.5 cm wide, with a whitish underside. The emerging young leaves are red to yellowish pink and drooping. The branches are slender, terete and glabrous. The bisexual flowers are 4–7.5 cm in diameter with four white petals and a center of numerous orange yellow stamens. The fruit is an ovoid to globose capsule with 1 to 2 seeds (Prasad et al., 1999). Part used: flowers Traditional use: tonic, cardio tonic, blood tonic (Wutthithammawet, 2002)

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2.3.11 Myristica fragrans Houtt.

Figure 2-19 Myristica fragrans Houtt. (Original picture)

Common name (Thai): Chan Tet Family: MYRISTICACEAE Botanical characteristics: Myristica fragrans is a small evergreen tree, usually 5–13 m tall but occasionally reaching 20 m. The alternately arranged leaves are dark green, 5–15 cm long by 2–7 cm wide with petioles about 1 cm long. The species is dioecious, i.e. "male" or staminate flowers and "female" or carpellate flowers are borne on different plants, although occasional individuals produce both kinds of flower. The flowers are bell-shaped, pale yellow and somewhat waxy and fleshy. Staminate flowers are arranged in groups of one to ten, each 5–7 mm long; carpellate flowers are in smaller groups, one to three, and somewhat longer, up to 10 mm (0.4 in) long. Carpellate trees produce smooth yellow ovoid or pear-shaped fruits, 6–9 cm long with a diameter of 3.5–5 cm. The fruit has a fleshy husk. When ripe the husk splits into two halves along a ridge running the length of the fruit. Inside is a purple-brown shiny seed, 2–3 cm long by about 2 cm across, with a red or crimson covering (an aril). The seed is the source of nutmeg, the aril the source of mace (Shafiei et al., 2012). Part used: stems Traditional use: tonic, cardio tonic, carminative (Wutthithammawet, 2002)

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2.3.12 Nelumbo nucifera Gaertn.

Figure 2-20 Nelumbo nucifera Gaertn. (Original picture)

Common name (Thai): Bua Luang Family: NELUMBONACAE Botanical characteristics: This plant is an aquatic perennial. The roots are planted in the soil of the pond or river bottom while the leaves float on top of the water surface or are held well above it. The flowers are usually found on thick stems rising several centimeters above the leaves. The plant normally grows up to a height of about 150 cm and a horizontal spread of up to 3 meters but some unverified reports place the height as high as over 5 meters. The leaves may be as large as 60 cm in diameter while the showy flowers can be up to 20 cm in diameter. An individual lotus can live for over a thousand years and has the rare ability to revive into activity after stasis. In 1994, a seed from a sacred lotus, dated at roughly 1,300 years old ± 270 years, was successfully germinated. The genome of the sacred lotus was sequenced in May 2013 (Zhang et al., 2014). Part used: pollens Traditional use: tonic, cardio tonic (Wutthithammawet, 2002)

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2.4 Biological activities of Prasachandaeng remedy and its plant ingredients

Table 2-1 Biological activities of Prasachandaeng remedy

Name Activities Detail on biological activities Prasachandaeng remedy Antibacterial The 95% ethanolic extract of Prasachandaeng remedy exhibited

antibacterial activity against S.aureus and MRSA with MIC values of 62

and 31 µg/ml. (Chusri et al., 2014) Cytotoxicity The 95% ethanolic extract of Prasachandaeng remedy had no cytotoxic effects on Vero cells. (Chusri et al., 2014) Phytochemical Prasachandaeng remedy exhibited the presence of alkaloid, triterpenoid, screening test condensed tannins and hydrolysable tannins by using Drangendorff reagent, Liebermann-Burchard reagent and ferric chloride reagent, respectively. (Chusri et al., 2014)

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Table 2-2 Biological activitives of Prasachandaeng remedy plant ingredients

Scientific name (Family) Activities Part of plant Detail on biological activities Bouea macrophylla Griff. Not found Root - (ANACARDIACEAE) Caesalpinia sappan L. Anti-inflammatory Stem The 70% ethanol extract had percent of inhibition of NO (FABACEAE) 94.27 ± 0.01 in LPS/IFN훾-stimulated RAW264.7 cells (Chen and Zhang, 2014). Phenolics including protosappanin A and 3- deoxysappanchalcone isolated from the heartwood showed strong inhibitory activities toward the LPS-induced NO

production in macrophage RAW264.7 cells with IC50 values of 12.5 and 8.1 μM, respectively (Min et al., 2012). Citrus aurantifolia Swing. Antibacterial Fruits Methanol, butanol and water extracts showed inhibition zone (RUTACEAE) against Haemopillus somnus about 13 – 17 mm. (Lee et al., 2014). Leaves Methanol and chloroform extracts showed inhibition zone against Burkholderia sp. about 13 – 17 mm (Lee et al., 2014).

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Table 2-2 Biological activitives of Prasachandaeng remedy plant ingredients (continued)

Scientific name (Family) Activities Part of plant Detail on biological activities Dracaena loureiri Gagnep. Anti-inflammatory Stem Stilbenoids including 4, 3′, 5′-trihydroxystilbene, 4, 3′- (AGAVACEAE) dihydroxy-5′-methoxystilbene and 4-hydroxy-3′,5′- dimethoxystilbene were isolated from stem. These compounds were evaluated for their inhibitory activity against the enzymes cyclooxygenase-1 and cyclooxygenase-2 with

IC50 1.29 - 4.92 μM (Likhitwitayawuid et al., 2002).

The 95% ethanolic extract of D. loureiri showed anti-

inflammatory effect by inhibition NO production (IC50 = 38.37±1.66 µg/ml) (Ruangnoo, 2012) Antibacterial Stem The 95% ethanolic extract of D. loureiri inhibited S. aureus and S. aureus MRSA with inhibition zone value of 9 and 16 mm, respectively by disc diffusion method. MIC value of 2.5 and 1.25 mg/ml. (Sattaponpan and Kondo, 2011) Helicia terminalis Kurz. Not found Root - (PROTEACEAE)

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Table 2-2 Biological activitives of Prasachandaeng remedy plant ingredients (continued)

Scientific name (Family) Activities Part of plant Detail on biological activities Jasminum sambac L. Antimicrobial Flower The methanol extract of the flowers showed weak activity (OLEACEAE) against Propionibacterium acnes MIC value ˃4 mg/ml (Tsung-Hsien T et al., 2010). Kaempferia galanga L. Antibacterial Rhizome Water extract showed inhibition zone against Bacillus pumilis (ZINGIBERACEAE) 9 mm (Sini and Malathy, 2005). Antinociceptive Rhizome In mice and rats using acetic acid-induced writhing, formalin, hot plate and tail-flick tests. The methanolic extract at test doses of 50, 100 and 200 mg/kg, p.o. clearly demonstrated antinociceptive activity in all tests. This activity was dose- and time- dependent. The methanolic extract administered at 200 mg/kg, p.o. had a stronger antinociceptive effect than aspirin (100 mg/kg, p.o.) but less than morphine (5 mg/kg, s.c.) (Ridtitid et al., 2008).

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Table 2-2 Biological activitives of Prasachandaeng remedy plant ingredients (continued)

Scientific name (Family) Activities Part of plant Detail on biological activities Ligusticum chuanxiong Anti-inflammatory Rhizome 70% ethanol extract have percent of inhibition of NO 91.13 ± (APIACEAE) 0.01 in LPS/IFN훾-stimulated RAW264.7 cells (Chen and Zhang, 2014). Ligustilides including chuanxiongnolide R1, chuanxiongdiolide R1, levistolide A and tokinolide B isolated from rhizome had percent of inhibition of NO 67.32, 61.31, 69.46 and 68.17% ,respectively (Huang et al., 2013). Mammea siamensis Kosterm. Anti-inflammatory Flower A methanol extract of the flowers was found to inhibit nitric (CLUSIACEAE) oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells. From the extract, coumarins, mammeasins

A (IC50 = 1.8 μM), kayeassamins G (IC50 = 0.8 μM) and

mammea A/AD (IC50 = 1.3 μM were found to inhibit induction of inducible nitric oxide synthase (iNOS) (Morikawa et al., 2012).

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Table 2-2 Biological activitives of Prasachandaeng remedy plant ingredients (continued)

Scientific name (Family) Activities Part of plant Detail on biological activities Mesua ferrea L. Antibacterial Flower The methanol extract of whole flowers was studied against various (CALOPHYLLACEAE) strains of gram-positive and gram-negative bacteria at concentration ranges of 100 to 50 mg/ml or even lower, as against vibrios and Escherichia coli (Mazumder et al., 2004). Anti-inflammatory Flower The 95% ethanolic extract of M. ferrea showed anti-inflammatory

effect by inhibition NO production (IC50 = 26.23±3.42 µg/ml) (Makchuchit, 2010) Myristica fragrans Houtt. Antibacterial Flesh Ethyl acetate extract against Streptococcus mitis ATCC 6249 (MYRISTICACEAE) showed MIC value 0.625 mg/mL and MBC value 1.25 mg/mL (Shafiei et al., 2012). Nelumbo nucifera Gaertn. Antibacterial Pollen Essential oil extract showed activity against gram negative (NELUMBONACAE) S.typhimurium ATCC 14028 and E.coli ATCC 25922; MIC values 10 and 40, and MBC values 20 and 80 ml L-1 resp. (Sittiwet, 2009).

The 95% ethanolic extract of N. nucifera inhibited S. aureus MRSA with inhibition zone value of 9 mm. by disc diffusion method. MIC

value of 10 mg/ml and MBC value of 10 mg/ml. (Sattaponpan and30

Kondo, 2011)

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CHAPTER 3 RESEARCH METHODOLOGY

3.1 Materials

3.1.1 Chemicals and reagents 3.1.1.1 Extraction Table 3-1 List of chemicals and reagents of extraction Name Source Ethanol 95%, commercial grad C.M.J Anchor company, Thailand Deionized water Milford, USA

3.1.1.2 Quality control (1) Acid insoluble ash Table 3-2 List of chemicals and reagents of quality control Name Source Hydrochloric acid (HCl) Merck, Germany Deionized water Milford, USA

(2) Extractive value Table 3-3 List of chemicals and reagents of extractive value Name Source Ethanol 95%, commercial grad C.M.J Anchor company, Thailand Deionized water Milford, USA

Chloroform (CHCl3), analytical grade RCI labscan, Thailand

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3.1.1.3 Antibacterial activities (1) Disc diffusion method Table 3-4 List of chemicals and reagents of disc diffusion method Name Source Brain Heart Infusion Agar Difco, USA Brain Heart Infusion Broth Difco, USA Dimethyl Sulfoxide (DMSO) RCL Labscan, Thailand Distilled water Milford,USA Mueller Hinton Agar Difco, USA Mueller Hinton Broth Difco, USA Nutrient Agar Difco, USA Sabouraud Dextrose Agar Difco, USA

(2) Microtitre plate-based antibacterial assay Table 3-5 List of chemicals and reagents of microtitre plate-based antibacterial assay Name Source Brain Heart Infusion Agar Difco, USA Brain Heart Infusion Broth Difco, USA Dimethyl Sulfoxide (DMSO) RCL Labscan, Thailand Distilled water Milford,USA Mueller Hinton Agar Difco, USA Mueller Hinton Broth Difco, USA Nutrient Agar Difco, USA Resazurin sodium salt Sigma-aldrich, USA Sabouraud Dextrose Agar Difco, USA

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3.1.1.4 Anti-inflammatory activities (1) Assay for NO inhibition effects Table 3-6 List of chemicals and reagents of assay for NO inhibition effects Name Source Dimethyl Sulfoxide (DMSO) RCL Labscan, Thailand Distilled water Milford,USA Fetal bovine serum (FBS) Biochem, Germany Hydrochloric acid (HCl) Univar, Australia Isopropanol RCI labscan, Thailand Lipopolysaccharide from E.coli O55:B5 (LPS) Sigma, USA N-(1-Naphthyl) ethylenediamine dihydrochloride Sigma, USA Penicillin-Streptomycin (P/S) Sigma, USA Phosphase buffered saline (PBS) Amresco, USA

Phosphoric acid 85% (H3PO4) Sigma, USA Prednisolone ≥90% Sigma, USA RPMI medium 1640 Gibco,USA

Sodium bicarbonate (NaHCO3) BHD,England Sodium hydroxide (NaOH) Univar, Australia

Sulfanilamide (H2NC6H4SO2NH2) Sigma, USA Thiazolyl blue tetrazolium bromide (MTT) Sigma, USA Trypan blue 0.4% Gibco,USA Trypsin-EDTA Gibco,USA

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3.1.2 Instruments Table 3-7 List of instruments, plastic and glass wares Name Source 75 cm2 plastic tissue culture flasks Costar Corning, USA 96-well microplates flat, bottom with lid Costar Corning, USA 96-well microplates flat, bottom without lid Costar Corning, USA 96-well microplates U, bottom with lid Costar Corning, USA Autoclave Hirayama, Japan Balance 0.01 mg-41 g Mettler-Toledo, Switzerland Balance 0.01 g-220 g Precica, Switzerland Balance 0.5 g-3100 g Mettle- Toledo, Switzerland Buchner Funnel Schott Duran, Germany Cell culture flask, canted neck 75 cm3 Costar Corning, USA Centrifuge Beckman Coulter, USA Centrifuge tube 15, 50 ml Costar Corning, USA

CO2 humidified incubator Shel lab, USA Crucibles Coorstex,USA Disposable pipette 2, 5, 10, 25 mL Costar Corning, USA Erlenmeyer flasks Schott Duran, Germany Eppendorfs Costar Corning, USA Examination glove Sritrang gloves, Thailand Filter paper no.1 (125 mmØ) Whatman, USA Filter paper no.40 (125 mmØ) Whatman, USA Freezer Sanyo, Japan Glass bottle 50, 250, 500, 1000 mL Schott Duran, Germany Glasswares 10, 25, 50, 100, 250, 600, 1000 mL Schott Duran, Germany Pyrex, USA Hemocytometer Boeco, Germany Hot air oven Memmert, Germany Hot plate Thermolyne, USA Incubated tabletop orbital shaker Thermo Sciencetific, USA

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Table 3-7 List of instruments, plastic and glass wares (continued) Name Source Inverted microscope Nikon, Japan Laminar air flow Boss tech, Thailand Liquid nitrogen tank Taylor-Wharton, USA Lyophilizer Telster, Spain Litmus paper pH-fix 4.5-10.0 Macherey-nagel, Germany McFarland densitometer Grant-Bio, England Membrane filter with pore-size rating of 0.22 micron Millipore, Germany Micropipettes 20 µl, 200 µl, 1000 µl Gibson, USA Microplate reader Bio Tek, USA Moisture analyzer Scaltec instrument,Germany Muffle furnace Nabertherm, Germany Multi-channels pipette Costar corning, USA Paper disc (0.6 cm diameter) Whatman, USA Petri dish Hycon, USA pH buffer Thermo Scientific, USA pH meter WTW inolab, Germany Pipette tips Costar corning, USA Pipette boy Integra biosciences, Switzerland Reagent reservoir (Sterile) Costar corning, USA Refrigerator (4ºC) Sharp, Japan Refrigerator (-20ºC) Sanyo, Japan Rotary evaporator Buchi, Switzerland Shaking incubator Vision Scientific, Korea Sonicator Elma, Germany Stability incubator Termarks, Norway Syringes Nipro, Thailand Vacuum desiccator Simax, USA Vacuum pump Rocker, Taiwan Vortex mixer Scientific industries, USA

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3.1.3 Plant materials Prasachandaeng remedy consists of 12 plants. Plant materials were purchased from Charernsook Osot pharmacy (Nakhornpathom, Thailand) and are shown in Table 3-8. Each plant ingredient was cleaned and dried by using a hot air oven at 45-50°C. The formulated remedy was ground to crude powder. The preparation was macerated in 95% ethanol and decocted in water. All crude extracts were stored at - 20°c and diluted on the day of use.

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Table 3-8 List of plant materials in Prasachandaeng remedy Voucher specimen % in Scientific Name Family Name Thai name Part used Flavour number remedy Bouea macrophylla Griff. ANACARDIACEAE SKP 009 02 13 01 มะปราง Root Flavourless 6.25 Caesalpinia sappan L. FABACEAE SKP 072 03 19 01 ฝาง Stem Bitter&Astringent 6.25 Citrus aurantifolia Swing. RUTACEAE SKP 166 03 01 01 มะนาว Root Bitter 6.25 Dracaena loureiri Gagnep. AGAVACEAE SKP 005 04 12 01 จันทน์แดง Stem Bitter 50 Helicia terminalis Kurz. PROTEACEAE SKP 157 08 20 01 เหมือดคน Root Bitter 6.25 Jasminum sambac L. OLEACEAE SKP 129 10 19 01 มะลิ Flower Cool&Scented 1.5625 Kaempferia galanga L. ZINGIBERACEAE SKP 206 11 01 01 เปราะหอม Rhizome Bitter&Spicy 6.25 Ligusticum chuanxiong Hort. APIACEAE SKP 199 12 19 01 โกฐหัวบัว Rhizome Bitter& Spicy 6.25 Mammea siamensis Kosterm. CLUSIACEAE SKP 083 13 19 01 สารภี Flower Cool&Scented 1.5625 Mesua ferrea L. CALOPHYLLACEAE SKP 083 13 06 01 บุนนาค Flower Cool&Scented 1.5625 Myristica fragrans Houtt. MYRISTICACEAE SKP 121 13 06 01 จันทน์เทศ Stem Bitter&Scented 6.25 Nelumbo nucifera Gaertn. NELUMBONACAE SKP 125 14 14 01 บัวหลวง Flower Cool&Scented 1.5625 ประสะ Bitter, Cool Prasachandaeng remedy - - - 100 จันทน์แดง & Scented

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3.2 Conceptual frame work

Plant collection

Dry and grind herbs Quality control

Extraction

Decoction (Water) Maceration (95%EtOH)

Antibacterial Anti-inflammatory Disc diffusion Nitric Oxide MIC MBC

Bacterial strains: Stability test Staphylococcus aureus Methicillin Resistant - S.aureus Streptococcus pyogenes Escherichia coli Shigella dysenteriae Salmonella typhimurium Klebsiella pneumoniae

Figure 3-1 Conceptual framework of research

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3.2.1 Preparation of crude extracts Each plant ingredient was cleaned and dried by using a hot air oven at 45-50°C. The formulated remedy was ground to crude powder, then macerated in 95% ethanol and decocted in water. All plants in this remedy were also macerated in 95% ethanol. All extracts were stored at -20°c and diluted it on the day of use. 3.2.1.1 Maceration The crude powder of Prasachandaeng remedy and each plant ingredient of this remedy (500g) were macerated in 95% ethanol for 3 days and maceration repeated 2 more times. Then, it was filtered and the filtrate dried using a rotary evaporator. These extracts are 95% ethanol extracts. 3.2.1.2 Decoction The crude powder of Prasachandaeng remedy and each plant ingredient of this remedy (500g) were boiled in distilled water for 15 minutes and decoction repeated 2 more times. Then it was filtered and the filtrate dried by lyophillizer. These extracts are water extracts. Weight of the extract (g) %Yield = ×100 Weight of dried powder (g)

3.2.2 Quality control The quality control methods of Prasachandaeng remedy and its plant ingredients was performed following Thai herbal pharmacopoeia protocols (Thai herbal pharmacopoeia, 2000). Moisture content, total ash, acid insoluble ash and extractive value were determined. These methods were carried out in triplicate. 3.2.2.1 Loss on drying Moisture content is necessary for standardization of plant material because it affects the quality of raw material and storage. This method analyzed loss on drying by electric moisture analyzer. The sample (2g) is put on moisture analyzer at 105°C. The weight of dried sample is displayed and loss on drying calculated using the following equation:

Weight of beginning sample-Weight of drying sample(g) % Moisture content= × 100 Weight of beginning sample (g)

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3.2.2.2 Total ash This method investigated the physiological ash and non- physiological ash or inorganic compound that may contaminate the raw material. First, the crucible is cleaned, dried and weighed until the weight of crucible is stable. After that, 2 grams of sample was weighed in crucible and burned in muffle furnace at 450°C until the ash changes to gray or white. It was then put in a desiccators to cool. Each crucible was then weighed and re-burned until the weight was stable. Finally, total ash was calculated using the following equation:

Weight of beginning sample-Weight of burned sample (g) % Total ash = x 100 Weight of beginning sample (g)

3.2.2.3 Acid insoluble ash This method was continued from total ash method. First, prepare 10% Hydrochloric acid and then boil ash with 25 ml of 10% HCl for 5 minutes. Next, filter boiled ash by Whatman paper no. 42 and dilute paper to pH 7 with distilled water. After that, dry paper and burn at 450°C in muffle furnace for 9 hours. Then, the sample with crucible was weighed and burned until the weight was stable. Finally, calculate acid insoluble ash using following equation:

Weight of beginning sample- Weight of burned sample (g) % Acid insoluble ash = x 100 Weight of beginning sample (g)

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3.2.2.4 Extractive value The ethanol soluble (ethanolic extract) and water soluble (water extract) was evaluated for extractive value. 5 g of the dried powder was macerated in Erlenmeyer flask. 100 ml of 95% ethanol was added for ethanolic extract and 100 ml of chloroform water added for water extract. The flask was shaken for 6 hours and allowed to stand at room temperature for 18 hours. 20 ml of each extract was filtered, evaporated and dried at 105°C. This process was repeated until the weight is constant. Percentage yields of all extracts was calculated using following equation:

Weight of the extract (g) % Yield = x 100 Weight of dried powder (g)

3.2.3 Antibacterial activity 3.2.3.1 Microbial strains Three types of gram positive bacteria were tested; Staphylococcus aureus (ATCC 25923), Staphylococcus aureus MRSA (DMST 20651) and Streptococcus pyogenes (ATCC 19615), and four types of gram negative bacteria; Escherichia coli (ATCC 25922), Shigella dysenteriae (DMST 151161), Salmonella typhimurium (DMST 22842) and Klebsiella pneumoniae (ATCC 700603). Bacteria were cultured in nutrient agar (NA) at 37°C for 24 hours. 3.2.3.2 Preparation of inoculums Separate colonies of each bacterium were cultured into Mueller- Hinton Broth (MHB) at 37°C for 2 hours. Then suspension turbidity were adjusted to 0.5 McFarland standards. 3.2.3.3 Preparation of test disc The extracts in 95% ethanol were dissolved in dimethysulfoxide (DMSO) to a final concentration 500 mg/ml and the water extracts were dissolved in distilled water to a final concentration of 100 mg/ml. Then, 10 µl of prepared extracts were used on 6 mm sterile paper discs.

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3.2.3.4 Disc diffusion method The agar disc diffusion method was used to screen antibacterial activity of the extracts according to Lorian V., 1996. Sterilized filter paper discs (6 mm in diameter) were contaminated with 10 µl of the extracts. Turbidity to 0.5 McFarland standards will be reached by diluting with Mueller-Hinton Broth (MHB). Then, the inoculum suspension was swabbed, excess fluid removed and the whole Muller-Hinton Agar (MHA) surface was swabbed equally in three directions with sterile cotton swab and left on the plate for 3 – 5 minutes. Then, the dried paper discs were put on the Mueller – Hinton Agar (MHA) with sterile forceps. Plates with bacteria and test samples were incubated at 37°C for 18 – 24 hours. Finally, measurement of the inhibition zone (clear zone) around the disc showed the sensitivity and resistance of the microorganism to test antibacterial activity. The inhibition zone was evaluated by measuring the diameter. Positive control is Gentamicin (Conc. 1 µg). 3.2.3.5 Minimum inhibitory concentration (MIC) The minimal inhibitory concentration (MIC) values were determined by microtiter plate-based assay by Sarker et al, 2007. The ethanolic extracts for testing were dissolved in dimethylsulfoxide (DMSO). Water extracts were dissolved in distilled water and filtered with Millipore filter 0.22 µm (Merck Milipore, Tullagreen). Then, samples were prepared to 10 mg/ml. concentration. S. aureus (ATCC 25923), S. aureus MRSA (DMST 20651), S. pyogenes (ATCC 19615), E. coli (ATCC 25922), S. dysenteriae (DMST 151161), S. typhimurium (DMST 22842) and K.pneumoniae (ATCC 700603) were prepared by culturing for 18 – 24 hours. The inoculum was adjusted for turbidity to 0.5 McFarland standard and diluted with sterile Mueller- Hinton Broth (MHB) at 1:200 to give a final concentration of 5 x 105 CFU/ml. 50 µl of extract solution was added to concentration 5 and 2.5 mg/ml in sterile 96 wells microtiter plates (Corning Inc., USA). The sample was diluted in serial two-fold. Then, 50 µl of the inoculums were added into sterile 96 wells microtiter plates. The plates were covered with a sterile plate and plastic wrap. The substances of the wells are then mixed using a plate shaker and incubated at 37°C for 18 – 24 hours

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Later, 10 µl of resazurin solution (blue compound, 7-hydroxy-3H- phenoxazin-3-one 10-oxide) was added at concentration 1 mg/ml into each well. The plate was incubated at 37°C for 3 hours. The result was a color change of resazurin. MIC value is the lowest concentration of crude extract solution that can inhibit the growth of bacterium by exhibiting a color change in sterile 96 well microtiter plates. The blue color of resazurin shows that the extract has inhibited the bacterium and when the color is purple or pink, the extract has not inhibited the bacterium. The assay was repeated in triplicate. Positive control, negative control and viable control were included. 3.2.3.6 Minimum bactericidal concentration (MBC) Bacterium from wells in microtiter plate were applied onto the Muller- Hinton Agar (MHA) plates. Cell viability was determined by the growth of bacterium after incubating at 37°C for 18 – 24 hours. The MBC values were recorded as the lowest concentration of the extract that prohibits any visible bacteria colony growth on the agar plate.

3.2.4 Anti-inflammation activities 3.2.4.1 Animal cell lines Murine leukemia macrophage cell line (RAW 264.7) was obtained from American Type Culture Collection (ATCC TIB-71). This cell line was cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, penicillin and streptomycin. The cells were incubated at 37°C in a 5% CO2 incubator and sub-passage every 4-5 days. 3.2.4.2 Preparation of sample solution The 95% ethanol extracts were dissolved in sterile dimethylsulfoxide (DMSO) to a final concentration of 50 mg/ml but the water extracts were dissolved in sterile distilled water to a final concentration of 50 mg/ml and filtered with Millipore filter 0.22 µm. Each extract was diluted with RPMI to obtain its final concentration between 1-100 µg/ml.

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3.2.4.3 Assay for NO inhibitory effects in RAW 264.7 cells (Tewtrakul and Itharat, 2007) This modified method was evaluated the inhibition of release of nitric oxide (NO) that is produced by mouse macrophage leukemia-like (RAW 264.7) in inflammation conditions. The cells (RAW 264.7) were cultured in a flask with RPMI 1640 medium containing 10% FBS, penicillin (100 unites/ml) and streptomycin (100 unites/ml). RAW 264.7 cells were washed by phosphate buffer saline (PBS) and suspended by 0.25% trypsin-EDTA. The cells were cultured in 96-well plate sterile

(1x105 cells/well) with 100 µl complete RPMI and incubated in 5% CO2, 37°C overnight. Complete RPMI (100 µl/well) containing 10 ng/ml of lipopolysaccharide (LPS) was replaced in control and only complete RPMI was replaced in normal. Next, 100 µl/well of each sample was added following the concentration, added 100 µl/well of complete RPMI in control medium, 100 µl/well of 0.2% DMSO in control solvent, then incubated overnight. Supernatant 100 µl was transferred to another sterile 96-well plate, followed by 100 µl of Griess reagent. The NO production was determined by measuring the accumulation of nitrite which interacted with Griess reagent. The absorbance was measured by spectrophotometer at wavelength 570 nm. This method was carried out in triplicate. The inhibition (%) was calculated using the following equation and IC50 value was calculated using Prism program.

C - S % Inhibition = x 100 C

Control (C) : [LPS (+), sample (-)] - [LPS (-), sample (-)] Sample (S) : [LPS (+), sample (+)] - [LPS (-), sample (+)]

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3.2.4.4 MTT assay (Tewtrakul and Itharat, 2007) MTT assay was determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5- diphenyl-2H-tetrazolium bromine (MTT) colorimetric method. This method continues from NO assay that was used to determine cytotoxicity against cells. Briefly, after incubated for 24 hours and tested with sample MTT solution (10 µl, 5mg/ml MTT in PBS) was added in each well and incubated 2 hours. After that, supernatant was removed and 100µl of isopropanol added containing 0.04 M HCl to dissolve the formazan production in cells. The density of formazan solution was measured by micro spectrophotometer at wavelength 570 nm. A density of treated sample less than 70% that was considered as toxic.

C - S % Toxicity = x 100 C

Control (C) : LPS (-), sample (-) Sample (S) : LPS (-), sample (+)

3.2.5 The stability test of Prasachandaeng extract The samples were put into transparent vials and kept in accelerated conditions (40±2°C, 75±5% RH) for 6 months period. Then, samples were withdrawn after 6 month periods (0, 15, 30, 60, 90,120,150 and 180 days) and each tested for biological activities.

3.2.6 Statistical analysis The results are completed in triplicate. Values of different variables are shown as the mean ± standard error of the mean. Statistical analysis was calculated using Prism Software.

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CHAPTER 4 RESULTS AND DISCUSSION

4.1 Preparation of crude extract

4.1.1 Percentage of yield The ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients were prepared by using maceration and decoction methods as reported in Chapter 3 (section 3.2.1). The extraction methods are related to Thai folk doctor’s uses. Most of the crude ethanolic extracts were gained in the forms of viscous liquid or viscous solid. Most of the crude water extracts were gained in the forms of powder or gluey mass. The percentages of yield of Prasachandaeng remedy and its plant ingredients are shown as percentage by weight in Table 4-1 The percentage yields of all ethanolic extracts were in range from 1.24% to 22.76%. The highest percentage yield of the ethanolic extracts was Dracaena loureiri (22.76%) and the lowest percentage yield of the ethanolic extracts was Myristica fragrans (1.24%). The percentage yields of all water extracts were in range from 0.85% to 27.29%. The highest percentage yield of the water extracts was Ligusticum chuanxiong (27.29%) and the lowest percentage yield of the water extracts was Myristica fragrans (0.85%). The percentage yield of the ethanolic and water extracts of Prasachandaeng remedy were 17.29% and 3.35%, respectively. Most of the ethanolic extracts were higher in percentage yield than the water extracts.

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Table 4-1 The percentage yields of the ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients

Thai Ethanolic extract Water extract Sample name Code %Yield Code %Yield Bouea macrophylla Griff. มะปราง MPE 2.30 MPW 1.32 Caesalpinia sappan L. ฝาง FSE 12.42 FSW 1.95 Citrus aurantifolia Swing. มะนาว MNE 1.70 MNW 3.52 Dracaena loureiri Gagnep. จันทน์แดง JDE 22.76 JDW 5 Helicia terminalis Kurz. เหมือดคน MKE 2.73 MKW 3.77 Jasminum sambac L. มะลิ MLE 8.18 MLW 25.78 Kaempferia galanga L. เปราะหอม PHE 5.25 PHW 9.83 Ligusticum chuanxiong Hort. โกฐหัวบัว KHBE 11.87 KHBW 27.30 Mammea siamensis Kosterm. สารภี SRPE 17.134 SRPW 14.35 Mesua ferrea L. บุนนาค BNE 18.61 BNW 12.20 Myristica fragrans Houtt. จันทน์เทศ JTE 1.24 JTW 0.85 Nelumbo nucifera Gaertn. บัวหลวง BLE 8.77 BLW 15.70 ประสะ Prasachandaeng remedy PCE 17.29 PCW 3.35 จันทน์แดง

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25 22.76

20 18.61 17.29 17.13

15 12.42 11.87

10 8.77

8.18 Yield of the extracts (%)the of extracts Yield 5.25 5 2.73 2.30 1.70 1.24

0 JDE BNE PCE SRPE FSE KHBE BLE MLE PHE MKE MPE MNE JTE

The ethanolic extracts of Prasachandaeng remedy and its palnt ingredients

Figure 4-1 The percentage yield of the ethanolic extracts of Prasachandaeng remedy and its plant ingredients

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30 27.29 25.78 25

20

15.71 15 14.35 12.20 9.83

10 Yield of the extracts (%)the of extracts Yield

5.00 5 3.77 3.52 3.35 1.95 1.32 0.85 0 KHBW MLW BLW SRPW BNW PHW JDW MKW MNW PCW FSW MPW JTW

The water extracts of Prasachandaeng remedy and its palnt ingredients

Figure 4-2 The percentage yield of the water extracts of Prasachandaeng remedy and its plant ingredients

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4.2 Quality control

Quality controls of Prasachandaeng remedy and its plant ingredients consist of loss on drying, total ash, acid insoluble ash and extractive values according to the standard values set by the Thai Herbal Pharmacopoeia (Thai herbal pharmacopeia, 2009)

4.2.1 Loss on drying The standard value of Thai herbal Pharmacopoeia (THP) shown that loss on drying is ≤10% after drying at 105ºC to steady weight. The results of loss on drying of Prasachandaeng remedy and its plant ingredients are in standard criteria of THP and show in Table 4-2. The percentage loss on drying of all plant ingredients ranged from 4.44% to 8.98%. The highest percentage loss on drying was Jasminum sambac (8.98±0.39%) and the lowest percentage loss on drying was Dracaena loureiri (4.44±0.37%). The percentage on loss on drying of Prasachandaeng remedy was 6.74±0.67%. 4.2.2 Total ash The standard value of Thai Herbal Pharmacopoeia (THP) shown that total ash content is ≤10% after burning at 450ºC to steady weight. The results of total ash content of Prasachandaeng remedy and its plant ingredients are in standard criteria of THP and show in Table 4-2. The percentage total ash of all plant ingredients range from 2.19% to 9.56%. The highest percentage total ash was Ligusticum chuanxiong (9.56±0.03%) and the lowest percentage total ash was Helicia terminalis (2.19±0.08%). The percentage on total ash of Prasachandaeng was 5.69±0.08%.

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4.2.3 Acid insoluble ash The standard value of Thai Herbal Pharmacopoeia shown that acid insoluble ash is ≤2% after burning at 500ºC to steady weight. The results of acid insoluble ash content of Prasachandaeng remedy and all its plant ingredients are in standard criteria of THP which was shown in Table 4-2. The percentage acid insoluble ash of all pant ingredients range from 0.44% to 1.79%. The highest percentage acid insoluble ash was Myristica fragrans (1.79±0.02%) and the lowest percentage acid insoluble ash was Mammea siamensis (0.44±0.02%). Prasachandaeng remedy of the percentage acid insoluble ash was 1.78±0.03% 4.2.4 Extractive value The percentage of ethanol-soluble extractive values range from 0.42% to 33.05%. The highest percentage of ethanol-soluble extractive values was Dracaena loureiri (33.05±0.12%) and the lowest percentage of ethanol-soluble extractive values was Kaempferia galangal (0.42±0.05%). The percentage of water-soluble extractive values range from 1.43% to 25.74%. The highest percentage of water-soluble extractive values was Caesalpinia sappan (25.74±0.33%) and the lowest percentage of water- soluble extractive values was Myristica fragrans (1.43±0.46%). Prasachandaeng remedy of the percentage ethanol and water-soluble extractive values were 30.38±0.14% and 17.81±0.17%, respectively. Most of ethanol-soluble extractive values were higher than water-soluble extractive values.

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Table 4-2 Results of quality controls of prasachandaeng remedy and its plant ingredients (mean±SD), (n=3) Thai %Loss on %Ash contents %Extractive values Herbals Code name drying Total ash Acid insoluble ash Ethanol-soluble Water-soluble Bouea macrophylla Griff. มะปราง MP 8.75±0.34 3.84±0.08 0.46±0.09 6.08±0.09 9.47±0.95 Caesalpinia sappan L. ฝาง FS 8.41±0.40 5.31±0.08 0.52±0.01 13.40±0.45 25.74±0.33 Citrus aurantifolia Swing. มะนาว MN 8.06±0.44 5.91±0.02 0.59±0.04 4.04±0.03 8.98±0.13 Dracaena loureiri Gagnep. จันทน์แดง JD 4.44±0.37 4.46±0.03 0.33±0.03 33.05±0.12 3.69±0.13 Helicia terminalis Kurz. เหมือดคน MK 8.17±0.56 2.19±0.08 0.41±0.08 9.62±0.06 9.67±0.42 Jasminum sambac L. มะลิ ML 8.98±0.39 8.36±0.09 0.73±0.01 7.58±0.13 13.73±0.46 Kaempferia galanga L. เปราะหอม PH 6.91±0.20 5.53±0.04 1.33±0.04 0.42±0.05 9.49±0.38 Ligusticum chuanxiong Hort. โกฐหัวบัว KHB 8.12±0.20 9.56±0.03 0.47±0.05 6.36±0.02 7.83±0.81 Mammea siamensis Kosterm. สารภี SRP 8.96±0.64 7.97±0.03 0.44±0.02 14.36±0.20 17.14±0.18 Mesua ferrea L. บุนนาค BN 8.08±0.56 5.02±0.02 1.50±0.04 9.73±0.13 6.59±0.26 Myristica fragrans Houtt. จันทน์เทศ JT 7.88±0.62 8.18±0.02 1.79±0.02 2.53±0.08 1.43±0.46 Nelumbo nucifera Gaertn. บัวหลวง BL 8.90±0.47 8.02±0.06 1.70±0.03 6.92±0.22 6.86±0.13 ประสะ Prasachandaeng remedy PC 6.74±0.67 5.69±0.08 1.78±0.03 30.38±0.14 17.81±0.17 จันทน์แดง

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12

10 8.98 8.96 8.90 8.75 8.41 8.17 8.08 8.06 7.88 8.12 8 6.91 6.74

6 4.44

Loss on dryingLoss(%) on 4

2

0 ML SRP BL MP FS MK KHB BN MN JT PH PC JD

Prasachandaeng remedy and its plant ingredients

Figure 4-3 The loss on drying (%) of Prasachandaeng remedy and its plant ingredients

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12

10 9.56

8.36 8.18 8.02 7.97 8

5.91 5.69 6 5.53 5.31

5.02 Total ash (%) ash Total 4.46 4 3.84

2.19 2

0 KHB ML JT BL SRP MN PC PH FS BN JD MP MK

Prasachandaeng remedy and its plant ingredients

Figure 4-4 The total ash contents of Prasachandaeng remedy and its plant ingredients 54

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2 1.79 1.78 1.8 1.70

1.6 1.50

1.4 1.33

1.2

1

0.8 0.73 0.59 0.6 0.52 0.46

Acid insoluble ash (%)insoluble Acid ash 0.47 0.44 0.41 0.4 0.33

0.2

0 JT PC BL BN PH ML MN FS KHB MP SRP MK JD

Prasachandaeng remedy and its plant ingredients

Figure 4-5 The acid insoluble ash contens of prasachandaeng remedy and its plant ingredients

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35 33.05 30.38 30

25.74 25

20 17.14 17.81 14.36 15 13.40 13.73 9.67 9.47 9.62 9.49 9.73 Extractive values(%) Extractive 10 8.98 7.83 6.92 7.58 6.86 6.08 6.36 6.59 4.04 5 3.69 2.53 1.43 0.42 0 MP FS MN JD MK ML PH KHB SRP BN JT BL PC

Ethanol-soluble extractive Water-soluble extractive

Prasachandaeng remedy and its plant ingredients

Figure 4-6 The extractive values of Prasachandaeng remedy and its plant ingredient 56

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4.3 Antibacterial activities

4.3.1 Disc diffusion method All extracts were tested for their antibacterial activity by disc diffusion method to define the inhibition zone against three gram positive bacteria (S. aureus ATCC 25923, S. aureus MRSA DMST 20651 and S. pyogenes ATCC 19615) and four gram negative bacteria (E. coli ATCC 25922, S. dysenteriae DMST 151161, S. typhimurium (DMST 22842) and K. pneumoniae ATCC 700603) that relate fever symptom. The results of disc diffusion method show in Table 4-3. For S. aureus, the ethanolic extract of FS showed the most against S. aureus with inhibition zone of 21.67±1.53 mm and the ethanolic extract of BN and the water extract of BL showed the lowest against S. aureus with inhibition zone of 7.33±0.58 mm. The ethanolic and water extracts of MP, MN, MK, ML, PH, KHB and JT had no activity against S. aureus. The ethanolic extract of FS showed the effective against S. aureus with inhibition zone as norfloxacin (positive control) 21.00±0.00 mm. For MRSA, the ethanolic extract of FS showed the most against MRSA with inhibition zone of 26.00±1.00 mm and the water extract of BL showed the lowest against MRSA with inhibition zone of 7.33±0.58 mm. The ethanolic and water extracts of MP, MN, MK, ML, PH, KHB, BN, JT and positive control had no activity against MRSA. For S. pyogenes, the water extract of FS showed the most against S. pyogenes with inhibition zone of 22.67±0.58 mm and the water extract of ML showed the lowest against S. pyogenes with inhibition zone of 7.00±0.00 mm. The ethanolic and water extracts of MP, PH and JT had no activity against S. pyogenes. The water extract of FS showed the effective against S. pyogenes with inhibition zone nearby clindamycin (positive control) 23.00±0.00 mm. For E. coli and S. typhimurium, FS had only activity while S. dysenteriae, FS and the water extract of JD had activity. The ethanolic extracts of FS showed the activity against with inhibition zone of 11.67±1.15, 14.33±1.15 and 21.00±0.00 mm, respectively.

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The ethanolic extract of FS showed the most effective against S. aureus, MRSA, E. coli, S. dysenteriae and S. typhimurium, except the water extract of FS showed the most effective against S. pyogenes. For K. pneumonia, neither ethanolic nor water extracts of Prasachandaeng remedy and its plant ingredients were activity to inhibit K. pneumonia while gentamicin and norfloxacin (positive control) showed antibacterial activity with inhibition zone of 12.00±0.00 and 19.00±0.00 mm, respectively. In addition, Prasachandaeng remedy had no activity against negative bacterial; E. coli, S. dysenteriae, S. typhimurium and K. pneumonia. However, the ethanolic extract of Prasachandaeng remedy had activity to against MRSA with inhibition zone of 11.67±0.58 mm while positive control had no activity.

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Table 4-3 Antibacterial activity of the ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients by disc diffusion method (mean±SD), (n=3) Inhibition zone (mm) Herbals Code S.aureus MRSA S.pyogenes E.coli S.dysenteriae S.typhimurium K.pneumonia MPE NI NI NI NI NI NI NI MP MPW NI NI NI NI NI NI NI FSE 21.67±1.53 26.00±1.00 17.00±0.00 11.67±1.15 21.00±0.00 14.33±1.15 NI FS FSW 18.00±1.00 20.00±1.00 22.67±0.58 7.00±0.00 13.00±0.00 9.00±0.00 NI MNE NI NI NI NI NI NI NI MN MNW NI NI 7.33±0.58 NI NI NI NI JDE 9.67±0.58 10.67±0.58 6.67±0.58 NI 7.67±0.58 NI NI JD JDW NI 8.67±0.58 11.33±0.58 NI NI NI NI MKE NI NI NI NI NI NI NI MK MKW NI NI 8.33±0.58 NI NI NI NI MLE NI NI NI NI NI NI NI ML MLW NI NI 7.00±0.00 NI NI NI NI PHE NI NI NI NI NI NI NI PH PHW NI NI NI NI NI NI NI NI = No inhibition zone

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Table 4-3 (continued) Inhibition zone (mm) Herbals Code S.aureus MRSA S.pyogenes E.coli S.dysenteriae S.typhimurium K.pneumonia KHBE NI NI NI NI NI NI NI KHB KHBW NI NI 10.67±0.58 NI NI NI NI SRPE 8.33±0.58 10.67±1.15 NI NI NI NI NI SRP SRPW NI NI 11.33±0.58 NI NI NI NI BNE 7.33±0.58 NI NI NI NI NI NI BN BNW NI NI 7.33±0.58 NI NI NI NI JTE NI NI NI NI NI NI NI JT JTW NI NI NI NI NI NI NI BLE NI NI NI NI NI NI NI BL BLW 7.33±0.58 7.33±0.58 7.33±0.58 NI NI NI NI PCE 10.00±1.00 11.67±0.58 7.67±0.58 NI NI NI NI PC PCW NI NI 12.67±0.58 NI NI NI NI Gentamicin Gen 22.00±0.00 NI 13.00±0.00 20.00±0.00 20.00±0.00 20.00±0.00 12.00±0.00 Ampicilin Amp 26.00±0.00 NI 27.00±0.00 18.00±0.00 20.00±0.00 20.00±0.00 NI Norfloxacin Nor 21.00±0.00 NI 17.00±0.00 30.00±0.00 30.00±0.00 33.00±0.00 19.00±0.00 Clindamycin Clin 25.00±0.00 NI 23.00±0.00 NI NI NI NI

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NI = No inhibition zone

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4.3.2 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by microtitre plate-based antibacterial assay All extracts were tested for antibacterial activity by microtitre plate- based antibacterial assay to define minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against three gram positive bacteria (S. aureus ATCC 25923, S. aureus MRSA DMST 20651 and S. pyogenes ATCC 19615) and four gram negative bacteria (E. coli ATCC 25922, S. dysenteriae DMST 151161, S. typhimurium (DMST 22842) and K.pneumoniae ATCC 700603). MIC and MBC values of all extracts are shown in Table 4-4. For S. aureus, the ethanolic extract of SRP showed the most against S. aureus with MIC and MBC values of 0.009 and 0.078 mg/ml, respectively and the ethanolic and water extracts of MN, ML, PH and JT had no activity against S. aureus. For MRSA, the ethanolic extract SRP showed the most against MRSA with MIC and MBC values of 0.004 and 0.019 mg/ml, respectively and the ethanolic and water extracts of MN, ML, PH and JT and gentamicin had no activity against MRSA. For S. pyogenes, the ethanolic extract SRP showed the most against S. pyogenes with MIC and MBC values of 0.001 and 0.019 mg/ml, respectively. All ethanolic extracts had activity against S. pyogenes. For E. coli, S. typhimurium and K.pneumoniae, only the ethanolic of FS had activity against E. coli, S. typhimurium and K.pneumoniae. The ethanolic extract of FS also showed the most against S. typhimurium with MIC and MBC values of 0.625 and 0.625 mg/ml, respectively. While, gentamicin had no activity against S. typhimurium. For S. dysenteriae, the ethanolic extract of FS, JD and Prasachandaeng remedy had activity against S. dysenteriae. The water extract of FS showed the most against S. dysenteriae with MIC and MBC values of 0.15 and 0.312 mg/ml, respectively. In gram positive bacterial, the ethanolic extract of SRP showed the highest activity against S. pyogenes with MIC and MBC of 0.001 and 0.019 mg/ml. In gram negative bacterial, the ethanolic extract of FS showed the highest activity against S. typhimurium with MIC and MBC values of 0.625 and 0.625 mg/ml. Although, Prasachandaeng remedy showed less effective to against both gram positive and gram negative, it had more efficiency than gentamicin to against MRSA.

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Table 4-4 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients by using microtitre plate-based antibacterial assay (n = 3) Minimum inhibitory concentration and minimum bactericidal concentration (mg/ml) Herbals Code S.aureus MRSA S.pyogenes E.coli S.dysenteriae S.typhimurium K.pneumonia MPE 5.00/5.00 NI 1.25/1.25 NI NI NI NI MP MPW NI 2.50/2.50 NI NI NI NI NI FSE 0.156/0.156 0.078/0.625 0.078/0.312 1.25/1.25 0.15/1.25 0.625/0.625 1.25/5.00 FS FSW 0.078/1.25 0.078/0.078 0.078/0.625 1.25/2.50 0.15/0.312 0.625/1.25 2.50/5.00 MNE NI NI 0.625/0.625 NI NI NI NI MN MNW NI NI 5.00/>5.00 NI NI NI NI JDE 5.00/5.00 1.25/1.25 0.625/1.25 NI 5.00/5.00 NI NI JD JDW 5.00/>5.00 1.25/1.25 2.50/5.00 NI 2.5/2.5 NI NI MKE 1.25/1.25 0.625/1.25 0.312/0.312 NI NI NI NI MK MKW NI NI NI NI NI NI NI MLE NI NI 0.625/0.625 NI NI NI NI ML MLW NI NI NI NI NI NI NI PHE NI NI 0.625/0.625 NI NI NI NI PH PHW NI NI NI NI NI NI NI NI = No inhibition

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Table 4-4 (continued) Minimum inhibitory concentration and minimum bactericidal concentration (mg/ml) Herbals Code S.aureus MRSA S.pyogenes E.coli S.dysenteriae S.typhimurium K.pneumonia KHBE 5.00/5.00 2.50/2.50 0.312/0.312 NI NI NI NI KHB KHBW NI NI NI NI NI NI NI SRPE 0.009/0.078 0.004/0.019 0.001/0.019 NI NI NI NI SRP SRPW NI NI NI NI NI NI NI BNE 0.625/2.50 0.156/0.625 1.25/2.50 NI NI NI NI BN BNW 5.00/>5.00 2.50/2.50 NI NI NI NI NI JTE NI NI 0.312/0.312 NI NI NI NI JT JTW NI NI NI NI NI NI NI BLE NI NI 0.625/0.625 NI NI NI NI BL BLW 5.00/>5.00 1.25/1.25 NI NI NI NI NI PCE 5.00/5.00 0.312/2.50 0.625/0.625 NI 5.00/5.00 NI NI PC PCW 5.00/>5.00 1.25/1.25 2.50/5.00 NI 5.00/5.00 NI NI Gentamicin* Gen 0.195/0.195 >100/>100 0.391/0.391 1.562/1.562 0.781/0.781 >100/>100 6.25/625 NI = No inhibition, * = positive control (µg/ml)

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4.4 Anti-inflammatory activity

4.4.1 Assay for NO inhibitory effects in RAW 264.7 cells Anti-inflammatory activity of the ethanolic and water extracts of Prasachandaeng remedy and its plant ingredients were tested by inhibitory effects on LPS-induced nitric oxide (NO) release from murine macrophages cell lines (RAW 264.7). Evaluation of nitric oxide (NO) production was performed by using Griess reaction and cytotoxicity was performed by MTT assay. The results were shown in Table 4-5.

The ethanolic extract of FS showed the highest IC50 value is 5.42 ±

0.24 µg/ml and the ethanolic extract of JD showed the lowest IC50 value is 69.97 ± 3.10 µg/ml. The ethanolic extracts of MP, MN, MK, ML, JT and BL had no activity on NO inhibitory effects in RAW 264.7 cells.

The water extract of FS showed the highest IC50 value is 6.13 ± 0.19

µg/ml and the water extract of KHB showed the lowest IC50 value is 60.54 ± 1.73 µg/ml. The water extracts of ML, BN, and JT had no activity on NO inhibitory effects in RAW 264.7 cells. Only, the ethanolic and water extracts of ML and JT had no activity on NO inhibitory effects in RAW 264.7 cells. In addition, the ethanolic extract of PC remedy showed the IC50 value is 39.7 ± 1.48 µg/ml less than the water extract of PC remedy showed the IC50 value is 16.87 ± 2.51 µg/ml.

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Table 4-5 Anti-inflammatory activities by Griess reagent and MTT assay on mouse leukemic macrophage cell lines (RAW 264.7).

% Inhibition of NO production / (% Toxicity) IC50 Herbals Code 0.1 µg/ml 1 µg/ml 5 µg/ml 10 µg/ml 30 µg/ml 50 µg/ml 70 µg/ml 100 µg/ml (µg/ml) 1.85±0.35 B. MPE ------>100 (-14.96±0.64) macrophylla 22.16±8.23 25.22±6.81 50.87±0.82 59.81±2.21 MPW - - - - 49.06±1.51 (-3.5±3.21) (2.00±2.13) (1.92±1.44) (3.13±1.06) 2.93±0.22 8.29±0.48 46.00±2.17 89.26±0.80 FSE - - - - 5.42±0.24 C.sappan (-0.75±0.23) (0.00±0.36) (0.15±0.41) (0.92±0.00) 26.22±0.77 65.55±0.99 81.89±0.93 96.58±0.71 FSW - - - - 6.13±0.19 (8.64±2.82) (17.68±1.65) (20.18±3.12) (25.18±2.49) 14.69±1.67 C. MNE ------>100 (28.17±0.93) aurantifolia 35.07±1.93 38.61±0.84 55.97±1.87 96.20±1.53 MNW - - - - 38.65±3.61 (6.26±3.04) (12.47±3.90) (17.94±2.60) (21.30±3.09) 14.91±16.05 9.18±6.43 39.51±2.22 74.59±2.73 JDE - - - - 69.97±3.10 D.loureiri (-7.96±8.93) (-6.03±8.27) (-0.16±5.96) (9.08±8.99) 37.34±0.74 44.35±1.47 73.35±2.43 83.51±1.63 JDW - - - - 18.00±3.39 (0.09±0.23) (7.42±5.05) (20.11±0.88) (28.26±0.45) 35.05±0.97 H.terminalis MKE ------>100 (-4.18±2.34) 29.22±5.11 46.59±1.67 73.00±5.78 83.60±2.69 MKW - - - - 11.98±0.71 (-8.62±2.15) (-2.98±2.16) (2.47±1.19) (8.23±2.45) 34.53±2.06 MLE ------>100 J.sambac (35.58±2.28) 15.31±2.67 MLW ------>100 (-2.30±3.33) Note: All data represents the mean ± SEM in triplicate experiments. 65

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Table 4-5 (continued)

% Inhibition of NO production / (% Toxicity) IC50 Herbals Code 0.1 µg/ml 1 µg/ml 5 µg/ml 10 µg/ml 30 µg/ml 50 µg/ml 70 µg/ml 100 µg/ml (µg/ml) 32.75±1.08 35.74±1.64 56.44±1.48 91.50±0.29 K. PHE - - - - 39.25±0.61 (-8.24±0.76) (-1.43±0.84) (5.31±0.33) (7.55±0.37) galanga 15.01±5.06 60.59±0.29 87.66±3.71 92.57±3.73 PHW - - - - 7.60±0.28 (1.55±0.25) (11.30±3.47) (14.37±4.09) (27.67±1.16) 0.89±0.39 7.37±1.01 55.37±1.56 94.727±0.25 KHBE - - - - 45.69±0.89 L. (-0.65±0.04) (1.52±0.64) (3.24±0.04) (4.71±0.52) chuanxiong 21.83±5.81 24.13±6.83 44.84±1.82 63.06±1.65 KHBW - - - - 60.54±1.73 (2.59±0.25) (3.13±0.35) (7.64±1.03) (11.72±1.78) 27.94±8.48 65.40±10.56 80.25±2.63 90.02±3.26 SRPE - - - - 6.53±2.68 M. (13.82±4.52) (19.63±0.12) (20.71±0.82) (24.87±2.10) siamensis 7.31±0.07 15.94±1.23 62.46±3.31 79.9±2.44 SRPW - - - - 38.44±2.65 (-14.58±2.28) (-7.77±3.51) (2.66±1.83) (6.24±0.78) 17.61±12.60 36.51±3.75 52.29±0.85 67.55±4.44 BNE - - - - 46.43±1.80 M.ferrea (-10.11±1.76) (-4.53±1.37) (-0.81±0.15) (7.51±2.26) 47.52±1.33 BNW ------>100 (13.61±2.36) 13.04±1.73 M. JTE ------>100 (-18.95±0.52) fragrans 33.83±1.60 JTW ------>100 (-5.75±1.49) 13.39±0.72 N. BLE ------>100 (-12.02±0.23) nucifera -2.46±0.10 3.57±0.20 53.49±1.41 74.57±2.12 BLW - - - - 34.13±0.26 (-0.75±0.12) (6.31±1.37) (14.94±2.39) (17.16±0.50) Note: All data represents the mean ± SEM in triplicate experiments. 66

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Table 4-5 (continued)

% Inhibition of NO production / (% Toxicity) IC50 Herbals Code 0.1 µg/ml 1 µg/ml 5 µg/ml 10 µg/ml 30 µg/ml 50 µg/ml 70 µg/ml 100 µg/ml (µg/ml) 25.60±6.21 27.83±5.58 57.48±0.34 69.76±8.67 PCE - - - - 39.70±1.48 PC (-2.81±1.13) (2.76±1.96) (7.24±2.42) (10.00±2.21) 13.40±0.81 37.22±2.59 75.12±1.11 85.23±1.96 PCW - - - - 16.87±2.51 (-0.47±0.97) (2.03±0.75) (10.71±0.06) (15.77±1.90) 36.33±4.22 46.85±4.40 56.95±2.37 73.91±3.19 Prednisolone Pred - - - - 2.12±1.08 (2.89±0.59) (7.21±0.93) (8.11±0.74) (8.57±0.80) Note: All data represents the mean ± SEM in triplicate experiments.

67

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100

90

80 69.97 70

60

50

(µg/ml) 45.69 46.43 50 39.25 39.7 IC 40

30

20

10 5.42 6.53 2.12 0 Pred FSE SRPE PHE PCE KHBE BNE JDE MPE MNE MKE MLE JTE BLE The ethanolic extracts of Prasachandaeng remedy and its plant ingredients.

Figure 4-7 Anti-inflammatory activity (IC50 (µg/ml) ± SEM) by Griess reagent on RAW 264.7 (N=3) of ethanolic extracts 68

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100

90

80

70 60.54 60

49.06

50

(µg/ml) 50

IC 38.65 40 38.44 34.13

30

20 16.87 18 11.98 10 6.13 7.6 2.12 0 Pred FSW PHW MKW PCW JDW BLW SRPW MNW MPW KHBW MLW BNW JTW The water extracts of Prasachandaeng remedy and its plant ingredients.

Figure 4-8 Anti-inflammatory activity (IC50 (µg/ml) ± SEM) by Griess reagent on RAW 264.7 (N=3) of water extracts 69

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4.5 The stability test of Prasachandaneng extracts

The ethanolic and water extracts of Prasachandaeng remedy were studied for stability according to Thai FDA guideline. The samples on the beginning day 0 (control sample), day 15, day 30, day 60, day 90, day 120, day 150 and day 180 were controlled under 40±2ºC with 75±5% RH as accelerated testing for 6 months period. The objective of this study was investigate whether the ethanolic and water extracts of Prasachandaeng remedy are stable when kept in a closed container protected from light and stored at the room temperature for at least 2 years. 4.5.1 The stability test of Prasachandaeng water extracts for anti- inflammatory by inhibition Nitric oxide production release from RAW264.7 cell lines The water extracts of Prasachandaeng remedy on the beginning day 0 (control sample), day 15, day 30, day 60, day 90, day 120, day 150 and day 180 were kept under accelerated condition at 40±2ºC with 75±5% RH and were tested for anti- inflammatory. The results showed that the water extract of Prasachandaeng remedy on day 15, 30 and 60 were not different by inhibition NO production release from RAW264.7 cell lines from day 0. The results were shown in Table 4-6.

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Table 4-6 Anti-inflammatory activity by Griess reagent and MTT assay on RAW 264.7 of the stability test of Prasachandaeng water extract (mean±SEM) (n=3) Inhibition (%) at different concentration (µg/ml)/ IC50 (µ Sample (Toxicity (%) at different concentration (µg/ml)) µg/ml) 1 10 50 100

Day 0 38.25±0.63 49.34±0.31 73.43±2.13 84.22±1.68 10.62±0.29 (1.58±0.10) (6.56±0.24) (14.10±1.76) (16.71±2.26) Day 15 30.85±0.25 46.36±0.78 71.47±0.72 86.08±1.34 12.58±0.65 (1.86±0.10) (6.51±0.17) (14.29±0.75) (16.92±1.29) Day 30 33.11±2.43 42.87±0.43 75.05±1.85 84.83±2.02 17.19±0.85 (1.21±0.03) (6.41±0.13) (15.02±1.63) (16.96±1.31) Day 60 39.92±2.19 43.67±0.78 77.89±1.51 86.88±2.69 19.15±0.24 (1.31±0.54) (4.42±1.67) (15.56±0.43) (16.83±0.88) Day 90 38.65±2.69 41.40±0.29 78.29±1.45 88.98±0.34 21.92±0.92 (1.05±0.37) (3.25±1.78) (15.78±1.64) (18.90±0.43) Day 120 10.10±4.03 12.96±2.60 58.65±0.27 75.01±1.40 42.56±0.26 (0.40±0.16) (0.70±0.06) (2.08±0.68) (2.63±0.76) Day 150 9.8±3.91 12.46±2.34 55.65±1.36 72.86±0.28 44.90±0.82 (0.39±0.16) (0.63±0.10) (1.66±0.46) (2.73±0.62) Day 180 11.88±4.79 14.31±3.03 52.91±0.39 75.20±0.70 47.03±0.33 (0.50±0.27) (0.82±0.10) (2.00±0.57) 2.81±0.69

60

47.03 50 44.9 42.56

40

30 (µg/ml)

50 50 21.92 19.15 IC 20 17.19 12.58 10.62 10

0 Day 0 Day 15 Day 30 Day 60 Day 90 Day 120 Day 150 Day 180 Time (Days) of accelerate conditions

Figure 4-9 Anti-inflammatory activities by Griess reagent and MTT assay on RAW 264.7 of the stability test of Prasachandaeng water extract (mean±SEM) (n=3)

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CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS

Prasachandaeng (PC) remedy has long been used for relief of fever and internal heat. It is composed with 12 plants as stem of Chan Daeng (Dracaena loureiri Gagnep.), root of Mueng Kon (Helicia terminalis Kurz.), root of Ma Now (Citrus aurantifolia Swing.), root of Ma Prang Wan (Bouea macrophylla Griff.), rhizome of Phro Hom (Kaempferia galanga L.), rhizome of Kot Hua Bua (Ligusticum chuanxiong Hort), stem of Chan Tet (Myristica fragrans L.), stem of Fang Saeng (Caesalpinia sappan L.), pollen of Bua Luang (Nelumbo nucifera Gaertn.), flower of Boon Nag (Mesua ferrea L.), flower of Sa Ra Pee (Mammea siamensis Kosterm.), and flower of Ma Li (Jasminum sambac Lour.). It is in the National List of Essential Medicines in Thailand, but there is no report for anti-inflammatory activity of this remedy. Some plant ingredients in this remedy were investigated for antibacterial and anti-inflammatory activities but there is no report of PC and some plant ingredients in PC remedy. Thus, the objectives of this research were to study on antibacterial and anti- inflammatory activities of PC remedy extracts by different extraction method and its plant ingredients. Plant raw material which are ingredients of PC remedy and PC were tested for standardization following Thai Herbal Pharmacopoeia (THP). The results of standard values of PC and its all plant ingredients were in standard criteria of THP (Total ash value, acid insoluble ash and loss on dying are less than 10, 2 and 10%, respectively). These results indicated that plant material less contamination from sand and heavy metal and less moisture content which is mention for less for contaminate with microbe. The extraction method of PC and plant ingredients were imitated from using in Thai traditional medicine as maceration in 95% ethanol as ethanolic extract and decoction by water as water extract. All extracts were tested antibacterial activity which is cause of fever was determined by using disc diffusion method to determine the inhibition zone and microtitre plate-based antibacterial assay to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Anti-inflammatory activity which is also cause of fever was also tested by using inhibition of NO product on RAW264.7 cell line. The results of this study on

Ref. code: 25595611031526QZI 73 antibacterial activity showed that the ethanolic extract of Prasachandaeng (PC) remedy can inhibit the growth of gram positive but no inhibit gram negative. Especially, this extract showed high inhibitory activity against S.aureus MRSA with MIC 0.312 mg/ml. MIC of gentamycin as positive control is more than 0.1 mg/ml. However, the water extract of PC had no antibacterial activity against all strain microbe. This result can be used to support using the ethanolic extract of PC remedy to be a good choice for against microbe as resistance strain. For plant ingredients of PC which showed the best inhibitory activity against all microbes was Mammea siamensis flowers and Caesalpinia sappan stems. The 95% ethanol extract of Mammea siamensis flowers has ever been reported the ability to inhibit the growth of gram positive bacterial, it showed antibacterial activity against S.aureus and S.aureus MRSA with MIC values as 1.25, 0.625 mg/ml, respectively (Sattaponpan, 2011). However, in this study, the 95% ethanol extract of Mammea siamensis flowers showed higher antibacterial activity than the previously reported against S.aureus and S.aureus MRSA with MIC values of 0.009 and 0.004 mg/ml, respectively. This result may be cause from collecting time, land of growing plant which are effect for ingredients and biological activity. The 95% ethanol and water extracts of Caesalpinia sappan stems showed the ability to inhibit the growth of both gram positive and negative bacterial. The previously work reports found that the chloroform, n-butanol, methanol and water extracts of the Caesalpinia sappan showed an antibacterial activity against standard methicillin-sensitive Staphylococcus aureus (MSSA) as well as MRSA (Kim et al,2004). The antibacterial result of Caesalpinia sappan in this study related with the previous results because both ethanolic and water extracts showed against all positive and negative bacterial. The water extract of Caesalpinia sappan showed higher antibacterial against S.aureus and S.aureus MRSA than ethanolic extract (MIC 78, 78, 156 and 78 µg/ml, respectively). Both Caesalpinia sappan extract also showed high antibacterial against gram negative bacterial cause of diarrhoea. These result can support using Caesalpinia sappan in form of maceration in ethanol to be tonic and postpartum, and boiling to be food for drink. For Dracaena loureiri stems which is an ingredient in PC remedy and up to 50% of remedy exhibit low antibacterial efficacy. Therefore, PC has also low antibacterial efficacy against gram positive and negative bacterial. Furthermore, some extracts showed no inhibition zone and MIC/MBC values showed less activity (MIC >1 mg/ml).

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This may be due to diffusion of extracts from paper disc perhaps depend on concentration of each extract. However, these result related with the previous report of Chusri et al., 2014, but the antibacterial against S.aureus and S.aureus MRSA of PC ethanolic extract in this study was less activity than Chusri report (MIC of this study = 5, 0.312 µg/ml) (MIC of Chusri = 62 and 31 µg/ml). This is report on S.pyogenes which is cause of fever, the ethanolic of PC showed good antibacterial activity (MIC = 0.625 µg/ml) However, this is the first report of Bouea macrophylla, Citrus aurantifolia, Helicia terminalis and Ligusticum chuanxiong on antibacterial assay. For anti- inflammatory effect, the water extract of PC showed better activity than its ethanolic extract as double time (16.87 and 39.70 µg/ml). Moreover, the water extract of PC remedy has no toxic for normal group which not stimulated by LPS. The major ingredient is Dracaena loureiri also active with antibacterial and anti-inflammation. The ethanolic extract of Dracaena loureiri showed antibacterial against MRSA, it related with previously study of Sattaponpan and Kondo, 2011 which this report showed that the same values. The ethanolic extract of Dracaena loureiri also showed anti-inflammatory effect by inhibition NO production, this study related with previously study of Ruangnoo, 2012. Including, the other plant ingredients such as the water extract of Caesalpinia sappan and the ethanolic extract of Mammea siamensis have effect to inhibition NO product with IC50 value nearby Prednisolone as anti-inflammatory drug. The ethanolic and water extracts of Caesalpinia sappan are the most effective on anti-inflammatory assay which related with the previously study of Cheng and Zhang, 2014. In addition, the anti-inflammatory activity of the water extract of PC remedy is quiet stable in 8 months after tested stability in accelerated condition. However, this research is the first of Bouea macrophylla and Helicia terminalis on anti- inflammatory assay. All of these results was supports the traditional used of PC remedy. The ethanolic extract of PC remedy used for treatment of infection fever especially MRSA pathogen and S.pyogenes which is cause of fever and the water extract of PC remedy used for treatment inflammation disorder cause of fever. The ethanolic extract of PC remedy should be uses instead of Gentamicin because Gentamicin is harmful side effect for overdoses. However, this results of PC remedy is inadequate for laboratory

Ref. code: 25595611031526QZI 75 because some plant ingredients have another activity such as in anti-viral activity such as; Caesalpinia sappan showed the most potent (neuraminidase) NAs inhibitory activities with H1N1, H3N2, and H9N2 virus (Liu et al., 2009 and Jeong et al., 2012) and the ethanolic and water extract of Dracaena loureiri (heartwood) were the potent against HIV-1 integrase (IN) by using the multiplate integration assay (MIA) with previously study of Bunluepuech et al., 2009 and anti-pyretic activity; D. loureiri extract exhibited antipyretic effect by suppressed yeast-induced fever in rats. The methanolic extract of N. nucifera showed significant decrease body temperature with previously study of Mukherjee et al., 1996. Finally, this is an in vitro investigation of PC remedy, thus, it should be include to continue to investigate TNF-α, IL-1 and prostaglandin that relate to fever, anti-viral, anti-pyretic and anti-cancer in in vitro and in vivo, the active compound should be isolate from both extracts to be antimicrobial and anti-inflammatory markers for analysis in quality control. Drug development for PC extract should be continuously studied for reduce fever in the future.

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REFERENCES

Books and Book Articles

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Penjor, T., Mimura, T., Matsumoto, R., Yamamoto, M. & Nagano, Y. (2014). Characterization of limes (Citrus aurantifolia) grown in Bhutan and Indonesia using high-throughput sequencing. Scientific Reports, 4, 4853. Poobrasert, O., Constant, H. L., Beecher, C. W., Farnsworth, N. R., Kinghorn, A. D., Pezzuto, J. M., Cordell, G. A., Santisuk, T. & Reutrakul, V. (1998). Xanthones from the twigs of Mammea siamensis. Phytochemistry, 47, 1661-3. Prasad, D. N., Basu, S. P. & Srivastava, A. K. (1999). Antispasmodic activity of the crude and purified oil of Mesua ferrea seed. Ancient Science of Life, 19, 74-5. Ridtitid, W., Sae-Wong, C., Reanmongkol, W. & Wongnawa, M. (2008). Antinociceptive activity of the methanolic extract of Kaempferia galanga Linn. in experimental animals. Journal of Ethnopharmacol, 118, 225-30. Ruangnoo, S., Jaiaree, N., Makchuchit, S., Panthong, S., Thongdeeying, P., & Itharat, A. (2012). An in vitro inhibitory effect on RAW 264.7 cells by anti- inflammatory compounds from Smilax corbularia Kunth. Asian Pacific Journal of Allergy and Immunology, 30(4), 268-274. Sarker SD, Nahar L, Kumarasamy Y. (2007). Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods. 42(4):321-4 Willer Eda, M., Lima Rde, L., & Giugliano, L. G. (2004). In vitro adhesion and invasion inhibition of Shigella dysenteriae, Shigella flexneri and Shigella sonnei clinical strains by human milk proteins. BioMedicines Center Microbiology, 4, 18. Zuo, G. Y., Han, Z. Q., Hao, X. Y., Han, J., Li, Z. S., & Wang, G. C. (2014). Synergy of aminoglycoside antibiotics by 3-Benzylchroman derivatives from the Chinese drug Caesalpinia sappan against clinical methicillin-resistant Staphylococcus aureus (MRSA). Phytomedicine, 21(7), 936-941.

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APPENDIX

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APPENDIX Chemical Reagents

1. Reagent for inhibitory of Nitric oxide (NO) assay  Griess reagent o Salfanilamide 1.0 g o N-(1-Naphyl)enediamine dihydrochloride 0.1 g o Phosphoric acid 2.5 g (Adjust volume with DI water to 100 ml)  MTT solution (5 mg/ml) o MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H- 200 mg tetrazoliumbromide or Thaizolyl blue tetrazolium bromide) o PBS 40 ml (Wrapped foil and stored in 4ºC)  0.04 M HCL in Isopropanol o HCL 0.83 ml (Adjust volume with Isopropanol to 250 ml and stored at room temperature) 2. Reagent for cell culture  FBS (inactivated) Slowly thaw the frozen FBS, heat inactivated (56ºC) (Aliquot and stored in -20ºC)  RPMI 1640 (incomplete media) o RPMI 1640 1X with L-glutamine 1 pack

o NaHCO3 2.0 g (Adjust volume with sterile water to 1000 ml pH 7.0-7.02 by 1 N NaOH or 1 N HCL and filtered at a pore size of 2.0 µM, and stored in 4ºC)

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 RPMI 1640 (complete media) o RPMI 1640 (incomplete media) 400 ml o 10% FBS 40 ml o 1% Penicillin-Streptomycin 4 ml (Stored in 4ºC)  PBS o PBS 1 tablet o DI water 100 ml (Autoclave 121ºC, 15 min and stored in 4ºC)  Penicilin-Streptomycin Slowly thaw the frozen P/S in water bath at 37ºC until complete thaw, aliquot and stored in -20ºC  Trypsin-EDTA Slowly thaw the frozen trypsin in water bath at 37ºC until complete thaw, aliquot and stored in -20ºC 3. Reagent for Antibacterial  Resazurin (1 mg/ml) 1 mg  0.1 M phosphate buffer (pH 7.9±0.1) 1 L  Gentamicin solution (10mg/ml) 100 mg  Mueller Hinton Agar (MHA) 38 g  Mueller Hinton Broth (MHB) 21 g  Nutrient Agar (NA) 28 g

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BIOGRAPHY

Name Miss Alisa Sangphum Date of Birth June 1st, 1988 Educational Attainment 2007-2011: Bachelor of Applied Thai Traditional Medicine, Mae Fah Luang University, Chiang Rai, Thailand Scholarship Funding by Faculty of Medicine, Thammasat University

Publications

Sangphum, A. and Itharat, A. (2017). Antibacterial activity of extracts from a Thai Traditional remedy called Prasachandaeng and its plant components. Journal of the Medicinal Association of Thailand- (In Press)

Conferences and Presentations

Sangphum, A. and Itharat, A. (2015). Antibacterial activity of extracts from a Thai Traditional remedy called Prasachandaeng and its plant components. Patient- Centered Care. Thammasat University. (Poster presentation) Sangphum, A. and Itharat, A. (2016). Antibacterial activity of extracts from a Thai Traditional remedy called Prasachandaeng and its plant components. Patient- Centered Care. Thammasat University. (Oral presentation)

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