Title of Invention

"PROCESS OF EXTRACTING ANTI WHITE SPOT SYNDROME VIRUS MOLECULES FROM MANGROVE PLANTS"

Abstract

A process of extracting antiwhite spot syndrome virus molecules from mangrove plants to protect shring comprising of shade drying leaves of Mangove plants, powdering, soaking 50-60gms in minimum quantity of double distilled water, freezing to -20°C, repeated thawing and freezing followed by extracting with 500 ml-600 ml distilled water in a warring blender, raising the temperature upto 40°C, seiving the extract through fine meshed (100 micron) cloth, centrifuging the extract at 10000-12000 rpm for 20 minutes to remove all particles, lyophilizing the supernatant and storing under airtight at -20°C, partitioning anti WSSV molecule from the crude aqueous extract by n-Butanol, wherein the crude aqueous extract is subjected for liquid-liquid partitioning in equal volume of n-Butanol and water, shaken and kept for a day on magnetic stirrer, separating two layers in a separating funnel and keeping butanol layer at 4°C, subjecting the aqueous layer for liquid-liquid partitioning and continuing the process till no colour could be extractd into n-Butanol, evaporating the pooled n-Butanol fractions in vacuum at 42°C in a rotary evaporator to obtain pasty mass under-20°C.

Full Text

Process of extracting anti white spot syndrome virus molecules from mangrove plants. Field of Invention The invention relates to the development of an aqueous extract from mangrove plant species having anti WSSV property in vitro and to protect shrimp from horizontal transmission of the virus. Background of Invention Shrimp culture has the dimensions of an industry but various diseases of the species standout as impediments in its progress. Among the diseases white spot disease caused by white spot syndrome virus is the most devastating one. Since its first report in Taiwan in 1992 it was spreading havoc both to the eastern and western hemisphere. Even though several methods and products have been attempted for the control of white spot virus, none of them have been successful and search for a viable prophylaxis has been continuing. Based on the literature it has been confirmed that there is no previous incidence of detection of specific anti WSSV property and preparation of an aqueous or any other kind of extracts of antiviral molecules/compounds from mangrove plant species. In this context screening for anti WSSV property was made among mangrove plants endemic to India based on the earlier reports on the presence of antiviral molecules related to human viruses. Right now there exists no process of preparing aqueous extracts containing anti WSSV molecules/compounds from mangrove plants. Moreover the presence of such property in the mangrove plant was also not known. Similar is the case with delivery of such molecules to the animals through diet by coating or surface dressing. The technology developed here is not an improvement of any already existing one, presently this is the first preparation of the kind ever made from mangrove plants having anti WSSV property /or having the protective effect on shrimp from WSSV on oral administration. Objects of Invention OBJECTS OF INVENTION Object of the invention is to develop a process of extracting molecules and compounds with anti white spot syndrome virus property from mangrove plants. Other object of the invention is to confirm their anti WSSV property in vitro in animal model. Further object is to evaluate their protective effect on animals on oral administration. Yet another object is to purify, characterize and elucidate the structure of anti-WSSV compounds detected in the aqueous extracts of the mangrove plants. STATEMENT OF INVENTION According to this invention, there is provided a process of extracting antiwhite spot syndrome virus molecules from mangrove plants to protect shring comprising: - i) shade drying leaves of Mangove plants, powdering, soaking 50-60gms in minimum quantity of double distilled water, freezing to -20°C, ii) repeated thawing and freezing followed by extracting with 500 ml-600 ml distilled water in a warring blender, raising the temperature upto 40°C, iii) seiving the extract through fine meshed (100 micron) cloth, iv) centrifuging the extract at 10000-12000 rpm for 20 minutes to remove all particles, v) lyophilizing the supernatant and storing under airtight at -20°C, vi) partitioning anti WSSV molecule from the crude aqueous extract by n-Butanol, wherein the crude aqueous extract is subjected for liquid-liquid partitioning in equal volume of n-Butanol and water, shaken and kept for a day on magnetic stirrer, vii) separating two layers in a separating funnel and keeping butanol layer at 4°C, viii) subjecting the aqueous layer for liquid-liquid partitioning and continuing the process till no colour could be extractd into n-Butanol, ix) evaporating the pooled n-Butanol fractions in vacuum at 42°C in a rotary evaporator to obtain pasty mass under~20°C. Detailed Description of the Invention Preparation of the Extract Seven species of mangrove plants brought under study are listed below (Table 1,2,3). Leaves of all the plants were shade dried and powdered. Quantity of 50 gm of the samples were soaked in minimum in quantity double distilled water and frozen to -20°C. After repeated thawing and freezing it was extracted with 500 mL double distilled water in a Warring blender. During the process temperature was permitted to rise up to 40°C. The above extract was sieved through a fine meshed (100 micron) cloth. The resultant extract was centrifuged at 10000 rpm for 20 minutes and all particles were removed. The extract was maintained at -20°C till used. In vitro inactivation of WSSV The aqueous plant extracts (0.5 mL) were mixed with equal volume of viral suspension (virus titer 1x10+13 69/ml) and incubated for 3 hrs at 25°C. Controls included mixtures of WSSV and PBS in the above preparation. From each of the preparations aliquots of 10 µL were intramuscularly administered to single spawner bread WSSV free animals and monitored for 15 days. The animals, which survived and died, were both subjected for nested PCR for the detection of WSSV using a PCR kit supplied by Bangalore Genei. Protection of Penaeus monodon from WSSV on oral administration of aqueous extracts of mangrove plants. Coating of feed with the extract All aqueous extracts were lyophilized and used for coating pelleted diet. Four grams each of the lyophilized materials were mixed with 36g shrimp feed and bound with 5 ml 4% aqueous gelatin prepared in distilled water. The above preparation was dried under vacuum in a vacuum desiccator, and used for feeding. Experimental animal Single spawner bread WSSV free juveniles (3-5g size) grown in a recirculation system was used for the study. Four animals each were fed with a diet coated with the mangrove plant extract for 22 days, which resulted in the administration of 1.462 gm dehydrate in each animal. The controls were the animals administered with the feed coated with gelatin and the feed as such. Challenge with WSSV Freshly generated infected tissue was used for oral challenge of the shrimps @ 0.6g per animal. Animals were observed for 10 days for mortality. During the period they were maintained on the respective diet. Those animals, which died during the period after the challenge were colleted and preserved for PCR assay of WSSV. Those animals, which survived the challenge, were sacrificed on the 10th day used for the PCR assay. The data generated on the in vitro anti WSSV activity of the aqueous mangrove extract in animal model indicated that the plants Rhizophora mucronata (Mng-4), Sonneratia caseolaris (Mng-6), Ceriops tagal (Mng-7) had anti - WSSV property (Table 1). This could be demonstrated through PCR, as the animals administered with the virus was inactivated with the aqueous extract from the plants Rhizophora mucronata (Mng-4) and Sonneratia caseolaris (Mng-5) as they were nested negative and those administered with Ceriops tagal (Mng-7) nested positive. This suggested that in the first two cases (Mng-4 and Mng-6) viral DNA could be eliminated from the body, and in the 3rd case the viral DNA was retained but never developed in to disease. In all other cases the animals turned out to be first step positive. On oral administration of the plant Sonneratia caseolaris (Mng-6) and Ceriops tagal (Mng-7) again showed protective effect to shrimp from WSSV. In addition, two more plants which did not have shown anti WSSV property in vitro in animal model, Excoecara agallocha (Mng-1) and Avicennia sp (Mng-3), also imparted shrimp protection from WSSV. On PCR of the samples it revealed that the animals fed with Sonneratia caseolaris (Mng-6) and Ceriops tagal (Mng-7) were all nested negative and in the case of he animals fed with Ming-3, the dead ones turned out to be nested positive and all others negative. Studies of each plant extract in according protection to shrimp form WSSV are summerised in Table 3. Accordingly aqueous extract Mng-6 (Sonneratia Caseolaris) and Mng-7 {Ceriops tagal) showed in vitro and in vivo anti WSSV activity and could accord protection to shrimp from WSSV on oral administration. Meanwhile Mng-4 [Rhizophora mucronata) aqueous extract exhibited only in vitro anti WSSV property in animal model and Mng-1 (Excoecaria agallocha) and Mng-3 [Avicennia sp.) extracts could protect shrimp from horizontal transmission of the virus on oral administration. Table 1. In vitro anti WSSV activity of the mangrove extracts in animal model (n=20) (Table removed) N.A: Not applicable; +: PCR Positive-: PCR Negative *The plant species recorded of having invitro anti WSSV property Table 2. Potential of the aqueous extracts in protecting P. monodon from WSSV on oral Administration (Table removed) NA: Not applicable: +: PCR Positive; -: PCR Negative *The plant species recorded of having in vitro anti WSSV property Table: 3 Status of each plant in according protection to Shrimps from WSSV (Table removed) Purification and Characterization 1. Preparation of Aqueous Extract The mangrove leaves (25 grams) of two plants, coded as MNG-6 and MNG-7, were dried, powdered, mixed with sterile distilled water (150ml) and macerated in a mixer - grinder, sieved through fine meshed muslin cloth and the extracted liquid kept under -20°C till used. The preparation was centrifuged at 10,000 rpm (16100g) and the supernatant lyophilized and stored air tight under -20°C till used. 2. Examination of in vitro anti WSSV property Aliquots of 100 µl of the above extracts (from MNG-7 and MNG-6) were mixed with equal volumes of the WSSV preparation in PBS and kept for 3 hours at 28°C. The control preparation contained 100µ1 PBS and 100 )µ1 virus suspension incubated under the same conditions and PBS alone. After the incubation the extracts were administered intramuscularly at 10µl aliquots in shrimps (24 animals in 4 batches) weighing 4-5 gms. The animals were observed for the disease signs. Moribund and dead shrimp during the experiment and the survived ones at the end of the experiment (15 days duration) were maintained under -20°C for confirming the infection by PCR. Result Result of administering WSSV preparation exposed to the aqueous extracts of the mangrove plants MNG-6 8B MNG-7 are given in Table-1. MNG-6 and MNG-7 aqueous extracts exhibited antiviral property in the animal model Penaeus monodon. Among the animals subjected for assay all animals which received WSSV suspension exposed to MNG-7 extract survived but were nested PCR positive. However, at the time of completing the experiment (15 days post challenge) they were apparently healthy without clinical signs of WSSV. This suggested that the viral DNA recorded in low copy number were not infective. However, among the animals which received WSSV exposed to MNG-6 exctract one died with clinical signs of the disease and turned out to be first step PCR positive. Among those survived three animals turned out nested positive for WSSV. (Table removed) 3. Infectivity of WSSV detected as nested positives in animals which have survived challenges with WSSV exposed to MNG-6 MNG-7 aqueous extract In the above experiment there were instances of animals surviving challenges with WSSV exposed to mangrove extracts, but turning out nested WSSV positive. In his context it was essentially required to know whether these virus particles were infective or not. To accomplish the task the above experiment was repeated with smaller number of animals. On completion of the experiment animals were subjected for PCR and those animals which survived with nested positive PCR were segregated and used for preparing tissue extracts for passage to fresh batch of animals. 250 mg tissue macerated in 1 ml PBS buffer and centrifuged at 10000 rpm for 30 minutes. The supernatant was filtered through 0.45 |im membrane filter and used for re-injection to healthy prawns. They were observed for white spot disease signs Result Table- 2. Evaluation of infectivity of nested positive tissue to WSSV of shrimp which survived cahallenges with WSSV exposed to he mangrove extracts (Table removed) The data presented above indicated that the nested WSSV positive tissue of shrimp which survived challenges with WSSV exposed to the mangrove extracts were not infective. This suggested that on exposure to the mangrove extracts the virus could be inactivated however, were not eliminated from the tissue. This situation needs more investigation for confirmation. 4. Focusing on MNG-7 crude aqueous extract Based on the above results the mangrove plant coded MNG-7 was selected for further study at present. i. Toxicity of MNG-7 crude aqueous extract on Penaeus monodon Crude extracts were prepared from MNG-7 in distilled water as mentioned above and lyophilized. Apparently healthy animal of uniform weight of 5g were acclimatized to sea water with salinity 20 for a day with adlibitum feeding. Animals in batches of 6 each were administered with 10 µL aliquots of different concentrations of the preparation (5 mg/ml, l0mg/ml, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, and 60mg/ml). Distilled water served as control. The animals were observed for any change in behaviors, rate of feeding and moulting pattern for 7 days. Result Table 3. Demonstration of toxicity of the crude aqueous extract in shrimp. P. monodon (Table removed) The result of the experiment indicated that the extract was non toxic to the animals at the concentration tested. ii. Minimal concentration of MNG-7 crude aqueous extract required for anti WSSV activity Aliquots of 50 µl of the different concentrations of the extracts prepared for toxicity assay were mixed with equal quantity of WSSV in PBS. All animal groups except control group were injected with 10 µl of the above mixture after incubation for three hours. Positive control was 10 µ1 PBS + Virus suspension and, negative control 10 |il PBS alone. All animals were monitored for 7 days for mortality. Result Table 4. Response of P.monodon to WSSV exposed to different concentrations of aqueous extracts of MNG-7 (Table removed) The lowest concentration, 5mg/ml, itself was exhibiting anti WSSV property. iii. Partitioning the anti WSSV molecule from the crude aqueous extract by n-Butanol n- Butanol was used for partitioning medium polar fractions from the aqueous extract retaining the polar fractions with the aqueous phase. Materials and Methods The crude aqueous extract prepared from MNG-7 was subjected for Liquid- Liquid partitioning in equal volume of n- Butanol and Water. The mixture was shaken well and kept for a day on a magnetic stirrer. The two layers were separated in a separating funnel and the n-Butanol layer kept at 4°C. The aqueous layer was again subjected for liquid- liquid partitioning and the process continued till no color could be extracted in to n-Butanol. The pooled n-Butanol fractions were evaporated in vacuum at 42°C in a rotary evaporator. The resultant pasty mass was collected and kept under -20°C till used. iv. Toxicity of MNG-7 n-Butanol fraction on Penaeus monodon A stock of 50-µg/ ml n-Butanol fraction was prepared in double distilled water. The different concentrations used for intramuscular administration per animal were 0.05 µg, 0.25)Lig, 0.5µg, 0.75µg, and 1µg prepared in double distilled water and administering at 20 µl aliquots. Double distilled water served as control. All animals were observed for behavioral changes and mortality for 7 days. Results Table 5. Response of shrimp to various concentrations of MNG-7 n- Butanol fraction (Table removed) Results of the experiment showed that the MNG-7 n-Butanol fraction was non-toxic to the animals at the concentration tested. V. Anti WSSV activity of MNG-7 n-Butanol fraction n-Butanol fraction prepared from fresh plant aqueous extract and lyophilized was used for the experiment. A stock solution of 40 µg /ml n-Butanol fraction was used for virus inactivation. From the above, l00µl n-Butanol fraction was mixed with equal volume of WSSV (effective concentration 0.020 µg / µL) and incubated at 28°C fro 3 hours from which 10 µl ach was injected to 10 apparently healthy shrimps. A positive control contained WSSV suspension mixed with PBS and negative control was PBS alone. The animals wee observed for clinical signs of WSSV infection and mortality. Results Table 6. Response of the animals administered with WSSV exposed to MNG-7 n- Butanol fractions (Table removed) The results suggest that the n-Butanol fraction at an effective concentration of 0.020 µg /µL has anti - WSSV property. It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims: - WE CLAIM: 1. A process of extracting antiwhite spot syndrome virus molecules from mangrove plants to protect shring comprising: - i) shade drying leaves of Mangove plants, powdering, soaking 50-60gms in minimum quantity of double distilled water, freezing to -20°C, ii) repeated thawing and freezing followed by extracting with 500 ml-600 ml distilled water in a warring blender, raising the temperature upto 40°C, iii) seiving the extract through fine meshed (100 micron) cloth, iv) centrifuging the extract at 10000-12000 rpm for 20 minutes to remove all particles, v) lyophilizing the supernatant and storing under airtight at -20°C, vi) partitioning anti WSSV molecule from the crude aqueous extract by n-Butanol, wherein the crude aqueous extract is subjected for liquid-liquid partitioning in equal volume of n-Butanol and water, shaken and kept for a day on magnetic stirrer, vii) separating two layers in a separating funnel and keeping butanol layer at 4°C, viii) subjecting the aqueous layer for liquid-liquid partitioning and continuing the process till no colour could be extractd into n-Butanol, ix) evaporating the pooled n-Butanol fractions in vacuum at 42°C in a rotary evaporator to obtain pasty mass under-20°C. 2. A process of extracting anti white spot syndrome virus molecules from mangrove plants as claimed in claim 1, wherein n-Butanol is partitioning medium and separated polar fractions from the aqueous extract retaining polar fractions with the aqueous phase. 3. A process of extracting anti white spot syndrome virus molecules from mangrove plants as claimed in claim 1, wherein Mngl (Excoecaria agallocha), Mng2 {Acanthus ilicifolius), Mng3 (Avicennia sp), Mng4 (Rhizophora mucronata), Mng5 (Rhizophora apiculata), Mng6 (Sonneratia caseolaris), Mng7 (Ceriops tagal) are used for extraction process and Mng6 [Sonneratia caseolaris) and Mng7 (Ceriops tagal) are used for purification process.

Documents:

2607-del-2006-abstract (05-12-2007).pdf

2607-DEL-2006-Abstract-(08-02-2012).pdf

2607-del-2006-claims (05-12-2007).pdf

2607-DEL-2006-Claims-(08-02-2012).pdf

2607-DEL-2006-Correspondence Others-(08-02-2012).pdf

2607-del-2006-Correspondence Others-(09-05-2012).pdf

2607-del-2006-correspondence-others (05-12-2007).pdf

2607-DEL-2006-Correspondence-Others-(15-07-2008).pdf

2607-del-2006-correspondence-others.pdf

2607-del-2006-description (complete) (05-12-2007).pdf

2607-DEL-2006-Description (Complete)-(08-02-2012).pdf

2607-del-2006-description (provisional).pdf

2607-DEL-2006-Form-1-(08-02-2012).pdf

2607-del-2006-form-1.pdf

2607-del-2006-form-18 (08-01-2008).pdf

2607-del-2006-form-2 (05-12-2007).pdf

2607-del-2006-form-2.pdf

2607-del-2006-form-5 (05-12-2007).pdf

2607-del-2006-GPA-(09-05-2012).pdf

2607-DEL-2006-GPA-(15-07-2008).pdf