In vitro anti-venom potential of various Jatropha extracts on neutralizing cytotoxic effect induced by phospholipase A₂ of crude venom from Indian cobra (Naja naja)

Abstract

In present study various species of Jatropha were evaluated for antidote nature induced by phospholipase A₂ (PLA₂) cobra venom. Although qualitative phytochemical analysis exhibited less variation across Jatropha species studied, substantial variability in terms of PLA₂ inhibition by various solvent extracts across the species and between different parts of same plant was observed. Among all samples methanolic extracts of J. gossypifolia leaf showed highest inhibition of PLA₂ toxicity while some aqueous extracts of J. foetida and all aqueous extracts of J. curcas, enhanced PLA₂ activity. Present results highlight Jatropha not only as rich source of secondary compounds with antidote property for snake bite but also potent toxic agents as revealed with increased hemolysis by some aqueous extracts of J. curcas and J. foetida. Our findings suggest that methanolic leaf extract of J. gossypifolia contain potent small molecular antagonist(s) to the snake venom PLA₂ which will be very useful to design adjuvant therapies in treatments of snake bites.

Introduction

Snakebite is one among vital public health issues of tropical countries including India. Most of the mortality cases come from farmers, hunter gathers of rural areas which lack proper sickbays and antivenin supply (Panfoli et al.,2010). Naja naja commonly called as Indian Elapid is one among the poisonous snakes with toxin subfractions like metalloproteases, phospholipases, hyaluronidases, neurotoxins, and myotoxins which wreak havoc in victim’s body. Phospholipase A₂ is one of well studied fraction of cobra venom with many pharmacological effects like presynaptic neurotoxicity (Kini and Iwanga, 1986), myotoxicity (Mebs and Samejima, 1980), edema (Vishwanath et al.,1987) cardiotoxicity and hemolysis (Condrea et al.,1980; 1981). Till date, antibodies produced in horses are the only source of antivenom, a process of time consuming. Moreover, antibodies need to store in low temperature conditions that lack in rural areas of developing countries. Snake bite victims sensitive to horse products produce hypersensitive side reactions when treated with antiserum of equine origin (Nazim et al.,2008). So, identification of alternative medicine with no risk and low temperature storage requirements became important task. Plant based antidote sources are known from olden ages for snake bites. Jatropha is one among reported to have antidote property and used in some tribal medicines with no scientific evaluation. Phytocompounds isolated from Jatropha plants are reported to have other therapeutic activities like anti-inflammatory, antitumor, molluscidal, insecticidal and fungicidal activities (Albuquerque, 2006).

The present study is aimed on systematic evaluation of antidote potentials of various solvent extracts of Jatropha species.

Materials and Methods

Plant selections, collections and material preparations

For the present study, three species of Jatropha viz., Jatropha gossypifolia (J.G), Jatropha foetida (J.F)and Jatropha curcas (3 lines)are selected. J. gossypifolia, J. foetida (collected from Seshachalam Hills, Tirupathi), were identified by Dr. A. Madhusudhana Reddy, Department of Botany, Yogi Vemana University, Kadapa, Andhra Pradesh, India. Three distinct varieties of J. curcas (these materials were generously provided by Dr. L. Sivarama Prasad, Naturole Bioenergy Ltd., Hyderabad, India, which were originally collected from Sai Petro Chemicals, Maharastra (J.C-1); Aleru, Warangal (J.C-2); and Uganda, Africa (J.C-3); the three varieties of J. curcus are being presently maintained in Yogi Vemana University, Kadapa) were selected based on genetic diversity shown by molecular genetic studies carried out using RAPD and ISSR profiling (Data not shown). Leaves, stems and roots of each variety were collected separately, thoroughly washed and shade dried.

Snake venom

Lyophilized crude venom of Naja naja (Indian cobra) was purchased from “The Irula Snake-Catchers Industrial Cooperative Society (ISCICS), Mamallapuram, Tamil Nadu and the crude venom was stored in airtight containers at 4ºC until used.

Plant materials sampling and soxhlet extractions

Equal amount of all dried plant materials (viz., leaf, stem and roots separately) were pulverized to fine powder and subjected to soxhlet extraction with chloroform, methanol and water solvents at elevated temperatures of 40°C, 45°C and 75°C respectively. The extract obtained was concentrated through rotavapour at a constant temperature of 38°C for chloroform, methanolic extracts and 46°C for aqueous extracts with the help of water bath. Solid extracts were weighed and stored in dry environment until further use in opaque containers in refrigerator.

Phytochemical screening and analysis

Equal amount of each extract (45 extracts in total) obtained from Soxhlet method was used for qualitative phytochemical estimations and identifications of various secondary metabolites like, alkaloids, flavonoids, terpenoids, saponins, tannins and phenolics present in the extracts by using standard methods as described elsewhere (Trease and Evans, 1996), Sofowora (Sofowora, 1993), and Harborne (Harborne, 1998). The positive results were noted as present (+) and negative results as absent (-).

Antivenom assay

Preparation of RBC cells from blood sample

Human blood was collected in glass tubes containing sodium citrate as anticoagulant. The blood samples were centrifuged at 800 rpm for 10 min and plasma was carefully removed by sterile Pasteur pipette. The pellet of red blood cells were washed three times by PBS buffer (0.15 M NaCl, 10 mM phosphate buffer, pH 7.0) and then suspended in a fresh PBS buffer at a density of 1.2 x 10⁹ cells/mL monitored by a Neubauer chamber. To this 1% of egg albumin was added (Habermann and Neumann, 1954; Gutierrez, 1988) and the resultant mixture was considered as substrate to snake venom PLA₂s in the hemolytic assay described below herein.

Hemolytic assay

Toxicity of the venom and its neutralization by the Jatropha extracts was determined through indirect hemolytic assay (Habermann, 1954; Gutierrez, 1988; Paula et al., 2010) by using human erythrocytes and hen’s egg yolk emulsion as substrate. The amount of venom (10 µg/mL) that can produce 70%hemolysis after the incubation was denoted as the Minimum Indirect Hemolytic Dose (MIHD). Experiments were performed by pre-incubating 25 µgof Jatropha extract with a MIH dose of Naja naja venom for 30 min at 37°C prior to evaluating the hemolytic activity. To this, 5 mL of prepared erythrocyte suspension was added and incubated for further thirty min at 37°C. After 30 min, venom activity on the substrate was stopped by adding 3 mL of ice cold PBS buffer to the reaction mixture. The extent of cell lysis/hemolysis caused by the venom on the RBC cells was measured by calculating the amount of hemoglobin released from the cells. The amount of released hemoglobin from the reaction mixture was estimated from the intensity measure-ed at 540 nm using UV spectroscopy. The hemolysis produced by the venom in the absence of Jatropha extracts was taken as 100% and the percentage of inhibition/activation by each Jatropha extract used in the present study against snake venom PLA₂s was calculated as shown in the following calculations. All measurements were made in triplicates and results are expressed as mean + standard deviation.

Results

Selection of Jatropha lines for present phytochemical and antidote analysis (Table I) was based on the phylogenetic tree constructed from molecular genetic studies of 35 lines using RAPD and ISSR marker profiling (data not shown here) was found to be helpful for initial screening. Our results strengthen the reports of Jatropha’s use as an antidote in folk medicine.

For the forty five extracts obtained (3 solvents x 5 plants x 3 parts of each plant– stem, leaf and root) qualitative phytochemical analysis exhibited less variation among the species of Jatropha viz., gossypifolia (J.G- stem, J.G- leaf and J.G- root), foetida (J.F- stem, J.F- leaf and J.F- root), and curcas (J.C- stem, J.C- leaf and J.C- root), and almost no varietal variation was found within the spe-cies (varieties – J.C-1) of curcas and also no significant variation of phytocompounds between different parts of same Jatropha plants is observed (Table I).

The selection of plants for the present study, based on phylogenetic analysis was found to be worth full as the same kind of variation has been reflected in both phytochemical composition and antiophidian property of various Jatropha extracts. Owing to the records of using Jatropha as antidote for snake bite in folk’s medicine, investigation on different species of Jatropha phytocompounds inhibiting PLA₂ was carried out. All the aqueous extracts of J. curcas, roots and stem extracts of J. foetida were found to be lethal as the extracts increased hemolysis when compared with positive control which has only venom (MIHD) (Figure 1). Interestingly, it was also found that these extracts alone did not cause any membrane lytic effect on red blood cells (results not shown). Of all the aqueous extracts, J. gossypifolia leaf, root and stem showed high hemolytic inhibition of 85.9%, 84.5% and 81% respectively (Figure 1). Variation was found in terms of antivenom activity across aqueous extracts of J. foetida plant parts viz., stem, leaf and root. Only leaf extract of J. foetida showed anti-venom property in terms of PLA₂ inhibition where-as stem and root aqueous extracts of the plant showed enhanced-hemolytic activity resembling J. curcas extracts when incubated with venom.

Methanolic extracts of all three varieties of J. curcas showed good inhibition within a range from 81% to 88.3% and the variation among different parts of a same plant was also negligible (Figure 2). However, extracts of various parts from J. gossypifolia and J. foetida showed high variations in the degree of their inhibition potentials against hemolytic activity of PLA₂. Roots of J. foetida (74.5%) and leaf of J. gossypifolia (89.8%) showed high inhibitions. Of all methanolic extracts examined in the present study, leaves extracts of J. gossypifolia showed strongest inhibition of PLA₂ (Figure 2).

All chloroform extracts of J. curcas showed very less inhibition of venom hemolysis which were in the range of 2.6% to 22.4%. In J. foetida, leaf showed very less inhibition of 9.1% and root extract showed high inhibition of 81.7%. In J. gossypifolia the leaf showed the highest of 87.5% PLA₂ inhibition followed by stem and root (Figure 3).

Of all three species of Jatropha (J. foetida, J. gossypifolia and J. curcas), and within three selected lines of curcas collected from diverse geographical conditions, J. gossypifolia leaf extracts was found to be good choice as antidote for Naja naja venom due to its high inhibition potential against PLA₂ activity vis-à-vis that of other extracts as demonstrated above. Among aqueous, methanolic and chloroform extracts of J. curcas, only methanolic extracts were showing high anti-venom properties as inhibition was less in case of extracts from chloroform and negative inhibition could be seen with aqueous extracts from all three varieties of curcas. The high inhibition exerted by the methanol extract of J. gossypifolia leaf against the Naja naja venom strongly suggest for presence of phytocompounds possessing therapeutic potentiality with respect to PLA₂ inhibitions in the extracts.

All chloroform extracts of J. curcas showed very less inhibition of venom hemolysis which were in the range of 2.6% to 22.4%. In J. foetida, leaf showed very less inhibition of 9.1% and root extract showed high inhibition of 81.7%. In J. gossypifolia the leaf showed the highest of 87.5% PLA₂ inhibition followed by stem and root (Figure 3).

Discussion

The present study is aimed to identify Jatropha species with potent antidote property. Of the three species selected for present study, our results clearly indicate all the species of Jatropha can’t be used as source of antidote for snake venom as indicated from our results based on increased hemolysis activity of snake venom when incubated with aqueous extracts of J. curcas and J. foetida. Though Jatropha is used in local / folk medicine as antidote for snake bite, our observations indicate that all species of Jatropha and all parts of the plant cannot be taken granted as antidote as a few of them are capable of enhancing venom activity in terms of hemolysis and consequently may lead to lethal. Our results highlight that overlooking or adulteration of plant materials with other plant materials at species level or with different parts of the same plant may not only reduce therapeutic efficiency of extracts but may also increase toxic effects as seen in the case of aqueous extracts of J. curcas and J. foetida. Moreover, the above antihemolytic studies and phytochemical analysis clarified that even the phytocompounds are qualitatively found similar in different parts (stem, leaf and root) of the same plant, their quantities may vary from part to part as extracts of different parts of a plant showed a great degree of variations in their venom inhibition potentials. Hence, choosing of a plant species, part of the plant and extract of the part are of great importance because of great variation in phytochemical distribution of which some are found to have therapeutic value as in present case of J. gossypifolia extracts and some exhibit lethal properties as seen in case of aqueous extracts from all J. curcas varieties and also stem and root extracts of J. foetida.

The observations clearly indicate that the aqueous extracts of J. curcas and J. foetida musthave micro/macro compounds or ions that may aid to enhance phospholipase A₂ activity of snake venom. There are reports on increased activity of phospholipase A₂ when the enzymes combine with some of the divalent ions like Ca²⁺, Sr²⁺ and Ba²⁺ etc., These ions are reported to enhance the activity of phospholipase A₂ by inducing conformational changes in the active site and substrate-binding site of the PLA₂ and the resultant conformation of the enzyme may either promote or suppress its biological activity (Jiang et al., 1989). The presence of quercetin-like compounds may also be a reason for increased hemolytic activity and they can form a lipid-raft like domains (Chiou et al., 2012).

From a scientific approach carried out in the present study to identifying potent plant-based antidote(s) to crude venom of Naja naja, J. gossypifolia leaf was found to be more effective source for compounds with anti-venom properties to inhibit PLA₂ that causes destabilizing the plasma membranes of cells and there by affecting cardiac, nervous and circulatory systems.

The extracts that were found to be a promising natural antidote, may presumably act by forming complexes with metal ions and proteins which in turn may change the conformation of active site and there by inhibiting the action of phospholipases A₂.The use of this kind of natural antidotes may reduce the risk of anaphylactic shocks in patients sensitive to equine products. Also, the natural antidotes have appreciable technical advantages when compared to commercial anti-venoms that need to be stored at very low temperatures (below 273 K) in a highly sophisticated refrigerator which are technically sound and they are presumably cost protective to have at all parts of rural areas of developing, undeveloped and even for developed countries. Work is in progress on identifying potent compounds responsible for anti-venom activities from methonalic leaf extract of J. gossypifolia at molecular/atomic level resolutions. In these backgrounds, we strongly believe that the outcomes will be very useful to design adjuvant therapies for treatments of snake bites.

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