J. Res. Educ. Indian Med 2013; XIX (1-2): 1-8 ISSN 0970-7700

COMPARATIVE PHARMACOGNOSTICAL EVALUATION OF LEAVES OF REGIONALLY ACCEPTED SOURCE OF AGNIMANTHA HARSHITHA KUMARI,1* SHRIKANTH P,2 RESHMI PUSHPAN,1 HARISHA C R3 AND NISHTESWAR K4 Department of Dravyaguna,1 SDMCA, Udupi () Department of Dravyaguna,2,4 Department of Pharmacognosy,3 Institute for PG Training & Research in Ayurveda, Gujarat Ayurved University, Jamnagar (India)

Abstract: In the vedic lore, the drug Agnimantha occupied an important place and was employed to ignite the agni (fire) while initiating yajna (ritual of sacrifice). In Ayurvedic literature, this drug is indicated in diseases like Shotha, Vatavyadhi, Prameha, Sthoulya, Agnimandhya, Arshas etc. It is also included in the group of Dashamula (group of ten drugs whose roots form the useful part) which has wider applications in therapeutics. But different botanical source plants are considered for Agnimantha in different regions of the country based on regional traditional practices. Premna obtusifolia R.Br., Premna latifolia Roxb, Premna tomentosa Willd, phlomidis Linn, Clerodendrum inerme (L) Gaertn. belonging to family are used as Agnimantha are somesuch sources. As leaves are indicated for certain conditions, a proper thought should be given for substituting root with its leaves. In the present study an attempt is made to carry out the Pharmacognostical evaluation of leaves of four selected species viz., Premna obtusifolia R.Br., Premna latifolia Roxb, Clerodendrum phlomidis Linn, Clerodendrum inerme (L) Gaertn. which showed the presence of stomata, crystals, warty trichomes, epidermal cells etc. Pharmaceutical analysis showed proteins, phenols and other compounds. The four source plants taken for the study have shown more similarities pharmacognostically. Phytochemical evaluation showed the presence of proteins and carbohydrates in all the source plants taken for the study. Keywords: Agnimantha, Pharmacognosy, Mesophyll, pharmaceutical. Introduction Ayurvedic pharmacopoeia of India identifies Agnimantha, one among the Dashamula Clerodendrum phlomides Linn.f, as the source plants of Ayurveda bears controversy in its botanical for Agnimantha.[8] identity. Premna obtusifolia R.Br., Premna latifolia Both roots and leaves are considered to be Roxb, Premna tomentosa Willd, Premna villosa the useful part of Agnimantha.[9] P. obtusifolia Cl., Clerodendrum phlomidis Linn and syn P. serratifolia roots and C. phlomides roots Clerodendrum inerme (L) Gaertn. are some of the (Arani moola) are included in the list of 70 source plants considered for Agnimantha by medicinal plant species of high trade sourced from Ayurvedic physicians of various regions in the tropical forests.[10] P. obtusifolia falls under the country.[1,2,3] The treatises like Charaka Samhita, endangered list in the IUCN red list of threatened Sushruta Samhita have not described any varieties species.[11] To address the problem of non of Agnimantha. But certain Nighantus and later availability of raw materials for manufacture of texts have described two varieties- Brihat medicines, a proper emphasis should be given for Agnimantha and Kshudra Agnimantha.[4,5] Premna the cultivation of the plant in larger proportion, obtusifolia R.Br is considered as Brihat variety substitution of other species with similar action and Clerodendrum phlomides Linn.f as Laghu and simultaneously research should be carried out variety by many of the later authors[6] whereas it to substitute the part used in order to prevent the is considered vice versa according to few.[7] extinction of the species.

1. Ph.D Scholars 2. Asst. Professor 3. Head, Department of Pharmacognosy 4. Professor and HOD, Dravyaguna 2 Harshitha et al.

Agnimantha leaf is used in the treatment Macroscopical evaluation of Arshsas (haemorhoides) for Avagaha Sweda Comparative Macroscopic evaluation of (sitz bath) to reduce inflammation and pain.[12] leaves of P. obtusifolia, P. latifolia, C. phlomidis, Ayurvedic scholars of modern era consider leaf and C. inerme were carried out. also as one of the useful part for internal and external use.[13] Leaves of Premna obtusifolia R.Br. Microscopical evaluation is reported to be used in the treatment of colic, Free hand sections of leaves were made rheumatism, neuralgia etc.[14] Premna latifolia and washed with chloral hydrate solution. Sections Roxb is antimicrobial, diuretic and indicated in were then stained with phloroglucinol and conc. dropsy, colitis and boils.[15] Clerodendrum HCl. Powder microscopy of shade-dried powder phlomidis Linn.f. is helpful in dyspepsia, dysentery, was also carried out. Photomicrograph were taken colic, debility and nervous disorders.[16] by Carl zeiss binocular microscope.[21,22] Clerodendrum inerme (L) Gaertn leaf juice is known to relieve muscular pain, stiffness of legs, Organoleptic characters rheumatism and arthritis.[17] Root decoction of P. Powders of individual leaves of the drugs obtusifolia has shown anti inflammatory potential are subjected to organoleptic evaluation ie. Evaluation in experimental study conducted on Albino rats.[18] of taste, colour and odour were carried out.[23] Pharmacognostical studies of the root of Agnimantha (P. obtusifolia& C. phlomidis) has Histochemical tests already been carried. Previous reported work on Thick sections were treated with various Powder microscopy of C. phlomidis root reports reagents to locate chemical constituents i.e. Tannin, fragments of cork cells; small, pointed, aseptate, mucilage, lignin and calcium.[24] lignified fibres; simple, pitted vessels; lignified cells packed with rhomboidal crystals of calcium Pharmaceutical evaluation oxalate and numerous starch grain having narrow Preliminary phytochemical tests were hilum.[19] P. obtusifolia root powder microscopy conducted on leaf powders of P. obtusifolia, P. reveals rectangular shaped, thick walled cork cells latifolia, C. phlomidis and C. inerme by using the with brownish yellow pigment; parenchymatous four different extracts (Aqueous, Petroleum ether, cortical cells, stone cells, starch grains and Methanol & Chloroform).[25] tracheids.[20] Pharmacognostical studies on the leaves of Results different sources used as Agnimantha is not Macroscopic characters of leaves available. In this regard comparative Premna obtusifolia- Leaves simple, opposite pharmacognostic evaluation of leaves of P. sometimes whorled up to 9×6 cm, exstipulate, obtusifolia, P. latifolia, C. phlomidis and C. inerme petiolated, broadly ovate, shortly obtusely was taken up to authenticate the substitution of acuminate at apex, rounded at base, entire or these source plants to Agnimantha. crenate- serrate towards the apex; membranous when young, coriaceous when mature, dark Material and Methods green shining above light green below, venation Collection of sample reticulate, petioles up to 1.3 cm long (Fig. 1). Fresh plant leaves of P. obtusifolia, P. Premna latifolia- Leaves simple, petiolated, latifolia, C. phlomidis and C. inerme were collected up to 9×6 cm, ovate to ovate lanceolate, from Udupi district, Karnataka, India during the exstipulate, acute at apex, serrate or less often month of November 2008 and the botanical identity entire, glabrescent, scented when crushed, was confirmed by the botanist of Udupi, Dr.K. petioles up to 3.5 cm long, usually drying blue or Gopalakrishna Bhat, Professor of Botany (Rtd.). black (Fig. 2). Pharmacognostical Evaluation of Leaves of Agnimantha 3

Clerodendrum phlomidis- Leaves simple, forming a horse-shoe shaped structure. The xylem petiolated, opposite, exstipulate, ovate or sub- is radially arranged with xylem parenchyma. rhomboid, obtuse or acute, crenate, dentate or Phloem is towards the upper epidermis. Some of entire, undulate, exstipulate, petiolate, puberlous. the mesophyll tissue consists prismatic crystals Petiole up to 6-8 cm in long (Fig. 3). of calcium oxalate crystals and browinish Clerodendrum inerme- Leaves simple, colouring matter (Fig. 6). 5.1 opposite or ternate, up to 5×3.8cm, elliptic or obovate, exstipulate, thick flesy leathery, entire, Powder microscopy of Premna latifolia obtuse or emarginated at apex, glabrous, dark green Organoleptic characters of leaf powder are above light green below, petioles up to 1 cm long dark green colour, aromatic odour and bitter taste. (Fig. 4). Diagnostic microscopical characters of the leaf powder are stomata, prismatic crystal, epidermal Transverse section of Premna obtusifolia cells, simple fibres of phloem and prismatic Anatomically, leaf structure is dorsiventral crystals of calcium oxalate crystals (Fig. 6.1- with clear dorsal surface and ventral surface. 6.3). Upper epidermis is single layered with multicellular uniseriate hairs. Lower epidermis is made of single Transverse section of Clerodendrum layer of cells with compact arrangement. Mesophyll phlomidis is differentiated into upper palisade layer and lower T.S. of leaf shows upper epidermis is made spongy layer. T.S. through midrib shows in many of single layer of compact cells except in the layered both below the upper and lower epidermis region of stomata and bear uniseriate multicellular and is consisting of collenchyma cells. The hairs. Lower epidermis is also made of single vascular bundles are well developed and is forming layer of compact cells and bear uniseriate a horse-shoe shaped structure. The xylem forms multicellular hairs. Mesophyll is dorsiventral in radially arranged with xylem parenchyma. Phloem structure, with upper palisade and lower spongy is towards the upper epidermis (Fig. 5). parenchyma are many layered. T.S. through midrib is well marked with bulged oval part bearing Powder microscopy of Premna obtusifolia uniseriate hairs. Hypodermis is of 3-4 layers Organoleptic characters of the leaf powder consisting of collenchyma cells followed by are dark green colour, strong pungent odour and parenchyma tissue. The vascular bundles are bitter taste. Diagnostic microscopical characters placed in 3-4 patches. The xylem is radially of the leaf powder are the presence of stomata, oriented with protoxylem towards upper epidermis Spiral vessel, Multicellular warty trichome, simple with xylem parenchyma. Phloem poorly developed fibres of phloem, fragments of spiral vessels of towords the upper epidermis. There are xylem, fragments of palisade and spongy sclerenchyma patches in the central part enclosed parenchyma of mesophyll, prismatic crystals of by the arch of vascular bundles (Fig. 7). calcium oxalate crystals (Fig. 5.1-5.3). Powder microscopy of Clerodendrum Transverse section of Premna latifolia phlomidis T.S. of leaf shows both upper and lower Organoleptic characters of leaf powder are epidermis made of single layer of compactly light green colour, characteristic odour and bitter arranged barrel shaped cells. Mesophyll is taste. Diagnostic microscopical characters of the differentiated into upper palisade layer and lower leaf powder are tannin, Prismatic crystals, Spiral spongy layer. T.S. through midrib both upper and vessels, simple fibres of phloem, fragments of lower epidermis and is consisting of collenchyma palisade and spongy parenchyma of mesophyll cells. Vascular bundles are well developed and is (Fig. 7.1-7.3) 4 Harshitha et al.

Figure 1. Figure 2.

Figure 3. Figure 4.

Figure 5. Premna obtusifolia R.Br. leaf Fig 5.1 Stomata Transverse section of Clerodendrum inerme uniseriate multicellular warty trichomes. Lower T.S. of leaf shows upper epidermis is made epidermis is also made of single layer of compact of single layer of compact cells and bear cells. Mesophyll made of upper palisade and lower Pharmacognostical Evaluation of Leaves of Agnimantha 5

Fig 5.2 Spiral vessel Fig 5.3 Multicellular warty trichome

Figure 6. Premna latifolia Roxb leaf Fig 6.1 Stomata

Fig 6.2 Prismatic crystal Fig 6.3 Epidermal cells spongy parenchyma. T.S. through midrib shows is on the lower side but not very prominent with well marked with bulged oval part. The few seive elements (Fig. 8). hypodermis is of 3-4 layers consisting of collenchyma cells followed by parenchyma tissue. Powder microscopy Clerodendrum inerme There are many vascular bundles arranged in the Organoleptic characters of leaf powder are form of a arch with the open end pointing the dark green colour, irritant odour and bitter taste. upper epidermis. The xylem is radially oriented Diagnostic microscopical characters of the leaf with protoxylem pointing upward. The phloem powder are prismatic crystal, multicellular warty 6 Harshitha et al.

Fig 7.1 Tannin

Figure 7. Clerodendrum phlomidis Linn.f.

Fig 7.3 Spiral vessels Fig 7.2 Prismatic crystal

Fig 8.1 Prismatic crystal

Figure 8 Clerodendrum inerme (L) Gaertn

Fig 8.2 Multicellular warty trichome Fig 8.3 Stomata Pharmacognostical Evaluation of Leaves of Agnimantha 7

Table 1. Histochemical tests

Sr. Results Reagents Observation Characteristics No. A B C D 1. Phloroglucinol+Conc. HCl Red Lignified cells ++ ++ ++ ++ 2. Iodine Blue Starch grains     3. Phloroglucinol+Conc. HCl Dissolved Calcium oxalate crystals ++ ++ ++ ++

4. Fecl3 solution Dark blue to black Tannin cells ++  ++ ++ 5. Ruthenium red Red Mucilage  ++   A-P. obtusifolia, B- P. latifolia, C- C. phlomidis, D- C. inerme

Table 2. Pharmaceutical evaluation

Sr Drug name Cold Petroleum Ethanol Chloroform No. Infusion ether 1. P. obtusifolia Proteins, Carbohydrate, Proteins, Proteins, Carbohydrate, Proteins, Anthocyanins, Carbohydrate, Anthocyanins, Carbohydrate Glycosides, Flavanoids, Glycosides Glycosides, Flavanoids, Phenols, Steroids Steroids 2. P. latifolia Carbohydrate, Saponin, Phenols Proteins, Phenols _ Phenols 3. C. phlomidis Proteins _ Carbohydrate Carbohydrate 4. C. inerme Proteins, Carbohydrate, Proteins Proteins, Carbohydrate Proteins, Phenols, Glycosides, Saponin Alkaloids trichome, stomata and simple fibres of phloem of the leaves shows epidermis with cuticle, (Fig 8.1-8.3). anomocytic stomata, well differentiated upper palisade and lower spongy parenchyma, Histochemical Test mesophyll with chloroplast. Prismatic crystals Various histochemical tests were of calcium oxalate were preset in all three species conducted on the leaf powders. The results are except P.obtusifolia. T.S. through midrib shows depicted in table 1. well developed vascular bundle radially arrandged metaxylem towards lower side, protoxylem Pharmaceutical evaluation towards upper epidermis along with few xylem The components of all four source plants parenchyma, oil globules, xylem bounded by in different extracts are enumerated in Table 2. phloem with some sieve elements. The powder Discussion microscopy shows the presence of prismatic In the present study, pharmacognostic crystals except in P.obtusifolia, multi cellular evaluation of all four major plant species being warty trichomes except in P.latifolia. stomata, considered as source plants of Agnimantha was spiral vessels, tannin common in all the species. carried out. All the samples Premna obtusifolia The Histochemical test reveals the presence of lignin, R.Br., Premna latifolia Roxb, Clerodendrum tannin and Calcium oxalate crystals. Phytochemical phlomidis Linn.f., Clerodendrum inerme (L) evaluation shows the presence of protein, Gaertn belong to the family Verbenaceae, carbohydrate in all the source plants in different considered as source plants for Agnimantha. exracts. Phenols are present in three species except Leaves of these plants are simple with irritant C. phlomidis, Glycosides present in P. otusifolia odour, pungent bitter taste. Transverse section and C. inerme. The pharmacognostical evaluation 8 Harshitha et al. of the source plants of Agnimantha show similar 7. Vanvarilal Mishra: Dravyaguna Hastamalaka, structural characters. Phytochemical analysis has Publication Scheme, Jaipur pp.391 shown the presence of Glycosides, Flavanoids, 8. Anonymous: Ayurvedic pharmacopoeia of India, Government of India 3(1):3 Steroids only in P. otusifolia. If the phytochemical 9. Bapalal Vaidya G: Nighantu Adarsha, constituents reported in this species show the Choukhambha Bharati Academy 2005; 2:251 scientific validation of the pharmacological activities 10. Ved D K and Goraya G S: Demand and Supply attributed to Agnimantha in Ayurvedic classics, it of Medicinal Plants in India, National Medicinal may be appropriate to consider P. obtusifolia as Plants Board, New Delhi 2008 the true source of classical Agnimantha. 11. http://www.iucnredlist.org/apps/redlist/details/ 106002678/0 12. Agnivesha: Charaka Samhita, Revised by Charaka Conclusion and Dhridhabala with Ayurveda Deepika Agnimantha, one of the dashamula groups commentary of chakrapanidatta, edited by Vaidya of plant is frequently indicated in the management Jadavji Trikamji Acharya, Choukhambha of several disorders. All the source plants taken for Publication Varanasi 2001; pp.504 the study belonging to the family Verbenaceae, 13. Sharma P.V: Dravyaguna Vijnana , Vol.5 , shows identical structural characteristics of the Choukhambha, Varanasi, Page no 8. leaves even though difference is found in the 14. Anonymous: Wealth of India. Council of Scientific and Industrial Research 1948-76; 8:240 pharmaceutical composition which may give rise 15. Agarwal V.S: Drug plants of India. Kalyani to varied degree of action. Further, comparative publishers 1997; 2:584 clinical study of these source plants is required to 16. Khare C.P: Indian Medicinal plants- An illustrated substantiate the substitution. dictionary, Choukhambha Publication 2007; pp.160 Acknowledgement 17. Anonymous: Wealth of India. Council of Scientific The authors acknowledge Dr. Chaithra S and Industrial Research 1948-76; 2:67 18. Yellappa G.K: Anti Inflammatory effect of Hebbar, Dr. T. Shridhara Bairy, SDM College of Agnimantha (Premna integrifolia), dissertation Ayurveda, Dr. K. Gopalakrishna Bhat, Professor of submitted to Rajiv Gandhi University of Health Botany (Rtd.), Dr. S. L. Karnik for their constant Sciences, Bangalore 2004 support during the study. 19. Anonymous: The Ayurvedic pharmacopoeia of References India, Government of India 2001; 3(1):4 1. Sarin Y.K: Illustrated manual of Herbal drugs used 20. Anonymous: Pharmacognosy of Ayurvedic drugs, in Ayurveda, A joint publication of council of scientific series 1, No.2, Department of pharmacognosy, and Industrial Research and Indian council of Medical Government Ayurveda college, Trivandrum 1978; Research 1996; pp.2 pp.25-27 2. Kamat S.D: Studies on medicinal plants and drugs 21. Trease G. E, Evans W.C: Pharmacognosy, 12th in Dhanvantari nighantu, Chaukhambha Sanskrit Ed. Bailliere Tindall, Eastbourne. U.K. 1983; prathishthan, Delhi 2002; pp.40 pp.95-99, 512-547 3. Yoganarasimhan S.N: Medicinal plants of India, 22. Wallis T.E: Text book of Pharmacognosy, 5th Vol 1, Regional Research centre, Interline publishing Ed, CBS Publishers, New Delhi 1985; pp.571- private ltd, Bangalore 1996; pp.128, 386 578 4. Raja Nighantu, Indradeeva Tripathi: Choukhambha 23. Kokate C K, Purohit A P, Gokhale S B: Orientalia, Varanasi, Prabhadradi varga 2003; pp.268 Pharmacognosy. 42nd Edition, Nirali Prakashan 5. K.C.C., Pandey G.S: Bhavamisra, Bhava Prakasha Pune 2008; Chapter 6.1, A 1 Nigantu, Choukhambha Bharati Academy,Varanasi. 24. Krishnamurty K.V: Methods in the plant Guduchydi varga 2006; pp.281 histochemistry, Vishwanadhan Pvt Limited, 6. K.C.C., Pandey G.S: Bhavamisra, Bhava Prakasha Madras 1988; pp.1-77 Nigantu, Choukhambha Bharati Academy,Varanasi. 25. Anonymous: The Ayurvedic pharmacopoeia of Guduchydi varga 2006; pp.281 India. Government of India. 2(1):189-262.

Address for correspondence: Dr. Harshitha Kumari, Resaerch Fellow, Department of Dravyaguna, Institute for PGTraining and Research in Ayurveda, Gujarat Ayurved University, Jamnagar (India). [email protected] 529_2012 Revised on 20-9-2012