GJRMI - Volume 2, Issue 12 - December 2013 by Dr. Hari Venkatesh.K.Rajaraman

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Honorary Members - Editorial Board Dr Farhad Mirzaei Mr. Harshal Ashok Pawar

INDEX – GJRMI, Vol. 2, Iss. 12, December 2013 MEDICINAL PLANTS RESEARCH Natural Resource CHARACTERISTICS OF ESSENTIAL OILS OF ROSMARINUS OFFICINALIS FROM EASTERN ALGERIA Takia Lograda, Messaoud Ramdani, Pierre Chalard, Gilles Figueredo

794–807

Pharmacology EVALUATION OF THERAPEUTIC POTENTIAL OF DIOSCOREA BULBIFERA TUBERS ON LEARNING AND MEMORY IMPAIRMENT IN HIGH FAT DIET (HFD) AND ICV STREPTOZOTOCIN (STZ) - INDUCED EXPERIMENTAL DEMENTIA IN MICE Bisht Neha, Kumar Arun, Kothiyal Preeti, Choudary Alka

808–823

INDIGENOUS MEDICINE Ayurveda – Dravya Guna PHARMACOGNOSTICAL EVALUATION OF THE ROOT OF OPERCULINA PETALOIDEA CHOISY - A SOURCE OF SHYAMA TRIVRUT Kolhe Rasika, Acharya Rabinarayan, Harisha C R

824–829

Ayurveda – Dravya Guna ETHNO-BOTANICAL SURVEY OF SOME MEDICINAL PLANTS IN JATASANKAR REGION OF GIRNAR FOREST, GUJARAT, INDIA Raval Nita D, Dhaduk Haresh L

830–841

Ayurveda – Stree Roga & Prasooti Tantra A CLINICAL STUDY ON MANSYADI VATI IN THE MANAGEMENT OF RAJONIVRITTI JANYA LAKSHANA (MENOPAUSAL SYNDROME) Dasondi Ami P, Donga Shilpa B, Rupapara Amit, Mistry I U

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF LATA KASTURI (ABELMOSCHUS MOSCHATUS MEDIK.), OF THE FAMILY MALVACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

842–848

Research article CHARACTERISTICS OF ESSENTIAL OILS OF ROSMARINUS OFFICINALIS FROM EASTERN ALGERIA Takia Lograda1*, Messaoud Ramdani2, Pierre Chalard3, Gilles Figueredo4 1, 2

Laboratory of Natural Resource Valorisation, SNV Faculty, Setif 1 University, 19000 Setif, Algeria Clermont Université, ENSCCF, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 ClermontFerrand, France 3 CNRS, UMR 6296, ICCF, F-63171 Aubiere, France 4 LEXVA Analytique, 460 rue du Montant, 63110 Beaumont, France *Corresponding author: Email: tlograda63@yahoo.fr; Phone: (213)36835894; Fax: (213)36937943. 3

Received: 07/10/2013; Revised: 23/11/2013; Accepted: 25/11/2013

ABSTRACT The analysis and identification of the essential oil components of six Rosmarinus officinalis populations, from Eastern Algeria, was performed using GC-MS. The average yield of essential oil samples is 0.23%, the highest rate was observed in the essential oil of Kherrata population (0.35%), while Agmeroual population was characterised by the lowest yield (0.10%). These analyses led to the identification of 43 components. The chemical composition of the essential oil was dominated by the presence of major products, camphor (9.1–42.7%), eucalyptol (6.6–42.2%), α-pinene (11.4– 25.2%), camphene (5.3–17.7%) and borneol (0.9–11.9%). Seven components were represented with average rates more than 1% in the essential oil, β-pinene, para-cymene, limonene, linalool, terpinene4-ol, α-terpineol and β-caryophyllene. This investigation allows us to support that the species Rosmarinus officinalis of eastern Algeria includes several chemotypes. The chemotype to (eucalyptol, Camphor, α-pinene and camphene) was located in the regions of Kherrata and Bibans. The regions of N’gaous, Agmeroual and Boussâada had favoured the development of chemotype to (camphor, camphene, α-pinene and eucalyptol). The chemotype to (α-pinene, camphor, camphene and eucalyptol) was located in the Boutaleb region. KEYWORDS: Rosmarinus officinalis, Lamiaceae, Essential oil, Chemotype, Algeria

Cite this article: Takia Lograda, Messaoud Ramdani, Pierre Chalard, Gilles Figueredo (2013), Characteristics of essential oils of Rosmarinus officinalis from Eastern Algeria, Global J Res. Med. Plants & Indigen. Med., Volume 2(12): 794–807

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 794–807

INTRODUCTION Rosemary (Rosmarinus officinalis L.) is a shrubby herb aromatic, typical of Mediterranean countries. Since ancient times, aromatic herbs and spices have been added to different types of foods industry as a natural antioxidant, for food conservation to improve the flavour and organoleptic properties (Ho et al., 2000, Naisheng et al., 2010). Dried rosemary leaves are used in fried chicken, salads, baked products, condiments, perfumes and soaps. Besides, essential oils of rosemary could also be used as functional ingredients (Viuda-Martos et al., 2010). The anti-inflammatory property of rosemary extracts was reported (Masuda et al., 2001; Bozin et al., 2007; Altinier et al., 2007; Poeckel et al., 2008; Viuda-Martos et al., 2010). The interest was also generated due to the anti-carcinogenic activity for the cancer chemopreventative potential (Cheung and Tai, 2001). The essential oils of R. officinalis showed antimytotic and antifungal activity (Yang et al., 2011; Mugnaini et al., 2012). All extracts of R. officinalis were effective in inhibiting bacterial growth (Abutbul et al., 2004; Bozin et al., 2007). The essential oil of rosemary contains mainly monoterpenes (Angioni et al., 2004; Diaz-Maroto et al., 2007). The principal volatile compounds in rosemary are camphor and 1,8-cineole (eucalyptol), followed by borneol, verbenone, α-pinene and camphene (Pino et al., 1998; Zaouali et al., 2005; DiazMaroto et al., 2007; Calin-Sanchez et al., 2011; Apostolides et al., 2013). The volatile compounds from rosemary samples could be grouped in chemical families; therefore, the predominant group was monoterpenoids (Bozin et al., 2007; Szumny et al., 2010; CalinSanchez et al., 2011). The chemical composition of essential oils of R. officinalis in the world is highly variable (Table 1). The major components, also having significant variability, are α-pinene (4.2– 61.2%), camphene (0–13.8%), eucalyptol (0– 61.4%), camphor (0–24%) and borneol (0– 15.6%). According to (Napoli et al., 2010),

rosemary essential oil can be classified into three chemotypes from a chemical point of view: cineoliferum (high content in 1,8-cineol); camphoriferum (camphor > 20%); and verbenoniferum (verbenone > 15%). The chemical composition and seasonal variations in rosemary oil from southern Spain were reported (Salido et al., 2003; Jordan et al., 2011, 2013). All the samples studied by Salido et al., (2003) belonged to the chemotype (αpinene-1,8-cineole-camphor). Jordan et al., (2013) identified five chemotype based on αpinene-1,8-cineole-camphor. Varela et al. (2009), reported the chemical polymorphism of the essential oil from Spanish wild rosemary populations, and defined different chemotypes depending on the geographical area. In the present study, the aim was to identify the chemical composition of the oils of R. officinalis and to compare the results to other composition of this species in the world, and re-evaluation of the geographical distribution of chemotypes. MATERIALS & METHODS Plant material Rosmarinus officinalis is collected from five localities in eastern Algeria, Kherrata (Bedjaia), Boutaleb (Setif), Bibans (BBA= Bourdj Bou-Arriridj), Agmeroual and N’gaous (Batna), and Boussâada (M’sila) (Figure 1). The plant identification was performed by Dr. Lograda Takia. Voucher specimen’s were preserved in the Herbarium at the Department of Biology and Ecology Vegetal, Setif-1 University, Algeria. Aerial parts were collected during the flowering stage in October 2012. Extraction of the essential oil 100 g of the air-dried aerial parts of five populations were subjected to hydro-distillation for 3 h with 500 ml distilled water using a Clevenger-type apparatus. The oil obtained was collected and dried over anhydrous sodium sulphate and stored in screw capped glass vials in a refrigerator at 4–5°C, prior to analysis. Yield based on dried weight of samples was calculated.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 794–807

Linalool

Camphor

Borneol

Terpinene-4-ol

α-terpeneol

Verbenone

Bornyl acetate

βcaryophyllene

26 27 28

Eucalyptol

22 23 24 25

Limonene

p-cymene

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Myrcene

β-pinene

1 2

Camphene

Portugal Spain 1 Spain 2 Spain 3 Spain 4 Spain 5 Spain 6 France Italy 1 Italy 2 Italy 3 Poland 1 Poland 2 Romania 1 Romania 2 Serbia Morocco 1 Morocco 2 Morocco 3 Morocco 4 Algeria (BBA) Algeria (Algers) Algeria (Setif) Algeria (Annaba) Tunisia 1 Tunisia 2 Tunisia 3 Tunisia 4 Tunisia 5 Tunisia 6 Turky 1 Turky 2 Turky 3 Turky 4 Iran 1 Iran 2 Iran 3 Iran 4 Iran 5 Iran 6 Pakistan

α-pinene

Pays

Authors (*)

Table 1: Chemical composition of Rosmarinus officinalis

14.1 15.6 19.2 14.6 16.2 14.1 21.0 13.1 23.4 6.7 14.5 11.0 33.3 61.2 18.4 4.2 6.2 18.3 15.3 8.4 5.2 44.1 12.6 19.7 8.7 8.5 13.3 10.2 10.1 12.0 10.2 11.5 14.2 12.6 14.9 21.7 43.9 46.1 21.5 48.7 11.5

7.4 8.4 8.0 7.7 7.4 6.5 9.2 6.9 0.0 2.3 3.8 5.2 13.8 11.1 3.5 4.1 4.1 5.0 3.4 5.1 3.0 6.1 4.3 2.6 2.9 3.1 11.6 5.9 4.3 4.6 3.4 2.6 3.4 4.4 3.3 0.3 8.6 9.6 6.3 13.7 6.0

4.0 1.1 1.1 1.2 1.6 2.2 5.5 2.2 3.8 0.7 2.5 9.2 7.9 2.8 8.2 0.3 6.2 4.6 0.9 7.3 5.7 2.6 2.2 1.6 2.2 2.1 0.6 1.6 3.7 1.1 1.1 0.7 0.8 5.2 0.0 0.1 1.9 1.9 3.5 2.0 6.5

23.7 1.6 3.7 2.7 3.3 3.4 0.0 0.0 0.0 0.3 1.6 1.2 4.1 2.7 0.0 2.6 0.0 0.0 1.3 2.2 1.7 1.9 1.2 0.0 0.6 0.8 0.7 0.7 1.4 1.3 1.5 1.3 1.5 1.8 2.1 1.4 3.9 3.9 1.6 4.5 0.7

0.0 2.9 2.5 2.3 2.3 1.5 1.3 2.4 0.0 1.4 0.0 1.3 0.2 1.6 0.0 0.0 1.5 2.0 2.0 0.2 2.2 1.3 0.0 2.8 0.8 1.1 1.6 1.2 2.3 0.2 2.3 2.4 1.1 1.2 0.0 0.3 0.4 0.9 3.0 0.5 0.0

4.2 2.9 3.9 3.3 3.4 3.1 3.6 0.0 4.7 0.0 0.0 1.0 4.2 2.9 0.0 0.0 2.3 3.6 3.8 0.0 0.0 5.5 1.2 5.1 1.2 1.3 1.5 1.3 0.1 1.1 2.4 2.6 3.6 2.5 0.0 0.0 1.2 0.0 2.8 0.0 4.5

10.6 22.1 19.1 19.7 21.1 18.9 24.5 13.7 27.5 7.3 50.9 46.4 12.3 6.0 40.6 52.2 49.1 5.3 11.6 27.1 52.4 0.0 13.5 7.9 37.9 38.7 22.7 33.1 35.8 46.4 61.4 27.9 12.1 44.4 7.4 23.5 11.1 11.1 15.2 13.7 29.2

0.2 0.7 1.2 1.1 1.2 0.9 0.0 0.0 0.0 2.2 0.6 0.5 1.9 0.0 0.0 0.0 0.0 0.2 5.4 0.0 1.1 2.0 3.1 0.0 0.5 0.7 0.2 0.5 0.7 0.7 0.6 2.9 3.4 1.1 14.9 1.5 0.0 0.0 2.6 0.1 3.4

12.8 18.5 15.0 20.2 17.7 24.0 19.2 15.6 7.1 14.6 12.1 11.4 6.7 3.8 11.4 1.4 12.6 0.0 11.6 11.2 12.6 7.8 11.8 12.6 11.7 14.4 28.0 18.0 14.5 4.9 5.8 10.2 16.1 1.4 5.0 7.2 2.4 5.3 6.8 2.7 17.2

1.1 7.1 6.6 5.8 5.2 4.1 2.7 3.9 0.0 10.4 3.6 3.1 4.0 0.3 0.3 0.0 3.7 3.1 15.6 6.6 3.4 2.6 9.4 11.2 13.1 6.7 4.1 8.0 9.2 11.8 0.0 0.0 0.0 0.0 3.7 3.4 0.0 0.0 8.6 0.3 6.3

0.0 1.0 1.2 1.2 1.2 1.0 1.6 1.2 0.0 0.0 1.0 0.4 2.2 0.0 0.0 0.0 0.7 2.0 0.0 0.0 0.7 2.1 1.0 2.3 9.5 6.6 1.6 6.0 1.7 0.0 0.0 0.0 0.0 2.8 1.7 1.1 0.0 0.0 0.0 0.2 0.0

0.0 2.8 2.8 2.8 2.8 2.7 0.0 1.4 0.0 2.4 4.1 0.0 1.1 0.0 0.3 0.0 0.2 2.9 2.0 4.8 2.1 0.8 1.3 0.5 3.9 4.5 2.2 3.6 4.4 3.2 0.0 1.0 2.8 0.0 0.8 1.8 0.6 0.6 0.0 0.2 0.0

1.4 5.4 5.0 4.8 5.1 4.0 1.2 10.7 0.0 21.8 0.0 0.0 3.5 0.0 0.0 0.0 0.0 0.0 11.2 0.4 0.0 6.4 8.3 1.5 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.0 1.9 0.0 2.6 2.3 0.0 2.1 3.8

0.9 0.7 1.1 0.8 1.0 1.2 1.3 12.5 0.0 12.3 0.4 1.0 14.8 0.0 2.6 0.0 1.2 4.4 1.4 2.6 0.7 0.8 4.2 6.1 1.2 1.9 2.0 0.0 1.6 0.0 0.2 0.6 1.5 3.8 3.1 0.0 0.0 0.0 6.1 1.9 0.0

0.2 0.4 0.8 1.2 1.0 1.6 2.3 1.4 0.0 0.0 0.9 3.5 0.8 0.0 10.2 1.9 3.5 2.6 0.9 6.9 4.2 1.0 2.8 3.1 0.0 0.0 0.0 0.0 3.7 2.5 0.0 0.0 0.0 1.2 2.7 0.0 0.0 0.0 1.7 0.0 3.2

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 794–807

βcaryophyllene

Bornyl acetate

Verbenone

α-terpeneol

Terpinene-4-ol

Borneol

Camphor

Linalool

Ecalyptol

Limonene

p-cymene

Myrcene

β-pinene

Camphene

α-pinene

Pays

Authors (*)

Table 1 : Continued

9.3 6.0 4.3 0.0 1.0 14.3 1.2 22.1 0.0 1.1 0.0 0.0 4.6 0.0 India 1 29 16.3 5.8 4.1 2.4 0.3 2.2 22.3 1.1 23.9 0.0 0.8 0.0 0.0 2.7 0.8 India 2 30 11.4 20.2 11.4 7.0 0.0 1.6 1.3 26.6 0.3 12.9 3.1 0.4 2.0 1.4 0.9 2.4 China 1 31 0.0 1.7 0.0 0.0 0.3 14.3 0.0 0.5 0.0 0.0 1.1 2.4 China 2 32 19.4 11.5 6.7 7.9 1.5 1.8 0.0 5.4 28.9 0.5 10.9 3.8 0.5 0.3 1.7 0.7 2.0 China 3 33 25.3 7.7 5.4 0.0 15.1 1.0 4.3 16.9 0.8 16.8 0.0 0.0 1.2 0.2 0.8 4.1 Chili 34 5.0 1.7 1.1 0.5 3.3 21.6 2.8 7.0 4.2 1.1 2.4 2.1 1.6 2.2 Argentina 35 31.2 5.7 1.1 1.3 0.0 0.0 11.9 2.0 16.6 5.7 1.4 0.0 17.4 9.2 0.0 South of Africa 36 11.5 Data of table 1 is used in the analysis of UPGMA (*) 1 = Martins et al., 2012; 2 = Jordin et al., 2013; 3 = Matsuzaki et al., 2013; 4 = Mugnaini et al., 2012; 5 = Sacchetti et al., 2005; 6 = Napoli et al., 2010; 7 = Sienkiewicz et al., 2013; 8 = Szumny et al., 2010; 9 = Socaci et al., 2010; 10 = Chifiriuc et al., 2012; 11 = Dimitrijeric et al., 2007; 12 = Matsuzaki et al., 2013; 13 = Derwich et al., 2011; 14 = Chebli et al., 2003; 15 = Fadli et al., 2011; 16 = Boutekedjiret et al., 2003; 17 = Bousbia et al., 2009; 18 = Zoubiri et al., 2011; 19 = Giordani et al., 2008; 20 = Zouali et al., 2008; 21 = Celiktas et al., 2007; 22 = Orhane et al., 2008; 23 = Gachkar et al., 2007; 24 = Jalali-Heravi et al., 2011; 25 = Jamshidi et al., 2009; 26 = Issabeagloo et al., 2012; 27 = Salehi et al., 2007; 28 = Hussain et al., 2011; 29 = Mudasir et al., 2012; 30 = Verma et al., 2011; 31 = Yang et al., 2011; 32= Wang et al., 2008; 33 = Sui et al., 2012; 34= Graber et al., 2010; 35 = Aderiana et al., 2013; 36 = Okoh et al., 2010

Figure 1: Populations of Rosmarinus officinalis studied

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 794–807

Essential oil analysis The essential oils were analysed on a Hewlett-Packard gas chromatograph Model 5890, coupled to a Hewlett-Packard model 5971, equipped with a DB5 MS column (30 m × 0.25 mm; 0.25 μm), programming from 50°C (5 min) to 300°C at 5°C/min, with a 5 min hold. Helium was used as the carrier gas (1.0 mL/min); injection in split mode (1:30); injector and detector temperatures, 250 and 280°C, respectively. The mass spectrometer worked in EI mode at 70 eV; electron multiplier, 2500 V; ion source temperature, 180°C; MS data were acquired in the scan mode in the m/z range 33–450. The identification of the components was based on comparison of their mass spectra with those of NIST mass spectral library (Masada, 1996; NIST, 2002) and those described by Adams, as well as on comparison of their retention indices either with those of authentic compounds or with literature values (Adams, 2001). Statistical analysis Cluster analysis (UPGMA) was carried out on the original variables and on the Manhattan distance matrix to seek for hierarchical associations among the populations. The cluster analyses were carried out using STATISTICA 10 software. RESULTS The hydro-distillation of the essential oil of Rosmarinus officinalis gave a viscous liquid with a whitish colour. The average yield of essential oil of the samples is 0.21%, the highest rate was observed in the essential oil of Kherrata population (0.35%), while the population of Agmeroual was characterised by the lowest yield (0.10%). The analysis and the identification of the components of the essential oil of R. officinalis were performed using the (GC-MS). The compounds identified in these oils and their relative abundances are presented in order of their appearance in Table 2. These analyses led to the identification of

43 components. The chemical composition of the essential oil of R. officinalis is dominated by the presence of major products, Camphor (9.13–42.73%), Eucalyptol (6.64–42.16%), αpinene (11.35–25.2%), Camphene (3.72– 22.68%) and Borneol (0.94–11.94%). The oil of Boussaâda and Agmeroual populations were characterised by camphor (42.73 and 38.84%). Kherrata and Bibans were characterised by high rates of eucalyptol (35.52 and 42.16%). While the population of Boutaleb, contained high levels of α-pinene, camphene and camphor (25.20, 22.68 and 24.08% respectively). The population of N'gaous contained similar rates of α-pinene (13.6%), eucalyptol (12.1%), camphor (16.9%) and borneol (11.91%). The chemical composition of this species contained other components of a lower rate, β-pinene, para-cymene, limonene, linalool, terpinene-4-ol, α-terpeniol and βcaryophyllene. The classification of our populations, according to their chemical kinship relations, was based on the construction of clades. The UPGMA based on the Unweighted pair-group average distance and the City-block (Manhattan) (Figure 2), reflected ecological relationships among the different population, and has divided the populations into two clades. The first clade included the populations of Kherrata and Bibans, containing high levels and equivalents of α-pinene, camphene, eucalyptol, camphor and borneol. The population of N'gaous was isolated in the second clade of the remaining populations with the presence of similar concentrations of αpinene, eucalyptol, camphor and borneol. In the second group, the Agmeroual and Boussâada populations were close by the presence of similar levels of α-pinene, camphene, eucalyptole, borneol and high rates of camphor; these populations opposed to the population of Boutaleb containing equivalent levels of αpinene (25.2%), camphene (22.68%), camphor (24.08) and very few eucalyptol. Thus it can be argued that the species R. officinalis of eastern Algeria includes several chemotypes (Table 3).

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 794–807

Table 2: Chemical composition of populations studied of Rosmarinus officinalis Compounds

Yield (v/v) Number of compounds Total (%) 926 Tricyclene 929 α-thujene 931 α-pinene 947 Camphene 957 verbenene 976 Sabinene 972 β-pinene 980 1-Octee-3-ol 992 Myrcene 1005 α-phellandrene 1018 α-terpinene 1026 Para-cymene 1031 Limonene 1030 Eucalyptol 1057 γ-terpinene 1068 Cis-sabinene hygratz 1088 Terpinolene 1100 Linalol 1102 Chrysanthenone 1144 Camphor 1148 1(7),5-Menthadien-2-ol 1162 Pinocarvone 1166 Borneol 1173 Cis- pinocamphone 1184 Terpinene-4-ol 1196 α-terpineol 1210 Verbenone 1245 L-carvone 1287 Bornyl acetate 1379 α-copaene 1403 Methyl eugenol 1423 β-caryophyllene 1446 Geranyl acetone 1454 α-humulene 1477 γ-muurolene 1480 Germacrene-D 1509 β-bisabolene 1510 γ-cadinene 1499 Δ-amorphene 1521 Cis-calamenene 1581 Caryophyllene Oxide 1640 Epi-α-cadinol 1668 α-Bisabolol

Kherrata

Bibans

Boutaleb

Agmeroual

N’gaous

Boussâada

0.35 27 98,29 0,28 0,41 11,35 8,03 0,00 0,08 4,02 0,28 1,06 0,16 0,58 1,97 0,00 35,52 0,85 0,12 0,33 0,38 0,00 14,47 0,00 0,00 5,28 0,00 1,38 3,36 0,00 0,00 1,03 0,00 0,00 5,05 0,00 0,23 0,00 0,00 0,57 0,07 0,00 0,00 1,20 0,23 0,00

0.30 29 97,47 0,18 0,14 13,81 5,33 0,00 0,06 2,82 0,16 1,50 0,15 0,27 2,88 0,00 42,16 0,50 0,00 0,00 0,76 0,10 9,13 0,13 0,00 7,29 0,00 1,14 5,22 0,00 0,00 1,85 0,00 0,00 0,54 0,00 0,09 0,05 0,00 0,00 0,10 0,11 0,00 0,72 0,18 0,10

0.20 31 99,37 1,20 0,00 25,20 22,68 0,00 0,00 1,61 0,06 0,78 0,30 0,50 2,29 4,57 8,83 0,35 0,00 0,26 0,10 0,00 24,08 0,23 0,13 0,94 0,58 0,58 0,62 0,00 0,00 0,23 0,17 0,00 0,66 0,00 0,14 0,16 0,00 0,00 0,11 0,21 0,08 0,18 0,00 1,54

0.10 32 98,36 0,59 0,00 16,91 13,83 0,25 0,00 0,91 0,11 0,68 0,33 0,42 1,88 4,45 5,43 0,30 0,00 0,33 1,27 0,27 38,84 0,46 0,15 3,42 0,55 1,11 1,67 1,58 0,00 0,58 0,20 0,00 0,48 0,00 0,00 0,17 0,00 0,00 0,10 0,24 0,00 0,39 0,00 0,46

0.25 39 89,94 0,00 0,23 13,61 3,72 0,90 0,23 0,91 0,34 1,11 0,19 0,32 2,21 3,81 12,13 0,31 0,13 0,49 5,10 0,44 16,88 0,14 0,40 11,94 2,26 1,34 2,57 3,92 0,11 1,25 0,11 0,28 0,83 0,16 0,14 0,12 0,08 0,00 0,08 0,17 0,08 0,90 0,00 0,00

0.15 30 99,72 0,82 0,00 15,13 17,67 0,00 0,00 1,37 0,18 0,40 0,22 0,32 1,68 3,16 6,64 0,27 0,00 0,19 0,09 0,13 42,73 0,46 0,00 3,34 0,00 1,27 1,26 0,00 0,00 0,18 0,06 0,06 0,24 0,00 0,07 0,00 0,00 0,00 0,06 0,08 0,00 0,16 0,16 1,32

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Figure 2: Cladogramme of Rosmarinus officinalis populations

Table 3: Volatile profile of rosemary essential oils selected by chemotype

Chemotypes Eucalyptol α-pinene Camphor Camphene Total β-pinene Para-cymene Limonene Linalol Borneol α-terpineol Verbenone βcaryophyllene

Kherrata

Bibans

E-Ca-αP-C

E-αP-Ca-C

35.52 11.35 14.47 8.03 69.37 4.02 1.97 0 0.38 5.28 3.36 0 5.05

42.16 13.81 9.13 5.33 56.62 2.82 2.88 0 0.76 7.29 5.22 0 0.54

Populations Boussâada Agmeroual Ca-C-αP-E

6.64 15.13 42.73 17.67 82.17 1.37 1.68 3.16 0.09 3.34 1.26 0 0.24

Ca-αP-C-E

5.43 16.91 38.84 13.83 75.01 0.91 1.88 4.45 1.27 3.42 1.67 1.58 0.48

N’gaous

Boutaleb

Ca-αP-E-C

αP-Ca-C-E

12.13 13.61 16.88 3.72 46.34 0.91 2.21 3.81 5.1 11.94 2.57 3.92 0.83

8.83 25.2 24.08 22.68 80.79 1.61 2.29 4.57 0.1 0.94 0.62 0 0.66

Chemotype classification in relation with the chemical compounds found in rosemary samples. E = eucalyptol; αP = α-pinene; Ca = camphor; C = camphene. Bold values signify relative concentration of the four major compounds that define the essential oil chemotype

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The first chemotype to eucalyptol has two variants. The first variant (E-Ca-αP-C) characterizes Kherrata population, the second (E-αP-Ca-C) characterizes the Bibans population. The difference of these two chemotype variants is the concentration of Eucalyptol and camphor.

chemotype (Ca-αP-E-C) was found in the region of N'gaous. The third chemotype to αpinene (αP-Ca-E-C), in R. officinalis of eastern Algeria, characterized the population of Boutaleb.

The second chemotype to camphor had three variants, the variant (Ca-C-αP-E) characterized the population of Boussâada. Agmeroual population contained the variant (Ca-αP-C-E), while the third variant of this

Our returns of the essential oil are low compared to those of the literature. This yield is between 0.6 and 0.8% (Bekkara et al., 2007). Ayadi et al. (2011) found a yield (0.71–2%) and show that the yield of rosemary varies according to geographical location.

DISCUSSION

Figure 3: UPGMA cluster of Rosmarinus officinalis populations

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The comparison of the chemical components of the essential oil of the samples with those of Rosmarinus officinalis oils shows that α-pinene, camphene, eucalyptol and camphor are the major products of the oil (Jordan et al., 2013; Mugnaini et al., 2012; Napoli et al., 2010; Sienkiewicz et al., 2013; Socaci et al., 2010; Dimitrijeric et al., 2007; Matsuzaki et al., 2013; Derwich et al., 2011; Fadli et al., 2011; Boutekedjiret et al., 2003; Bousbia et al., 2009; Celiktas et al., 2007; Orhane et al., 2008; Jalali-Heravi et al., 2011; Jamshidi et al., 2009; Issabeagloo et al., 2012; Salehi et al., 2007; Hussain et al., 2011; Mudasir et al., 2012; Verma et al., 2011; Yang et al., 2011; Wang et al., 2008; Sui et al., 2012; Graber et al., 2010; Aderiana et al., 2013), with few exceptions, the verbinone is found with a heavy concentration in the sample of Italy 2 (21.8%) (Sacchetti et al., 2005), that of France (10.7%) (Matsuzaki et al., 2013.), Morocco 3 (11.2%) (Chebli et al., 2003) and South Africa (17.4%) (Okoh et al., 2010). The rate of myrcene is important in Portugal (23.1%) (Martins et al., 2012) and in Chili (15.1%) (Graber et al., 2010). The sample of Iran 1 contains 14.9% of linalool (Gachkar et al., 2007).The population of Italy 2 (Sacchetti et al., 2005), Morocco 3 (Chebli et al., 2003), Algeria (Setif and Annaba) (Zoubiri et al., 2011 and Giordani et al., 2008) and of Tunisia 1 and 6 (Zouali et al., 2008) contain significant concentrations of borneol. The bornyl acetate is found in the populations of France (Matsuzaki et al., 2013), of Italy 2 (Sacchetti et al., 2005), of Poland 2 (Szumny et al., 2010) and South Africa (Okoh et al., 2010). The sample of Romania 2 contains a high level of βcaryophyllene (Chifiriuc et al., 2012).

The comparison of our populations to those in the world, using the UPGMA, allowed us to divide the populations into nine distinct groups (Figure 3). The observation of several sets of populations means we are dealing with in heterogeneous ensembles, but there were no clear distinctions between the separations of chemotypes.

The UPGMA confirmed the observations of (Salido et al., 2003; Jordan et al., 2011, 2013) on the effect of seasonal variations in rosemary essential oils. The chemical polymorphism of rosemary populations and the different chemotypes have a relationship with the geographical area (Varela et al., 2009; Ramdani et al., 2013, Lograda et al., 2013).

ACKNOWLEDGEMENT

This analysis revealed the presence of heterogeneity in the chemical composition of the samples, as well as mix in the distribution and classification of populations. The only populations which have shown certain homogeneity are grouped by geographical area. The populations of Spain are mixed with other populations, Portugal, Tunisia and Morocco. Natural populations of Algeria are confined in the group 5 and that are well separated from the subgroup formed by the populations of Italy 1 and 3, Serbia, Pakistan, South of Africa and Iran 1. CONCLUSION Analysis of the chemical composition of the essential oil of Rosmarinus officinalis has allowed identifying 43 compounds. The majority compounds are the α-pinene, Camphene, eucalyptol and camphor. Although the study of R. officinalis led to identification of same natural products as minor components, it can be concluded from a comparative analysis with previously reported studies that the content of terpenoid compounds in the world species does not generally present significant quantitative variations. In this study we have identified three chemotypes in R. officinalis, with six variants possible, localised in eastern Algeria.

The works were supported by Algerian MESRS and Chemical Laboratory of carbohydrates Heterocyclic of Clermont Ferrant, France

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Source of Support: Algerian MESRS and Chemical Laboratory of carbohydrates Heterocyclic of Clermont Ferrant, France

Conflict of Interest: None Declared

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Research article EVALUATION OF THERAPEUTIC POTENTIAL OF DIOSCOREA BULBIFERA TUBERS ON LEARNING AND MEMORY IMPAIRMENT IN HIGH FAT DIET (HFD) AND ICV STREPTOZOTOCIN (STZ) - INDUCED EXPERIMENTAL DEMENTIA IN MICE Bisht Neha1*, Kumar Arun2, Kothiyal Preeti3, Choudary Alka4 1,2,3,4

Department of Pharmacology, Division of Pharmaceutical Sciences, Shri Guru Ram Rai Institute of Technology and Science, Dehradun- 248001, INDIA. *Corresponding Author: Email: nhbisht@gmail.com; Mob: +917579129175 Received: 08/11/2013; Revised: 03/12/2013; Accepted: 05/12/2013

ABSTRACT The present study was undertaken to investigate the potential effect of Dioscorea bulbifera L., tubers in experimental dementia of Alzheimer Disease type. Streptozotocin [STZ, 3 mg/kg, injected intracerebroventricular (i.c.v), and high fat diet (HFD, administered for 90 days)] were used to induce dementia in separate groups of Swiss albino mice. Morris water maze (MWM) test and Elevated plus maze (EPM) test was performed to assess learning and memory of the animals. Extent of oxidative stress was measured by estimating the levels of brain reduced glutathione (GSH) and thiobarbituric acid reactive species (TBARS). Brain acetylcholinesterase (AChE) activity and serum cholesterol levels were also estimated. i.c.v STZ and HFD produced a marked decline in MWM and EPM performance of the animals, reflecting impairment of learning and memory. Higher levels of brain AChE activity and TBARS and lower levels of GSH were observed in i.c.v STZ- as well as HFD-treated animals. HFD-treated mice also showed a significant increase in total serum cholesterol levels. Treatment of DB (250, 500, 1000 mg/kg p.o., respectively) significantly reversed i.c.v STZ and HFD- induced learning and memory deficits along with significant attenuation those- induced rise in brain AchE activity and brain oxidative stress (increase in TBARS and decrease in GSH) levels. HFD treated mice also produced a significant reversal of elevated serum cholesterol levels and memory impairment on administration of DB. Thus, the present study constitutes the first report documenting the beneficial effect of Dioscorea bulbifera tubers in memory deficits of mice possibly through its multiple actions including potent antioxidative effect. KEY WORDS: Dioscorea bulbifera, Learning, Memory, Dementia, Alzheimer disease, Cholesterol, Polyphenols.

Cite this article: Bisht Neha, Choudary Alka, Kumar Arun, Kothiyal Preeti (2013), EVALUATION OF THERAPEUTIC POTENTIAL OF DIOSCOREA BULBIFERA TUBERS ON LEARNING AND MEMORY IMPAIRMENT IN HIGH FAT DIET (HFD) AND ICV STREPTOZOTOCIN (STZ) - INDUCED EXPERIMENTAL DEMENTIA IN MICE, Global J Res. Med. Plants & Indigen. Med., Volume 2(12): 808–823

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 808–823

INTRODUCTION Brain is the organ that is responsible for what we call the mind. It is the basis for thinking, feeling, wanting, perceiving, learning and memory, curiosity and behaviour (Abel T and Lattal KM, 2001). Learning is defined as the acquisition of information and skills whereas, Memory is the process by which acquired knowledge is retained. Learning and memory can be conceived as both a psychological process, as well as change in synaptic neural connectivity. They are the basic constituents of cognitive behaviour (Ahmed T and Gilani AH, 2009). Alzheimer‟s disease (AD) represents the most common cause of dementia, affecting more than 15 million individuals worldwide (Costantini C et al., 2005). It is characterized by progressive memory loss, cognitive impairments, and personality defects accompanied by diffuse structural abnormalities in the brain (Parle M et al., 2004a; Parle M et al., 2004b). The main histological hallmarks of AD are extracellular “amyloid plaques”, consisting of amorphous extracellular deposits of a β- amyloid protein (known as Aβ), and intraneuronal “neurofibrillary tangles”, comprising filaments of a phosphorylated form of microtubuleassociated protein (Tau) (Davies P and Maloney AJF, 1976). One of the most consistent and profound change associated with AD is diminished central cholinergic neurotransmission (Puglielli L et al., 2003). Besides reducing cholinergic activity, oxidative stress an imbalance between free radicals and antioxidant system, plays a critical role and is one of the major causes for memory loss in AD (Markesbery WR et al., 1997; Lovell MA et al., 1995). Substantial evidences accumulating in the literature specifically prove that high cholesterol diets increase the risk of sporadic AD (Mandrekar-Colucci S and Landreth GE, 2011; Andersson S et al., 2005). Despite intensive advancement in research, available therapeutic options are limited, thus, increasing demand for new drugs.

Medicinal herbs are indispensible part of traditional medicine and in the recent past, attracted attention due to their potential role in dementia. Polyphenols constitute a large group of naturally occurring substances in the plant kingdom, which include the flavonoids. Several epidemiological studies suggest that inclusion of antioxidant-rich foods in the diet is helpful to improve cognitive performance in humans (Singh M et al., 2008; Luchsinger JA and Mayeux R, 2004). Furthermore, dietary intake of flavonoids has been inversely related to the risk of dementia (Commenges D et al., 2000; Letenneur L et al., 2007). The increasing realization that polyphenol-rich diets may directly benefit human health, fuels continued research interest in these important compounds. Dioscorea bulbifera L., the Aerial yam, commonly known as „air potato‟ is unique among 600 species of the genus Dioscorea. It is a major staple food crop widely distributed around the world in tropical and subtropical regions. It possesses profound therapeutic potential and is widely used in traditional Indian and Chinese medicine as a valuable herb in the process of rebuilding and maintaining kidney function, in treating diseases of the lungs and spleen and used to lower glycemic index, providing a better protection against obesity and diabetes (Ahmed Z et al., 2009). Dioscorea bulbifera accomplishes high nutritive value and is natural store of antioxidants (Suriyavathana M and Indupriya S, 2011) making them an excellent addition to the human diet. Studies reported that Dioscorea bulbifera tubers contain phytoconstituents like alkaloids, steroids, fats and fixed oils, flavanoids, phenols, resins, tannins, protiens and carbohydrates (Shubhash C et al., 2012). The tuber extract is rich in polyphenolic compounds, especially flavonoids such as kaempferol -3, 5- dimethyl ether, caryatin, catechin, myricetin, quercetin-3-Ogalactopyranoside, myricetin -3- O-galactopyranoside, myricetin -3- O- glucopyranoside (Gao HY et al., 2002; Wang G et al., 2009). Studies also reported its potent antioxidant (Suriyavathana M and Indupriya S, 2011), antihyperglycemic and antidyslipidemic

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 808â&#x20AC;&#x201C;823

(Ahmed Z et al., 2009), analgesic and antiinflammatory (Mbiantcha M et al., 2011), antitumour (Gao HY et al., 2002; JM et al., 2012) and reversal of bacterial resistance (Shrirama V et al., 2008). Natural polyphenols significantly attenuated cognitive impairments and amyloidbeta burden. Beneficial effects of natural polyphenolic might attribute to direct scavenging free radicals and increasing antioxidant capacity and anticholinesterase activity (Gsell W et al., 1996; Tumiatti V et al., 2008) which certainly contribute to their neuroprotective effect. However, to date, there are no reports on memory enhancing activity of Dioscorea bulbifera tubers and in the light of above background, the present study was undertaken to investigate the potential of Dioscorea bulbifera tuber extract in memory dysfunctions. MATERIALS AND METHODS Experimental Animals Swiss albino mice, weighing 15â&#x20AC;&#x201C;20gm of either sex (procured from the animal house facility of Shri Guru Rai Institute of Technology & Sciences, Dehradun) were employed in the present study. They were maintained on standard laboratory pellet chow and water ad libitum. The high fat diet groups of animals were subjected to a standard diet enriched with fat ad libitum for 90 days. The mice were exposed to a 12-h light and 12-h dark cycle and were acclimatized to the laboratory conditions five days prior to the behavioral study. The experimental protocol was duly approved by the institutional animal ethical committee and care of the animals was carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India (Reg.No. M.PH/IAEC/01/2012/ECC-7). Drugs and Reagents Streptozotocin (STZ) was purchased from Sigma Aldrich, St. Louis, USA. Piracetam and Atorvastatin were obtained as gift samples from Akums Pharmaceuticals Ltd., Haridwar. Bovine Serum Albumin (BSA) standard,

Reduced Glutathione (GSH) standard, Thiobarbituric acid, Tris Hydrochloric acid, Tricholoroacetic acid, 5,5-Dithiobis 2nitrobenzoic acid (DTNB), Ethylene diamine tetra acetic acid (EDTA), Acetylcholine iodide (ATC) were provided by Himgiri Traders, Dehradun. All the reagents and chemicals used in this study were of analytical grade. Streptozotocin was dissolved in freshly prepared artificial cerebrospinal fluid (ACSF) (147 mM NaCl; 2.9 mM KCl; 1.6 mM MgCl2; 1.7 mMCaCl2; and 2.2 mM dextrose). Standard Cholesterol estimation kit was used to estimate total serum cholesterol level. Plant material The fresh tubers of Dioscorea bulbifera (DB) were collected from the local areas of Dehradun, Uttarakhand. These tubers were then identified and authenticated by the taxonomist at Forest Research Institute (FRI), Dehradun. The voucher Specimen no. Dis/ 1050/ 2011Bot-15-1/ (Rer. Gen) was then provided. Preparation of Plant Extract The fresh tubers of DB were collected and washed well in tap water first and then with the distilled water. The cleaned tubers were sliced and allowed for the complete shade drying and then made to fine powder with homogenizer. A crude extract of enriched with polyphenolic compounds was prepared by extracting residue with aqueous ethanol (50:50 and 90:10) solution at room temperature for extraction time of seven days. The crude extracts were then concentrated in vaccum rotatory evaporator. The percentage yield from 50:50 hydroalcoholic solvent and from 90:10 hydroalcoholic solvent was obtained. The presence of polyphenols was confirmed by Ferric chloride test for tannins. Interoceptive behavioral models High Fat Diet (HFD) - induced experimental dementia. Animals were subjected to a cholesterolrich diet for 90 days and allowed free access to the HFD 24 h/day for 90 days to induce memory impairment (Dalla Y et al., 2010). High fat diet was prepared by properly mixing the ingredients mentioned in Table 1.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 808–823

Table 1. Composition of High Fat Diet (HFD) Feed Contents Powdered pellet diet (Standard lab diet) Lard Casein Cholesterol Sodium cholate DL- methionine Yee-sac powder Vitamins and minerals Sodium chloride Total

Mass (g) 365 310 250 10 5 3 1 55 1 1000

*Composition of vitamins and minerals mixture per kg of HFD: vitamin A (120000 I.U.), vitamin D 3 (24000 I.U.), vitamin B2 (48 mg), vitamin E (18 units), vitamin K (24 units), calcium pantothenate (60 mg), nicotinamide (240 mg), vitamin B12 (144 mg), calcium (18 mg), magnesium (660 mg), iodide (24mg), iron (180 mg), zinc (360 mg), copper (48mg), cobalt (108 mg).

Intracerebroventricular (i.c.v) Streptozotocin (STZ) - induced experimental dementia.

artificial cerebrospinal fluid (ACSF) via i.c.v injection in similar manner (Dalla Y et al., 2010).

Mice were anesthetized with anesthetic ether for i.c.v administrations. Ether has been preferred here due to its ultrashort action and fast reversibility. Moreover, brief extent of ether exposure for i.c.v injection has been reported to exert no significant effect on learning and memory in animals. Intracerebroventricular injections were made with a 0.4 mm external diameter hypodermic neddle attached to a 10 μl Hamilton microlitre syringe (Top Syringe, Mumbai, India). The needle was covered with a polypropylene tube except for 3 mm of the tip region, which was inserted perpendicularly through the skull into the brain of mouse. The injection site was 1 mm to the right or left of the midpoint on the line drawn through to the anterior base of the ears. Injections was made into the right or left ventricle on alternate days. Two doses of STZ (3 mgkg-1, i.c.v, 10 μl each) administered bilaterally. The second dose was administered 48 h after the first dose. The STZ concentration was adjusted to deliver 10 μl per injection. The injection was made in two locations due to the difficulty of administering 10 μl to a single site. STZ was dissolved in artificial CSF (25 mgml1 ) solution which was made freshly just before the injection. Control mice were administered

Exteroceptive behavioral models Morris water maze (MWM) Test. Morris water maze test was employed to assess learning and memory of the animals (Morris R, 1984). MWM is a swimming based model where the animal learns to escape on to a hidden platform. It consisted of large circular pool (150 cm in diameter, 45 cm in height, filled to a depth of 30 cm with water at 28 ± 1ºC. The water was made opaque with white colored non-toxic dye. The tank was divided into four equal quadrants with help of two threads, fixed at right angle to each other on the rim of the pool. A submerged platform (10 cm2), painted in white was placed inside the target quadrants of this pool, 1 cm below surface of water. The position of platform was kept unaltered throughout the training session. Each animal was subjected to four consecutive training trials on each day with inter trial gap of 5 min. The mouse was gently placed in the water between quadrants, facing the wall of pool with drop location changing for each trial, and allowed 120 s to locate submerged platform. Then, it was allowed to stay on the platform for 20 s. If it failed to find the platform within 120 s, it was guided gently onto platform and allowed to remain there for

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 808â&#x20AC;&#x201C;823

20 s. Day 4 escape latency time (ELT) to locate the hidden platform in water maze was noted as index of acquisition or learning. Animal was subjected to training trials for four consecutive

days, the starting position was changed with each exposure as shown in Table 2, and target quadrant (Q4) remained constant throughout the training period.

Table 2. Sequence of training trials on Morris water - maze. Day 1 Day 2 Day 3 Day 4

Q1 Q2 Q3 Q4

Q2 Q3 Q4 Q1

On fifth day, platform was removed and each mouse was allowed to explore the pool for 120 s. Mean time spent in all four quadrants was noted. The mean time spent by the animal in target quadrant searching for the hidden platform was noted as index of retrieval or memory. The experimenter always stood at the same position. Care was taken that relative location of water maze with respect to other objects in the laboratory serving, as prominent visual clues were not disturbed during the total duration of study. Elevated Plus Maze Test Acquisition and retention of memory process was assessed using elevated plus maze on day 4, 5 of the study. The methodology followed was according to the method of Kulkarni, 2009. The plus maze consisted of two open (16 5 cm2) and two enclosed (16 5 12) arms. The arms extended from a central platform (5 5 cm2) and the maze was elevated to a fixed height (25 cm) from the floor. On the first day, each mice was placed at the end of an open arm, facing away from the central platform. Transfer latency (TL) was the time taken by mouse with all its four legs to move into one of the enclosed arms. TL was recorded on the first day. If the animal did not enter into one of the enclosed arms within 90 sec, it was gently pushed into one of the enclosed arms and the TL was assigned as 90 sec. the mouse was allowed to explore the maze for another 10 sec and then returned to its home cage. Retention of this learned task was examined 24

Q3 Q4 Q1 Q2

Q4 Q1 Q2 Q3

hour after the first day trial (Sharma AC and Kulkarni SK, 1992). Biochemical Parameters Animals were sacrificed by cervical dislocation, brains were removed and homogenized in phosphate buffer (pH = 7.4). The homogenates were than centrifuged (Remi cooling centrifuge; C- 24BL) at 3000 rpm for 15 min. The supernatant of homogenates were used for biochemical estimations as per the methods described below. Blood sample was collected by retro-orbital puncture just before sacrificing the animal. The blood was then kept at room temperature for 30 min after which it was centrifuged at 4000 rpm for 15 min to separate serum. Serum was used to estimate the level of serum total cholesterol. Estimation of brain acetyl cholinesterase (AChE) activity. The whole brain AChE activity was measured by the method of Ellman et al. (1961) with slight modifications. The esterase activity is measured by providing an artificial substrate, acetylthiocholine (ATC). Thiocholine released because of the cleavage of ATC by AchE is allowed to react with the -SH reagent 5,5â&#x20AC;&#x;dithiobis-(-2-nitrobenzoic acid) (DTNB), which is reduced to thionitrobenzoic acid, a yellow coloured anion with an absorption maxima at 412 nm. The extinction coefficient of the nitrobenzoic acid is 1.36 104/molar/centimeter. The concentration of thionitrobenzoic acid detected using a UV

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 808–823

spectrophotometer is then taken as a direct estimate of the AchE activity. Results were expressed as μmol of Ach hydrolysed/ min/ mg of protein.

procedure as mentioned in the standard cholesterol estimation kit. The absorbance was measured against blank at 540 nm using spectrophotometer.

Estimation of brain thiobarbituric acid reactive species (TBARS) level.

EXPERIMENTAL PROTOCOL

The whole brain TBARS level was measured by the method of Slater et al., (1971) with slight modifications. The method estimates Malondialdehyde (MDA), a product of lipid peroxidation. One molecule of MDA reacts with two molecules of Thiobarbituric acid (TBA) under mildly acidic conditions to form a pink colour chromogen, whose intensity is measured in spectrophotometer at 535nm. Results were expressed as nmol/ mg of protein. Estimation of brain reduced glutathione (GSH) level. The whole brain GSH level was estimated by the method of Moran et al., (1979) with slight modifications. Glutathione present in RBC consists of some sulfhydryl groups. 5,5 dithio bis 2-nitrobenzoic acid (DTNB), a disulphide compound gets easily attacked by tissue sulfhydryl groups and formed a yellow coloured anion which is measured at spectrophotometer at 412 nm. Results were expressed as nmol/ mg of protein. Estimation of brain total protein. For the estimation of total protein in brain, method of Lowry et al., (1951) with slight modifications. The absorbance was determined spectrophoto- metrically at 750 nm against suitably prepared blank. A standard curve using 200 mg of BSA was plotted. The amount of total protein was expressed in mg. Estimation of total serum cholesterol. The total serum cholesterol level was measured by Allain method (Allain CC et al., 1974) with slight modifications by employing commercially available standard cholesterol estimation kit. The blank, standard and test sample prepared according to the standard

Group I: Control. Mice were kept on free access to standard food pellets chow diet and water. Then normal saline (10 ml/kg, i.p.) was administered intraperitoneally 30 min before conducting the trials in MWM and EPM test. Group II: Artificial cerebrospinal fluid (ACSF) control. Mice were injected intracerebroventricularly ACSF (25 mg/ml, 10 μl, i.c.v) in two dosage schedules i.e. on first day and third day followed by exposure to MWM and EPM test after 15 days. Group III: Streptozotocin (STZ) control. Mice were injected intracerebroventricularly Streptozotocin (3 mg/kg, 10 μl, i.c.v) in two dosage schedules i.e. on first day and third day followed by exposure to MWM and EPM test after 15 days. Group IV: High fat diet (HFD) control. Mice were allowed free access to the HFD 24 hours/day for 90 days and then subjected to MWM and EPM test. Group V: HFD + Piracetam. HFD fed mice were administered Piracetam (400 mg/kg, i.p.) for 15 days and then for 5 days during exposure to MWM and EPM test. Group VI: HFD + Atorvastatin. HFD fed mice were administered Atorvastatin (10 mg/kg, p.o.) for 15 days and then for 5 days during exposure to MWM and EPM test. Group VII: HFD + DB (250 mg/kg). HFD fed mice were administered DB (250 mg/kg p.o.) for 15 days and then for 5 days during exposure to MWM and EPM test. Group VIII: HFD + DB (500 mg/kg). HFD fed mice were administered DB (500 mg/kg, p.o.) for 15 days and then for 5 days during exposure to MWM and EPM test.

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Group IX: HFD + DB (1000 mg/kg). HFD fed mice were administered DB (1000 mg/kg, p.o.) for 15 days and then for 5 days during exposure to MWM and EPM test. Group X: STZ + Piracetam. STZ mice were administered Piracetam (400 mg/kg, i.p.) for 15 days and then for 5 days during exposure to MWM and EPM test. Group XI: STZ + Vit E. STZ mice were administered Vit E (100 mg/kg, p.o.) for 15 days and then for 5 days during exposure to MWM and EPM test. Group XII: STZ + DB (250 mg/kg). STZ mice were administered DB (250 mg/kg, p.o.) for 15 days and then for 5 days during exposure to MWM and EPM test. Group XIII: STZ + DB (500 mg/kg). STZ mice were administered DB (500 mg/kg, p.o.) for 15 days and then for 5 days during exposure to MWM and EPM test. Group XIV: STZ + DB (1000 mg/kg). STZ mice were administered DB (1000 mg/kg, p.o.) for 15 days and then for 5 days during exposure to MWM and EPM test. STATISTICAL ANALYSIS All results were expressed as mean ± SEM. Data was analyzed by using one way ANOVA followed by Tukey‟s test and Bonferroni test. p<0.05 was considered to be statistically significant. RESULTS Effect on Escape Latency Time (ELT) and Time Spent in Target Quadrant (TSTQ) using Morris Water Maze. Control group showed significant decrease in day 4 ELT as compared to its ELT on day 1 (Table 3). Further, these mice spent significantly more time in the target quadrant

(Q4) in search of missing platform as compared to the time spent in other quadrants (Q1, Q2, Q3) during the retrieval trial on day 5 (Fig. 2). Treatment of vehicle artificial cerebrospinal fluid (ACSF) did not show any significant effect on day 4 ELT (Table 3) and day 5 TSTQ as compared to control group (Fig 2).Administration of STZ (3 mg/kg,10 μl, i.c.v) and HFD for 90 days, significantly prevented the decrease in day 4 ELT as compared to control group (Table 3) and markedly diminished TSTQ (Q4) observed in the retrieval trial on day 5 (Fig. 2). Further, administration of DB (250, 500 and 1000 mg/kg, p.o.) significantly attenuated the day 4 rise in ELT (Table 3) and decrease in day 5 TSTQ (Fig.2) in i.c.v. STZ as well as HFD treated mice, indicating reversal of i.c.v. STZ and HFD-induced learning and memory deficits. Treatment with Piracetam and Atorvastatin significantly attenuated the day 4 rise in ELT (Table 3) and decrease in day 5 TSTQ (Fig.2) in HFD treated mice. Also treatment with Piracetam and Vit E significantly attenuated the day 4 rise in ELT (Table 3) and decrease in day 5 TSTQ (Fig.2) in i.c.v STZ treated mice. Effect on Transfer Latency (TL) using Elevated Plus Maze. Initial transfer latency (ITL) did not differ significantly in any of the groups. Retention transfer latency (RTL) of HFD control and i.c.v STZ control group significantly increased as compared to control group, indicating impairment in learning and memory (Table 4). Further, administration of DB (250, 500 and 1000 mg/kg, p.o.) significantly lowered the RTL in HFD as well as STZ- treated mice (Table 4), indicating reversal of HFD and i.c.v STZ- induced learning and memory deficits. Treatment with Piracetam and Atorvastatin significantly lowered the RTL in HFD- treated mice. Also, treatment with Piracetam and Vit E significantly lowered the RTL (Table 4) in i.c.v STZ- treated mice.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 808–823

Table 3: Effect of Dioscorea bulbifera on High fat diet and i.c.v Streptozotocin- induced changes on Escape latency time (ELT) using Morris water maze. S.No. I II III IV V VI VII VIII IX X XI XII XIII XIV

Group

Day 1 ELT Mean ± S.E.M.(sec) 93.2 ± 2.8 92.8 ± 2.5 105.1 ± 2.3 101.2 ± 2.3 89.8 ± 2.8 87.2 ± 2.7 100.0 ± 2.8 99.8 ± 2.8 95.6 ± 2.8 90.8 ± 2.8 92.6 ± 2.7 102.2 ± 2.9 100.0 ± 2.7 99.2 ± 2.9

Control (10 ml/kg, i.p.) ACSF Control (25 mg/ml; 10 μl) STZ Control ( 3mg/kg, i.c.v) HFD Control HFD + Piracetam (400 mg/ kg, i.p.) HFD + Atorvastatin (10 mg/kg, p.o.) HFD + DB (250 mg/ kg, p.o.) HFD + DB (500 mg/kg, p.o.) HFD + DB (1000 mg/ kg, p.o.) STZ + Piracetam (400 mg/kg i.p.) STZ + Vit E (100 mg/kg p.o.) STZ + DB (250 mg/ kg, p.o.) STZ + DB (500 mg/kg, p.o.) STZ + DB (1000 mg/ kg, p.o.)

Day 4 ELT Mean ± S.E.M.(sec) 38.5 ± 2.4a 38.0 ± 2.5 70.5 ± 2.6b 60.1 ± 2.6b c 41.9 ± 2.9 43.1 ± 2.8c c 44.9 ± 2.4 41.3 ± 2.1c 38.8 ± 2.6c 46.4 ± 2.8d 49.8 ± 2.8d 47.8 ± 2.7d 43.8 ± 2.9d d 39.2 ± 2.6

Each group (n=6) represents mean ± standard errors of means (S.E.M), a = p< 0.05 as compared to Day 1 ELT in control group, b = p< 0.05 as compared to the day 4 ELT in control, c = p< 0.05 as compared to the day 4 ELT in HFD control, d = p< 0.05 as compared to the day 4 ELT in STZ control.

Fig 2: Effect of Dioscorea bulbifera on High fat diet and Streptozotocin- induced changes on

Total time spent in target quadrant in seconds (TSTQ) using Morris Water Maze. 100 a

Time Spent in Target Quadrant (TSTQ) (in sec)  S.E.M.

d c

c d

H FD ro l H C FD on t + ro H FD Pir l ac + H et A FD a to rv m + as D H ta FD B(2 50 tin + m H D g B( FD 50 /kg + ) 0 D m B( 10 g/k g ST 00 m ) Z g /k + g) Pi ra ce ST ta ST m Z Z + + D V ST B( 2 5 it E Z 0m + D ST g/ B( Z 5 0 kg ) + 0 D B( mg/ kg 10 ) 00 m g/ kg )

ro l

on t

on t C

C SF A

ST Z

on tr

Each group (n=6) represents mean ± standard errors of means S.E.M. a = p< 0.05 Vs time in other quadrants in control group, b = p< 0.05 Vs time spent in target quadrant in control group, c = p< 0.05 Vs time spent in target quadrant in HFD control group, d = p< 0.05 Vs time spent in target quadrant in STZ control group.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 808–823

Effect of Dioscorea bulbifera (DB) in brain AchE activity. i.c.v. STZ and HFD control group produced a significant increase in brain AchE activity (Fig. 3) when compared to control group. Further, treatment with Dioscorea bulbifera (250, 500 and 1000 mg/kg p.o.) and Atorvastatin significantly lowered the HFDinduced rise in brain AchE activity as compared to HFD control group. Treatment with Piracetam produced no statistical significant effect on HFD- induced rise in brain AchE activity (Fig.3). Treatment with Vit E significantly lowered the i.c.v STZ- induced rise in brain AchE activity as compared to STZ control group. Treatment with Piracetam produced no statistically significant effect on i.c.v STZ- induced rise in brain AchE activity (Fig.3). Effect of Dioscorea bulbifera (DB) in brain Thiobarbituric acid reactive species (TBARS) levels. i.c.v STZ and HFD control group produced a significant increase in brain Thiobarbituric acid reactive species (TBARS) levels (Fig.4) when compared to control group. Further, treatment with Dioscorea bulbifera (250, 500

and 1000 mg/kg p.o.) significantly lowered the i.c.v STZ and HFD- induced rise in brain TBARS levels (Fig.4) as compared to i.c.v STZ and HFD control group. Treatment with Piracetam and Atorvastatin also produced significant effect on HFD- induced rise in brain TBARS levels as compared to HFD control group (Fig.4). Effect of Dioscorea bulbifera (DB) on Reduced Glutathione levels (GSH) levels in brain. i.c.v. STZ and HFD control group produced a significant increase in GSH levels in brain (Fig.5) when compared to control group. Further, treatment with Dioscorea bulbifera (250, 500 and 1000 mg/kg p.o.) significantly lowered the i.c.v STZ and HFD- induced rise in brain GSH levels (Fig.5) as compared to i.c.v STZ and HFD control group. Treatment with Piracetam and Atorvastatin also produced significant effect on HFD- induced rise in brain GSH levels as compared to HFD control group (Fig.5). Treatment with Piracetam and Atorvastatin also produced significant effect on i.c.v STZ- induced rise in brain GSH levels as compared to i.c.v STZ control group (Fig.5).

Table 4: Effect of Dioscorea bulbifera on High fat diet and i.c.v Streptozotocin- induced changes in Transfer latency (LT) using Elevated plus maze. S.No. I II III IV V VI VII VIII IX X XI XII XIII XIV

Group Control (10 ml/kg, i.p.) ACSF Control (25 mg/ml; 10 μl) STZ Control ( 3mg/kg, i.c.v) HFD Control HFD + Piracetam (400 mg/ kg, i.p.) HFD + Atorvastatin (10 mg/kg, p.o.) HFD + DB (250 mg/ kg, p.o.) HFD + DB (500 mg/kg, p.o.) HFD + DB (1000 mg/ kg, p.o.) STZ + Piracetam (400 mg/kg i.p.) STZ + Vit E (100 mg/kg p.o.) STZ + DB (250 mg/ kg, p.o.) STZ + DB (500 mg/kg, p.o.) STZ + DB (1000 mg/ kg, p.o.)

TL on 1st day Mean ± S.E.M.(sec) 50.2 ± 2.2 49.6± 2.4 61.8 ± 2.1 59.0 ± 2.3 42.6 ± 1.5 41.8 ± 1.7 39.6 ± 1.8 38.4 ± 1.8 40.4 ± 1.7 52.8 ± 1.9 54.7 ± 1.4 51.9 ± 1.6 52.6 ± 1.9 50.1 ± 1.6

TL after 24 h Mean ± S.E.M.(sec) 40.3 ± 2.1a 39.7 ± 2.1 55.0 ± 2.2b b 50.0 ± 2.1 30.6 ± 1.6c 33.6 ± 1.6c 28.0 ± 1.8c 29.6 ± 1.7c 30.8 ± 1.9c 44.8 ± 1.6d 45.6 ± 1.9d 40.6 ± 1.5d 42.8 ± 1.7d 40.9 ± 1.6d

Each group (n=6) represents mean ± standard errors of means (S.E.M), a = p< 0.05 as compared to day 1 transfer latency in control group, b = p< 0.05 as compared to the day 2 transfer latency in control, c = p< 0.05 as compared to the day 2 transfer latency in HFD control, d = p< 0.05 as compared to the day 2 transfer latency in STZ control.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 808–823

Fig 3: Effect of Dioscorea bulbifera on High fat diet and Streptozotocin- induced changes in brain AchE activity.

Brain AChE activity (M of Ach hydrolysed/min/mg of protien)

250

200

b c

c d

c c

150

100

C SF ont ro ST Con l Z tr H C ol H FD ont F r H D + Co ol FD P nt r H FD + A irac ol e t H + D orv tam FD B a s H + (25 tat FD D 0m in B( g + D 500 /kg B( m ) ST 100 g/k Z 0 m g) + Pi g/k ST g r Z ST ace ) t + ST D Z a m Z B + ST + D (25 Vit Z B( 0m E + g D 500 /kg B( 10 mg ) 00 /kg m ) g/ kg )

Each group (n=6) represents mean ± standard errors of means (S.E.M), a = p< 0.05 as compared to control group, b = p< 0.05 as compared to control, c = p< 0.05 as compared to HFD control, d = p< 0.05 as compared to STZ control.

Fig.4: Treatment with Piracetam and Atorvastatin also produced significant effect on i.c.v STZ- induced rise in brain TBARS levels as compared to i.c.v STZ control group 20

TBARS levels (nano mol/mg of protien)

b c

c c

d d

A C

C SF ont r ST Con ol Z tr H C o H FD on l t FD C ro H FD + on l P t H FD + A ira rol ce t + ta o H FD DB rva m H + (25 sta FD D 0 tin m + B(5 g/ D 00 kg B m ) ST (100 g/k Z 0 m g) + Pi g/k ST g r Z ST ace ) + t a Z ST D m Z B + ST + (25 Vit Z DB 0m E + ( g D 500 /kg B( m ) 10 g/ 00 kg m ) g/ kg )

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Fig 5: Effect of Dioscorea bulbifera on High fat diet and Streptozotocin- induced changes in Reduced Glutathione levels (GSH) levels in brain. 25 a GSH levels (nano mol/mg of protien)

20 c c

15 b

d d

C SF ont ro ST Con l Z tr H C ol H FD ont F r H D + Co ol FD P n tr H FD + A irac ol e t H + D orv tam FD B a H + (25 stat FD D 0m in + B(5 g/k D 00 B( m g) ST 100 g/k Z 0 m g) + Pi g/k ST g r Z ST ace ) t + ST D Z + am Z B V ST + (25 it Z DB 0m E g ( + D 500 /kg B( m ) 10 g/ 00 kg m ) g/ kg )

Effect of HFD and Dioscorea bulbifera (DB) on Total serum cholesterol levels. Mice subjected to HFD for 90 days showed a significant increase in total serum cholesterol levels compared to control group. However, treatment with Dioscorea bulbifera (250, 500 and 1000 mg/kg p.o.) and Atorvastatin significantly attenuated HFD induced rise in total serum cholesterol levels (Fig 6). Effect of HFD on Body weight of mice (gm) ± S.E.M. There was a significant increase in the body weight of animals over the period of 90 days in mice receiving normal diet or High fat diet (HFD), when compared to the body weights of mice on day 1. Furthermore, HFD treatment for 90 days produced a significant increase in body weight of mice as compared to those receiving normal diet for 90 days (Table 6).

DISCUSSION Morris Water Maze test employed in present study is one of the most widely accepted models to evaluate learning and memory of the animals (Morris R, 1984). A significant decrease in day 4 escape latency time (ELT) of control animals during ongoing acquisition trials denoted normal acquisition of memory and an increase in time spent in target quadrant (TSTQ), in search of missing platform during retrieval trial indicated, retrieval of memory. Elevated plus maze test, which is a very useful test to investigate the ant anxiety agents, has been also helpful to measure the cognitive performance, basically to evaluate the spatial long term memory in rodents. Transfer latency (TL) of the first day is associated with the acquisition of information and memory whereas TL of the second day reflects retention of learning and memory (Parle M and Singh N, 2004).

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Fig 6. Effect of High fat diet and Dioscorea bulbifera on Total serum cholesterol levels. 200

Total serum cholesterol(mg/dl)

Day 90

Day 111

150 a a

c c

100

l to r FD va st + at D in B( 2 50 H FD m g/ + kg D ) B( H 50 FD 0m + g/ D kg B( ) 10 00 m g/ kg )

on tr o

Each group (n=6) represents mean ± standard errors of means (S.E.M), a = p< 0.01 Vs control group, b = p< 0.01 Vs control, c = p< 0.05 Vs HFD control group.

Table 6: Effect of HFD on Body weight of mice (gm) ± S.E.M. S.No.

I II

Group

Control (Normal diet) HFD Control

Day 1 Body weight (g)

Day 30 Body weight (g)

Day 60 Body weight (g)

Day 90 Body weight (g)

20.2 ± 1.0a

22.6 ± 1.2a

23.6 ± 1.0a

24.4 ± 1.2a

22.0 ± 1.1b

25.1 ± 1.1b

27.4 ± 1.2b

29.9 ± 1.3b

Each group (n=6) represents mean ± standard errors of means (S.E.M), a = p< 0.05 Vs day 1 body weight, b = p< 0.05 Vs day 90 body weight in control (normal diet) group.

The STZ i.c.v. model has been described an appropriate animal model of dementia closely related to Alzheimer‟s disease, typically characterized by progressive impairment of learning abilities and memory capacities (Lannert H and Hoyer S, 1998). Recent animal studies have demonstrated that STZ i.c.v. produces brain changes that are hallmark of human AD (Grunblatt E et al., 2006; SalkovicPetrisic M et al., 2006). This observation is in line with various previous reports whereby intracerebroventricular administration of

streptozotocin at sub-diabetogenic dose has been shown to induce memory deficits along with increase in brain oxidative stress levels and brain AChE activity( Sharma B et al., 2008; Kaur B et al., 2009). Studies have documented that administration of cholesterol rich high fat diet induces memory deficits in rodents. HFD model has been a well described animal model of dementia typically characterised by increased expression of cytokines, chemokines and increased reactive astrocytosis and

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microgliosis triggering brain inflammation. Increased brain cholesterol has also been documented to raise the β- amyloid peptide, PGE2 production, activation of NF-kβ in brain eventually culminating in neuronal damage and dementia (Sodhi RK and Singh N, 2013). Cholinergic neurotransmission also plays a crucial role in regulating learning, memory, and cortical organization of movement. Literature has demonstrated one of the most important mechanisms responsible for correct cholinergic function is performed by enzyme acetylcholine esterase (AChE) that hydrolyses the acetylcholine. Acetylcholine (Ach) is a classic mediator of learning and memory. The degeneration and dysfunction of cortical cholinergic neurons is closely associated with cognitive deficits of AD (Kumar R et al., 2010). In line with above discussed studies, STZ and HFD treated mice in our study performed poorly on Morris water maze indicating impairment in their learning abilities and memory capabilities. STZ and HFD induced impairment produced impairment of acquisition and retrieval of memory as reflected by significant increase in day 4 ELT and decrease in day 5 TSTQ respectively. Intracerebroventricular streptozotocin (STZ, i.c.v.) in our study has also impaired learning and memory along with significant rise in brain oxidative stress levels and brain AchE activity of mice. Further there was a significant rise in brain acetyl cholinesterase (AChE) activity and brain oxidative stress levels (indicated by an increase in TBARS and decrease in GSH levels).The results of the present investigation indicate that intracerebroventricular (i.c.v) administration of streptozotocin (STZ, 3 mg/kg) has produced cognitive deficits, abnormal biochemical alterations in the mice brain similar to that of dementia of AD type. High fat diet (HFD) for 90 days also accentuated the body weight and serum cholesterol levels in a significant manner. In the present study, Administration of Dioscorea bulbifera significantly attenuated HFD- induced rise in ELT and HFD- induced decrease in time spent in target quadrant in MWM, indicating reversal of HFD -induced

learning and memory deficits. Further, Administration of Dioscorea bulbifera significantly attenuated STZ - induced rise in ELT and STZ - induced decrease in time spent in target quadrant in MWM, indicating reversal of STZ -induced learning and memory deficits. It showed significant attenuation of i.c.v. STZ and HFD- induced rise in brain AchE activity, which may attribute to its anti-cholinesterase activity. It also showed significant attenuation of i.c.v. STZ and HFD- induced rise in brain oxidative stress (increase in TBARS and decrease in GSH) levels, which may attribute to its anti-oxidant activity. HFD treated mice also produced a significant reversal of elevated serum cholesterol levels and memory impairment, which may attribute to its cholesterol lowering effect. These findings in our study, demonstrate the potential of Dioscorea bulbifera tuber extract rich in polyphenolic compounds, as a safe and effective indigenous drug in memory dysfunctions. CONCLUSION It is concluded that, (DB) as shown its potential as a memory preserving/ curing and restorative agent in mice. The study also supports an important concept that onset of neurodegenerative disease may be delayed or mitigated with the use of dietary polyphenols that protects against oxidative stress and neurodegeneration. Perhaps, the present study constitutes the first report documenting the beneficial effect of Dioscorea bulbifera tubers on learning and memory impairment in High Fat Diet (HFD) and Streptozotocin (STZ) induced experimental dementia in mice; nevertheless in depth further studies are required to explore full potential and precise mechanism behind its beneficial role in memory dysfunctions. ACKNOWLEDGEMENT The authors wish to express their profound gratitude and appreciation to the Department of Pharmaceutical Sciences, Shri guru ram rai institute of technology and sciences for providing their support at each and every step.

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REFERENCES Abel

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Ahmed T, Gilani AH (2009). Inhibitory effect of curcuminoids on acetylcholinesterase activity and attenuation of scopolamine induced amnesia may explain medicinal use of turmeric in Alzheimer‟s disease. Pharmacology, Biochemistry and Behaviour. 91: 554–559. Ahmed Z, ChishtiMZ, Johri RK, Bhagat A, Gupta KK, Ram G (2009). Antihyperglycemic And Antidyslipidemic Activity Of Aqueous Extract Of Dioscorea Bulbifera Tubers. Diabetologia Croatica. 38(3): 63–72. Allain CC, Pool LS, Chan CS, Richmond W, Paul CF (1974). Enzymatic determination of total serum cholesterol. Clin Chem. 20: 470–475. Andersson S, Gustafsson N, Warner M, Gustafsson JA (2005). Inactivation of liver X receptor beta leads to adultonset motor neuron degeneration in male mice. Proc Natl Acad Sci USA. 10: 3857–3862. Commenges D, Scotet V, Renaud S, JacqminGadda H, BPrBerger-Gateau P, Dartigues JF (2000). Intake of flavonoids and risk of dementia. Eur J Epidemiol. 16: 357–63. Costantini C, Kolasani RMK, Puglielli L (2005). Ceramide and cholesterol: possible connections between normal aging of the brain and Alzheimer‟s disease. Just hypotheses or molecular pathways to be identified? Alzheimer‟s and Dementia. 57: 43–50.

Dalla Y, Singh N, Jaggi AS, Singh D (2010). Memory restorative role of statins in experimental dementia: an evidence of their cholesterol dependent and independent actions”, Pharmacological Reports. 62: 784–796. Davies P, Maloney AJF (1976). Selective loss of central cholinergic neurons in Alzheimer's disease. Lancet. 2: 1403. Ellman GF, Courthey KD(1961). Biochem Pharmacology. 7: 88–95. Gao HY, Kuroyanagi M, Wu LJ, Kawahara N, Yasuno T, Nakamura Y (2002). Antitumor promoting constituents from Dioscorea bulbifera L. in JB6 mouse epidermal cells. Biol. Pharm. Bull. 25: 1241–1243. Grunblatt E, Koutsilieri E, Hoyer S, Riederer P (2006). Gene expression alterations in brain areas of intracerebroventricular streptozotocin treated rat. J Alzheimers Dis. 9: 261–271. Gsell W, Strein I, Riederer P (1996). The neurochemistry of Alzheimer type, vascular type and mixed type dementias compared. J Neural Transm. 47: 73– 101. JM, Ji LL, Branford-White CJ, Wang ZY, Shen KK, Liu H, Wang ZT (2012). Antitumor activity of Dioscorea bulbifera L. rhizome in vivo. Fitoterapia. 83: 388–394. Kaur B, Singh N, Jaggi AS (2009). Exploring mechanism of pioglitazone induced memory restorative effect in experimental dementia. Fundam Clin Pharmacol. 23: 557–566. Kumar R, Jaggi AS, Singh N (2010). Effects of Erythropoietin on Memory Deficits and Brain Oxidative Stress in the Mouse Models of Dementia. Korean J Physiol Pharmacol. 14: 345–352.

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Lannert H, Hoyer S (1998). Intracerebroventricular administration of Streptozotocin causes long term diminutions in learning and memory abilities and in cerebral energy metabolism in adult rats. Behav Neurosci. 112: 1199–1208. Letenneur L, Proust-Lima C, Le Gouge A, Dartigues JF, BPrBerger-Gateau P (2007) Flavonoid intake and cognitive decline over a 10-year period. Am J Epidemiol. 165: 1364–71. Lovell MA, Ehmann WD, Butler SM and Markesberg WR (1995). Elevated Thiobarbituric Acid Reactive Substances and Antioxidant Enzyme Activity in the Brain in Alzheimer‟s disease. Neurology. 45(8): 1594–1608. Lowry OH, Rosebrough NJ, Far AL, Randall RJ (1951). Protein measurement with folin-phenol reagent. J Biol Chem. 193: 265–275. Luchsinger JA, Mayeux R (2004). Dietary factors and Alzheimer‟s disease. Lancet Neurol. 3: 579–587. Mandrekar-Colucci S, Landreth GE (2011). Nuclear receptors as therapeutic targets for Alzheimer‟s disease. Expert Opinion in Therapeutic Targets. 15: 1085–1097. Markesbery WR (1997). Oxidative stress hypothesis in Alzheimer‟s disease. Free Radical Biology and Medicine. 23(1): 134–147. Mbiantcha M, Kamanyi A, Teponno RB, Tapondjou AL, Watcho P and Nguelefack TB (2011). Analgesic and anti-inflammatory properties of extracts from the bulbils of Dioscorea Bulbifera L.var sativa(Dioscoreaceae) in mice and rats. Evidence-Based Complementary and Alternative medicine. 1–9.

Moron MS, Depierre JW, Mannervik B (1979). Levels of glutathione, glutathione reductase and glutathione transferase activities in rat lung and liver. Biochemica at Biophysica Acta. 582: 67–78. Morris R (1984). Developments of water- maze procedure for studying spatial memory in the rat. Journal of Neuroscience. 11: 47–60. Parle M, Dhingra D, Kulkarni SK (2004a). Neurochemical basis of learning and memory. Indian Journal of Pharmaceutical Sciences, 66: 371–376. Parle M, Dhingra D, Kulkarni SK (2004b). Neuromodulators of learning and memory. Asia Pacific Journal of Pharmacology.16: 89–99. Parle M, Singh N (2004). Animal models of testing memory. Asia pacific J Pharmacol. 16: 101–120. Puglielli L, Tanzi RE, Kovacs DM (2003). Alzheimer's disease: the cholesterol connection. Nature Neuroscience. 6: 345–351. Salkovic-Petrisic M, Tribl F, Schmidt M, Hoyer S, Riederer P (2006). Alzheimerlike changes in protein kinase B and glycogen synthase kinase-3 in rat frontal cortex and hippocampus after damage to the insulin signalling pathway. J Neurochem. 96: 1005– 1015. Sharma AC, Kulkarni SK (1992). Evaluation of learning and memory mechanisms employing elevated plus-maze in rats and mice. Progress in Neuropsychopharmacology Biology and Psychiatry. 16(1): 117–125.

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Sharma B, Singh N, Singh M (2008). Modulation of celecoxib and Streptozotocin induced experimental dementia of Alzheimer‟s disease type by pitavastatin and donepezil. J Psychopharmacol. 22: 162–171. Shrirama V, Jahagirdar S, Lathac C, Kumara V, Puranikd V, Rojatkard S, Dhakephalkar PK, Shitolea MG (2008). A potential plasmid-curing agent, 8-epidiosbulbin E acetate, from Dioscorea bulbifera L. Against multidrug-resistant bacteria. Int. J. Antimicro. Agents. 32: 405–410. Shubhash C, Sarla S, Mishra PA, Anoop B (2012). Nutritional profile and phytochemical screening of Garhwal Himalaya medicinal plant Dioscorea Bulbifera. Int Res J Pharm. 3(5): 289– 294. Singh M, Arseneault M, Sanderson T, Murthy V, and Ramassamy C (2008). Challenges for research on polyphenols from foods in Alzheimer‟s disease: bioavailability, metabolism, and cellular and molecular mechanisms. Journal of Agricultural and Food Chemistry. 56(13): 4855–4873.

Source of Support: NIL

Slater TF, Sawyer BC (1971). The stimulatory effect of carbontetrachloride and other halogenalkane or peroxidative reaction in the rat liver fraction in vitro. Biochem Journal. 123: 805–814. Sodhi RK, Nirmal Singh N (2013). Defensive Effect of Lansoprazole in Dementia of AD Type in Mice Exposed to Streptozotocin and Cholesterol Enriched Diet. Plos One, 8(7): e70487. Suriyavathana M and Indupriya S (2011). Screening of antioxidant potential of Dioscorea Bulbifera. International Journal of Pharmacy And Life Sciences. 2(4): 661–664. Tumiatti V, Bolognesi ML, Minarini A, Rosini M, Milelli A, Matera R (2008). Progress in acetylcholinesterase inhibitors for Alzheimer‟s disease: an update. Expert Opinion on Therapeutic Patents. 12: 45–56. Wang G, Liu JS, Lin BB, Wang GK, Liu JK (2009). Two new furanoid norditerpenes from Dioscorea bulbifera. Chem Pharm Bull. 57: 625– 627.

Conflict of Interest: None Declared

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Research article PHARMACOGNOSTICAL EVALUATION OF THE ROOT OF OPERCULINA PETALOIDEA CHOISY - A SOURCE OF SHYAMA TRIVRUT Kolhe Rasika1*, Acharya Rabinarayan2, Harisha C R3 1

PhD scholar, Dept. of Dravyaguna, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India Associate Professor, Dept. of Dravyaguna, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat – 361008. India 3 Head, Pharmacognosy laboratory, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, India *Corresponding Author: Email:dr.rasika_kolhe@yahoo.com 2

Received: 11/10/2013; Revised: 31/11/2013; Accepted: 03/12/2013

ABSTRACT Operculina petaloidea Choisy (Convolvulaceae) [Synonym: Ipomoea petaloidea, Merremia crispatula Prain] is a large shrubby climber with shallowly cordate leaves and yellow flower. It is known as kali tihudi by the traditional practitioner of Odisha and also considered as source of drug for two ayurvedic classical drugs, shyma trivrut and vruddhadaru. The present study deals with the detailed morphological and micro-scopical profile of O. petaloidea root including its powder microscopic characters. Roots of O. petaloidea are light brown in colour with rough exterior. Root can be identified by the presence of lignified cork; oil globules, laticiferous cells and pitted stone cells. Interxylary phloem, intervascular pitting, latex cells and cluster crystals, Simple and compound starch grains with hilum, concentric line were some of the diagnostic charactrers. Organoleptic characters showed light brown colour, smooth texture with characteristic taste and tingling sensation. Lignin, calcium oxalate crystals, starch and tannin were present in histochemical test. Simple fibre, rosette crystals, scleroids, laticiferous cell, tracheids and tannin were diagnostic characters of powder microscopy KEY WORDS: Pharmacognosy, Operculina petaloidea Choisy, Ipomoea petaloidea, root, Shyama Trivrut

Cite this article: Kolhe Rasika, Acharya Rabinarayana, Harisha C R (2013), PHARMACOGNOSTICAL EVALUATION OF THE ROOT OF OPERCULINA PETALOIDEA CHOISY -A SOURCE OF SHYAMA TRIVRUT, Global J Res. Med. Plants & Indigen. Med., Volume 2(12): 824–829

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INTRODUCTION The family Convolvulaceae comprises nearly of 1650 predominantly tropical species. Operculina petaloidea Choisy (Convolvulaceae) [Synonym: Ipomoea petaloidea, Merremia crispatula Prain] is an important species of Operculina genus differentiated from O. turpethum by yellow flower, glabrous outer sepals and minutely velvety seeds with subvillous margin. (H O Saxena, 1995). It is considered as the botanical source of ayurvedic classical drug Shyama Trivrit (C P Khare, 2007) and Vruddhadaru (Anonymous AFI, 2000). It is also known as Kali Tihudi by the traditional practitioners of Odisha and reported as an adulterant for vruddhadaru (http://shodhganga.inflibnet.ac.in/bitstream/106 03/2576/9/09_chapter1.pdf). Shyama Trivrut is a variety of Trivrut known for its purgative action, used in near about 190 formulations like kushtha (skin disease) gulma (abdominal lump), prameha (diabetes), jwara (fever), pandu (anemia) etc. in different dosage form like Kwatha (decoction), churna (powder), taila (oil) etc. both internally and externally (Kolhe Rasika et al., 2013). Though the plant, Shyama trivrut is used extensively in ayurvedic system of medicine, the detail pharmacognostical characters including powder microscopy and histochemical test are not reported for its botanical source (Prasad S et al, 1974; K Raghunathana, 2005). The present study incorporates the detailed morphological characters of root along with the microscopical profile of T S of O. petaloidea root including its powder microscopical characters. MATERIAL AND METHODS

the pharmacognosy laboratory of institute. Matured roots were separated and cleaned thoroughly with running water. Few pieces of root were stored in solution of AAF. (70% Ethyl alcohol: Glacial acetic acid: Formalin) in the ratio of (90:5:5) to utilize them for further studies. Collected roots were chopped; shade dried and pulverized using an electric blender. The powder was sieved through mesh size 60 and stored in an air tight food grade plastic container for further use. Pharmacognostic studies Morphological characters like surface, margin, rootlets etc. were studied by observing the roots with naked eyes and also with the dissecting microscope. Its organoleptic characters like colour, texture, smell etc. were noted down. Thin transverse sections of fresh root were taken for detailed microscopic observation following standard procedure (Anonymous, 2004; Trease and Evans, 2009). The sections were cleared with chloral hydrate and observed under the microscope for the presence of any crystals, then were stained with phloroglucinol and hydrochloric acid to notice the lignified element like fibres, vessels etc. of the meristele and other parts. Photographs of the sections were taken with the help of camera. Histo-chemical tests were carried out by taking thick sections following the standard procedure methods (Krushnamurthy, 1988). The sections were stained with various reagents like phloroglucinol followed by HCL for lignified elements, iodine for starch grains etc. Powder microscopy of root powder was carried out following standard procedures (Wallis TE, 1985). RESULT AND DISCUSSION

Plant material Macroscopic characters of root Matured fresh drug identified as kalitihudi by local herbal experts was collected from Gandhamardana hill ranges, Balangir district of Odisha, India, during the month of November 2012. The collected plant samples were cleaned to remove adherent soil and dirt. The material was also identified and confirmed as Operculina petaloidea Choisy (Convolvulaceae) at Botanical Survey of India, Pune and a voucher specimen of herbarium. (Phm-6069/8th April 2013) was deposited in

Roots stoloniferous, cylindrical, branched, elongated with thin rootlets, light brownish coloured, 20–60 cm in length and 5–10 cm in width and could be easily peeled off into pieces. Central portion was light yellowish in colour, very fibrous & difficult to break. Dark brown coloured latex was accumulated at the cut portion. Root was having ridges and rough exterior due to the presence of numerous lenticels and remains of rootlets (fig. 1.2).

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Plate 1: Morphology and transverse sections of root of O. petaloidea

Fig. 1.1 Leaf and flower of O petaloidea

Fig. 1.2 Root of O petaloidea

Fig. 1.3Cork cell in tangential view

Fig. 1.4 Lysogenous cavity

Fig. 1.5 Intervascular pitting in xylem

Fig. 1.6 Tannin content

Fig. 1.7 Stone cells in group with oil globule and starch grains

Fig. 1.8 Rosette crystal

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Plate 2: Powder microscopy of root O. petaloidea

Fig. 2.1 Cluster crystal

Fig. 2.3 Border pitted vessel

Fig. 2.5 Stone cell

Microscopic characters of root Thin transverse section of the matured root showed circular structure composing cork, cortex, phloem and xylem crossed with the medullary rays. Outer most layer was formed

Fig. 2.1 Latex content

Fig. 2.4 Simple fibre

Fig. 2.6 Lignified fibres

by compactly arranged lignified cork, composed of 8–10 layered tangentially arranged barrel shaped and elongated cells stratified into 2–3 strata. Parenchymatous cells formed wide zone of cortex, heavily filled with simple and compound starch grains, clustered

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crystals and oil globules. Most of parenchymatous cells were filled with yellow brown content which might be tannin. Large cavities of laticiferous cells were distributed all over the cortex region filled with yellowish latex (fig.1.4). Wide lumen of pitted stone cells was found scattered all over the cortical region along with the group of pericyclic fibres. Secondary phloem was a wide zone composed of sieve elements and large amounts of phloem parenchyma was accumulated above the xylem, consisting of fibres, starch grains, cluster crystals and also latex containing cells arranged in discontinuous rings crossed by somewhat elongated uni to biseriate medullary rays rising from the centre and reached upto inner layers of the cortex. Xylem occupied most of the central portion made up of 3–5 group of xylem vessels, consisting of large number of xylem fibres and its parenchyma. Interxylary phloem formed in the stellar portion. Intervascular pitting was observed in some of the xylem.

Latex cells, rosette and cluster crystals were rarely observed inside the xylem vessel (fig. 1.8). Starch grains both simple and compound along with concentric line and hilum were observed. Compound starch grains consisting of 2–4 components of oval to circular shape size vary from 5–44 μ in diameter was found scattered in cortex, phloem parenchyma, xylem parenchyma and medullary rays (plate 1). Organoleptic characters Organoleptic evaluation of the powder revealed light brown colour, smooth powder with characteristic taste and tingling sensation on the tongue. Histo-chemical evaluation Various histochemical tests were carried out using different chemicals to detect lignin, calcium oxalate crystals, starch and tannin. The results are depicted in table no. 1

Table No 1: Showing the result of Histo-chemical evaluation Material Section of root Section of root Section of root Section of root

Reagent Phloroglucinol +Conc HCL Phloroglucinol +Conc HCL Iodine Ferric chloride solution

Powder microscopy Diagnostic characters of the powder include the presence of simple fiber, rosette crystals, scleroids, laticiferous cells, parenchyma, tracheids and tannin in microscopical observations (plate 2). CONCLUSION Transverse section of the root of Operculina petaloidea showed the presence of

Test for Lignin

Result Present

Calcium oxalate crystal Present Starch Tannin

Present Present

laticiferous cells and pitted stone cells distributed all over the cortex region along with the presence of cluster crystal in the secondary xylem region. Powder microscopy showed the diagnostic characters like simple fiber, rosette crystals, scleroids, laticiferous cells, parenchyma and tracheids. Thus this detailed morphological and microscopical study along with the photomicrographs will definitely help for the identification and authentication of the root of Operculina petaloidea.

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Res Med Plants & indigen Med,Vol 2(11):772–784

REFERENCES Anonymous (2004), Ayurvedic Pharmacopoeia of India.Part I, Vol II, Appendix 2.1.1 2.1.2: Government of India publication, New Delhi p.no.184

Krishnamurty K.V. (1988), Methods in the plant histochemistry. Vishwanadhan Pvt Limited, Madras, p. no 1–77.

Anonymous (2000), The Ayurvedic Formulary of India Part II, List of single drugs plant origin New Delhi: Government of India Publication; p no.339

Prasad,

H.O.Saxena (1995), Flora of the Orissa,1st edition, Regional research laboratory, Bhuvneshwar, p no.1198

Raghunathana, Roma Mitra (2005), Pharmacognosy of indigenous drugs, Vol II, CCRAS, New Delhi, p.no.1085

http://shodhganga.inflibnet.ac.in/bitstream/106 03/2576/9/09_chapter 1.pdf

Trease

G. E and Evans W.C. (2009), Pharmacognosy, 16th Ed. Saunders, Elsevier. P no. 309–10.

Wallis

T.E. (1985), Text book of Pharmacognosy, 5th Ed, CBS Publishers, New Delhi, 1985. P no.572–78.

Khare CP (2007), Indian medicinal plants, 1st edition. E book, Springier reference. Delhi, p no.334 Kolhe rasika, Acharya R N (2013). Shyama trivrut, less known but frequently used drug of ayurveda: a review, Global J

Source of Support: NIL

Singh (1974). Pharmacognostical studies on `vidhara', Ipomoea petaloidea, J Res Ind Med 1974, 9 (2): 50–56;

Conflict of Interest: None Declared

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Research article ETHNO-BOTANICAL SURVEY OF SOME MEDICINAL PLANTS IN JATASANKAR REGION OF GIRNAR FOREST, GUJARAT, INDIA Raval Nita D1*, Dhaduk Haresh L2 1

Lecturer, Department of Dravyaguna, Government Ayurved College, Junagadh, Gujarat. Associate Professor, Department of Agricultural Botany, B.A.College of Agriculture, Agriculture University, Anand, Gujarat. 388110. *Corresponding author: Email - drnitadraval@yahoo.in; Mobile: +919898340450 2

Received: 03/10/2013; Revised: 29/11/2013; Accepted: 03/12/2013

ABSTRACT The present study deals with the ethno botanical survey of some important medicinal plants from Jatasankar, area of Girnar forest Junagadh. These medicinal plants comprise of 45 tress, 27 herbs, 15 shrubs and 25 climbers belongs to different families. Fabaceae, Euphorbiaceae, Apocynaceae, Asclepiadaceae, Moraceae, Acanthaceae and Solanaceae are the dominant families with high species diversity. The botanical name, vernacular name, family, part used and medicinal uses of these plants were narrated. During the study, emphasis was given on herbal treatment for everyday common ailments and diseases particularly used by local people of Gujarat state. The study gives an account on the diversity and uses of medicinal plants and priority medicinal plants for conservation. KEY WORDS: Medicinal plants, Girnar forest, Ethnobotany

Cite this article: Raval Nita D, Dhaduk Haresh L (2013), ETHNO-BOTANICAL SURVEY OF SOME MEDICINAL PLANTS IN JATASANKAR REGION OF GIRNAR FOREST, GUJARAT, INDIA, Global J Res. Med. Plants & Indigen. Med., Volume 2(12): 830–841

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INTRODUCTION

MATERIALS AND METHODS

India is one of the 12 mega-diversity countries of the world, due to the species richness and level of endemism recorded in the various agro climatic zones of the country. Here several medicinal systems have evolved like Ayurveda, Siddha and the Unani Systems of Medicine. In different civilizations the contribution of floral biodiversity to health care has been well documented (Posey 1999). According to Schippmann et al. 2002 more than 50000 species are used for medicinal purposes worldwide, of which almost 13 per cent are flowering plants. Over 8000 plant species are used in traditional and modern medicine in India (Planning Commission, 2000), and 90 to 95 per cent collection of medicinal plants is from the wild source, of which more than 70 per cent collection involves destructive and unscientific extraction. In this era over exploitation of trade species, destructive way of collection, vulnerability due to anthropogenic pressure are some of the major threats to medicinal plants. Because of the accelerated local, national and international interest in recent years the demand for medicinal and aromatic plants has increased in both developing and developed countries. Around 50,000 herbal drug formulations have been developed by traditional medicine systems.

Study Area

Human intervention in the biosphere has resulted in the widespread loss of the unique ecosystems and contributed to the extinction of biotic resources. Therefore, conservation of threatened spices of plants is needed for the sustainable development of the society. In order to achieve sustainable harvest of medicinal plants a multi disciplinary approach must be consider which includes ecological, biological, socio cultural and economical aspects of the species. (Ghimire et al 2004). The present study includes status, part(s) use and medicinal uses of available plants from Jatashankar area of Girnar forest, Junagadh, Gujarat, India.

Junagadh is one of the oldest city of Gujarat states. Girnar forest is located at the periphery of Junagadh city and is spread over 181.3 square km area. Girnar forest lies between parallel of latitude 21.25’’ N Latitude and meridian of 70.30’’ and 70.40’’ E Longitude. Jatashankar is one of the area located in Girnar forest. We have selected this particular area for the survey of medicinal plants because it is having maximum diversity of plants. The survey was conducted in the year 2012. During the investigation plant specimens were collected and sample vouchers were deposited in the department of Dravyaguna, Government Ayurved Collage, Junagadh. Sample vouchers numbers 1 to 112/2012(JS) was identified by Dr.H.L.Dhaduk, Associate Professor, Department of Agricultural Botany, B.A.College of Agriculture, Agriculture University, Anand, Gujarat. 388110. with the help of Flora of Gujarat State, Flora of the Presidency of Bombay, Flora of Saurashtra and by using online web resources. RESULT AND DISCUSSION In the present study, 112 medicinal plants encountered belonging to 41 families from the selected study area used by the local people and herbal practitioner in various diseases. In the present study 45 were trees, 15 shrubs, 27 herbs and 25 were climbers. Different parts of medicinal plant species were used for curing different diseases and mostly leaves were used followed by bark, fruit etc. From this collection, 14 medicinal plants belonging to 10 families are categorized as highly prioritized medicinal plants as they are of immense value in curing various diseases. Five parasitic plants like Cuscuta were also reported from this belt by Khwaja salahuddin et al. 2013.

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A few species such as Tectona grandis, Operculina terpethum, Gardinia gumifera, Sterculia urens, Wrightia tinctoria, Morinda citrifolia, Cissampelos pareira, Achyranthes aspera, Butea monosperma, Pongamia pinnata,

Alstonia scholaris, Cassia fistula, Cassia tora, Balanites egyptica were abundantly available in this area of Girnar forest.

Fig.1 Total medicinal plants 50

45 40 35 30

25 medicinal plants

15 10 5 0 CLIMBERS

TREES

HERBS

SHRUBS

Total 112 medicinal plants were observed during this survey. Out of them 45 species were trees, 27 species were herbs, 25 species were climbers and 15 species were shrubs. This is shown in Fig. 1.

Fig.2 Plant part used for medicinal purposes 1 11

Lv 51

Rt Fr Bk

Sd Wp Fl

La St/Tu/Gum

The plant part used for various ailments is shown in Fig. 2. The leaves (51 species), is used followed by roots (45 species), fruit (38 species), bark (29 species), seeds (24 species), whole plant (15 species), flower (11 species), latex (5 species), stem, tuber and gum (3 species) and extract (1 species) for various ailments.

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Table 1 Medicinal plant species of Jatasankar Junagadh, Girnar forest, Gujarat India.

Species

Local name

Tinospora cordifolia (Wil Guduchi ld.) Miers

Habit

Family

Menispermaceae

Part use St, Lv

Medicinal use Urinary troubles, general debility, leprosy, fever Antidote to snake and scorpion, constipation, gastric trouble, and urinary troubles, abdominal diseases Body swelling, wounds and joint Pain Mental disorders anaemia, backache gout, headache, paralysis, stomachache, wounds, rheumatism, leprosy, diarrhoea, cold, cough, eczema fever, digestive complaints Mental illness, liver problem, memory problem

Cissampelos pareira L.

Veni vel(Patha)

Menispermaceae

Lv, Rt

Cocculus hirsutus (L.) W.Theob. Celastrus paniculatus Wi lld.

Patala garudi

Menispermaceae

Jyotishmati

Celastraceae

Clitoria ternatea L.

Aprajita

Fabaceae

Abrus precatorius L.

Gunja

Fabaceae

Lv, Sd, Rt, Wp Sd

Mucuna pruriens (L.) DC.

Kapikacchu

Fabaceae

Sd,R t

Pain, parkinsonism, general debility

Operculina turpethum (L. Trivrut ) Silva Manso

Convolvulaceae

Purgative

Pueraria tuberosa Dc

Vidarikanda

Fabaceae

Caesalpinia crista L.

Latakaranja

Fabaceae

Asthma and abdoman pain, ache, chest pain, cholera, swelling Malaria, pain, diarrhea

Cayaponia laciniosa (L.) C.Jeffrey

Shivalingi

Cucurbitaceae

Rt,B k,Lv, Sd Sd Infertility

Trichosanthes dioica Roxb

Patola

Cucurbitaceae

Argyreia nervosa (Burm. f.) Bojer

Vrudhdaru

Convolvulaceae

Lv, Fr, Rt Rt

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Stops conception

Fever, skin disease, purgative, hyperacidity Joint pain, piles, oedema, diabetes, indigestion

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Hemidesmus indicus (L.) R. Br. ex Schult. Cryptolepis dubia (Burm. f.) M.R.Almeida Leptadenia reticulata (Re tz.) Wight & Arn. *Jasminum sambac (L.) Sol.

Sariva

Asclepiadaceae

Blood purifier

Krushna sariva C

Asclepiadaceae

Jivanti

Asclepiadaceae

Rt, La Rt

Fever, rheumatism, stomach ache, syphilis, stop bleeding Wounds, night blindness

Jati

Oleaceae

Wounds, disease

Cuscuta reflexa Roxb.

Amarvel

Convolvulaceae

Rt, Lv, Fl Wp

Tylophora asthmatica (L. f.) Wight & Arn. Dioscorea bulbifera L.

Arkapatri

Asclepiadaceae

Varahikanda

Dioscoreaceae

Lv,R t Tu

Asparagus racemosus Wi lld. *Piper longum L.

Shatavari

Liliaceae

Pippali

Piperaceae

Coccinia grandis (L.) Voigt

Bimbi

Cucurbitaceae

Luffa echinata Roxb.

Vrutkosha

Cucurbitaceae

*Momordica charantia Linn.

Karavellaka

Cucurbitaceae

Thespesia populnea (L.) Sol. ex Corrêa Aegle marmelos (L.) Corrêa

Parisha

Malvaceae

Rt, Fr Lv, Rt, Fr Lv, Fr Lv, Fr, Rt Bk

Bilva

Rutaceae

*Ailanthus excelsa Roxb.

Aralu

Simarubaceae

*Azadirachta indica A.Ju ss. *Melia azedarach L.

Nimba

Meliaceae

Mahanimba

Meliaceae

Butea monosperma (Lam. Palasha ) Taub.

Fabaceae

Fl, Sd

Pongamia pinnata (L.) Pierre

Karanja

Fabaceae

Dalbergia sissoo DC.

Shimshapa

Fabaceae

Lv, Bk, Sd St, Lv

Rt, Lv, Fr Bk Fr, Lv Fr, Lv

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ear

and

eye

Joint pain, indigestion, worms Bronchitis, cold and cough Diabetes, skin disease, worms Amenorrhoea, piles, diarrhoea, weakness Liver disease, diabetes, skin disease Jaundice, swelling, anemia

Abdominal disease Liver disease, fever, diabetes, gout, skin disease Diabetes, skin disease, leucorrhoea Diarrhoea, oedema, diabetes, indigestion Diarrhoea, skin disease, worms Antiseptic, skin diseases, lice, diabetes and spermicid Abortifacient, wormicides, antiseptic, rheumatic pain, skin disease Anthelmintic, antiseptic, blood purifier, tonic, indigestion Leucorrhoea, skin disease, worms, piles, oedema Gonorrhoea, headache, dysentery, leprosy, and skin disease

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Cassia fistula L.

Aragvadha

Fabaceae

Lv, Fr

Blood purification, asthma, antifertility, antiseptic, burn cough, leprosy, jaundice, liver problem, ringworm, stomachache, tooth ache, swelling of throat, pimples Indigestion, jaundice

Tamarindus indica L.

Amlika

Fabaceae

Bauhinia racemosa Lam.

Kanchnara

Fabaceae

Acacia nilotica (L.) Delile

Babula

Fabaceae

Acacia catechu (L.f.) Willd. Sapindus laurifoliusVahl.

Khadira

Fabaceae

Aristaka

Sapindaceae

Fl, Sd,Fr Lv,B Leprosy, piles, wounds, k dysentery, indigestion, worms Bk, Joint fracture, diabetes, Fr, leucorrhoea Gum Bk Luecoderma, skin disease, dental disease, fever, cough Fr Headache, skin disease

Albizzia lebbeck Benth.

Shirisha

Fabaceae

Terminalia chebula Retz.

Haritaki

Combretaceae

Terminalia bellirica (Gae Bibhitaki rtn.) Roxb. Terminalia arjuna (Roxb. Arjuna ex DC.) Wight & Arn. Syzygium rubicundum Wi Jambu ght & Arn.

Combretaceae

Myrtaceae

Mitragyna parvifolia (Ro xb.) Korth.

Kadamba

Rubiaceae

Nyctanthes arbortristis L.

Parijata

Oleaceae

Lv,B k,Fr

*Alstonia scholaris (L.) R. Br.

Saptaparna

Apocynaceae

Bk,L v

*Nerium indicum Mill

Karvira

Apocynaceae

*Thevatia peruviana Merrill.

Pita karvira

Apocynaceae

Rt, Bk Rt, Bk

Bk, Cough, skin disease, Lv, wounds, anti poison Fl,Sd Fr Bronchitis, cold, constipation, dysuria, eczema, dysentery, measles, pneumonia, stomach and spleen problem Fr Cough, asthma, bronchitis, diabetes, gastric disease Bk Heart disease Lv,S d,Fr, Bk Bk,F r

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Anaemia, diarrhoea, diabetes, piles, digestive problem Skin diseases, pimples, dysentery, sores, fever, snake bite Bone fracture, cough, fever, dysentery, indigestion, injury, malaria, rheumatism skin disease, sores, ulcer, sciatica, obesity Fever, malaria, diarrhea, headache, sinusitis, leprosy, wounds. Skine disease, hair loss, syphilis Cough, bronchitis,

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Phyllanthus emblica L. Syn.

Amalaki

Euphorbiaceae

Fr, Lv, Bk

Ficus racemosa L.

Udumbara

Moraceae

Ficus lacor Buch.-Ham.

Plaksha

Moraceae

Bk, La, Fr, Rt Bk

Ficus carica Linn

Anjira

Moraceae

Bambusa bambos (L.) Voss

Vansha

Poaceae

Cordia dichotoma G.Fors t.

Shleshmataka

Ehretiaceae

Extre ct,Lv Rt,sd Sd, Lv, Fr

Morinda citrifolia L.

Ala

Rubiaceae

Alangium salviifolium (L. f.) Wangerin

Ankol

Alangiaceae

Ficus religiosa L.

Ashwatha

Moraceae

Mangifera indica L.

Amra

Anacardiaceae

Bk,S d

Mimusops elengi L.

Bakula

Sapotaceae

Tooth ache, chronic fever, headache

Kavalama urens (Roxb.) Raf. Syn. Sterculia urens Roxb. Tectona grandis L.f.

Kadayo

Sterculiaceae

Bk, Fr, Fl Gum

Shakha

Verbenaceae

*Citrus lemon Linn.

Nimbuka

Rutaceae

Bk,S d, Fl Fr

Balanites aegyptiaca (L) Delile

Ingudi

Simarubiaceae

Headache, bleeding, skin disease, indigestion Diarrhoea, dysentery, indigestion, worms Purgative, burns, skin disease

Emblica officinalis Gaertn.

Fr,L v Bk, Lv, Sd Fr,B k

Bk, Fr

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Bronchitis, asthma, burns, constipation, headache, stomach ache, dropsy, liver problems, diabetes, acidity, dysentery Diarrhoea, dysentery, bleeding, diabetes

Wounds, oedema Purgative

leucorrhoea,

Cough, asthma, skin disease, burning maturation Urticaria, ulcer, dyspepsia, expectorant, stomach ache, urinary complaints, jaundice cholera, cold, cough, chest infection, lung diseases Liver disease, urinary disease Skin disease, common cold, liver disease Skin ailment, bronchitis, abortifacient, asthma, carbuncle, cholera, sores in mounth, toothache, small pox, urinary complain whooping cough, tonic, gonorrhoea Haemorrhage, diarrhoea

Mild purgative, throat disease

cough,

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Zizyphus mauritiana Lam Badara .

Rhamnaceae

Fr,L v,Rt

Wrightia tinctoria R.Br.

Kutaja

Apocynaceae

Gardenia gummifera L.f.

Nadihingu

Rubiaceae

Gum

Ficus benghalensis L.

Vata

Moraceae

La, arial root

Putranjiva roxburghi Wall. Gmelina arborea Roxb.

Putranjivak

Euphorbiaceae

Gambhari

Verbenaceae

Elephantopus scaber L.

Bhopathari (Gojihwa)

Asteraceae

Sd, Lv Fr, Bk,R t Wp

Azanza lampas (Cav.) Alef.

Jangli bhindi

Malvaceae

Tiliaceae

Cuts, stomachache

Kakajangha

Acanthaceae

Rt, Fr, Lv Wp, Rt, Fr,L v

Shalaparni

Fabaceae

Dysentery, toothache, snake

Sharapunkha

Fabaceae

Liver disease

Chakramarda

Fabaceae

Wp, Rt Lv, Rt, Sd, St

Fabaceae

Triumfetta rhomboidea Ja Zizgirita cq. Syn.Triumfetta bartramii L.

Dicliptera paniculata (Fo rssk.) I.Darbysh. Syn. Peristrophe paniculata (F orssk.) Brummitt Desmodium gangeticum ( L.) DC. Tephrosia purpurea (L.) Pers. Senna tora (L.) Roxb. Syn. Cassia tora L.

Senna occidentalis (L.) Kasamarda Link Syn.Cassia occidentalis L

Lv, Sd, Fl

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Dysentery, headache, indigestion, rheumatism, cough, wounds, fever, eye diseases, diarrhoea cholera, colic, blood purification, spleen disease Skin disease Indigestion, fever, worms, skin disease Dysentery, sores, boils, diruretic, epilepsy, fever, head ache, hydrocycle, leucorrhoea Urinary disease, infertility, burning disease Indigestion, fever, piles, vertigo Intermittent fever, headache, heart diseases, liver disease, pimples, rheumatism, swelling, dysentery, cough, dropsy, syphilis Pneumonia dysentery,

Wounds, gout, rheumatism, bone fracture,

Night blindness, eczema, antiseptic, cuts, cold, stomachache, wounds, scabies, ring worm, jaundice, skin diseases, bone fracture Eczema, gastric trouble, lactation, ringworm, skin diseases, dysentery

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Cyanthillium cinereum (L Sahadevi .) H.Rob. Syn. Vernonia cinerea (L.) Less.

Asteraceae

Lv, Wp. Rt

Plumbago zeylanica L.

Chitraka

Plumbaginaceae

Solanum americanum Mi ll. Syn.

Kakamachi

Solanaceae

Lv,F r

Datura metel L.

Dhatura

Solanaceae

Barleria cristata L.

Sareyak

Acanthaceae

Sd,L v,Rt Wp, Rt,L v

Martynia annua L. Syn. Martynia diandra Gloxin Ocimum tenuiflorum L. Syn. Ocimum sanctum L.

Kakanasa

Pedaliaceae

Lv ,Fr

Tulsi

Lamiaceae

Lv, Wp

Achyranthes aspera L.

Apamarga

Amaranthaceae

Euphorbia hirta L.

Dugdhika

Euphorbiaceae

Euphorbia thymifolia L.

Laghu dugdhika Ajagandha

Euphorbiaceae

Cleomaceae

Lv, Wp Sd

Bhumiamalaki

Euphorbiaceae

Solanum nigrum L.

Cleome viscosa L Phyllanthus amarus Schu mach. & Thonn.

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Dysentery, impotance, constipation, leucorrhoea, malaria, piles, night blindness, skin disease wounds, spleen complaints, insect bite Skin diseases, wounds, eczema, diarrhoea, dyspepsia, fever, spleen complaints, wounds, indigestion Diarrhoea, fever, lever complains, dysentery, piles skin diseases, stomach ache throat trouble, cough, eye complains, goiter, ulcer in mouth, urinary complains Fever, skin disease, pain and inflammation, asthma Bronchitis, swelling on legs toothache, bodyache, anaemia, pneumonia, wounds, Skin disease

Antiseptic, cold, cough and fever, urinary troubles, vomiting, bronchitis, chicken-pox, ear complaints, malaria, colitis gastric complaints, live complaints, wounds, Malarial fever, dropsy, bleeding, cold, cough, colic dysentery, headache, Warts, lactification, bronchial infection and asthma Menorrhagia, diarrhoea, dysentery Rheumatic pain and wounds Dropsy, gonorrhoea, urinogenetic disorder, kidney stone, gallbladder stone, hepatitis, liver complaints

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Jatropha gossypiifolia L.

Nepalo

Euphorbiaceae

Purgative

Cyperus rotundus L.

Musta

Cyperaceae

Rt, Bb,

Boerhavia diffusa L.

Punarnava

Nyctaginaceae

Rt, Lv

Trianthema portulacastru Swet punarnava m L.

Aizoaceae

Astringent, diaphoretic, diuretic, jaundice, wounds,sores, urinary complaints, stomach disorders, dysentery, bowel complaints, heat stroke, snake bite Tonic, eye complaints, asthma bronchitis and liver trouble Asthma, liver disease, jaundice

Oxalis corniculata L.

Changeri

Oxalidaceae

Merremia emarginata (B urm. f.) Hallier f. Syn. Ipomoea reniformis (Rox b.) Choisy Acalypha indica L.

Musakarni

Convolvulaceae

Indigestion, rectum proleps, piles Skin disease, worms

Haritamanjari

Euphorbiaceae

Skin disease, asthma

Abutilon fruticosum Guil. & Perr. Helicteres isora L.

Atibala

Malvaceae

Joint pain, diabetes

Avartani

Sterculiaceae

Woodfordia fruticosa (L.) Dhataki Kurz

Lythraceae

Rt, Lv, Fr Lv, Bk, Fl

Catunaregam spinosa (T Madanphala hunb.) Tirveng. Syn. Randia dumetorum (Retz. ) Lam. Wrightia antidysenterica Kutaja (L.) R.Br. Syn. Holarrhena antidysenteri ca (L.) Wall.

Rubiaceae

Diarrhoea, spasmodic pain, intestinal worms, stomach ache Haemorrhoides, cough, dysentery, fever, injuries, night blindness, bleeding in pregnancy, loss of appetite in pregnancy, skin diseases, sores, spleen complaints, sprain, syphilis ulcer, stop bleeding Emetic, respiratory disease, oedema, wounds

Apocynaceae

Bk, Lv, Sd

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Dysentery, asthma, colic, diarrhoea

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Rauwolfia tetraphylla

Sarpagandha variety

Apocynaceae

Carissa carandas L.

Karmarda

Apocynaceae

Calotropis procera (Aito n) Dryand.

Arka

Asclepiadaceae

Calotropis gigantea (L.) Dryand.

Rajarka

Asclepiadaceae

Withania somnifera (L.) Dunal Adhatoda vasica Nees

Ashwagandha

Solanaceae

Rt,L v,Fr Rt, La, Lv Rt, La, Lv Rt

Vasa

Acanthaceae

Vitex negundo L.

Nirgundi

Verbenaceae

Clerodendrum phlomidis L.f. Ricinus communis L.

Arani

Verbenaceae

Eranda

Euphorbiaceae

Senna auriculata (L.) Roxb. Syn. Cassia auriculata L.

Avartaki

Fabaceae

Lv, Fr, Fl Lv, Rt, Fr, Fl

Bk, Lv Sd, Lv, Rt

Fl,B k,Sd

Epilepsy, intestinal disorder, nervous disorders, malaria, insomnia, blood pressure, anxiety, vomiting Dysuria, heart disease, malaria gingivitis Asthma, cold and cough, rheumatism, skin diseases, Dropsy, eczema, leprosy

Debility, leucoderma, oedema, oligospermia Purgative, emetic, ulcer in mouth, cough, cold Rheumatism, sprains, headache, bone fracture, blisters, boil, cold, bodyache, cough, diarrhoea, dysentery, gout, itching, paralysis, pneumonia, skin diseases, Joint pain, fever, skin disease, eye disease Skin diseases, sores, gum trouble, cholera, boils, constipation, hydrocle, joint pain, muscular pain, headache, burns Diabetes, diarrhoea, skin disease

* Cultivated; Note: La- Latex, Lv-Leaves, Bk-Bark, Rt-Root, St-Stem, Sd-Seeds, Fl-Flowers, Fr-Fruits H-Herb, S-Shrub, T-Tree, Tu â&#x20AC;&#x201C; Tuber, C-Climber, Wp-Whole Plan

CONCLUSION The survey indicated that, the study area has magnificent plant diversity with plenty of medicinal plants to treat a wide spectrum of human ailments. The investigation concluded

that there is an urgent need to assess the biodiversity of the local forests, and conserve the biodiversity as well as the traditional knowledge by proper documentation and conservation strategies.

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REFERENCES Chunekar K.C., (1999), G.S. Pandey (eds) Bhavprakash nighantu, Chaukhambha orientalia publication. Database on Medicinal Plants Used in Ayurveda, (2001), Volume 1 to 5, Published by The central council of Research in Ayurveda & Siddha, New Delhi. Ghimire, S., McKey, D. and AumeeruddyThomas, Y. (2004), Heterogeneity in ethnoecological knowledge and management of medicinal plants in the Himalayas of Nepal: implications for conservation. Ecology and Society 9(3):6. Gyanendra Pandey (2005), Dravyaguna Vijnana, 3rd ed Varanasi: Krishnadas Academy. J.L.N. Shastry (2005), Dravyaguna Vignana, Vol.II, 2nd ed.Varanasi: Chaukambha Orientalia;

Source of Support: NIL

Khwaja Salahuddin, Gor Suresh, Visavadia Manish, Soni Virendra and Tatmia Nalin. (2013), Ethnobotanical survey of some parasitic plants growing in girnar forest of Junagadh District of Gujarat, India. Int. Res. J. Biological Sci., Vol.2 (4), 59â&#x20AC;&#x201C;62. Planning Commission. (2000), Conservation and Sustainable Use of Medicinal Plants. Task Force Report, Planning Commission, New Delhi. Posey, D.A. (1999), Cultural and Spiritual Values of Biodiversity A Complementary Contribution to the Global Biodiversity Assessment, UNEP. Schippmann, U., Leaman, D.J. and Cunningham, A.B. (2002) Impact of cultivation and gathering of medicinal plants on biodiversity: global trends and issues. In: Biodiversity and the Ecosystem Approach in Agriculture, Forestry and Fisheries. Food and Agriculture Organization (FAO), Rome.

Conflict of Interest: None Declared

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Research article A CLINICAL STUDY ON MANSYADI VATI IN THE MANAGEMENT OF RAJONIVRITTI JANYA LAKSHANA (MENOPAUSAL SYNDROME) Dasondi Ami M1, Donga Shilpa B2*, Rupapara Amit3, Mistry I U4 1

Medical Officer, (Ayu), Aolpad, Dist. Surat, Gujarat, India Asst Prof., Department of Streeroga & Prasutitantra, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. 3 Ph.D. Scholar, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. 4 Ex. Professor and Head of Department, Department of Kaumarbhritya, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. *Corresponding Author: E-mail: drshilpadonga@yahoo.com; Mob: +919825646796 2

Received: 10/09/2013; Revised: 10/11/2013; Accepted: 20/11/2013

ABSTRACT Rajonivritti (Menopause) is the aging manifestation in female affects 1/3 population, among them 50–60% seek medical help for their physical and related psychological problems. This condition produced by degenerative changes in the twitting period between the active and inactive ovarian function. These types of degenerative processes are being well managed by Medhya (brain tonic) drugs and Rasayana Chikitsa (rejuvenation therapy). For the present study Mansyadi Vati (Jatamansi 1part, Ashwagandha ¼ part, Parasikayavani 1/8 part) 3 tablets (500 mg each) thrice a day by orally were given for duration of one month with Luke warm water. In this interventional open clinical trial, 15 patients who fulfilled the diagnostic criteria of Rajonivritti Janya Lakshana had been selected for the study. The signs and symptoms were assessed by the specially designed assessment criteria before and after treatment. Significant outcome was observed in both somatic as well as psychological symptoms. KEY WORDS: Menopausal syndrome, Rajonivritti, Medhya Drug, Rasayana Chikitsa, Mansyadi Vati

Cite this article: Modi Ami P, Donga Shilpa B, Rupapara Amit, Mistry I U (2013), A CLINICAL STUDY ON MANSYADI VATI IN THE MANAGEMENT OF RAJONIVRITTI JANYA LAKSHANA (MENOPAUSAL SYNDROME), Global J Res. Med. Plants & Indigen. Med., Volume 2(12): 842–848

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 842–848

INTRODUCTION Menopause is generally defined as cessation of periods for 12 months or a period equivalent to three previous cycles or as time of cessation of ovarian function resulting in permanent amenorrhea. (Howkins & Bourne, 2005). This period is usually associated with inevitable manifestation of aging process in women (Mashiloane CD, 2001). This failure often begins in the late 30s and most of the women experience near complete loss of production of estrogen by their 50 years (AACE Menopause Guidelines, 2006). During reproductive years, women are protected from degenerative process by female hormones i.e. estrogen and progesterone. In menopause, women enter an estrogen deficient phase which accelerates the ageing process resulting in to inevitable scars of menopause. With increasing life expectancy, women spends one third of her lifespan after menopause. Hot flushes, sweating, changes in mood and libido are some important symptoms affecting the quality of life (QoL). Quality of life covers physical, functional, emotional, social and cognitive variables up to 85% of menopausal women (Blumel JE, 2000). According to Ayurveda, Menopause relates with ‘Jara Pakva Avastha’ (Acharya J.T., 1980) of body. Jara (aging) and Rajonivritti (menopause) are manifested due to progressive reduction in the functional ability of Agni (metabolism), which results into an inadequate tissue nutrition. According to Sushruta (Ambika Dutta Shastri, 2006) and various other references (Kavi Atridev Gupta, 2007) 50 years is mentioned as the age of Rajonivritti. In conservative management, Hormone Replacement Therapy (HRT) is only alternative for this health hazard by which one can get amazing achievement in combating the disease, but it has a wider range of secondary health complications like vaginal bleeding, breast cancer, endometrial cancer, gallbladder diseases etc. (Anklesaria BS, 2006). On the other hand, this therapy is not much effective in the psychological manifestations.

Rajonivritti Janya Lakshana is a group of symptoms produced by degenerative changes. So, it can be well managed with certain Ayurvedic therapeutics having Medhya (brain tonic) and Rasayana (rejuvenation) properties. Rasayana Chikitsa (rejuvenation therapy) is described by Acharyas to check the degenerative process of our body tissues can be very useful in the management of disorder like menopausal syndrome. Hence, in this study, Mansyadi Vati had been selected for the management of menopausal syndrome. MATERIAL AND METHODS 1. Patients attending the Out Patient Department of Stree Roga & Prasooti Tantra, Institute for Post Graduate Teaching and Research in Ayurveda (I.P.G.T. & R.A.), Jamnagar who fulfilled the diagnostic criteria of Rajonivritti Janya Lakshana were registered for present study. 2. The test drug “Mansyadi Vati” was prepared in pharmacy of Institute for Post Graduate Teaching and Research in Ayurveda (I.P.G.T. & R.A.), Jamnagar. Preparation of Drug Mansyadi Vati (Anubhuta): The ingredients of Mansyadi Vati i.e.1part Jatamansi root (Nordostachys jatamansi DC), 1/4part Ashwagandha root (Withania somnifera Linn.), and 1/8 part Parasikayavani seeds (Hyoscyamus niger Linn.) was procured and formulated as Vati in pharmacy of Gujarat Ayurved University, Jamnagar. CRITERIA PATIENTS

FOR

SELECTION

Criteria for selection of patients The patients who fulfilled the diagnostic criteria of Menopausal syndrome were selected for the present study. Inclusion criteria 1. Women aged between 35–55 years. 2. Amenorrhea for 12 months or more.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 842–848

3. The associated symptoms like Dhatukshyatmaka Lakshana (symptoms of diminution of body tissue elements) were also taken into consideration. Exclusion criteria 1. Patient with evidence of malignancy 2. Surgical menopause 3. Crippling conditions like hyper tension, diabetes mellitus, coronary artery disease, hepatic disorder, etc. Investigations 1. Blood: Hemoglobin (Hb), Total count (TC), Differentiate count (DC), Erythrocyte sedimentation rate (ESR), Packed cell volume (PCV), Random blood sugar (RBS). 2. Urine: Routine and microscopic examination. Ethical Approval The study was started after getting approval by the Institutional Ethics Committee, IPGT & RA, Jamnagar. Study Design Present study was designed as open clinical trial to evaluate the efficacy of trial drugs on Rajonivritti Janya Lakshana. Treatment Protocol After proper diagnosis, all patients were given Haritaki Churna 03 gm at night with warm water for 03 days for Koshtha Shuddhi (mild purgation)and then Mansyadi Vati was given in a dose of 3 tablets (each of 500 mg) thrice a day for one month with Luke warm water. Follow up The patients were observed for two weeks after completion of treatment. Assessment Criteria Based on improvement in signs and symptoms reported by the patient, relief in

physical and mental health was assessed on the basis of score developed for grading these clinical features followed by statistical analysis. Overall effect of therapy  Completely cured (75–100% relief in sign and symptoms).  Markedly improved (51–75% relief in sign and symptoms).  Improved (26–50% relief in sign and symptoms).  No improvement (0–25% relief in sign and symptoms). Statistical test: Based on observations, the data obtained were statistically analyzed in terms of mean, standard deviation, standard error and unpaired „t‟ test was considered at the level of p<0.001 as highly significant, p<0.05 or p<0.01 as significant and p>0.05 as insignificant to assess the result. RESULTS The observation made on 15 patients of Menopausal syndrome showed the maximum number of patients were between the age group 35–40 i.e. 46.67% suggests that in this particular age group climacteric changes are gradually developing., 86.67% were Hindus, being dominancy of Hindu population in the area, 86.67% of patients belong to Urban locality, 86.67% house wives, 73.33% were from middle class family. Effect of therapy Effect on menopausal symptoms The productive result of Mansyadi Vati was observed in all the features of menopause syndrome. The result was observed statistically highly significant (P<0.001) in hot flushes (78.40%), headache (73.24%), sleep disturbance (79.09%), excessive sweating (69.01%), depression (57.98%), palpitation (70.33%), pain in joints (67.83%), backache (67.87%), loss of appetite (82.24%) and irritability (68.86%) (Table No.-1).

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 842â&#x20AC;&#x201C;848

Table -01 Effect of Mansyadi Vati on cardinal symptoms Sr. Symptoms No

Mean Score BT

No of % Relief pts.

Hot flushes

2.13

0.46

78.40%

0.60 0.15 10.83 <0.001

Headache

1.57

0.42

73.24%

0.41 0.11 11.07 <0.001

Sleep disturbances

2.20

0.46

79.09%

0.57 0.15 11.70 <0.001

Excessive sweating

2.13

0.66

69.01%

0.50 0.13 11.39 <0.001

Depression

2.38

1.00

57.98%

0.63 0.18 07.99 <0.001

Palpitation

2.46

1.73

70.33%

0.57 0.15 11.71 <0.001

Pain in joints

2.58

0.83

67.83%

0.72 0.20 08.40 <0.001

Backache

2.21

0.71

67.87%

0.63 0.17 08.95 <0.001

Loss of appetite

2.14

0.38

82.24%

0.47 0.13 14.62 <0.001

Irritability

2.28

0.71

68.86%

0.50 0.13 11.88 <0.001

Effect on Dhatukshyatmaka Lakshana

Total effect of therapy

The productive relief was observed in Dhatukshyatmaka Lakshana. The result was observed in Rasa (nutrient value of plasma) (86.96%), Rakta (Blood) (73.21%), Mansa (Muscles/ tissues) (69.29%), Meda (Fat or Adipose tissues) (70.00%), Asthi (Bone) (61.19%), Majja (Bone marrow) (66.66%) and Artava (Menstrual blood/ Ovum) (09.52%) Dhatukshyatmaka Lakshana (Table No.-2).

The total effect of therapy had been carried out, which had shown that marked improvement was found in 67.67% of the patients, improvement and mild improvement was found in 26.67% and 06.67% of the patients respectively, while none of the patients remained unchanged (Figure- 1).

Table -02 Effect of Mansyadi Vati on Dhatukshyatmaka Lakshana Mean Score Sr. No.

Dhatukshaya

1 2 3 4 5 6 7

Rasa Rakta Mansa Meda Asthi Majja Artava

BT 92 56 62 30 67 18 42

AT 12 15 24 09 26 06 38

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% 86.96 73.21 69.29 70.00 61.19 66.66 09.52

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 842â&#x20AC;&#x201C;848

Figure-1 Total effect of Mansyadi Vati on menopausal Syndrome

66.67

60 50 %

40 30

26.67

20 6.67

0 Marked Improvement Mild No improvement improvement improvement

DISCUSSION According to Ayurveda, Menopause relates with â&#x20AC;&#x2DC;Jara Pakva Avasthaâ&#x20AC;&#x2122; (Acharya J.T., 1980) of body. Jara and Rajonivritti are manifested due to progressive reduction in the functional ability of Agni, which results into an inadequate tissue nutrition. Though Rajonivritti is physiological phenomenon but due to the rapid migration, stress, strain, hurry-worry, repeatedly leads to Dhatukshyavastha (symptoms of diminution of body tissue elements) which stimulates the aging process in early age. Due to this aging process and incapability to bear the condition it becomes a state of pathology. Probable mode of action of Mansyadi Vati The drug, Mansyadi Vati which possesses the Sangya Sthapana (resuscitative), Vedana Sthapana (sedatives), Balya (bulk promoting), Medhya (brain tonic) and Bhutaghna (Antimicrobial and insecticidal) properties,

affects the Manasika Bhava more than Sharirika Bhava. It is known that Sharirika Bhavas (physical elements) and Manasika Bhavas (psychological elements) are interrelated and affect on each other (Acharya J.T., 1994). Thus ultimately Manasika Bhavas also help in Prashastha Dhatu (high quality of body tissue elements) formation. Ashwagandha and Parasikayavani which are ingredients of Mansyadi Vati having Ushna Virya (hot potential) property, which may be stimulate the Sadhaka Pitta [a sub type of Pitta (one among three humors)] resulting in promotion of Medha (intelligence). It achieves this function by dispelling the Kapha and Tamasha (the inert-one type of psychological attributes or factors) from Manovaha Strotas (channels of mind). Thus, it helps in psychological factors. All the ingredients of this drug are Vatakapha Shamaka which also corrects the aggravated Vata Dosha. Thus all these properties help to break the pathogenesis of Rajonivritti (Chart1).

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 842â&#x20AC;&#x201C;848

Chart-1 Mode of action of Mansyadi Vati

Effect on the inflammatory response Prostaglandins cause inflammation and are implicated in the generation of Hot flush. Enzymes such as cyclooxygenase-2 (COX-2), synthesize prostaglandins from cellular lipids. Vanisree Mulabagal and co-workers at Michigan State University showed that Ashwagandha inhibited the enzyme activity of COX-2 in laboratory tests (Mansi B. Modi, 2013). Effect on the immune system IL-8 (Interleukin-8) is a potent vasodilator released by macrophages under stressful conditions. According to study conducted at the University of Tokushima, IL-8 was significantly increased in 179 women with hot flashes and it suggests that macrophages sense the decline in estrogen and respond by secreting IL-8. Working with cultured cells, researchers at the University of Buffalo found that

Ashwagandha decreased the genetic expression of IL-8 (Mansi B. Modi, 2013). CONCLUSION Rajonivritti (Menopause) is natural aging manifestation in women. Rasayana therapy (rejuvenation therapy) is a unique preventive Ayurvedic health care measure that deals with Jara (Aging process). Mansyadi Vati had significant effect in duration of one month. But better results could have been gained if treatment was continued for long period, as disease is Yapya in nature. There was no any drug reaction found. So, it is safe in menopause. ACKNOWLEDGEMENT The authors are thankful to the authorities of IPGT & RA and Gujarat Ayurved University, Jamnagar, Gujarat for providing a material.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 12 | December 2013 | 842–848

REFERENCES AACE Menopause Guidelines (2006), Endocr Pract.; 12 (No. 3) ENDOCRINE PRACTICE Vol 12 No.3 May/June; page No.317 Acharya J.T (1980), Charaka Samhita, 4th edition, Varanasi, Chaukhamba Sanskrit Sansthan publication, page No. 254. Acharya J.T (1980), Sushruta Samhita, 1st edition, Varanasi, Chaukhamba Orenatila publication, page No. 54 Ambika Dutta Shastri (2006), Sushruta Samhita, „Ayurveda-Tattva-Samdipika‟ Vyakhya, Reprinted 2nd edition, Varanasi, Chaukhamba Samskrit Samsthan, page No. 48 Ambika Dutta Shastri (2006), Sushruta Samhita, „Ayurveda-Tattva-Samdipika‟ Vyakhya, 2nd edition, Varanasi, Chaukhamba Samskrit Samsthan, Dalhana commentary. Anklesaria BS, Soneji RM.(2006), “Risk – Benefit Balance” in Management of Menopause in Menopause Current Concepts by C.N. Purandare, federation Source of Support: NIL

of Obstetric and Gynaecological Society of India. Reprint New Delhi: Jaypee, p. 194–205 Blumel JE, Castelo-Branco C, Binfa L (2000) Quality of life after the menopause: a population study. Maturitas; 34: 17–23. Howkins & Bourne (2005), Shaw's a Textbook of Gynecology. Menopause, Reprinted, edit., Published by ELSEVIER, PP.56–67 Kavi Atridev Gupta (2007), Asthanga Hridaya, Reprinted Varanasi, Chaukhamba Surbharti Prakashan; page No. 170 Mansi B. Modi (2013), Clinical evaluation of Ashokarist, Aswagangha Churna and Pravala Pisti in the management of menopause syndrome. (Article available on internet) Available fromwww.ayujournal.org Mashiloane CD, Bagratee J, Moodley J. (2001) Awareness of an attitude toward menopause and hormone replacement therapy in an African Community, International Journal of Gynecology and Obstetrics,;76:91–93.

Conflict of Interest: None Declared

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