Cobra venom neutralization by gold nano particle-2-hydroxy-4-methoxy benzoic acid.

Sir,

Snake envenomation have been treated by ASVS, with many limitations/side effects[1] but from ancient time herbs and herbal compounds were in use against snake bite. Herbs/herbal compounds may overcome the drawbacks of ASVS.[2] 2-Hydroxy-4-methoxy benzoic acid (HMBA), from Indian sarsaparilla had effective neutralization potential against Russell's viper venom.[2] HMBA conjugated with nano-sized gold particle enhanced efficacy against Russell's viper venom.[34] Naja kaouthia (cobra) found in south-east Asian countries, including India, causing fatal bites.[5] This study was an attempt to establish experimental neutralization potential of a herbal-nano compound (Gold nanoparticle [GNP]-HMBA) against Naja kaouthia venom (NKV).

GNP was conjugated with HMBA as reported earlier.[4] NKV was purchased and processed.[5] Albino Swiss male mice of 18–22 g were used as per the guidelines of IAEC and grouped (n = 6): (1) control, (2) NKV treated, (3) NKV + HMBA treated, (4) NKV + GNP-HMBA treated, and (5) NKV + GNP treated. The minimum lethal dose (MLD) amount of NKV was incubated with HMBA/GNP-HMBA/GNP stored at 37°C and injected (intravenous [iv]) and efficacy of NKV neutralization was evaluated up to 24 h. Group 1, 2, 3, 4, and 5 animal's paw were injected (subplanter) with 0.9% saline, NKV, NKV + HMBA, NKV + GNP-HMBA and NKV + GNP, respectively, and the paw diameters were measured at 0 and 8 h for the minimum edema dose (MED) of NKV, i.e., 2 µg. For NKV induced oxidative toxicities and inflammatory changes, Group 2, 3, 4, and 5 animals were injected with 5 µg NKV (s. c.), after 1 h followed by injections (IV) of 0.9% saline (100 µl), HMBA (100 µl), GNP-HMBA (200 µl) and GNP (100 µl), respectively, and observed till 24 h and sacrificed for blood collection, serum preparation. The degree of neutralization of NKV Phospholipase was determined by mixing the venom (1–5 units) with HMBA/GNP-HMBA/GNP (37°C/15 min) and evaluating the PLA2 activity. NKV-induced plasma recalcification neutralizing potential of HMBA/GNP-HMBA/GNP and hemolytic activity of NKV was tested by standard protocol. The significant differences between data (mean ± standard error of mean) were measured using one-way ANOVA at P < 0.05.

MLD of NKV in IV route was 5 µg. GNP and HMBA offered no protection against 1 MLD dose but GNP-HMBA showed protection against 1 MLD dose. NKV-induced MED was 2 µg. Protection offered by GNP-HMBA was 4 MED, as compared with HMBA (3MED) and GNP (1MED). Serum reduced glutathione (GSH), superoxide dismutase (SOD), catalase were decreased (35.39%, 57.42%, and 33.24%, respectively) and lipid peroxidation (LPO) was increased (147.4%) (significance at P < 0.05) in when compared with venom control group [Table 1]. Treatment with HMBA increased (significance at P < 0.05) GSH, SOD, and catalase (61.36%, 53.85%, and 62.54%, respectively) and decreased LPO (55.17%) when compared with venom control group. GNP-HMBA treatment increased (significance at P < 0.05) GSH, SOD, catalase (65.16%, 87.85%, and 101.39%, respectively) and decreased (significance at P < 0.05) LPO (66.32%) when compared with venom control group. Treatment with GNP offered no protection against venom-induced changes in oxidative stress and inflammatory markers when compared with venom control animals. NKV injection in venom control animals increased serum cytokines (pro-inflammatory), i.e., tumor necrosis factor-alpha, interleukin 1 (IL1) β, and IL 17 and decreased serum antiinflammatory cytokine (IL-10) when compared with control animal. HMBA and GNP-HMBA decreased proinflammatory cytokines (significance at P < 0.05) and increased antiinflammatory cytokine when compared with venom control animals [Figure 1]. In vitro NKV PLA2 activity was neutralized by HMBA and GNP-HMBA. The degree of neutralization of NKV PLA2 was GNP-HMBA (3-fold) >HMBA (2-fold) >GNP (1-fold). NKV (4 µg) increased the plasma (goat) recalcification time by 60 s when compared with control group. The degree of neutralization of plasma recalcification time was GNP-HMBA (1fold) > HMBA and GNP (no protection). 1 HD50 of NKV was found to be 4 µg. The degree of neutralization of HD50 was GNP-HMBA (1.5-fold) >HMBA (1-fold) >GNP (no protection).

Table 1

Effect of gold nanoparticle -2-hydroxy-4-methoxy benzoic acid on antioxidant parameters in venom induced stress on Swiss male albino mice

Animal group	Antioxidant parameters
	GSH (µM/mg protein)	SOD (IU/mg protein)	Catalase (IU/mg Hb)	LPO (MDA/mg protein)
Group 1	120.15±11.05	51.06±3.05	38.71±6.25	7.97±9.27
Group 2	42.53±9.59#	29.32±8.92#	12.87±4.94#	19.72±7.28#
Group 3	110.08±6.03*	45.11±2.4*	20.92±4.11	8.84±2.43*
Group 4	122.08±3.13*	55.08±2.9*	25.92±4.32*	6.64±1.20*
Group 5	58.19±6.54	35.45±4.87	12.62±1.12	16.89±2.68

Group 1: Sham control, Group 2: Venom control, Group 3: HMBA treated, Group 4: GNP-HMBA treated, Group 5: GNP treated. Values were expressed as mean±standard error of mean (n=6), statistical analysis was done with one way ANOVA, # P<0.05 when compared to sham control group, *P<0.05 when compared to venom control group. GSH=Glutathione, SOD=Superoxide dismutase, LPO=Lipid peroxidation, GNP-HMBA=Gold nanoparticle -2-hydroxy-4-methoxy benzoic acid

Figure 1

Effect of gold nanoparticle -2-hydroxy-4-methoxy benzoic acid on serum proinflammatory and antiinflammatory cytokines in Naja kaouthia venom-induced Swiss male albino mice. Gr. 1, sham control, Gr. 2 Venom control, Gr. 3 2-hydroxy-4-methoxy benzoic acid treated, Gr. 4 Gold nanoparticle -2-hydroxy-4-methoxy benzoic acid treated, Gr. 5 Gold nanoparticle treated. Values were expressed as mean ± standard error of the mean (n = 6), statistical analysis was done with one way ANOVA, #P < 0.05 when compared to sham control group, *P < 0.05 when compared to Venom control group

NKV PLA2 activity was neutralized by GNP-HMBA, which may contribute to the protection of NKV-induced hemolysis, oxidative stress. NKV-induced decrease of GSH, SOD and catalase, and increase in LPO, suggesting oxidative stress. ASVS did not neutralize venom-induced oxidative stress.[5] Treatment with GNP-HMBA increased the GSH, SOD, catalase and decreased LPO, indicating the production of antioxidants inside the body and neutralizing the venom-induced prooxidant effects. Cobra envenomation increased the release of serum pro-inflammatory cytokines from T-cells and activated macrophages, and decrease in anti-inflammatory cytokine release leading to inflammation.[5] GNP-HMBA treatment significantly inhibited proinflammatory cytokines release and stimulated antiinflammatory cytokine production, resulting in restoration of serum cytokine equilibrium. GNP alone could not neutralize venom-induced inflammation and oxidative stress, but it may be suggested that GNP conjugation with HMBA leads to increase in the availability, uptake, and utilization of HMBA at local sites.

In conclusion, the present study indicated the increased NKV neutralization potential of HMBA after the conjugation with GNP, i.e., GNP-HMBA significantly neutralized NKV-induced toxicity in experimental in vivo and in vitro models which may be utilized for designing supportive treatment against snake (Naja) bite.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.