Plant Archives Volume 21, No 1, 2021 pp. 1661-1666 e-ISSN:2581-6063 (online), ISSN:0972-5210 Plant Archives Journal home page: www.plantarchives.org DOI Url: https://doi.org/10.51470/PLANTARCHIVES.2021.v21.no1.228 MACROSCOPIC AND MICROSCOPIC CHARACTERIZATION OF RAW HERBAL DRUG MAMAJJAKA [ENICOSTEMA AXILLARE SUBSP. LITTORALE (BLUME) A. RAYNAL] Pankaj Kumar1,2, Bushan Kumar1, Sumeet Gairola1,2* 1 Plant Sciences Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu – 180001, UT of J&K, India 2 Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India *Email: sumeetgairola@iiim.res.in (Date of Receiving-12-01-2021; Date of Acceptance-18-04-2021) ABSTRACT Enicostema axillare subsp. littorale (Blume) A. Raynal belonging to the family Gentianaceae, known as Mamajjaka in Ayurveda, is a medicinally important plant used to treat Madhumeha or diabetes mellitus. The plant has high demand in the Indian herbal drug market and is used in various herbal preparations. The present study was conducted to characterize dried herbal samples of E. axillare subsp. littorale botanically. The botanical characterization involved macroscopic and microscopic studies on the herbal samples. Qualitative and quantitative microscopic characters were studied, and digital photographs of transverse sections (T.S.) and powder cell structures were taken. In T.S. of the stem, the vascular region characters such as the appearance of xylem tissue, lumen diameter of xylem vessels, and powder characters of aerial parts such as abundance and size of starch grains, prismatic crystals, presence of stomata, and trichomes were studied. The characters identified in the present study can be used to identify the herbal samples of E. axillare subsp. littorale in fresh as well as in dry forms. Keywords: Raw herb, anatomical characters, Chota Chirayta, Mamajjaka, qualitative characters, quantitative characters. INTRODUCTION Enicostema axillare subsp. littorale (Blume) A. Raynal belonging to the family Gentianaceae, occurs as a weed in diverse natural habitats such as savannas, grasslands, forests to beaches, from wet to very dry habitats, and also survives well in a very saline environment. This species is globally distributed in South America, Africa, and Asia. It is found almost throughout India up to 450 m asl, commonly in coastal areas (Praveena et al., 2014). As per www.theplantlist.org version 1.1 (TPL, 2013), E. axillare subsp. littorale is known by various synonyms such as Enicostema littorale Blume, Adenema hyssopifolium G.Don, Coutoubea verticillata G.Don, Ericoila verticillata Borkh., Gentiana hyssopifolia L. ex Spreng., Gentiana lignosa Murray ex Steud., Gentiana octoflora Vitman, and Hippion littorale (Blume) Miq. It is commonly known as ‘Bitter tonic,’ ‘Chota Chirayta,’ ‘Indian Gentian,’ or ‘Indian Whitehead.’ In Ayurveda, it is known by various names such as Naagjhvaa, Maamajjaka, Naahi, and Tikshnapatra; in Unani, it is known as Naai, and Naahi, and in Siddha, it is known as ‘Vellargu’ (Khare, 2007). The plant is reported to be used in Indian Systems of Medicine (ISM), including Ayurveda, Unani, and Siddha. Dried roots and the aerial parts of the plant are used as a laxative, anthelmintic, in curing several health problems such as fever, rheumatism, vata diseases, skin diseases, abdominal problems, obesity, regulating blood sugar levels, and also in snake bite (Kirtikar and Basu, 1935; Saranya et al., 2013). In Ayurveda, it is used to treat diabetes, typhoid, control arthritis, and as a cooling agent (Mishra et al., 2017; Thondaiman and Saha, 2017). The plant is known to have several phytoconstituents and secondary metabolites such as alkaloids, sterols, volatile oils (Natarajan and Prasad, 1972), betulin, a triterpene sapogenin (Dymock et al., 1893), monoterpene alkaloids, flavonoids (Ghosal et al., 1974), catechins, saponins, steroids, sapogenin, triterpenoids, flavonoids, xanthones, and flavone C-glucoside (Verticilliside) (Jahan et al., 2009). The plant is reported to have ophelic acid, which is also present in ‘Chiretta’ as a hydrolytic product of ‘Chiratin’ (Khare, 2007). It is considered a potential hypolipidemic plant (Gopal et al., 2004) and used as a substitute for Swertia chirayita (Roxb.) Buch.-Ham. ex C.B.Clarke (Nadkarni, 1976; Khare, 2007). The plant is also known to have many pharmacological properties such as hepatoprotective (Dash et al., 2000; Gupta and Singh, 2007; Vaijanathappa et al., 2008; Gite et al., 2010), antimicrobial (Katewa and Arora, 2001; Leelaprakash and Dass, 2012), hypoglycaemic (Ravi et al., 2000; Vishwakarma et al., 2010), antihelminthic (Mishra and Shukla, 2011), antinociceptive (Jaishree et al., 2009), antioxidant (Mukundray et al., 2011; Thirumalai et al., 2011), antiulcer and anti-inflammatory (Roy et al., 2010), antihyperlipidaemic (Gopal and Udayakumar, 2008; Vaidya et al., 2009; Gopal et al., 2011), and anti-diabetic (Shah and Gopal, 1985; Upadhyay and Goyal, 2004). The estimated annual trade of E. axillare is between 50- Pankaj Kumar, Bushan Kumar, Sumeet Gairola 100 MT (NMPB, 2020). Dried herbal samples do not show many characteristic morphological features useful for identification. Besides, similar and confusing common names create an identification problem in dried herbal samples. For quality assurance of plant samples in herbal medicines, correct identification of raw herbal samples is required (Sahoo et al., 2010). Microscopic characterization of herbal drugs is considered significant for taxonomic identification of fragmented herbal samples (Metcalfe and Chalk, 1957). In the present study, macroscopic and microscopic studies were performed on aerial plant samples of E. axillare subsp. littorale to develop botanical references, which can be used for correct identification of its samples in both fresh and dried forms. Macroscopic characters The plant is a perennial herb, 5-30 cm tall with few branches at the base having a cylindrical, glabrous stem, sessile leaves, and pentamerous flowers in axillary clusters (Fig. 1A-1F). Leaves are linear to lanceolate or narrowly oblong, sessile, glabrous, with entire margin, mucronate apex, lamina narrow, lanceolate, and 3-nerved (Fig. 1D). Flowers are pentamerous, sepals 5 with acute apex and fused base; petals 5, fused at base, appear funnel-shaped, five epipetalous stamens, and ovary superior (Fig. 1E & 1F). Flowers white with greenish lines and turn yellowish after drying (Fig. 1B & 1C). The plant is known to produce many seeds but reported with low germination rates in natural conditions. Root suckers are used for vegetative propagation. Dried plant material consists of the light yellowish-brown, quadrangular thin stem (0.1-0.2 cm), with nodes and internodes (Fig. 1A). Nodes bear opposite decussate leaves and inflorescence in leaf axils (Fig. 1B & 1C). MATERIAL AND METHODS Authentic plant material was collected from Chota Udaypur, Rajasthan, and dry herb material was procured from Khari Boali Herbal Market Delhi. Duly identified herbarium specimens were submitted to the internationally recognized Janaki Amal Herbarium (RRLH) at Indian Institute of Integrative Medicine (CSIR-IIIM), Jammu (accession nos RRLH-23379 and RRLH-23380), and dried raw herb samples were submitted to the Crude Drug Repository at CSIR-IIIM Jammu (accession nos CDR4035 and CDR-4046). Microscopic characters The stem’s T.S. appeared irregular circular or angular in outline with wing-like protuberances (Fig. 1H & 1I). The outermost zone is formed of the single-layered, cuticularised epidermis with thin-walled, square, or rectangular-shaped cells. Next to the epidermis, the parenchymatous cortex zone (3-8 layered) of variable thickness (46.64 to 111.20 µm) with loosely arranged cells is present. Cortex is followed by a thin, continuous phloem zone (21.29±2.18 µm) present over a broad xylem zone (192.88±10.63 µm). Xylem tissue formed a continuous ring-like structure and appeared nearly circular in outline on the outer side (near phloem region), whereas it appeared irregular to angular (quadrangular) on the inner side. Xylem consisted of thick-walled xylem fibers and few xylem vessels, being present in a linear arrangement. The central part formed of broad hollow pith, mostly with disintegrated cells in dried stem samples (Fig. 1H & 1I). Pith formed a major broad (408.50±35.41 µm) hollow zone in the center compared to other tissue zones. Phloem was observed to form the smallest zone relative to the radius of studied T.S. of the root (Table 1). Macroscopic and microscopic studies were performed on dry herbal samples. The macroscopic study involved the study of surface characters, color, texture, and appearance. For the anatomical study, dried samples were rehydrated in water for 24 h, and thin transverse sections (T.S.) were obtained by freehand sectioning using a razor blade. Fine T.S. was stained as per Kumar et al., (2018) with minor modifications. The T.S. were dehydrated in series of alcohol gradients (30%, 50%, and 70% alcohol, each for 10-15 min), stained in safranin stain for 5-7 min, decolorized in 70% alcohol for 5 min, stained in fast green for 3-5 min. The T.S. was again decolorized in 70% alcohol for 3-5 min and then dehydrated in 90% alcohol, followed by absolute alcohol for 5-7 min. The T.S. were cleared in xylene, mounted in Canada balsam, and were examined under a compound microscope (LEICA DM 750) with the associated camera (LEICA ICC50E). Also, a microscopic examination of powder samples was done on the aerial drug powder. The powder was passed through a fine sieve, and the water mounted slides were observed under a compound microscope to study the various cell types and cell contents. Powder characteristics Aerial plant powder of E. axillare subsp. littorale is fine textured, light greenish to brown colored with characteristic odor and bitter taste (Fig. 1G). Microscopic examination of aerial powder sample under the compound microscope showed the presence of few xylem vessel fragments, several prismatic crystals of variable size, few fragments of parenchyma cells, few anisocytic stomata, unicellular trichomes, leaf epidermal fragments, and few starch grains (elongated to oval in shape) (Table 1, Figs. RESULT AND DISCUSSION The macroscopic and microscopic studies’ observations have been given in Table 1 and Figures 1 & 2. 1662 Macroscopic and microscopic characterization of raw herbal drug Mamajjaka [Enicostema axillare subsp. Littorale (blume) a. Raynal] Table 1: Quantitative anatomical characters of the T.S. of the stem of E. axillare subsp. littorale. Character Min. Max. Mean (±S.D.) The thickness of various zones The radius of studied T.S. 651.12 778.72 717.96 (±13.15) Cortex zone thickness 46.64 111.20 67.03 (±6.32) Phloem zone thickness 10.07 32.14 21.29 (±2.18) Xylem zone thickness 121.25 236.34 192.88 (±10.63) Pith radius 364.55 553.71 408.50 (±35.41) Epidermal cell size Length 13.16 26.33 20.11 (±1.17) Breadth 9.41 21.13 14.18 (±1.10) Xylem vessel lumen diameter 13.08 35.73 21.34 (±2.49) Powder characteristics Starch grains (Length) 20.61 65.63 35.47 (±4.25) Starch grains (Breadth) 16.32 34.54 24.70 (±2.16) Prismatic crystals (Length) 26.78 42.65 35.01(±1.83) Prismatic crystals (Breadth) 14.57 42.08 24.31 (±2.32) 2A-2I). other genera like S. chirayita. Distinguishing herbal drug samples of species with confusion in botanical characters requires a reference standard. Sanmugarajah et al., (2013) performed Phyto-physicochemical standardization for correct identification and authentication of whole plant samples of E. axillare. In some botanical studies, macroscopic and microscopic characters of fresh leaf, stem, and root samples were studied (Laxman et al., 2010; Praveena et al., 2014). In an anatomical study of the stem, Laxman et al., (2010) observed around ten vascular bundles in the stem, however, Praveena et al., (2014) observed vascular region in T.S. of the stem as a single continuous ring-like structure. In an anatomical study, Praveena et al., (2014) observed T.S. of the stem as fourangled with the short stumpy wing-like structure on four edges, few stomata in the epidermal region, and a central broad, parenchymatous pith. In the present study, a single continuous ring-like vascular bundles were observed in T.S. of the stem with compact, deformed parenchyma cells of the cortex and the pith region’s disintegrated cells giving pith a hollow appearance. For botanical identification of dried herbal samples, parenchymatous cells may be prone to disintegration and may not be suitable for species characterization in dried form. However, the vascular region cells were observed as intact and can be considered suitable characters for species distinction. The anatomical characters of the vascular bundle (the type of xylem cells, arrangement of xylem cells, and lumen diameter of xylem vessels) were found to be of taxonomical value. Discussion Several earlier studies used qualitative and quantitative anatomical characterization for botanical identification of raw herbal samples. The macroscopic and microscopic study of aerial drug samples, including qualitative descriptions combined with quantitative data, can help dry samples’ correct identification. Li et al., (2008) used semi-quantitative and quantitative micromorphological characters to authenticate Halenia elliptica (Gentianaceae). Hassan et al., (2015) described the thickness of various tissue zones and T.S.’s total thickness as a quantitative character for the characterization of stem, root, and leaf samples of Calotropis procera. Singh et al., (2020) used some quantitative characters such as the size of epidermis, hypodermis, cortex cells, starch grains, number and distribution of vascular bundles, presence or absence of specific cells in cross-section, etc. for the anatomical description of the stem of F. cirrhosa. Kumar et al., (2018) observed the shape and size of starch grains, presence, type, shape, and size of calcium oxalate crystals to distinguish closely related Astavarga species. Enicostema axillare subsp. littorale is reported to have many pharmacological properties and several important traditional uses. However, its some synonyms and common names are confusing and reported to be interchangeably used with other species of Enicostema like E. verticellatum (L.) Engl., and also with species of 1663 Pankaj Kumar, Bushan Kumar, Sumeet Gairola 1664 Macroscopic and microscopic characterization of raw herbal drug Mamajjaka [Enicostema axillare subsp. Littorale (blume) a. Raynal] Figure 1: Enicostema axillare subsp. littorale, A). Dry aerial herb sample, B). Aerial part, (C), Flowers in clusters, D). A 3-nerved leaf, E). Inner pentamerous flower view showing stamens and pistil, F). Outer pentamerous flower view showing sepals and petals, G). Powder of aerial part, H). T.S. of the stem, I). T.S. of stem- an enlarged portion. Gite V.N., R.D. Pokharkar, V.V. Chopade and S.B. Takate (2010). Hepato-protective activity of Enicostema axillare in paracetamol induced hepato-toxicity in albino rats. J. Pharmacol., 1(2): 50–53. CONCLUSION In the present study, qualitative and quantitative microscopic characters were studied, and digital photographs of transverse sections (T.S.) and powder cell structures were taken, which can be used as a reference for identifying samples of E. axillare subsp. littorale in both fresh and dried forms. Powder study of aerial parts also revealed some organoleptic and microscopic features such as shape and size of starch grains, prismatic crystals, type of stomata and trichomes, and abundance of other cell structures, which are useful for species identification. Gopal, R. and R. Udayakumar (2008). Enzymatic and nonenzymatic antioxidant activity in p- DAB induced hepatocarcinoma in rats. Int. J. Pharmacol., 4 (5): 369–375. Gopal, R., A. Gnanamani, R. Udayakumar and S. Sadulla (2004). Enicostema littorale Blume- A potential hypolipidemic plant. Nat. Prod. Rad., 3(6): 401–405. Gopal, T.K., S. Vidyadhar, U.M. Reddy, Chamundeeswari, S, Reddy, A. Saidulu, et al., (2011). In vitro antifungal activity of various extracts of Enicostema littorale. J. Biotech. Biother., 1:2. ACKNOWLEDGMENTS The authors thank Director, IIIM Jammu for providing necessary facilities to carry out the work. The authors are thankful to the Council of Scientific and Industrial Research (CSIR), Government of India, for financial assistance under the Major Lab Project (MLP-1007). PK acknowledges the financial support provided by CSIR in the form of JRF/SRF fellowships. Gupta R.S. and D. Singh (2007). Hepatomodulatory role of Enicostema littorale Blume against oxidative stress induced liver injury in rats. Afr. J. Agri. Res., 2(4): 131–138. REFERENCES Hassan, L.M., T.M. Galal, E.A. Farahat and M.M. El-Midany (2015). The biology of Calotropis procera (Aiton) W.T., Trees. 29: 311–320. Dash, G.K., A. Samanth and S.K. Kannungo (2000). Hepatoprotective activity of Indian medicinal plants. Indian J. Nat. Prod., 16: 2. Jahan, E., S. Perveen and A. Malik (2009). Verticilliside, a new flavones C-glucoside form Enicostema verticillatum. J. Asi. Nat. Prod. Res., 11(3): 257–260. Dymock, W., C.J.H. Warden and D. Hooper (1893). Pharmacographica Indica. Calcutta: Thacker, Spink & Co., 2: 516. Jaishree, V., S. Badami, M.R. Kumar and T. Tamizhmani (2009). Antinociceptive activity of swertiamarin isolated from Enicostema axillare. Phytomedicine, 16: 227–232. Ghosal, S., A.K. Singh, P.V. Sharma and R.K. Chaudhuri (1974). Chemical constituents of Gentianacceae IX: Natural occurrence of erythrocentaurin in Enicostema hissopifolium and Swertia lawii. J. Pharm. Sci., 63(6): 944–945. Katewa, S.S. and A. Arora (2001). Indian Drugs. 38: 6. Khare, C.P. (2007). Indian medicinal plants: An illustrated dictionary, Springer. Kirtikar, K.R. and B.D. Basu (1935). Indian medicinal plants 1665 Pankaj Kumar, Bushan Kumar, Sumeet Gairola Roy, S.P., C.M. Niranjan, T.M. Jyothi, M.M. Shankrayya, K.M. Vishawanath, K. Prabhu, V.A. Gouda and R.S. Setty (2010). Antiulcer and anti-inflammatory activity of aerial parts of Enicostema littorale Blume. J. Young Pharm., 2 (4): 369–373. Vol. III. Saujanya Books, Dehradun, India. Kumar, P., B. Kumar, K. Singh and S. Gairola (2018). Morphoanatomical standardization of six important RET medicinal plants of Astavarga group from Western Himalaya, India. Res. Rev. Biotech. & Biosci., 5(1): 1–14. Sahoo, N., P. Manchikanti, S. Dey (2010). Herbal drugs: standards and regulation. Fitoterapia, 81(6): 462–471. Laxman, S., P. Bala, K. Yusuf and P. Nancy (2010). Pharmacognostical standardization of Enicostema littorale Blume. Pharmacogn. J., 2(16): 15–24. Sanmugarajah, V., I. Thabrew, S.R. Sivapalan (2013). Phyto, Physicochemical standardization of medicinal plant Enicostema littorale, Blume. IOSR-J. Pharm., 3(2): 52–58. Leelaprakash, G. and S.M. Dass (2012). Antimicrobial activity and phytochemical screening of methanol extract of Enicostema axillare. Int. J. Pharm. Pharm. Sci., 4(1): 342–348. Saranya, R., T. Thirumalai, M. Hemalatha, R. Balaji and E. David (2013). Pharmacognosy of Enicostema littorale: A review. Asian Pac. J. Trop. Biomed., 3(1): 79–84. Li, J., T. Yi, H.S. Lai, D. Xue, H. Jiang, H.C. Peng and H. Zhang (2008). Application of microscopy in authentication of traditional Tibetan medicinal plant Halenia elliptica. Microsc. Res. Techniq., 71 (1): 11-19. Shah, G.L. and G.V. Gopal (1985). Ethnobotanical notes from the tribal inhabitants of the North Gujarat (India). J. Econ. Tax. Bot., 6(1): 193–201. Singh, K., P. Kumar, B. Kumar, J.F. Lone, P.R. Sharma and S. Gairola (2020). Morpho-anatomical and palynological standardization and DNA barcoding of Fritillaria cirrhosa D. Don (Syn. Fritillaria roylei hook.). Plant Arch., 20(2): 1304–1313. Metcalfe, C.R. and L. Chalk (1957). Anatomy of the dicotyledons. Vol I. Clarendon Press, Oxford., 502– 535. Mishra, N., K. Kaushal, R.C. Mishra and A.K. Sharma (2017). An ayurvedic herb: Enicostema littorale Blume-A review article. J. Med. Pl. St., 5(1): 78–82. Thirumalai, T., V.S. Therasa, E.K. Elumalai and E. David (2011). Hypolipidemic and antioxidant effect of Enicostema littorale Blume. Asian Pac. J. Trop. Biomed., 1(5): 381–385. Mishra, S. and P. Shukla (2011). In vitro anthelmintic activity of Enicostema littorale Blume. Int. J. Pharma. Sci. Res., 2(5): 1193–1196. Thondaiman, V. and A. Saha (2017). A bitter plant with sweet future. Kerala Karshakan, 4(11): 30–31. Mukundray, N.B., K. Chauhan, S. Gupta, P. Pillai, C. Pandya, J.V. Thaikoottathil and S.S. Gupta (2011). Protective effect of Enicostema littorale Blume methanolic extract on Gentamicin induced Nephrotoxicity in rats. Am. J. Inf. Dis., 7(4): 83–90. TPL (2013). www.theplantlist.org (accessed on 18th March 2018). Upadhyay, U.M. and R.K. Goyal (2004). Efficacy of Enicostema littorale in Type 2 diabetic patients. Phytother. Res., 18(3): 233–235. Nadkarni, A.K. (1976). Indian Materia Medica, Vol. 1. Bombay: Popular Prakashan Private Ltd.. Vaidya, H., M. Rajani, V. Sudarsanam, H. Padh and R. Goyal (2009). Swertiamarin: A lead from Enicostema littorale Blume for antihyperlipidaemic effect. Eur. J. Pharmacol., 617(1-3): 108–112. Natarajan, P.N. and S. Prasad (1972). Chemical investigation of Enicotemma littorale. Planta Med., 22: 42–46. NMPB (2020). Traded Medicinal Plant Database. http:// envis.frlht.org/traded-medicinal-plants-database.php (accessed 28th July 2020). Vaijanathappa, J., S. Badami and S. Bhojraj (2008). In vitro antioxidant activity of Enicostema axillare. J. Heal. Sci., 54(5): 524–528. Praveena, P., D. Sudarsanam, J.M. Ambrose and Jayaraman (2014). Phytochemical and Pharmacognostic Value of Enicostema littorale. Int. J. Pharmacog. Phytochem. Res., 6(4): 795–800. Vishwakarma, S.L., S.D. Rakesh, M. Rajani and R.K. Goyal (2010). Evaluation of effect of aqueous extract of Enicostema littorale Blume in streptozotocin- induced type 1 diabetic rats. Ind. J. Exp Bio., 48(1): 26–30. Ravi, V., K. Monika and G. Sarita (2000). Hypoglycaemic effect of aqueous extract of Enicostema littorale Blume (Chhotachirayata) on alloxan-induced diabetes mellitus in rats. Indian J. Exp. Biol., 38: 781–784. 1666