WORK ON WILD TUBER CROPS: WHAT I AM GOING TO START VERY SOON

Isolation, purification and characterization of pharmacological agents from Dioscorea species collected from Simlipal Biosphere Reserve Forest, Odisha

SANJEET KUMAR

Clinical microbiology has been facing new problems that pathogens are being resistant to particular synthetic drugs due to mutation, overdose, inappropriate diagnosis and treatment. Drug resistance is a big problem for pharmacological industries and health care. Drug’s potential is going on declining and it’s being difficult to fight against microbial attacks. Therefore, there is urgent need to screen of new compounds from plants and animal wealth which are unexploited and have little scientific documentation. Among those plants Dioscorea species are unexploited and having very little clinical validation of their extracts against pathogens is reported. They are major food staple among aboriginals and rural mass in 3^rd world particularly in Africa and India. The ethnobotanical survey from literature revealed the potent pharmacological values of these unexploited wild tubers. Therefore an attempt will be taken to investigate the phytochemicals, anti-microbial, toxicity and other pharmacological activity.

Objectives:

1.     Documentation of therapeutic practices of different extracts of Dioscorea species against microbial infections and other diseases.

2.     Evaluation of pharmacological values using clinical trials with microbes and experimental animals.

3.     Isolation, purification and characterization of desired bioactive compounds using chromatography and NMR.

Output:

1.     Help in the gene conservation of these valuable wild tubers.

2.     Formulation of new bioactive compounds.

3.     Manufactures of new drugs against specific pathogens and diseases.

Evaluation of antidiabetic activity of selected wild tuber crops of Odisha

SANJEET KUMAR

Diabetes is a very common and chronic endocrine disease caused by defect in production of insulin in human body which promote to increase the concentration of glucose in the blood leading damage blood vessels and nerves. More than 150 million people are suffering from diabetes worldwide and it is likely to increase to 300 million by the year 2025. International Diabetes Federation has declared that India is the “Diabetic capital of world”. From ancient medicinal plants continue to provide valuable therapeutic agents as natural and synthetic drugs in traditional and modern medicines. Many wild plant species are reported to be useful in Diabetes and they are considered to be less toxic and free from side effects than synthetic one. Among those plants wild tuber crops play an important role. Wild tuber crops have been used as traditional medicine against Diabetes among rural and tribal communities of different regions in Odisha.  Thus, an attempt will be made to evaluate the antidiabetic activity of wild tuber crops in alloxan induced experimental animals.

Objective of Research

·         Documentation and validation of therapeutic practices or claims of wild tuber crops among tribal communities of Odisha.

·         Study the impact of the extracts of these tubers and their plant parts on alloxan induced animals.

·         Assessment of bioactive compounds of wild tubers and their mechanism of action.

·         Isolation, purification and characterization of desired bioactive compounds.

Future Prospects

·         Study will help in elucidation the mechanism of antihyperglycemic effect.

·         Study will help to isolate the lead bioactive compounds responsible for the antidiabetic activity.

·         Estimation of bioactive compounds and antidiabetic activity of plant parts (in vivo and in vitro) will facilitate the maximum formulation in the manufacture of synthetic drugs to fight against chronic diabetic.

Study of diversity, nutritional and ethno-pharmacological activity of available Ban Aalu (Dioscorea species) in Simlipal Biosphere Reserve Forest (SBR), Odisha

SANJEET KUMAR

Simlipal Biosphere Reserve Forest is located in the central part of the district Mayurbhanj of Odisha having a compact mass of natural mixed forests with hub of different types of tribal communities. In developed 21^st century, synthetic medicines are very common against every disease throughout the world still they depend on wild plants for their food and medicines. Among those wild plants, Dioscorea species are more important as per the high consumption rate among rural and aboriginals during critical periods in SBR of the state Odisha. Dioscorea bulbifera, Dioscorea alata, Dioscorea pentaphylla and Dioscorea puber are very common in SBR forest. Tubers of Dioscorea species are rich in starch, fibre, carbohydrate and also having diverse bioactive compounds such as Diosgenin and others steroidal saponins. There is very little documentation on ethnobotany, nutritional and pharmacological values of Dioscorea species available in SBR. Therefore an attempt will be made to evaluate genetic diversity, ethnobotanical practices, nutritional and pharmacological activity of Dioscorea species available in SBR forest.

Objective of Research

1.      Standardize Passport Data Form and documentation of ethnic knowledge on Ban Aalu available in SBR of Odisha.

2.      Evaluation of diversity richness index of Dioscorea species in SBR forest.

3.      Estimation of primary metabolites, anti-nutritional factors and toxicity of Dioscorea tuber.

4.      Validation of ethno-pharmacological claims using experimental trials against selected diseases.

Future Prospects:

1.      Diversity index help in gene pool conservation and can protect the minor indigenous tuber crops from extinction helping in conservation of wide biodiversity available in SBR forest of Odisha.

2.      Ethnobotanical documentation will help to evaluate the pharmacological activity of the plants.

3.      Nutritional values will help to promote mass cultivation of these wild plants for eradication of malnutrition among inhabitants of SBR.

4.       Validation of tribal claims will lead to isolate new synthetic drugs against particular diseases.

DOWN STREAM PROCESSING

Sanjeet Kumar
Ravenshaw University

sanjeet.biotech@gmail.com

·        Downstream processing refers to the recovery and purification of biosynthetic products. The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing (DSP) or product recovery.

·        DSP is as complex and important as fermentation process.

·        The methodology adopted for downstream processing depends on the nature of the end product, its concentration, stability and the degree of purification required, besides on the presence of other products.

·         The desired products for isolation by DSP are most frequently metabolites which may be present as follows:-

1.      Intracellular metabolites- Vitamins

2.      Extracellular metabolites- amino acids

3.      Both Intra- and Extra- cellular- Flavomycin

Stages in Downstream processing

Downstream processing of metabolites is a multistage operation and broadly divided into the following stages:

1.      Release of intracellular products

2.      Solid-liquid separation

3.      Concentration

4.      Purification

5.      Formulation

Solid-liquid Separation

The first step in product recovery is the separation ofwhole cells and other insoluble ingradients from the culture broth.

Several methods are useful for solid- liquid separation such as:

a)      Flotation

b)      Filtration

c)      Flocculation

Filtration

Filtration is the most commonly used technique for separating the biomass and culture filtrate. Several types of filters are in use.

1)      Depth Filters:- Filamentous fungi are removed

2)      Absolute Filters:- Removal of bacteria from culture medium

3)      Rotary Drum Vacuum Filters:- Filtration of yeast cells

4)      Membrane Filters:- Reduces Clogging

Ø  Static Flow

Ø  Cross Flow

In cross-flow filtration, the culture broth is pumped in a crosswise fashion across the membrane.

Ther are 3 types of filtration used:

a)      Microfiltration

b)      Ultrafiltration

c)      Reverse Osmosis

Type	Applications
a)      Microfiltration	Separation of cells or cell fractions, viruses
b)      Ultrafiltration	Separation of compounds with molecular weight greater than 1000 μg
c)      Reverse Osmosis	Separation of compounds with molecular weight less than 1000 μg

Release of Intracellular Product

·         There are several biotechnological products such as vitamins, enzymes etc. are located within the cells. Such compounds have to be first released for their further processing and final isolation.

·         The selection of a particular methods for released of intracellular products, depends on the nature of the cells.

·         Gram-negative bacteria and filamentous fungi can be more easily broken compared to Gram-positive bacteria and yeasts.

·         In order to increase the efficiency of cell disintegration in a cost-effective manner, a combination of physical, chemical and enzymatic methods are employed.

Concentration

The filtrate that is free from suspended particles usually contains 80-98 % of H₂O. The desired product is a very minor constituent. The H₂O has to be removed to achieve the product concentration. The commonly used techniques are:

a)      Evaporation

b)      Liquid-liquid extraction

c)      Membrane filtration

d)      Precipitation

e)      Adsorption

    Evaporation can be done by evaporated such as plate evaporators, Falling film evaporator, Forced film evaporators and Centrifugal forced film evaporators.

Liquid-liquid extraction is useful for partial purification of a product, which is categorized as:

a)      Extraction of low molecular weight products using Dissociation, supercritical fluid etc.

b)      Extraction of high molecular weight compounds using Aqueous 2-phase systems (ATPs), and reverse miceller systems.

Precipitation is the most commonly used technique for the concentration of macromolecules using neutral salts, organic solvents etc.

Purification

The biological products of fermentation are very effectively purified by chromatography. It is basically an analytical technique dealing with the separation of closely related compounds from a mixture.

The different types of chromatographic techniques are used for such as

a)      Gel-filtration chromatography

b)      Ion-exchange chromatography

c)      Affinity chromatography and

d)      Hydrophobic interaction chromatography

CHROMATOGRAPHY	PRINCIPLE
Gel-filtration	Size and Shape
Ion-exchange	Net charge
Affinity	Molecular recognition
Hydrophobic Interaction	Polarity
Immobilized metal-ion affinity	Metal ion-binding

Formulation

Formulation broadly refers to the maintenance of activity and stability of biotechnological products during storage and distribution.

·         The formulation of low molecular weight products can be achieved by concentration them with removal of most of the water.

·         For certain small molecules such as citric acid, antibiotic etc., formulation can be done by crystallization by adding salts.

LIGAND	TYPES OF PROTEIN
Antibody	Antigen
Cofactor	Enzyme
Hapten	Antibody
Lectins	Glycoproteins

                       (Ligands use for separation of proteins by Affinity Chromatography)

Drying is an essential component of product formulation. It basically involves the transfer of heat to a product for removal of moisture. Freeze-drying or Lyophilization is the most preferred method for drying and formulation of wide-range of products such as food stuffs, pharmaceuticals, bacteria, viruses. Lyophiization is based on the principle of sublimation of liquid from a frozen state. In this technique, the liquid containing the product is frozen and then dried in a freeze-dryer under vacuum. The vacuum released and the product containing vials are sealed.  Penicillin can be freeze dried directly in ampoules. For good output can integrate the fermentation and downstream processing to finally get the desired product.

Medicinal, Pharmacological and Toxic values of Amorphophallus spp. of Odisha

Sanjeet Kumar
sanjeet.biotech@gmail.com
Ravenshaw University
Cuttack, India

A Review on Pharmacological aspects of Amorphophallus species

A. Behera & Sanjeet
sanjeet.biotech@gmail.com

Introduction

Millions of people in many developing countries do not get enough food to meet their daily requirement and many more suffer deficiency of micronutrients. The rural and tribal communities even today depend on wild resources including wild edible tuber(s) to meet their daily food requirement and more particularly during the period of food crisis. The value of wild edible plants in food security has not been given sufficient attention in India and particularly in Odisha. During last few years some attempt has been made to document the rich indigenous knowledge on the medicinal use of wild plants but socio-economic, traditional nutritional aspect of wild food plants still lack adequate attention. There are about 1532 edible wild food species available in India mostly in Eastern Ghats, Western Ghats and Himalayan Hill ranges (Reddy et al., 2007). Tubers play a major role in supplementing staples foods with micronutrients and can constitute a “Safety Net” during the period of food shortage (Chattopadhyay et al., 2010). Knowledge on wild tubers is gradually declining and even disappearing with increased modernization and migration to urban area due to poverty and other socio-economic factors. Documentation and revalorisation indigenous knowledge on wild tubers is urgently needed to promote nutritional health of the local inhabitants and to conserve genetic and cultural diversity as well. Almost all tribal groups of Odisha have been using Amorphophallus species (Oluā) as a food that are generally taken as vegetables and used as medicine in different formulations. Amorphophallus tubers (corms) constitute an economically important staple food and medicine for millions of people in the world (Sen et al.,1995).

Ethnobotanical Values

Amorphophallus are long been used in China, Japan and South East Asia as a food source and traditional medicine. In traditional Chinese medicine (Tem), a gel prepared from the flower has been used for detoxification, tumour- suppression, blood stasis alleviation and phlegm liquefaction and for treatment of asthma, cough, hernia, breast pain, burns as well as haematological and skin disorders (Chua et al., 2010). Amorphophallus campanulatus tuber is used for the treatment of enlarged spleen, rheumatism and tumour (Tripathi et al., 2010). The tubers are known to treat stomach ailments, has medicinal properties against fever, swelling, diarrhoea, used in piles and given as restorative in dyspepsia enlargement, asthma and rheumatism. Rhizomes are used for boils and opthalmia, also as an emmenagogue. Petioles used in scorpion bites, dysmenorrhea and are applied externally as an irritant to treat rheumatism (Hasan et al., 2009). Corms are also used in case of ear aches, swelling of throat, pimples, intereostal neuralagia, fever and also useful in cases of elephantiasis, tumours, inflammations, constipation, seminal weakness, fatigue, anaemia and general disability (Ravi et al., 2009).

Pharmacological Activity

Anti-Inflammatory Activity

Antioxidant activity of methanolic and aqueous extract of Amorphophallus campanulatus tuber was studied for its free radical scavenging property (Sahu et al., 2009). The anti-inflammatory activity of the hydroalcohol extract of Amorphophallus  bulbifer (whole plant) was studied in Wistar rats and mice at dose levels of 100 and 200 mg/kg p.o. with a plethysmograph  paw volume difference of the animals pre- and post-treatment. Ibuprofen (10 mg/kg) was employed as reference standard. The extract exhibited anti-inflammatory activity of 56.5 (p< 0.001) and 57.1 % (p< 0.001) inhibition compared to the control group in the carrageenan and histamine-induced inflammation model at a dose of 200 mg/kg (Reddy et al., 2012).

Analgesic Activity

Analgesic activity in Amorphophallus bulbifer (whole plant) was evaluated using tail flick and tail immersion techniques by measuring the reaction time of the animals (rats) treated with either standard or extract. Pentazocin (30 mg/kg) was used as reference which showed significant anti-inflammatory at the two test dose levels at the 4th hour (p< 0.001). The extract showed reaction times of 7.33 (p< 0.001) and 7.83 (p< 0.001) min in the tail flick and tail immersion models at a dose of 200 mg/kg while the normal and reference groups exhibited reaction times of 2.16, 2.66 and 8.16 (p< 0.001) and 8.5 (p< 0.001) in the tail flick and tail immersion methods, respectively, this lends some support for its use in traditional medical practice (Reddy et al., 2012).

Anti-Cancer Activities

Epidemiological evidence suggests that dietary factors play an important role in human health and in the treatment of certain chronic diseases including cancer (Trichopoulos et al., 1996). Some dietary sources of Amorphophallus species contain anti - tumour compounds and such compound are candidates for chemo preventive agents against cancer development (Dorai et al., 2004). The anticancer  property of nutrients derived from plants as well as non - nutritive plant derived constituents have been proved indifferent in vitro and in vivo models , which has led to an increased emphasis on cancer prevention strategies in which these dietary factors are utilised (Barnes et al.,1995). Epidemiological evidence suggests that dietary factors play an important role in human health and in the treatment of certain chronic diseases including cancer (Madhurima et al., 2012). Amorphophallus campanulatus has anticancer properties (Madhuri et al., 2008). Antioxidant   and anti- tumor activity of ethanolic extract of Amorphophallus paeonifolius tubers against 7,12- dimethyl benz (a) anthracene (DMBA) induced mammary tumour in rats were studied in-vitro and in-vivo methods and the plant extract was evaluated for mammary tumour activity by chemical induced tumour. The results showed total flavanoids compound in ethanolic tuber extract of Amorphophallus paeonifolius was found to be 8.8 g/100g calculated as Quercetin equivalent, effect of ethanolic extract on RBC, WBC, Hb & Neutrophils as a-P< 0.001,b-P<0.01,c-P<0.05, ns-non significant, effect of AP-extract on tumor latency and tumour burden were found as extremely significant at P<0.001. One-way ANOVA Tukey method was followed for statistics. It was concluded that ethanolic extract of Amorphophallus paeonifolius has shown significant antitumor and antioxidant effect in animals. The flavonoid in tuber extract of Amorphophallus paeoniifolius showed significant antitumour and antioxidant effect (Jagatheesh et al., 2010).

Hepatoprotective Properties

The pretreatment with dried tuber of Amorphophallus campanulatus (MAC) reduced the biochemical markers of hepatic injury like serum glutamate pyruvate transaminase (SGPT), serum oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), bilirubin (BRN) and total protein shows hepatoprotective activity. There is increase in the levels of superoxide dismutase (SOD), Catalase (CAT), and glutathion peroxidise (GPx) shows that the plant may possess hepatoprotective and antioxidant property (Singh et al., 2011). The hepatoprotective activity of methanol and aqueous extracts of Amorphophallus paeoniifolius tubers was confirmed against paracetamol induced liver damage in rats which was induced by paracetamol. The phytochemical investigation of the extracts showed presence of carbohydrates, proteins, steroids and flavonoids. Pre-treatment of the rats with methanol and aqueous extract prior to paracetamol administration caused a significant reduction in the values of sGOT, sGPT, sALP and sB (P<0.01) almost comparable to the silymarin and Liv-52. Thus was confirmed by histopathological examination of the liver tissue of control and treated animals (Hurkadale et al., 2012).

Anti-bacterial Activity

The flavonoid, 3,5-diacetyltambulin isolated from Amorphophallus campanulatus was studied for  in-vitro antibacterial, antifungal and cytotoxic activities which showed significant antibacterial activites against four gram positive bacteria   (Bacillus subtilis, Bacillus megaterium, Staphylloccous aureus, Streptococcus β – hemolyticus ) and six gram negative bacteria (Escherichia coli, Shigella dysenteria, Shigella sonnei, Shigella flerneri, Pseudomonas aeruginosa, Salmonella typhii). The MIC values against these bacteria ranged from 8 to 64 μg/ml but had weak antifungal activity against a number of fungi. In cytotoxicity determination, LC50 of the compound against brine shrimp nauplii was 10.02 μg/ml (Khan et al., 2009). The aqueous and methanolic extracts of spinach (Spinacea oleracia), pumpkin (Cucurbita pepo), suran (Amorphophalus campanulatus) and ghuiya (Colocasia esculenta) were evaluated for antimicrobial activity against bacterial strains (Bacillus cereus, Bacillus subtilis, Escherichia coli, Enterobacter aerogenes, Enterobacter agglomerans, Salmonella enteritidis, Salmonella cholerasius, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Penicillium chrysogenum, Enterobacter faecalis, Klebsiella pneumonia, B.sphericus, B.thruengiensis and Cryptococcus meningitis and was performed by Agar well diffusion method on Nutrient Agar medium and Muller Hinton Agar medium. The methanolic extracts of all the vegetables showed moderate to high activity against all the investigated microbial strains (Dubey et al., 2010). The Amorphophallus species showed remarkable antibacterial activity against gram positive (Bacillus subtilis, Staphylococcus aureus) gram negative (E. coli, P. aeruginosa ) bacteria and antifungal test showed significant activity of Candida albicans (Maharajan et al.,2012).

Antifungal Activity

The complete genome sequence of Bacillus subtilis strain BSn5, isolated from Amorphophallus species, calli tissue show strong inhibitory activity to Erwinia carotovora subsps- carotovora, which causes Amorphophallus soft rot disease and affects the industry development of this organism (Deng et al., 2011). Amorphophallus campanulatus showed good antifungal activity against Aspergillus flavus, Aspergillus nizer and Rhizopus aryzae (Khan et al.,2008).

Anti-Diabetic Activities  

The Amorphophallus species can be used as anti-diabetic agent (Li et al., 2004). The effect of the acetone extract of elephant-foot yam (Amorphophallus paeoniifolious Dennst.) at 0.1 and 0.25% in the diet of streptozotocin-induced male Wistar diabetic rats was studied which involved a comparison between control and diabetic groups: starch-fed control/diabetic (SFC/SFD), 0.1% acetone extract fed control/diabetic (AEFC0.1/AEFD0.1), 0.25% acetone extract fed control/diabetic (AEFC0.25/AEFD0.25) and aminoguanidine fed control/diabetic (AFC/AFD). The rats were examined for water intake, diet intake, urine output, gain in body weight, urine sugar, fasting blood sugar (FBS) and glomerular filtration rate (GFR). A concentration-dependent amelioration of the diabetic status was observed with respect to all the above studied parameters. FBS of AEFD0.1 and AEFD0.25 groups showed a 23% and 37% reduction, respectively whereas the AFD group showed a 45% reduction relative to the SFD group. The GFR of experimental rats in AEFD0.1 and AEFD0.25 groups showed a 28% and 41% reduction, respectively whereas the AFD group showed a 54% reduction compared to the SFD group which clearly indicate that the acetone extract of elephant foot yam is an effective for streptozotocin-induced diabetic rats (Arva et al., 2012).

Immunomodulatory Activity

Amorphophallus campanulatus tuber is used for the treatment of enlarged spleen, rheumatism and tumour. The effect of the methanol extract (ME) of Amorphophallus campanulatus tuber on immunological function in mice was studied using charcoal clearance, spleen index and delayed-type hypersensitivity (DTH) response models. The extract was administered orally at doses of 250 and 500 mg/kg, thus exhibited immunomodulatory activity by causing a significant decrease in charcoal clearance, spleen index and delayed-type hypersensitivity (DTH) response (Tripathi et al., 2010).

Anti-helminthic Activity

Corm was extracted with petroleum ether, chloroform and methanol. Crude tannins were isolated from methanol extract. The extracts and crude tannins were evaluated for anthelmintic activity. Chloroform, methanol extracts and crude tannins showed very good anti-helmintic activity (Ramalingham et al., 2010).

Toxicity

The cytotoxic property of different solvent extracts of Amorphophallus paeoniifolius tuber using Allium cepa L. root tip cells and HEp-2 cell line as two model in vitro systems. The seven extracts of Amorphophallus tuber were tested; the mitotic index and cytotic index were found to be high in petroleum ether and ethanol fractions. The magnitude of cytotoxicity displayed a dose dependent antiproliferative activity on HEp-2 cells which confirms the cytotoxic property (Angayarkanni et al., 2007).