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Evaluation of seeds of Celastrus paniculatus Willd. – A potential medicinal plant of Pharmacopoeia

Abstract : Celastrus paniculatus Willd. (Celastraceae) is a potential medicinal plant extensively used in ayurvedic medicinal systems. The seeds oil of these medicinal plants is used for the treatment of large numbers of diseases. The seeds of this plant were collected from four regions of Jharkhand state and the various constitution and oil were compared with the parameters described in the Ayurvedic pharmacopoeia of Govt. of India. The percentage of oil contents of Celastrus paniculatus Willd. ranges from 43.2 to 44.67 % while the contents of the oil mentioned in pharmacopeia is 45%. The similarities in other contents like foreign matter, total ash, acid insoluble ash, alcohol soluble extract and water soluble extract too is identical. It is, therefore, suggested that the Celastrus paniculatus Willd. should be protected and awareness should be generated amongst public private sector for the production of seed oil at cheap rate for medicinal use.

Keywords: Celastrus paniculatus, Seed oil, Pharmacopoea, Quality evaluation

Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 1-2

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Kaushal Kumar, S. G. Abbas and M.H. SiddiquiFaculty of Forestry, Birsa Agricultural University, Kanke, Ranchi-834006Email: [email protected]

Introduction

Celastrus paniculatus Willd. is a large climbing deciduous shrub, yellowish wood, with lenticelled branches. It belongs to family Celastraceae. The plant mostly occurs in sub Himalayan tract up to 6000 ft. It is also found in Central India, Western and Eastern Ghats. It is extending from Rajmahal hills and Chhotanagpur plateau of Jharkhand and Orissa states. It is one of the potential medicinal plants of India. The plant is known in Sanskrit and ayurveda as 'Jyotishmati'. It is principally a 'Medhya drug'. In ayurvedic medicine it is used as a brain tonic and also used in treatment of some the nervous disorders. The plant is utilized as ethnomedicine among the tribal people and they call it 'Kujri' 'Malkangni', 'Konjri' 'Kusur', 'Rangud' etc. The seeds oil is utilized topically as well as internally among the tribal people ((Jain, 1991). It is also used for bodyache, rheumatism, leprosy, eczema and skin diseases (Asolkar et al. 2005; Atal et al., 1978 ; Handa, 1988; Rekha et al., 2005). According to Bhanumathy et al, (2010). the seeds have great potentials as acrid, bitter, thermogenic, emollient, stimulant, intellect promoting, digestive, laxative, emetic, expectorant, appetizer, aphrodisiac, cardio tonic, anti-inflammatory, diuretic, diaphoretic, febrifuge and tonic, abdominal disorders, leprosy, skin diseases, paralysis, cephalalgia, arthralgia, asthma, leucoderma, cardiac debility, inflammation etc. The oil contains fatty acids composition, sesquiterpene alkaloid viz. celapanin, celapanigin and celapagin and a number of sesquiterpene esters namely malkanguine- I to VIII and sterol. The present work is based to confirm the contents of quality of seeds and their oil of Celastrus paniculatus useful for the use of illness as reported by Department of AYUSH, Ministry of Health and Family Welfare, Govt. of India, New Delhi.

Methodology of works

The seeds of Celastrus paniculatus Willd were collected from four different places from the district of Ranchi (Jonha), Gumla (Bishunpur), Dumka (Baghraidih) and Pakur (Satia) and in Jharkhand state. They were collected with the help of native people in the months of December- January. The above seeds sample were brought

o oto laboratory and dried in an oven at 45 C to 50 C. The opowdered of the dried seeds was made at 25 C and kept in

airtight container. The identity, purity and strength of samples of seeds have been evaluated according to the methods upon which the standards of Pharmacopoeia depend.

Results

The samples were collected from four sites of Ranchi (Jonha), Gumla (Bishunpur), Dumka (Baghraidih) and Pakur (Satia) designated as SS-1, SS-2, SS-3 and SS-4 respectively. The % of foreign matter, the total ash, acid soluble ash, alcoholic soluble extract, water-soluble and oil contents from sample of Ranchi (Jonha, SS-1), Gumla (Bishunpur, SS-2), Dumka (Baghraidih, SS-3) and Pakur (Satia, SS-4) were presented in Table1. The seeds were separated in the laboratory and the color and identical seeds in structure and diameter were recorded (Fig.1). The seeds were processed for the foreign matter, total ash, acid insoluble ash, alcohol soluble extract, water soluble extract and oil contents were studied and presented in Table 1. The table also contains the data available in Ayurvedic Pharmacopoea for comparison to know the purity and strength of the contents of the seeds obtained from four locations of Jharkhand. It has been observed that all parameters are similar with the reported parameters in the Ayurvedic pharmacopoea.

Discussion

In the present investigation, genetic resources of Celastrus paniculatus available in Jharkhand has almost standard in quality for the extraction of oil contents for the preparation of ayurvedic medicine at a cheaper cost as the contents of oil of this plant is identical with the contents as per Ayurvedic Pharmacopoeia. The mean of samples taken from four different places is about 44.92, which is similar

Parameters/ Samples

Foreign Matter

(%)

Total ash (%)

Acid-insoluble Ash (%)

Alcohol soluble extractive

(%)

Water soluble extractive (%)

Oil contents (%)

Ayurvedic Pharmacopoea

2.0 6.0 1.15 20.0 9.0 45.0

SS-1 2.1 5.1 1.16 18.3 9.1 44.1 SS-2 1.9 5.2 1.17 19.2 8.95 43.2 SS-3 2.2 5.4 1.10 19.6 8.73 44.4 SS-4 1.8 6.1 1.14 19.5 8.5 44.67 Mean 2.0 5.7 44.92

to 45% described in Ayurvedic Phamacopoea, Government of India. The above studies have great significant towards understanding of seed samples of the plant occurs in Jharkhand state for bioprospection in perspectives to pharmaceutical standards. The above data analyzed in the present work is most useful for manufactures of herbal drugs and other products based on the Celastrus paniculatus.

Table 1. : Identity, purity and strength of samples of seeds

Acknowledgements

The authors are grateful to the native peoples of different districts of Jharkhand who have provided the samples of seeds used in the evaluation of present works.

References

Asolkar L.V., Kakkar K.K. and Chakre O.J. (2005): Second supplement to Glossary of Indian Medicinal Plants with active principles, NISCARE, New Delhi.188-189.

Atal C.K, Srivastava J.B., Wali B.K., Chakravarthy R., Dhawan B.N. and Rastogi R.R. (1978): Screening of medicinal plants for Pharmacological activity Part III Indian Journal of Experimental Biology 16: 330-349.

Bhanumathy M., S. B. Chandrasekar S.B., Chandur U, Somasundaram T. (2010): Phyto-pharmacology of

Celastrus paniculatus: An Overview. International

Fig-1. : Seeds of Celastrus paniculatus

Journal of Pharmaceutical Sciences and Drug Research 2 (3): 176-181

Handa S.S. (1988): Indian Herbal PharmacopoeiaCelastrus paniculatus, IDMA, Mumbai and RRL, Jammu, 1998, II: 26-34.

Jain S.K. (1991): Dictionary of Indian Folk Medicine and Ethnobotany, (Deep Publication, New Delhi), 48.

Rekha K., Bhan M .K, Balyan S.S. and Dhar A.K. (2005): Cultivation prospects of endangered species Celastrus paniculatus Willd. Natural Product Radiance 4(6):482- 486.


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Foraminiferal studies along the littoral zone of the east coast of India and its zoogeographical affinity: an overview

Abstract: An overview of the studies, mostly related to the taxonomy, ecology of the littoral Recent foraminifera (Protozoa) and the zoogeographical affinity of the east coast of India has been attempted on the basis of the published literature. The study shows that the Recent foraminifera along the east coast of India show affinity with the Indo-Pacific faunal province.

Keywords: Foraminifera, Taxonomy, East coast beaches, India

P. K. Kathal and V. K. SinghCentre of Advanced Study in GeologyDr. Harisingh Gour University, Sagar 470003, IndiaE-mail : [email protected]

Introduction

Foraminifera, the shelled protozoans thrive as the most diverse group in the modern oceans. They derived their name from 'foramen' the 'pores' connecting the chambers in their tests. Varying in size/diameter size from less than a millimetre (micro-foraminifera) to 100 mm (mega-foraminifera they also occur as fossils in the marine Phanerozoic rocks (560 Million Years to Recent) and a wide range of applications in geological, biological, environmental and oceanographic studies.

Some of foraminifera are agglutinated that acquire foreign-material to make a test, but most of them form calcareous 'test' (shell) by secreting calcium carbonate present in dissolved state in the marine waters. Being unicellular, highly abundant in sea and very sensitive to even minute changes in the environmental parameter, their ecological studies help deducing the paleoenvironmental conditions of the sediments/rocks besides wide geological applications, like- oil exploration, higher resolution biostratigraphy, environmental /paleoenvironmental interpretations. Their 'planktic' forms display 'surface water-' while the 'benthic' species help studying the 'bottom water' conditions of the oceans of the present/past.

Although the studies on Recent foraminifera along the east coast of India started quite early indeed (Schwager, 1857; Chapman, 1895; Hofker, 1927 & 1933; and Cushman, 1939) yet it received less attention in comparison to the studies carried out, elsewhere.

It has been endeavoured here to make a review of the work conducted on the Recent foraminiferal fauna obtained from the littoral-zone along the east coast of India in order to pave way for future studies in this field of research.

Observations

Sastry (1963), Shetty (1982) and Khare et al. (2007) have reviewed the literature but no detailed account on studies made exclusively on the Recent foraminifera from the exposed littoral-zone of the eastern

coast of India has been given.

So far as the beaches of 5800 km long coastal stretch of the east coast of India are concerned, a total of 27 papers have been published (Table 1) covering taxonomy, ecology, geographical distribution of the Recent foraminifera and establishing the zoo-geographical affinity of the east coast of India based on the Q-mode cluster analysis (Kathal, et al., 2000 and Kathal and Bhalla, 2001). Besides, paleoenvironment/climate studies (Kathal, 1996 and Kathal and Bhalla, 1996a) show their utility in petroleum exploration.

Taxonomic and ecological studies

The taxonomic and ecological studies on the Recent foraminifera in the beach sediments beaches along the east coast of India (Fig 1) include the work carried out by Sarojini (1958), Bhatia and Bhalla (1959), Bhalla (1968, 1970), Gosh (1966), Ameer Hamsa (1971, 1973), Kathal (1989, 1991, 1999, 2002a, 2002b, 2004), Bhalla and Kathal (1998), Kathal and Bhalla (1996b, 1998), Kathal and Matoba (2001), Kathal and Singh 2010), Singh (2009) and Singh and Kathal (2010).

Singh and Kathal (2011) compared foraminiferal species from littoral-zone along east coast of India with that of the east coast of Japan.

Foramgeographical affinity of the east and west coasts of India

The cluster analysis of the Recent foraminiferal assemblages obtained from the 26 beaches from the two coasts was carried out to delineate their foramgeographical affinities. The study shows that the east coast belongs to the warm water 'Indo-Pacific' realm while the west coast shows close affinity to the 'East African' realm with a 'mixed-zone' in between (Kathal et al., 2000) and Kathal and Bhalla (2001).

Morphological aspects of foraminifera

Importance of various morphological features and their response to ambient physico-chemical conditions has been well recognized. Along the east coast of India


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Fig.1 : Beaches, Littoral-Zone and bathymetry, east cost of India

Location map

east coast of India


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Kathal and Bhalla (1996b) studied migratory trends and morphological variations in Rotorboides granulosum – a less known Recent foraminifera of tropical region that lived in 'low oxygen' conditions between latitudes 25 N° to 25° S in shallow warm water conditions since Middle Miocene. (23.03 million year ago).

Table 1 - Major work carried out on littoral sediments region along the East coast of India.

Environmental/climatic inferences from foraminifera

Studies have been made to infer/reconstruct paleoclimatic changes (Kathal, 1996 and Kathal and Bhalla, 1996c). Accordingly, these micro-organisms have emerged as reliable proxies of paleoclimatic/ paleoenvironmental conditions. However, studies on the role of foraminifera in addressing various issues related to climatic/ environmental changes especially along the littoral regions of east coast of India still has a lot of scope.

Sr. Year Author Area Remarks

1. 1958 Sarojini Bay of Bengal Studies on littoral foraminifera

2. 1959 Bhatia & Bhalla

Beach sand of Puri Recorded and illustrated 14 species of Recent foraminifera

3. 1966 Ghosh Digha beach Southern Bengal

Asterorotalia trispinosa (Thalmann) aspinose rotalid

4. 1968 Bhalla Visakhapatnam Recent foraminifera and its relation to foramogeographical province in Indian Ocean

5. 1970 Bhalla Marina beach (Madras)

Identified 15 species of foraminifera

6. 1971 Ameer Hamsa

Palk Bay, Gulf of Mannar

Reported some foraminifera

7. 1973 Ameer Hamsa

Palk Bay, Gulf of Mannar

Listed 34 species from the beach sands and illustrated 12 species

8. 1989 Kathal East coast of India Recent foraminifera from the beach sands

9. 1991 Kathal Puri to Vishakhapatnam

Recent foraminifera from the beach sands of the east coast of India (Puri toVishakhapatnam)

10. 1996 Kathal Kakinada Bay Significance of ecophenotypes inpaleolatitudinal interpretations

11. 1996a Kathal, & Bhalla

East coast of India Distribution of Recent foraminifera from littoral zone

12. 1996b Kathal, & Bhalla

East coast of India Intraspecific variation and Palaeolatitudinal significance of Rotorboides granulosum

13. 1996c Kathal & Bhalla

East coast of India Migratory trends and paleolatitudinalsignificance of Rotorboides granulosum

14. 1998 Bhalla & Kathal

Gulf of Mannar Reported 43 species of Recent foraminifera discussed the ‘mixed-zone’ of the East African and Indo-Pacific realms


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Sr. Year Author Area Remarks

15. 1998 Kathal & Bhalla

Palk Strait & Kakinada Bay

Presented the taxonomic observations of Rotorboides granulosum

16. 1999 Kathal Gulf of Mannar Foramgeographical affinity of the Indian Ocean during Quaternary

17. 2000 Kathal et al.

East and West coasts of India

Statistical treatment of 26 beaches, coasts of India showed ‘Indo -Pacific’ affinity to the east coast of India (up to Vedranniyam) and the east African affin ity to the west coast of India

18. 2001 Kathal & Bhalla

Indian coasts Reported 56 species and discussed the foramgeographical affinity of east coast of India with the known foramgeographical provinces

19. 2001 Kathal & Matoba

East coast of India and Japan

Reported 85 common species of the two distantly located areas within the warm -water Indo-Pacific province

20. 2002a Kathal Sixteen stations Taxonomy and distributional pattern of Recent foraminifera

21. 2002b Kathal Sixteen stations Distribution and ecology of Recent species

22. 2004 Kathal Kakinada bay Biostratigraphic and paleolatitudinalimplications of ecophenotypes

23. 2009 Singh East coast of India and Japan

Morphological comparison 41 common species out 120 species.

24. 2010 Kathal & Singh

Seven beaches First reported of 12 foraminiferal species from the Indian water

25. 2010 Singh & Kathal

Five beaches First reported of 12 foraminiferal species from the Indian water and 1 from east coast

26. 2011 Singh & Kathal

East coast of India and Japan

Out of 41species, 20 show narrow; 17 show medium; and 4 show medium to wide ranges of intraspecific variations

References

Ameer Hamsa K. M. S. (1971): Some foraminifera from Palk Bay and Gulf of Mannar. In: Sym. Ind. Ocean Adjacent Sea, Sec.10. J. Mar. Biol. Assoc., Ind. 28.

Ameer Hamsa K. M. S. (1973): Foraminifera of the Palk Bay and Gulf of Mannar. J. Mar. Bio. Assoc.,14: 418-423.

Bhalla S. N. (1968): Recent foraminifera from Vishakhapatnam beach sands and its relation to the known foramgeographical provinces in the Indian

Ocean. Bull. Nat. Inst. Sci. Ind. 376-392.

Bhalla S. N. (1970): Foraminifera from Marina beach sands of Madras and faunal provinces of the Indian Ocean, Contri. Cushman Found. Foram. Res. 21: 156-163.

Bhalla S. N. and Kathal, P. K. (1998): Recent foraminiferal thanatocoenoses from the Gulf of Mannar, India. N. Jb. Geol. Paläont. Abh. 207: 419-431.

Bhatia S. B. and Bhalla, S. N. (1959): Recent foraminifera from beach sand at Puri, Orissa. J. Pal. Soc. Ind. 4: 78-81.


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Chapman F. (1895): On some foraminifera obtained by the Royal Indian Marine Survey's SS Investigator from the Arabian Sea, near Laccadive Islands. In: Proc. Zool, Soc., London, 1-55.

Cushman J. A. (1939): Notes on some Pliocene foraminifera described by Schwager from Car Nicobar. J. Geol. Soc., Japan, 46: 149-154.

Ghosh B. K. (1966): 'Asterorotalia trispinosa' (Thalamann), A spinose rotaliid foraminifera from Digha Beach, southern Bengal. Contri. Cushman Found. Foram. Res. 17: 104-108.

Hofker J. (1927): Foraminifera of the Siboga Expedition, Part1 Siboga Exped. Monogr. 4: 1-78.

Hofker J. (1930): Foramenifera of the Siboga Expedition, Part 2 Siboga Exped. Monogr. 4(A): 79-170.

Kathal P. K. (1989): A study of Recent foraminifera from the sandy beaches of east coast of India. Ph.D. Thesis, Agra University.

Kathal P. K. (1991): Studies on Recent foraminifera from sandy beaches of the east coast of India (Puri to Vishakhapatnam). Project Completion Report, UGC/8/3[1]88[SR-II], 22p., pls. 6.

Kathal P. K. (1996): Foraminiferal ecophenotypes in paralic environment: significance in paleolatitudinal interpretations. In: Proc. IX Ind. Geol. Congr. (1993): 120-123.

Kathal P. K. (1999): Foramgeographical affinity of Indian Ocean during Quaternary based on the Recent foraminiferal thanacoenoses, Gulf of Mannar India. Tiwari, M. P. & Mohabay, D. M. eds. Gond. Geol. Mag., spl. pub. 4: 281-292.

Kathal P. K. (2002a): Taxonomy, distribution pattern and ecology of the Recent littoral foraminifera of the east coast of India. N. Jb. Geol. Paläont. Abh. 224(1): 115-160.

Kathal P. K. (2002b): Distribution and ecology of Recent foraminifera from littoral sediments of eastern India. J. Geol. Soc. Ind. 60: 429-454.

Kathal P. K. (2004): Ecophenotypes in Recent foraminifera, Kakinada bay, east coast of India: biostratigraphic and paleolatitudinal implications in Tertiaries. In: 4th Inter. Conf. Environ. Micropal. Microbio. Meioben. (EMMM 2004), Turkey, 101-102.

Kathal P. K. and Bhalla, S. N. (1996a): On the first report from the Indo-Pacific region, migratory trends and paleolatitudinal significance of Rotorboides granulosum – a tropical water foraminifer since middle Pliocene (late Miocene?), In: Proc. XV Indian Colloq., Micropal, Stratigr. Ed. (J. Pandey, R.J. Azmi, A. Bhandari and A. Dave), pp. 317-320.

Kathal P. K. and Bhalla, S. N. (1996b): A note on recent

foraminifera from littoral zone along east coast of India. Bull. Ind. Geol. Assoc. 29(1-2): 89-94.

Kathal P. K. and Bhalla S. N. (1996c): Intraspecific variation and Palaeolatitudinal significance of Rotorboides granulosum- less known recent foraminifera of tropical waters. Rev. de Paleobio. 15(10): 79-85.

Kathal P. K. and Bhalla S. N. (1998): Taxonomy and paleolongitudinal significance of Rotorboides granulosum – a less known Recent foraminiferal of tropical region, J. Geol. Soc. Ind. 51: 799-802.

Kathal P. K. and Bhalla S. N. (2001): Blending of Recent foraminiferal biogeoraphical provinces of India waters- a statistical approach. N. Jb. Geol. Paläont., Abh. 4: 250-256.

Kathal P. K. and Matoba Y. (2001): Morphological variations in the common elements of Recent 'Indo-Pacific' and 'Atlanto-Caribbean' foraminifera, off the eastern Japanese and Indian coasts. In: Neogene Climate, Indian Institute of Technology, Kharagpur, India. (Ed) A.K. Gupta, IIT Kharagpur Pp. 20-21.

Kathal P. K. and Singh V. K. (2010): First report of some Recent benthic foraminifera from the east coast of India. J. Geol. Soc. Ind. 76(1): 69-74.

Kathal P. K. Bhalla S. N. and Nigam R. (2000): Foramgeographical affinities of the west and east coast of India: An approach through cluster analysis and comparison of taxonomic, environmental and ecological parameters of Recent foraminiferal thanatotopes, In: Proc. XVI Indian Colloq. Micropal. Stratigr., NIO, Goa. R. Nigam and A. K. Bhandari ed. (Bull. ONGC), 37: 65-75.

Khare N., Chaturvedi S. K. and Mazumdar A. (2007): An overview of foraminiferal studies in near shore regions off eastern coast of India and Andaman and Nicobar Islands. Ind. J. Mar. Sci. 36: 288-300.

Sarojini D. (1958): Studies on littoral foraminiferal from the Bay of Bengal. Ph. D. Thesis, Andhra Univ.

Sastry V. V. (1963): Bibliography of papers published since 1939 on foraminifera from the Indian region. Micropal. 9: 107-110.

Schwager C. (1866): Fossile foraminiferen von Kar Nikobar, Reise der Osterreichischen Fregatte Novara um Rede inden Jahren 1857, 1858, 1859. Unter den Befehlem des Commodore B. Von Wullerstorf-Urban Geo log i sche The i l , geo log i sche Beobach tungen , no .2 , pa laon to log i sche Mitheilungen, 2: 187-268.

Setty M. G. A. P. (1982): Recent marine microfauna from the continental margin, west coast of India. J. Sci, Ind. Res. 6: 674-679.

Singh V. K. (2009): Study of comparative morphology of common Recent benthic foraminifera from the east


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coast of India and off Choshi Province, east coast of Japan. Ph.D. Thesis, Dr. Haringh Gour Univ. Sagar, India.

Singh V. K. and Kathal P. K. (2010): First report of twelve Recent benthic foraminifera from the Indian waters. Gond. Geol. Maz. 25(2): 259-266.

Singh V. K. and Kathal P. K. (2011): Morphological variations in common Recent benthic foraminifera from the east coast of India and the southern east coast of Japan. J. Pal. Soc. Ind. 57(1): 67-84.

Stubbing H. G. (1933): Stratification of biological remains of marine deposits of the Arabian Sea, British Mus. (Nat. Hist.). John Murray Expd. 3: 159-192.


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Effect of growth regulatory hormones on the germination stored seeds of Withania somnifera

Abstract : In the present study the effect of gibberellic acid (GA ) and kinetin (KIN) on the germination and 3

dormancy on the stored seeds of medical plant, Withania somnifera (Var. WS 20) has been studied. Seeds of Withania somnifera (Var. WS 20) were stored at constant temperature in desiccators and the germination data was taken up to 2 years with interval of 6 months. The parameters include are speed of germination (QI) , average root length (SVI) and average total length (SVI – II). In addition to these parameters, the measurements of average root length, average shoot length, average total length, average fresh weight, and average dry weight, average root length, average shoot length, average total length, average fresh weight, average dry weight were also calculated.

-5 -7 The quantity/concentration of both plant growth regulators was used between 10 M- 10 M. The presoaking -5treatment was given to seeds. The findings of the study show that gibberellic acid (GA ) with 10 M and kinetin 3

-7(KIN) 10 M are better for acquiring % germination (%G), the quality index and seedling vigour index (SVI-1 and SVI-11) in fresh and partially aged (1 year and 2 year) seeds . Comparative data suggest that the storage potential of seeds, Withania somnifera (Var. WS 20) can be improved significantly with the help of PGRs.

Keywords : Regulatory hormones, Germination, Withania somnifera

Introduction

The term plant hormone for growth regulating substances was introduced by Fitting (1909). The seed is one of the astonishing innovations of nature and it is now established that plant hormones affect seed germination by acting on different parts of the seed (Srivastava, 2002). Recent advances in agricultural research has suggested that the improvement in crop productivity and quality can be further improved by incorporating new technologies into traditional breeding programs and influence harvesting stages on seed vigour (Anonymous, 1985 & Mhatre and Rao, 1998). Grove et al. (1979) reported that brassinolide is a plant growth-promoting steroid, which they isolated from Brassica napus pollen. Tsai F-Y et al. (1997) made a comparative study of the effects of abscisic acid and methyl jasmonate, plant growth regulators, on seedling growth of rice. They found that growth regulators promote flowering, cellular division, and in seeds growth after germination. Gopikumar and Moktan (1994) studies the effects of plant hormones on seed germination and growth of true seedlings in the nursery. Tsai et al. (1997) studied the metabolism of gibberellins and suggested that gibberellins are important in seed germination affecting enzyme production that mobilizes food production used for growth of new cells. Saxena (1974) described the presowing hardening treatments improves crop production. Ethylene upregulate auxin biosynthesis in Arabidopsis seedlings to inhibition of root cell elongation (Swarup et al, 2007). Nitric oxide signaling in plants and help in the synthesis in the chloroplast (Shapiro, 2005 & Roszer, 2012). Keeping in view the aforesaid facts a study was taken to observe the seedling vigour of fresh and partially aged seeds (6 month to 2 year old seeds and to

Monika ChristianTissue culture & Seed Technology laboratory, Botany Department, School of Sciences, Gujarat University, Ahmedabad-380009, Gujarat, IndiaE-mail : [email protected]

improve the seedling vigour with presoaking treatment of Gibberellic Acid (GA ) and Kinetin (KIN) on germination.3

Methodology

Studies of germination are divided into different steps during 2005 as per the procedure followed by Agrawal and Dadlani (1987).

Plant Growth Regulators (PGRs) were procured from Sigma laboratory. The Seeds were treated with PGRs for experimental purpose. The seeds were dried and soaked in different concentrations of gibberellic acid (GA ) 3

-5 -7ranging between 10 M to 10 M. Similarly the same concentration was taken for kinetin (KIN) in distilled water. Soaking period of seeds was for 5 hours. Then seeds were removed from the solution and transferred to the tray, which was covered with filter paper and dried at room temperature till than original weight was measured. Dry seeds were put for germination up to the final count.

Hormones preparation–3Stock solution of GA (10 M): Accurately 3

weighed 34.64 mg GA was dissolved in 2 ml acetone and 3

final volume was made up to 100 ml DW. Different – 5 – 7concentrations (10 Mto 10 M) were prepared from stock.

–3Stock solution of KIN (10 M): Accurately weighed 21.52 mg KIN was dissolved in 2 ml 1N NaOH and final volume was made up to 100 ml DW.

Seeds were germinated in sterilized chamber. Intensity of light was maintained at 500 lux from tube light.

0e: Constant temperature 25 + 2 C was maintained during germination, the relative humidity of the room was kept at 50-55%.


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Germination parameters were recorded as suggested by ISTA (1985 a,b):

Normal seedling count, abnormal seedling count were counted. Germination percentage (%G) was calculated as follows on the basis of the number of normal seedlings as suggested by Agrawal (1987).

%G = N / T X 100,

N = Number of normal seedlings, T = Total number of seeds kept for the germination

Seedling length was recorded in cm and divided into root length and shoot length.

Fresh and dry weight of seedlings: Average fresh weight (per seedling) was recorded. Normal seedlings were

0packed in blotting paper and kept in oven at 80 + 2 C till constant dry weight was recorded.

The formula quality Index (QI) or Speed of germination was calculated as follows (Maguire, 1962);

Number of seeds germinated QI = ----------------------------------- Day of inspection

Seedling Vigour Index (SVI): SVI-I and SVI-II were calculated according to method given by Jayraj and Karivartha Raju (1992) and Abdul Baki, James and Anderson (1973): The formulae are as follows:

SVI-I = %G X Dry weight of seedling

SVI-II = %G X Total length of seedling

Results and Discussion

Plant hormones are considered effective molecule in development of seeds. The hormones affect seed germination and dormancy by acting on different parts of the seed. Walz et al. (2002) stated that there is a

correlation of auxins and cytokinins in plant, known as a A/C= constant. They further held that a gene encoding a protein modify by the phytohormones, indoleacetic acid acting by modulating chromosomal transcription. Gibberellins include a large range of chemicals that are produced naturally within plants and are important in seed germination, affecting enzyme production that mobilizes food production used for growth of new cells (Agrawal and Dadlani,1987). Gopikumar and Moktan (1994) studies on the effects of plant hormones on seed germination and growth of true seedlings in the nursery and found that plant hormones are suitable to cover the dormancy due to storing of seeds and initiate germination. Plant hormones like GA3

and KIN act upon a responsive plant system by interaction the molecules and effect the morphological, physiological and biochemical responses. Chinoy (1942, 1967) had first time tried presowing treatment of PGRs in wheat seeds. The critical studies were made on proper concentration, soaking volume and application of PGRs on Indian plants (Saxena, 1974, 1990; Murlikrishna, 1993). In the present

-5 investigation the pretreated stored seeds were with 10 M -7to 10 M of GA and KIN kept in distilled water for five 3

hours gave good (Table 2 & Table 3). The results were better in comparison to control (Table 1). The present study is in conformity with the observations of earlier investigation in the field (Chinoy, 1942 & 1967; Saxena, 1974 & 1990; Murlikrishna, 1993). Out of three

-5 -6 -7concentration of GA and Kin (10 M, 10 M, 10 M), GA 3 3-5 (10 M) gave more germination percentage (%G, 60),

seedling length (3.3), fresh and dry weight, quality index (QI, 2.6), Seedling Vigour Index (SVI-1,40.20) and(SV1- II,330.0) are more as compare to control. On the other

-7hand, KIN (10 M) is better results in r fresh and partial (1 year and 2 year) aged seeds in % G , Q.I., SVI-I and SVI-II. The PGRs are known to break seed dormancy in a number of plants (Gopikumar and Moktan, 1994).

Table 2: GA treatment to Fresh and Partially aged seeds of WS 100 variety of Withania somnifera3

Treatments

No. of seeds

Normal seedlings

Abnormal seedlings

% G

Average root length (cm)

Average shoot length (cm)

Average total length (cm)

Average fresh weight (mg)

Average dry weight (mg)

Q.I. SVI - I SVI - II

Treatment to 0 Month Old seeds (Fresh seeds)

GA3

10-5

M

25

15

0

60

3.3

2.2

5.5

34.60

0.67

2.67 40.20 330.0GA3

10-6

M

25

15

0

60

2.0

1.5

3.5

27.33

0.40

2.67 40.20 210.0GA3

10-7

M

25

12

3

48

1.2

1.2

2.4

25.90

0.36

2.12 17.28 115.2Treatment to 12 Month Old seeds

GA3

10-5

M

25

12

0

48

1.4

1.2

2.6

25.00

0.62

1.85 29.76 124.8GA310-6M 25 9 0 36 1.2 1.5 2.7 27.77 0.55 1.28 19.80 97.2GA310-7M 25 6 0 24 1.0 1.5 2.5 42.50 0.48 0.86 11.52 60.0Treatment to 24 Month Old seedsGA310-5M 25 5 1 20 3.0 1.0 4.0 52.00 0.70 0.20 14.00 80.0GA310-6M 25 6 4 24 1.8 1.2 3.0 40.00 0.42 1.45 10.08 72.0GA310-7M 25 6 0 24 1.0 1.2 2.2 28.33 0.52 0.69 12.48 52.8


10

Treatments

No. of seeds

Normal seedlings

Abnormal seedlings

% G

Average root length (cm)





Q.I. SVI - I SVI - II

Treatment to 0 Month Old seeds (Fresh seeds)

KIN 10-5

M

25

8

3

32

2.6

1.9

4.5

36.9

0.50

1.01 16.00 144.0KIN 10-6M 25 9 3 36 3.3 2.3 5.6 31.7 0.33 1.54 11.88 201.6KIN 10-7M 25 14 1 56 1.2 2.2 3.4 33.6 0.38 2.21 21.28 190.4Treatment to 12 Month Old seedsKIN 10-5M 25 6 4 24 1.8 1.3 3.1 40.0 0.43 1.40 10.32 74.4KIN 10-6M 25 5 1 20 3.0 1.1 4.1 52.0 0.71 0.23 14.20 82.0KIN 10-7M 25 12 3 48 1.2 1.0 2.2 25.9 0.36 2.12 17.28 105.6Treatment to 24 Month Old seedsKIN 10-5M 25 5 0 20 0.8 1.0 1.8 27.2 0.21 0.20 4.20 36.0KIN 10-6M 25 6 2 24 0.7 1.1 1.8 28.9 0.25 0.38 6.00 43.2KIN 10-7M 25 7 0 28 0.9 1.6 2.5 35.0 0.40 0.52 11.20 70.0

Table 3: KIN treatment to Fresh and Partially aged seeds of WS 100 variety of Withania somnifera

Table1: Seed vigour and germination data of WS 100 variety of Withania somnifera

Storage period No. of seeds

Normal seedlings

Abnormal seedlings

% G Average root length (cm)





Q.I. SVI - I SVI -II

0 Month

25

14

0

56

1.5

1.0

2.5

21.73

0.65

1.35 36.40 148.06 Month

25

14

0

56

1.0

1.0

2.0

21.73

0.50

1.83 25.00 112.012 Month

25

14

0

56

0.7

1.2

1.9

21.42

0.39

1.54 21.84 106.418 Month 25 10 0 40 0.9 1.0 1.9 29.00 0.45 1.23 18.00 76.024 Month 25 9 0 36 0.9 1.0 1.9 29.44 0.49 0.93 17.64 68.4

Referance

Abdul Baki A. A., James and Anderson (1973): Vigour estimation in soyabean by multiple criteria, Crop Sci., 13: 630-633

Agrawal P. K. (1987): Germination test under controlled conditions and its evaluation, In: Techniques in seed science and technology. (Eds.) Agrawal, P. K. and Dadlani, M. (South Asian Publishers, New Delhi)

Agrawal P. K. and Dadlani, M. (Eds.) (1987): Techniques in seed, science and technology, (second edition, South Asian publishers, New Delhi)

Anonymous (1985): International rules for seed testing, Seed Sci. & Technol., 13 (2): 307-520

Chinoy J. J. (1942): Presowing treatment and phasic development, Curr.Sci., 11, 400

Chinoy J. J. (1967): Role of ascorbic acid in crop production, Poona Agr. College Magaz., 57: 1-6

Fitting H. (1909): Die Bee influssung der orchideenbluten durch die bestaubung und durch andere umstande, Z. of Bot., 1: 1

Gopikumar K. and Moktan M. R. (1994): Studies on the effects of plant hormones on seed germination and growth of true seedlings in the nursery, J. Tropical Forestry, 10: 45-50

Grove M. D.; Spencer G. F.; Rohwedder W. K.; Mandava

N.; Worley, J. F.; Warthen J. D.; Steffens G. L.; Flippen-Anderson J. L. et al. (1979): Brassinolide, a plant growth-promoting steroid isolated from Brassica napus pollen. Nature. 281, 5728

International Seed Testing Association (ISTA) (1985 a): International rules for seed testing, rules 1985, Seed Sci. & Technol., 13: 299-355

International Seed Testing Association (ISTA) (1985 b): International rules for seed testing, annexes 1985, Seed Sci. & Technol., 13: 358-513

Jayraj T. and Karivartha Raju T. V. (1992): Influence of harvesting stage on seed vigour in groundnut cultivars, Seed Research, 20: 41-44

Maguire J. D. (1962): Speed of germination - a new vigour test for crop seeds, In: Seed production. (Ed.) Habblethwaite (Butterworth, London). pp, 647-660

Mhatre M. and Rao P. S. (1998): Plant tissue culture - current trends and future prospects, In: Advances in plant physiology, (Ed.) Hemantranjan A. (Sci. Publ., Jodhpur, India) pp: 72-101

Murli Krishna S. (1993): Physiological studies on castor and cotton, Ph.D. Thesis, Gujarat University, Ahmedabad

Roszer T. (2012): Nitric Oxide Synthesis in the Chloroplast. In: The Biology of Subcellular Nitric Oxide (Ed) Roszer T. (Springer New York London)


11

Heidelberg).

Saxena O. P. (1974): Presowing hardening treatments and crop production – a review, Planta, 4: 1-11

Saxena O. P. (1990): Some aspects of seed germination, In: International Symposium Environmental Mechanism. In Recant Advances in Research and Technology, Jodhpur. Jan. 27-28

Shapiro A. D. (2005): Nitric oxide signaling in plants. Vitam Horm. 72:339-98.

Srivastava, L. M. (2002). Plant growth and development: hormones and environment. (Academic Press). p. 140.

Swarup R., Perry P., Hagenbeek D. (2007): Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation

Tsai F-Y.; Lin C.C.; Kao C.H. (1997): A comparative study of the effects of abscisic acid and methyl jasmonate on seedling growth of rice. Plant Growth Regulation 21 (1): 37–42.

Walz A, Park S, Slovin JP, Ludwig-Müller J, Momonoki Y. S, Cohen J. D (2002). "A gene encoding a protein modified by the phytohormone indoleacetic acid". Proc. Natl. Acad. Sci. U.S.A. 99 (3): 1718


12

Evaluation of antimicrobial activity of Azadirachta indica, Syzygium aromaticum and Cinnamomum zeyalnicum against oral microflora.

Abstract :

For several years, varieties of plants have been used throughout India to treat oral care issues and are used till date in rural as well as urban areas. The inhibitory effects of aqueous extracts and essential oil of three plants, Neem (Azadirachta indica), Clove (Syzygium aromaticum) and Cinnamon (Cinnamomum zeylanicum) against clinical isolates identified as Staphylococcus auricularis, Micrococcus species, Acinetobacter lwoffii and Candida albicans, were studied. Cinnamon extract successfully inhibited all four organisms showing largest zones of inhibition. Clove oil inhibited S. auricularis, A. lwoffii and C. albicans but not Micrococcus. Neem oil did not show any significant activity against any organisms. Toothpastes were then formulated using effective plant extract showed the antimicrobial activity.

Key words: Aqueous extract, Azadirachta indica, Syzygium aromaticum, Cinnamomum zeylanicum, Staphylococcus auricularis, Micrococcus species, Acinetobacter lwoffii, Candida albicans.

Introduction

Microflora refers to the collective bacteria and other microorganisms present in a particular ecosystem. The ecosystem can be an animal or human host or a single part of its body. Our body is a host to billions of bacteria of many kinds. The mouth harbours many microorganisms and is an ecosystem of considerable complexity that has not been fully investigated yet and the mechanism/ chemistry has also not been completely understood. More than 30 genera of bacteria have been detected in human mouth. The oral microflora can be beneficial or detrimental. They supply some nutrients and digestive enzymes such as amylase, lipase and proteases and contribute to the hosts defences by protecting from exogenous microbes (Marsh et al., 2006). Any change in the host's oral environment or immune system can cause a number of different oral problems. Use of plants for maintaining oral hygiene is common till date in rural areas where toothpastes, mouthwashes, massage gels and other oral care products are not available. These products contain synthetic substances such as triclosan, phenol, benzydamine hydrochloride, zinc chloride, stannous fluoride etc. as antimicrobial agents. However, use of leaves, stem, fruits, seeds and bark of some plants (Neem/babool etc.,) for oral hygiene is common.Azadirachta indica has anti-inflammatory, antibacterial, antimalarial, antiulcer, antiparasitic, antifungal, antiprotozoal and antiviral properties (Biswas et al., 1973; Siddique et al., 1992; Ian et al., 1994). All parts of the tree have been used traditionally to cure various disorders as stated by Subapriya et al., 2005. Buds of Syzygium aromaticum are used as an analgesic, antiseptic and a carminative. Cinnamomum zeylanicum is known to inhibit

Haripriya Parthasarathy and Smruti ThombareDeptt. of Biological Sciences, Ramniranjan Jhunjhunwala College, Ghatkopar (W) - Mumbai 86E-mail : [email protected]

the growth of Candida hence preventing & curing oral thrush. It has been proven to be active against many pathogens (Suresh et al., 1992).

Materials and Methods

Collection of plant Material: Azadirachta indica twigs, Syzygium aromaticum buds and Cinnamomum zeylanicum bark were obtained from local market. Plant essential oil was obtained from commercial outlet of Dr. Urjita Jain Herbal ltd, Mumbai.

Collection and maintenance of oral flora: Mouth swabs from 6 people were collected and isolated on sterile Nutrient Agar and Sabouraud's Agar plate and incubated at 37ºC and room temperature respectively for 24hrs. The colonies obtained were transferred to sterile slants and sent to Metropolis Labs, Worli for identification.

Preparation of plant Extracts: The plant parts were sun-dried and ground into a course powder in a blender. 2 g of powdered plant material was added to 10 ml of sterile distilled water and heated to 70-80ºC for 2 hours. The mixtures were then allowed to cool, filtered through muslin cloth and centrifuged. The supernatant were then passed through a 0.45µm millipore filter to obtain sterile extracts which were stored at 4ºC (Nazia et al., 2006).

Determination of antimicrobial activity: Kirby Bauer disc diffusion method was used to determine the effect of aqueous extracts and plant essential oil on the isolates. The broth culture of each of the isolate was swabbed on sterile Mueller-Hinton agar. Sterile discs were dipped in plant extracts and placed onto the agar plates. Plates were incubated at 37ºC for 24 hours. The zone of inhibition was observed after 24 hours.


13

Formulation of herbal toothpaste:

The herbal toothpaste was prepared as per the formulation given by Jadge, 2008. The ingredients and quantities were mixed(Table 1).

Evaluation of toothpaste's activity against the isolates

Agar cup Method was used for testing the antimicrobial activity of the toothpastes prepared using the essential oil. The broth culture of the isolates was swabbed on Mueller-Hinton agar plates. Wells were bored using sterile borer. Toothpaste formulations were added in the wells and plates were incubated at 37ºC for 24 hours. The zone of inhibition was observed after 24 hours. For each strain a control was maintained using a formulation without the active ingredient.

Results and discussion

Asikainen and Chen ( 2000) stated that the oral ecology is different from person-to-

person in transmission of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

Smith (1993) studied the oral streptococcal colonization of infants while Pearce (1995) found viridans streptococci in the oral cavity of human neonates. In the present study the isolates form the oral cavity were identified as Staphylococcus auricularis, Micrococcus species, Acinetobacter lwoffii and Candida albicans.

Aqueous extract of Azadirachta indica and Syzygium aromaticum could not inhibit any of the culture. Essential oil of Azadirachta indica showed the activity only against S. auricularis. The essential oil of Syzygium aromaticum was found to be effective against Staphylococcus auricularis, Acinetobacter lwoffii and Candida albicans. Aqueous extract and the essential oil of Cinnamomum zeylanicum were found to be effective against all the four isolates. These results have been compiled in Table 2.

The toothpastes formulated were found to be effective against the isolates. The zone sizes observed after 24 hrs incubation are tabulated in Table 3.The activity of cinnamon is due to the presence of cinnamaldehyde that

inhibits amino acid decarboxylase activity as suggested by Wendakoon and Sakaguchi (1995). Cinnamon oil contains benzoic acid, benzaldehyde and cinnamic acid whose lipophyllic part is responsible for its antimicrobial properties (Ramos-Nino et al., 1996). Both clove and cinnamon contain eugenol, which is known to be bacteriostatic and bactericidal depending on the concentration used (Pelczar et. al., 1988). Essential oil from cinnamon bark also contains cinnamyl acetate (8.7%), which increases the activity of the parent compound (Gupta et al., 2008). The mechanisms or modes of action of the compounds on the bacteria and fungi include cytoplasmic granulation, cytoplasmic membrane rupture and inhibition of intracellular and extracellular enzymes. The mechanism of oils is generally hydrophobicity leading to partition in the lipid bilayer of the cell membrane, leading to alteration in the permeability and consequent leakage of cell contents. It also inhibits respiration in the cell and causes potassium ion leakage.

The present study suggests that the aqueous extracts were not able to inhibit the isolates. However, essential oil can inhibit the growth at larger extent. Further study of the components responsible for the biological activity is required and possibly a combination of these components would yield better results. The tooth pastes also were found to be effective in controlling the growth of these organisms hence such an herbal product may have a potential to replace the standard formulations.

Ingredient Quantity in %

Calcium Carbonate 35.00

Sodium Lauryl Sulphate 01.50

Glycerin 30.00

Sodium Alginate 01.00

Sodium Benzoate 00.12

Sodium saccharine 00.30

Plant Extract 02.50

Purified water q.s.

Table 1: Formulation of Toothpaste

Table 2: Evaluation of antimicrobial activity of plant extracts

Plant Average Zone size in mm S.auricularis Micrococcus spp A.lwoffii C.albicans

A.E E.O A.E E.O A.E E.O A.E E.O A. indica No

Zone 13.30 ± 1.5 No

Zone No Zone

No Zone

No Zone

No Zone

No Zone

S. aromaticum No Zone

26.0 ± 1 No Zone

No Zone

No Zone

18.0 ± 1 No Zone

26.3 ± 0.5

C. zeylanicum 11.00 ± 1 45.3 ± 3 11.6 ± 0.5 23.6 ± 1.5 11.3 ± 0.5 22.3 ± 2.5 11.0 ± 0 45.6 ± 1 Key: AE: Aqueous extract, EO: Essential oil


14

Plant Average Zone size in mm

S.auricularis Micrococcus spp A.lwoffii C.albicans

A. indica 11.6 ± 0.5 - 10.6 ± 0.5 11 ± 1

S. aromaticum 16 ± 1 - 15 ± 1 11.6 ± 1.5

C. zeylanicum 13.3 ± 0.5 13.6 ± 1.5 13.6 ± 0.5 13 ± 0

Control No Zone No Zone No Zone No Zone

Table 3: Evaluation of toothpaste's activity

References

Asikainen S. and Chen C. (2000): Oral ecology and person-to-person transmission of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Periodontol, 20, 65–81.

Biswas Kausik, Ishita Chattopadhyay, Ranajit K.Banerjee and Uday Bandyopadhyay. (2002): Biological activities and medicinal properties of Neem (Azadirachta indica). Current Science. 82(11), 1336-1345.

Gupta Charu, Garg Amar, Uniyal ramesh, Archana Kumari. (2008): Comparative analysis of the antimicrobial activity of cinnamon oil and cinnamon extract on some food-borne microbes. African Journal of Microbiology Research. 2(9): 247-251.

Ian W. Jones, Alastair A. Denholm Steven V. Ley, Helen Lovell, Anthony Wood, Robert E. Sinden (1994): Sexual development of malaria parasites is inhibited in vitro by the Neem extract Azadirachtin, and its semi-synthetic analogues. FEMS Microbiology Letters. 120(3): 267–273.

Jadge D. R. et al. (2008): Formulation of toothpaste from various forms and extracts of tender twigs of neem. Journal of Pharmacy Research. 1(2):148-152.

Marsh P. and Percival R. (2006): The oral microflora — friend or foe? Can we decide?. International Dental Journal. 56, 233–239.

Nazia Masood Ahmed Chaudhry and Perween Tariq. (2006): Anti-microbial activity of Cinnamomum cassia against diverse microbial flora with its nutritional and medicinal impacts. Pak. J. Bot. 38(1): 169-174.

Pearce C., Bowden G.H., Evans M., Fitsimmons S.P., Johnson J,Sheridan M.J. (1995): Identification of pioneer Viridans streptococci in the oral cavity of human neonates. J Med Microbiol.42: 67–72

Pelczar M. J., Chan E.C.S., Krieg N.R. (1988): Control of microorganisms, the control of microorganisms by physical agents In:Microbiology, (New York: McGraw-Hill International), pp. 469 509.

Ramos-Nino M. E., Cliford M.N., Adams M.R. (1996): Quantitative structure activity relationship for the effect of benzoic acid, cinnamic acids and benzaldehydes on Listeria monocytogenes. J. Appl. Microbiol. 80: 303-310.

Siddiqui Salimuzzaman, Shaheen Faizi, Beena Siddiqui, Ghiasuddin (1992): Constituents of Azadirachta indica: Isolation and Structure Elucidation of a New Antibacterial Tetranortriterpenoid, Mahmoodin, and a New Protolimonoid, Naheedin. Journal of Natural Products (55) 3, 303-310.

Smith D.J., Anderson J.M., King W.F., van Houte J, Taubman M.A., (1993): Oral streptococcal colonization of infants. Oral Microbiol Immunol ; 8: 1-4.

Subapriya R., Bhuvaneswari V., Nagini S. (2005): Ethanolic Neem (Azadirachta indica) Leaf Extract Induces Apoptosis in the Hamster Buccal Pouch Carcinogenesis Model by Modulation of Bcl-2, Bim, Caspase 8 and Caspase 3. Asian Pacific Journal of Cancer Prevention. 6, 515-520.

Suresh P., Ingle V.K. and Vijayalakshima V. (1992): Antibacterial activity of eugenol in comparison with other antibiotics. J. Food Sci. Technol. 29: 254-256.

Wendakoon C.N., Sakaguchi M. (1995): Inhibition of amino acid decarboxylase activity of Enterobacter aerogenes by active components of spices. J. Food Prot. 58: 280-283.


15

The Study of Nidification Behavior In Red Wattled Lapwings, Vanellus indicus

Abstract : The present study focuses on the nidification and courtship behavior of Red Wattled Lapwings. During breeding season the male bird select the territory. Both male and female take part in building nest. The nest is generally a circular depression encircled by stones or pieces of hard clay. Four eggs are led in each nest. During the mid-March and June the birds exhibit courtship behavior. Male first gives signals of courtship. The female respond by emitting a mating call. The incubated is completed by both parents. Eggs hatch into nidifugous chicks in 28-30 days. Both parents protect territory and protect their own plumage completely (3-5 weeks) tills become good fliers.

Key Words- Nidification, Courtship, Clutch size, Nidifugous

V.L. Saxena and A. K. Saxena*Department of Zoology, D.G.College, Kanpur-208001* Department of Zoology, D.A.V.College, Kanpur-208001E-mail id. - [email protected]

Introduction

The description about Indian birds has been well documented by Whistler (1948) and Salim Ali and Ripley (1980). Birds possess note worthy tendency to build a home to raise their young ones (Holway,1965 & Collias,1997). The incubation and broody behavior in Pheasant (Phasianus colchicus) been studied (Breihenback et al., 1965). Rhythm of incubation from egg laying to hatching in mountain white-crowned sparrow has been reported by Zebra and Morton (1980). The nest and detailed nidification activities of the spoonbill, Platalea leucorodia has been described by Dayanadi and Hosetti (2008 & 2009). They stated that casting of nest is an important part of bird's biology as it plays a key role in shaping the relationships between parents with their offspring. The procedure of site selection for nidification in Savannah sparrow and procedure of nest has been critically described by Wheelwright et al.( 1997). The parental building roles of male and female under different environmental has been studied in Western Gull and several species of birds ( Plerotti and Ridley.,1981; Bryant &Tatner,1990;Conrad & Robertson,1993;Collias,1997; Brawn et al., 2011). The activities and pattern of behavior are successful courtship, acquiring of a nesting area, building a safe and protected nest (Clark and Shulter,1999) laying of the clutch, taking care of the hatchlings, feeding and protecting them (Krebs, 1987) till they become good fliers. This behavioral pattern finds its origin in the direction towards self and racial survival. Nidification behavior is also very important for maintaining the ecological and genetically balances. Saxena et al.(2008) stated that breeding and nidification in Colonbia livia and Stretopelia chinensis undergoes throughout the year, although pigeon breeds preferably during January to May while Dove breeds during January to August. Recently, Vaithianathan problems faced by the species due to changes in environment as a result of pollution. The use of territory has been traced by early as 1941 by Nice

Material and Method The study was conducted on 6 pairs of birds in

Kanpur city, including one nest located at the rooftop of D.G.College, Kanpur. The period of study was during the months of March 2010 to June 2010, which is the time of spring followed by hot summers till the onset of monsoons.

The observations were taken with from full precautions without disturbing birds. For the concealment of human activities without the knowledge of birds. A 6 feet height frame was constructed which was covered by a cloth with two openings small enough to observe the nidification, courtship behavior etc needed for the study. This 'hide' was left near the nest for 2-3 days to acclimatize

Fig.1 : Hide used for observation


17

the birds (Figure 1).

The observations were taken on daily basis by naked eye starting in early hours of dawn and ending with dusk. The photographs were taken through a Nikon camera.

The eggs that were laid by the birds were measured for size with the help of a thread and a scale and average were calculated. The permission from ethical committee of the college has been obtained for the study of these endangered birds.

Observations

Courtship Behavior

The birds were spotted moving in pairs between 18-22 March, 2010 in Kanpur and its suburbs. The male initiated the courtship at all the station. Male reaches first towards the female with its fanned tail and then stood erect keep the neck in stretched position. The breast of the male was fully puffed out and to sought attention of the female, the male bird showing tumbling flights and produce songs. Female responded to these antics of the male by giving short, quickly repeated calls. Pair formation included display flights with tail fanned; shallow rapid wing beats and gives a number of calls.

Number of mates

The male and female were observed to jointly take up the responsibility of rearing the young.

Territory

The breeding pair maintained a territory in the surrounding of their nest. It is treated as a special area and intrusion in this area by strangers is protected by parents.

Male first acquires a territory and then gives a call note to the female. The male greets the arrival of the female joyfully by wheeling in the air and squawking happily. These birds give alarming calls protesting intrusion; they sing a territory song to get it distinguished and noticed to avoid strangers.

Nest construction and nesting materialndNest building started on 2 April 2010. Nest was

built during early morning up to noon and then in late afternoon on successive days. Nest building is a joint effort of both the parents with almost equal contribution.

Clutch Size

The clutch size in Lapwing was observed to be of four eggs. The eggs were laid on alternate day starting from 8 April 2010 till 14 April 2010. Eggs were laid during afternoon hours. The eggs were so arranged by the bird that their small ends meet in the center, making for even sitting and easier incubation by the parent. The bird was observed to rearrange the disarranged eggs.

Eggs

The eggs were of plover type, broad at one end and much pointed towards the other. They were pyriform with color varying from a pale olive green to a reddish buff.

There were deep brownish black markings or spots, which are distributed all over the surface of the eggs. The egg size varied from 1.2 inches-1.6 inches on an average. (Figure 2)

Incubation

Lapwings incubated the eggs by sitting on them. Incubation started with the laying of the first egg. Both the sexes shared the duty of sitting on the eggs. Mostly female did the duty but male assisted her a lot. Incubation took 3 weeks and 4 days. The season of summer made the birds wet their breast feathers regularly to keep the eggs wet. (Figure 3, 4)

Fig.2. Eggs of lapwing

Fig.3. Incubation of eggs

Fig.4. Incubation of eggs


18

Hatching pattern

Young hatched out one after the other starting on 4 May 2010, at an interval of 46-48 hours, in the order in which they were laid. Hatching was synchronous. (Figure 5)

Physical features of hatchlings

The hatchlings were covered with brown coloured down feathers. They had upper parts grayish brown, mottled black. They had a broad white collar, a black pectoral band, the chin and rest of the under parts were white tinged whereas the belly and the flanks were buff colored. The newly born chickens were nidifugous. The parents cared for these precocial young. The young left the nest as soon as they were dry and were able to move about with great ease. Their brown color helped them to blend in the surroundings. They were able to fly within three-five weeks. (Figure 6, 7)

Brood care and feeding

The brooding mother kept freshly hatched young warm at night. During the day the male and both the female protected them from the hot sun taking turns. Day brooding stops after 5 days of first hatching. Night brooding stopped only after the chicks were partly fledged which took about 16 days.

On the appearance of any danger or an intruder the brooding pair slowly creeped away to some distance and began calling to lure the enemy away from the nest.

If birds of prey flew overhead the brooding bird crouched silently on the eggs or the young to conceal them.

(Figure-8)

Behavior of the nestlings

The nestlings were nidifugous and precocial. They start running about as soon as they emerged out of egg shell. They were born feathered and could feed themselves. They needed their parents only for their protection till they could learn to defend themselves.

The nestlings spread over an area to feed, ran a few steps, stopped, pecked and then stood up straight. The chicks fed themselves easily but were safely protected by

Fig.5. Hatching pattern of lapwing

Fig.6. Physical feature of hatching lapwing

Fig.8. Brood care

Fig.7. Physical feature of lapwing hatching


19

the parents. They ran for cover in some plant or debris whenever their parents uttered a warning note or an alarm call.

Nest sanitation

Lapwings kept the nest clean and tidy. The eggshells after hatching were removed from the nest providing both sanitation and concealment.

Nestlings were nidifugous and precocial. They start running about as soon as they emerged out of egg shell. They were born feathered and could feed themselves. They needed their parents only for their protection till they could learn to defend themselves.

The nestlings spread over an area to feed, ran a few steps, stopped, pecked and then stood up straight. The chicks fed themselves easily but were safely protected by the parents. They ran for cover in some plants or debris whenever their parents uttered a warning note or an alarm call.

Nest sanitation

Lapwings kept the nest clean and tidy. The eggshells after hatching were removed from the nest providing both sanitation and concealment.

Discussion

According to IUCN (2009 ), Red Wattled Lapwings new zoological name is Vanellus indicus instead of Lobivanellus indicus .The species has been spread over in a large range. It maintains a well guarded and a well-distinguished territory which is in the interest for the welfare of their young ones. Territories selection has been started to select the site for the construction of nest. The site selection has been studied in birds (Clark and Shulter,1999). Saxena et al.(2008) reported that in pigeons and dove the territory is selected and claimed by mutual consent from both the parent only after pair formation. They further stated that individual breeding pairs maintain a territory surrounding the nest. In pigeon the male defends its territory by holding its wings aloft, landing on its rival at times or by clubbing the rivals head with bent wings. In dove male as well as female both defend the territory by chasing the intruders out of defined space. The present study is in confirmation to above referred authors that the breeding pair maintained a territory in the surrounding of their nest. It is special area protected by parents in this area strangers are not permitted by the parents. These birds give alarming calls protesting intrusion; they sing a territory song to get it distinguished and noticed to avoid strangers. Male first acquires a territory and then gives a call note to the female. The male greets the arrival of the female joyfully by wheeling in the air and squawking happily.

These birds show courtship behavior by giving mating call. The mating call is in the form songs, show flights and mock displays by the male (Singh, 2004). Similar behavior has been reported in American Avocets

(Sordhl, 2001). Monogamy assures better care for the young, these birds practice monogamy to assist in parental care (Ali and Ripley, 1980). Parental care has also been observed in Western Gulls (Collias, 1997; Plerott and Ridly, 1981). Lapwings maintain a well guarded and a well-distinguished territory to aid the welfare of their young ones The present study supports the observation of earlier author for the nest site selection as proposed by Clark and Shulter., 1999). Territory also aids in concealment and protection of young ones ( Holway, 1991) . Lapwings build their nest in open while selection of site places or even at the roof of the building keeping the concealing coloration for protection of eggs. They scoop a shallow hallow in the ground and line it with pebbles. Vicinity of water is preferred. The male scrapes the ground for the nest and the female lines it with local materials when she finds it satisfactory. Lapwings construct their nests early in the mornings and late in the afternoon during April, similar to nest building of passerine birds. (Collias, 1997).

Nest and Nidification Activities of the Spoonbill Platalea leucorodia in Westerghat Region of Shimoga, Karnataka is also being studied and The nesting activity, nest site selection, nesting habitat, nesting material, nest size, and nidification of Platalea leucorodia are discussed (Dayananda and Hosetti, 2009). Such studies on nidification is done on different birds including Indian grey horn bill,black headed ibis pig (Charde et al., 2011), pegion and dove (Saxena et al., 2008), Spot-billed Pelican (Vaithianathan and Jeganathan, 2012), tropical birds (Jeffrey et al., 2011) etc. are done in recent years. Lapwings kept the nest clean and tidy. The eggshells after hatching were removed from the nest (Smith, 1993). The hatchlings start running a few steps, then stopped, pecked and then stood up straight as soon as they emerged out of egg shell. It is critically noticed that new born bears feathered and could feed themselves. They need their parents only for their protection till they could learn to defend themselves.

Clutch size of Lapwings is four in the present study, however, some workers have also reported three to five eggs (Conrad and Robertson,1993). The present investigation the incubated is 25 days in natural conditions without using hormone treatment (Smith, 1993) and both the sexes share in the incubation simultaneously. Studies of incubation behavior have been done as early as 1965 on Pheasants (Breitenbach et al., 1965). The young hatchlings of Lapwings are grayish brown with white on the underside. Sibling competition and behavior of siblings in Swiftlets and Bee-eaters has been observed (Bryant and Tatner.1990) while behavior of adults and young of Acadian Flycatcher has also been reported in detailed( Whitehead and Taylo, 2001; Wesolowski, T. 1994). The eggs are often collected by people and used in traditional remedies for asthma and typhoid. (Negi,et .al .2007).


20

References

Ali S and Ripley S.D. (1980): Handbook of the birds of India and Pakistan. (2 Ed.). (Oxford University Press). 212–215.

Bird Life International. Vanellus indicus. In: IUCN (2009). IUCN Red List of Threatened Species.

Brawn Jeffrey D., Angehr George, Davros Nicole, Robinson W. Douglas, Styrsky Jennifer N., Tarwater Corey E.(2011): Sources of variation in the nesting success of understory tropical birds. Journal of Avian Biology. 42 (1), 61–68.

Breitenbach R.P., Nagra C.L. and Meyer R.K.( 1965): Studies of Incubation and Broody behaviorin the Pheasant (Phasianus colchicus). Anim. Behav., 13: 143-148.

Bryant D.M. and Tatner P. (1990): Hatching asynchrony, Sibling competition and Siblicide in nestling birds. Studies of Swiftlets and bee-eaters. Anim. Behav, 39:657-671.

Charde Pravin, Kasambe Raju and Tarar Jeevan L. (2011): BREEDING BEHAVIOUR OF INDIAN GREY HORNBILL IN CENTRAL INDIA.THE RAFFLES BULLETIN OF ZOOLOGY. Supplement No. 24: 59–64.

Clark R.G. and Shulter D. (1999); Avian habitat selection pattern from process in nest site use by ducks. Ecology. 80, 272-287.

Collias N.E. (1997); On the Origin and Evolution of Nest Building by Passerine Birds. Condor, 99: 253-270.

Conrad K.F. and Robertson, R.J. (1993):Clutch size in eastern Phoebes (Sayornis phoebe). I. The cost of nest building. Canadian Journal of Zoology. 71:1003-7.

Dayananda G.Y. and Hosetti B.B.(2009): Nest and Nidification Activities of the Spoonbill Platalea leucorodia in Westerghat Region of Shimoga, Karnataka . Our Nature. 7: 26-31.s

Dayananda G. Y.; Hosetti, B. B.(2008): Nesting and nidification activities of the Black-headed Ibis (Threskiornis melanocephalus) in mid-western ghat region of Karnataka, South India. Current Biotica. 2 (4), 439-452.

Holway D.A. (1991): Nest site selection and the importance of nest concealment in the Black throated Blue Warbler. Condor. 93:575-581.

Hume, Allan O (1889-90) The nests and eggs of Indian Birds.:3.

Kannan Vaithianathan and Pandiyan Jeganathan (2012): Nesting Ecology of the Spot-Billed Pelican Pelecanus Philippensis in Southern India. World Journal of Zoology. 7 (4): 295-302.

Krebs J.R. (1987) Parental care and mating system. An

Introduction to Behavioural Ecology. 9, 191-220. LaPergola Joshua B., Mortensen Jennifer L., and Curry

Robert L.(2012): Nest, Eggs, and Nesting Behavior of the Gray Trembler (Cinclocerthia gutturalis) on St. Lucia, West Indies. The Wilson Journal of Ornithology .123(2):390-395.

Naik.RM et al (1961): For behavior when incubating. JBNHS. 58,222-230.

Negi Chandra S : Traditional Uses of Animal and Animal Products in Medicine and Rituals by the Shoka Tribes of District Pithoragarh, Uttaranchal, India. Ethno-Med. 1 (1): 47–54.

Plerotti R. and Ridly (1981): Male and female parental roles in the Western Gull under different environmental conditions. Auk. 81:532-549.

Saxena V.L., Pandey Eshita, Agarwal Sona and Saxena A.K.(2008): Execution of Breeding and Nidification Behaviour in Pigeon (Columba livia) and Dove (Streptopelia chinensis). Asian J. Exp. Sci. 22(3), 405-410.

Silverin B. and Goldsmith A. (1983): The effects of modifying incubation on prolactin secretion in free living Pied-Flycatchers. Gen.Comp.Endocrinol. 55:239-244.

Singh R. (2004);. Behavior of Silver Pheasant (Lophura nycthmera) Ph.D. Thesis C.S.J.M.Univ. Kanpur.

Smith Staart (1993): The instinctive nature of nest sanitation. Part II Brit. Birds. 36:186-188.

Sordhl T.A. (2001) Copulatory behavior of American Avocets and Black Necked Stills. Auk. 118(4): 1072-1076.

Wesolowski, T. (1994): On the Origins of early evolution of male and female parental roles in birds. American Naturalists. 43:39-58.

Wheelwright, N.T., Lawler J.J. and Weinstein J.H. (1997) : Nest site selection in Savannah Sparrows: using Gulls as scarecrows? Anim. Behav.; 53:197-208.

Whistler H. (1948): Population Hand Book of Indian Birds. (Natraj Publication, Dehradoon). 459-461.

Whitehead D.W. and Taylor T. (2001): Acadian Flycatcher (Empidonax virescens). In the birds of North America. (Eds). A.Poole and F. Gill, (Birds of North America Inc. Philadelphia). No.614

Zerba E. and Morton, M.L. (1983): The rhythm of incubation from egg laying to hatching in mountain White-crowned Sparrows. Ornis Scand, 14:188-197.


21

Histochemical characterization of protocell-like supramolecular assemblies “Jeewanu”, synthesized in a irradiated sterilized aqueous mixture of some inorganic and organic substances

Abstract

The present study shows the occurrence of autoreplicative protocell-like model “Jeewanu” for the origin of life in the possible prebiotic atmosphere. The Jeewanu has been prepared in the laboratory in a sunlight exposed sterilized aqueous mixture of some inorganic and organic substances(Bahadur and Ranganyaki;1970). An attempt has been made to study histology of these artificially prepared particles with acidic and basic dyes. The investigation revealed that Jeewanu has orderly structural organization with metabolic characteristics. In prebiotic atmosphere possibly energy transducing systems similar to Jeewanu existed which had an ability to convert solar energy into useful forms.

Keywords : Jeewanu, Protocell, Supramolecular assemblies, Abiogenesis, Origin of life , Chemoton model.

Vinod Kumar Gupta and Indira ChaturvediDepartment of ZoologyC.M.D. Post Graduate College, Bilaspur -495001 (Chattisgargh) IndiaEmail: [email protected]

Introduction

One of the most fundamental problems of origin of life is to know of functional cells in prebiotic times (Maynard 1986 &, 1987; Dawkins 1976). The origin of life presumably occurred by self-assembly of organic compounds on the prebiotic earth into encapsulated molecular systems capable of catalyzed polymer synthesis. Such a self-assembly might possess a membrane bound self reproducing molecular cell systems (Deamer, 2002). The self assembly of molecular systems within a variety of cell-sized has been extensively studied by workers in the field (Deamer 1997, Segre et al., 2001). It is suggested that mineral surfaces have an important role into pre-cellular evolution. Life began as a series of reactions resembling metabolism. The autocatalytic pathways were established perhaps on mineral surfaces in aqueous phases as suggested by Bernal (1967). Pinto et al. (1980) was of the opinion that endogenous synthetic stages need to be investigated in detail. Overtime the systems became increasingly complex to the point that self reproducing polymers may be synthesized with cellular compartments in course of time

(Wachtershäuser,1988, Cody 2000). Prior to the evolution of biochemical machinery the growth and division of simple primitive cells ( protocells) must have been driven by environmental factors (Zhu et al., 2012). Self assembly processes can produce supramolecular complex structures with certain properties of living state. Such structures are able to capture energy available in the environment and initiate primitive reactions associated with metabolism, growth and replication (Deamer,2007). The self assembly and replication of membrane, the nature of potential polymers and the nonenzymatic template directed copying nucleic acid sequences postulated

formation of vesicles by various scenarios of self-organization (Hanczyc Szostak, 2004; Orgel,2004; 2006;Eschenmoser, 1999). The synthesis of amino acids might be initiated by spark discharge in a mixture of reduced gases (Miller, 1953). The abiogenesis of organic compounds on the early earth became central point in the postulation for the origin of life (Willis and Bada, 2000). Life has been defined as a chemical system capable of Darwinian evolution (Joyce and Orgel, 1998). Prior to evolution of biochemical machinery the growth and simple primitive cells (protocells) must have been driven by environmental factors ( Zhu et al., 2012).

Laboratory model of protocell systems should be helpful in modeling various hypotheses of origin of life. Assuming if protocell reproduction can be achieved and the adaptive innovations are possible in the spontaneous evolution it will then be simple chemical system. The nature of such adaptations may provide clues as how modern cells evolved from their earliest ancestors (Zhu et al., 2012 ).

Chemical evolution would have produced primitive self maintaining chemical systems with rudimentary mechanisms of energy transduction but without (replication and mutable) records. We call such systems as “Infrabiological Systems” (Szarthmáry, 2005).

Several concepts of models of cells have been produced to test various theories for the origin of cellular life (Cavalier-Smith, 1987; Luisi, 1998; Szostak et al.,2001; Pohorille and Deamer, 2002).

“Chemoton model” of living system was given by Gánti (1971). He emphasized combination of a metabolic cycle and a membrane was also proposed called a self reproducing microsphere (Gánti, 2003). In contrast, a


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protocell-like entity with a boundary and template replication but no metabolic system was conceived earlier (Szostak, 2001). Based on Chemoton concept protocells have been visualized as the functional integration of metabolism, containment and information processing (Rasmussen et al., 2003). The photochemical, formation of protocell-like microstructures “Jeewanu” in a laboratory simulated prebiotic atmosphere capable of showing multiplication by budding, growth from within by actual synthesis of material and various metabolic activities has been reported (Bahadur et al., 1963;1964; 1966;1967; 1970; 1975;1980). Jeewanu have been analysed to contain a number of compounds of biological interest viz. amino acids in free as well as in peptide combination (Bahadur et al., 1954; 1961;1965; 1970; Briggs;1965; Maurya;1977), sugars as ribose as well as deoxyribose (Bahadur et al. 1963; 1964; 1966; 1967; 1970 ), nucleic acid, bases as purines as well as pyrimidines (Bahadur et al., 1970, 1972, Ranganayaki et al.,1976), phospholipids ( Bahadur et al., 1970; Singh, 1975) and ferredoxin -like material ( Rao et al., 1978; Bahadur et al., 1980) in them. The presence of various enzyme like activities viz., phosphatase, ATP-ase, esterase ( Briggs, 1965; Bahadur et al., 1970; Singh, 1973; Gupta 1980 ), nitrogenase ( Smith et al., 1975; Bahadur et al., 1980 ) have been also been detected in Jeewanu mixture. It was found that under certain specific conditions Jeewanu can catalyse photolytic decomposition of water utilizing sunlight as a source of energy. Further it was observed that H thus released is utilized in photochemical 2

fixation of nitrogen, and carbon dioxide. These findings 14 15were confirmed using C , N and D O ( Smith et al ; 2

Bahadur et al., 1980). Gáinti (2003) discussed that Jeewanu posseses a promising configuration similar to protocell-like model. Therefore, an attempt has been made made to investigate histological characteristics of Jeewanu to characterize their structural organization. The present investigation is aimed to acertain the possible nature of earliest living system on the earth.


The high mineral Jeewanu were prepared by the method as described by Bahadur and Ranganyaki (1970) and Verma (1980). The histochemical localization of acidic material in the Jeewanu was studied by Eosin (Baker, 1969) while the basic materials were stained with by Gentian Violet (Baker, 1969) and Methyl Green (basic dye) (Brachet, 1944; Pearse,1961). The histochemical localization of RNA-like activity was studied by Pyronin Y staining technique (Brachet, 1944; Pearse,1961). The staining procedure employed in the present study is routine procedure.

The High Mineral Jeewanu mixture was exposed to sunlight and the photochemical formation of Jeewanu was studied at regular intervals under high power and oil immersion (1500x) of optical microscope.

The ingredients of the solution for the preparation of Jeewanu are as follows :

Solution (i) :

Ammonium molybda te 4 .00 gm and diammonium hydrogen phosphate 12.00 gm were dissolved in 100 ml of distilled water.

Solution (ii) :

Mineral solution : It was prepared by dissolving the following salts in 100 ml of distilled water. Each salt was added when one salt was dissolved completely.

NaCl 3.00 gm

Ca(CH COO) 0.30 gm3 2

K SO 0.30 gm2 4

MgSO 0.50 gm4

FeSO 0.50 gm4

Solution (i) and (ii) were mixed, as a result of which, a white precipitate was formed, which was digested in the least quantity of HCl by boiling.

After cooling, the volume of the solution was made up to 300 ml by distilled water. Mixture was cotton plugged and then sterilized in an autoclave at 15 lb pressure for 30 minutes.

After cooling, 3 volume of above mixture and 1 volume of 36% formaldehyde were aseptically added in a conical flask.

A part of mixture was kept in dark as control. Another part was taken in a separate conical flask, cotton plugged and covered with black cloth and was kept as control.

The mixtures were exposed to sunlight for varying periods of exposure as per requirement of the experiment. The control mixtures covered with black cloth were examined for the formation of photoproducts at regular intervals.

Observations

The Jeewanu mixture kept in dark as control showed negative results. It is colourless and did not show any initiation of photochemical reaction.

The mixture after exposure to sunlight immediately became bluish in colour. The intensity (bluish) of colour increases on further increasing the exposure time of sun light. The photochemical formation of Jeewanu becomes visible under high power of microscope even in few minutes (2-5 minutes) of exposure to sunlight.

Morphologically, Jeewanu are spherical in shape and exhibit bluish colour. The microscopic (1500x magnification) observation of Jeewanu showed that they became yellowish in colour with a definite boundary wall and possesses intricate internal structures. The number of Jeewanu formed increases in number on further exposure to sunlight. The size of Jeewanu forms varies from 0.5µ to


24

3.5µ in diameter (Fig. 1 & 2)

With Gentian Violet staining, the central structure of high mineral Jeewanu presence of basic material-like substance. In some particles bluish colour was seen diffused in the peripheral region (Figs 3 & 4).

The microscopic examination of slides under high power and oil immersion (1500 X) showed that extra central region of High Mineral Jeewanu was stained pink showing the localization of acidic structure in the extra central region.

With Methyl Green (basic dye) the central structure ofl Jeewanu take intense green stained and diffused greenish colouration was also seen in the peripheral region showing the presence of a basic material-like substance in the Jeewanu mixture (Figs 5 and 6).

The bright red colour of Pyronin Y was diffused throughout the High Mineral Jeewanu showed the presence of RNA like activity in the mixture (Figs 7 &8).

Fig 1 & 2 : Micrograph showing photochemical formation of high mineral Jeewanu showing multiplication by budding and growth from within under optical microscope (1500 X)

Fig 3 & 4 : High mineral Jeewanu stained by Gentian Violet histochemical localization of acidic in the central region

Ó showing


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Fig 5 & 6 : High mineral Jeewanu stained by Eosin (micrograph c, d) showinghistochemical localization of basic material in the extracentral region

Fig 7 & 8 : High mineral Jeewanu stained by Pyronin Y (micrograph c,d,) histochemical localization of RNA-Like activity diffused throughout the micro structure

showing

Discussion

One of the major challenges is to artificially synthesize protocell which can ensemble organic and inorganic substances into self-reproducing system of life ( Szostak et al., 2001; Hanczyc et al.,2004). Eigen and Shuster (1979) described certain organic molecules which can spontaneously organize into larger structures. Haldane (1928) and Oparin (1957& 1968) gave theory of coacervation of colloidal material as the beginning of growth material characterization of life. Molecular evolution refers to the ultimate formation of molecules by chemical transformation of substances with the condition that earliest living systems were madeup of the same material of which the present day living system is madeup of (Bahadur, 1964 & 1966). Bahadur postulated that first

forms of metabolism arose inside closed compartments and became more complex later. At certain concentration additional molecules no longer dissolve but instead begin to associate into small aggregates called “micelles” (Bahadur, 1967). The abiogenesis of biogenic materials viz. amino acids, nucleic acid bases, sugars, phospholipids have been discussed for the origin of life. He has formed photochemically self- sustained protocells- like suprarmolecular assemblies called Jeewanu responsible for the origin of life. The present study is in conformity to the observations of Bahadur (1964 & 1966) that sunlight exposed sterilized aqueous high mineral mixture of ammonium molybdate, diammonium hydrogen phosphate, biological minerals and formaldehyde confirms photochemical formation of protocell-like molecular associations.They have a definite boundary wall


26

and intricate internal structure. Their size varies from 0.5 µ to 3.5 µ in diameter. It is also held that formation of Jeewanu is strictly an outcome of a photochemical reactionin the possible prebiotic atmosphere.

The present cytochemical studies of Jeewanu with basic dyes Gential Violet, Methyl Green and Pyronin Y showed the presence of acidic material-like substance in the central region. Staining with Pyronin Y suggested that RNA-like activity is possibly diffused throughout High Mineral Jeewanu. The histochemical staining of High Mineral Bahadur et al Jeewanu with acidic and basic stains showed that Jeewanu possesses an ordered structural organization. The present investigation confirms the observations of Bhadur et al. (1970, 1980, 1972,1973, 1976) that RNA monomers are more readily photochemically synthesized in Jeewanu mixtureas The also supported the view that primitivity of RNA in the possible primitive atmosphere (Joyce and Orgel, 2006). The microscopic examination of Jeewanu, the autoreplicative protocell-like abiogenic microstructure shows that it possesses a definite ordered structure (Bahadur,1975; Bahadur and Gupta, 1972; Gupta,1980; Gupta,2002).

All cellular life today incorporates two processes – (a) self-assembly; and (b) directed assembly. The directed assembly involves the formation of covalent bonds by energy dependent synthetic reactions and require a coded sequence; while spontaneous self-assembly occurs when certain compounds interact through non-covalent, hydrogen bonds, electrostatic forces and non-polar interactions to form closed membrane bounded micro-environment (Pohorille and Deamer, 2002). It is also suggested that it is possible that in the primitive atmosphere there may nonlinear collaboration of photo-redox transformations at mesoscopic level possibly led to emergence of supramolecular assemblies similar to Jeewanu, showing properties of biological order, viz. multiplication by budding, growth from within by actual synthesis of material and metabolic activities. It may be postulated that earliest energy transducing systems were possibly a photoautotroph similar to Jeewanu which had an ability to convert solar energy into useful forms.

Acknowledgement

V.K. Gupta gratefully acknowledges University Grant Commission, Central Regional Office, Bhopal– M.P.,India for financial assistance under a minor research project.

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Barriers to Breast Cancer Screening In Young Indian Women:A Tale of Two Cities

Abstract : Screening for breast cancer reduces mortality by 30 – 40% but there are many psychological barriers to screening. A comparative case study was undertaken in young well educated Indian women to map the barriers to screening and their relation to the socio-cultural milieu. Volunteers from two premier educational institutes of Ranchi and Chennai were required to respond to a self completion questionnaire recording their attitudes to breast cancer screening. The responses were marked on a scale of 1-5. The two cohorts were from similar social and economic backgrounds and had equally fair knowledge of Breast cancer but differed in their access to healthcare and their choice of dress. The significant differences in outcome variables of embarrassment, fear and barrier in the two cohorts were found to correlate to the centre of study only. The authors postulate that this may be the due the influence of their present social environment i.e. 'neighbourhood'.

Key Words: Breast cancer, Screening, Barriers, Socio-cultural parameters, Knowledge of breast cancer

! #A.Vidyarthi*, A.Soumya**, S.Choudhary , B.K.Sinha*Onco-Surgeon, Guru Nanak Hospital and Research Center**Fashion Designer, Shri Arvind Mills, Banglore!Department of Psychology, RLSY College, Ranchi#Department of Zoology,SSM College, Ranchi

Introduction

Regular screening for breast cancer reduces mortality by about 30 – 40 % (Vaino et al., 2002; Tabar et al., 2003; Swedish Organized Service Evaluation Group, 2006). But there are many psychological, social and economic barriers to screening e.g. embarrassment, lack of access to health-care, or lack of physician referral. (McGarvey et al., 2005; Secginli et al., 2006; Ansnik et al., 2008)

Patients undergoing mastectomy have a worse “Quality-of-Life” (The WHOQOL Group, 1994; Fleck et al., 1999; Kluthcovsky et al., 2007;) compared to those undergoing Breast conserving surgery, especially the physical and social components (Ganz et al., 2004; Pandey et al., 2006;), with a poorer body image (Falk Dahl et al., 2010).

Screening uptakes in US and UK are 75 – 80 % (Cole and Bryant, 1997; Patnick, 2008 ;), but it is almost nil in India. This study attempts to define the barriers that prevent Indian women from volunteering for breast cancer screening and compare the magnitude of those barriers in young women from a metropolitan city and a state capital. The authors have interviewed several educated women with a fair knowledge of Breast cancer who have been advised breast self-examination but refuse to practice even that. What prevents them from undergoing screening?

In this backdrop the present study aims to:

1. Map the barriers to screening in young, educated, well-off Indian women with good access to healthcare facilities and,

2. Define the influence of the socio-cultural

environment of the subject on the barriers to screening.


A comparative case study (Hantrais, 1996) was designed, selecting women from two premier educational institutes of Chennai and Ranchi with the following inclusion criteria:

1. Women less than 30 years with a graduate's degree.

2. Women with no breast diseases

38 females from Chennai and 40 from Ranchi, from two premier institutes, volunteered for the study. Embarrassment felt to activities relating to breast examination, fear of loss of breast and knowledge of breast cancer were tested through a self-completion questionnaire. They were also tested for their knowledge of breast cancer through ten questions answered in true/false format. A pilot study was carried out to establish the internal reliability of the questionnaire and Cronbach alpha scores of the various sections measured 0.919, 0.816 and 0.765 respectively (Cronbach, 1951).

Independent variables included age, annual family income, family history of breast cancer, and access to healthcare (access to family physician, history of previous gynecological consultation and access to Health insurance). The socio-cultural parameters included the preferred style of dress and the place of residence, now and as a child.

The pilot survey showed that some subjects who had consented to the study had not answered one or more questions. The possible reasons may be:


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1. They did not know the answer

2. Perceptual Defense (Blum , 1955; Minard, 1965; Loiselle and Williamson, 1966;) caused them to ignore questions that evoked strong negative emotions

Therefore, “barrier”, a third dependent variable was added to account for the unanswered questions. All variables were marked on a scale of 1-5 and final scores were obtained by taking the section mean.

The results were analyzed using PAST 2.17b (Hammer, Harper, and Ryan 2001) software. The statistical tests included Chi-square test, ANOVA, and Kruskal – Wallis test.

Results:

The demographic characteristics of the sample have been presented in table 1.The mean age of the respondents from Chennai and Ranchi were 20 years and 22 years respectively.78 per cent were graduates and 22 per cent post-graduates. The two cohorts were from comparable economic backgrounds (Family income, p = 0.051;).They were significantly different in their access to a family physician (p < 0.001) and access to health insurance (p < 0.0001) but similar in the number of volunteers who had visited a gynecologist (p = 0.16). 12/38 volunteers from Chennai had seen breast cancer in their family compared to 5/40 from Ranchi (p = 0.041).

Socio-cultural background

63 per cent respondents from Chennai were residents of a non – metropolitan city and 60 per cent had spent their childhood in one. In comparison, 95 per cent respondents from Ranchi had spent their childhood in a non – metropolitan city and 92 per cent were now staying in one. The difference did not reach statistical significance (Place of residence as a child, p = 0.80; present place of residence, p = 0.86). 87 per cent Chennai respondents preferred western dresses compared to 53 per cent from Ranchi (p < 0.001). Respondents from Ranchi also displayed a markedly heightened 'perceptual defense' response to uncomfortable questions – either due to embarrassment or lack of knowledge. In fact, 38 per cent Ranchi volunteers didn't know the size of their brassier cup compared to 0 per cent Chennai volunteers!

That, two cohorts of women of similar age groups, educational and economic backgrounds who have spent their lives in similar urban social environments should display such varied response to embarrassing questions points to the effect of their social environment i.e. the effect of the 'neighborhood'.

Knowledge of Breast cancer

The average score was 4.57/10 ranging from 0 – 9.The mean score of respondents with a family history of breast cancer was 5.12 and those without a family history was 4.41; the difference was not significant (p =

0.22).There was no correlation between the knowledge scores of the respondents and their embarrassment, fear and barrier scores. The difference in the knowledge scores between the respondents from Chennai and Ranchi did not reach statistical significance (Chennai = 4.79 Ranchi = 4.35, p = 0.36).

Measures of Embarrassment

Five questions measured embarrassment and the responses are shown in figure 1.Briefly, 35 per cent would refuse breast examination by a male doctor, 46 per cent will hide a breast lump from their sons, 40 per cent would refuse a Mammogram because it is too embarrassing, and 76 per cent may ignore any advice regarding breast examination due to embarrassment. The mean Embarrassment score in Chennai was 2.24 and in Ranchi was 3.01(p < 0.01).This score did not vary in relation to any of the independent variables except the center of study.

Measures of Fear of mastectomy

The absence of fear of loss of breast (mastectomy) was used as a surrogate measure of resistance to screening and the responses are shown in table 2 and figure 2. Briefly, 49 per cent would not demand breast prosthesis, 62 per cent would not feel any restriction in their choice of dress, 77 per cent would not feel deformed, and 71 per cent fear the cancer more than the deformity after mastectomy. The mean Fear scores in Chennai were 2.50 and those in Ranchi were 3.14 (p < 0.001). None of the recorded independent variables correlated with the fear score except the center of study.

It is implicit from figure 3 that a significantly greater number of respondents from Ranchi than Chennai avoided consenting to mammograms (Chi^2 = 16.56; p < 0.001).Only 50 per cent respondents from both centers chose regular mammograms! The mean barrier scores in Chennai were 1.81 and Ranchi were 2.31 (p < 0.001).

Discussion

This survey attempted to define the emotional and psychological barriers against breast cancer screening in Indian women. Therefore, the respondents were chosen from a population which would face minimum practical or logistic problems in accessing healthcare services. The questions, too, were designed to strike deep emotional chords in educated women and were based on situations commonly encountered by the first author in his clinical practice.

The results revealed deep emotional distress to situations commonly encountered by breast cancer patients. For example, a woman often discovers a lump in her breast when she is a widow and dependent on her son. Our survey shows that in such situations 46 per cent respondents would hide the lump from their son and allow the disease to progress. When asked to react to the slogan 'if only women paid as much attention to their breasts as men do'; one out of four respondents ignored the question and a


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similar number opined that it is a 'necessary evil' - men will ogle at female breasts! Very few respondents really understood that the slogan refers to breast-self examination as a means of cancer screening.

These deep –seated emotional barriers have been found in highly educated women. The conventional wisdom is to overcome them through education about cancer. Therefore, the respondents were also tested for their knowledge of breast cancer through ten questions answered in true/false format. The median score was five with most scoring between 3 and 6.Thus the respondents displayed a high level of emotional avoidance combined with fair knowledge of the disease and minimal practical or logistic barriers. Forbes et al. (2011) surveyed the health seeking attitudes of women from various ethnic communities in East London. They found that women of Indian origin reported emotional rather than logistic or practical barriers to seeking medical help. 59 per cent reported embarrassment as a barrier, 46 per cent worried about what the doctor might find, and 53 per cent reported not feeling confident talking about their symptoms. Another survey amongst South Asian women living in Canada (Bottorff et al., 1998) found that many respondents would not visit a doctor unless accompanied by a friend or relative.

These studies and the present study show that barriers to health – seeking behavior, in Indian women, in general and cancer screening in particular are predominantly emotional and may not be influenced by the subjects' health – related knowledge. Embarrassment, Fear and Barrier scores of the respondents were not associated with either their having a patient in the family or their knowledge of cancer (p values; 0.15, 0.79 and 0.14 respectively). Forbes et a.l (2011) and Scanlon and Woods (2005) both found that South Asian women have better knowledge of age-related risks of breast cancer but are less likely to examine their breasts than their western counterparts, same as the findings of our survey.

The volunteers for this survey were young women given to wearing western dresses and those from Chennai were students at a national fashion designing institute. It is expected that such women would pay more attention to their body image than the general Indian female. Cash, Melnyk and Hrabosky (2004) have postulated that body image includes an attitude of satisfaction or dissatisfaction in one's body that varies with two factors-self-evaluation and investment in appearance or the respondent's view of the importance of her appearance. Assuming that younger women are likely to invest more in their appearance we hypothesized that fear of disfigurement by loss of breast may prompt them to choose regular screening, but the results failed to support our hypothesis. Surveys in Indian women from low socio – economic strata (Khan et al., 2010) did find that such patients did not pay much importance to their appearance, but finding the same result in a group of young educated

women was surprising. When specifically questioned if they would suffer the embarrassment of mammograms to avoid the deformity due to loss of breast 70 per cent respondents chose to 'get the tumor out anyhow'! This indicates that fear of a diagnosis of cancer overrides the fear of deformity. Therefore, fear of being detected with cancer would prove a barrier to screening rather than fear of deformity motivating them to undergo screening. A similar result was obtained by Tejeda et al. (2009) and Watts et al. (2009) when surveying women to find barriers to mammography and pap smears respectively.

The second aim of the study was to define the influence of socio-cultural differences on screening attitudes. Therefore, two groups of volunteers of comparable educational, economic and cultural backgrounds were chosen from a metropolitan city and a state capital. In spite of their comparable backgrounds more respondents from Ranchi preferred ethnic dresses than those from Chennai. Given that dress choices are vulnerable to peer pressures this change in style reflects the influence of 'neighborhood'. In the authors' opinion the differences in their embarrassment, fear and barrier scores are also due the effect of 'neighborhood', since we could not detect any correlation to either family history of breast cancer, ease of access to health care or knowledge of breast cancer.

Many authors have linked perceptions of neighborhood to general health status (Ross and Mirowsky, 2001; Hill, Ross and Angel, 2005; Wen, Hawkley and Cacoppo, 2006) .Schempf, Strobino and O'Campo (2009) were able to show that neighborhood structures and processes shaped maternal behavioral risks thus impacting infant birth weight. In the authors' opinion the social environment of a metropolitan city helps reduce the barriers to screening thus influencing health-risk behavior of respondents. At the same time, as more and more urban centers develop there is hope that tier two cities like Ranchi will also develop social environments like Chennai in the future to positively impact the health behavior of its residents.

Conclusions

The study seems to indicate that barriers to cancer screening are an emotional response of the subject and that 'neighborhood' i.e. place of residence may have an impact on the risk behavior of the residents.

Limitations of the study:

Being a comparative study, the results of this study can't be generalized. But the first author has come across many instances in his clinical practice when women of this socio-economic group avoided screening. The question; why are they avoiding screening, seemed to beg an answer!

The study asks young women to respond to situations they have not faced. But a majority has rightly said that mastectomy would result in a poorer body image


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and dress restriction, similar to the results of Falk Dahl et al. (2010) and Ohsumi et al. (2009) Therefore the other responses are unlikely to differ significantly.

The study also suffers from small sample size but in a conservative society such as India it is very difficult to persuade women to answer intimate questions about their breasts.

Acknowledgments:

The authors wish to express their gratitude to the students and faculty of National Institute of Fashion Technology, Chennai and Xavier Institute of Social Sciences, Ranchi for their cooperation.

Demographic characteristic Chennai Ranchi

Total number of respondents 38 40

Mean age (range) 20(17 – 23) 22(20 – 27)

Education

Graduate (%) 37(97.4) 22(56.4)

Post – Graduate (%) 1(2.6) 17(43.6)

Annual Family Income (%)

< INR 15000(%) 0 1(2.7)

INR 15000 – 500,000(%) 23(60.5) 27(73.0)

INR 500,000 – 1,000,000(%) 4(10.5) 9(24.3)

> INR 1,000,000(%) 10(26.3) 0

Positive history of Breast Cancer in Family (%) 12(31.6) 5(12.5)

Respondents having Family Physician (%) 27(71.1) 16(40)

Respondents who have seen Gynecologist (%) 13(34.2) 20(50)

Respondents with Health Insurance (%) 33(86.8) 9(22.5)

Dress Style

Ethnic +/- dupatta (%) 5(13.2) 17(42.5)

Western +/- dupatta (%) 33(86.8) 21(52.5)

No response (%) 0 2(5.0)

Present place of residence

Tier 1 city (%) 14(36.8) 3(7.5)

Tier 2 city (%) 9(23.7) 23(57.5)

Tier 3 town ((%) 15(39.5) 14(35.0)

Residence during childhood

Tier 1 city (%) 14(36.8) 2(5.0)

Tier 2 city (%) 6(15.8) 19(47.5)

Tier 3 town (%) 17(44.7) 19(47.5)

Mean knowledge of cancer score(CI)* 4.79(4.13 – 5.42) 4.35(3.7 – 5.0)

* F (1, 73) = 0.8399p (same) = 0.3624Not Significant

Table 1.Comparison of the demographic characteristics of the respondents from Chennai and Ranchi


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Table 2: A Comparison of Mean Embarrassment, Fear and Barrier scores of

respondents from Chennai and Ranchi.*

*The respondents from Ranchi have significantly higher scores in spite of having similar scores when tested for knowledge of Breast cancer (ref: Table 1)

Chennai Ranchi P value

Embarrassment 2.237 3.096 <0.01

Fear 2.504 3.143 <0.001

Barrier 1.81 2.31 <0.001

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Improved Maturation of Wild and Pond-reared Black Tiger Shrimp Penaeus monodon ( Fabricius) using different Combinations of Live and Wet Feeds

Abstract : The impact of various combinations of live feeds and frozen feeds such as polychaete, squid meat, crab meat and beef liver was tested on the maturity and spawning of the shrimp Penaeus monodon. Females with weight ranges 86g to 96g were selected for the present experimentation. The shrimps were fed with five different combinations at the rate of 15% of their body weight. Feed combinations selected for current study was crab and polychaete, oyster and polychaete, squid and clam, beef liver and squid, and oyster and beef liver. We applied different ablated methods to evaluate the reproductive ability of wild and pond reared Penaeus monodon The great reproductive performance was achieved when shrimps fed with Feed-I, the reproductive performance based on fecundity was identified by the number of eggs in gravid females. Future research efforts focused on the artificial diets which contain both live and frozen feed combinations will be essential for the improved egg quality in tank-reared Penaeus monodon. Thereby increasing the consistency and reliability of maturation diets for better quality nauplii production.

Key Words: Penaeus momodon, Spawning, Fecundity, Hatching, nauplii, Survival rate

K. Ramesh BabuDepartment of Marine Living Resources, College of Science and Technology Andhra University, Visakhapatnam- 530 003. A.P., India.Corresponding author Email Id: [email protected].

Introduction

In addition to eyestalk ablation, various naturally occurring and formulated feeds are proved fertile in inducing maturation in pond reared and naturally collected penaeid shrimps. Fish meal, Vitamin-C, and fish oil play important role in growth and reproduction in aquatic animals (Jia-Yuan Xu, et al., 2010). In general the fresh or frozen diets currently used for maturation of shrimps under captivity have (1) rich animal protein (approximately 60% on dry weight basis) with amino acid profiles similar to that of shrimps (e.g. shrimp and some bivalves such as Tapes sp.) (2) High cholesterol and (3) Phospholipids, with Phosphatidylcholine (Lecithin) fraction which is probably of major importance. Other than these generalities, little is known of maturation diets. Nevertheless, the inhibition of ovarian development under captive conditions was overcome by eye stalk ablation (Beard and Wickin, 1980). Unilateral eye stalk ablation is simple techniques for seed production (Villaluz et al., 1972).

Food is an important factor for sexual maturation (Browday, 1992) and male reproductive performance (Meunpol et al., 2005) Many advances were made in the development of technology for maturation and reproduction of penaeid shrimp (Bray and Lawrence, 1992 and Browday, 1992). The present study was undertaken to demonstrate the larval survival of ablated pond – reared P. monodon by using five different practical diets.

Materials and methods

For studies on the impact of fresh feeds on fecundity & hatching rates, the brooders , raised in shrimp farms were transferred to 10 ton capacity epoxy resin coated rectangular RCC tanks of 52.51 m for further

development as spawners. The initial weight of females and males ranged from 86.18 to 96.52 g and from 50.61 to 65.21 g respectively. The shrimps were fed with five different feeds combinations as crab and polychaete, oyster and polychaete, squid and clam, beef liver and squid, and oyster and beef liver. Shrimp were fed at the rate of 15% of their body weight. It took about 60 days for the conversion of pre-vitellogenic female into the vitellogenic stage. The developed spawners were transferred to a hatchery to assess the efficiency of spawners in terms of spawning, hatching and survival of larvae.

The gravid females were transferred to hatchery, where they were gradually acclimatized to the hatchery conditions and were disinfected with 10 ppm of formaldehyde and 50 ppm of Treflon (Trifluralin) respectively for 20 minutes. Spawners were fed at the rate of 15-20% of their bio-mass at regular interval of time both during the day and night. Water temperature ranged from 28-31C and sea water salinity 29-32.5 ppt in the hatchery and also in the laboratory. Prior to initiation of the trail, each spawner was weighed and the weight was recorded.

2Spawners were stocked at the rate of 2-3 m in female to male ratio of 2:1. Females were checked for the presence of spermatophores in the thelycum. Unilateral and bilateral eyestalk ablation was carried out in the early morning hours during the intermoult stage by cutting the eyestalk with sterilized scissors. After eyestalk ablation and a period of five to seven days, females were checked by with under water torchlight (sourcing light) for ovarian maturation by external observation. Based on the relative size of the ovary gravid females in maturation stage IV were transferred to round fibre tanks having 0.5 ton of filtered sea water of 30 ppt for spawning. After spawning, the


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water was drained using fine meshed net (100 µ mesh) to retain the eggs. The spawned eggs were washed with treated seawater containing Treflon as anti-fungal reagent, concentration ranging from 0.02 to 0.05 ppm. After 2 to 3 washings, the eggs were stirred and three one ml aliquots were drawn from the beaker to count the number of eggs. Then the eggs were transferred to 500 litre capacity black paint coated hatching tanks. After 40-45 minutes of spawning and upon the appearance of hatching envelop, the number of nauplii in 50 ml aliquots drawn from the hatching tanks was enumerated. The hatching rate was calculated by the following formula.

Total naupliiHatching rate (%) = ------------------ X 100

Total eggs spawned

Morbid larvae and Dead larvae were removed from larval rearing tanks by siphoning. A water temperature of 29-31C, pH 7.8, and salinity of 29-32.5 ppt were maintained at the optimum levels throughout out the study period. For studies on larval diet and larval development, the treated seawater as said above was used in the laboratory and hatchery. The larval rearing tanks (LRT) have capacities of 14-16 tons, which are parabolic in their structure and design.

The larvae were maintained in 1.2 m 7.5 m 1.8 m size tanks. The water level in each tank was maintained at 16 tones. Tanks with a water holding capacity of 14 tonnes were used for rearing the post larvae. A haemocytometer was used for algal cell counting in the medium. A salinity refractrometer was used for regular checking of the water salinity. Algal culture was done in 250 ml, 500 ml, 1000 ml and 2000 ml conical flasks (Borosil glass) and 20 litre carboys were used for indoor algal culture. 100 litres plastic cans, 1000 litre to 1200 litre FRP tanks, and 4 tonnes capacity rectangular concrete outdoor tanks were used for outdoor algal culture. Round, clylindro-conical fibre glass tanks of 500 litres capacity were used for artemia culture.

The various prophylactic measures taken with treatment groups are presented in Table I. In all the treatments, the water quality parameters such as temperature and salinity were in optimal range of 29-31C, 28-32 ppt respectively.

Results

An important aspect of the present study is on the influence of feeding condition on the growth and maturation of shrimps under captivity. This study is aimed in filling up the gap in aquaculture industry. Considerable variations were noticed when the shrimps were fed with combined fresh feeds. The feeds have remarkable influence on the growth body weight, and also on fecundity rates.

The maximum Average daily growth (ADG) of 0.401 g was recorded when the female shrimps were fed with crab and polychaete. In males the highest average daily growth (ADG) of 0.301 g, was observed when the shrimps were fed with crab and polychaete, while the lowest average

daily growth (ADG) of 0.257 g in female shrimps was recorded when the shrimps were fed with oyster and beef liver. The lowest average daily growth (ADG) of 0.183 g was recorded in male shrimps fed with oyster and beef liver (Fig.1 & 2).

Crab+Polychaete Oyster+Polychaete Squid+Clam

Beaf Liver + Oyster Squid + Beaf Liver



Fig. 1. ADG of Female shrimps (P. monodon)fed with different feed combinations

Fig. 2. ADG of Male shrimps (P. Monodon)fed with different feed combinations

A notable difference in body weight gain was recorded among the shrimps fed with different combinations of feed. The maximum weight gain of 24.06 g was noticed in females fed with crab and polychaete in 60 days of time, whereas in males the weight gain obtained was 18.12 g in the same duration of feeding condition and with the same feed. The results with respect to the average daily growth (ADG) and net body weight are summarized as follows.

Females

Average Daily Growth (ADG): Crab and polychaete, 0.401 g; oyster and polychaete, 0.364 g; squid & clam, 0.340 g: beef liver and squid, 0.298 g; oyster and beef


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liver 0.257 g. (Fig.1)

Net weight gain in 60 days: Crab & polychaete, 24.06 g; oyster and polychaete, 21.84 g; squid & clam, 20.40 g; beef liver and squid, 17.88 g; beef liver & oyster, 15.42 g; (Fig.3).

ADG of Males:

Crab and polychaete, 0.302 > oyster and polychaete 0.273 > squid and clam 0.234 > beef liver and squid 0.191 g > oyster and beef liver, 0.183 g. (Fig. 2).

Net weight gain: Crab and polychaete 18.12 > oyster and polychaete 16.38 > squid and clam 14.04 > beef liver and squid 11.46 g > oyster and beef liver 10.98 g. (Fig. 4)

Maturation

Ovarian maturation in shrimps fed with different combinations of feeds was found to vary significantly between the treatments. The minimum elapsed period recorded among wild unilaterally ablated was 6-9 days in shrimp fed with crab and polychaete followed by 5-10 days in shrimp fed with oyster and polychaete and the maximum of 10-16 days was recorded in the case of shrimp fed with oyster and beef liver (Table 1)

The minimum elapsed period recorded among captive unilateral ablated was 9-15 days in shrimp fed with crab and polychaete, and the maximum of 19-27 days was recorded in case of shrimp fed with oyster and beef liver. The minimum elapsed period in recorded among wild bilateral was 10-13 days in shrimp fed with crab and polychaete, and the maximum of 20-24 days was recorded in case of shrimp fed with oyster and beef liver (Table 1)





Fig. 3. ABW of Female shrimps (P. monodon)fed with different feed combinations

Fig.4. ABW of Male shrimps (P. Monodon)fed with different feed combinations

Feed combinations

Wild unilaterally ablated

Captive unilaterally ablated

Wild Bilaterally ablated

Crab and polychaete 6 to 9

9 to 15

10 to 13

Oyster and polychaete

5 to 10

11 to

16

12 to 18

Squid and clam

6 to 13

13 to 20

15 to 20

Beef liver and squid

8 to 15

16 to 24

18 to 22

Oyster and beef liver 10 to 16 19 to 27 20 to 24

Table: 1. Efficiency of different fresh feed combinations on maturation of shrimps

(elapsed time) from first stage to spawning (days).

Wild unilaterally ablated

Experiments on eyestalk ablation were conducted on shrimps collected from the wild (from the Sea) and the following observation ware made. Crab and polychaete 6-9 days > oyster and polychaete 5-10 days > squid and clam 6-13 days > beef liver and squid 8-15 days > oyster and beef liver 10-16 days (Table 1).

Captive unilateral ablated:

Eyestalk ablation was conducted on shrimps initially reared in aquaculture ponds and the results are like this. Crab and polychaete 9-15 days > oyster and polychaete 11-16 days > squid and clam 13-20 days > beef liver and squid 16-24 days > oyster and beef liver 19-27 days (Table 1).

Wild bilateral ablated:

Experiments on eyestalk ablation were conducted on shrimps collected from the wild (from the Sea) and following observation were made. Crab and polychaete 10-13 days > oyster and polychaete 12-18 days > squid and clam 15-20 days > beef liver and squid 18-22 days > oyster and beef liver 20-24 days (Table 1).


39

Spawning rate:

A comparison is made on the number of eggs laid per each brood in the shrimps raised under captivity and also shrimps caught in the sea. For this care is taken to include the same weight groups of animals and it is clearly evident that the number of eggs laid by the shrimps caught in the sea are significantly higher when compared to those that were raised under captivity Shrimp fed with crab ad polychaete and oyster and polychaete showed interesting results of higher spawning.

Hatching rate:

Hatching rate was more or less the same for successive spawning in ablated females. This could be related to batch availability and variability of sperm in the thelycum of the female. Hatching rate was more in shrimps collected from the Sea. Among wild ones the maximum hatching rate of 90.56 was recorded in unilateral ablated wild shrimp fed with crab and polychaete and the minimum of 69.85 in the unilateral ablated wild shrimp fed with oyster beef liver. The maximum number of nauplii hatched was 71.05 in the unilateral ablated captive shrimp fed with crab and polychaete and minimum of 32.25 captive shrimp fed with oyster and beef liver. In the wild bilateral ablated shrimps fed with crab and polycheate maximum 72.75 and minimum of 27.45 shrimp fed with beef liver and oyster respectively (Table 2).

Discussion:

Feed combinations

Wild unilaterally

ablated

Captive unilaterally

ablated

Wild bilaterally

ablated

Crab and polychaete 90.46 ±

1.34

71.05 ±

1.72

72.75 ±1.20

Oyster and polychaete

84.25 ±

2.44

53.13 ±

1.05

65.20 ±2.83

Squid and clam

72.66 ±

1.95

42.83 ±

1.42

51.99 ±1.56

Beef liver and squid

78.65 ±

1.93

40.88 ±

1.27

45.82 ±1.68

Oyster and beef liver 69.85 ±1.57 32.25 ±1.16 27.45 ±1.17

Table: 2. Percentage of hatching rate in wild and captive spawners

*Mean values standard deviation

As far as hatchery production of shrimp seed is concerned, continuous production of high quality nauplii has certainly remained an elusive goal. In this backdrop, the greater and better ovarian maturation and spawning success in captive spawners developed by feeding crab and polychaete was better, this argues in general with the findings of (Brown et al., 1979, Primavera et al., 1979, Beard and Wickins, 1980, Chamberlain & Lawrence 1981, Menasveta et al., 1994 and Sangpradub et al., 1994). In order to estimate the ecosystem support area that is needed to supply hatcheries with shrimp spawners, the supporting ecosystem or habitat type must first be assigned. Present studies will argue that the mangrove ecosystem is the

critical habitat for Penaeus monodon in Andhra Pradesh, India.

In present studies argue that the mangrove ecosystem is crucial prerequisition for the recruitment success of many penaeid shrimp species, particularly P.monodon. In the state of Andhra Pradesh, the Godavari river delta should be a key nursery ground, since this delta alone harbours 56% of the total mangrove cover in the state. This is also supported by the fact that the coastal areas off the Godavari delta constitute the main trawling grounds for both fin and shell fish in Andhra Pradesh.

Numerous investigations made on shrimp reproduction pointed out that various factors as photoperiod, light intensity, physico - chemical parameters of waters and color of maturation tanks play a vital role (Chamberlain and Lawrence, 1981 and Wyban et al., 1987) and eyestalk ablation (Chamberlain and Lawrence, 1981) was reported to be of much help in inducing maturation. When all the requirements are provided, eyestalk ablation is of immense use in inducing gonadal maturation. In the present study, brood stock developed from wild grown shrimps was converted as spawners making use of various feed combinations. Their reproductive performance was tested in a commercial hatchery. Detailed comparison is made with naturally caught shrimps with respect to survival rate, maturation, spawning rate and hatching rate to drive home the advantage of developing brood stock and spawners from farm grown ones due to the inherent merits.

In the feeding experiments, importance has been given to live polychaetes in the present study towards maturation and spawning as suggested by (Brown et al., 1979 and Middleditch et al., 1979), Next to polychaetes, squid was found to be the best single - food diet, as it produced higher levels (although not significantly higher in every case) of growth, molting, maturation and spawning than the other single food diets, the growth promoting characteristics of squid have earlier been reported (Deshimaru & Shigeno, 1972 and Fenucci et al., 1980). The high maturation performance may be related to the high percentage (98%) of sterol in the form of cholesterol (Lytle and Lytle, 1989) have pointed out that crustaceans such as Artemia and Mactra chinensis (Teshima et al., 1988) are considered as good diets with essential feed ingredients for maturation of captive shrimp. Nevertheless, it would appear that mussels are a much more productive in inducing maturation than clams. (Brown et al., 1979) found that Penaeus setiferus accepted, mussel more readily than oysters (Crassotrea sp.). suggested that food organisms for maturation should have ripe gonads for ingestion by brood stock and spawner. This would agree well with the present study where the various food items were not reproductively active when they were offered as food and therefore, were less effective than polychaetes which were reproductively active and rich in C 20: C 20 6, C 20:5 3 and C 22:6 3 compounds. Combined diets were proved to be more effective than a single diet.


40

The relationship between reproduction and growth is dependent up on the division of energy resources. Due to the division of energy towards reproduction a gradual decline in weight of females occurred.

Presently shrimp fed with crab and polychaete showed higher growth rates when compared to others. The next highest growth rate was recorded in shrimps fed with crab and polychaete and oyster and polychaete. The same trend was found in males also, that maximum growth rate in shrimps fed with crab and polychaete and in shrimps fed with oyster and beef liver. Santiago (1977) reported that P. monodon gained an ABW of 16 g. after four months of pond culture. Eyestalk ablation induces maturation in shrimps, whether they are caught in the sea or they are reared in the pond. Elapsed time from eyestalk ablation up to first maturation will be less initially compared to subsequent maturation; more elapsed time also leads to failure of hatching because of low fertilization. (Beard and Wickins, 1982,) found that 22 days of elapsed time in pond-reared females, but with less fertilization rate. Presently, highest elapsed time was recorded in captive animals fed with oyster and cow liver which showed the lowest percentage of hatching. The lowest elapsed time was recorded in both wild and captive spawners fed with crab and polychaete which showed higher hatching rates. Unilateral ablated shrimps showed better hatching rates with low elapsed time compared to bilateral ablated captive shrimps. (Emmerson and Andrews,1983; Hiller, 1984) recorded a higher spawning of 2,57,650 to 5,50,330 eggs per spawn from wild ablated P. monodon and 60,000 to 7,47,500 eggs from non-ablated females. The wide range in egg numbers obtained could be due to variations in weight of females (50 - 200g) and inclusion of eggs in counts from both partial and complete spawning (Alikunhi et al., 1975; Muthu & Laxminarayana, 1977). However, (Sangpradub et al., 1994) found that the average number of eggs per spawner fed with a fresh diet was 1,47,000 contrarily to shrimps with an average mean weight of 136.5 gr. fed with a fresh and pelleted diet yielded 1,39,000 eggs. (Menasveta et al., 1994) stated that the total egg production ranged from all 9, 11,460 in smaller (86.68.20 g) to 5, 73,086 in larger ones (135.715.989) reared in the pond.

The result obtained in the present findings indicated highest spawning rate of 4, 45,440 per shrimp in wild unilateral shrimps fed with crab and polychaete. The next highest egg production of 3, 56,487 per spawner was obtained in wild bilateral ablated shrimps fed with the same feed combination. Lowest egg production of 1, 52,945.6 was recorded in shrimp fed with oyster and beef liver of captive unilateral. In pond reared shrimps fecundity and hatching rate were 2, 04,000 eggs. In the present study the hatching rate was found to be low in captive spawners compared to wild ones. The differences in hatching rates may be due to nutrition, sex ratio, water depth and other bio-physical factors. It is also important to maintain certain male: female ration in the maturation tanks.

The highest rate, 90% hatching was recorded in wild unilateral eyes talk ablated shrimps fed with crab and polychaete and lowest hatching rate, 69% was obtained in wild unilateral ablated shrimps fed with oyster and beef liver. The hatching rate in captive raised spawners, 71% as higher and unilateral ablated shrimps fed with crab and polychaete, and minimum lowest of 32% was obtained in shrimps fed with oyster and beef liver. Hatching rate was more or less similar for successive spawning of wild and captive shrimps. Effective fertilization is possible only with males, weighing > 40 g showing signs of full maturity. (Primavera et al., 1979, Beard and Wickins, 1980 and Alfaro, 1993), recorded less hatching rate due to insufficient quantity of sperms. Captive and wild shrimp males weighing > 60 g were used in the treatment groups and there were good spawning rates from captive reared spawners as of wild ones. However the hatching rate was low in captive spawner and the reason might be long elapsed time, poor sperm quality and over stocking during spawner development. These should be overcome through further study. In the present work combination of crab and polychaete, and oyster and polyhchaete showed better result in terms of lower elapsed time, high rate of spawning and hatching than other feeds. Improvement of quality of water used in maturation tanks, spawning and hatching tanks through UV radiation found to reduce the levels of specific pathogenic micro organisms in the incoming sea water may be of help in the overall improvement of the reproductive performance. Efforts should also be made to investigate the male shrimps, which will be of help in enhancing the fertilization and hatching rates. Poor water parameters also affect reproductive quality (Perez-Velazquez et al., 2001). Nevertheless, the water quality for all treatments was considered ideal for the species (Peixoto et al., 2005), with temperature between 24.5 °C to 29 °C and salinity of 33 to 35 ppt. Thus, food probably was important factor for aggrieved the spermatophore degeneration reported in the results of the present study.

Conclusion

Our present findings gave satisfactory results on ovarian maturation and spawning in captive spawners, among five different feed combinations crab and polychaete combinations was better which elicit a greater success in the captive and wild caught spawners , further research should continue to establish other good combinations of feeds which will helpful for the production of high quality of nauplii, as hatchery production of shrimp seed is concerned, this will definitely fulfil the gaps in aquaculture.

Acknowledgement

The author thankful to “Pavan Aqua Pvt Ltd” located at Timmapuram, Visakhapatnam.


41

References

Alfaro J.J., Palacios A., Tito M.A. and Angulo R.A. (1993): Reproduction of the shrimp Penaeus occidentalis (Decapoda: Penaeidae) in the gulf of Nicoya, Costa Rica. Revistta de Bilogia Tropical. 41(3 part A): 563-572.

Alikunhi K.H., Poernomo A., Adisukresno S., Budiono M. and Busman S. (1975): Preliminary observations on induction of maturity and spawning in Penaeus monodon ( Fabricius) and Penaeus merguiensis de Man by eyestalk extirpation. Bull. Shrimp. Cult. Res. Cont. Jepera. 1(1): 1-11

Beard T.W., and Wickins J.F. (1980): Breeding of Penaeus monodon in a laboratory recirculation system. Acquaculture. 20: 79-89

Bray W.A. and Lawrence A.L. (1992): Reproduction of Penaeus species in capacity. In: A. Fast and L.J. Lester (eds.) marine shrimp culture: Principles and practices, (Elsevier Science Publishers), B.V. Bredene. The Netherlands.

Browday C.L. (1992): A review of the reproductive biology of Penaeus species: perspectives on controlled shrimp maturation system for high quality nauplii production. In J. Wyban (Ed.) Proceedings of the special session on shrimp farming, World Acquaculture Society, Baton Rouge, Louisiana, U.S.A.

Brown A., Mc Vey J.P., Middleditch B.S. and. Lawrence A.L. (1979): Maturation of white shrimp Penaeus setiferus in capacity. Proc. World Maricult. Soc.11: 488-499.

Chamberlain G.W. and Lawrence A.L. (1981): Maturation, reproduction and growth of Penaeus vannamei and P.stylirostris fed different diets. J. World Maricult. Soc. 12(1): 209-224.

Deshimaru O. And Shigeno K. (1972): Introduction to the artificial diet for Penaeus japonicus. Aquaculture. 1-12: 115.

Emmerson W.D. and Andrews B. (1983): The effect of stocking density on the growth and development and survival of Penaeus indicus (Milne Edwards) larvae. Acquaculture. 23: 45-47

Fenucci J.L., Zein Eldin Z.P. and Lawrence A.L. (1980): The nutritional response of two Penaeid species to various levels of squid meal in a prepared feed. Proc. World Maricult. Soc.11: 403-409

Hiller D.D. (1984): Artificial conditions influencing the maturation and spawning of sub adult Penaeus monodon (Fabricius). Aquaculture, 36: 179-184

Jia-Yuan Xu, Ting-Ting Wang. Yu-Feng Wang. Yu Peng. (2010): Effect of combined fish meal; soyabean meal ratio, Vitamin-C, and Fish oil supplementations in diet on the growth and reproduction of red shrimp. Cray fish, Procambarus clarkii (Crustacea-

Decapoda), Aquaculture 41, e 252-e 259.

Lytle J.S. and Lytle T.F. (1989): Fatty acid composition and variation in individual blood worms. Gulf coast research laboratory, P.O. Box. 7000, Ocean springs, Mississippi 39564, USA

Menasveta P., Sangpradub S., Piyatiratitivorakul S. and Fast A.W. (1994): Effects of broodstock size and source on ovarian maturation and spawning of Penaeus monodon (Fabricius) from the gulf to Thialand. J. World Aquaculture Soc. 25(1):41-49.

Meunpol O., Meejing P. and Piyatiratitivorakul S. (2005): Maturation diet based on fatty acid content for male Penaeus monodon (Fabricius) broodstock. Aquac. Res. 36, 1216–1225

Middleditch B.S., Missler S.R., Ward P.G., Mc Vey J.B., Broun A. and Lawrence A.L. (1979): Maturation of Penaeid shrimp: dietary fatty acids. Proc: World Maricult. Soc: 10: 472-476

Muthu M.S., and Laxminarayana A. (1977): Induced maturation and spawning Indian Penaeid prawns. Indian J. Fish. 24:172-180.

Peixoto S., Cavalli R.O. and Wasielesky W. (2005). Recent developments on broodstock maturation and reproduction of Farfantepenaeus paulensis. Braz. Arch. Biol. Technol. 48 (6), 997–1006.

Perez-Velazquez M., Bray W.A., Lawrence A.L., Gatlin D.M. and González-Félix M.L. (2001): Effect of temperature on sperm quality of captive Litopenaeus vannamei broodstock. Aquaculture 198, 209–218.

Primavera J.H., Lim C. and Bolongan E. (1979): Feeding regimes in relation to reproduction and survival of ablated Penaeus monodon, Klikasan. J. Biol. 8:227-235.

Sangpradub S., Fast A.W., Piyatiratitivorakul S. and Menasveta P. (1994): Effect of different feeding regimes on ovarian maturation and spawning reared gaint tiger prawn in Thialand. Acquaculture. Int. 2(1):49-58.

Santigo A.C.J.R. (1977): Successful spawning of cultured Penaeus monodon (Fabricius) after eyestalk ablation, Aquaculture, 11: 195-196

Teshima S., Kanazawa A. and Kakuta Y. (1988): Role of Dietary phospholipids in the transport of 14C tripalmitia in the prawn, Bull. Jpn. Soc. Sci. Fish. 52: 519-524.

Villaluz D.K., Villaluz A., Ladrera B., Sheik M. and Gonzaga A. (1972): Production, larval development and cultivation of sugpo (Penaeus monodon Fabricius). Phillip. J. Sci. 98: 205-236.

Wyban J.A., Lee C.S., Sweeney J.N. and Richards W.K Jr. (1987). Observations on development of a maturation system for P. vannamei. J. World Aquaculture. Soc. 18(3) 198-200.


42

Inhibitory effects of aqueous leaf extracts of Lantana camara on the growth of P.hysterophorus in fruiting stage.

Abstract : Lantana camara is notorious weed and a popular ornamental garden plant belonging to the family verbenaceae. L.camara is native to tropical and subtropical America. An experiment was conducted to understand inhibitory effects of aqueous leaf extracts derived from L.camara on growth of P.hysterophorus in fruiting stage. Bioassay also indicated that the inhibitory effect was proportional to the concentrations of the extracts and higher concentration had the stronger inhibitory effect.

Keywords : Allelochemicals, Leaf extract, Fruiting stage

Arpana Mishra Department of Botany, Govt. Chhatrasal P. G. College, Panna (M.P.), India Corresponding author, e-mail: [email protected]

Introduction

The term allelopathy is derived from two Greek words Allelon means each other and Pathos means to suffer, the term refers to effects that are both detrimental and beneficial among the interacting organisms (Rice, 1984). These effects have been observed in all classes of plants and also extend to microorganisms. Plants produce chemicals that directly of indirectly influence the environment. These chemicals are called allelochemicals.

th The origin of Parthenium weed dates back to 4 century BC in the Greek city of Parthenia. This weed has been named so according to the name of Greek city. It was distributed throughout the world along with wheat and belonging to the family Asteraceae. Parthenium is not only harmful to crop but also causes several diseases to man e.g. asthama, contact dermatitis and hay fever. Lantana camara is a most common weed belonging to the family Verbenaceae. L.camara has an allelopathic potential because it contains a number of phenolic compounds (Narwal, 1994) . The weed is aggressively growing in forest, agriculture, tea garden and wastelands of all over the country (Ahmed, 1997) . Allelochemicals of L.camara has potential in the development of green herbicides.

The present work was undertaken to study the effect of L.camara on growth and metabolism of Parthenium and to evaluate the allelopathic potential of the former.


The study area Shakti nagar lies in the Banda district of Uttar Pradesh in between Latitude 24º 53' and 25º 55' N, Longitude 80º 07' and 81º 34' E, the geographical area of the district is 4114.20 sq. km. Leaves of Lantana camara were collected from Chitrakoot region of Madhya Pradesh. Collection of raw material and preparation of extract in two days advance for each spray.

The preparation process undertaken for Lantana

camara leaf aqueous extract is as. 100gm under of leaf chopped in small pieces and crushed in the mixture grinder after grinding the material of leaf paste were soaked in 200 ml of distilled water for 24 hrs then prepare the following concentrations 100%, 50%, 33%, 25% and water as a control treatment. The extract of each specimen was filtered with muslin cloth. The concentration volume of each specimen was maintained by adding distilled water. Foliar treatment of fruiting Parthenium hysterophorus with different concentration aqueous leaf extract of Lantana camara on alternate days but control quadrates sprayed only distil water .

Plants samples were analyzed for shoot and root length, leaf area. Leaf area was measured with the help of a leaf area meter (Model No. 211 Systronice).


1. Effect of Lantana camara aqueous leaf extract on shoot and root length of Parthenium hysterophorus in fruiting stage

The different concentration of aqueous leaf extract of Lantana camara had significant effect on shoot and root length of Parthenium hysterophorus fruiting plant. The plant growth inhibit after aqueous leaf extract spray on Parthenium hysterophorus. Plant shoot and root length were decreased over control with the increasing concentration of extract. Plant shoot and root length were

th th rd stcontrol after 5 spray, 4 spray, 3 spray and 1 spray of 25%, 33%, 50% and 100% concentration of aqueous leaf extract of Lantana camara respectively. Finally plant were

th th th rddead after 7 , 5 , 4 and 3 spray of 25%, 33%, 50% and 100% concentration of aqueous leaf extract of Lantana camara respectively.

Maximum growth of shoot and root were observed 54.13% and 54.4% increased respectively in control. Plant shoot and root length were decreased over control with the increasing concentration of extract. The


43

plant growth inhibit after aqueous leaf extract spray on Parthenium hysterophorus fruiting plant. In 25% extract the plant growth were observed 8.27% increased in shoot and 6.4% increased in root over control. In 33% extract the plant growth were observed 4.78% increased in shoot and 33% increased in root over control.

Shoot and root length (cm) at the time of spray

Number of spray at the alternate days

Concentration in %

Plant growth

(cm)

1

2

3

4

5

6

7

Total Days (24)

% Increase 24 days

Over BT

% Decrease 24 days

Over BT

Control Shoot 29.0 30.9 32.7 34.7 36.9 39.3 41.9 44.7 54.13 Root 25.0 26.7 28.2 29.9 31.8 33.9 36.2 38.6 54.4

25 % Shoot 29.0 29.9 30.6 31.1 31.4 31.4 31.4 - 8.27 45.86 Root 25.0 25.7 26.2 26.5 26.6 26.6 26.6 - 6.4 48

33 % Shoot 23.0 23.6 24.0 24.2 24.2 - - - 4.78 49.35 Root 21.0 21.4 21.6 21.7 21.7 - - - 3.33 51.07

50 % Shoot 28.0 28.4 28.6 28.6 - - - - 2.14 51.99 Root 24.0 24.2 24.3 24.3 - - - - 1.25 53.15

100 % Shoot 29.4 29.4 29.4 - - - - - 0 0 Root 25.3 25.3 25.3 - - - - - 0 0

Table-1: Effect of Lantana camara leaf extract on growth (cm) of Parthenium hysterophorus in fruiting stage .

BT = Before treatment; - = Dead the Parthenium weed.

Minimum percentage increased 2.14% in shoot length and 1.25% in root length were recorded in 50% concentration, but in 100% extract concentration the plant growth was completely suppressed after single spray. Detail result showed in Table 1.

2. Effect of Lantana camara aqueous leaf extract on leaf area of Parthenium hysterophorus in fruiting stage

The different concentration of aqueous leaf extract of Lantana camara had inhibitory effect on leaf area of Parthenium hysterophorus fruiting plant. Plant leaf area was decreased over control with the increasing concentration of extract. The leaf area was decreased after aqueous leaf extract spray on plant. Maximum leaf area of Parthenium hysterophorus was observed 40.74% increased in control. In 25% concentration aqueous leaf extract the leaf area were observed 6.42% increased and in 33% concentration leaf area were observed 3.43% increased over control. Minimum percentage 1.70% increased was recorded in 50% concentration, but in 100% extract concentration, the leaf area was found constant as first spray. observation showed in fig1.

The effect of different concentration of Lantana camara leaf aqueous extracts were recorded and compared with control (i.e., distil water). Result showed, different concentration of aqueous leaf caused inhibitory effect on shoot and root elongation, leaf area of Parthenium hysterophorus fruiting plant. Bioassays also indicated that the inhibitory effect was proportional to the concentration of the extracts and higher concentration had the stronger inhibitory effect. The reason of inhibition may be the

Fig.1: Effect of different concentration of Lantana camara leaf extract on leaf area of

Parthenium hysterophorus in fruiting stage.

40.74

6.423.43 1.7 0

0

5

10

15

20

25

30

35

40

45

Control 25% 33% 50% 100%

Concentration

%In

cre

ase

inle

af

are

a

Leaf

presence of allelochemicals. The L. camara leaves contain allelochemicals like phenolic compounds, mono- and sesquiterpenes, triterpenes, triterpenoids, quinines, essential oils, flavonoids, biocides etc (Raghavan, 1976).

The water soluble allelochemicals of Lantana camara inhibited the initial growth of both the agricultural ( Oryza sativa, Triticum aestivum, Vigna sinensis, Cucurbita pepo, Abelmoschus esculentus, Amaranthus tricolor and forest crops (Acacia auriculiformis, Paraserianthes falcataria, Albizia procera) in the laboratory conditions (Hossain & Alam, 2010). The phenolic compounds extracted from the leaves of Lantana camara were found to be phytotoxic to rice, wheat and three grass seedlings. The extracts of leaf, stem, flower and


44

fruit of Lantana camara inhibited the seed germination of Parthenium hysterophorus (Mishra & Singh, 2010). The extracts of Lantana camara leaves and their fractions reduced the biomass of Eichhornia crassipes and Microcystis aeruginosa within 7 days under laboratory conditions (Kong et al., 2006). Leaf extract showed pronounced inhibition of shoot length, root length, leaf area, fresh and dry weight of the seedling Parthenium (Mishra & Singh, 2012). Thus, the phytotoxicity of L.camara can be considered as a source of potent green herbicide to control P. hysterophorus fruiting stage.

References

Ahmed N. (1997) : Wild Flowers of Bangladesh. Dhaka, Bangladesh. (The University Press Ltd.) p.142.

Hossain M K and Alam, NMD. (2010): Allelopathic effects of Lantana camara leaf extract on germination and growth behavior of some agricultural and forest crops in Bangladesh. Pak.J.Weed Sci .Res. 16(2), 217-226.

Mishra A. and Singh R. (2010): Comparative study of effect of Lantana camara extract of different parts on seed germination of Parthenium hysterophorus L. International Journal of Plant Sciences 5 (1), 74-75.

Mishra A. (2012): Allelopathic interaction of L. camara leaf of extract on growth of P. hysterophorus in seedling stage. International journal of plant sciences. 7 (2), 259- 262.

Kong C.H: Wang P., Zhang C.X., Zhang M.X. and HU F. (2006): Herbicidal potential of allelochemicals from Lantana camara against Eichhornia crassipes and the alga Microcystis aeruginosa. Weed Research 46(4), 290-295.

Narwal S. S. (1994) : Allelopathy in crop production. (Scientific publishers, Jodhpur India) p.288.

Raghavan V. (1976): In Recent Advances in Botany. (Ed.) Kachroo P., Bishan Singh and Mahendra Pal Singh, (Dehradun) p.264

Rice E. L. (1984): Allelopathy. II edition. (Academic Press, New York) 422.


45

Inhibitory efficacy of Chlorpyriphos and Datura stramonium on Acetylcholinesterase activity, Kinetics and Histology of Brain of Catla catla

Abstract : Influence of ethanol leaf extract of Datura stramonium, an indigenous plant used in ayurvedic medicine in India and commonly used organophosphorus pesticide; chlorpyriphos on acetylcholinesterase (AChE) activity, kinetics and histology were investigated in the brain of Catla catla. The recovery of inhibited AChE by both compounds was also determined. It was observed that 96 hrs exposure of Datura stramonium leaf extract (100 mg/L) produces 47.2% AChE inhibition and chlorpyriphos (0.00073 mg/L) elicits 33% AChE inhibition. A considerable synergistic inhibitory effect was found in the brain AChE with pre-treatment of Datura stramonium leaf extract followed by chlorpyriphos exposure. This treatment could yield 60% inhibition. The recovery of Datura stramonium extract-induced AChE inhibition was ranged from 1.6% (48 hrs) to 11.7% (120 hrs) while chlorpyriphos-induced AChE inhibition was recovered to 8.1% (48 hrs) and 23.8% (120 hrs). Kinetic study of

-3AChE also showed inhibitory potential of these compounds. The Km of control group was 0.41 x 10 M. This was -3 -3 -3increased to 0.9 x 10 M (Datura stramonium extract), 0.76 x 10 M (chlorpyriphos), and 1.1 x 10 M (pre-treated

with Datura stramonium extract followed by chlorpyriphos exposure). The Vmax values were constant i.e. 0.11 activity/min/mg protein in control and treated groups showing competitive AChE inhibition. The cyto-architectural profile of treated brain shows histological alterations indicated by varying degree of necrosis and vacuolation in the molecular and granular layers.

Keywords: Acetylcholinesterase, Brain, Catla catla, Chlorpyriphos, Datura stramonium Inhibition, Kinetics, Histology.

Namdeo, A., Tembhre, M.,* Banerjee, S., Gour, S. and Ahirwar, S. MLB Govt Girls (Autonomous) PG College, Bhopal, IndiaM K Ponda College, Bhopal, India. *Email: [email protected]

Introduction

The acetylcholinesterase enzyme (AChE; E.C 3.1.1.7) is most acceptable target for the assessment of inhibitory action of various organophosphate (OP) pesticides. Hence, determination of inhibition of AChE is widely employed in bio-monitoring studies and considered as most reliable biomarker of pesticide pollution (Carr et al., 1995; Somnuek et al., 2007, Kumar and Tembhre, 2010 and Tembhre et al., 2012). The primary mode of action of organophosphorus pesticide is inhibition of AChE activity, the enzyme that degrades the neurotransmitter acetylcholine in cholinergic synapses in the vertebrate nervous system (Silver, 1974). The use of large number of new generation pesticides is still in progress, which leads to adverse synergistic implications on non-target species (Susan et al., 2010). Many of these pesticides may linger on in the environment and lead to detrimental effects. Inhibitory effects of dimethoate on AChE activity of fish have been reported by Satyadevan et al. (1993), Tembhre and Kumar (1994), Singh and Kumar (2000). Methylamine is also known to produce significant inhibition in fish AChE. (Tembhre and Kumar, 1995 & 1997). The chronic exposure to sub lethal concentration of Diazinon shows significant inhibitory effect on AChE in the brain of Clarius gariepinus (Adedeji, 2011). Chlorpyriphos (CPF), an organophosphate pesticide is widely used for pest control on cotton, corn, almond and fruit trees including oranges and apples. CPF binds with acetylcholinesterase in cholinergic nerves at synaptic vesicles. This results in

inhibition of AChE causing impairment of hydrolysis of neurotransmitter acetylcholine (Tembhre et al., 2006; Somnuek, 2007). Chlorpyriphos have been reported to inhibit acetylcholinesterase activity in the brain of fish (Rao et al., 2005; Halappa and David 2009; Wang et al., 2010).

Study of recovery of pesticide-induced acetylcholinesterase inhibition is important as it reveals status of pesticide-AChE complex. Carr et al. (1995) reported that after 60 days, the brain AChE of mosquito fish was almost fully recovered from inhibition by chlorpyriphos. Tembhre et al. (2006) observed recovery of chlorpyriphos-induced AChE inhibition in Cyprinus carpio. They reported 83%, 88% and 90% recovery in fore-, hind- and mid brain on seventh day after chlorpyriphos exposure. Complete recovery of inhibited brain AChE was observed in mosquito fish after 60 days exposure of chlorpyriphos (Russel et al., 1997)

The kinetic study on the brain AChE inhibited by malathion in Tilapia mossambica reveals that malathion competitively inhibits AChE (Sahib et al., 1980). Dembele et al. (1999) investigated AChE kinetics in the brain of Cyprinus carpio exposed to chlorfenvinphos and carbofuran. They reported that both the pesticide induced competitive inhibition in the brain AChE of the fish.

Scientists have investigated a large number of medicinal plants for screening their pesticides properties (Tiwari and Singh, 2004; Crowch and Okello, 2009). Some


47

medicinal plant extract contain various phyto-chemical known as alkaloids, tannin, saponin, nicotine etc. These active ingredients are toxic to fish (Murthy et al., 2010; Kumar and Sikarwar, 2003). Since, synthetic pesticides create various environmental problems because of slow rate of their degradation. The plant based insecticides are considered to be eco friendly because of their easy degradability. Datura stramonium has been reported to be used as medicine in reducing pain and as a narcotic and local anaesthetic drug in many places (Abena et al., 2003). Some findings of investigation revealed that Datura stramonium extract predominately contained alkaloids viz. atropine, hyoscyamine, and scopolamine (Adekomi et al., 2011). In all species of genus Datura the concentration of alkaloid varies depending on species and on the part of the plant (Duez et al., 1985). Friedman and Levin, (1989) reported Datura stramonium seeds as neuroinhibitor. Several investigators have discussed the effect of ethanol leaf extract of Datura stramonium on animals (Gidado et al., 2000 & 2007).

Organophosphate pesticides are well known to adversely affect the histological components of various organs including brain of fish (Gupta and Guha, 2006; Butchiram et al., 2009). Exposure of sub lethal concentrations of malathion and dimethoate to Catla catla showed vacuolation in brain with eccentric nuclei, necrosis in molecular and granular layers (Singh M, 1998). Sarma et al. (2010) also reported mild necrosis in the apical lobe of cerebrum of brain of Channa punctatus intoxicated with endosulfan.

The present study was undertaken to investigate that whether the use of synthetic pesticides can have a botanical alternative which may proved to be more effective and less persistent in the environment due to its rapid rate of degradation. Nevertheless, Datura stramonium (D.s) has never been tried for anticholinesterase activity in fish. Therefore, in the present study, we compared the effects of efficacy of an organophosphorus pesticide, chlorpyriphos and a medicinal herb Datura stramonium leaf extract on AChE activity, kinetics, and recovery of inhibited AChE and histology of brain of Catla catla.


Chemicals

Chlorpyriphos (O,O,Diethyl-O-(3,5,6 trichloro-2-pyridyl) phosphorothioate) 94% purity technical grade, Acetylthiocholine Iodide (ATChI) and DTNB (5-5'dithiobis-2-nitrobenzoic acid) (Himedia, India), Bovine Serum Albumin (BSA) (Loba Ceremic), Folin's reagent (Merck).

Plant Material

Fresh leaves of Datura stramonium were collected from botanical garden, authenticated, thoroughly washed in water and shade dried. Leaves were powdered and extracted in Soxhlet apparatus with 90% ethanol as

solvent. The extract was kept at room temperature for evaporation of ethanol till semi solid mass left. This was

okept stored at 4 C for further use.

Experimental Animal

Fingerlings of Catla catla collected from the fishpond near Kolua village, Raisen road, Bhopal were used in this experiment. They were acclimatized for 15 days prior to the experiment. Fishes were fed daily with commercial dry feed pellets (Tokyu, Spirulina, Japan). The fishes had median weight 100 ± 10 gm. The fishes were stocked in glass aquaria of 60 liters supplied with tap water

0(temperature 22.7 + 0.61 C, hardness as CaCO 212 + 4.8 3

ppm, pH 7.3 + 0.05, chlorides 87.62 + 2.39, total alkalinity as CaCO 165 + 1.15 ppm). A Physico-chemical property 3

of water was constantly checked according to APHA/AWWA/WEF (2005). Oxygen content was maintained with the help of aerator. The feeding was stopped before 24 hrs prior to and during the exposure period, which extended 96 hrs.

Fishes were divided into four groups of thirty each. Group-I: served as Control; Group-II: exposure to sub lethal concentration 0.00073 mg/ L was based on the 96 h LC value (0.0034 mg/ L) of CPF for Catla catla was 50

selected for test. The fishes were exposed to this concentration daily for 96 hrs with replenishment of water at every 24 hrs. Group-III: Fishes were exposed to 100 mg/L of Datura stramonium leaf extract for 96 hrs. ; Group- IV: The fishes were pre-treated with 100 mg/L of Datura stramonium extract for 96 hrs followed by the exposure to 0.00073 mg/L chlorpyriphos for 96 hrs. At the end of the experiment, five fish were removed from each group to study the effect of CPF and D. s extract. However, remaining fishes were transferred to toxicant free water to study recovery of AChE. Water was changed after every 24 hrs. Five fishes from this stock were removed and dissected at the end of 24 hrs, 48 hrs, 72 hrs, 96 hrs and 120 hrs.

Sample Preparation

Treated fish were euthanized, dissected and the brains were removed quickly and washed in 0.9% saline. A 10% (w/v) tissue homogenate was prepared in Elvehjem-potter homogenizer and centrifuged at 5000 rpm for 20 min

0in cooling centrifuge (Remi) at 4 C. The supernatants were kept in deep freeze for AChE assay, kinetics and Protein content.

Enzyme Assay

AChE activity was measured spectrophoto-metrically according to the method of Ellman's et al. (1961) in the Brain of Catla catla. Samples of homogenate were diluted with 2.6 ml 0.1 M Sodium phosphate buffer (pH 7.4) to which 100 mM DTNB and 75 mM ATChI was added. The rate of color production was measured at 412 nm in SL 164 UV-VIS spectrophotometer. All measurements were done in duplicate. Specific activity was expressed in nmol/min/mg protein.


48

Protein estimation

Protein was estimated by the method of Lowry et al. (1951) using BSA as the standard. Samples of homogenate were diluted with reagents then 0.5 ml Folin's reagent was added and after 20 min read at 620 nm against a reagent blank. Measurements were done in duplicate.

AChE Kinetics

Kinetic parameters (Km & Vmax) were determined by using four different concentrations of substrate i.e 0.66 mM, 0.44 mM, 0.33 mM, 0.26 mM. Lineweaver-Burk plot was made by plotting the reciprocals of velocity and substrate concentration.

Histology

Brain was removed from three fishes from each group after treatment. Tissues were fixed in freshly prepared aqueous Bouin's fluid in glass vials for 24 hrs, washed in running tap water, dehydrated in graded series of alcohol, cleared in xylene, infiltrated and embedded in paraffin wax. Multiple sections were cut at 5-6 micron thickness and stained with Haematoxylin and Eosin. Slides were examined using binocular microscope (Olympus) and the selected fields were microphotographed at 100X and 400X with computer-aided microscope (Leica).

Statistical analysis

Graphs of results were prepared by applying Excel 2007 software. For the data of statistical comparison between different treatments and control, data were analyzed by Student's t-test to determine the effect of the treatment. The level for the accepted statistical significance was p>0.05.


The effect of sub lethal concentration of CPF (0.0034 mg/ L) for 96 hrs demonstrated significant (p > 0.01) reduction in acetylcholinesterase activity in the brain of Catla catla. The brain AChE activity was declined to 9.45 + 2.69 nmole/ min/ mg protein against the control fishes in which AChE activity was 14.14 + 8.41 nmole/ min/ mg protein. CPF produced -33% inhibition (Table-1: Fig-1). The treatment of ethanol leaf extract of Datura stramonium (100 mg/L) also caused depletion in AChE activity to 7.46 + 3.68 nmole/ min/ mg protein showing 47.2% brain AChE inhibition. However, 96 hrs pretreatment of fish to D.s extract (100 mg/L) followed by (0.0034 mg/ L) CPF exposure for 96 hrs produced significant (p > 0.01) inhibition in AChE activity to 5.65 + 2.11 nmole/ min/ mg protein indicating 60% inhibition (Table-1: Fig-1).

Our previous study reported that the AChE activity of Cyprinus carpio intoxicated with CPF displayed differential inhibition i.e. 66.6% in fore- , 40% in mid- and 50% in hind brain (Tembhre et al., 2006). Balint et al. (1995) reported that the exposure of 2 mg/L methidathion for 5 days caused 90-92% decrease in AChE activity in Cyprinus carpio. A dose dependent reduction in AChE

activity in the brain of Clarius gariepinus was demonstrated up to 85% (Adedeji, 2011). The decrease in brain AChE activity in Cyprinus carpio exposed to sub lethal concentration 7.5 µg/L of quinalphos was recorded with 75.2% AChE inhibition (Chebbi and David, 2009). The present study revealed that CPF produced significant brain AChE inhibition in Catla catla. The neurotoxic effect of CPF is potentiated by its biotransformation to a more potent oxon metabolite that inhibits AChE substantially (Fukuto, 1990).

It has been reported that various parts of Datura stramonium being medicinal plant, were observed to be poisonous (Devi et al., 2012). There is a little information in the literature regarding its anticholinergic property to the animals. The present investigation is a first attempt towards the understanding of the neurotoxic effect of Datura stramonium leaf extract by observing brain AChE inhibition in Catla catla. The exposure of plant extract at 100mg/L concentration was found to induce significant 47.2% AChE inhibition.

Suganthy et al. (2009) reported 50% AChE inhibition by methanolic leaf extract of Rhizophora lamarkii, Suaeda monica, Avicennia officinalis and Sesuvium portulacastrum. An active compound mahanimbine, a carbazole alkaloid isolated from Murraya koengii displayed a dose-dependent AChE inhibition (Kumar et al., 2010). Ethanol extract of Bacopa monnieri and Ginkgo biloba possess AChE inhibitory power as suggested by Das et al. (2002). They reported that 100 mg/kg and 300 mg/kg extract of Bacopa monnieri and Ginkgo biloba respectively showed dose-dependent in vitro AChE inhibition in brain.

Recently it has been revealed that ethanol extract of Bacopa monnieri noticeably inhibit AChE activity in various regions of the brain (Ahirwar et al., 2012). They reported 72% AChE inhibition in hippocampus, 58% in brain stem, 50% in pons, 46.5% in cerebellum, 44% in thalamus, 40% in Cerebral cortex and 33.3% straitum. The preliminary phytochemical study showed the presence of alkaloids including atropine, hyoscyamine and scopolamine that can all elicit anticholinergic poisoning (Ertekin et al., 2005). The analysis also showed that these anticholinergic alkaloids are responsible for blocking of AChE activity in cholinergic nerves (Friedman, 2004). We observed that pre-treatment of D.s leaf extract followed by CPF exposure substantiate AChE inhibition in brain further to 60%. It might be causing synergistic physiological effect when used in continuous exposures.

Our result showed that AChE activity was recovered gradually to 8.1% at 48 hrs; 17.7% at 72 hrs; 21.6% at 96 hrs & 23.8 % at 120 hrs after exposure to chlorpyriphos. Recovery of inhibited AChE after exposure to leaf extract of Datura stramonium was found to be 1.6% at 48 hrs; 2.4% at 72 hrs; 5.2% at 96 hrs & 11.7% at 120 hrs. Our findings revealed that there is considerable difference in the extent of recovery of chlorpyriphos and Datura


49

stramonium treated fish (Table-2: Fig-2). Brain AChE inhibition in Cyprinus carpio exposed to sub lethal concentration of quinalphos was recovered up to 35% after

th14 day (Chebbi and David, 2009). Parathion-induced brain AChE inhibition in Gasterosteus aculeatus was significantly recovered after 48 hrs of exposure (Wogram et al., 2000). It is evident from observation of various investigations that recovery period vary with tenure after exposure, degradability of inhibitor and degeneration of acetylcholinesterase (Russel et al., 1997). Tembhre et al., (2006) reported 83%, 88% and 90% recovery of AChE in fore-, hind- and mid brain respectively after 168 hrs of exposure of chlorpyriphos in Cyprinus carpio. Our findings indicate that leaching of D. s extract is slow than CPF in the brain.

We have compared AChE Kinetics also to study the nature of inhibition. The Km value of control brain was

-30.41 x 10 M. Our study showed that Km increased in -3chlorpyriphos treated Brain to 0.76 x 10 M and in D. s

-3extract treated to 0.9 x 10 M. However, in group IV it was -3further enhanced to 1.1 x 10 M. However, Vmax

remained constant i.e. 0.11 activity/min/mg protein in control and treated fishes (Table-1). Therefore, increasing pattern of Km and constant Vmax showed that both the CPF and D.s extract inhibit the Brain AChE in a competitive manner (Fig-3). Similar findings were reported with malathion in Catla catla (Singh and Kumar, 2000), with methyl paraoxon in Prochilodus lineatus (Silva Filho et al., 2004) and with monocrotophos in Channa punctatus (Rahman et al., 2004). There is lack of information on kinetics of enzyme inhibition in fish by herbal extracts. Our study has demonstrated for the first time that Datura stramonium extract is able to display brain AChE inhibition kinetics similar to those of OP pesticides. Therefore, we reported for the first time that it has competitive inhibitory property of AChE. Acacia

niloticus and Rhamnus prinoides showed mixed inhibitory effects viz. non competitive-uncompetitive type (Crowch and Okello, 2009).

The histological observations of the control brain (Fig-4, 5) showed the molecular layer, granular layer and purkinje cell layer. The components of these layers were architecturally normal. CPF treatment mildly affected molecular and granular layers causing necrosis, which is evidenced by the appearance of narrow spaces and vacuolation (Fig-6, 7). These changes were moderately increased in the brain treated with leaf extract of Datura stramonium (Fig-8, 9). However, it was slightly greater in the brain of fish pre-treated with Datura stramonium leaf extract followed by CPF exposure (Fig-10, 11). Intoxication of 0.35 ppm hexachlorocyclohexane to Labeo rohita produced mild vacuolar changes with small spaces in brain, whereas 1.73 ppm exposure showed severe necrosis (Das and Mukherjee, 2000). Chronic exposure of 0.6 and 1.3 µg/L endosulfan to rainbow trout did not cause lesions in brain (Altinok and Capkin, 2007). Severe damage in brain cells and neural cells with broken neural bundles were observed in 100 ppm malathion treated Ophiocephalus punctatus (Pugazhvendan et al., 2009). Recently, above mentioned alterations were found in common carp exposed to atrazin and chlorpyriphos (Houjuan et al., 2012).

Thus, present investigation leads us to conclude that leaf extract of Datura stramonium induced more neurotoxicity in the brain as compared to chlorpyriphos. The study revealed that Catla catla brain AChE is more sensitive to herbal extract. Thus, leaf extract of Datura stramonium may be considered as potential herbal pesticide for the control of predatory fishes. However, further isolation and purification of phytochemicals are necessary to investigate the presence of active ingredient.

-

-33%

-47.2%

-60%

Parameters

Groups

Specifi c

activity of

AChE

% inhibition of

AChE Km x 10

-3 M

Vmax

(activity/min/mg

protein)

Control 14.14 ± 8.41 0.41 x 10 -3 M 0.11

CPF 9.45 ± 2.69** 0.76 x 10 -3 M 0.11

D. s 7.46 ± 3.68* 0.9 x 10 -3 M 0.11

Pre -treatment of

D. s followed by

exposure of

CPF

5.65 ± 2.11** 1.1 x 10 -3 M 0.11

Table-1: Effect of 96 hrs exposure of chlorpyriphos and Datura stramonium on acetylcholinesterase activity, inhibition, Km and Vmax of the Brain of Catla catla. The Specific activity is expressed in nmole/ min/ mg protein. Each value is mean ± SD of five individual observations. Significant: P > 0.05*; Highly significant: P > 0.01**


50

Group Parameter

Control CPF (0.00073 mg/ L)

Recovery of inhibited AChE D.s (100 mg/L)

Recovery of inhibited AChE

48 hrs 72 hrs

96 hrs

120 hrs

48 hrs

72 hrs 96 hrs

120 hrs

AChE activity (nmole/min/ mg protein)

14.14± 8.41

9.45± 2.69 **

10.2±4.0**

11.1±2.3 ***

11.5± 0.68

***

11.7± 0.86 ***

7.46± 3.68*

7.58± 0.50 ***

7.6± 0.81 ***

7.85±

0.48 ***

8.34± 0.26

***

% inhibition of AChE

- 33% 27.7 21.2 18.6 17.2 47.2% 46.3 45.9 44.4 41

% recovery of AChE

- - 8.1 17.7 21.6 23.8 1.6 2.4 5.2 11.7

Table-2: Recovery of inhibited AChE due to chlorpyriphos and Datura stramonium exposure in the Brain of Catla catla. Each value is mean ± SD of five individual observations. Significant: P > 0.05*; Highly significant: P > 0.01**; Very highly significant: P > 0.001***

Fig-1: Percentage inhibition of AChE activity in the Brain of Catla catla of control group, CPF group (0.00073 mg/ L), D.s group (100 mg/L) and pre-treatment of D.s leaf extract followed by CPF.

0

33

47.2

60

0

10

20

30

40

50

60

70

Control Chlorpyriphos Datura stramonium

Datura stramonium and chlorpyriphos

BRAIN

% i

nh

ibit

ion

of

AC

hE


51

Fig: 2 - Recovery of inhibited AChE after 48,72, 96 and 120 hours in the Brain of Catla catla exposed to chlorpyriphos (0.00073 mg/ L) and Datura stramonium (100 mg/L) for 96 hours.

Fig-3: Lineweaver-Burk plot showing competitive inhibition in the Brain AChE of Catla catla exposed to CPF (0.00073 mg/ L), D. s leaf extract (100 mg/L) and pre-treatment of D.s leaf extract followed by CPF for 96 hrs.

8.1

17.7

21.623.8

1.6 2.4

5.2

11.7

0

5

10

15

20

25

48 hrs 72 hrs 96 hrs 120 hrs

Duration (in hours)

BRAIN ChlorpyriphosDatura stramonium

% R

ecov

ery


52

Fig-4: Microphotograph of T. S. of the Brain of Catla catla (Control group) showing molecular layer (ML); granular layer (GL); purkinje cell layer (PCL) (H & E; 100 X)

Fig-5: Microphotograph of T. S. of the Brain of Catla catla (Control group) showing molecular layer (ML); granular layer (GL) (H & E; 400 X)

Fig-6: Microphotograph of T. S. of the Brain of Catla catla exposed to CPF (0.00073 mg/L) for 96 hrs showing necrosis in molecular layer (NML); necrosis in granular layer (NGL) (H & E; 100 X)

Fig-7: Microphotograph of T. S. of the Brain of Catla catla exposed to CPF (0.00073 mg/L) for 96 hrs showing necrosis in molecular layer (NML); necrosis in granular layer (NGL) (H & E; 400 X)

Fig-8: Microphotograph of T. S. of the Brain of Catla catla exposed to 100 mg/L D.s extract for 96 hrs showing necrosis in molecular layer (NML); necrosis in granular layer (NGL) (H & E; 40 X)

Fig-9: Microphotograph of T. S. of the Brain of Catla catla exposed to 100 mg/L D.s extract for 96 hrs showing necrosis in molecular layer (NML); necrosis in granular layer (NGL); vacuole formation (VF) (H & E; 100 X)


53

Figure-10: Microphotograph of T. S. of the Brain of Catla catla pretreated with 100 mg/L ethanolic leaf extract of D.s followed by CPF (0.00073 mg/L) for 96 hrs showing necrosis in molecular layer (NML); necrosis in granular layer (NGL); vacuole formation (VF) (H & E; 100 X)

Figure: 11 - Microphotograph of T. S. of the Brain of Catla catla pretreated with 100 mg/L D.s extract followed by CPF (0.00073 mg/L) for 96 hrs showing necrosis in molecular layer (NML); necrosis in granular layer (NGL); vacuole formation (VF) (H & E; 400 X)

Acknowledgement

The authors are thankful to Principal, MLB Govt Girls College, Bhopal, India for the research facility and to Head of the Department of Biotechnology, Barkatullah University, Bhopal, India for providing facility for microphotography.

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57

An Advance Technique for Solving Assignment Problems

P.R. Sharma*, S. Shukla** *Department of Mathematics, University of Rajasthan, Jaipur (India) **S. S. Jain Subodh P.G. College, Jaipur (India)

Email Id.: [email protected] , [email protected]

Abstract: The assignment problem is a typical combinatorial optimization problem. Classical Assignment Problem (AP) is a well-known topic world-wide. In this problem cij denotes the cost for assigning the jth job to the ith person. This cost is usually deterministic in nature. But in realistic situations, it may not be practicable to know the precise values of these costs. We consider many classical problems from location theory which may serve as theoretical models for several logistic problems such that some linear or quadratic function attains its minimum. It turns out that linear objective function yields a linear assignment problem, which can be solved easily by several primal-dual methods like Hungarian method, Shortest augmenting path method etc. Aim of the paper is to investigate a new approach to solve assignment problems of different types. This method proposes momentous advantages over similar methods. Keywords: Assignment Problem, Branch, Bound Technique.

Introduction:

The assignment problem is one of the earliest applications of linear integer programming problem. A

diversity of practical problems turn out to be a special illustration of the assigning problem, i.e. a problem,

where one looks for an assignment of members of set A to members of set B such that some function attains

its optimum. We may assume that the numbers of elements in sets A and B are equal and that we want to

assign exactly one element from A to each element from B. Solving such problem often means that we have

to evaluate some function for each assignment. Since there are n! Possible assignments, where n is the

number of elements in A, this may lead to a very hard problem. Different methods have been presented to

solve assignment problem by Goel and Mittal (1982), Bazarra et al. (2005) and Hamdy (2007).

A considerable number of methods have been so far presented for assignment problem in which, the best

known, most widely used, and most written about method for solving the assignment problem is the

“Hungarian Method”, originally suggested by Kuhn in 1955. Ford and Fulkerson (1957) provided vital ideas

for the untimely approaches used in solving network flow problems, extended to solve the transportation

problem [Munkres (1957)] and generalized to solve the linear programming problem [Dantzig et al. (1956)].

It is a dual method with a feasible assignment being obtained only at the last computational step.

Aim of the paper is to apply Branch and Bound technique for all type of assignment problems. The proposed

method is a methodical process, easy to apply and can be exploited for all types of assignment problems with

maximize or minimize objective functions.


58

Mathematical Formulation of Assignment Problem:

Let there be n persons and n jobs. Each job must be done by exactly one person and one person can do, at

most, one job. If the cost of doing thj job by thi person is ijc , then the cost matrix is given by the following

table:

jobs � Person �

1 2 3 ………. J ………..n

1 11c 12c 13c ………. 1 jc ……….. 1nc

2 21c 22c 23c ………. 2 jc ……….. 2nc

3 31c 32c 33c ………. 3 jc ……….. 3nc

……….. ………..

i

…… ……

1ic

….. …..

2ic

….. …..

3ic

………… ………… ……….. ijc

………… …………

………… inc

……….. ………..

n

….. …..

1nc

….. …..

2nc

…. ….

3nc

……….. ……….. ……….. njc

…………. ………….

…………. nnc

Table 1

The problem is to assign the persons to the jobs so that the total cost of completing all jobs become

minimum.

We introduce ,ijx where

1, if the person i is assigned the job j; i,j = 1, 2, ....., n

0, otherwiseijx⎧

= ⎨⎩

………. (1)

Corresponding to the ( )thi j event of assigning person i to job j, the constraint

1

1, 1, 2,....,n

iji

x j n=

= =∑ i.e. each job must be done by exactly one person, and the constraint

1

1, i 1, 2,....,n

ijj

x n=

= =∑ means each person must be assigned at most one job.

Thus the model for crisp Assignment Problem (AP) is given by

Model 1:

1 1

n n

ij iji j

Min z c x= =

=∑∑ ………. (2)


59

1

1

1, 1, 2,....,

subject to 1, i 1, 2,....,

0 or 1, , 1, 2,...,

n

iji

n

ijj

ij

x j n

x n

x i j n

=

=

⎧ = =⎪⎪⎪

= =⎨⎪⎪ = =⎪⎩

∑

∑ ............ (3)

This cost ijc is usually deterministic in nature. But in real situations, it may not be practicable to know the

precise values of these costs. In such an uncertain situation, instead of exact values of costs, if the preferences

for assigning the thj job to the thi person is known in the form of composite relative degree ( ijd ) of

similarity to ideal solution (maximum degree indicates most preferable combination), then ijc can be replaced

by ijd in the classical assignment problem in the maximization form which can be solved by any standard

procedure ( e.g. Hungarian method or by any software) to get the optimal assignment. In that case the model

for the preference AP becomes

Model 2:

1 1

n n

ij iji j

Max z d x= =

=∑∑ ………. (4)

1

1

1, 1, 2,....,

subject to 1, i 1, 2,....,

0 or 1, , 1, 2,...,

n

iji

n

ijj

ij

x j n

x n

x i j n

=

=

⎧ = =⎪⎪⎪

= =⎨⎪⎪ = =⎪⎩

∑

∑ ……… (5)

Method of Solution:

In this paper, the assignment problem is solved by Branch and Bound Algorithm. Using curtailed

enumeration technique. The terminologies of the branch and bound technique applied to the assignment

problems are presented below:

Let k be the level number in the branching tree (for root node, it is 0), σ be an assignment made is the

current node of a branching tree. kpσ be an assignment at level k of the branching tree, A be the set of

assigned cells (fractional assignment) up to the node kpσ from the root node (set of i and j values with respect


60

to the assignment cells up to the node kpσ from the root node), and vσ be the lower bound of the fractional

assignment A up to kpσ , such that

,

ij iji j A i X j Y

v c Min cσ∈ ∈ ∈

⎛ ⎞= + ⎜ ⎟

⎝ ⎠∑ ∑ ∑ ………. (6)

where ijc is the cell entry of the cost matrix with respect to the thi row and thj column, X is the set of rows

which are not removed up to the node kpσ from the root node in the branching tree, and Y is the set of

column which are not removed up to the node kpσ from the root node in the branching tree.

Branching guidelines

1. At level k, the row marked as k of the assignment problem will be assigned with the best column of the

assignment problem.

2. If there is a tie on the lower bound, then the terminal node at the lower most level is to be considered for

further branching.

3. Stopping rule: - If the minimum lower bound happens to be at any one of the terminal nodes at

the (n 1)th− level, the optimality is reduced, and then the assignment on the path from the root node to that

node along with the missing pair of row column arrangement will from the optimum solution.

Types of Assignment Problems:

There are three types of Assignment Problems.

Type-I: In maximization problems jobs effectiveness is frequently measured by profit instead of cost. When

a worker is assigned to different jobs, the profit contribution often differs from jobs to jobs. This difference

arises from workers capability and experience on a particular jobs, as well as the different nature of jobs to be

assigned. Therefore job effectiveness of the worker is expressed in terms of profit matrix.

Except for one transformation an assignment problem in which the objective is maximize total payoff

measures can be solved by the Branch and Bound Algorithm.

The transformation involves subtracting all the entries of the original payoff table from the maximum entry

of that table. The transformed entries give us the relative costs and the problem then becomes a minimization

problem. Once the optimal assignment for the transformed problem is obtained. The total measure of the

original payoff matrix can be found by those cells to which the assignment has been made. In these types of

problem the number of persons to be assigned and number of jobs were assumed to be the same. Such as

assignment problem is known as balanced assignment problem.

Type-II: If the number of persons is different from number of jobs, the assignment problem is said to be

unbalanced problem. If the number of jobs is less than the number of persons, some of the persons cannot be

assignment any job. In such problems one or more dummy jobs of zero duration are introduced to make the

assignment problem balanced. On the other hand if the number of persons is less than the number of jobs


61

than we add one or more dummy persons with duration time zero to balance the assignment problem. The

balanced problem then can be solved by using the Branch and Bound Techniques.

Type-III: In some cases, a certain worker cannot be assigned a particular job. The reasons for impossible

assignments are numerous. Back of required skills, deficiency in technical know-how, improper training and

physical inability are only a few many reasons. For solving such type of assignment problems, infinite cost is

put in the cell where no assignment is possible. The remaining procedure is exactly the same as the ordinary

assignment problems.

Numerical Examples:

Example–1. The jobs 1, 2, 3 are to be assigned three machines 1, 2,3 the processing cost (Rs/-) are as given

in the matrix shown below, find the allocation which will minimize the overall processing cost

Machine 1 2 3

1 Jobs 2

3

Solution: Initially, no job is assigned to any machine so the assignment(σ ) at the root node(level 0)of the

following branching tree is a null set and the corresponding lower bound vσ is also 0, as shown is Figure 1:

Figure1. Branching tree at the root node

Further branching: The three different sub-problems under the root node are shown in the Figure 2, the

lower bound for each of the sub-problems is shown by the right side of it.

Figure 2. Branching tree after from 0pϕ

Calculation for lower bound:

Lower bound for 111p

vσ =,i j A∈∑ ijc +

i X∈∑ (

j y∈∑ min ijc ). ………. (7)

Where σ = {(11)}, A = {(11)}, X= (2, 3), Y= (2, 3).

19 28 31 11 17 16 12 15 13


62

Then

11v = 11c + (2,3)i∈∑ (

(2,3)j∈∑ min ijc ) ………. (8)

= 11c + 23c + 33c = 19+16+13=48.


σ = {(12)}, A = {(12)}, X= (2, 3), Y= (1, 3).

Then

12v = 12c + (2,3)i∈∑ (

(1,3)j∈∑ min ijc ) ………. (9)

= 12c + 21c + 31c = 28+11+12 = 51.


σ = {(13)}, A = {(13)}, X= (2, 3), Y= (1, 2).

Then

13v = 13c + (2,3)i∈∑ (

(1,2)j∈∑ min ijc ) ………. (10)

= 13c + 21c + 31c = 31+11+12 = 54.

Further branching: Further branching is done from the terminal node which has the least lower bound. At

this stage, the nodes 1 111 12, ,p p and 1

13p are the terminal nodes. Among these nodes, the node 111p has the least

lower bound. Hence, further branching from this node is shown in Figure 3. The lower bound of each of the

newly created node is shown by the right side of it.

Figure 3. Branching tree after from 111p


63



σ = {(22)}, A = {(11), (22)}, X= (3), Y= (3).

Then

22v = 11c + 22c + 3i∈∑ (

3j∈∑ min ijc ) ………. (11)

= 11c + 22c + 33c = 19+17+13 = 49.


σ = {(23)}, A = {(11), (23)}, X= (3), Y= (2).

Then

23v = 11c + 23c + 3i∈∑ (

2j∈∑ min ijc ) ………. (12)

23v = 11c + 23c + 32c =19+16+15=50.

Further branching: At this stage, the nodes 112p , 1

13p , 223p and 2

22p are the terminal nodes. Among these

nodes, there is one node with the least lower bound of 49. So the node 222p which is at the bottom-most level

is considered for further branching. Since this node lies at (n 1)th− (level k=2) of the branching tree, where

n is the size of the assignment problem, optimality is reached. The corresponding solution is traced from the

root node to the node 222p along with the missing pair of job and operator combination (3, 3), so optimal

solution is presented through the following table.

Hence total cost = 19+17+13 = 49 Rs.

Example-2. A methods engineer wants to assign four new methods to three works centers. The assignment

of the new methods will increase production and they are given below. If only one method can be assigned to

a work center, determine the optimum assignment:

Increase in (production) unit

Job Machine Cost 1 1 19 2 2 17 3 3 13


64

Methods

Solution: First we will convert maximum matrix into minimum matrix, for this subtracting all the elements

of given matrix from maximum element (12), then we have minimum matrix as following:

Now we will introduce a dummy column for making a square matrix, which is balanced assignment problem

is given below:

Initially no method is assigned to any production so the assignment (σ ) at the root node of the following

branching tree is a null set and the corresponding lower bound vσ is also 0, as shown in Figure 4.

Figure 4. Branching tree at the root node

Further branching: The four different sub-problems under the root node are shown in the Figure 5, the

lower bound for each of the sub-problems is by the right side of it.

Figure 5. Branching tree at the root node 0pφ

A B C 1 10 7 8 2 8 9 7 3 7 12 6 4 10 10 8

A B C 1 2 5 4 2 4 3 5 3 5 0 6 4 2 2 4

A B C D 1 2 5 4 0 2 4 3 5 0 3 5 0 6 0 4 2 2 4 0


65



vσ =,i j A∈∑ ijc +

i X∈∑ (

j y∈∑ min ijc ). ………. (13)

Where σ = {(11)}, A = {(11)}, X= (2, 3, 4), Y= (2, 3, 4).

11 11(2,3,4) (2,3,4)

iji j

v c Min c∈ ∈

⎛ ⎞= + ⎜ ⎟

⎝ ⎠∑ ∑ ………. (14)

= 11c + 24c + 34c + 44c =2+0+0+0=2.


σ = {(12)}, A = {(12)}, X= (2, 3, 4), Y= (1, 3, 4).

Then

12v = 12c + 24c + 34c + 44c ==5+0+0+0=5. ……….. (15)


σ = {(13)}, A = {(13)}, X= (2, 3, 4), Y= (1, 2, 4).

Then

13v = 13c + 24c + 34c + 44c =4+0+0+0=4. ………. (16)


σ = {(14)}, A = {(14)}, X= (2, 3, 4), Y= (1, 2, 3).

Then

14v = 14c + 22c + 32c + 42c =0+3+0+2=5. ……….. (17)

Further branching:-Further branching is done from the terminal node which has the least lower bound. At

this stage, the nodes 1 111 12, ,p p 1

13p and 114p are the terminal nodes. Among these nodes, the node 1

11p has the

least lower bound of 2. Hence, further branching from this node is shown as Figure 6. The lower bound of

each of the newly created node is shown by the right side of it.


66

Figure 6. Branching tree after branching from 111p



σ = {(22)}, A = {(11), (22)}, X= (3, 4), Y= (3, 4).

Then

22v = 11c + 22c + 34c + 44c =2+3+0+0=5. ……….. (18)


σ = {(23)}, A = {(11), (23)}, X= (3, 4), Y= (2, 4).

Then

23v = 11c + 23c + 34c + 44c =2+5+0+0=7. ………… (19)


σ = {(24)}, A = {(11), (24)}, X= (3, 4), Y= (2, 3).

Then

24v = 11c + 24c + 32c + 42c =2+0+0+2=4. ……….. (20)

Further branching: Further branching is done from the terminal node which has the least lower bound. At

this stage, the nodes 112p , 1

13p , 114p , 2

22p , 223p and 2

24p are the terminal nodes. Among these nodes, two nodes

have the least lower bound of 4. So the node 224p which is at the bottom most level is considered for further

branching from this node is shown as Figure 7. The lower bound of newly created node by the right side of

it.


67




σ = {(32)}, A = {(11), (24), (32)}, X= (4), Y= (3).

Then

32v = 11c + 24c + 32c + 43c =2+0+0+4=6. ……….. (21)


σ = {(33)}, A = {(11), (24), (33)}, X= (4), Y= (2).

Then,

33v = 11c + 24c + 33c + 42c =2+0+6+2=10 .......... (22)

Further branching: At this stage, the nodes 112p , 1

13p , 114p , 2

23p , 222p , 3

32p and 333p are the terminal nodes.

Among these nodes, there is one node 113p with the least lower bound but not bottom-most node and 3

32p is

bottom-most node. Hence, further branching from 332p node. Since this node lies at (n 1)th− level of the

branching tree, where n is the size of the assignment problem, optimality is reached. Hence, optimal solution

is


68

Hence total units =10+0+12+8=30.

Example-3. Secretary of a school is talking bids on the city’s four school bus routes. Four companies have

made the bids as detailed in the following table.

Company �

Bids A B C D

C1 7000 8000 --- --- C2 --- 7000 --- 7000 C3 6000 --- 5000 --- C4 --- --- 7000 8000

Suppose each bidder can be assigned only to one route. Use the assignment model to minimize the school’s

cost of running the four bus routes.

Solution: First we are taking highest cost (�) for unassigned routes. The problem can be represented as

1000 ⇒ ×

Initially no company is assigned to any bidder, so the assignment ( )σ at the root node (level 0) of the

and the corresponding lower bound vσ is also following branching tree is a null set

0, as shown in Figure 8.

Figure 8. Branching tree at the root node

Further Branching: The four different sub-problems under the root node are shown in Figure 9. The lower

bound for each of the sub-problems is shown by the right side of it.

Method Production Unit 1 A 10 2 B 0 3 C 12 4 D 8

A B C D C1 7000 8000 � � C2 � 7000 � 7000 C3 6000 � 5000 � C4 � � 7000 8000

7 8 � � � 7 � 7 6 � 5 � � � 7 8


69

Figure 9. Branching tree after branching from 0pφ



( ){ } ( ){ }11 11 22 33 43

11 ; 11 ; (2,3, 4), (2,3, 4).

7 7 5 7 26.

A x y

v c c c c

σ = = = =

= + + + = + + + = ………. (23)


( ){ } ( ){ }12 12 24 33 43

12 ; 12 ; (2,3, 4), (1,3, 4).

8 7 5 7 27.

A x y

v c c c c

σ = = = =

= + + + = + + + = ………. (24)

Further Branching: At this stage, the nodes 1 1 1 111 12 13 14, , and p p p p are terminal node. Among these nodes,

the node 111p has the least lower bound. Hence, further branching from this node is shown in Figure 10. The

lower bound of newly created nodes by the right side of it.



70



( ){ } ( ){ }22 11 22 33 43

22 ; 11 , (22) ; (3, 4), (3, 4).

7 7 5 7 26.

A x y

v c c c c

σ = = = =

= + + + = + + + = ………. (25)


( ){ } ( ) ( ){ }24 11 24 33 43

24 ; 11 , 24 ; (3, 4), (2,3).

7 7 5 7 26.

A x y

v c c c c

σ = = = =

= + + + = + + + = ………. (26)

Further Branching: At this stage, the nodes 1 1 1 2 212 13 14, 22, 23, ,p p p p p and 2

24p are terminal node. Among these

nodes, two nodes have least lower bound of 26. The node 222p is the first least lower bound so that further

branching considering from this node shown in Figure 11. The lower bound of newly created nodes by the

right side of it.





71

( ){ } ( ){ }33 11 22 33 44

33 ; 11 , (22), (33) ; (4), (4).

7 7 5 8 27.

A x y

v c c c c

σ = = = =

= + + + = + + + = ………. (27)

Further Branching: At this stage, the nodes 1 1 1 212 13 14, 23, ,p p p p , 2

24p , 333p and 3

34p are terminal node. Among

these nodes, the node 224p has least lower bound but this is not bottom most and after branching from this

node we have nodes 332p and 3

33p . These nodes have highest lower bound so that this not for further

branching. Hence 333p is second least lower bound and bottom most node at level three. Since this node

lies at (n 1)th− level of the branching tree, where n is the size of the assignment problem, optimality is

reached. Hence optimal solution is

Company route Cost C1 A 7000 C2 B 7000 C3 C 5000 C4 D 8000

Hence total cost= (7+7+5+8) ×1000=27000 Rs.

Conclusion

In this paper, Branch and Bound technique is used to solve assignment problem. This method is applicable

for all kind of assignment problems, whether maximize or minimize objective function. This technique is

easy to apply and consume less time comparative to another techniques.

Acknowledgement: The authors are thankful to Dr. Sandeep gupta for his valuable help in preparation

of the paper.

References

Goel B.S., Mittal, S. K. (1982): Operations Research. (Pragati Prakashan). 2405-2416.

Dantzig G. B., Ford, L. R., Jr. and Fulkerson D. R., (1956): A Primal-Dual Algorithm for Linear Programs, in Linear Inequalities and Related Systems. Annals of Mathematics Study No. 38, (Eds) Kuhn and Tucker, (Princeton University Press, Princeton).

Ford L. R., Jr. and Fulkerson D. R. (1957): A Simple Algorithm for Finding Maximal Network Flows and an Application to the Hitchcock Problem. Canadian Journal of Mathematics. 9, 210-218.

Hamdy A. Taha (2007): Operations Research, an introduction. (Pesarson Publication).

Kuhn H.W. (1955): The Hungarian Method for the Assignment Problem, Naval Research Loqistics Quarterly, 2,83-97.

Bazarra M.S., Jarvis J. J. and Sherali H.D. (2005): Linear programming and network flows, Wiley.

Munkres, J. (1957): Algorithms for the Assignment and Transportation Problems. Journal of the Society for Industrial and Applied Mathematics. 5,32-38.


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