Advances in Biomedicine and Pharmacy (An International Journal of Biomedicine, Natural Products and Pharmacy)

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1 ISSN: Adv. Biomed. Pharma. 3:2 (2016) Advances in Biomedicine and Pharmacy (An International Journal of Biomedicine, Natural Products and Pharmacy) Original Article Histological, Histochemical and Phytochemical studies of the Raw drug Hallakam from different Raw Drug Markets of Kerala Sereena K 1 * and Rema Shree AB 1 1 Post Graduate Dept. of Botany, MES Kalladi College, Mannarkkad, Palakkad (Dt), Kerala, India *Corresponding Author: Sereena K Post Graduate Dept. of Botany, MES Kalladi College, Mannarkkad, Palakkad (Dt), Kerala, India address: sereenamajeed@gmail.com Running Title: Histological and phytochemical studies of Hallakam Received: 06 March, 2016; Revised: 16 April, 2016 Accepted: 29 April, 2016 Available online at Abstract The accepted source of the drug Hallakam is Kaempferia rotunda and some peoples use Lagenandra toxicaria as a substitute. This paper deals with the study of Histological, Histochemical and Phytochemical studies of the Raw drug Hallakam from different Raw Drug Markets of Kerala. From anatomical and phytochemical analysis, which we carried out, for the market samples revealed that the raw drugs which are available under the name Hallakam in the raw drug markets of Kerala are the genuine K. rotunda. From the Kerala market survey it is concluded that 100% of the drug Hallakam was found to be K. rotunda. Keywords: Mucormycosis, immuno compromised, immuno competent, anterior chamber Debulking, Amphotercine-B. Introduction Hallakam is mentioned earliest in the famous Ayurvedic lexicon [1]. In the text, the names Kalhara (water plant), Sougandhika (aromatic), and Raktasandhyakam (reddish) and Hallakam (which attracts bees) together apply to one drug. According to Dalhana, a famous commentator of Sushrutasamhita (2005) [2], Kalhara is one of the varieties of Utpala (water lilly), more precisely, Raktolpala or Sougandhikotpala (the red aromatic water lilly). Kalhara has been mentioned in another famous lexicon- Bhavaprakasanighantu, written around 16 AD, being included in its Puspavarga (group of flowers). In this nighantu, the above mentioned names including Hallakam do apply to Kalhara which its commentator, Chunekar [3] has also identified as water lilly. The Ayurvedic expert Thakur Balwant Singh in his Glossary of vegetable drugs in Brihattrayi [4] has opined that everywhere in Brhattrayi Sougandhika is considered to be variety of Utpala or Kumuda (water lilly). So Hallakam evidently denotes water lilly itself, the Nymphea sp. Eventhough rarely, there has been one difference of opinion to this. Some of the Ayurvedic experts and writers of Kerala has considered Chengazhi as Utpala which is identified botanically as Kaempfera rotunda. Moosad, in his commentary on Amarakosa, (2008) [1] has equated Hallakam with the Chengazhinirkizhangu of Kerala physicians, which has been identified as Kaempferia rotunda of Zingiberaceae. This has been accepted by recent authors like Nadkarni, (1954) and Chopra et al., (1956) [5,6]. So Utpala has been considered as Chengazhi by some in Kerala. However there is controversy regards to the source plants. Sivarajan and Balachandran (1994) [7] equated the source plants of Hallakam are Kaempferia rotunda and some peoples use Lagenandra toxicaria also. Kaempferia rotunda L. is an aromatic perennial herb with tuberous rhizomes which produce numerous tuberous roots; leaves simple ligulate, few, erect, lanceolate, acute, 124

2 Advances in Biomedicine and Pharmacy Vol. 3 (2) 2016 variegated green above and tinged with purple below; up to 45 cm long and 10 cms wide. Materials and methods Raw drugs were collected from different raw drugs markets, wholesalers / retailers, raw drug collectors from 11 Districts of Kerala (Calicut, Eranakulam, Idukki, Kollam, Kottayam, Kuttyadi, Palakkad, Payyannur, Thrissur, Trivandrum and Wynad). The useful parts were fixed in Formalin Acetic acid Alcohol mixture for further study. The plant materials were also collected for the phytochemical studies. Histological and histochemical studies The useful parts of the genuine plants/ substitutes/ adulterants and the market samples were fixed in Formalin Acetic acid Alcohol mixture. Cellular details of genuine plant and their substitutes/ adulterants were studied by taking sections using microtomes and double staining with appropriate staining procedure [8] and also using histochemical techniques [8] Double staining Specimens were double stained with safrannin and fast green as per the procedure given by Johansen (1940) [8]. Procedure for double staining Safranin Fast green Double staining is carried out using saffranin and fastgreen according to Johansen (1940) [8]. The following steps were used for double staining. Kept the sections in watch glass containing safranin prepared in water for min and then washed Dehydrated through graded series of alcohol ie, 50%, 60%, 70%, 80%, 90% and 100% taken in cavity blocks covered with lids. The interval in each series was 3 minutes Counter stained the section with fast green dissolved in clove oil for less than 15 seconds Washed excess stain with clove oil Cleaned the section for a few seconds in a mixture of clove oil and xylene followed by 3 changes in xylene and mount in DPX. Sereena and Rema Shree. Histochemical characterization of raw drugs were carried out to found out the presence of starch, tannin; presence, nature and position of laticifers, resin ducts/ oil ducts; identification of depositions like cutin, lignin, suberin, wax; inclusions like calcium oxalate, calcium phosphate, cystolyth etc. using appropriate histochemical techniques (Johansen,1940) [8]. For examine the presence of starch the specimen was stained with Iodine. The blue colour indicates the presence of starch content. Lignified cells For examining the lignified cells or cell walls, the specimen was stained in phloroglucinol and allowed to drying. Then added one or two drops of hydrochloric acid. The presence of lignin indicated by the pink or cherry red colour. Phytochemical studies General method of extraction The first step in the phytochemical evaluation of the plant material was the extraction. The choice of extraction method depends on the nature of the plant material and the compounds to be isolated. Dried material was usually powdered before extraction. Extraction was carried out by the methods involving heating of the drug with suitable solvent. TLC Identity test Chromatography refers to the separation of chemical compounds by partitioning them between a stationary phase and a mobile phase. Basic TLC techniques involves application of sample at one end of the pre-coated plate, developed the plate in the solvent system in a closed chamber to the specified height and visualize the plate for the compounds in visible light, under UV 254, 366nm and/ or by derivatisation of the plate with suitable reagents. The R f values and the colour of the bands were recorded and the finger print profiles were established. Identification of the chemical marker was done by comparison of R f values. Thin layer Chromatography (TLC) studies Thin Layer Chromatographic (TLC) studies conducted using pre-coated plates of silica gel 60 F 254 (E. Merck) of uniform thickness of 0.2mm. Histochemical studies 125

3 Histological and phytochemical studies of Hallakam Advances in Biomedicine and Pharmacy Vol. 3 (2) 2016 Results and Discussion Details of the Market sample studies Materials collected from 11 districts of Kerala the author made an attempt to check the extent of variability of genuine hallakam from different markets of Kerala. Differences were observed in the depositions of starch according to the place of collection but the chemical profiling shows that the compounds are similar. It is clear that the amount of starch is inversely proportional to the chemical compounds present in the samples. From the TLC profiles, in the genuine sample, in addition to the bands which are present in the market samples, two additional bands are also present which indicates the presence of volatile compounds. The absence of those volatile compounds in the market samples may be due the evaporation of volatile oils during storage. From anatomical and phytochemical analysis, which we carried out for the market samples revealed that the raw drugs which are available under the name hallakam in the raw drug markets of Kerala are the genuine K. rotunda. From the Kerala market survey it is concluded that 100% of the drug hallakam were found to be K. rotunda. (Table 1 & 2; Plate 1-5). It is clear that the amount of starch is inversely proportional to the chemical compounds present in the samples. From the Thin Layer Chromatographic profiles, in the genuine sample, in addition to the bands, which were present in the market samples, two additional bands were also present which indicates the presence of volatile compounds. The absence of those volatile compounds in the market samples may be due the evaporation of oils during storage. Table 1: Market sample comparison of Hallakam from different districts of Kerala Sl.No. Name Hallakam 1 Calicut Genuine, K. rotunda. grains less 2 Ernakulam Genuine, K. rotunda. grains plenty 3 Idukki Genuine, K. rotunda. grains plenty 4 Kollam Genuine, K. rotunda. grains comparatively less 5 Kottayam Genuine, K. rotunda. grains plenty 6 Kuttyadi Genuine, K. rotunda. grains absent. 7 Payyannur Genuine, K. rotunda. grains comparatively less 8 Palakkad Genuine, K. rotunda. grains plenty 9 Thrissur Genuine, K. rotunda. grains comparatively less 10 Trivandrum Genuine, K. rotunda. grains plenty 11 Wynad Genuine, K. rotunda. grains comparatively less 126

4 Advances in Biomedicine and Pharmacy Vol. 3 (2) 2016 Sereena and Rema Shree. Table 2: R f values of market samples of Hallakam from different district s of Kerala Rf value of bands in 1 CLT EKM IDK KLM KTM KTD PYR PKD TSR TVM WYD Colour of the band Dark pink Light pink Brown Dark pink Violet Light rose Dark blue Pink Light red Light pink GEN - Genuine CLT - Calicut EKM- Ernakulam IDK - Idukki KLM - Kollam KTM - Kottayam KTD - Kuttyadi PYR - Payyannur PKD - Palakkad TSR - Thrissur TVM - Trivandrum WYD - Wayanad Lignin Calicut Ernakulam Idukki Plate 1: Anatomical comparisons of samples from different raw drug markets 127

5 Histological and phytochemical studies of Hallakam Advances in Biomedicine and Pharmacy Vol. 3 (2) 2016 Lignin Payyannur Palakkad Thrissaur Plate 3: Anatomical comparisons of samples from different raw drug markets 128

6 Advances in Biomedicine and Pharmacy Vol. 3 (2) 2016 Sereena and Rema Shree. Lignin Trivandrum Waynad Plate 4: Anatomical comparisons of samples from different raw drug markets Plate 5: Phytochemical comparisons of samples from different raw drug markets 129

7 Histological and phytochemical studies of Hallakam Advances in Biomedicine and Pharmacy Vol. 3 (2) 2016 Conclusion Using histological, histochemical and phytochemical studies to check the extent of variability of source plant of hallakam from different market samples of Kerala. From anatomical and phytochemical analysis, which we carried out for the market samples revealed that the raw drugs which are available under the name hallakam in the raw drug markets of Kerala are the genuine K. rotunda. From the Kerala market survey it is concluded that 100% of the drug hallakam were found to be K. rotunda. Acknowledgement Kerala State Council for Science, Technology and Environment (KSCSTE) Financial Assistance Kerala State Council for Science, Technology and Environment (KSCSTE) Conflict of Interest Authors declare that there is no conflict of interest to reveal References [1] Amarakosa., Amarakosam with Parameswari Vyakhyanam, Vachaspati T.C Paramesvaran Moosad. Kerala Sahitya Acadamy, Thrissur, 278, [2] Sushrutasamhita With Dalhana s commentary and English translations by P. V. Sharma. Chaukhambha visvabharati, Varanasi & Delhi, [3] Bhavaprakasanighantu., Bhavaprakasanighantu of sri Bhavamisra, Commentary by K. C Chunekar and edited by Dr. G S Pandey. Chaukambha Bharat Academy, Vranasi, : , 484, [4] Singh TB and Chunekar KC., Glossary of vegetable drugs in Brihatrayi, Chaukambha Sanskrit series office, Varnasi, 457, [5] Nadkarni AK., Indian Materia Medica, Popular Prakashan Pvt. Ltd., Bombay, Maharashtra, India. 1: , 716, 723, , 1229, [6] Chopra RN, Nayar SL and Chopra IC., Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research (CSIR), New Delhi, India 39, [7] Sivarajan VV and Balachandran I., Ayurvedic drugs and their Plant Sources, IBH Publishing Co. Pvt. Ltd, New Delhi , , , [8] Johansen DA., Plant Microtechnique, (McGraw Hill, New York, USA), ABP 2016 Reproduction is free for scientific studies This work is licensed under a Creative Commons Attribution 4.0 International License. ADVANCES IN BIOMEDICINE AND PHARMACY is indexed in Google scholar, Index Copernicus International (ICV: 66.16), Eurasian Scientific Journal Index, Cite Factor, Open Academic Journals Index, International Scientific Indexing, InfoBase Index (IBI Factor: 3.1), Global Impact Factor (GIF: 0.435) Advanced Science Index, Universal Impact Factor, International Society of Universal Research in Sciences, Scientific Indexing Services, Polish Scholarly Bibliography, Directory of Research Journal Indexing, Electronic Journals Library, Genamics Journal Seek, Chemical Abstracts Services, African Index Medicus, Academic Resource Index, Journal Index.net, Scientific Journal Impact Factor. 130

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