QUALITY CONTROL ANALYSIS OF CRUDE DRUGS IN PRASA-KANPHLU FORMULA Jirapat Saetan1,*, Seewaboon Sireeratawong2, Nongluck Ruangwises3, Noppamas Soonthornchareonnon4,# 1Master of Science program in Pharmaceutical Chemistry and Phytochemisty, Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand 2Division of Pharmacology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Bangkok, Thailand. 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand 4Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand *e-mail: [email protected], #e-mail: [email protected]

Abstract In today’s society, people are becoming more conscious regarding health in order to take better care of themselves. Thai herbs are an alternative which have been popular both in domestic and in other foreign countries. However, the use of herbal medicine for treatment or prevention of diseases in Thailand today requires the resource and development of the concept of ancestral wisdom that has been passed down from generation to generation. In order to achieve acceptance, factual evidence must be proved. Prasa-Kanphlu formula was created from old traditional knowledge and has been used for long periods. Now Prasa-Kanphlu formula has been selected in the National List of Essential Drugs [1] for the treatment of the gastrointestinal system. Prasa-Kanphlu formula is composed of twenty-seven Thai herbal medicines. All of them were authenticated by an applied Thai traditional medicine practitioner. The present research aimed to conduct the quantitative and qualitative determination of active constituents as well as to ensure their safety and efficiency before a further screening of pharmacological activity. As a result from the quality control of raw materials by physicochemical methods: test of loss on drying; total ash and acid-insoluble ash, eighteen herbal medicines (as 66% of the whole formula) were considered to meet the requirements according to the reference pharmacopoeia [2-7]. From the determination of volatile oil [4,5], sixteen herbal medicines were found with different amounts of volatile oil. Curcuma longa was found with the most outstanding volatile oil composition, 6.18% v/w. Polysaccharides[8] could be found in every herbal medicine of the formula: Saussurea lappa was found with obviously the highest amount of total polysaccharides, 74.69% w/w. From the phytochemical screening test [9], alkaloids and coumarins were detected as the main constituents of the raw materials of this formula.

Keywords: Prasa-Kanphlu, Syzygium aromaticum, pharmacopoeia, quality control and phytochemical screening.

Introduction Thai Traditional medicine (TTM) is a system of methods and practices, such as herb lore, that have been handed down from monks. In Thailand, a century ago, it was outlawed as quackery in favour of western medicine. It is now making a comeback. In present, the Ministry of Public Health launch 71 items of National List of Essential Medicine 2012[1] (Herbal Medicine) and Prasa-Kanphlu is one of those items. It is used to alleviate dyspepsia and flatulence which follows Thai traditional usage. Prasa-Kanphlu is composed of 27 medicinal plants and 2 kinds of minerals (sulfur and camphor). Syzygium aromaticum is the major component of this formula, it is 50% by weight and the others are 50%. Twenty seven medicinal plants are Acorus calamus, Albizia procera, Amomun testaceum, Angelica dahurica, Aquilaria crassna, Boesenbergia rotunda, Cissampelos pareira, Coriandrum sativum, Cuminum cyminum, Curcuma longa, Kaempferia galangal, Maerua siamensis, Myristica fragrans, Nigella sativa, Oroxylum indicum, Laurus nobilis, Piper interruptum, Piper nigrum, Piper retrofractum, Piper sarmentosum, Plumbago indica, Saussurea lappa, Syzygium aromaticum, Vetiveria zizanioides, Terminalia triptera, Zingiber montanum and Zingiber officinale. The precaution is not to use in the pregnant and in people who have fever. The warning is to be careful of using continuously especially in patients with liver abnormality because of camphor accumulation. Quality control analysis of crude drugs indicates quality assessment of raw material during production and herb product. The objective of quality control analysis of raw material is to ensure the authenticity of crude druds, the regular amount of active ingredient and the state of being not contaminated from any adulterants. The determination of quality control analysis of crude drugs is specified by Pharmacopoeia[2-7] of each country. WHO has introduced the determination of quality control analysis of crude drugs in order for setting up their standardization[2]. Most importantly, the good activity depends on the quality of crude drugs. This study is concentrated on quality control of crude drugs in Prasa-Kanphlu formula. The objective of this study is to present quality research of crude drugs in Prasa- Kanphlu formula, following pharmacopoeia. The determination of Pharmacopoeia as follows by WHO and USP 29[2,3] compose of plant name, source, part of use, character, chemical markers, foreign organic matter, total ash, acid-insoluble ash, water-soluble ash, loss on drying or azeotropic method, extractive values, microbial contamination, heavy metals contamination and insecticide contamination. There are 14 categories of the Pharmacopoeia standard [2, 3] and the quality control is compared with that of the standard. Presently, 7 categories have been tested which are total ash, acid-insoluble ash, loss on drying, volatile oil, total polysaccharide, phytochemical screening, chemical markers and plant name.

Methodology Prasa-Kanphlu is composed of 27 medicinal plants and 2 kinds of minerals which were authenticated by an applied Thai traditional medical practitioner. 1. Quantitative Total ash, acid-insoluble ash, loss on drying/azeotropic, volatile oil, total polysaccharide content Total ash Place a 2- to 4-g sample of the ground substance, accurately weighted, or the quality specified in the monograph, in a suitable tared crucible (usually of platinum or silica), previously ignited, cooled and weighted. Incinerate the sample by gradually increasing the temperature, not exceeding 450˚, until free from carbon; cool and weigh. If a carbon-free ash cannot be obtained in this way, cool the crucible and moisten the residue with about 2 ml of water or a saturated solution of ammonium nitrate. Dry on a water-bath and then on a hot plate and, incinerate to constant weight. Calculate the percentage of total ash with reference to the air-dried substance[4,5]. Acid-Insoluble ash Use Method I unless otherwise indicated in the monograph. Method I Boil the total ash for 5 minutes with 25 ml of dilute hydrochloric acid, collect the insoluble matter on and ashless filter paper, wash with hot water until the filtrate is neutral, and ignite at about 500˚. Calculate the percentage of acid-insoluble ash with reference to the air-dried substance. Method II Place the total ash or the sulfated ash, as directed in the monograph, in a crucible, add 15 ml of water and 10 ml of hydrochloric acid, cover with a watch glass, and boil for 10 minutes; allow to cool. Collect the insoluble matter on an ashless filter paper, wash with hot water unit the filtrate is neutral, ignite to dull redness ( 500˚ to 700˚ ), cool in the desiccators and weigh. Reheat until the difference between two successive weightings is not more than 1 mg. Calculate the percentage of acid-insoluble ash with reference to the air-dried substance[4,5]. Loss on drying The procedure set forth in this section determines the amount of volatile matter of any kind that is driven off under the conditions specified. For substances appearing to contain water as the only volatile constituent, the procedure given in “Determination of water”, is appropriate, and is specified in the individual monograph. Unless otherwise directed in the monograph, conduct the determination on 1 to 2 g of the substance (2 to 5 g in case of crude drugs), previously mixed and accurately weighed. If the test specimen is in the form of large crystals, reduce the particle size to about 2 mm by quickly crushing. Tare a glass-stoppered, shallow weighing bottle that has been dried for 30 minutes under the same conditions to be employed in the determination. Put the test specimen in the bottle, replace the cover, and accurately weigh the bottle and the contents. By gentle, sidewise shaking distribute the test specimen as evenly as practicable to a depth of about 5 mm generally, and not over 10 mm in the case of bulky materials. Place the loaded bottle in the drying chamber, removing the stopper and leaving it also in the chamber. Dry the specimen at the temperature and for the time specified in the monograph. The temperature of heating is within the range of ±2˚ of the stated figure in the monograph. Upon opening the chamber, close the bottle promptly and allow it to come to room temperature in a desiccators before weighing. If the substance melts at a lower temperature than the specified for the determination of Loss on drying, maintain the bottle with its contents for 1 to 2 hours at a temperature 5˚ to 10˚ below the melting temperature, the dry at the specified temperature. Where the individual monograph directs that loss on drying be determined by thermogravimetric analysis, a sensitive electrobalance is to be used[4,5]. Azeotropic Distillation Method Clean the receiving tube and the condenser of the apparatus by a suitable method, thoroughly rinse with water, and dry.Introduce 200 ml of toluene and about 2 ml of water into the dry flask. Distil for about 2 hours, allow to cool to room temperature and read the water volume to an accuracy of 0.05 ml. Place in the flask a quantity of the substance, weighed to the nearest centigram, excepted to give about 2 to 3 ml of water. If the substance is of a pasty character, weight it in a boat of metal foil. Add a few pieces of porous material and heat the flask gently for 15 minutes. When the toluene begins the boil, distil at the rate of 2 drops per second until most of the water has distilled over, and then increase the rate of distillation to about 4 drops per second. When the water has all distilled over, rinse the inside of the condenser tube with toluene. Continue the distillation for 5 minutes, remove the heat, allow the receiving tube to cool to room temperature, and dislodge any droplets of water which adhere to the walls of the receiving tube. When the water and toluene have completely separated, read the volume of water and calculate the percentage present in the substance using the formula[4,5]: 100 (n´-n) P where p = the weight in g of the substance to be examined, n = the volume in ml of water obtained in the first distillation, and n´= the total volume in ml of water obtained in the two distillations. Determination of volatile oil Carry out the determination according to the nature of the drug to be examined. Place the prescribed quantity of the drug in the round-bottomed flask of suitable capacity as specified in the monograph and add the prescribed volume of distillation liquid. Set up the apparatus, inserting a reflux condenser. Fill the graduated tube with water to the standard line and add the prescribed volume of xylene. Heat the liquid in the flask in an oil-bath between 130˚ and 150˚ to boiling and adjust the distillation rate as prescribed in the monograph. Unless otherwise specified, continue boiling for 5 hours at the same rate. Allow it to stand for some time, open the stopper of the apparatus and draw off the water slowly until the level of the xylene and volatile oil mixture corresponds to the preparation line, and allow it to stand for more than 1 hour at cool temperature. Then lower the level of the xylene and volatile oil mixture to the zero line, and read the volume in milliliter of the mixture of xylene and volatile oil at cool temperature. Subtract the volume in milliliter of xylene from the volume of the mixture of xylene and volatile oil and calculate the percentage of volatile oil content in the sample[4,5]. Total polysaccharide content Phenol-sulfuric method: The calibration curve of glucose which was used as the benchmark for the yield determination was obtained as follows. A stock 1 mg/ml glucose solution was used to prepare 5 or 6 dilution plots of standard ranging from 0 to 20 µg/ml. Each standard was subjected to the reaction procedure as described in Table1, transferred to 96-wells microplate and measured at 490 nm. The sample for determination of total poly saccharide content was prepared from lyophilized water extract of crude drugs in Prasa- Kanphlu formula[9].

Table 1. Preparation of glucose reference standard by phenol/H2SO4 assay

Glucose 1 mg/ml (µl) H2O (µl) Total (µl) 0 200 200 5 195 200 10 190 200 15 185 200 20 180 200

2. Qualitative Phytochemical Screening[8]. Alkaloids Alkaloids tests could be classified into 2 tests. The first one was to test alkaloids in free base form by using conc.NH4OH. So alkaloids could be dissolved in chloroform. Indication of alkaloids was done by a color change to orange with Dragendorff spray reagent. The second one was to test alkaloids in acid salt form by using 5%HCl. So alkaloids could be dissolved in aqueous solvents. Identification of alkaloids was done by precipitating with heavy metal reagents, such as Dragendorff’s, Hager’s, Marme’s, Mayer’s, Tannic acid, Valser’s and Wagner’s. Cardiac glycosides Cardiac glycosides were tested with 3 reagents composed of Kedde’s for Lactone part, Keller-Killiani’s for Deoxy sugar part and Liebermann Burchard’s for steroids part. When the positive results of 3 parts were displayed, the presence of cardiac glycosides was indicated. Flavonoids Flavonoids were observed by shinoda test. The positive result was shown by a change to yellow to red color. This could be further identified as leucoanthocyanins, anthocyanins and other types of flavonoids. Tannins One property of tannins was that they could precipitate gelatin salt and gelatin. Few drops of ferric chloride solution would identify the type of tannin. Occurrence of a blue- black (Hydrolyzable tannin), green or blue-green(Condense tannin) precipitate indicated the presence of tannins. Anthraquinones About 0.2 g of each portion to be tested was shaken with 10 ml of benzene and then filtered. Five millilitres of the 10% ammonia solution was then added to the filtrate and thereafter shaken. Appearance of a pink, red or violet colour in the ammoniacal (lower) phase was taken as the presence of free anthraquinones. Lactone glycosides About 2 g of drug powder was placed in a tube which was closed by a filter paper previously dipped in 20% NaOH. Let the tube stand on water bath for 5 minutes. Lactone glycosides could be fluorescent in blue, green and yellow shades under a UV light condition. Cyanotic glycosides About 2 g of drug powder was placed in a flask. Few drops of H2O were added for moistness followed by dropping 1-2 drops of CHCl3 and closing the flask by sodium picrate paper. Let it stand on water bath. The positive result which was observed by a change to brick red color of the sodium picrate paper referred to the presence of cyanotic glycosides. Saponins One gram of each portion was placed in a tube. Boiled, distilled water was added before shaking vigorously for about 5 minutes. Frothing which persisted on warming was taken as an evidence for the presence of saponins.

Results 1. Quantitative Total ash, acid-insoluble ash, loss on drying/azeotropic, volatile oil and polysaccharide content of all herbs in Prasa-Kanphlu formula are described in table 2. Table2. Quantitative of crude drugs in Prasa-Kanphlu formula

Crude drug Loss on drying Azeotropic Total Ash Acid-insoluble ash Volatile oil Total polysaccharide A. calamus - 9.5d 5.3 d 0.6 d 1.7 d 4.0 A. procera 9.7 - 10.1 0.3 - 0.7 A. testaceum - 9.2c 5.1 0.7 0.5 c 0.6 A. dahurica 12.8 - 3.6 c 0.5 c - 14.3 A. crassna - 7.2 2.0 0.2 0.1 0.5 B. rotunda - 9.3 5.6 2.2 1.4 1.6 C. pareira 8.4 - 5.0 1.1 - 1.5 C. sativum - 5.5b 7.2 b 0.2 a 0.1 d 2.1 C. cyminum - 6.5d 7.5 d 0.4 a 3.5 d 2.6 C. longa - 4.8 d 7.0 d 0.2 d 6.2 d 2.6 K. galangal - 8.0 8.0 1.7 3.9 4.3 M. siamensis 8.9 - 2.1 0.2 - 0.6 M. fragrans - 6.3 a 1.7 d 0.1 a 4.1 d 2.0 N. sativa - 4.8 d 4.4 d 0.1 c 0.5 d 2.8 O. indicum 8.9 - 11.4 0.9 - 2.3 L. nobilis - 4.5 5.7 0.3 1.0 0.6 P. interruptum 7.4 - 9.5 1.9 - 1.4 P. nigrum - 4.8 d 4.3 d 0.2 d 0.8 d 0.1 P. retrofractum - 6.8 d 7.3 d 0.2 d 1.0 d 5.1 P. sarmentosum - 9.0 8.2 0.6 - 4.3 P. indica 8.1 - 10.0 2.4 - 12.0 S. lappa 11.3 - 4.2 a 0.5 a - 74.7 S. aromaticum - 8.2 e 6.0 e 0.2 e 13.2 d 2.5 V. zizanioides 5.7 - 8.0 4.0 - 36.2 T. triptera 9.4 - 10.7 0.2 - 0.1 Z. montanum - 8.0 d 7.1 d 1.9 d 1.4 d 3.0 Z. officinale - 7.7 b 9.1 e 6.5 * 0.8 c 1.1

a Ayurvedic pharmacopoeia of India[7]

b British pharmacopoeia[3]

c Pharmacopoeia of the people’s republic of China[6] 2. Qualitative Phytochemical Screening of all herbs in Prasa-Kanphlu formula are described in table 3.

Table 3. phytochemical screening tests of crude drugs in Prasa-Kanphlu formula

Crude drug Alkaloids Cardiac Flavonoids Tannins Anthraquinones Lactone Cyanotic Saponins glycosides glycosides glycosides A. calamus  - - - -  - - A. procera - - -  -  -  A. testaceum  - - - -  - - A. dahurica  - - - -  - - A. crassna - - - - -  - - B. rotunda - - - - -  - - C. pareira  - - - -  - - C. sativum  - - - -  - - C. cyminum - -  - -  - - C. longa - -  - -  - - K. galangal - - - - -  - - M. siamensis  -  - -  - - M. fragrans - -  - -  - - N. sativa  - - - -  - - O. indicum - - - - -  - - L. nobilis - - -  -  - - P. interruptum  - - - -  - - P. nigrum  - - - -  - - P. retrofractum  - - - -  - - P. sarmentosum  - - - -  - - P. indica - - - - -  - - S. lappa  - - - -  - - S. aromaticum - - -  -   - V. zizanioides  - - - -  -  T. triptera - - -  -  - - Z. montanum - -  - -  - - Z. officinale - - - - -  - - Discussion and Conclusion

As a result from the quality control of crude drugs by physicochemical methods: test of loss on drying; total ash and acid-insoluble ash, eighteen herbal medicines (as 66% of the whole formula) were considered to meet the requirements according to the reference pharmacopoeia [2-7]. There were 12 herbs qualified for the pharmacopoeial standard except 2 herbs: S. lappa and Z. officinale had total ash weight higher than the pharmacopoeial standard. The 15 remaining herbs were not included in monographs of any pharmacopoeias and 3 of them had total ash weight higher than 10% including A. procera, O. indicum and T. triptera, two of them (B. rotunda and V. zizanioides) had acid insoluble weight higher than 2% and two of them (A. dahurica and S. lappa) had loss on drying weight higher than 10%. From the determination of volatile oil [4-5], sixteen herbal medicines were found with different amounts of volatile oil. The most outstanding volatile oil composition, 6.18% v/w, was found in Curcuma longa. Twelve herbs had pharmacopoeial standard requirements, whereas the remaining 4 herbs did not. Among twelve herbs which had their own pharmacopoeial standard requirements, four of them (A. testaceum, C. sativum, P. nigrum and Z. montanu ) were determined with the lower amounts of volatile oils than in pharmacopoeial standard. Polysaccharides[9] were found in every herbal medicine of the formula: Saussurea lappa was found with obviously the highest amount of total polysaccharides, 74.69% w/w. From the phytochemical screening test [8], alkaloids and coumarins were detected as the main constituents of the raw materials of this formula. The results showed that thirteen samples were positive to alkaloids test including A. calamus, A. testaceum, A. dahurica, C. pareira, C. sativum, M. siamensis, N. sativa, P. interruptum, P. nigrum, P. retrofractum, P. sarmentosum, S. lappa, V. zizanioides. For lactones glycosides, the result showed that all of the samples had fluorescent compounds after adding 20% NaOH in samples and heat. Cardiac glycosides tests showed that each sample did not contain these compounds. Flavonoids were observed by Shinoda test. The result showed that five samples have flavonoids including C. cyminum, C. longa, M. siamensis, M. fragrans and Z. montanum. For anthraquinones, the result showed that no sample was positive for anthraquinones. For cyanotic glycosides, only S. aromaticum showed a positive result. In conclusion, the crude drugs of Prasa-Kanphlu Formula still have been being studied to provide optimum quality evidence-based activities. As stated before, Prasa- Kanphlu formula has main activities including dyspepsia and flatulence. Other activities are possible due to its twenty seven-herb combination which makes this formula a highly interested topic. References 1. National Drug Committee. National List of Essential Medicine 2012.: Press of The Agricultural Cooperative Federation of Thailand, 2012. 2. World Health Organization. Quality Control Methods for Medicinal Plant Materials. Geneva: World Health Organization, 1998. 3. The United States Pharmacopoeia 29/The National Formulary 24, MD: The United States Pharmacopeial Convention, 2006. 4. Department of Medical Sciences. Thai Herbal Pharmacopoeia. Vol. I. Bangkok: Prachachon Co.,1995. 5. Department of Medical Sciences. Thai Herbal Pharmacopoeia. Vol. II. Bangkok: Prachachon Co.,1995. 6. China Pharmacopoeia Commission. Pharmacopoeia of The People’s Republic of China. Vol. 1. Beijing: People’s Republic of China, 2005. 7. Government of India, Ministry of Health and Family Welfare. The Ayurvedic Pharmacopoeia of India. Part-1. 1st ed. Vol. 1. Delhi: Government of India, Ministry of Health and Family Welfare, Department of India Systems of Medicine & Homoeopathy, 2001. 8. Ponsuda poomsing. Qualitative and Quantitative determination of Active constituents from Ganoderma lucidum(FR.) Karst Cultivated in Thailand, 2012. 9. Farnsworth NR. Biological and phytochemical screening of plants. J Pharm Sci 1966;55(3):225-277.

Acknowledgements: We would like to give particular thanks to the Department for Development of Thai Traditional and Alternative Medicine for funding the grant of this research.