PHCOG J. ORIGINAL ARTICLE Microscopic Characterization as a Tool for Separation of Stemona Groups Sumet Kongkiatpaiboon1, Vichien Keeratinijakal2,3 and Wandee Gritsanapan1,* 1 Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, 10400 2National Center for Agricultural Biotechnology, Kasetsart University, Bangkok, 10900 3Agronomy Department, Faculty of Agriculture, Kasetsart University, Bangkok, 10900, Thailand A B S T R A C T: Introduction: Stemona plants have been traditionally used as an insecticide, scabicide and pediculocide, and for the treatment of skin and respiratory diseases. Stemona can be separated into two groups according to their morphological characters and bioactive components i.e. stichoneurine and protostemonine groups. Protostemonine group contains alkaloids that possess potent insecticidal activity while stichoneurine group accumulates alkaloids with antitussive activity. In Thailand, a vernacular name “Non Tai Yak” refers to the roots of different species of Stemona, making it confusing to discern different species. The purposes of this study are to investigate the microscopic characteristics of the roots of seven species of Stemona growing in Thailand and to distinguish and identify these groups of Stemona. Methods: Cross-sectional histology of fresh root samples and powdered drug characteristics of 7 species of Stemona were studied under a microscope. Results: The roots of Stemona in the stichoneurine group (S. tuberosa and S. phyllantha) contained a non-lignified large pith while the roots of protostemonine group (S. burkillii, S. cochinchinensis, S. collinsiae, S. curtisii and S. kerrii) had a small lignified one. The powder of stichoneurine group contained numerous thin-walled parenchyma, but only few thick-walled parenchyma and lignified fibers and vessels were present. In contrast, thick-walled parenchyma and lignified fibers and vessels were frequently found in the powdered roots of protostemonine Stemona. These characteristics could be used to discern between Stemona in the stichoneurine and protostemonine groups. Conclusions: The microscopic characterizations can be used as a primary tool to categorize and separate 2 main Stemona groups. Key words: Non Tai Yak, protostemonine, Stemona, Stemonaceae, stichoneurine INTRODUCTION Since ancient time, Stemona plants have been traditionally used as an insecticide, scabicide, pediculocide; used for treating skin and respiratory diseases, and also for killing head lice.[1-3] “Non Tai Yak” is a Thai vernacular name that refers to various species of Stemona in Thailand[4] and some other plants such as Asparagus sp. of the family Asparagaceae and Clitoria sp. of the family Leguminosae in some locations.[1] This plant has been used to protect plants against insect attack, the infection of fermented fish “Pla Raa” or fermented shrimp “Ka Pi” from housefly larvae. The inconsistency when providing and using the proper Stemona plant materials has led to the confusion in the scientific identification and in agricultural and *Address for correspondence: E-mail: pywgs@mahidol.ac.th DOI: **** Pharmacognosy Journal | December 2010 | Vol 2 | Issue 17 pharmaceutical uses.[1] Recent taxonomic revision of the family Stemonaceae indicates that Stemona in Thailand comprises of 11 known species i.e. S. aphylla Craib, S. burkillii Prain, S. cochinchinensis Gagnep., S. collinsiae Craib, S. curtisii Hk. F., S. involuta Inthachub, S. kerrii Craib, S. phyllantha Gagnep., S. pierrei Gagnep., S. rupestris Inthachub and S. tuberosa Lour.[5] They can be separated into two main groups according to their morphological characters and bioactive component accumulation i.e. stichoneurine or tuberosa group and protostemonine or non-tuberosa group.[6] Stichoneurine group comprises of S. tuberosa and S. phyllantha and they are different from other Stemona plants because of their large and thick tuberous roots, large perianths and scented flowers.[5,6] However, the three dominant species of Stemona (S. tuberosa, S. collinsiae and S. curtisii) and some other species of Stemona in Thailand are still called “Non Tai Yak”, making it confusing and causing misuses of these plants. Phytochemical investigations of Stemona species revealed the presence of alkaloids, stilbenoids and chromenols. The 1 Kongkiatpaiboon, et al.: Microscopic Characterization as a Tool for Separation of Stemona Groups. alkaloids display a remarkable accumulation trend in Stemona species.[1,7] Stemona alkaloids constitute a unique chemical feature of the family Stemonaceae and cannot be detected in any other plant families thus far.[1,2] Classification of Stemona alkaloids based on biosynthetic considerations confirms three skeleton types i.e. stichoneurine- (tuberostemonine-), protostemonine-, and croomine-type alkaloids.[1] Stichoneurine group contains stichoneurine- and croomine-type alkaloids while protostemonine group contains protostemonine-type alkaloids.[1,7] Potent insect toxicity of Stemona plants is attributed to the derivatives of protostemonine-type alkaloids, especially the stemofoline derivatives,[1,8,9] whereas stichoneurine-type alkaloids possess only a remarkable insect repellance.[8] In contrast, stichoneurine- and croomine-type alkaloids were reported to be associated with antitussive activity.[10-14] Herbal extracts from various Stemona plants have been used for over a century. Stemona has been developed into commercial products for bioinsecticide or antitussive drug. Since the variation of authentic Stemona raw materials affects their promised biological properties, the confusion stems out when Stemona roots as well as their powdered drug are used. Identification of these plants via morphological characteristics is limited by the presence of flowers and it cannot be determined in the form of powdered drug. Authentication of Stemona species using DNA-based techniques is effective but it is also expensive and time-consuming. This study presented a simpler method to identify and distinguish Stemona in stichoneurine or tuberosa group from protostemonine or non-tuberosa group by exploiting the dissimilarities between their microscopic characteristics. Microscopic methods Cross-sectional histology of fresh root samples and powdered drug of the seven species of Stemona were examined under a microscope (Olympus, Japan) using mounting reagents. The characteristic tissues were photographed using a camera and drawn using a camera lucida (Olympus, Japan). RESULTS The tuberous roots of Stemona in stichoneurine group (S. tuberosa and S. phyllantha) were large and thick, 10-50 cm long, pale yellowish-brown in color, while the protostemonine group (S. burkillii, S. cochinchinensis, S. collinsiae, S. curtisii and S. kerrii) had slender pale yellowish-brown roots with varied length (4-50 cm). Cross-sectional histological characteristics of the fresh root samples of Stemona showed that the stichoneurine group had a larger pith compared to those in the protostemonine group. After applying aniline sulfate solution, the pith of the protostemonine Stemona turned yellow, making it a lignified pith, while the stichoneurine Stemona roots contained a non-lignified pith (Figure 1). MATERIALS AND METHODS Plant materials The samples of 7 Stemona species (S. burkillii, S. cochinchinensis, S. collinsiae, S. curtisii, S. kerrii, S. phyllantha and S. tuberosa) were collected from various locations in Thailand. Each sample was cultivated at National Corn and Sorghum Research Center, Nakorn-Ratchasima province in the NorthEast of Thailand. All root samples at the age of 4 year old were collected in August, 2009. The plant specimens were identified by Dr. Vichien Keeratinijakal, Kasetsart University, and the voucher specimens (VKS001-VKS007) were deposited at Pharmacognosy Department, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand. Each fresh sample was dried at 60°C then powdered by an electronic mill. The powder was passed through a sieve no. 60 to yield fine powder. The powder of each sample was separately kept in a tightly-closed vial until used. Other portions of fresh root were used for histological inspection. 2 Figure 1: Cross-sectional histology of fresh root samples of various Stemona species applied with aniline sulfate showing characteristic features : (A) a large non-lignified pith of stichoneurine group, or tuberosa (B) a small lignified pith of protostemonine or non-tuberosa group. Pharmacognosy Journal | December 2010 | Vol 2 | Issue 17 Kongkiatpaiboon, et al.: Microscopic Characterization as a Tool for Separation of Stemona Groups. Powders of all Stemona species appeared as creamishwhite to creamish-yellow with a faint distinct odor and a sweet and bitter taste. The diagnostic characteristics of the powders of both groups of Stemona are shown in Figure 2 and are compared in Table 1. These characteristics are as follows. (1) Abundant starch granules that are simple, small, spherical to ovoid, or compound with two, three, four or occasionally up to six components. (2) Abundant parenchyma from the cortex and stele. The cells are fairly large and vary from rounded to elongated rectangular in outline with thin wall. The cells are almost filled with starch granules. (3) The lignified vessels of xylem occur in groups of interlocking cells. The vessels contain numerous bordered pits. (4) Long fibers, fragmented. (5) Thick-walled parenchyma of the xylem and medullary ray. DISCUSSION Stemona in stichoneurine group (S. tuberosa and S. phyllantha) had larger, thicker and longer tuberous roots than the protostemonine group (S. burkillii, S. cochinchinensis, S. collinsiae, S. curtisii and S. kerrii). The colors of the roots of both groups were the same pale yellowish-brown. Figure 2: Powdered drug characteristics of Stemona roots. Tuberosa group comprised of: A, part of fibers; B, xylem vessels which arranged bordered pits; C, numerous small starch granules; D, part of cork and cortex in sectional view; E, parenchyma cells; F, parenchyma cells containing starch granules; G, sclereids. Non-tuberosa group was composed of : I, part of fibers; II, xylem vessels which arranged bordered pits and occasionally occurred in groups of interlocking cells; III, starch granules; IV, part of cork and cortex in sectional view with sclereids (Sc.); V, small part of fiber (Fb) connected to parenchyma cells (Pc); VI, thick walled parenchyma cells. Cross-sectional histology of the roots of stichoneurine Stemona showed a non-lignified large pith containing numerous thin-walled parenchyma cells while the protostemonine Stemona roots had a small liginified pith with less abundant thin-walled parenchyma cells. The powdered drugs of the roots of stichoneurine group contained several thin-walled parenchyma but only few thick-walled parenchyma cells were found. The thick-walled parenchyma cells were frequently spotted in the powdered roots of the protostemonine group. The lignified fibers and vessels were frequently found in the powdered roots of protostemonine Stemona but rarely found in the root powders of the stichoneurine group. Numerous simple TABLE 1 : Comparison of powdered drug characteristics of the roots of Stemona spp. in stichoneurine and protostemonine groups Stemona species Amount found Parenchyma Stichoneurine gr. S. tuberosa S. phyllantha Protostemonine gr. S. burkillii S. cochinchinensis S. collinsiae S. curtisii S. kerrii Liginified cells Starch granules Vessels Fibers numerous thin-wall, rarely thick-wall few few small, numerous moderate thin-wall and thick-wall moderate moderate small, numerous Pharmacognosy Journal | December 2010 | Vol 2 | Issue 17 3 Kongkiatpaiboon, et al.: Microscopic Characterization as a Tool for Separation of Stemona Groups. and compound types of small starch granules were observed in both groups of Stemona. CONCLUSION Cross-sectional histology and powdered drug characteristics of the roots of various Stemona species growing in Thailand verified that the roots of the stichoneurine or tuberosa group had non-lignified larger pith containing numerous thin-walled parenchyma cells. The protostemonine Stemona roots had smaller liginified pith and were less abundant in thin-walled parenchyma. Thick-walled parenchyma cells were frequently found in the protostemonine Stemona roots, but rarely found in the roots of the stichoneurine group. The lignified fibers and vessels were frequently found in the root powders of protostemonine Stemona but rarely found in the stichoneurine group. These microscopic characterizations could be used as a primary tool to clearly identify groups of Stemona, and it could confirm their macroscopic characteristics. However, these characteristics could not distinguish each Stemona species. 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Lin LG, Li KM, Tang CP, Ke CQ, Rudd JA, Lin G, et al. Antitussive stemoninine alkaloids from the roots of Stemona tuberosa. J Nat Prod. 2008; 71:1107-10. 13. Lin LG, Leung HP, Zhu JY, Tang CP, Ke CQ, Rudd JA, et al. Croomine- and tuberostemonine-type alkaloids from roots of Stemona tuberosa and their antitussive activity. Tetrahedron. 2008; 64:10155-61. 14. Zhou X, Leung PHH, Li N, Ye Y, Zhang L, Zou Z, et al. Oral absorption and antitussive activity of tuberostemonine alkaloids from the roots of Stemona tuberosa. Planta Med. 2009; 75:575-80. ACKNOWLEDGEMENTS This study is a part of a Ph.D. thesis on Stemona, which is financially supported by the Thailand Research Fund (Royal Golden Jubilee Ph.D. Program Grant No. PHD/0139/2550). The authors thank Mr. Panupon Khumsupan for his kind help in proofing the manuscript. 4 Pharmacognosy Journal | December 2010 | Vol 2 | Issue 17