499 Darius (Identification of Secretory).cdr IDENTIFICATION OF SECRETORY STRUCTURE, HISTOCHEMISTRY AND P C HYTOCHEMICAL OMPOUNDS OF EDICINAL LANT qM P Hyptis capitata Jac . DARIUS RUPA , YOHANA SULISTYANINGSIH , DORLY DIAH RATNADEWI 1 2* 2 2 C. and 1 Universitas Department of Biology Education, Faculty of Teacher Training and Education, Borneo Tarakan, Tarakan 77123, Indonesia 2 t of y c of h cs and Natural Sciences, Departmen Biolog , Fa ulty Mat emati Institut Pertanian Bogor, Bogor 16680 a , Indonesi Received 29 June 2015/Accepted 13 June 2016 ABSTRACT Hyptis capitata Jacq. (common name: Knobweed or False Ironwort) belongs to Lamiaceae family and is among known traditional medicine. The Anak Dalam Tribe of Jambi Province uses the leaves of H. capitata to cure external and internal wounds. This study was aimed at identifying and analyzing secretory structure, histochemistry and phytochemical content of H. capitata leaves. The results showed that H. capitata leaves have secretory structures in the form of peltate, capitate and uniseriate glandular trichomes on the upper and lower leaf surfaces, with idioblast cells scattered throughout the leaf mesophyll. Histochemical tests indicated that the peltate trichomes have four head cells, containing alkaloids and terpenoids. This study classified capitate trichomes into two types: 1. type I has one stalk cell and two head cells, all containing alkaloids, terpenoids and lipophilic compounds; 2. type II has long stalk cells consisting of seven to ten cells with one head cell, all containing alkaloids and terpenoids. Uniseriate glandular trichomes of H. capitata leaves consist of four to eight cells containing alkaloids and terpenoids. The idioblast cells are round-shaped and contain lipophilic compounds. GC-MS analysis showed that H. capitata leaves contain terpenoid compounds assumed to serve as anti-infective agents, including l-limonene, eugenol, farnesol isomers A, d-nerolidol, hexahydrofarnesol and neophytadiene. Keywords: Glandular trichome, Hyptis capitata, idioblast, medicinal plants, phyto-compounds INTRODUCTION Rapid deforestation in Jambi area will cause a great loss of medicinal plants. It is, therefore, important to preserve the tribal knowledge on herbal medicine. is one of various Hyptis capitata plant species used as herbal medicine by Anak Dalam Tribe, which is native to Jambi Province in Sumatera, Indonesia. Hyptis capitata an erect herbaceous plant, is native to Central and tropical South America. The plant is usually abundantly found in open fields and roadsides. In Indonesia, particularly in Jambi Province H. capitata, the leaves of (Fig. 1) are used by the tribe as herbal medicine to Anak Dalam cure external and internal wounds. The tribe crushes or squeezes the leaves prior to using them to cure wound. The lea can be f sap consumed to treat inner injuries (T menggungu Tarip, Anak Dalam Tribe, pers. comm. 2012). This plant is also used as traditional medicine in India, Jamaica and Bangladesh (Michell & Ahmad 2006; Biswas et al. 2010 phytochemical study ). A conducted by Okach et al. (2013) reported that several other species of the Lamiaceae family, such as (E.Mey. Ex Benth) Becium obovatum . N.E.Br., Leucas calostachys Oliv, Ocimum kilimandscharicum Baker Ex. Gurke, Plectranthus barbatus Satureja and biflora (Ham Ex. Andrews D. Don) Brig., contain sterols, terpenoids, alkaloids, saponins, glycosides, flavonoids and tannins. Dickison (2000) reported that most medicinal plants have secretory structures that play a part in metabolite production. Various chemical * Corresponding author: yo_sulistya@yahoo.com 94 BIOTROPIA 4 2 7 94 103 Vol. 2 No. , 201 : - DOI: 10.11598/btb.201 .2 . .7 4 2 499 compounds, such as essential oils, resins, latex, mineral salts, alkaloids and glycosides, are produced by secretory structures. Werker (1993) stated that phytochemical substance of terpenoids, alkaloids and various volatile compounds are generally produced by the glandular trichomes contained in the species of Lamiaceae. Scientific study to identify the secretory structures which might be the site of accumulation and/or biosynthesis of metabolite and phytochemical compounds in will H. capitata improve the production of its desirable phytochemical compounds through tissue or cell culture. This study was aimed at identifying the secretory structure analyz , ing the accumulated metabolite compounds in the secretory structure and analyzing the H. phytochemical content in capitata . This research also examined the leaves efficacy of H. capitata plant, so that the natural substance contained in the plant can be scientifically justified for its pharmaceutical properties. MATERIALS AND METHODS Plant Material Collection The study was carried out in tropical lowland rain forest of Bukit Dua Belas National Park in Jambi Province, Sumatera, Indonesia (01°96'109" S, 102°58'630" E, 87 m asl). Leaf samples were collected from areas of jungle-rubber agroforest, which is an agroforestry system that combines rubber trees with natural wild plant species grown surrounding the rubber trees. Leaves were harvested in the dry season of July 2013 at average temperature of 29 °C and average relative humidity of 84%. Microscopic observation and histochemical analysis were performed on leaves from the third node of the plant, with three replications. A composite of leaves taken from plant several samples was subjected to phytochemical analysis. For microscop observation leaves ic purpose, were 70% or preserved in ethanol. F observation using Scanning Electron Microscope , (SEM) the leaves were in FAA solution preserved (formaldehyde, glacial acetic acid and 70% ethanol ; 1 : 1 : 18). Fresh leaves were collected for the histochemi or GC-MS analysis cal analysis. F the samples were dried for three days and sun- then dried at 60 for three days oven- °C . Observation using Light Microscope Paradermal leaf sections were prepared as semi-permanent slides, following a procedure suggested by Sass (1951). The preserved leaves were washed with distilled water for five minutes, soaked in 50% HNO solution for five minutes, 3 rinsed with distilled water for one minute, and then the adaxial and abaxial surfaces of the leaves were peeled. The samples were observed using a light microscope (Olympus CX21) at 100x and 400x magnifications. Identification of medicinal properties of Hyptis capitata Jacq. – Rupa et al. 95 Figure 1 H. capitata grows wildly in the jungle-rubber agroforest at Bukit Duabelas National Park, Jambi Province 70% ethanol and observed using light microscope at 400x magnification. The presence of lipophilic compound was by the production of s indicated red, yellow, or orange colors in the leaf tissue, following et al ( Boix . 2011). Phytochemical iAnalys s phytochemical components The presence of in the leaf tissue was identified using GC-MS technique. Two milligrams of powdered sample was placed in the chamber of the GC-MS instrument Shimadzu QP2010 . To analyze ( - ) the chemical composition, the sample went through the following processes: pyrolysis temperature 00 , oven temperature , o o 4 C 50 C injector temperature 280 , inter-surface o C temperature 280 o C and ion source temperature 200 . The carrier gas was helium. The o C spectrogram mass resulting from those processes was calculated then automatically by the GC-MS instrument. The calculation was based on the similarity of mass number/charge number pattern referring to the GC-MS instrument's database NIST Wiley databases. / RESULTS AND DISCUSSION In the leaves of H. capitata, there were two types of secretory structure observed i.e. glandular trichomes and idioblast cells. Glandular trichomes were found on the upper (adaxial) surface and lower (abaxial) surface of the leaves. Idioblast cells were found throughout the leaf mesophyll, from the palisade to the spongy parenchyma. There were one peltate, two types of capitate and one uniseriate glandular trichome identified in this species (Fig. 2). Members of the Lamiaceae family, such as Ocimum canum, Mentha spicata as well as Scutellaria linearis Benth., S. baiacalensis, S. gallericulata L. and S agrestis (Shang et al. 2010; Venkateshapp & . Sreenath 2013; Oliveira et al. 2013), generally have glandular trichomes. Several species have both peltate and capitate trichomes, while others have either only peltate or only capitate trichomes (Huang . 2008). Species of Lamiaceae family et al having various types of glandular trichomes include Salvia chrysophylla, Egyptian ballota, Isodon rubescens, Pogostemon cablin Benth., Scutellaria agrestis and Rosmarinus officinalis (Kahraman et al. 2009; Observation using Scanning Electron Microscope (SEM) The leaf samples were washed in caccodylate buffer for two hours using an ultrasonic cleaner (Sibata SU-6THE, Japan), pre-fixed in a 2.5% glutaraldehyde solution for two days at 4 °C, and then fixed in 2% tannic acid solution for 6 hours. The samples were then washed twice in caccodylate buffer for 10 minutes each time, rinsed with distilled water for 15 minutes, and dehydrated in a graded ethanol series of 70%, 80% and 90% for 10 minutes each, followed by twice dehydration in 100% ethanol for 10 minutes each time. The samples were then soaked twice in tert-butanol for 10 minutes each time, dried in vacuum drier for 20 minutes, coated with gold, and finally observed using a Scanning Electron Microscope (JEOL JSM 5310 LV Hitachi) at 20 kV ( 200x, 750x and 1,000x magnifications). Histochemical Analysis For histochemical analysis, fresh leaves were transversely sectioned at 15 - 20 µm, using a dual purpose microtome (Yamato RV-240). A small piece of fresh leaf lamina was inserted in the specimen holder. The specimen holder was then filled with water and frozen using the freezing system. The specimen embedded in the frozen water was then sectioned directly using a microtome blade. Leaf sections were then treated with specific reagents to identify the presence and specific locality of terpenoids, alkaloids and lipophilic compound . Terpenoid in the s presence leaf tissues was identified by soaking leaf section a in 5% cupric acetate solution, as suggested in Harbone 1993 . A positive test for terpenoids ( ) was indicated by the appearance of yellow or brownish yellow color in the tissue. Alkaloid - presence was lamina tested by soaking leaf sections in Wagner reagent. A positive result was indicated by the presence of reddish brown or - yellow deposits. For negative control, the leaf sections were pre treated with 5% tartaric acid in - 95% ethanol for 48 hours at room temperature, as suggested in ( Furr & MahIberg 1981). The presence of slipophilic compound was tested using sliced samples washed in 70% ethanol for one , which were . minute then soaked in 0 03% sudan IV for 30 minutes at 40 C in a water bath. o The sliced sample w then washed quickly in s ere 96 BIOTROPIA Vol. 24 No. 2, 2017 Salmaki et al. 2009; Liu & Liu 2012; Rusydi et al. 2013; Oliveira et al. 2013; Boix et al. 2011). Species belong to genus having peltate and capitate Hyptis glandular trichomes include andH. caespitosa H. suaveolens (Rudall 2007; Jelani & Prabhakar 1991). has peltate glandular trichomes H. capitata in a submerged (sunken) position in the epidermal tissue. This type of glandular trichomes was also found in several species, such as Oci um basilicum, Oci um irvinei H. caespitosam m and (Ogunkule & Oladele 2000; Rudall 2007). Glandular Trichomes Peltate glandular trichomes, found on the leaves of H. capitata, consisted of four head cells and one basal cell. The head cells of peltate glandular trichomes in Lamiaceae family vary widely in terms of cell numbers. Some species have three to six head cells, for example those of Isodon rubescens (Liu & Liu 2012). Several species of genus Ziziphora have 12 to 18 head cells, namely Ziziphora clinopodioides, Z. tenuior, Z. taurica and Z. taurica subsp. cleonioides (Kaya et al. 2013). There are 4 head cells in the peltate glandular trichomes of both H. capitata and H. caespitosa (Rudall 2007). Capitate glandular trichomes in Lamiaceae family usually consisted of one to two head cells with short or long stalks consisted of one to three cells. These are found in many species, such as Lavandula pinnata, Thymus lykae, Isodon rubescens and Rosmarinus officinalis (Huang et al. 2008; Marin et al. 2008; Liu & Liu 2012; Boix et al. 2011). All three types of glandular trichome were found scattered on both leaf surfaces of H. capitata. Type I of capitate glandular trichomes in H. capitata had a short stalk with one cell and one head consisted of two cells. Type II of capitate glandular trichomes had a long stalk consisted of seven to ten cells and one head cell. Uniseriate glandular trichomes consisted of four to eight cells and had a tapered tip (Fig. 2). Peltate glandular trichomes were generally shorter than other trichomes. However, t heir head was much larger than that of capitate size glandular trichomes. The size of each trichome in the adaxial and abaxial surfaces was similar (Table 1). In fact, most trichome dimensions in adaxial and abaxial surfaces showed no significant differences, except for the width of type I capitate glandular trichome which was greater in the abaxial surface than that in adaxial surface. 97 Figure 2 Glandular trichome types of H. capitata; images obtained using SEM (A, B, C) p = peltate, c1 = capitate glandular trichome type I, c2 = capitate glandular trichome type II and u = uniseriate (Note: Bar = 50 µm) Table 1 Size and density of glandular trichomes of H. capitata leaf Trichome types Length of trichome stalk (µm) Trichome head (µm) Density (mm-2) Length Width Adaxial Abaxial Adaxial Abaxial Adaxial Abaxial Adaxial Abaxial Peltate - - 27.7±0.3 28±0.5 ns 43.6±0.3 44.4±1.2 ns 9.6±0.7 32.3±1.9 s Capitate type I 7.5±0.5 7.8±0.9 s 12.8±0.5 13.3±0.9 ns 25.1±0.5 27.5±0.9 s 12.6±3.3 18.9±1.3 s Capitate type II 351±14.7 354.7±8.1 ns 27.5± 0.7 28.3±0.3 ns 19.1±0.5 19.4±0.9 ns 3.9±0.3 4.5±0.2 s Uniseriate 343.7±32.6 342.7±44.2 ns - - - - 3.9±0.7 4.4±0.3 ns Idioblast Diameter (µm) Diameter (µm) Palisade 5.9±0.4 Sponge 4.2±0.3 s Palisade 261.2±.15.4 Sponge 271.7 ±33.2 ns Note: s = significant; ns = non-significant (based on t-test at α = 5%) Identification of medicinal properties of Hyptis capitata Jacq. – Rupa et al. Peltate and capitate glandular trichomes were randomly scattered in the adaxial and abaxial surfaces of leaves. The density of peltate glandular trichomes was much higher in the abaxial than that in the adaxial . surface The distribution densities of both types of capitate glandular trichomes were similarly higher in abaxial than in adaxial he iseriate surfaces. T un trichomes were distributed equally in the adaxial and abaxial (Table 1)surfaces . S species tudies on various showed that glandular trichomes are usually found to be more densely distributed surface of the leaf in abaxial . Only in certain species that glandular trichomes are such as H. more abundant in the adaxial side, in suaveolens O. basilicumand (Ogunkule & Oladele 2000). The peltate and capitate glandular trichomes found in the leaves of are H. capitata more abundant surface in abaxial than in adaxial . This distribution pattern is observed in several also species belong to Lamiaceae family i.e. Mentha piperita, Salvia chrysophylla Rosmarinus officinalisand (Turner . ; Kahraman . 20 ; Boix et al 1999 et al 10 et al 1 . 201 ). The same pattern is also observed in several other families, such as Dipterocarpaceae (Parashorea (Gossypium hirsutum spp); Malvaceae L.) and Fabaceae L.) (Dahlin . (Phaseolus vulgaris et al 1992; Noraini & Cutler 2009; Bondada & Oosterhuis 2000). Idioblast cells H. capitataIdioblast cells in the leaves of were round-shape and the mesophyll of the d found in leaf, from the palisade to the spongy .parenchyma Idioblast cells in the leaves of different plant species are not always found throughout the mesophyll. In some cases idioblast cells are found only in specific tissues. I Litsea euosma, n Litsea praecox Actinodaphne trichocarpa the and idioblast cells were only present in the palisade parenchyma the leaves of , while in Phoebe forrestii, they were found in the spongy parenchyma Hai (Gang & 1999). Idioblast cells found in the palisade parenchyma of were larger than similar H. capitata cells located in the spongy parenchyma, while the cell densities showed no significant difference (Table 1). species a variety of Different exhibit sizes and densities of idioblast cell. For example, in , several species of Machilus and Persea genera various sizes and densities of idioblast cells have been previously reported i.e. the size of idioblast cells in palisade and spongy parenchyma of Machilus leptophylla were 30 - 40 µm with a density of 3 mm n M. yunnanensis the idioblast -2; i cells were 25 - 50 µm with a density of 60 mm ; -2 in M. salicoides the idioblast cells were 30 - 40 µm with a density of 24 mm he largest idioblast -2; t cells were found in Persea americana at 35 - 45 µm with a density of 89 mm (Gang & Hai 1999). -2 Referring to these data the size of idioblast , cells in palisade and spongy parenchyma the of H. capitata were much smaller in size than those in Machilus and Persea genera, while the density was much higher than those of the two generas. Histochemical Analysis of Glandular Trichome and Idioblast Cells Histochemical testing of type I capitate glandular trichomes gave a positive result for terpenoids, as confirmed by the yellow-colored response to a cupric acetate reagent. The presence of was indicated by alkaloids the formation of a brownish yellow deposit, when treated with Wagner's reagent. Type II capitate glandular trichomes were shown to terpenoids, as contain indicated by the formation of a yellow color in the resultant mixture. This type II also contained alkaloids brown color as indicated by response as well as s as indicated by lipophilic compound yellow-colored reaction. Peltate glandular trichomes were tested positive for terpenoids and alkaloids indicated by yellow to brownish-yellow color. iseriate-type trichomes were tested Un positive for terpenoids as indicated by brown color as well as for alkaloids indicated by yellow to brownish-green color (Fig. ).3 Various studies reported on the metabolites produced by the glandular trichomes in Lamiaceae plants. Nazzar et al. (2010) reported that peltate and capitate glandular trichomes in Lamiaceae generally secrete essential oils in the form of terpenoid compounds. According to Gang et al. (2002), terpenoids in Ocimum basilicum (Lamiaceae) were produced by the peltate glandular trichomes. Glandular trichomes in Mentha piperita and Mentha spicata produced terpenoids in form of monoterpenes (Fahn 1979). In Salvia sclarea L., capitate glandular trichomes play a role in producing essential oil 98 BIOTROPIA Vol. 24 No. 2, 2017 compounds like diterpene and monoterpenes in form of linalool and linalyl acetate compounds. Peltate glandular trichomes of Salvia sclarea L. produce sesquiterpene compounds (Schmiderer et al. 2008). Liu and Liu (2012) reported that the glandular trichomes in the leaves of Isodon rubescens (Lamiaceae) contain a combination of phytochemical compounds, consisting of phenolic compounds, terpenoids, flavonoids, carbohydrates and alkaloids. 99 Figure 3 Glandular trichomes in H. capitata. Histochemical tests using cupric acetate for terpenoid (A, E, I and M); Sudan IV test for lipophilic substance (B, F, J and N); glandular trichome in water (control) (L); Wagner test for alkaloid (C, G, K and O); negative control for alkaloid test using tartaric acid reagent (D, H and P) (Note: Bar = 50 µm) A B D C H E G F I J L K N P O M Figure 4 Histochemical results for idioblast cells in leaf transversal section; using sudan IV for lipophilic compounds (A) and water as a control (B) Ep = Epidermis, Pl = Palisade parenchyma and Sp = Spongy parenchyma (Note: Bar = 50 µm) A Ep Pl Sp B Identification of medicinal properties of Hyptis capitata Jacq. – Rupa et al. No R Time Metabolite Group RI Relative area (%) Constituent 1 2.950 Alcohol 85249549 10.02 (O-D)ethenol 2 3.185 Amide 113186810 13.30 Acetamide, 2,2-dichloro- (CAS) Dichloroacetamide 3 4.501 Acetic acid 37932716 4.46 Acetic acid (CAS) Ethylic acid 4 5.228 Acetal 38862858 4.57 2-Propanone, 1-hydroxy- (CAS) Acetol 5 13.313 Ketone 17521339 2.06 1,2-CYCLOPENTANEDIONE 6 15.039 Phenol 13550624 1.59 Phenol (CAS) Izal 7 15.633 Terpenoid (Monoterpene) 159030886 18.69 L-Limonene 8 15.881 Ketone 16614244 1.95 2- Cyclopenten-1-one, 2-hydroxy-3-methyl- (CAS) Corylon 9 17.108 Phenol 37470245 4.40 Ph enol, 4-methoxy- (CAS) Hqmme 10 17.448 Aldheida alkyl 17828416 2.10 Pentanal (CAS) n-Pentanal 11 18.631 Aromatic aldehyde 9097365 1.07 Benzaldehyde, 2-methyl- (CAS) o-Tolualdehyde 12 19.688 Ester 13182646 1.55 2-Propenoic acid, 2-methyl-, ethyl ester (CAS) Ethyl methacrylate 13 19.849 Acetic acid 15135388 1.78 2,3-dihydro-benzofuran 14 21.122 Aromatic heterocyclic 31783131 3.74 1H-Indole (CAS) Indole 15 21.198 Phenol 29786120 3.50 Phenol, 4-ethenyl-2-methoxy- 16 21.813 Terpenoid 8964401 1.05 Phenol, 2-methoxy-4-(2-propenyl)- (CAS) Eugenol 17 22.552 Alkaloid 29383206 3.45 1H-Indole, 3-methyl- (CAS) 3-Methylindole 18 22.705 Terpenoid (Sesquiterpene) 8814772 1.04 Farnesol isomer A 19 22.915 Terpenoid (Sesquiterpene) 10811896 1.27 d-Nerolidol 20 23.496 Polycyclic aromatic 12729349 1.50 2H-1-Benzopyran-2-one (CAS) Coumarin 21 26.417 Terpenoid (Sesquiterpene) 8734907 1.03 1-Dodecanol, 3,7,11-trimethyl- (CAS) Hexahydrofarnesol 22 27.717 Terpenoid (diterpene) 24454424 2.87 Neophytadiene 23 27.999 Terpenoid 17043839 2.00 dodeca-1,6-dien-12-ol, 6,10-dimethyl- 24 28.207 Terpenoid (diterpene) 11252199 1.32 Neophytadiene 25 28.671 Fatty acid 13380605 1.57 Tetradecanoic acid, 12-methyl-, methyl ester (CAS) Methyl 12- methyltetrade 26 29.191 Fatty acid 12112786 1.42 9-Octadecenoic acid (Z)- (CAS) Oleic acid 27 29.537 Vitamin 10213731 1.20 Vitamin d2-7,8-diol 28 30.656 Fatty acid 9972296 1.17 Tricyclo[3.2.2.0e2,4]non-8-en-exo-6,exo-7-dicarboximid, 3,3-dic 29 31.789 Fatty acid 19635391 2.31 Hexadecanamide (CAS) Amide 16 30 43.507 Fatty acid 17189048 2.02 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl- (CAS) Squ Total 850925187 100 Histochemical tests conducted on the idioblast cells in the leaves of H. capitata did not detect terpenoids and alkaloids in these structures. However, these cells demonstrated the presence of indicated by lipophilic compounds, as yellow to orange colors (Fig. ). found 4 Such cell types were in (L.) (Lamiaceae) (Lersten & Physostegia virginiana Curtis 1998), but the idioblast cells containing such compounds are not specific to this family. Similar cell types are present also in various families, such as Lauraceae, Asteraceae, Caprifol iaceae, Rosaceae, Leguminosae ( ) and Bixaceae (Caesalpinia echinata Cochlospermum rhegium et al et al) (Lersten . 2006; Teixeira . 2007; Filho et al . . 2014) Gas Chromatograph Mass Spectrometer - ( - )GC MS GC-MS analysis revealed the presence of alkaloids, terpenoids, fatty acids and phenolic compounds (Table 2). L-limonene, eugenol, f a r n e s o l i s o m e r s A , d - n e r o l i d o l , hexahydrofarnesol, neophytadiene and dodeca- 1,6-dien-12-ol, 6,10-dimethyl were the predominant terpenoids found in the leaf tissue. L-limonene was terpenoid detected in the largest quantity (18.69%). The alkaloids were detected in only one substance i.e. 1H-Indole, 3-methyl-(3- Methylindole). Those compounds support the healing of wound or infection and also act as antibacterial agents. According to Pei et al. (2009), 100 Table 2 Phytochemical compounds of H. capitata leaves using GC/MS BIOTROPIA Vol. 24 No. 2, 2017 eugenol (a member of terpenoids) also acts as antibacterial agent. Eugenol is able to inhibit the growth of gram-negative and gram-positive bacteria (Catherine et al. 2012). Antibacterial tests of eugenol proved that this substance can inhibit the growth of Salmonella typhi a gram-negative , bacteria (Devi et al. 2010). D'Alessio et al. (2014) reported that d-limonene acts as an anti- inflammatory that can signifcantly heal skin inf lammation caused by murine 12-O- Tetradecanoylphorbol-13-acetate (TPA). This substance can also heal wounds by reducing neovascularization in mice. Kusma et al. (2009) reported that essential oil of Salvia sclarea leaves is used as analgesic, anti-inflammatory, antioxidant, antifungal and antibacterial agents. This oil is toxic to various pathogenic microbe including s, Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans fungus. Curvelo et al. (2014) reported that nerolidol extracted from Piper claussenianum (Miq.) C. DC. is able to inhibit the development of C albicans, a fungus which causes . candidiasis. According to Ragasa et al. (2009), neophytadiene can inhibit the growth of some fungi and bacteria including C. albicans, Aspergillus . niger, Trichophyton mentagrophytes, E coli and. P s eudomonas a e r ug ino sa . Neophytad iene compounds serve as antipyretic, analgesic, anti- inflammatory, antimicrobial and antioxidant agents (Raman et al. 2012). leaf of H. suaveolens is The extract used as traditional medicine in Brazil to treat inflammation, peptic ulcers, infections can and reduce gastric lesions in oral application in mice ( et al et alJesus . 2013). According to Shaikat . (2012), he extract can cure diarrhea t leaf in mice previously treated with H. suaveolenscastor oil. contains a combination of terpenoids, consisting of eucaliptol, gamma-ellemene, beta-pynene, (+)- 3-Carene, trans-beta-cariophyl lene and germacrene (Moreira . 2010)et al . According to Malar . (2012) et al H. suaveolens also contains alkaloids, glycosides, saponins, tannins and flavonoids he leaf extract of can ; t H. suaveolens inhibit the growth of various bacteria such as Aeromonas formicans, Aeromonas hydrophila, Bacillus subtilis, E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa ( et al as well as Malar . 2012), the growth of pathogenic fungi Aspergillus flavus, A. parasiticus, A. ochraceus, A. fumigatus A. niger et aland (Moreira . 2010). H. verticillata Jacq. is used as traditional medicine in Central America. Th plant containsis lignans, triterpenes, diterpenes, sesquiterpenes, monoterpenes, flavonoids, polyphenols and alkaloids. The plant extracts and isolated crude phytochemicals play a role in antibacterial and anti-fungal treatment of infectious diseases (Picking . 2013)et al . CONCLUSIONS H. capitata leaves have secretory structures in the form of glandular trichomes and idioblast cells. The secretory structures produce secondary metabolite compounds. 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