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ISSN 2277 – 4289 www.gjrmi.com Editor-in-chief 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. 9, September 2013 MEDICINAL PLANTS RESEARCH Natural Resources CHARACTERISTICS OF ESSENTIAL OILS OF JUNIPERUS PHOENICEA FROM EASTERN ALGERIA Messaoud Ramdani, Takia Lograda, Azzedine Zeraib, Pierre Chalard, Gilles Figueredo, Meriem Bouchaala, Samra Zerrar 613–623

Agricultural Bio-Technology DIRECT SOMATIC EMBRYOGENESIS IN FENUGREEK (TRIGONELLA FOENUM-GRAECUM L.) Ashrafzadeh Seyedardalan, Khosrowshahli Mahmood, Bozorgipour Reza

624–629

Bio-Technology STUDY OF ANTIMICROBIAL, ANTI-OXIDANT AND ANTI-QUORUM SENSING PROPERTIES OF EUPHORBIA TRIGONA Upadhyay M, Nashikkar N, Begde D, Bundale S, Pise M, Rudra J , Upadhyay A

630–641

Micro-Biology FUNGAL PROTEIN PRODUCTION BY SMALL SCALE FERMENTATION TECHNOLOGY USING ASPERGILLUS NIGER Chouhan Pawan Kumar, Das Prakash

642–647

Review Article THERAPEUTIC PLANTS AND THEIR ANTIOBESITY PROPERTIES Mohan Reddy N, Kalyani P, Nagalaksmi K, Kaiser Jamil

648–655

INDIGENOUS MEDICINE Ayurveda – Review Article REVIEW ON THE CONTRIBUTION OF DASHAPUSHPA, A TRADITIONAL MEDICINE IN THE MANAGEMENT OF CANCER Arun Raj GR, Shailaja U, Rao Prasanna N, Sharanesh T, Gokul J

656–663

Ayurveda – Review Article CATEGORIZATION OF AYURVEDIC SPECIALITIES – PRESENT & FUTURE WITH SPECIAL REFERENCE TO KRIYA SHAREERA Kamath Nagaraj, Kulkarni Pratibha, Chiplunkar Shivprasad

664–668


Unani – Review Article A REVIEW ON POLYGONUM BISTORTA L. WITH REFERENCE TO ITS PHARMACOLOGY & PHYTOCHEMISTRY Adiba Mehar, Hussain I Mohammed Tabarak

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – JYOTISHMATI (CELASTRUS PANICULATUS WILLD.), OF THE FAMILY CELASTRACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

669–674


Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 613–623 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article CHARACTERISTICS OF ESSENTIAL OILS OF JUNIPERUS PHOENICEA FROM EASTERN ALGERIA Messaoud Ramdani1, Takia Lograda2*, Azzedine Zeraib3, Pierre Chalard4, Gilles Figueredo5, Meriem Bouchaala6, Samra Zerrar7 1,2,3,6,7

Laboratory of Natural Resource Valorisation, Sciences Faculty, Ferhat Abbas University, 19000 Setif, Algeria 4 Clermont Université, Université Blaise Pascal, BP 10448, F-63000 Clermont Ferrand 5 LEXVA Analytique, 460 rue du Montant, 63110 Beaumont, France *Corresponding author: Email: tlograda63@yahoo.fr; Phone: (213)36835894; Fax: (213)36937943.

Received: 23/07/2013; Revised: 17/08/2013; Accepted: 20/08/2013

ABSTRACT The analysis and identification of the components of the essential oil of five populations of Juniperus Phoenicea, from Eastern Algeria, was performed using the GC-MS. The average yield of essential oil of the samples is 0.82%, the highest rate is observed in the essential oil of T'kout and Elhadjaz populations (0.92%), while the population of Menâa is characterised by the lowest yield (0.70%). These analyses led to the identification of 72 components. The chemical composition of the essential oil of J. Phoenicea is dominated by the presence of a major product, α-pinene (36.3– 55.9%). Three components are represented with large concentrations in the essential oil; terpinolene (12.95%), Δ3-carene (0–12.38%) and the β-phellandrene (0–7.3%). Our investigation allows us to support the presence of several chemotypes in J. Phoenicea. The chemotype to Terpinolene is located in the region of Boussâada; T'kout is the region that has favoured the development of Δ3carene chemotype. The chemotype in β-phellandrene is located in the Boutaleb region. KEYWORDS: Juniperus phoenicea, Cupressaceae, Essential oil, Chemotype, Algeria

Cite this article: Messaoud. R., Takia. L., Azzedine. Z., Pierre C, Gilles. F., Meriem. B., Samra. Z. (2013), Characteristics of essential oils of Juniperus phoenicea from Eastern Algeria, Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 613–623

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 613–623

INTRODUCTION The genus Juniperus is an important element of arid and semi-arid ecosystems throughout the northern hemisphere (Farjon, 1992; Adams, 2008). Species of the genus Juniperus are considered medicinally important as they are widely used in traditional medicine. The seed decoction of Juniperus is used as folk medicine for kidney diseases, and as a diuretic (Karryev, 1967; Bellakhder, 1997). The isolation and anti-inflammatory activity of some diterpenoids of J. polycarpus (El-Sayed, 1998) and several studies about the essential oil of J. seravschanica have been published (Adams, 1999). Previously, from the genus Juniperus some terpenoids have been isolated (De Pascual Teresa et al., 1980; Fang et al., 1992, 1996; Barrero et al., 2000; Lee and Cheng, 2001; Nakanishi et al., 2005; Martin et al., 2006; Okasaka et al., 2006; Mansouri et al., 2010), neolignans (Nakanishi et al., 2004) and flavonoids (Yuldashev and Rasulova, 2001; Inatomi et al., 2005). Juniperus phoenicea is an evergreen tree indigenous to North Africa and belongs to the family Cupressaceae, with about 70 species distributed over the Northern Hemisphere. The leaves of J. phoenicea species are used in the form of decoction to treat diarrhoea, rheumatism (Bellakhder, 1997) and diabetes (Bellakhder, 1997; Allali et al., 2008). The mixture of leaves and berries of this plant is used as an oral hypoglycaemic agent (Amer et al., 1994), whereas the leaves are used against broncho-pulmonary disease and as diuretic (Bellakhder, 1997). There are many paper reports on chemical compositions of leaves, berries and essential oils of J. phoenicea grown in north Mediterranean basin (Adams et al., 1996; Rezzi et al., 2001; Ennajar et al., 2010). In Morocco (Barrero et al., 2004; Derwich et al., 2010,

2011; Mansouri et al., 2011a, b; Ait Ouazou et al., 2012); in Egypt (El-Sawi et al., 2006, 2007), in Tunisia (Akrout 1999; Bouzouita et al., 2008; Ennajar et al., 2007; Medini et al., 2007), in Algeria (Dob et al., 2008; Kilani et al., 2008; Bouzebata and Hadef, 2009; Mazari et al., 2010; Bekhechi et al., 2012), in the Canary Islands and Madeira (Adams et al., 2009), in Portugal (Cavaleiro et al., 2001), in North Africa (Barrero et al., 2006) (table 1). All oils of Juniperus phoenicea have a high content of α-pinene. The population of Mehdia is individualized by the presence of significant levels of β-pinene, ∆3-carene, limonene, terpinolene and the α-terpinyl acetate. The population of Spain is isolated by a high rate of manoyl oxide (22%), as well as Tarifa population in Spain with a rate of 6.6% of myrcene (Adams et al., 2009). The essential oil composition of J. phoenecea depends on organs, seasons and methods (Ennajar et al., 2007, 2010). In the present study, the aim was to identify the chemical composition of the oils of J. phoenicea and to compare our results with other reports of this species from different parts of the world, and re-evaluation of the geographical distribution of chemotypes. MATERIALS & METHODS Plant material Juniperus phoenicea is collected from five localities in eastern Algeria, Boutaleb (Setif), Boussâada (M’sila), Menâa and T’kout (Batna), and Elhadjab (Biskra) (Figure 1). Aerial parts were collected during the flowering stage in October 2012. The air dried materials were subjected to hydro-distillation for 3 h using a clevenger apparatus type (Lograda et al., 2013). Voucher specimens were deposited in the herbarium of the Department of Ecology and Biology, Setif University, Algeria.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 613–623

Table 1: Chemical composition of Juniperus phoenicea in Mediterranean Authors Adams et al., 2009 Mansouri Adams et al., 2009 et al., 2011 Country Por Sp Mor Tun Can Regions 1 2 Sp 3 4 5a 6a 7 8 5b 6b α-pinene β-pinene Myrcene ∆3-carene Limonene β-phellandrene terpinolene Linalool piperitone α-terpinyl acetate germacrene B germacrene-D4ol manoyl oxide

57.8 1.2 3.3 0 0.9 8.0 1.0 1.0 0 5.0 0 0.5

76.0 1.4 2.8 0 1.9 1.3 0.6 0 0 0 0 9.3

41.2 2.1 3.2 1.5 0.6 4.9 0.7 1.0 0.2 0.1 0.6 0.2

25.8 1.3 6.6 0 0 31.5 1.8 0.1 0.3 13.1 0.2 0.2

65.4 0.8 1.7 2.3 0.9 0.6 0 0.3 0 0 0 0.1

34.2 4.7 0.2 20.6 14.6 0 4.1 0 0.9 6.8 0 0

79.1 3.1 0 5.7 3.9 0 1.1 0 0 0.9 0 0

74.0 0.8 1.8 0 0.4 0.9 0 1.0 1.4 0.4 0 0

89.3 2.1 0 0.1 0.7 0 0 0 0 0.3 0 0

59.1 0.6 1.1 0 1.1 0 0 3.3 0 0 3.2 1.6

67.9 1.6 2.7 0.3 1.9 1.2 0.6 0.4 0 0.2 0 0.2

57.3 1.5 2.3 0 0.6 0.5 0.6 0 0 0.1 0 0.6

0

0

22.0

0.4

2.6

0

0

0

0

0

1.1

2.4

Por: Portugal; Sp: Spain; Mor: Morocco; Tun: Tunisia; Can: Canary; 1-Madeira; 2-Palma; 3-Tarifa; 4-Morocco; 5Mehdia; 6- Assif Almal; 7-Tenerif; 8-Gomera; a- leaves; b- branches ; Table data are used in the UPGMA analysis

Figure 1: Populations of Juniperus phoenicea studied

* Populations of Juniperus phoenicea studied

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 613–623

Extraction of the essential oil

RESULTS

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 sulfate and stored in screw capped glass vials in a refrigerator at 4–5°C prior to analysis. Yield based on dried weight of the samples was calculated.

The hydro-distillation of the essential oil of Juniperus phoenicea gave a viscous liquid with a yellowish color and strong odor of juniper. The average yield of essential oil of our samples is 0.82%, the highest rate is observed in the essential oil of the populations of T'kout and Elhadjaz (0.92%), while the population of Menâa is characterised by the lowest yield (0.70%).

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, 1976; NIST, 2002) and those described by Adams, 2001 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.

The analysis and identification of the components of the essential oil of J. Phoenicea was 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 73 components. The chemical composition of the essential oil of J. Phoenicea is dominated by the presence of a major product, α-pinene with a average (48.08%), the highest content was observed at the Elhadjaz population 55.9% and the lowest was recorded at station of T'kout with 36.5%. Three components are represented with large concentrations in the essential oil. The oil of Boussaâda is characterised by terpinolene (13%). The Δ3-carene caracterise the populations of T’kout, Menâa and Elhadjaz, with a rate of 12.4%, 5.4% and 3%, respectively. The populations of Boutaleb and T’kout contains a percentage of (7.3–4.4%) of β-phellandrene. The chemical composition of this species contains other components of a lower rate, linalool tetrahydro- in Menâa, Elhadjaz and T’kout. Our populations contain a low rate, but more than 1%, of βcaryophyllene, germacrene-D and germacreneB. The classification of our populations, according to their chemical kinship relations, is based on the construction of clades. The UPGMA based on the Unweighted pair-group average distance and the City-block (Manhattan) (Figure 2), has divided our populations into four clades.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 613–623

0.75 47 85 0.2 47.1 1.5 −

Yield (v/w) Number of Compounds Total (%) Pseudopinene α-Terpinyl acetate GermacreneD Citronellyle propanoate

92 0 93 5 98 8 10 06

Boussâda

0.8 47 85 0.3 53.7 2.4 −

Boutaleb

0.92 44 86 0.2 36.5 1.9 12.4

T’kout

0.92 44 91.4 0.2 56 1.8 3

Elhadjaz

Boussâda

0.7 53 88.5 0.3 47.2 1.8 5.4

KI

Menâa

Boutaleb

Populations

T’kout

Yield (v/w) Number of Compounds Total (%) Tricyclene α-Pinene Myrcene ∆-3-Carene

Elhadjaz

Populations

Menâa

Table 2: Chemical composition of Juniperus phoenicea populations

0.7 53 88.5 0.8 − 1.5 −

0.92 44 91.4 0.8 − 1.6 −

0.9 2 44 86 3.3 − 1.5 −

0.8 47 85 − 0.7 1.4 0.3

0.7 5 47 85 − 0.4 1.5 0.1

14 51 14 58 14 94

0.6 1.1 −

0.6 0.9 −

0.6 0.7 −

− 0.6 0.5

− 0.8 0.5

KI

13 47 13 49 14 38 14 46

Isosylvestrene Para cymene Limonene

10 09 10 22 10 27

− 0.4 1.3

− 0.3 0.7

− 0.4 −

1.6 0.8 0.6

0.4 0.5 0.8

α-cubebene α-humulene

β-Phellandrene

10 28

0.8

4.4

7.3

1.7

Epi-bicyclosesquiphellandre ne

14 95

1.6

1.1

1.3

γ-Terpinene Linalool oxide (trans) Terpinolene Cymenene Linalool Linalool tetrahydro− α-campholene aldehyde Trans-Pinocarveol Trans-Verbenol α-Phellandren8-ol

10 56 10 69 10 85 10 88 10 97 10 99 11 25 11 40 11 45 11 50

0.3 − − 0.1 − 3.6 0.2 0.2 0.2 −

0.3 − − − − 3.2 0.3 0.4 0.9 −

0.1 0.1 − 0.2 − 1.8 0.3 0.5 0.8 0.1

0.3 0.3 0.1 0.5 1.7 0.5 0.2 − − 0.3

0.2 0.2 13 0.2 0.8 0.1 0.3 − − 0.1

Cadina-1,4-Diene

Valencene Elemol Muurol-5ene-4-α-ol cis

14 96 14 97 14 99 15 11 15 14 15 20 15 23 15 39 15 50 15 61

− 1.2 0.7 0.3 0.2 1.8 1.8 0.3 0.7 −

− 1.1 0.4 0.2 3.2 − 1.4 0.2 0.6 −

− 0.7 0.4 0.3 0.1 3 1 0.1 0.8 −

0.1 − 0.3 0.1 − 0.2 − − 0.4 1.5

0.2 − − 0.2 − 0.2 − − 0.8 2.3

p-mentha-1,5dien-8-ol

11 72

0.1

0.2

0.4

Germacrene B

15 62

1.7

1.9

1.7

0.9

1.2

Pinocamphone cis Terpinene-4-ol

11 75 11 80

− 0.1

− 0.2

− 0.3

0.2 0.2

0.3 0.1

α-amorphene Citronellyl propionate

15 66 15 72

0.5 0.2

0.3 0.5

0.2 0.2

− −

− −

α-terpineol Safranal Nopol β-Fenchyl acetate

11 95 11 97 12 03 12 17

0.5 − 0.1 0.1

0.7 − − −

1 − − −

− 0.1 0.3 0.7

− 0.9 0.2 0.4

Germacrene D-4ol Caryophyllen e oxyde Ethyl laurate Humulene1,2-epoxyde

15 79 15 85 15 93 16 13

0.2 0.6 0.5 −

0.2 0.5 0.5 −

0.3 0.6 0.9 −

0.2 0.4 − 0.2

0.2 0.8 − 0.3

Citronellol

12 26 13 14 13 32 13 36 13 43

− 0.5

− 0.2

− 0.4

0.4 −

0.3 −

α-Cedrene α-gurjunene

2.8 0.2

− −

− 0.2

− −

− 0.2

0.2 0.3

− 0.3

0.3 0.3

− −

− −

(+)-β-guaiene

0.8 0.5

0.5 0.6

0.7 0.8

− 0.2

− 0.6

0.6

0.2

Manoyl oxide

16 31 16 35 16 58 16 98 19 97

0.2

2,4-Decadien-1-ol

γ-Terpinene ∆-Elemene Piperitone

Muurola 4,14,5-dienecis

Calarene (+) α-muurolene α-selinene α-amorphene ∆-cadinene Cis-calamenene

Hexenyl cyclopentanone

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0.1

1.6


Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 613–623

Figure 2: Cladogramme of Juniperus phoenicea populations

Figure 3: UPGMA cluster of Juniperus phoenicea populations

We noted the individualization of our populations studied. The population of T'kout is rich in Δ3-carene (12.4%); Boussâada is characterized by a high rate of terpinolene (13%), while the β-phellandrene with a rate (7.3%) characterizes the population of Boutaleb. Both populations, Menâa and Elhadjaz, are grouped by the presence of appreciable levels of Δ3-carene and linalool-

tetrahydro, thus we can conclude that the Juniperus phoenicea of Eastern Algerian includes several chemotypes. The terpinolene chemotype to be found in the Boussâada region, while T'kout is the region that has encouraged the development of chemotype to Δ3-carene. The β-phellandrene chemotype is localized in the Boutaleb region. The stations of Mena and Elhadjez are characterized by the

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 613–623

presence of low levels of Δ3-carene and linalool-tetrahydro-. DISCUSSION Our returns of the essential oil are low compared to those of the literature. This yield is 1.96% in Egypt (El-Sawi et al., 2007), in Morocco, the yield is 1.62% (Derwich et al., 2011), in Portugal, Spain and Greece the yield is low (Adams et al., 1996). Bouzouita et al., (2008) found a yield of 0.5% in Tunisia. This difference in essential oil content is related to several factors, such as the geographical area of collection, climate, stage of development and the season. The comparison of the chemical components of the essential oil of our samples with those of Juniperus phoenicea oils shows that α-pinene is the major product of the oil. The highest rate of α-pinene is found in the population of Spain (Palma) and Morocco (Assif Almal and Mahdia) (Adams et al., 2009; Mansouri et al., 2011), while the lowest rate is found in the population of Spain (Tarifa) (Adams et al., 2009). The β-phellandrene is the second product; the population of Tarifa in Spain is individualized by a rate of 31.5% (Adams et al., 2009). The Δ3-carene is substituted by low levels except for the population of Morocco (Mahdia) (20.64%) (Mansouri et al., 2011) and the Algerian population (T'kout) with a rate of 12%. the terpinolene, limonene oxide and manoyl characteriz, with a high rate, each one of populations, Boussaâda (Algeria), Mehdia (Morocco) and Spain. The comparison of our populations to those in the world, using the UPGMA, allowed us to divide the entire population into three distinct groups (Figure 3). The observation of several sets of populations means that we are dealing with in heterogeneous ensembles.

T'kout). These populations are biogeographically contiguous. The second group consists of populations from Spain, while in the third group; we found a mix of populations, which is divided into three subgroups. In the first subgroup, at its end, although separated from the populations, we find our study populations (Boutalab and Boussâada). This separation, based on the linkage distance, indicates the presence of important terpenoids variability, within our populations, which indicates the presence of an heterogeneity of terpenoids concentration, and the existence of several chemotypes. CONCLUSION Analysis of the chemical composition of the essential oil of Juniperus phoenicea has allowed identifying 73 compounds. The majority compounds are the α-pinene, Δ3carene, β-phellandrene, myrcene, linalooltetrahydroxy-, germacrene-D and βphellandrenedrene. Although the study of J. phoenicea 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 European and African species does not generally present significant quantitative variations. In this study we have identified three chemotypes in Juniperus phoenicea localised in eastern Algeria. The terpinolene chemotype to be found in the Boussâada, region; chemotype to Δ3-carene in T'kout region β-phellandrene chemotype is localized in the Boutaleb region. ACKNOWLEDGEMENTS The work was supported by Algerian MESRS and Chemical Laboratory of carbohydrates Heterocyclic of Clermont Ferrant, France

The first group is represented by the Algerian populations (Menâa, Elhadjaz and

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REFERENCES Adams RP (1999). Systematics of multi-seeded eastern hemisphere Juniperus based on leaf essential oils and RAPD DNA fingerprinting. Biochemical Systematics and Ecology. 27: 709–725. Adams RP (2001). Identification of essential oils components by Gas Chromatography-Mass spectroscopy., Allured Publishing Corporation Carol Stream, Illinois USA. Adams RP (2008). Junipers of the World: the genus Juniperus, 2nd Ed.. Vancouver, BC, Canada: Trafford Publishing. Adams RP, Barrero AF, Lara A (1996). Comparisons of the leaf essential oils of Juniperus phoenicea, J. phoenicea subsp. eu-mediterranea Lebr. & Thiv. and J. phoenicea var. turbinata (Guss.) Parl. The Journal of essential oil research. 8(4): 367–371. Adams RP, Rumeu B, Nogales M, Fontinha SS (2009). Geographic variation and systematics of Juniperus pheonicea L. from Madeira and the Canary Islands: Analyses of leaf volatile oils., Phytologia. 91(1): 40–53. Ait Ouazzou Abdenour, Loran Susana, Arakrak Abdelhay, Laglaoui Amin, Rota Carmen, Herrera Antonio, Pagan Rafael, Conchello Pilar (2012). Evaluation of the chemical composition and antimicrobial activity of Mentha pulegium, Juniperus phoenicea, and Cyperus longus essential oils from Morocco., Food research international. 45(1): 313–319. Akrout A (1999). Etude des huiles essentielles de quelques plantes pastorales de la région de Matmata (Tunisie). Institut des Régions Arides, 4119 Medenine, Tunisie

Allali H, Benmehdi H, Dib MA, Tabti B, Ghalem S, Benabadji N (2008). Phytotherapy of diabetes in West Algeria. Asian J. Chem. 20: 2701–2710 Amer MMA, Wasif MM, Abo-Aytta AM (1994). Chemical and biological evaluation of Juniperus phoenicea as a hypoglycaemic agent. J. Agric. Res. 21: 1077–1091. Barrero AF, Herrador MM, Arteaga P, Quilez Del Moral JF, Sanchez Fernandez E, Akssira M, Aitigri M, Mellouki F, Akkad S (2006). Chemical composition of the essential oil from the leaves of Juniperus phoenicea L. from North Africa., The Journal of essential oil research. 18(2): 168–169. Barrero AF, Quılez del Moral, J Lara, A (2000). Sesquiterpenes from Juniperus thurifera L. Stereochemistry in unusual cedrane and duprezianane series. Tetrahedron. 56: 3717–3723. Barrero Alejandro F, José F Quı́ lez del Moral , M Mar Herrador, Mohamed Akssira, Ahmed Bennamara, Said Akkad, Mohamed Aitigri (2004). Oxygenated diterpenes and other constituents from Moroccan Juniperus phoenicea and Juniperus thurifera var. Africana. Phytochemistry. 65(17): 2507–2515. Bekhechi Chahrazed, Atik Bekkara Fewzia, Consiglio Danaë, Bighelli Ange, Tomi Félix (2012). Chemical Variability of the Essential Oil of Juniperus phoenicea var. turbinata from Algeria, Chemistry & biodiversity. 9(12): 2742– 2753. Bellakhder J. (1997). La pharmacopée marocaine traditionnelle. Ed. Ibis Press, Paris, p 271–272.

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Bouzabata A, Hadef Y (2009). Variability of the Yield and the Chemical Composition of Essential Oils of Juniperus Phoenicea L. coming from two regions of Algeria. TJMPNP. 2: 1– 9. Bouzouita N, Kachouri F, Ben Halima M, Chaabouni MM (2008). Composition chimique et activité antioxydante, antimicrobienne et insecticide de l'huile essentielle de Juniperus phoenicea. Société Chimique de Tunisie. 10: 119– 125. Cavaleiro C, Rezzi S, Salgueiro L, Bighelli A, Casanova J, Proença da Cunha A (2001). Infraspecific chemical variability of the leaf essential oil of Juniperus phoenicea var. turbinata from Portugal. Biochemical Systematics and Ecology. 29(11): 1175–1183. Davies N. W., 1990; Gas chromotographic retention indices of monoterpes and sesquiterpenes on methylsilicone and carbowaxe 20 m phases. J. Chromatogr., 503: 1–24 De Pascual Teresa J, Barrero AF, Muriel L, San Feliciano A, Grande M (1980). New natural diterpene acid from Juniperus communis. Phytochemistry. 19: 1153– 1156. Derwich E, Benziane Z, Boukir A (2011). Chemical composition of leaf essential oil of Juniperus phoenicea and evaluation of its antibacterial activity. Int. J. Agric. Biol. 12: 199–204. Derwich E, Z Benziane, Taouil R, Senhadji O, Touzani MA (2010) Comparative Study of The Chimical Composition of The Leaves Volatil Oil of Juniperus phoenicea and Juniperus oxycedrus . Middl-East J. Res. 5(5): 416–424. Dob Tahar, Dahmane Dahmane, Chelghoum Chaabane (2008). Chemical Composition of the Essential Oil of Juniperus phoenicea L. from Algeria,

The Journal of essential oil research, 20(1): 15–20. El-Sawi SA, Motawae HM (2006). Chemical composition and cytotoxic activities of essential oils of leaves and berries of Juniperus phoenicea L grown in Egypt. Planta medica. 72: 990–990. El-Sawi SA, Motawae HM, Amal MA (2007). Chemical Composition, Cytotoxic Activity and Antimicrobial Activity of Essential oils of leaves and berries of Juniperus phoenicea. Grown in Egypt. African J. of Traditional, Complementary and Alternative Medicines. 4(4): 417–426. El-Sayed AM (1998). Diterpene constituents of Juniperus polycarpos and their antimicrobial and anti-inflammatory activities. Zagazig J. Pharm. Sci. 7: 80– 86. Ennajar Monia, Bouajila Jalloul, Lebrihi Ahmed (2010). The influence of organ, season and drying method on chemical composition and antioxidant and antimicrobial activities of Juniperus phoenicea L. essential oils. Journal of the science of Food and agriculture. 90(3): 462–470. Ennajar Monia, Romdhane Mehrez, Abderrabba Manef (2007). Influence de la période de récolte sur la teneur et la composition de l'huile essentielle du Genévrier de Phénicie (Juniperus phoenicea L.). Revue des régions arides (Tunis). 2: 647–651. Fang JM, Chen YC, Wang BW, Cheng YS (1996). Terpenes from heartwood of Juniperus chinensis. Phytochemistry. 41: 1361–1365. Fang JM, Lee CK, Cheng YS (1992). Lignans from leaves of Juniperus Chinensis. Phytochemistry. 31: 3659–3661.

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Farjon A (1992). The taxonomy of the multiseed junipers (Juniperus sect Sabina) in southwest Asia and east Africa. Edinb. J. Bot. 49: 251–283. Inatomi Y, Iida N, Murata H, Inada A, Murata J, Lang FA, Iinuma M, Tanaka T, Nakanishi T (2005). Pair of new atropisomeric cupressuflavone glucosides isolated from Juniperus communis var. depressa. Tetrahedron Lett. 46: 6533–6535. Karryev MO (1967). Comparative characteristics of the essebtial oils from Central Asian specier of Juniperus. Izv. Acad. Nauk Turkm. SSR Ser. Biol. Nauk. 1: 40–43. Kilani S, Abdelwahed A, Ben Ammar R (2008). Chemical Composition of the Essential Oil of Juniperus phoenicea L. from Algeria. Journal of essential oil. 20: 695–700. Lee CK, Cheng YS (2001). Diterpenoids from the leaves of Juniperus chinensis var. kaizuka. J. Nat. Prod. 64: 511–514. Lograda Takia, Messaoud Ramdani, Abderazak Kiram, Pierre Chalard, Gilles Figueredo (2013). VARIATION OF ESSENTIAL OILS COMPOSITION OF PITURANTHOS SCOPARIUS IN ALGERIA. Global J Res. Med. Plants & Indigen. Med. 2(1): 1–9. Mansouri N, B Satrani, M Ghanmi L. El Ghadraoui A. Aafi A. Farah (2010). Valorisation des huiles essentielles de Juniperus thurifera et de Juniperus oxycedrus du Maroc. Phytothérapie. 8: 166–170. Mansouri Nazik, Badr Satrani, Mohamed Ghanmi, Lahsen El Ghadraoui, Abderrahman Aafi (2011). Étude chimique et biologique des huiles essentielles de Juniperus phoenicea ssp. Lycia et Juniperus phoenicea ssp.turbinata du Maroc. Biotechnol. Agron. Soc. Environ. 15(3): 415–424.

Mansouri Nazik, Satrani Badr, Ghanmi Mohamed, Lahsen EL-Ghadraoui, Boukir Abdellatif, Aafi Abderrahman (2011). Effet de la provenance sur le rendement, la composition chimique et l'activité antimicrobienne des huiles essentielles des rameaux de Juniperus phoenicea L. du Maroc. Acta botanica gallica. 158(2): 215–224. Martin

AM, Queiroz EF, Marston A, Hostettmann K (2006). Labdane diterpenes from Juniperus communis L. berries. Phytochem. Anal. 17: 32–35.

Masada Y (1976). Analysis of essential oils by Gas Chromatography and Mass Spectrometry. J. Wiley & Son’s, Inc. New York Mazari K, Bendinerad N, Benkhechi C, Fernandez X (2010). Chemical Composition and Antimicrobial Activity of Essential Oil Isolated from Algerian Juniperus phoenicea L and Cupressus sempervirens. Medicinal Plants Research. 4(10): 959–964. Medini H, Elaissi A, Chraif I, BannourF, Farhat F, Ben salah M, Khoudja ML, Chemli R (2007). Composition and variability of the essential oils of the leaves from Juniperus phoenicea L. from Tunisia. Revue des régions arides (Tunis). 1: 185–189. Nakanishi T, Iida N, Inatomi Y, Murata H, Inada A, Murata J, Lang FA, Iinuma M, Tanaka T, Sakagami Y (2005). A monoterpene glucoside and three megastigmane glycosides from Juniperus communis var. depressa. Chem. Pharm. Bull. 53: 783–787. Nakanishi T, Iida N, Inatomi Y, Murata H, Inada A, Murata J, Lang FA, Iinuma M, Tanaka T (2004). Neolignan and flavonoid glycosides in Juniperus communis var. depressa. Phytochemistry 65: 207–213.

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NIST (2002). Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library, vers. 2.0. fiveash data, USA. Okasaka Mamoru, Yoshihisa Takaishi, Yoshiki Kashiwada, Olimjon K. Kodzhimatov, Ozodbek Ashurmetov, Ai J Lin, L Mark Consentino, Kuo-Hsiung Lee (2006). Terpenoids from Juniperus polycarpus var. seravschanica. Phytochemistry. 67: 2635–2640.

Rezzi S, Cavaleiro C, Bighelli A, Salgueiro L, Cunha AP, Casanova J (2001). Intraspecific chemical variability of the leaf essential oil of Juniperus phoenicea subsp. turbinata from Corsica. Biochem. Systematics Ecol. 29: 179– 188. Yuldashev MP, Rasulova LKh (2001). Flavonoids of Juniperus seravschanica. Chem. Nat. Compd. 37: 226–227.

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

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 624–629 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article DIRECT SOMATIC EMBRYOGENESIS IN FENUGREEK (TRIGONELLA FOENUM-GRAECUM L.) Ashrafzadeh Seyedardalan1*, Khosrowshahli Mahmood2, Bozorgipour Reza3 1, 2

Department of Agricultural Biotechnology, Science and Research Branch, Islamic Azad University, Tehran, Iran 3 Seed and Plant Improvement Institute, Karaj, Iran *Corresponding author: Email: ardalan.ash@gmail.com; Phone: 0064-221917600; Present address: School of Biological Sciences, University of Canterbury, Christchurch, New Zealand

Received: 06/07/2013; Revised: 20/08/2013; Accepted: 28/08/2013

ABSTRACT The present study was conducted to develop a high efficient protocol for direct somatic embryogenesis, which does not require the intervening callus phase, in fenugreek. Fenugreek seeds were sterilized and placed on wet filter papers in plates for 7−10 days to sprout. The hypocotyls were excised from the germinated seedlings as the explants and cultured for embryogenesis initiation on Murashige and Skoog (MS) media supplemented with different combinations and concentrations of plant growth regulators. A MS medium containing 3 mg/l Picloram and 0.5 mg/l 6-benzyl amino purine (BAP) was chosen as the optimal medium for induction of globular embryos. In the next stage, globular embryos were cultured on the same medium for two weeks which is the time needed for embryo maturation to the heart-stage and finally the fully-matured cotyledonary embryos. KEYWORDS: Plant growth regulator, Embryo, Fenugreek, Somatic embryogenesis of fenugreek

Cite this article: Ashrafzadeh. S., Khosrowshahli. M., Bozorgipour. R., (2013), DIRECT SOMATIC EMBRYOGENESIS IN FENUGREEK (TRIGONELLA FOENUMGRAECUM L.), Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 624–629

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 624–629

INTRODUCTION Fenugreek (Trigonella foenum-graceum L.) is an annual medicinal plant belonging to the Leguminosae family. According to Acharya et al. (2008), it is known as one of the oldest medicinal plants. Fenugreek is used to treat digestive diseases, diabetes, hypercholesterolemia and metabolism disorder. In addition to these properties, antioxidant and antibacterial activities of fenugreek, make it a highly-important plant in pharmaceutical industry. Nowadays, it is also used as a rich source of natural dietary fiber in modern food industry. It can be consumed as a spice, vegetable and fodder as well (Aasim et al., 2009; Basche et al., 2003; Hegazy, 2011; Kumar and Maliakel, 2008). Somatic or asexual embryogenesis could be named as a phenomenon in plant reproduction. It causes the development of a large number of new plant cells from embryo-like somatic cells without any gamete fusion within a short period. Although some difficulties exist in identification of origin cells of somatic embryos, but many distinct advantages has made somatic embryogenesis an outstanding pathway. One of the most significant benefits of somatic embryos is their potential to be used as synthetic seeds. Moreover, it can cause clonal propagation, virus elimination, polyploid plant production and germplasm preservation (Quiroz-Figueroa et al., 2006; Kamle, 2011; Patil and Paikrao, 2012; Puhan and Rath, 2012).

has two advantages. Firstly, it is faster and secondly, chance of mutation occurrence is much less than indirect pathway since it does not require callogenesis stage (Gholami et al., 2013; Shaijee et al., 2006). In this report, direct somatic embryogenesis in fenugreek was studied for the first time due to lack of efficient and rapid protocols and also unique significance of direct pathway (Aasim et al., 2010, Kamle, 2011; Anita and Hariprasad, 2012). The main objective was investigation of different hormonal combination and concentration effects on the relative efficiency of embryo induction and maturation in Fenugreek. MATERIALS AND METHODS The seeds of cv. Hamedan of fenugreek were obtained from the Iranian Institute of Medicinal Plants. Uniform shape and size seeds were chosen and the surface was sterilized by soaking them in 70% (v/v) ethanol and then 2.5% sodium hypochlorite solution with 1 drop of Tween 20 solution for 30 seconds and 20 minutes respectively. Subsequently, seeds were rinsed three times with autoclaved distilled water and then allowed to germinate on wet filter papers in petri dishes (Figure 3: A).

Afshari et al. (2011) studied indirect somatic embryogenesis in fenugreek. To develop their protocol, they investigated the effects of two basal media with some hormonal combinations. Previously, shoot regeneration was investigated in fenugreek using combinations of cytokinins-auxin from different explants (Aasim et al., 2010).

Hypocotyl explants were excised from 7−10 days-old germinated seedlings and cultured under in vitro conditions on MS basal medium (Murashige and Skoog, 1962) supplemented with 2−5 mg/l Picloram or αnaphthalene acetic acid (NAA) and 0.25−1 mg/l 6-benzylaminopurine (BAP) or Kinetin (KIN). All the media containing 3% (w/v) sucrose and were solidified with 0.8% (w/v) agar. The pH was adjusted to 5.7−5.8 with 1 N NaOH or 1N HCL followed by media autoclaving at 121ºC, 14 psi for 15 minutes. All the cultures were subjected to a photoperiod of 16 h light at 25 ± 1ºC for four weeks.

The frequency of direct somatic embryogenesis within plants is usually very lower than indirect method which makes it a specific pathway. The main difference between indirect and direct pathways is origins of embryo cells which are calli and propagules repectively. Moreover, direct embryogenesis

Each treatment had 3 replicates with 8 explants. Significance was determined by analysis of variance (ANOVA) and the differences between the means were compared by Duncan's range test using SPSS for Windows computer program (Steinmacher et al., 2007).

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 624–629

Figure 3: Different stages of direct somatic embryogenesis in fenugreek:

A. Germinated plantlet derived from a fenugreek seed. B. snowy pre-embryo cells on a hypocotyl explant. C. Globular embryo. D. Development of a globular embryo to a heart-stage embryo. E. Heart-stage embryo F. Fully-matured cotyledonary embryo

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 624–629

As it is depicted in figure 3, maturation trend of the embryos was followed well. Figure 3.A-D demonstrates how an embryo distinguished from unipolar shape (globular) to bipolar shape (cotyledonary).

cells appeared on explants before induction of globular embryos, on the six media shown in Table 1 (Figure 3: B). The other 42 media induced calli which are not cells of interest in direct somatic embryogenesis or had no impact on explants therefore after few weeks; explants necrosed without any regeneration. Globular embryos were observed after the second week (Figure 3: C) but interestingly they did not developed from pre-embryo cells and as it is shown in Figure 1, they emerged on different area from pre-embryos on the explants. It seems more investigation needed about the embryo origin which if is not pre-embryos, impact of preembryo cells should be figured out as well.

RESULT AND DISCUSSION To initiate embryogenesis, the explants were cultured on MS media containing the 48 combination of either Picloram or Alphanaphthalene acetic acid with Kinetin or 6Benzylaminopurine in different concentrations (2, 3, 4 and 5 mg/l for Auxins and 0.25, 0.5 and 1 mg/l for Cytokinins). After the first week, epidermis of hypocotyls lysed and it followed by appearing of pre-embryo cells: the snowy

Table 1. Plant growth regulator combination and concentrations of media used for induction and development of somatic embryos Growth regulators (mg l-1)

S1

S2

S3

S4

S5

S6

PIC

3

3

4

2

3

3

1

0.5

1

KIN 0.5

BAP

1

0.5

* Media are named as S1-S6 ** PIC: Picloram; KIN: Kinetin; BAP: 6-Benzylaminopurine

Table 2. Effect of the different media on the different stages of somatic embryo development. Mean ± SE of three replicates with 8 explants per treatment Media

Mean number of globular embryos ± SE

Mean number of heart stage embryos ± SE

Mean number of cotyledonery stage embryos ± SE

S1

4.88 ± 0.22*

4.25 ± 0.25*

4.13 ± 0.29*

S2

2.13 ± 0.22

1.88 ± 0.22

1.63 ± 0.18

S3

1.25 ± 0.36

1.13 ± 0.29

0.88 ± 0.29

S4

1.13 ± 0.39

0.75 ± 0.31

0.63 ± 0.26

S5

1.25 ± 0.41

1.00 ± 0.32

0.88 ± 0.35

S6

1.38 ± 0.46

1.25 ± 0.41

1.56 ± 0.20

*It is significantly different according to Duncan’s range test at p < 0.01

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Figure 1. Pre-embryo and globular embryo cells on different parts of explants

Figure 2: The influence of media on embryogenesis stages 6 5 4 Globular stage

3

Heart-shape stage Cotyledonery stage

2 1 0 S1

S2

S3

S4

Subsequently, for embryo maturation, globular embryos were sub-cultured on the same media for two more weeks (Figure 3: D, E, F). As it is clear in Table 2, S1 was the most efficient medium for embryo initiation and maturation. Figure 2 illustrates the interaction effect of plant growth regulators on embryogenesis trend in fenugreek. Comparison of chart bars demonstrates that 3 mg/l Picloram is the optimized concentration as an Auxin source. Benzyl amino purin as a Cytokinin source also shows more efficiency in fenugreek embryogenesis rather than Kinetin.

S5

S6

CONCLUSION The present report has been introduced as the first protocol of direct somatic embryogenesis in fenugreek. Embryo proliferation by this two-step protocol is much more efficient and faster than multi-step indirect somatic embryogenesis protocols which could be affected by somaclonal variation as well. Matured somatic embryos can be applied in a wide spectrum of plant biotechnology such as regeneration, transformation and synthetic seeds.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 624–629

REFERENCES Aasim M, Hussain N, Umer EM, Zubair M and Bilal S (2010) In vitro shoot regeneration of fenugreek (Trigonella foenum-graecum L.) using different cytokinins. Afr. J. Biotechnol. 9: 7174– 7179. Aasim M, Khawar KM, Sancak C and Özcan S (2009) In vitro shoot regeneration of fenugreek (Trigonella Foenum graecum L.). Amer. Eurasian J. Sustainable Agri. 3: 135–138. Acharya SN, Thomas JE and Basu SK (2008) Fenugreek, an alternative crop for semiarid regions of North America. Crop Sci. 48: 841–853. Afshari E, Ranjbar GA, Kazemitabar SK, Riasat M and Kazemi poshtmasari H (2011) Callus induction, somatic embryogenesis and plant regeneration in fenugreek (Trigonella foenumgraecum L.). Iranian J. Med. and Aro. Plants. 27: 147−160. Basche E, Ulbricht C, Kuo G, Szapary P and Smith M (2003) Therapeutic applications of fenugreek. Clin. Thera. 8: 20–27. Dodeman VL, Ducreux G and Kreis M (1997) Zygotic embryogenesis versus somatic embryogenesis. J. Exp. Bot. 48: 1493– 1509. Gholami AA, Alavi SV, Majd A and Fallahian F (2013) Plant regeneration through direct and indirect somatic embryogenesis from immature seeds of citrus. Euro. J. Exp. Bio. 3: 307–310. Hegazy AI (2011) Influence of using fenugreek seed flour as antioxidant and antimicrobial Source of Support: Nil

agent in the manufacturing of beef burger with emphasis on frozen storage tability. World J. of Agric. Sci. 4: 391–399.

Kamle M, Bajpai A, Chandra R, Kalim SH and Kumar R (2011) Somatic embryogenesis for crop improvement. GERF Bulletin of Biosci. 2: 54–59. KUMAR IM K and MALIAKEL BP (2008) Fenugreek dietary fiber a novel class of functional food ingredient. Supplement to Agrofood Indus. hi-tech. 19: 18–21.

Murashige T and Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Plant Physiol. 79: 988−991. Patil AS and Paikrao HM (2012) A Novel regeneration system for a wild passion fruit species (Passiflora Foetida L.) based on direct somatic embryogenesis from leaf explants. Global J. Res. Med. Plants & Indigen. Med. 1(10): 458–495. Puhan P and Rath SP (2012) Induction, development and dermination of somatic embryos from in vitro grown seedling explants in Desmodium gangeticum L.: a medicinal plant. Research J. of Med. Plant. 6: 346–369. Quiroz-Figueroa FR, Rojas-Herrera R, GalazAvalos RM and Loyola-Vargas VM (2006) Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell Tiss. Org. 86: 285–301. Shaijee K, Tehranifar A, Naderi R and Khalighi A (2006) Somaclonal variation induiced De Novo leaf chimeric mutants during in vitro propagation of african violet (Saintpaulia Ionantha Wendl). Acta Hort. 725:337–340. Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 630â&#x20AC;&#x201C;641 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article STUDY OF ANTIMICROBIAL, ANTIOXIDANT AND ANTIQUORUM SENSING PROPERTIES OF EUPHORBIA TRIGONA Upadhyay M1, Nashikkar N2, Begde D3, Bundale S4, Pise M5, Rudra J6 Upadhyay A7* 1,2,4,5,6,7

Hislop School of Biotechnology, Hislop College, Temple Road, Civil Lines, Nagpur, Maharashtra, India-440 001. 3 Department of Biochemistry, Dr. Ambedkar College, Deekshabhoomi, Nagpur, Maharashtra, India-440 001. *Corresponding Author: E-mail: avinash.upadhyay6@gmail.com; Phone: +91-9822573332, +91-712-2532004, +91-712-2520158, +91-712-2558460; Fax: +91-712-2527760

Received: 24/07/2013; Revised: 30/08/2013; Accepted: 04/09/2013

ABSTRACT Euphorbia trigona is well known in India for its medicinal properties. In this study, organic solvent extracts of its aerial parts were evaluated for their antimicrobial, antioxidant and antiquorum sensing properties in order to validate their use for the treatment of several diseases in Ayurveda. The antibacterial and antifungal properties of the extracts were determined by well diffusion method using Mueller-Hinton and potato dextrose agar respectively. Although conventional antibacterial activity was not observed in any of the extracts, the chloroform extract was found to possess antifungal activity. Urease and hemolysin production by Proteus mirabilis in the presence of the E. trigona whole plant extracts was assayed spectrophotometrically by estimating the urea and hemoglobin respectively. The production of these quorum sensing controlled virulence factors by P. mirabilis was observed to be adversely affected in the presence of the extracts. The effect of E. trigona whole plant extracts on biofilm formation by P. mirabilis was assayed using the microtiter plate method. Growth of P. mirabilis in the presence of the extracts was monitored turbidometrically for 24 h. Biofilm formation and growth was not affected by the extracts. The antioxidant properties of E. trigona whole plant extracts were evaluated by the 2, 2 Diphenyl-1-picryl hydrazil radical scavenging assay and Fe3+ reducing power assay. The extracts were found to possess significant antioxidant activity. The medicinal efficacy of E. trigona may be attributed to its anti-QS and antioxidant properties. The emergence of recalcitrant infections and chronic ailments has renewed interest in the traditional plant based medicinal systems. Hence E. trigona, possessing a diverse range of bioactivities, can be exploited to derive pharmaceuticals with a novel, multidimensional mode of action. KEY WORDS: antioxidant, urease, hemolysin, antifungal, Euphorbia trigona

Cite this article: Upadhyay. M., Nashikkar. N., Begde. D., Bundale. S., Pise. M., Rudra. J., Upadhyay. A., (2013), STUDY OF ANTIMICROBIAL, ANTIOXIDANT AND ANTIQUORUM SENSING PROPERTIES OF EUPHORBIA TRIGONA, Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 630â&#x20AC;&#x201C;641

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INTRODUCTION Medicinal plants have been an essential part of the traditional health care systems the world over. However, with the increased ease of production and usage of synthetic and semisynthetic drugs, knowledge about these plants has become fragmented and is slowly vanishing (Zahin et al., 2010). Also, the misuse and/ or overuse of synthetic drugs often results in undesirable effects such as the development of multiple drug resistant pathogens or hypersensitivity reactions. Hence, with an increasing difficulty in treating such infections and physiological diseases, there has been a growing interest in alternative therapies and the development of novel pharmaceuticals derived from plants. The specific attenuation of bacterial virulence, which can be attained by targeting key regulatory systems that mediate the expression of virulence factors (Yeo and Tham, 2012) instead of killing or inhibiting growth of pathogenic bacteria, is being recognized as a viable therapeutic strategy. One of the target regulatory systems is quorum sensing (QS), or bacterial cell-to-cell communication. QS is a mechanism of gene regulation in which bacteria coordinate the expression of certain genes including those responsible for virulence, in response to the presence or absence of small signal molecules, mainly acyl homoserine lactones (Lazdunski et al., 2004). Several patho-physiological processes such as inflammation, diabetes, genotoxicity, and cancer (Gülçin et al., 2010) are the result of excessive generation of reactive oxygen species, induced by various stimuli and which exceed the antioxidant capacity of the body. The search for free radical scavengers is thus an important component in drug discovery and extensive research has been carried out on the antioxidant properties of traditional medicinal plants. Several Euphorbia species (E. hirta, E. antiquorum, E. nerifolia, E. tirucalli, E. trigona) have been used in Ayurveda to treat infection and inflammation in general (Kumar

et al., 2010). Euphorbia spp. is also prescribed to treat jaundice, blood disorders, tumors, rheumatoid arthritis, gout, insect bites and hepato-splenomegaly. The use of this plant for the treatment of such a diverse range of diseases suggests that the latex and whole plant extracts contain antioxidant principles apart from anti-infective and immuno-modulatory properties. These plants are known to contain several bioactive compounds which possess medicinal properties (Madureira et al., 2003, Biswas and Mukherjee, 2003), but the mode of action is not yet clear. This work aims to study the antimicrobial, anti-QS and antioxidant properties of E. trigona whole plant extracts for potential drug development. Proteus mirabilis, selected in the present study of the anti-QS properties of E. trigona, is an opportunistic pathogen, responsible for causing urinary tract infection, especially among hospitalized and/or catheterized patients and menopausal women. Its pathogenicity is known to be QS controlled (Wang et al., 2006) and swarming along with the associated expression of virulence factors enables it to successfully counter the host defense mechanisms and establish disease. MATERIALS AND METHODS Microorganisms, media and conditions: Microorganisms:

growth

Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Proteus mirabilis, Candida albicans, Aspergillus niger and Rhizopus were obtained from Institute of Microbial Technology (IMTech), Chandigarh, and all bacterial cultures were routinely maintained on nutrient agar slants while fungal cultures were maintained on potato dextrose agar at 4°C. An overnight liquid culture of the bacteria in Luria Bertani (LB) broth and a 48 h old culture of the fungi in potato dextrose broth were used in the experiments. Plant material and extract preparation: The aerial parts of Euphorbia trigona were washed, air dried in the shade and ground to a

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fine powder. 5 g of the powder was subjected to successive extraction in a Soxhlet apparatus using solvents (150 ml each) with increasing polarity, viz. petroleum ether, chloroform, ethyl acetate, and methanol. All the extracts were dried in a vacuum concentrator; the residues were resuspended in 20 % dimethyl sulfoxide (DMSO) and used for the assays. Controls were set up with each assay using an equal volume of 20 % DMSO. Antimicrobial activity: The antibacterial activity of the extracts was determined by well diffusion method using Mueller-Hinton agar. The antifungal properties of the extracts were determined by disc diffusion method using potato dextrose agar and compared to Amphotricin B, a standard antifungal agent. Anti-QS properties of E. trigona whole plant extracts:

Hemolysin assay: Hemolysin assay was performed as described by Felmlee et al., with some modifications (Felmlee and Welch, 1988). P. Mirabilis cells exposed to the E. trigona whole plant extracts (10 µg/ml) were washed with phosphate-buffered saline/10 mM CaCl2 (hemolysin buffer), suspended in the same buffer and the optical density was adjusted to 0.5 at 600 nm. Approximately 2 × 106 freshly isolated human RBCs were added to this cell suspension. After incubation for 60 min at 37°C the hemoglobin content of the supernatant was evaluated spectrophotometrically at 540 nm to measure the extent of hemolysin expression by P. mirabilis in the presence and absence of E. trigona whole plant extracts (10 µg/ml). Percentage hemolysis was calculated from the formula. OD test - OD blank × 100 OD total lysis Biofilm formation assay:

P. mirabilis, an opportunistic pathogen often implicated in urinary tract infection was selected to study the effect of E. trigona whole plant extracts on the production of some of its QS controlled virulence factors viz, production of urease and hemolysin and capacity to form biofilms.

The assay was performed using the microtiter plate method (Merritt et al., 2005) in the presence of E. trigona whole plant extracts (10 µg/ml). Wells containing an equal volume of 20 % DMSO served as solvent control.

Urease assay:

To study the effect of E. trigona whole plant extracts on growth of P.mirabilis, growth was monitored turbidometrically for 24 h in LB broth in the presence of the extracts (10 µg/ml). A 20 % DMSO solvent control was also set up and both were compared to a positive control.

Urease activity in the presence and absence of the E. trigona whole plant extracts was assayed by estimating the unhydrolyzed urea in urea-LB broth cultures (Lin et al., 2005). Briefly, urea-LB broth containing the extracts (10 µg/ml) was inoculated with 100 µl of an overnight culture of P. mirabilis. At the end of an incubation period of 48 h at 37°C, the amount of urea hydrolyzed was compared to a positive control set-up, lacking the extracts. An uninoculated urea-LB medium containing the extracts was used to adjust blank and as a negative control, to rule out non-enzymic degradation of urea.

Bacterial growth assay:

Antioxidant Assay: 2, 2 Diphenyl-1-picryl hydrazil (DPPH) radical scavenging activity: The antioxidant activities of the E. trigona whole plant extracts were measured in terms of hydrogen-donating or radical-scavenging ability, using the DPPH method (Molyneux, 2004, Szabo et al., 2007) with a minor

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modification. Briefly, 1.0 ml of DPPH solution (0.004 % in 95 % Methanol) was mixed with 1.0 ml of the extracts at various concentrations (0.2–1.0 mg/ml) to be tested. The reaction mixture was shaken well and incubated in the dark for 30 min at room temperature. The control was prepared as above using 20 % Scavenging effect (%)

=

DMSO. The absorbance of the solution was read at 520 nm against a blank i.e. 95 % Methanol. The radical scavenging activity was measured as a decrease in the absorbance of DPPH and was calculated using the following equation

(Absorbance of control − Absorbance of test sample) × 100 (Absorbance of control)

Reducing power assay: The reducing power of E. trigona extracts was determined according to the method of Oyaizu, 1986. Briefly, 1.0 ml of sample with different concentrations (0.1–0.8 mg/ml) was mixed with 1.0 ml of a 0.2 M phosphate buffer (pH 6.6) and 1.0 ml of potassium ferricyanide solution (10 g/l). The mixture was incubated in a water bath at 50°C for 20 min. The reaction was terminated by adding 1.0 ml of trichloroacetic acid solution (100 g/l) and the mixture was centrifuged at 3000 rpm for 10 min. Finally, 2.0 ml of the supernatant layer solution was mixed with 2.0 ml of distilled water and 0.5 ml of ferric chloride (1 g/l), and the absorbance of the reaction mixture was measured at 700 nm. Three replicates were run for each sample. Increased absorbance of the reaction mixture indicated increased reducing power of the sample.

gram negative bacteria such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at whole plant extract concentrations of 10–50 µg/ml. None of the E. trigona whole plant extracts was found to possess antibacterial activity whereas substantial anti-swarming activity was observed against P. mirabilis and P. aeruginosa at these concentrations as reported in our earlier work (Nashikkar et al., 2011). Upon testing the antifungal properties of E. trigona it was observed that the chloroform extract was effective against Candida albicans, Aspergillus niger and Rhizopus. The activity was comparable to 0.5–1.0 mg/ml Amphotericin B. Anti-QS properties: Biofilm formation and the expression of virulence factors (urease and hemolysin) which are controlled by QS were evaluated in the presence of E. trigona whole plant extracts.

Statistical analysis: All experiments were performed in triplicate. Results were expressed as mean + standard error. Statistical significance was analyzed using Students t test. A P value less than 0.05 was considered statistically significant. RESULTS Antimicrobial properties: The antibacterial properties of E. trigona were evaluated against gram positive as well as

Effect of E. trigona on Urease production by P. mirabilis: The activity of the extracellular urease produced by P. mirabilis in the presence and absence of E. trigona whole plant extracts was assayed by estimating urea hydrolysis in ureaLB broth cultures inoculated with P. mirabilis after an incubation period of 48 h. The amount of urea hydrolyzed is indicative of the urease activity present. As seen in Fig. 1, percent urea hydrolyzed by P. mirabilis cells exposed to petroleum ether (PE) and chloroform (CL) extracts was 4 and percent urea hydrolyzed by

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cells cultured in the presence of methanol (ML) extract was 11, whereas per cent urea hydrolyzed in the dimethyl sulfoxide (DMSO) control set-up was around 28. PE and CL extracts were thus found to be much more effective in inhibiting urease than the MT extract as the urease activity was found to be significantly (P < 0.01) reduced in the presence of the extracts at a concentration of 10 μg/ml. Effect of E. trigona on hemolysin production by P. mirabilis: In the present study, hemolysin activity was found to be present in both cell free supernatant

and in total cell cultures of P. mirabilis. It was seen however that the activity was highly unstable in case of cell-free supernatant and was not considered in this study. A reduction in hemolysin activity is reflected by a reduction in the extent of hemolysis, indicated by a decrease in the absorption values of the supernatant. As depicted in Fig. 2, hemolysin activity was reduced 5.62 times in the presence of the E. trigona CL extract. The other extracts also proved to inhibit the lysis of RBCs by hemolysin as evident from the optical density (OD) values.

Fig. 1: Effect of Euphorbia trigona on urease production by Proteus mirabilis.

A statistically significant (**P < 0.001) inhibition of urease activity observed in the presence of E. trigona extracts (10 μg/ml)

Effect of E. trigona on biofilm formation by P. mirabilis: The absorbance of the extracted crystal violet is proportional to the cell biomass in the biofilm. The average absorbance levels of the extracted crystal violet were found to be 0.1211 ± 0.0065, 0.1632 ± 0.0034, 0.1753 ± 0.0038, 0.1468 ± 0.0021 in the presence of methanol, chloroform, petroleum ether and ethyl acetate extracts respectively and 0.1127 ± 0.0087, 0.1627 ± 0.0015, 0.1781 ± 0.0028, 0.1431 ± 0.0011, in the

respective solvent control wells. The absorbance levels in wells containing E. trigona extracts are comparable to the solvent control wells containing the respective solvents. Thus the biofilm forming capability does not seem to be affected by the bioactive components in any of the extracts. However, in our earlier study, biofilm formation was found to be significantly reduced in the presence of the ethanolic latex extracts (Nashikkar et al., 2011) of E. trigona.

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Effect of E. trigona whole plant extracts on growth of P. mirabilis: The growth of P. mirabilis was monitored for 24 h in the presence of E. trigona whole plant extracts at a concentration of 10 μg/ml in liquid culture at 37°C and compared to a solvent control as well as a positive or normal control set up (Fig. 3). It was observed, that in the initial stages (upto 6 h), the growth rate in

the presence of all the extracts was at par with the positive control as well as the solvent control with a slight decrease in the growth rate in the mid-stages. However, the organisms grew to similar cell densities at 24 h .in the presence/absence of either solvent or extracts at concentrations at which virulence factors viz urease and hamolysin are significantly inhibited.

Fig. 2: Effect of Euphorbia trigona on hemolysin production by Proteus mirabilis.

A statistically significant (*P < 0.01) inhibition of hemolysin activity observed in the presence of E. trigona extracts (10 μg/ml).

Fig. 3: Effect of Euphorbia trigona on growth of Proteus mirabilis.

Growth of P. mirabilis is not affected by E. trigona extracts (10μg /ml). Each point represents a mean of three independent observations ± S.D.

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Fig. 4: 2, 2 Diphenyl-1-picryl hydrazil radical scavenging activity of Euphorbia trigona.

Dose dependent DPPH radical scavenging activity of Euphorbia trigona extracts (200–1000 μg/ml) compared to ascorbic acid. Each point represents a mean of three independent observations ± S.D.

Fig. 5: Reducing antioxidant power of Euphorbia trigona.

Dose dependent Fe3+ reducing antioxidant activity of E. trigona extracts (100–800 μg/ml) compared to ascorbic acid. Each point represents a mean of three independent observations ± S.D.

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Antioxidant properties of E. trigona whole plant extracts: The antioxidant properties of E. trigona whole plant extracts were evaluated by the DPPH radical scavenging assay and Fe3+ reducing power assay. DPPH radical scavenging activity: Fig. 4 shows the dose-response curve of DPPH radical scavenging activity of the E. trigona whole plant extracts, compared with ascorbic acid. The highest activity (99% scavenging) was observed in the MT extract even at lower concentrations (0.6–0.8 mg/ml) and is comparable to ascorbic acid, a wellknown antioxidant, while the other extracts also showed good scavenging effects. Reducing power activity: When the activity of all the E. trigona whole plant extracts was compared they appeared to reduce the ferric ions in the reaction mixture in a concentration dependent manner, as depicted in Fig. 5. The comparative reducing power of the extracts is as follows: EA extract (OD = 1.7) > MT extract (OD value = 1.6) > CL extract (OD = 1.1) > PE extract (OD = 0.2). The ethyl acetate extract seems to be the most potent, although less than ascorbic acid, a standard antioxidant used in this assay. DISCUSSION The use of E. trigona for the treatment of infections is well documented in the traditional medicinal system of Ayurveda. However, in the present study we observed no bactericidal or bacteriostatic properties in any of the extracts, although the chloroform extract does possess some antifungal activity. This led to an investigation of the anti-QS properties of the E. trigona whole plant extracts Virulence factor expression, biofilm formation and swarming motility are controlled by QS and coordinately regulated in several pathogens. P. mirabilis is known to produce

virulence factors such as proteases, hemolysins, urease etc, whose expression is up-regulated when the cells differentiate into hyperflagellated swarm cells (Verstraeten et al., 2008). Hence molecules that quench QS and disrupt the production of virulence factors by pathogens can be used as alternative therapy, especilly in case of multiple drug resistant strains. Effect of E. trigona on Urease production by P. mirabilis: Urease-mediated urea hydrolysis by P. mirabilis is responsible for its ability to cause urolithiasis and blockage of catheters. Ammonia, per se, is also known to be toxic to kidney epithelium cells (Mobley and Chippendale, 1990). A urease negative mutant, in fact, has been shown to cause UTI at a significantly lower rate (Jones et al., 1990). The activity of urease was found to be significantly inhibited in the presence of the extracts. Inhibition of urease activity in the presence of the bioactive compounds in the extracts probably helps to maintain the pH of the urine in vivo, precluding the formation of struvites and facilitating the clearance of infection by the host defense mechanism. Effect of E. trigona on hemolysin production by P. mirabilis: Hemolysins are exotoxins produced by several pathogenic bacteria which cause lysis of red blood cells in vitro. Their ability to target other cells, including white blood cells, often accounts for the effects of hemolysins in the host (Kaca and Rozalski, 1991). The phenomenon of hemolysis in P. mirabilis, P. vulgaris and P. penneri has been studied by several researchers (Fraser et al., 2002, Rozalski and Kotełko, 1987, Senior and Hughes, 1988) and was shown to be cellassociated and could be demonstrated in the actively growing cells. The activity of hemolysin produced by P. mirabilis was found to be reduced several fold in the presence of the extracts in vitro. The hemolysin is cytotoxic to human blood leukocytes and causes release of inflammatory mediators from granulocytes and

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mast cells. These events may result from insertion of the hemolysin into the membrane of the target cell. Thus, the inhibition of hemolysin may reduce the virulence of this pathogen in the patient. Effect of E. trigona on biofilm formation by P. mirabilis: The ability to form biofilms is also under QS control. This enables extensive colonization of kidney and urinary tract endothelium apart from the catheters, resulting in the creation of persistant foci of infection not easily removed by the flushing action of urine (Ali, 2012). Cells growing in biofilms are known to be highly resistant to conventional antibiotics as well as to the host defense mechanisms. Although the E. trigona whole plant extracts were not found to be inhibitory to biofilm formation by P. mirabilis, the latex extracts were observed to be highly effective in controlling biofilm formation as reported in our earlier work. Effect of E. trigona on growth of P. mirabilis: The growth and metabolism of P. mirabilis does not seem to be adversely affected by any of the extracts. Hence, the inhibitory effect on all the QS-related attributes in this study is certainly not due to its toxic or growth inhibitory action. Also, it is well known that the presence of compounds that challenge growth facilitates the selection of resistant strains. Since the bioactive compounds of E. trigona do not inhibit growth, per se, the likelihood of the emergence of resistant strains is minimal. Antioxidant properties of E. trigona whole plant extracts: Free radicals formed during normal cellular metabolism have been widely implicated with the etiology of several pathological processes, including cancer, diabetes, atherosclerosis, neurodegenerative disorders and arthritis. Antioxidants of plant origin (Durga et al., 2010) are believed to help protect the cells from free radical damage. These benefits are thought to result from the antioxidant

components of plants such as vitamins, polyphenols, flavonoids, and carotenoids (Urquiaga and Leighton, 2000). Hence, considerable attention has been focused on the natural antioxidants as potential disease preventing agents. The antioxidant properties of E. trigona whole plant extracts were evaluated by the DPPH radical scavenging assay and Fe3+ reducing power assay. DPPH assay has been extensively used for screening antioxidant activity because it is sensitive enough to detect bioactive components at low concentrations (SĂĄnchez-Moreno, 2002). The ferric reducing capacity of a compound serves as an indicator of its potential antioxidant activity. Natural antioxidants are considered to be multifunctional and their activity depends on various parameters such as the multiplicity and heterogeneity of the matrix, the experimental conditions and mainly the reaction mechanism (Dorman et al., 2003). It is now known that the antioxidant properties of plant extracts cannot be evaluated by one single method due to the complex nature of phytochemicals. Hence the antioxidant properties of the extracts were evaluated by two different tests and were compared with ascorbic acid, a standard antioxidant. The data generated in this study indicates that the E. trigona whole plant extracts possess considerable radical scavenging activity as well as Fe3+ reducing power, which may be partly responsible for its medicinal properties. Thus, the extracts could serve as free radical scavengers, acting possibly as primary antioxidants. A relatively high percentage of effective plant derived antioxidants have been reported to belong to the flavonoids, phenolics, quinines and saponins, all of which were found to be present in some or other E. trigona extracts as reported in our earlier work where we also observed that the E. trigona extracts possess considerable immunostimulant (Nashikkar et al., 2012) properties as well as the ability to elicit the formation of Neutrophil Extra cellular Traps or NETs.

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CONCLUSION Plant derived compounds are often large complex molecules, possessing several bioactivities and thereby exhibiting a multipronged mode of action on pathological conditions. Accordingly, E. trigona extracts too have been shown to possess multiple

bioactivities such as antifungal, antioxidant and anti-QS properties. It is, therefore likely that the well established efficacy of E. trigona for the treatment of bacterial infections including UTI and several other ailments may be attributed to the synergistic action of its biomolecules, validating its use in Ayurveda.

REFERENCES ALI, O. A. U. (2012). Prevention of Proteus mirabilis Biofilm by Surfactant Solution. Egyptian Academic Journal of Bilogical Sciences, 4, 1–8. BISWAS, T. K. & MUKHERJEE, B. (2003). Plant Medicines of Indian Origin for Wound Healing Activity: A Review. The International Journal of Lower Extremity Wounds, 2, 25–39. DORMAN, H. J. D., PELTOKETO, A., HILTUNEN, R. & TIKKANEN, M. J. (2003). Characterisation of the antioxidant properties of de-odourised aqueous extracts from selected Lamiaceae herbs. Food Chemistry, 83, 255–262. DURGA, K. R., KARTHIKUMAR, S. & JEGATHEESAN, K. (2010). Isolation of potential antibacterial and antioxidant compounds from Acalypha indica and Ocimum basilicum. African Journal of Plant Science, 4, 163–166. FELMLEE, T. & WELCH, R. A. (1988). Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion. Proceedings of the National Academy of Sciences USA, 85, 5269–5273.

FRASER, G. M., CLARET, L., FURNESS, R., GUPTA, S. & HUGHES, C. (2002). Swarming-coupled expression of the Proteus mirabilis hpmBA hemolysin operon. Microbiology, 148, 2191–2201. GÜLÇİN, İ., KİREÇCİ, E., AKKEMİK, E., TOPAL, F. & HİSAR, O. (2010). Antioxidant, antibacterial, and anticandidal activities of an aquatic plant: duckweed (Lemna minor L. Lemnaceae). Turkish Journal of Biology, 34, 175–188. JONES, B., LOCKATELL, C., JOHNSON, D., WARREN, J. & MOBLEY, H. (1990). Construction of a urease-negative mutant of Proteus mirabilis: analysis of virulence in a mouse model of ascending urinary tract infection. Infection and immunity, 58, 1120–1123. KACA, W. & ROZALSKI, A. (1991). Characterization of cell-bound and cellfree hemolytic activity of proteus strains. European Journal of Epidemiology, 7, 159–165. KUMAR, S., MALHOTRA, R. & KUMAR, D. (2010). Euphorbia hirta: Its chemistry, traditional and medicinal uses, and pharmacological activities. Pharmacognosy reviews, 4, 58.

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LAZDUNSKI, A. M., VENTRE, I. & STURGIS, J. N. (2004). Regulatory circuits and communication in Gramnegative bacteria. Nature Reviews Microbiology, 2, 581–592. LIN, Y., KWON, Y., LABBE, R. & SHETTY, K. (2005). Inhibition of Helicobacter pylori and associated urease by oregano and cranberry phytochemical synergies. Applied and environmental microbiology, 71, 8558–8564. MADUREIRA, A. M., ASCENSO, J. R., VALDEIRA, L., DUARTE, A., FRADE, J. P., FREITAS, G. & FERREIRA, M. J. U. (2003). Evaluation of the Antiviral and Antimicrobial Activities of Triterpenes Isolated from Euphorbia segetalis. Natural Product Research, 17, 375– 380. MERRITT, J. H., KADOURI, D. E. & O'TOOLE, G. A. (2005). Growing and Analyzing Static Biofilms. Current protocols in microbiology. John Wiley & Sons, Inc. MOBLEY, H. L. T. & CHIPPENDALE, G. R. (1990). Hemagglutinin, Urease, and Hemolysin Production by Proteus mirabilis from Clinical Sources. Journal of Infectious Diseases, 161, 525–530. MOLYNEUX, P. (2004). The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Journal of Science and Technology, 26, 212–219. NASHIKKAR, N., BEGDE, D., BUNDALE, S., MASHITHA, P., RUDRA, J. & UPADHYAY, A. (2012). Evaluation of immunomodulatory properties of Euphorbia trigona- An in vitro study. International Journal of Institutional Pharmacy and Life Sciences. , 2, 88– 105.

NASHIKKAR, N., BEGDE, D., BUNDALE, S., PISE, M., RUDRA, J. & UPADHYAY, A. (2011). Inhibition of swarming motility, biofilm formation and virulence factor expression of urinary pathogens by Euphorbia trigona latex extracts. International Journal of Pharmaceutical Science and Research., 2, 558–566. OYAIZU, M. (1986). Studies on products of the browning reaction. Antioxidative activities of browning reaction products prepared from glucosamine. Japanese Journal of Nutrition [Eiyogaku Zasshi], 44, 307–315. ROZALSKI, A. & KOTEŁKO, K. (1987). Hemolytic activity and invasiveness in strains of Proteus penneri. Journal of Clinical Microbiology, 25, 1094–1096. SÁNCHEZ-MORENO, C. (2002). Review: Methods Used to Evaluate the Free Radical Scavenging Activity in Foods and Biological Systems. Food Science and Technology International, 8, 121– 137. SENIOR, B. W. & HUGHES, C. (1988). Production and properties of hemolysins from clinical isolates of the Proteeae. Journal of Medical Microbiology, 25, 17–25. SZABO, M. R., IDIŢOIU, C., CHAMBRE, D. & LUPEA, A. X. (2007). Improved DPPH determination for antioxidant activity spectrophotometric assay. Chemical Papers, 61, 214–216. URQUIAGA, I. & LEIGHTON, F. (2000). Plant polyphenol antioxidants and oxidative stress. Biological Research, 33, 55–64.

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VERSTRAETEN, N., BRAEKEN, K., DEBKUMARI, B., FAUVART, M., FRANSAER, J., VERMANT, J. & MICHIELS, J. (2008). Living on a surface: swarming and biofilm formation. Trends in microbiology, 16, 496–506. WANG, W.-B., LAI, H.-C., HSUEH, P.-R., CHIOU, R. Y.-Y., LIN, S.-B. & LIAW, S.-J. (2006). Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol. Journal of Medical Microbiology, 55, 1313–1321.

Source of Support: Nil

YEO, S. S. M. & THAM, F. Y. (2012). Antiquorum sensing and antimicrobial activities of some traditional Chinese medicinal plants commonly used in South-East Asia. Malaysian Journal of Microbiology, 8, 11–20. ZAHIN, M., HASAN, S., AQIL, F., AHMAD KHAN, M. S., MABOOD HUSAIN, F. & AHMAD, I. (2010). Screening of certain medicinal plants from India for their anti-quorum sensing activity. Indian journal of experimental biology, 48, 1219.

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 642–647 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article FUNGAL PROTEIN PRODUCTION BY SMALL SCALE FERMENTATION TECHNOLOGY USING ASPERGILLUS NIGER Chouhan Pawan Kumar1*, Das Prakash2 1

Department of Microbiology, Rajiv Gandhi Govt. P.G. College, Mandsaur, M.P., India 2 Department of Zoology, Rajiv Gandhi Govt. P.G. College, Mandsaur, M.P., India *Corresponding Author: pawan_chouhan30@yahoo.com; Mobile: +919826932237

Received: 06/07/2013; Revised: 20/08/2013; Accepted: 28/08/2013

ABSTRACT Protein is a nitrogenous compound and made up of 20 different amino acids. It is required to all living organisms for their multiplication. On the industry level protein produced by fermentation technology using many microorganisms including fungi and bacteria. In this study isolates Aspergillus niger from soil and protein produced by small scale fermentation process using two different culture medium during various incubation times. Crude protein was harvested by filtration method and estimated via Spectrophotometer using Lowry reagents.

KEYWORDS:

Aspergillus niger, Protein production, Lowry method, Incubation time,

Fermentation technology.

Cite this article: Chouhan Pawan Kumar, Das Prakash (2013), FUNGAL PROTEIN PRODUCTION BY SMALL SCALE FERMENTATION TECHNOLOGY USING ASPERGILLUS NIGER, Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 642–647

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INTRODUCTION: Generally fungi are filamentous multicellular achlorophyllus organism grown by tacking nutrients through absorption from dead and living cells. They multiply on surface or submerge condition during fermentation process and produce their metabolites intracellularly as well as extracellularly which are harvested, purified and used as supplements. Similar proteins are produced by A. niger extracellularly and quantitatively estimated by Lowry method (Lowry et al., 1951). Metz and Kossen (1977) reported that many parameters influence fungal pellet formation including inoculum level, initial pH of medium, agitation, medium composition and use of polymer additives or surface-active agents. Nielsen and Carlsen (1996), Jimenez-Tobon et al. (1997) studied fungal growth and reported that filamentous growth of fungi observed at high initial spore levels whereas increased pellets size were usually formed with reduced inoculum level. Fungi have been used in a variety of industries e.g. food, chemical, detergent, textiles and paper industries for production of protein, enzyme and other products (Moreira et al., 1999 & 2001; Kathiresan and Manivannan, 2006). Filamentous fungi have been widely used in the fermentation industry as it becomes a principal source of protein, enzymes and other metabolites. Therefore, fungi have been widely investigated by various researchers due to low cost and high productivity which attracted many other researchers to improve fungal strains by molecular techniques and also bioprocess (Finkelstein and Ball, 1992; Banerjee et al., 2003). Whitaker & Long (1973) and Wainwright et al. (1993) reported that the initial pH of medium plays an important role in fungal morphology as the higher pH values (5.0 to 6.0) produce pellets while low pH values (2.0 to 3.0) leads to filamentous mycelium growth,

meant the surface properties of the spores are influenced by pH. Proteins are nitrogen or amino acid supplements and most of the enzymes are made of proteins. Besides energy source, protein required for fungi, bacteria, actinomycetes and other unicellular and multicellular cells for their membrane and enzyme synthesis. Protein present in cells as lipoprotein, glycoprotein and other forms. Therefore, proteins are produced by fermentation technology and used in a lot of industries such chemical, detergent and food as nutrient supplements for human diet (Moreira et al., 2001). The objectives of our study to isolate fungal strain from soil for protein production in laboratory via small scale fermentation technology using two different types culture. Fungal isolate was inoculated in both production media and incubated for various incubation times at 30ºC. Total protein was harvested by filtration method and quantitatively estimated by spectrophotometer using Lowry reagents. MATERIAL AND METHODS: Isolation, Purification & Identification of fungi: Soil samples were collected from Botanical garden of Govt. RGPG College, Mandsaur, Madhya Pradesh, India. All soil samples were mixed together then formed one composite sample. Isolation of fungi was done by serial dilution technique. 0.1 ml sample was transferred from 10-3 dilution to sterilized Petri plates and poured melted Potato dextrose agar (PDA) medium then solidified at room temperature. All plates were incubated at 30ºC for 5 days and purified by streak plate method. After purification, fungal cultures were observed on the basis of morphological structures by microscopic method (Fig.-1) and identified using laboratory manual of filamentous soil fungi (Gilman, 1944 and Smith et al., 1983). Identified cultures were transferred to PDA slant and incubated at 30ºC for 5 days then stored in refrigerator for further experiments.

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Media preparation: An amount of 34.501 g and 102.2 g of Czapeks Dox medium and Starch peptone medium was weighted into Erlenmeyer conical flasks respectively. One thousand milliliter of glass distilled water was added to conical flasks. The mixtures were poured into conical flasks, 100 ml each and plugged with cotton wool. The cotton wools were covered with brown paper and the conical flasks were autoclaved at 15 lb steam presser for 15 min. The Czapeks Dox medium and Starch peptone medium were cooled at room temperature before use. Protein production technology:

by

fermentation

and inoculation loop. Spores were inoculated to Czapeks Dox medium and Starch peptone medium respectively (Fig.- 2). All inoculated conical flasks were incubated at 30ÂşC for 3, 6, 9 and 12 days. Protein estimation by Lowry method: After incubation time, the Lowry method (Lowry et al., 1951) was used for total protein estimation. Culture filtrates were obtained by filtration method using Whatman filter paper No 1 and then 1 ml culture filtrate of Czapeks Dox medium and Starch peptone medium was mixed with Lowry reagents. The color density was recorded at 750 nm by Spectrophotometer and compared to standard curve of protein (BSA 1mg /ml).

Aspergillus niger spores were obtained from PDA slants using sterilized distilled water Fig.- 1 Aspergillus niger on PDA plate (A) and microscopic photo (B).

A

B

Fig.- 2- Aspergillus niger inoculated in production medium (A- Czapeks Dox medium and B- Starch peptone medium).

A

B

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RESULT AND DISCUSSION:

2. Total protein estimation from culture filtrate of Starch peptone production medium:

1. Total protein estimation from culture filtrate of Czapeks Dox production medium:

It is evident from table- 2 and Fig.-4 that 1.23 mg/ml protein was produced by Aspergillus niger within 3 day incubation and also 2.73 mg/ml protein was recorded in 9 day incubation. The amount of protein was recorded maximum on 9 (2.82 mg/ml) day incubation period. Starch peptone medium have starch, a polysaccharide, which was initially not metabolized. Therefore, the growth of Aspergillus niger was slow which affect protein production initially on three day incubation time.

After inoculation and incubation, culture filtrates were obtained by filtration method using Whatman No.1 filter paper and 0.1 ml culture filtrate was used for total protein estimation by Lowry method at 750 nm. It is evident from table-1 and Fig.- 3 that 15.4 mg/ml protein was produced by Aspergillus niger within 3 days incubation period and also 2.15 mg/ml protein was recorded in 9 day incubation. Further table- 1 indicates that synthesized protein was used by fungi for their building blocks. Hence, the amount of protein was decreased on 6 and 12 days.

Table-1 Total protein estimation from Czapeks Dox medium by Lowry method. S. No. Incubation days Total Protein (mg/ml) 3 6 9 12

1 2 3 4

15.4 1.92 1.01 2.15

Fig.- 3 Estimation of total protein from culture filtrate of Aspergillus niger. Czapex Dox medium

Total protein (mg/ml) 16 14 12 10

Total protein (mg/ml)

8 6 4 2 0 3

6

9

Incubation days

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12


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Table-2 Total protein estimation from starch peptone medium by Lowry method. Serial No. Incubation days Total Protein (mg/ml) 3 1 1.23 6 2 2.72 9 3 2.82 12 4 2.73 Fig.- 4 Estimation of total protein from culture filtrate of Aspergillus niger. Starch peptone medium

Total protein (mg/ml) 3

2.5

2

Total protein (mg/ml)

1.5

1

0.5

0 3

6

9

12

Incubation days

These data are supported by Finkelstein and Ball (1992); Moreira et al. (1999 and 2001); Banerjee et al. (2003) and Karthiresan and Manivannan (2006) which were reported that fungi have been used for their metabolite production. A. niger produced maximum amount of protein (15.4 mg/ml) in Czapeks Dox medium while minimum (1.23 mg/ml) in Starch peptone medium within three incubation time. In Czapeks Dox medium the amount of protein was decreased with increased incubation time and slightly increased on 12th day while amount of protein was increased till 9th incubation time in starch peptone medium and slightly decreased on 12th day. The growth of Aspergillus niger was fast in Czapeks Dox medium as compared to starch peptone medium due to availability of

oligosaccharide instead of polysaccharide as carbon source. Starch is polysaccharide and initially not metabolized, it metabolized after protein (amylase enzyme) synthesis. Therefore, the amount of protein was more in Czapeks Dox medium as compared to starch peptone medium. CONCLUSION: It is suggested that microbial proteins are easily produced by fermentation technology on small and industrial scale. It is available on low cost for humans, animals as well as other living cells where it completes nutrient requirement of cells. Further it is suggested that Czapeks Dox medium was better for protein production by fungi where it produced maximum amount of protein within 3 days incubation time.

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REFERENCES: Banerjee AC, Kundu A, Ghosh SK (2003). Genetic manipulation of filamentous fungi. In: Roussos S, editor. New horizons in biotechnology. Dordrecht (Neth)Kluwer Academic Publishers. 193–8. Finkelstein DB, Ball C (1992). Biotechnology of filamentous fungi. Boston Butterworth-Heinemann. 221–416. Gilman JC, (1944). A manual of soil fungi, Revised 2nd edition, Oxford and IBH publishing Co. Jimenez-Tobon GA, Penninckx MJ, Lejeune R (1997). The relationship between pellet size and production of Mn(II) peroxidase by Phanarochaete chrysosporium in submerged culture. Enzyme Microb. Technol. 21: 537–42. Kathiresan K, Manivannan S (2006). αAmylases production by Penicillium fellutanum isolated from mangrove rhizoshere soil. Afr. J. Biotechnol. 5 (10): 829–832. Lowry OH, Rosbrough NJ, Farr AL, Randall RJ (1951). J. Biol. Chem. 193: 265. Moreira FG, De Lima FA, Pedrinho SRF, Lenartovicz V, De Souza CGM, RM Peralta CGM (1999). Production of

Source of Support: Nil

amylases by Aspergillus tamari. Rev. Microbiol. 30: 157–162. Moreira FG, Lenartovicz V, De Souza CGM, Ramos EP, Peralta RM (2001). The use of α-methyl-D-glucoside, a synthetic analogue of maltose, as inducer of amylase by Aspergillus sp. In solidstate and submerged fermentation. Braz. J. Microbiol. 32: 15–19. Metz B, Kossen NWF (1977). Biotechnology review: the growth of molds in the form of pellets- A literature review. Biotechnol Bioeng. 19: 781–99. Nielsen J, Carlsen M (1996). Fungal pellets In: Willaert RG, Baron GV, De Backer L editors. Immobilised living cell systems: modeling and experimental methods. Chichester (NY) John Wiley & Sons. 273–93. Smith JE, Berry DR, Kristiansen B (1983). Filamentous fungi Vol. IV, Fungal Technology. Edward Arnold. London. Whitaker A, Long PA (1973). Fungal pelleting. Proc Biochem, 8: 27–31. Wainwright MP, Trinci APJ, Moore D (1993). Aggregation of spores and biomass of Phanerochaete chrysosporium in liquid culture and the effect of anionic polymers on this process. Mycol Res. 97: 801–6.

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 648â&#x20AC;&#x201C;655 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article THERAPEUTIC PLANTS AND THEIR ANTIOBESITY PROPERTIES Mohan Reddy N1, Kalyani P2, Nagalaksmi K3, Kaiser Jamil4* 1, 2, 3

Research Scholar, Department of Genetics, Bhagwan Mahavir Medical Research Center, Mahavir Marg, A.C.Guards, Hyderabad - 500004, Andhra Pradesh, India. 4 Head, Department of Genetics, Bhagwan Mahavir Medical Research Center, Mahavir Marg, A.C.Guards, Hyderabad - 500004, Andhra Pradesh, India. *Corresponding author: E-mail: kjmohan.bmmrc@gmail.com; Tel: +919849706385; fax: +914066631500

Received: 22/07/2013; Revised: 22/08/2013; Accepted: 29/08/2013

ABSTRACT Obesity has reached epidemic levels in industrialized countries. However, the prevalence of obesity is also rising in other less developed parts of the world, like Asia and Africa. For centuries people across the countries have been using natural products and plant based dietary supplements for weight control. The discovery of bioactive compounds from herbs is one possible way to control obesity and to prevent or reduce the risks of developing various obesity-related diseases. The present review covers the taxonomy, habitat, distribution, extraction and identification of active principle of potential medicinal plants used in obesity treatment.

KEY WORDS: Obesity, Therapeutic plants, Phyto medicine.

Cite this article: Mohan Reddy. N., Kalyani. P., Nagalaksmi. K., Kaiser J. (2013), THERAPEUTIC PLANTS AND THEIR ANTIOBESITY PROPERTIES, Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 648â&#x20AC;&#x201C;655

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INTRODUCTION The world health organization has described obesity as one of todayâ&#x20AC;&#x;s most neglected public health problems, affecting every region of the globe (Serena Low, 2009). Obesity in children and adolescents is gradually becoming a major public health problem in India. Totally 5% of the Indian population has been affected by obesity (Kumar NVRTP, 2008). Overweight and obesity are associated with an increased burden of diabetes, hypertension, cardiovascular diseases, some types of cancers and premature mortality but also with the social and psychological effects of excess weight (Bell CG, 2005). Different types of obesity-treatment drugs are currently available in the market. Antiobese drugs, which reduce intestinal fat absorption through inhibition of pancreatic lipase, and some of the drugs, are appetite suppressants. These drugs may have side-effects, including increased blood pressure, dry mouth, constipation, headache, and insomnia (Karamadoukis L et al., 2009; Tziomalos, K et al., 2009). A number of anti-obesity drugs are currently undergoing clinical development, including centrally-acting drugs; targeting peripheral episodic satiety signal drugs; fat absorption and blocking drugs (Halford J.C., 2006). Herbal plants or their constituents could provide a new paradigm in development of drugs. It was agreed that herbal medicines are much safer, less toxic, and have fewer harmful side effects than chemically synthesized pure drugs. Thus, many scientists have proposed that herbal medicines can be used to provide much safer and better drugs for personal health care in the near future (Wang J. F., 2008; Singh Balpreet et al., 2012). In this perspective, both hypocaloric diets (decreased energy intake) and increased physical activity (increased energy output) result in loss of body weight and body fat. With these traditional approaches to weight loss, potential therapeutic agents could be important tools in preventing and/or treating obesity and associated metabolic diseases. Some plants have been utilized as medicinal agents from

ancient to modern times. These medicinal agents initially took the form of crude drugs such as tinctures, teas, poultices, powders and other herbal formulations. The specific plants to be used and the methods of application for particular ailments were passed down through oral history (Samuelsson G, 1991). In this paper the current status of herbal plants, including their clinical features in treatment of obesity are reviewed and discussed. HERBAL PLANTS ANTI-OBESITY PROPERTIES Prickly Chaff Flower (Achyrathus aspera) (family: Amaranthaceae) It is an annual or perennial herb. Popularly known as Apamarga, is a commonly available plant in India and had claims in the treatment of hyperlipidaemia in Ayurveda, an Indian system of medicine (Anil M and Mahesh CS, 2009). Saponins from different plant sources were proved to have hypolipidemic activity (Rachh PR et al., 2010) and the presence of saponins was reported in A. aspera (Michl G et al., 2000). The alcoholic extract of this plant at 100 mg/kg dose reduces total serum cholesterol (TC) and phospholipid (PL), triglyceride (TG) and total lipid (TL) levels. This cholesterol lowering activity might be due to rapid excretion of bile acids causing low absorption of cholesterol (B. Pushpa Latha et al., 2010, Khanna A.K et al., 1992). The methanolic leaf extract at 1g/kg showed significant lowering of serum lipids such as total cholesterol, triglyceride, HDL, LDL (Workineh Shibeshi et al., 2006). T Malarvili et al. (2011) study results demonstrate that, seed extract of Achyranthes aspera exhibit potent hypolipidimic activity and active against High fructose fed rats. This may be due to aneroxic property activated by phyto-chemical components like saponins which might prove useful in the treatment and/or prevention of obesity. Garlic (Allium sativum)(family: Alliaceae) Allium sativum is a small herb. It is a native of central Asia and occurs all over India.

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Extracts of garlic contains various biologically active compounds such as alliin, allicin, ally methane thiosulfinate, ajoene, diallyl disulfide, diallyl trisulfide, and S-allylycysteine (Agarwal KC., 1996). Garlic and its extracts when supplemented with diet have been proven beneficial in animals. Lee MS et al. (2011) study results suggest that, the anti-obesity effects of garlic were moderately mediated via activation of AMPK this system increased thermogenesis and up regulation of the expression of mitochondrial uncoupling protein and decreased expression of multiple genes involved in adipogenesis. Black garlic and its fermented version have shown promising results in protecting high fat diet induced obesity, hyperglycemia, liver and kidney damage in mice (Jung YM et al., 2011; Inhye Kim et al., 2011). Sweet flag Araceae)

(Acorus

calamus)

(family:

Acorus calamus is a semi-aquatic, perennial, aromatic herb with creeping rhizomes (N. Tiwari et al., 2010). It is found throughout India in damp marshy places.The common name of Acorus calamus in Telugu is VASA (A.E. Raja et al., 2009).Investigations revealed that the presence of tannins in this plant is responsible for hypolipidemic activity. In calamus oil α and β- asarone compounds were found to be the major constituents. 50% alcoholic extract of A. calamus rhizomes in combination with saponins has been found to produce hypolipidemic activity in the tested rats. α-asarone exhibits the hypolipidemic activity in hypercholesterolaemic rats and βasarone inhibit the differentiation of 3T3-L1 adipocytes and decreased the expression of PPARγ, C/EBPα and C/EBPβ and also the phosphorylation of p-ERK in 3T3-L1 cells. βasarone which is isolated from the calamusoil has been found to inhibit the differentiation of adipocytes and hence possess the potential for the treatment of obesity and other obesityassociated insulin resistance (M.H. Lee et al., 2011).

Veld Grape (Cissus quadrangularis) (family: Vitaceae) Cissus quadrangularis occurs throughout India, it has been used by common folk in India for promoting the fracture healing process. The whole plant is used in oral re-hydration, while the leaf, stem, and root extracts of this plant are important in the management of various ailments. It has been prescribed in Ayurveda as an alterative, anthelmintic, dyspeptic, digestive, tonic, analgesic in eye and ear diseases, and in the treatment of irregular menstruation and asthma. Earlier works on C.quadrangularis report its effectiveness on the management of obesity and complications associated with metabolic syndrome (Julius Oben et al., 2006). Various formulations now contain extracts of C.quadrangularis in combination with other compounds, used for the purpose of management of overweight and obesity (Mehta M et al., 2001).Proprietary extract of this plant (CQR-300) at a dose of 300 mg daily and proprietary formulation containing CQR-300 (CORE) at a dose of 1028 mg daily showed significant (p < 0.001) reductions in plasma thiobarbituric acid reactive substances (TBARS) and carbonyls. They also brought significant reductions in weight, body fat, total cholesterol, LDL-cholesterol, triglycerides, and fasting blood glucose levels over the respective study periods in a double-blind placebo controlled design, involving initially 168 overweight and obese persons. These changes were accompanied by a significant increase in HDL-cholesterol levels, plasma 5-HT, and creatinine (Julius Oben et al., 2007, 2008). Blue spur flower (Coleus forskohlii) (family: Lamiaceae) C. forskohlii is a perennial plant a member of the mint family. It is indigenous to India found mostly on the dry and barren hills and is recorded in Ayurvedic Materia Medica under the Sanskrit name „Makandi’ and „Mayani” (Shah v et al., 1996; C. Kavitha et al., 2010). Forskolin is the unique adenylate cyclase activating phytonutrient. Adenylate Cyclase is an enzyme that activates Cyclic Adenosine

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Monophosphate (cAMP) in the cell. Cyclic AMP promotes the breakdown of stored fats in animal and human fat cells. It regulates the body's thermogenic response to food, increases the body's basal metabolic rate, and increases utilization of body fat. It may also release fatty acids from adipose tissue, which results in increased thermogenesis, loss of body fat, and theoretically increased lean body mass (Himesh Soni & Akhlesh Kumar Singhai, 2012).This plant extract (10% forskolin) at a dose of 500 mg showed significant decrease in body weight (66.33 ± 3.00 to63.96 ± 3.10, p=0.0038), fat content (29.64 ± 2.19 to 27.77 ± 2.27, p=0.0038), lean body mass (44.34 ± 2.98 to 43.93 ± 3.01, p=0.0044) and basal metabolic rate (1379.1 ± 74.4 to 1363.9 ± 77.5, p=0.0254) when given to fifteen volunteers twice a day with meals in an 8-weeks open-label study. This indicates that C. forskohlii extract has potential effect as an adjunct therapy in the treatment of obesity (Seika Komhara, Somboon Nopara, 2006). Guggul (Commiphora Burseraceae)

mukul)

(family:

Guggul is an oleo gum resin. The plant contains essential oil,mainly consisting of myrecene, dimyrecene and polymyrecene,Zguggulusterone, E-guggulusterone, guggulusterone-I, guggulusterone-II, and guggulusterone-III. These isolates have been found useful in curing many diseases like rheumatism, arthritis, hyperlipidemia, obesity, inflammation. Guggul earlier used to grow abundantly in the states of Karnataka, Gujarat and Rajasthan (Jain Anurekha & Gupta VB, 2006). A recent study demonstrates that guggulsterone upregulates the bile salt export pump (BSEP), an efflux transporter responsible for removal of cholesterol metabolites, bile acids from the liver. Such up regulation of BSEP expression by guggulsterone favors cholesterol metabolism into bile acids, and thus represents another possible mechanism for its hypolipidemic activity (Deng R. 2007). Guggul an extract from the resin of this tree at a dose of 2160 mg (4 capsules) daily showed significant reduction in mean levels of total

cholesterol and HDL-C in a double-blind, randomised, placebo controlled trial in Norwegian general practice including 43 women and men, age 27–70, with moderately increased cholesterol. However, the mean levels of LDL-C, triglycerides, and total cholesterol/HDL-C ratio did not change significantly (Nohr LA et al., 2008). Brindal Berry (Garcinia cambogia) (family: Clusiaceae) Garcinia cambogia is a small or mediumsized tree with diminutive purple fruit. It occurs in the evergreen and shola forests of Western Ghats in India. Garcinia and its active ingredient, HCA (hydroxycitric acid) have been extensively studied for over thirty years and found to be effective in inhibiting lipogenesis, suppressing appetite, and encouraging weightloss in humans (Deepak Shrivastava, 2012). Hydroxycitric acid is the active ingredient of the fruit and the rind of this plant. It competitively inhibits the extra mitochondrial enzyme adenosine triphosphate–citrate (pro3S)-lyase. This enzyme ensures the supply of acetyl-CoA, which is used by acetyl-CoA carboxylase, the regulatory enzyme of lipogenesis in the liver. This is also important in the regulation of biosynthesis of endogenous lipids, levels of plasma lipoproteins VLDL (very-lowdensity lipoprotein), LDL (lowdensity lipoprotein) and HDL (high-density lipoprotein) and the distribution of the lipid in extra hepatic tissues, especially adipocytes, the storage site of body fats(Ateş, A et al., 2012). Gudmar (Gymnema Apocynaceae)

sylvestre)

(family:

It is a woody climber. It is found in the Deccan Peninsula and western India. The active compound of this plant is a group of acids termed as gymnemic acids. It has been observed that there could be a possible link between obesity, Gymnemic acids and diabetes (Parijat Kanetkar et al., 2007). Maji et al. (2000) have reported that water soluble portion of the alcoholic extract of G. sylvestre leaves

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has a significant glucose lowering effect compared with standard hypoglycaemic agents. Vinaykumar et al. (2010) concluded that the possible mechanism for an anti-obesity effect of G. sylvestre extract may be via suppression of levels of leptin, insulin, dyslipidemia, apolipoproteins, lipids, visceral fat pad weights, and oxidative stress in obese rats fed with high fat diet. Indian Sarsaparilla (Hemidesmus indicus) (family: Apocynaceae) It is prostrate or semi-erect shrub. It is distributed throughout India. Indian Sarsaparilla is used in traditional medicine as one of the Rasayana plants of Ayurveda, for its anabolic effect. Rasayana plants are characteristically anabolic in nature they stimulate protein synthesis and other metabolic activities. The roots of the plant are woody, sweet in taste and possess cooling effect. Roots are one of the well known drugs in the Ayurvedic system of medicine (Gayathri Mahalingam, Krishnan Kannabiran, 2009). Cell culture extract of H.indicus (2, 4 and 16 mg/kg, p.o) showed significant treatment reduction (p<0.01) of lipid levels in serum of rats which received atherogenic diet for first 30 (l−30) days. When atherogenic diet and cell culture extract were given together for 60 (l−60) days there was significant (p<0.01) reduction of lipid in liver, heart and serum fecal excretion of cholesterol and phospholipids were significantly increased (p<0.01 in rats fed with cell culture extract (Bopanna K. N et al., 1997). Roselle (Hibiscus Malvaceae)

sabdariffa)

(family:

It is an annual/Perennial shrub. It is native ofWest Indies and is now cultivated in Uttar Pradesh, Andhra Pradesh, West Bengal, Bihar, Punjab, Assam and Tamil Nadu (Gautam RD, 2004). Hibiscus acid and its 6-methyl ester were respectively isolated as active principles from the 50% methanol and acetone extracts of rosella tea. Kaempferol-3-O-rutinoside,

kaempferol-3-O-glucopyranoside, quercetin, 3O-rutinoside were isolated from 70% aqueous ethanol extract of leaves (Akiyoshi S et al., 2005). A total daily dose of 100 mg Hibiscus sabdariffa extract powder (HSEP) was orally administered in capsules for one month. Total cholesterol, LDL-c, HDL-c, VLDL-c, triglycerides, glucose, urea, creatinine, AST, and ALT levels in the blood were determined in all individuals pre- and post-treatment. The metabolic syndrome (MeSy) patients treated with HSEP had significantly reduced glucose and total cholesterol levels, increased HDL-c levels, and an improved TAG/HDL-c ratio. Therefore, use of HSEP in individuals with dyslipidemia associated with MeSy (GurrolaDíaz CM et al., 2010). Aqueous extract of dried calyces of H. sabdariffa at 0.8 ml/kg body weight showed significant decrease in plasma glucose and cholesterol in rats fed with 99% growers mash and 1% cholesterol. Same results were obtained when rats were subjected to an aqueous extract of H. sabdariffa and Zingiber officinale at 1 ml/Kg body weight. Extracts of H. sabdariffa and Z. officinale apart from being hypocholesterolemic and hypoglycemic, they control blood sugar especially in those prone to diabetes mellitus (Agoreyo F. O et al., 2008). CONCLUSION We roofed only ten plants in this review. Many more plants are there which have effect not only on obesity but on every possible disorder. Present review summarizes various plant derived extracts which are widely prescribed worldwide and are considered natural, safe and beneficial for the treatment of obesity. The potential of natural products to treat obesity is still largely unexplored and might provide an excellent alternative strategy for the development of safe and effective antiobesity drugs. However biotechnology provides methods to try the possibility of enhancing the natural level of desired active principle in intact plant itself. This may open a new window for rewarding research to biotechnologists.

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REFERENCES A.E. Raja., M. Vijayalakshmi & G., (2009) Devalarao.,Acorus calamusLinn.: Chemistry and Biology. Research J. Pharm and Tech.2 (2). Agarwal KC., (1996). Therapeutic actions of garlic constituents.Med Res Rev. 16:111–24. Agoreyo F. O., Agoreyo B. O., and Onuorah M. N., (2008). Effect of aqueous extracts of Hibiscus sabdariffa and Zingiber officinale on blood cholesterol and glucose levels of rats. African Journal of Biotechnology. 7 (21): 3949– 3951. AkiyoshiS.,Chiaki N., Masanori M., Sadayoski M., Yoshiharu M and Sadao K., (2005) Glycosides in African dietary leaves, Hibiscus sabdariffa, J Oleo Sci. 54(3),185–191. Anil M., Mahesh CS., (2009). Evaluation of certain medicinal plants for antiobesity properties.Ind. J. Trad. Know. 8(4): 602–605. Ateş, A., Esen Gürsel, F., Bilal, T and Altıner, A., (2012). Effect of dietary Garcinia cambogia extract on serum lipid profile and serum enzymes in rats fed highlipid diet. Iranian Journal of Veterinary Research,Vol. 13, No. 1, Ser. No. 38.

of hypercholesterolemia innormal and hyperlipidemicrats.Indian J Pharmacol. 29: 105–109. C.Kavitha., K. Rajamani and E. Vadivel., (2010). Coleus forskohlii: A comprehensive review on morphology, phytochemistry and pharmacological aspects. Journal of Medicinal Plants Research. Vol. 4(4) pp. 278–285, Deepak Shrivastava., (2012). Formulation of Sustained Release Tablet of Anti Obesity Drug Garcinia cambogia. World Journal Of Pharmacy And Pharmaceutical Sciences. Vol.1, Issue 2, 731–743. Deng R., (2007). Therapeutic effects of guggul and its constituent guggulsterone: cardiovascular benefits.Cardiovasc Drug Rev.Winter . 25(4):375–90. Gautam RD., (2004). Sorrel-A lesser-known source of medicinal soft drink and food in India,Nat Prod Rad. 3(5), 338–342. Gayathri Mahalingam., Krishnan Kannabiran., (2009). Hemidesmus indicus root extract ameliorates diabetes-mediated metabolic changes in rats.International Journal of Green Pharmacy. Vol: 3, Issue: 4: 314–318.

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Inhye Kim., Jin-Young Kim., Yu-Jin Hwang., Kyung-A Hwang., Ae-Son Om., JaeHyun Kim and Kang-Jin Cho., (2011). The beneficial effects of aged black garlic extract on obesity and hyperlipidemia in rats fed a high-fat diet.Journal of Medicinal Plants Research. Vol. 5(14), pp. 3159–3168. Jain Anurekha & Gupta VB., (2006).Chemistry and pharmacological profile of guggulA review.Indian J Traditional Knowledge.Vol.5 (4), pp.478–483. Julius E Oben., Damaris Mandob Enyegue., Gilles I Fomekong., Yves B Soukontoua and Gabriel A Agbor., (2007).The effect of Cissusquadrangularis (CQR-300) and a Cissus formulation (CORE) on obesity and obesity-induced oxidative stress. Lipids in Health and Disease.6:4. Julius E Oben., Judith L Ngondi., Claudia N Momo., Gabriel A Agbor and Caroline S Makamto Sobgui., (2008). The use of a Cissus quadrangularis/Irvingia gabonensis combination in the management of weight loss: a doubleblind placebo-controlled study. Lipids in Health and Disease.7:12. Julius Oben., Dieudonne Kuate., Gabriel Agbor., Claudia Momo and Xavio Talla.,(2006).The use of a Cissus quadrangularis formulation in the management of weight loss and metabolic syndrome Lipids in Health and Disease.5:24. Jung YM., Lee SH., Lee DS., You MJ., Chung IK., Cheon WH., Kwon YS, Lee YJ, Ku SK (2011). Fermented garlic protects diabetic, obese mice when fed a high-fat diet by antioxidant effects. Nutr Res. 31(5):387–96. Karamadoukis, L., Shivashankar, G.H., Ludeman, L., Williams, A.J., (2009). An unusual complication of treatment with orlistat.Clin.Nephrol. 71, 430–432.

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Nohr LA., Rasmussen LB., Straand J., (2008). Resin from the mukul myrrh tree, guggul, can it be used for treating hypercholesterolemia? A randomized, controlled study. Complement Ther Med. Parijat

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of non-timber forest products in agroforestry systems. FAO corporate document repository. Singh Balpreet, Kaur Rajvir, Kumar Manoj, Singh Amarjeet., (2012). Need and Relevance of Formation of Indian Systems of Medicine and Homoeopathy (ISM & H) Policy 2002 in India. Global J Res. Med. Plants & Indigen. Med. 1(11): 612–619. T Malarvili., RM Veerappan and V. Hazzena Begum., (2011).Journal of Pharmacy Research. 4(6): 1769–1771. Tziomalos, K., Krassas, G.E., Tzotzas, T., (2009). The use of sibutramine in the management of obesity and related disorders: an update. Vasc.Health Risk Manag. 5, 441–452. Vinay Kumar., Uma Bhandari., Chakra Dhar Tripathi., Geetika Khanna., (2012). Evaluation of antiobesity and cardioprotective effect of Gymnema sylvestre extract in murine modal. Ind J Pharmacology.Vol. 44; Issue 5. Wang, J. F., D. Q. Wei and K. C. Chou., (2008). Drug candidates from traditional Chinese medicines. Curr.Top. Med. Chem. 8: 1656–1665. Workineh Shibeshi., Eyasu Makonnen., Legesse Zerihun., Asfaw Debella., (2006). Effect of Achyranthes aspera L. on fetal abortion, uterine and pituitary weights, serum lipids and hormones. African Health Sciences. 6(2):108–112.

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 656â&#x20AC;&#x201C;663 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article REVIEW ON THE CONTRIBUTION OF DASHAPUSHPA, A TRADITIONAL MEDICINE IN THE MANAGEMENT OF CANCER Arun Raj GR1*, Shailaja U2, Rao Prasanna N3, Sharanesh T4, Gokul J5 1

PG Scholar, Department of PG Studies in Kaumarabhritya, SDM College of Ayurveda and Hospital, Hassan Professor and Head, Department of PG Studies in Kaumarabhritya, SDM College of Ayurveda and Hospital, Hassan 3 Principal and Head, Department of PG Studies Salya Tantra, SDM College of Ayurveda and Hospital, Hassan 4 Reader, Department of PG Studies in Agada Tantra, SDM College of Ayurveda and Hospital, Hassan 5 PG Scholar, Department of PG Studies in Samhita, SDM College of Ayurveda and Hospital, Hassan, Karnataka, India *Corresponding author: Email address: drdrarunraj26@gmail.com 2

Received: 05/07/2013; Revised: 20/08/2013; Accepted: 27/08/2013

ABSTRACT Cancer is a generic term for a large group of diseases that can affect any part of the body. In Asian and African countries, 80% of the population depend on traditional medicine for primary health care. Even though modern science is very much advanced in the field of cancer treatment, the role of traditional medicine in the same field is highly appreciated. Dashapushpa, a prominent traditional medicine is a group of ten herbs which is culturally and medically important to the people of Southern India, especially in Kerala state. These ten herbs are widely used in treating different varieties of cancer and other malignant conditions by the Visha Vaidyas of the state. Moreover the methodology implemented in the study was the thorough literary research through different traditional Ayurvedic books where the utility of these herbs in cancer like conditions are mentioned in addition to the review through various pharmacological studies. Almost all the ingredients of Dashapushpa have been extensively studied for their pharmacological efficacy as anti-cancer drugs. This paper is a review of various folklore, therapeutic uses and pharmacological studies conducted for the anti-cancer activity of these ten herbs. KEY WORDS: cancer, Dashapushpa, traditional medicine, Visha

Cite this article: Arun Raj. G.R., Shailaja. U., Rao. P. N., Sharanesh. T., Gokul. J., (2013), REVIEW ON THE CONTRIBUTION OF DASHAPUSHPA, A TRADITIONAL MEDICINE IN THE MANAGEMENT OF CANCER, Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 656â&#x20AC;&#x201C;663

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INTRODUCTION Traditional medicine has a long history of serving people all over the world. It is the sum total of knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures that are used to maintain health, as well as to prevent, diagnose, improve or treat physical and mental illnesses (Patrick OE et al., 2008). In Asian and African countries, 80% of the population depend on traditional medicine for primary health care (WHO media centre, 2013). Herbal medicines are the most lucrative form of traditional medicine, generating billions of dollars in revenue (Thomas D, 2010). In recent years, the use of traditional medicine information in cancer research received considerable interest (Rajesh et al., 2011). Cancer is responsible for millions of deaths each year worldwide (Siveen et al., 2011). Cancer is a generic term for a large group of diseases that can affect any part of the body. One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs (WHO media centre, 2013). This process is referred to as metastasis. Metastases are the major cause of death from cancer (WHO media centre, 2013). There are more than 200 different types of cancer (Cancer Research UK, 2013). GLOBOCAN 2008 estimated that about 12.7 million cancer cases and 7.6 million cancer deaths have occurred in 2008 and among that 56% of the cancer cases and 64% of the deaths occurred in the economically developing world (Jemal et al., 2011). Through this paper the authors makes review of various folklore, therapeutic uses and pharmacological studies conducted for the anticancer activity of Dashapushpa, a traditional medicine in the management of cancer. And also to draw the attention of research scholars and research institutes to these ten drugs. Thorough literary search was made through various Ayurvedic classical texts such as Arogya Kalpadruma, Sarvaroga Chikitsaratna,

Abhidana manjari, Kodasheri margam, Prayoga samuchayam, Chikitsa kauthukam, Vaidya manorama and Agastya marmashastra and also various research papers. Methodology comprised of a concept about the role of traditionally used medicine, i.e. Dashapushpa in the management of cancer on the basis of its clinical practice by some traditional Visha Chikitsaka (traditional Vaidyas practising toxicology). Dashapushpa – The group of ten medicinal flowers Dashapushpam literally means ‘ten flowers’ („Dasham‟ refers to ten and „Pushpam‟ refers to ‘flowers’). In the present context, the word “Dashapushpam” refers to ‘ten species of plants.’ Dashapushpas are culturally and medically significant to the people of Kerala. All are used as ingredients in various Ayurvedic formulations (Sindhu et al., 2009). The plants referred to as Dashapushpas are listed in table: 1 (Mini VN et al., 2010). Cultural view on Dashapushpas As per the tradition of Kerala, women wear Dashapushpa garlands on the head. In front of the household shrine, the ten sacred plants of Dashapushpa were displayed in a gleaming brass plate in the Malayalam month of Karkkidakam (the monsoon season in Kerala when diseases are more prominent and the body has little resistance against diseases) in the olden days. It is also used with “Ashtamangalyam” (group of eight auspicious materials which are carried on a large bronze vessel for offerings) during marriage functions. It was also prescribed by the Rajavaidyas (doctors for the king) to the ladies to wear these plants on their head, probably due to the medicinal value imparted by them. Dashapushpas are been eaten in the form of Karkkadaka kanji in Karkkidaka masam to get better health in the upcoming monsoon season (Jiny et al., 2010). They are considered auspicious and each herb is associated with a deity in Hindu mythology. It has been shown in table: 2 (Mini VN et al., 2010).

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Table 1: General Description of Dashapushpa – The group of ten Medicinal flowers Sl No.

SANSKRIT NAME

LATIN NAME

1

Bhadra

Aerva lanata (L.) Juss.

2

Viparitha lajjalu

Biophytum sensitivum (L.) DC.

3

Indravalli

4

Musali

5

Durva

6

Bhringaraja

7

Akhukarni

8

Harikrantha

Cardiospermum halicacabum (Linn.) Curculigo orchioides Gaertn. Cynodon dactylon (Pers.) Eclipta alba (L.) Hassk. Emilia sonchifolia (L.) DC. Evolvulus alsinoides (Linn.) Linn.

9

Lakshmana

10

Sahadevi

Ipomea sepiaria Roxb. Vernonia cinerea L.

POPULAR ENGLISH NAME Indian water lily

FAMILY NAME

PARTS USED

Amaranthaceae

Whole plant

Sensitive wood Sorrel

Oxalidaceae

Whole plant

Balloon vine

Menispermaceae

Shoots, leaves

Black musali

Amaryllidaceae

Tuber

Bermuda grass

Poaceae

Leaves

Asteraceae

Shoots, leaves Shoots, leaves Whole plant

Canada Flea-bane Slender dwarf morning glory Ipomea

Convolvulaceae

Ash coloured Flea-bane

Asteraceae

Convolvulaceae

Convolvulaceae

Table 2: Association of Dashapushpa with Hindu deities Sl No. SANSKRIT NAME DEITY IN HINDU RELIGION Yama Dev 1 Bhadra Shree Parvathy 2 Viparitha lajjalu Lord Indra 3 Indravalli Bhumi Devi 4 Musali Surya 5 Durva Lord Shiva 6 Bhringaraja Kamadeva 7 Akhukarni Lord Vishnu 8 Harikrantha Shree Bhagavathy 9 Lakshmana Brahma 10 Sahadevi

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Whole plant Whole plant


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Fig 1: Aerva lanata (L.)

Fig 3: Cardiospermum halicacabum

Fig 6: Eclipta alba (L.) Hassk.

Fig 2: Biophytum sensitivum (L.) DC.

Fig 4: Curculigo orchioides Gaertn.

Fig 7: Emilia sonchifolia (L.) DC.

Fig 9: Ipomea sepiaria Roxb

Fig 5: Cynodon dactylon (Pers.)

Fig 8: Evolvulus alsinoides (Linn.) Linn.

Fig 10: Vernonia cinerea L

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Therapeutic use of Dashapushpas

DISCUSSION

Dashapushpas are therapeutically active against fever, dysentery, haemorrhage, constipation, lithiasis, strangury, headache, dyspepsia, jaundice, liver congestion, pneumonia, typhoid, insomnia, tumours etc (Radhamani and Muralidharan, 2000). Herbs play a significant role in pharmaceutical industries as natural sources of life saving drugs (Khanna et al., 2008). Healthcare sectors around the world, more frequently than ever are facing the problems of combating the entry of novel, mutant pathogenic strains of microorganisms and their resistance against synthetic drugs (Khanna et al., 2008). This calls for the discovery of new antimicrobial compounds with diverse chemical structures and novel mechanisms of action. Natural products either as pure compounds or as standardized plant extracts is the right solution because of their unmatched display of chemical diversity (Parekh and Chanda, 2007). Essential oils from some plants like Allium sp. having antimicrobial properties can be used as natural antimicrobial additives in food production (Benkeblia, 2004). But for the development of safe and effective antimicrobials based on indigenous knowledge and ethno medicine, the screening of enormous, untapped plant wealth is inevitable (Amit and Shailendra, 2006). The wild flora of Peninsular India, one among the mega diversity centres in the world still remains to be exploited for its medicinal properties. These plants are therapeutically very active for various diseases and ailments. These are used from time immemorial in Visha Chikitsa. Some of them are scientifically validated for various bioactivities related to anti-cancer therapy. Dashapushpas has been used by traditional Vaidyas of Visha Chikitsa.

Understanding cancer in the perspective of Visha In Susruta samhita Kalpasthana, Sushrutacharya explained how the Visha properties imply to fatal stage of life. Turning in on this it implies that the same pathology might be happening in cancer. Cancer, the most fatal and rapidly growing disease can be better understood on comparison with the Visha Gunas. The properties of Visha have been detailed in different Ayurvedic classics (Srikantha Murthy, 2006). The different symptoms and stages of different types of cancer can be understood in the purview of Visha Gunas. Laghu guna imply the Laghavatva of Shareera or in simple words the emaciation or decrease in body weight. This feature can be noted invariably in almost all types of cancers. Anorexia and elevated energy expenditure contributes to weight loss in cancer patients. The emaciation of cancer is called cachexia and is very dangerous on its own. Ruksha or dryness is noted as a significant feature in Squamous cell carcinoma or the cancer of squamous cells of the skin. Ashukari refers to the fastness or rapidity in spreading. Certain tumors like astrocystoma are known for its rapid progress while tumors like low grade gliomas take a slow and gradual path of progression. Vyavayi refers to the spreading nature of tumor or to be precise the Metastasis. it is the process of spreading of cancer cells from its primary site to the secondary sites. A carcinoma of breast may cause secondaries in lungs or bones. Teekshna as the word denotes, the penetrating nature of tumor cells or infiltration to deeper tissues. The penetrating property can be understood by the entry of carcinogen cells in the blood stream by penetrating the basement membrane and epithelial tissue by releasing metallo protenosis. Vikashi denotes the instability or the weakness of body caused by the effect of tumor. Sukshma connotes the capability of

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tumor cells to travel through minute channels. This is best appreciated in cases of lymphomas. Ushna even though does not directly lead us to a significant feature, may imply the fever caused by various infection in the later stages of different carcinomas. This can also be regarded as a feature in basal cell carcinoma which present with burning sensation of the skin. Apaki as the word indicates does not undergo suppuration. Except a few condition like a carcinoma of oral cavity, all type of carcinomas are non suppurative in nature. Vishada as it implies its Kledakara nature more here ie may be taken as discharge like in cervical cancer, nipple discharge in breast cancer etc. For the same reason, it can be useful while dealing with the cancers, especially Dashapushpas the most popular medicine of Visha Chikitsa in Kerala.

cancer cell lines was studies and it was compared with normal, Vero cell line using MTT assay (Kanimozhi, 2012). The hydroalcoholic extract of Eclipta alba (L.) Hassk. was evaluated for its anticancer potential (Chaudhary et al., 2011). Methanolic extract of Emilia sonchifolia (L.) DC. was found to be cytotoxic to Daltons lymphoma (DL), Ehrlich ascites carcinoma (EAC) and mouse lung fibroblast (L-929) cells, but not toxic to normal human lymphocytes, under in vitro conditions (Shylesh and Padikkala 2000). Evolvulus alsinoides (Linn.) and Ipomea sepiaria Roxb. Also shows anti-cancer activity (Auddy et al., 2003; Jiny et al., 2010). The effect of various extracts of Vernonia cinerea Linn. Against Daltonâ&#x20AC;&#x2122;s Ascitic Lymphoma (DAL) in Swiss Albino mice was also studied Sangeetha and Venkatarathinakumar, 2011).

Research works done on plant drugs in Dashapushpa group

CONCLUSION

Methanolic extract of Aerva lanata Linn. has shown antioxidant and anticancer effect against Ehrlich Ascites Carcinoma (EAC) in vivo (Obayed et al., 2012). Anticancer activity of aerial parts of Aerva lanata Linn Juss ex Schult against Dalton's Ascitic Lymphoma has also been studied (Rajesh et al., 2011). While the ethanolic extract of whole plant of Aerva lanata Linn. exhibited immune-modulatory and anti-tumour activity. Biophytum sensitivum (L.) DC inhibits tumor cell invasion and metastasis through a mechanism involving regulation of MMPs, prolyl hydroxylase, lysyl oxidase, nm23, ERK-1, ERK-2, STAT-1, and proinflammatory cytokine gene expression in metastatic lung tissue (Guruvayoorappan et al., 2008). Cardiospermum halicacabum (Linn.) and Curculigo orchioides Gaertn. is also shown to exhibit anti-tumour activity (Venkatesh Babu and Krishnakumari, 2006) (Sigh and Gupta, 2008; Raman et al., 2009). The effect of invitro anticancer activity of the ethanolic extract of Cynodon dactylon (Pers.) against HEP-2 laryngeal, HELA cervical and MCF-7 breast

The global scenario has shown a great increase in herbal medicine research as anticancer therapy. Some of the research activities have been carried out on these plants during the past few decades which give sufficient motivation among the scientific community in exploring more information about these sacred plants. So there is an ultimate need to screw out the anticancer properties of Dashapushpa with the help of allied sciences. Some traditional Vaidyas have shown evidently that the Dashapushpas are effective in curing cancer patients, but to have more effective and sustainable results, a great deal is required in analysis and standardization of these ten sacred plants, assessment of pharmacological effect and designing the treatment protocol. Research and development must be encouraged for developing new pharmaceutical preparations from these plants. A detailed investigation of its standardization, pharmacological activity and clinical trials may help to develop new formulation for controlling various types of cancers.

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Source of Support: Nil

World Health Organization Media centre (2013). Traditional Medicine [www] World Health Organization. Available from: http://www.who.int/mediacentre/factshe ets/fs134/en/ [Accessed 18/08/2013]. World Health Organization Media centre (2013). Cancer [www] World Health Organization. Available from: http://www.who.int/mediacentre/factshe ets/fs297/en/ [Accessed 18/08/2013].

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 664–668 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article CATEGORIZATION OF AYURVEDIC SPECIALITIES – PRESENT & FUTURE WITH SPECIAL REFERENCE TO KRIYA SHAREERA Kamath Nagaraj1*, Kulkarni Pratibha2, Chiplunkar Shivprasad3 1

P.G. Scholar, 2Reader, 3Associate Professor & Head, Department of Kriya Shareera, Sri Dharmasthala Manjunatheshwara College of Ayurveda & Hospital, Hassan-573201, Karnataka, India *Corresponding author: Email address: nagaraj.kamath1989@gmail.com

Received: 11/07/2013; Revised: 25/08/2013; Accepted: 30/08/2013

ABSTRACT Various researches are being conducted to re-establish the concepts of Ayurveda, there is a scope for reframing and redefining some of the trends that are being followed in Ayurvedic education system. The common practice of categorization of speciality education in Ayurveda as clinical and non clinical streams is one amongst them. The basis for this classification is that the present curriculum of some of the specialities of Ayurveda requires continuous interaction with patients and some of the specialities like Kriya Shareera are not prescribed this continuous interaction and follow up with the patients. The concepts of Kriya Shareera like Dosha, Dhatu, Mala Vrudhi-Kshaya, Prakruti Pariksha, Agni Parikshana, etc are applicable clinically and every Ayurvedic physician has to use these tools to diagnose and treat the disease. Hence the basis used for categorization mentioned above can be reframed and institutes can follow newer trends in future i.e Fundamental and Applied branches of Ayurvedic specialities. Fundamental specialities like Kriya Shareera will be dealing more with the basic principles of Ayurveda and practical knowledge required for the concerned speciality. Whereas the application of the basic Principles of Ayurveda will be carried out by the applied branches of Ayurvedic specialities in diagnosing and treating patients. KEY WORDS: Kriya Shareera, clinical, non-clinical, fundamental, applied

Cite this article: Kamath. N., Kulkarni. P., Chiplunkar. S., (2013), CATEGORIZATION OF AYURVEDIC SPECIALITIES – PRESENT & FUTURE WITH SPECIAL REFERENCE TO KRIYA SHAREERA, Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 664–668

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INTRODUCTION Ayurveda is a science which deals with all the aspects related to life (Acharya YT, 2009). Ayurveda is considered as the Upaveda of Atharvaveda containing lakhs of verses and thousands of chapters (Acharya YT, 2009). Eight branches/specialities of Ayurveda as told in Ayurvedic Samhitas are Kaya (General Medicine), Bala (Paediatrics), Graha (Astrology), Urdhwanga (ENT, Opthomology), Shalya (Surgery), Damshtra (Toxicology), Jara (Geriatrics), Vrasha (dealing with impotency, sterility etc) (Paradhara HSS, 2005). The practice, prevalence popularity and status of Ayurvedic education have had many ups and downs in the time line of Ayurvedic education. In 1970, the Indian Medical Central Council Act which aimed to standardize qualifications for Ayurveda and provide accredited institutions for its study and research was passed by the Parliament of India (Wujastyk, 2008). In India, over 100 colleges offer degrees in traditional Ayurvedic medicine. The Indian government supports research and teaching in Ayurveda through many channels at both the national and state levels, and helps institutionalize traditional medicine so that it can be studied in major towns and cities. The state-sponsored Central Council for Research in Ayurveda & Siddha has been set up to research the subject (Central Council of research in Ayurveda and Siddha, 2013). To fight biopiracy and unethical patents, the Government of India, in 2001, set up the Traditional Knowledge Digital Library (TKDL) as repository of 1200 formulations of various systems of Indian medicine, such as Ayurveda, Unani and Siddha. (Wikipedia, 2013) The library also has 50 traditional Ayurveda books digitalized and available online. Central Council of Indian Medicine (CCIM) a statutory body established in 1971, under Department of Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homoeopathy (AYUSH), Ministry of Health and Family Welfare, Government of India, monitors higher education in Ayurveda (Wikipedia, 2013). Many new branches started for speciality studies by CCIM among which

Kriya Shareera (Ayurvedic Physiology) is one of the vital basic specialities. Kriya Shareera is the science which deals with the study of human body in relation to its physiological norms. This subject is of greater importance for medical students especially of Ayurveda to understand most of the clinical and paraclinical subjects. Even though many developments took place in the field of Ayurveda, there are some trends which can be reframed, among that, trend of classification of speciality branches in institutes as Clinical and Non clinical is the one. Specialities like Kaya chikitsa (General Medicine), Salya Tantra (Surgery), Salakya Tantra (ENT, opthomology) are considered as Clinical branches and specialities like Shareera Rachana (Ayurvedic anatomy), Dravya Guna (study of medicinal plants), Rasashastra (study of minerals), Bhaishajya Kalpana (pharmaceutical preparations) are considered as Non clinical specialities including Kriya Shareera. The study of Ayurveda requires the study of shareera (body), indriya (sense organs), satva (mind), atma (soul). This can only be done by systematic and continuous interaction with the subjects and patients. Therefore the practice of classification of streams of Ayurveda into clinical and non clinical will be contradictory with the traditional form of Ayurvedic education. This article intends to focus on usage of some of these terminologies and the relevance of their usage. The article also intends to look into some possible solutions and corrections to these discrepancies. DISCUSSION The main basis for development of trend to classify the specialities into clinical and non clinical is that, the faculties of the clinical branches will be taking history, examining and treating the patients whereas non clinical faculties including Kriya Shareera will not be doing this in the same frequency as that of the clinical specialities in almost all of the Ayurvedic academic institutes. The basis used for the classification and the classification trend

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can be proved as off the beam to certain extent by taking into account of various quotations mentioned in our classics and topics or syllabus allotted under the subject Kriya Shareera. Dosha (body humors), Dhatu (body tissues), Mala (by products) are considered as the basis for body (Paradhara HSS, 2005). This is the concept used by every physician when deals with patients i.e. by various techniques he will be examining the status of Dosha (bodily humors), Dhatu (tissues, organs), Mala (by products of various metabolisms) (Paradhara HSS, 2005). The status of Dosha is Vrudhi (increase), Kshaya (decrease), Prakopa (excessive increase) and of Dhatu and mala are Vrudhi and Kshaya. (Paradhara HSS, 2005). The Vrudhi, Kshaya Lakshanas (features) of Dosha, Dhatu, Mala quoted by different Acharyas comes under the purview of subject Kriya Shareera, in which each and every Lakshanas are understood systematically and scientifically as per CCIM curriculum (Central Council of Indian Medicine, 2013b). The study of these fundamentals requires continuous interaction and followup with the patients as the doshas can change based on time, food season and likewise. This requires a student of Kriya Shareera to be involved clinically too. This is even more necessary because the dosha and its variations cannot be done entirely in a laboratory without involvement with the patients. Every Ayurvedic physician has to use the concept of Vrudhi-Kshaya Lakshanas of Dosha, Dhatu, Mala while examining the patients. The physician who is unable to measure the status of biological humors can not intervene or stop the disease pathogenesis/can not cure the disease (Acharya YT, 2007). The techniques used to examine the status of disease and diseased are Trividha (three type) (Acharya YT, 2009), Astavidha (eight types of examination), Dashavidha Pariksha (ten types of examination) (Acharya YT, 2007). Among these Dashavidha Pariksha is widely used by the Ayurvedic physicians to examine the status of disease and diseased. The factors included in the Dashavidha Pariksha like Prakruti (body constitution), Sara (well

nourished body tissues), Samhana (physical functions), Satmya (compatability), Satva (mental functioning), Ahara (diet) & Vyayama Shakti (physical strain tolerance capacity) are the points that are dealt understood practically and systematically in the subject Kriya Shareera as per CCIM curriculum (Central Council of Indian Medicine, 2013a). Prakruti, its types, features, disease proneness and its importance in judgement of prognosis of the disease and therapeutic role are dealt in the subject Kriya Shareera. Prakruti is the major factor which has to be observed and recognised properly while examining each and every patient (Acharya YT, 2009). This will help to understand the disease proneness to that particular patient and also regarding his Kosta (gastro intestinal tract), Agni (Digestive fire) etc factors which aids in diagnosing the disease and also in fixing the dosage, time and medicine for administration. The other major factor is Sara Pariksha (examination of essence of particular tissue) which aids in the examination of the status of Dhatus and the predominance of the Dhatus in patients (Acharya YT, 2007). Sara is the topic which is dealt practically and scientifically in Kriya Shareera. Since Dosha and Dhatu are the prime factors involved in disease formation it should be examined. So the concept Sara pariksha used by the physician while dealing with patients is the topic dealt or learnt in the subject Kriya Shareera. The another important tool used by the physicians during history taking and examination is Pariksha Bhava (confounding factors) in which Dosha (body humors), Dushya (body tissues), Bala (physical strength), Anala (digestive capacity), Ahara (diet) etc things are included (Acharya YT, 2009). Among all these Agni (digestive fire) is an important factor which is responsible for the healthy and diseased state (Acharya YT, 2007). Agni is the concept which is dealt in detail practically and systematically under the purview of Kriya Shareera as per CCIM syllabus. Each and every Ayurvedic physician has to assess the status of Agni in the patient

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and application is by the knowledge that he got from Kriya Shareera. Similarly Ahara is an important factor which acts as cause for the disease and even can acts as medicine. The detail knowledge regarding the Ahara (food), its Parinamakara Bhavas (factors responsible for digestion), Ayatanas (rules) etc are dealt in Kriya Shareera. The practical application of Ahara and other factors related to it is very essential to understand the cause of the disease and to advice proper Pathya. Human being is a part of the eco system and he will be continuously interacting with the environment. As changes takes place in the environment including day to day changes and seasonal changes, accordingly changes takes place in the body, especially status of the Dosha. Predominance of particular Dosha in a particular time interval of the day and night, Sanchaya (accumulation), Prakopadi Avastha (state of excessive increase) of the Dosha in different Rutu (season), concept of Shat Kriyakala (six stages of disease formation) are the topics dealt under the purview Kriya Shareera and these concepts mentioned above every physician has to be observed in each and every patients before administrating medicines, especially concept of Shat Kriyakala should be applied during examination of the patient then only he will be successful in diagnosing the disease, treating it and will become successful physician (Acharya YT, 2009). Karya Samarambha and Pravruti are considered as the synonyms of Kriya (Acharya YT, 2007). Pravruti is Chikitsa (Acharya YT, 2007). Chikitsa is given to the Shareera. Hence in broader sense we can even consider administering medicine to the patient/diseased body as Kriya Shareera. Even the curriculum stipulated by CCIM for Kriya Shareera too emphasises on this and has suggested regular clinical examination of patients as a part of the syllabus (Central Council of Indian Medicine, 2013b). There are various developments taken place in the field of medicine from small thermometer to big CT, MRI, PET scans. So

the concepts from the contemporary science are also included in the Ayurvedic graduation and Post graduation courses. Some of the concepts like Pulse, Auscultation, Stethoscope, sphygmomanometer, Thermometer, ECG, various haematological and urinary investigations are studied systematically along with its practical implications under the subject Kriya Shareera. This is done to enable a post graduate of kriya shareera to express and validate some of the traditional Ayurvedic concepts in modern terminologies and methods. Every Ayurvedic physicians applies the knowledge of following concepts which help in diagnosing and treating the disease easily. Almost all the concepts that are dealt in Kriya Shareera are applicable clinically/while dealing with patients to find out the cause of the disease, diagnosis and treatment. Hence considering Kriya Shareera speciality under the category of Non-clinical branch needs to be addressed. Hence the basis of categorization of Ayurvedic speciality branches into clinical and non-clinical can be reframed. Instead of clinical and non-clinical categorization it is better to follow the new trend in future as- Fundamental and Applied branches of Ayurvedic specialities. Fundamental specialities are those which deals with basic principles, original, essentials and primary form of Ayurveda, it aims at going to the root of matter. It is more concentrated on the theoretical aspects and concepts related to Ayurveda along with the practical knowledge concerned to that particular subject. Specialities like Shareera Rachana, Kriya Shareera, Dravya Guna etc can be included. Those specialities which apply the fundamentals while dealing with patients in knowing the cause, diagnosing the disease and treating can be included under the applied branches. Specialities like Kaya Chikitsa, Salya and Salakya Tantra etc branches can be included under applied branches. CONCLUSION Understanding of Kriya Shareera will not be complete without regular follow up and interactions with the patients. Therefore there is a need to increase the frequency of patient

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interaction for the students of Kriya Shareera. Non clinical teaching may not serve this particular purpose.

that come under the purview of Dravya Guna, Shareera Rachana are also applicable clinically.

The basis for classification of specialities can be reframed since the concepts of Kriya Shareera like Dosha, Dhatu, Mala vrudhikshaya (increase-decrease of humors, tissues, by product of metabolism), Prakruti pariksha (examining body constitution), Agni Parikshana (examining digestive fire), Ahara (food), Rutu (season) and Vyadi Kriya Kala (stages of disease) etc are applicable clinically and every Ayurvedic physician has to use these tools to diagnose and treat the disease of the patients. In broader sense we can even consider administering medicine to the patient/diseased body as Kriya Shareera. Similarly the concepts

The basis for categorization can be reframed and a new trend can be followed in future as - Fundamental and Applied branches of Ayurvedic specialities in future. Fundamental specialities will be dealing more with the concepts and basic principles related to Ayurveda along with the practical knowledge required for the concerned specialities. Whereas the application of the fundamentals and basic Principles of Ayurveda will be carried out in the applied branches of Ayurvedic specialities i.e in diagnosing and treating the patients.

REFERENCES Acharya YT (2007). Charaka Samhita with Ayurveda Dipika commentary of Chakrapani Datta: Chowkambha Orientalia, Varanasi, Reprint ed.187,273â&#x20AC;&#x201C;4,512.

Central Council of research in Ayurveda and Siddha, (2013) Retrieved from http://www.ccras.nic.in/index.htm (Accessed on 28/06/2013).

Acharya YT (2009). Sushrutha Samhita with Nibandhasangraha commentary of Dalhana: Chowkambha Orientalia,Varanasi, Reprint ed. 2,106,148,255.

Paradhara HSS (2005). Ashtanga Hrudaya with Sarvangasundara commentary of Arunadatta and Ayurvedarasayana commentary of Hemadri: Chowkabha Orientalia publisber,Varanasi, Reprint ed.5,67,183, 201 .

Central Council of Indian Medicine (2011a). Retrieved from http://ccimindia.org/ (Accessed on 01/07/2013)

Wikipedia (2013), http://en.wikipedia.org/wiki/Ayurveda (Accessed on 01/07/2013)

Central Council of Indian Medicine (2011b). Retrieved from http://Central Council of Indian Medicine.2013/syllabus/first_year_sylla bus.html (Accessed on 01/07/2013)

Wujastyk (2008). Retrieved from http://www.zoominfo.com/p/DominikWujastyk/41514036 (Accessed on 01/07/2013)

Source of Support: Nil

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 9 | September 2013 | 669–674 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article A REVIEW ON POLYGONUM BISTORTA L. WITH REFERENCE TO ITS PHARMACOLOGY & PHYTOCHEMISTRY Adiba Mehar1*, Hussain I Mohammed Tabarak2 1

PG Scholar, Dept of Ilmul Advia, NIUM, Bangalore, Karnataka, India Lecturer, Dept of Ilmul Advia, HMS Unani Medical collge, Tumkur, Karnataka, India *Corresponding/ main author: Email Id: drmeharadiba@gmail.com Mobile: +919019676981 2

Received: 20/07/2013; Revised: 22/08/2013; Accepted: 31/08/2013

ABSTRACT Herbal medicine is defined as a plant-derived product used for medicinal and health purposes. Polygonum bistorta Linn is a plant origin drug used for many ailments by the traditional practitioners. Many of the phytochemical and pharmacological studies have also been carried out scientifically to evaluate and explore its pharmacological potentials in the recent 2 decades. This is a brief review of its botanical description, its scientific studies, biological activities, some of their compounds isolated, pharmacological actions and plausible medicinal applications of Polygonum bistorta Linn along with its safety evaluation KEYWORDS: Polygonum bistorta Linn, Anjabar, Phyto-chemistry, Pharmacology, Unani Drug,

Cite this article: Adiba. M., Hussain I. M. T., (2013), A REVIEW ON POLYGONUM BISTORTA L. WITH REFERENCE TO ITS PHARMACOLOGY & PHYTOCHEMISTRY, Global J Res. Med. Plants & Indigen. Med., Volume 2(9): 669–674

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INTRODUCTION: Humans have been using plant products for medicinal purposes since the Neanderthal period, i.e. 6000 years ago. Herbal medicines include a wide spectrum of substances ranging from home-made teas prepared from collected herbs to medicinal products that are approved by national regulatory bodies (Hodges et al., 2002). Polygonum bistorta Linn, belonging to the family Polygonaceae is also an herbal medicine used as a haemostyptic drug by traditional practitioners in many diseases associated with bleeding (Wealth of India, 2003). Unani physicians have many claims regarding its therapeutic uses such as Habis dam (haemostyptic), Qabis (astringent), Mudir (diuretic), Munaffis (expectorant), Muqee (emetic) etc (Wealth of India, 2003; Prajapathi et al., 2005; Hakim 2002; Pillai KM, 2006) few of its medicinal properties are already been scientifically evaluated and many are yet to be evaluated. Taxonomical classification (Wealth of India, 2003)

base, and becoming smaller higher up the stem with winged petioles; topped by a cylindrical cluster of small pink-rose to white colored flowers; fruit small triangular black and shiny; Stem solitary, simple, erect, straight leafy, 1.5−2 feet height, round striated and smooth (Lindley, 2008) from which raises rhizomes which are rough and blackish red colored (Kabiruddin, 2007); Rootstock is somewhat flattened, hard with annual thickenings and traces of rootlets . Habitat and Distribution: Bistort is native to Europe, Asia and North America and in India it is distributed in the Himalayas from Kashmir to Sikkim and in the hills of Assam at the altitudes of 2,700– 4,500 m. Parts used medicinally: Root, rhizome (Wealth of India, 2003; Kabiruddin, 2007; Baitar, 2000), leaves (Baitar, 2000) Temperament:

Kingdom- Plantae; order- Caryophyllales; Family- Polygonaceae; Genus- Polygonum; Species- bistorta.

Cold and Dry 3º (Hakim, 2002), Cold and Dry 1º (Kabiruddin, 2007, Baitar, 2000)

Vernacular names (Hakim, 2002; Kabiruddin, 2007),

Pharmacological actions: (Wealth of India, 2003; Kabiruddin, 2007; Prajapathi, et al., 2005; Baitar, 2000)

English- Bistort, Snake root, Adder wort, Dragon wort; Persian- Angabar; Arabic, UnaniAnjabar Botanical Description: Polygonum bistorta Linn commonly known as Bistort or Snakeroot belongs to the family Polygonaceae (Manoharan KP, 2005; Pillai KM, 2006). It is a perennial herb usually grows between grasses near water (Baitar, 2000). It is tall with slender stems, grows upto 60 cm of height with a thick, twisted rootstock; Leaves are produced near the lower end of the flowering stems. They are radical, lanceolate, cauline, oblong-ovate or triangular-ovate in shape, bluish green in colour, longer near the

Astringent, Haemostatic, Antidiarrhoeal, Antidysenteric, Febrifugal, Diuretic, Expectorant, Antiseptic, Antihelminthic, Gastric Tonic, Haematuria, Emetic. Therapeutic Uses Haemopoitic system P. bistorta is a very good haemostyptic hence it is used in the diseases with bleeding as a symptom such as menorrhagia, bleeding haemorrhoids, haematuria, epistaxis, haemoptysis, dysentery etc (Baitar, 2000; Prajapathi et al., 2005; Kabiruddin, 2007)

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Gastro-intestinal tract

Scientific reports

It is one of the strongest herb astringents. It is excellent for the treatment of cholera, diarrhea and dysentery. It is also used in jaundice, irritable bowel syndrome, peptic ulcers, ulcerative colitis and worm infestations. Since it is an astringent it is also used in bleeding haemorrhoids (Manoharan KP, 2005; Pillai KM, 2006, Kabiruddin, 2007)

Phytochemical reports on P. bistorta

Urogenital system

It is a very good astringent hence used in cystitis and haematuria (Manoharan KP, 2005; Pillai KM, 2006)

Skin When applied to a wound, it stops bleeding. It has been used in traditional Chinese medicine as a remedy for smallpox, measles, and pimples (Pillai KM, 2006) Antidote

It has been used in traditional Chinese medicine as a remedy for Insect stings and snake bites (Pillai KM, 2006) Adverse Effect (Kabiruddin, 2007) 

For cold temperament individuals Correctives (Kabiruddin 2007) Habbul Aas (Myrtus communis Linn) Substitutes (Kabiruddin 2007) Habbul Aas (Myrtus communis Linn) Dose (Baitar, 2000; Hakim, 2002; Kabiruddin, 2007) 3−4 gram Unani Compound Formulations 2000; Hakim 2002; Kabiruddin 2007)

(Baitar, 

Sharbat Anjabar, Safoof Istehaza, Majoon Tewaj

Tannin compound 15−22% usually mixed type; catechol, phloroglucinol, gallic acid and phlobaphene in roots, and 5−10% in leaves. Methyl anthraquinone, calcium oxalate (1.1%), starch 30%. albumin 10% and emodin in traces. (Wealth of India 2003). Caffeic, chlorogenic and protocatechnic acid and Ascorbic acid; flower: 746.6, leaves: 722.3 and rootstocks 132.2 mg/ 100 g (Wealth of India 2003). Catechin, epicatechin, flavonoids, aromatic compounds, flavonoid glycosides, sitosterols, a few pentacyclic triterpenoids, 5-glutinen-3-one (alnusenone) and friedelinol (Pillai 2005, Pillai, 2006) B -sitosterol, friedelin and 3 b –friedelinol and seven known compounds were isolated through 2D NMR spectroscopy (Pillai, 2005; Pillai, 2006) A new tannin-related compound named bistortaside A and a new compound was elucidated as 3-methyl-gallic acid 4-O-β-D(6′-O-3″-methyl-galloyl)-glucopyranoside and a known compound was quercetin-3′O-β-D-glucopyranoside on the basis of spectroscopic analysis (Liu et al., 2006). Essential oils of rhizome of PB were extracted by hydro distillation and analyzed by GC-MS. 81 compounds were obtained and 77 were successfully identified. The percentage yield ranged between 0.11−0.29 %. A significant difference in their chemical composition was also found. Major constituents and their percentage ranges were determined as: furfural (0.4−16.4 %), oleic acid (4.3−8.9 %), oleic acid methyl ester (0.3−8.6 %), palmitic acid (4.8−6.6 %), 5-methyl furfural (0.5−6.5 %), linoleic acid (0.6−4.2 %), linoleic acid methyl ester (0.2−4.0 %) and cosanes (tetracosane to nonacosane) (Intisar et al., 2012). Coagulant Compounds from powder of Rhizomes of P. bistorta were separated from the flour and identified chemically.

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Two of these were identified as flavonol of flavonoids. They were 2, 3’,4’,4,6pentahydroxy flavone and 2,5’,6trihydroxy-4,2’-dimethoxy flavones (Partovia and Zabihia, 2012). Various Pharmacological activities reported on P. bistorta Anti-inflammatory activity The ethanolic extract showed strong antiinflammatory effect (Duwiejua et al., 1994); alnusenone and 3bfriedelinol were identified as active constituents for such effect (Duwiejua et al., 1999).

50 percent growth inhibition (GI50) values for five most active fractions were calculated and results were in a range of 86.5 (±3) −126.8 (±3) µg/mL. Hence P. bistorta showed a definite dose dependent relationship between amount of fractions and cytotoxic activity (Intisar et al., 2013). Hepatoprotective activity P. bistorta showed hepatoprotective activity in Albino Rats with Carbon Tetrachloride and Paracetamol induced Hepatotoxicity (Kumar et al., 2012a). PB is also found to be effective in treatment of hepatic damage induced by DNA damage by comet and MTT-assay in liver (Kumar et al., 2012b).

Antiarthritic activity CNS inhibitory and hypnotic activity Aqueous and ethanolic extracts of root have shown very good effect in arthritis in experimental animals (Pillai KM, 2006). Mutagenic activity It was also reported that the aqueous extract strongly inhibits the mutagenicity of Trp-P-1 (Miki et al., 1994; Pillai KM, 2006). Anticancer and cytotoxic activity Chloroform and hexane fractions and a few sub-fractions of P. bistorta showed moderate to very good activity against P388, HL60 and LL2 cancer cell lines (Manoharan KP, 2005). Anticancer phenolic compounds such as gallic acid, proto-catechuic acid, phydroxybenzoic acid, chlorogenic acid, vanillic acid, syringic acid, catechol, 4-methyl catechol, syringol and pyrogallol and fatty acids such as linoleic acid, myristic acid and palmitic acid were separated from different fractions from HPLC which were evaluated for their cytotoxic activity on a rarely studied human hepatocellular carcinoma cell line (HCCLM3). 11 fractions showed good to strong cytotoxicity in a range of 200−800 µg/mL, whereas 2 fractions did not show any activity even at 800 µg/mL and no anticancer constituent was detected from them.

n-Butyl alcohol extract of P. bistorta inhibited the spontaneous activity in mice. It also shortened the hypnagogic time of pentobarbital sodium and prolonged its hypnotic effect with pentobarbital sodium (Jing et al., 2003). Analgesic activity n-Butyl alcohol and water extract of P. bistorta could reduce the writhing times of the mice induce by acetic acid and raised threshold of pain induced by hot and electric stimulation. The antagonist naloxone could not reverse the analgesic effect of Polygonum bistorta L. nButyl alcohol extract (Yu-shan et al., 2004, Jing et al., 2005). Antimicrobial activity P. bistorta L. shows antimicrobial activity against Staphylococcus aureus and E. coli (Chuen et al., 2006). Antioxidant activity n-butyl alcohol extract of P. bistorta has obvious protective effects on myocardium in a dosage-dependent manner by increasing the superoxide dismutase and decreasing malondialdehyde in myocardial tissue,

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decreasing serum lactate dehydrogenase and creatine phosphokinase, eliminating free radicals, and preventing lipid peroxidation (Heyang et al., 2005). CONCLUSION The information regarding the herbal Drug P. bistorta is in accordance with the literature available in both Unani and modern books and also the publications reports available online. Since past decade the natural compounds and

the herbal medicine has received more attention towards their scientific development with respect to safety and efficacy. This is a classical approach to explore novel medicines for various diseases affecting human beings worldwide. P. bistorta is such an herbal drug which is being used by Unani, Ayurveda and the traditional practitioners for its medicinal values. Some of the therapeutic properties of it are been already explored by the researchers and many are yet to be explored.

REFERENCES Anonymous., (2003). The wealth of India; A Dictionary of Indian Raw materials and Industrial products. NISCAIR, New Delhi. 8: 193−196 Baitar I., (2000). Jameal mufradath wal Advia wal Aghzia, CCRUM, New Delhi.1: 138 Chuen

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


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Source of Support: Nil

Conflict of Interest: None Declared

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