# Naja naja karachiensis Envenomation: Biochemical Parameters for Cardiac, Liver, and Renal Damage along with Their Neutralization by Medicinal Plants

Authors: Muhammad Hassham Hassan Bin Asad; Ghulam Murtaza; Muhammad Ubaid; Durr-e-Sabih; Ashif Sajjad; Rubada Mehmood; Qaisar Mahmood; Muhammad Muzzmil Ansari; Sabiha Karim; Zahid Mehmood; Izhar Hussain
Journal: BioMed Research International
 (2014)

---

## Abstract

Naja naja karachiensis envenomation was found to hit more drastically heart, liver, and kidneys. 400 μg/kg of venom-raised moderate serum levels of ALT (72
±
4.70 U/L, 0.1
>
P
>
0.05), AST (157
±
24.24 U/L, 0.1
>
P
>
0.05), urea (42
±
3.08 mg/dL, 0.05
>
P
>
0.02), creatinine (1.74
±
0.03 mg/dL, 0.01
>
P
>
0.001), CK-MB (21
±
1.5 U/L, 0.05
>
P
>
0.02), and LDH (2064
±
15.98 U/L, P
<
0.001) were injected in experimental rabbits. However, lethality was enhanced with 800 μg/kg of venom in terms of significant release of ALT (86
±
5.0 U/L, 0.05
>
P
>
0.02), AST (251
±
18.2 U/L, 0.01
>
P
>
0.001), urea (57.6
±
3.84 mg/dL, 0.02
>
P
>
0.01), creatinine (2.1
±
0.10 mg/dL, 0.02
>
P
>
0.01), CK-MB (77
±
11.22 U/L, 0.05
>
P
>
0.02), and LDH (2562
±
25.14 U/L, P
≪
0.001). Among twenty-eight tested medicinal plant extracts, only Stenolobium stans (L.) Seem was found the best antivenom (P
>
0.5) compared to the efficacy of standard antidote (ALT =
52.5
±
3.51 U/L, AST =
69.5
±
18.55 U/L, urea =
31.5
±
0.50 mg/dL, creatinine =
1.08
±
0.02 mg/dL, CK-MB =
09
±
0.85 U/L, and LDH =
763
±
6.01 U/L). Other plant extracts were proved less beneficial and partly neutralized the toxicities posed by cobra venom. However, it is essential in future to isolate and characterize bioactive compound(s) from Stenolobium stans (L.) Seem extract to overcome the complications of snake bite.


---

## Body

## 1. Introduction

One of the animals which represent Pakistan and India throughout the world is cobra; in particular it is rearing out of a wicker basket and dancing to the sound of turban-wearing snake charmer music. In fact, they are deadly venomous among all species of the snakes due to high rate of mortality which strongly reasoned for their notoriety [1]. At the end of the nineteenth century, all cobra populations have been merged into single species naja (formerly known as naia); however, subsequently ten subspecies ofNaja naja were identified [1]. Later on, Deraniyagala classifiedNaja naja (India) into several subspecies and consideredNaja naja karachiensis one of them widely distributed in Southern Punjab province of Pakistan [2]. They are extremely toxic due to their severe side effects in the victims. Major signs and symptoms of cobra envenomation are edema, necrosis, pain, respiratory paralysis, vomiting, headache, hypotension, cardiac arrest, coagulopathies (elevated PT, aPTT, and TT), bleeding wounds, hematuria, mucus discharge, proteinuria, and increased creatinine and urea levels along with altered consciousness [3, 4].Indeed snake venom is a complex mixture of various sizes of polypeptides [5, 6] (<1.5 kDa, 5 to 10 kDa, and 10 to 150 kDa), carbohydrates, lipids, metal ions, organic molecules, purines, and amines [7, 8]. Snake bite envenomation has been attributed to pose drastic changes in the physiology of the victims that could be accessed clinically by measuring various biochemical parameters. Among toxic components, phospholipases A2 (cell destroying enzymes cause edema, lipolytic or myolytic) [9, 10], phosphodiesterases (nucleic acid degrading enzyme causes hypotension/shock) [11, 12], 5′ nucleotidases (digest the sugar moities causing the delay in coagulation) [13], alkaline phosphatases (hydrolyze phosphate esters nonspecifically cause multiple toxicities via adenosine generation) [14], hyaluronidases (spreading factor causes digestion, necrosis, and leakage of blood vessels) [15], L-amino oxidases (deamination leads to cell damage/apoptosis) [16], and proteases (hydrolyze various proteins) [17] are included.Laboratory animals, particularly rabbits, have been selected previously to monitor various biochemical changes related to liver, heart, and renal toxicities [18, 19]. Aspartate aminotransferase (GOT/AST) and alanine aminotransferase (GPT/ALT) are surrogate markers for liver toxicities while creatine kinase isoenzyme (CK-MB) is used exclusively to access cardiac tissue damage [20, 21]. Elevated creatinine and urea levels are indicators of 75% renal damage [20], whereas high level of lactate dehydrogenase (LDH) implies general toxicity related to the liver, heart, kidneys, and skeleton muscles [22].Diverse immunological properties of different cobras have fascinated protein chemists and other researchers to carry out efforts for the development of new antidotes. Due to this reason, for the first time, venom fromNaja naja karachiensis is selected to evaluate its toxicityin vivo and to search out effective as well as cheap treatment from folk herbal remedies. Pakistan is a hub of medicinal plants where mostly rural communities rely on natural herbs to treat their problems of snake bite [6, 23]. It is therefore necessary to evaluate scientifically their folklore claims as anti-snake venom in traditional system of medicine. Due to this reason, various medicinal plants (Albizia lebbeck(L.) Benth.,Allium cepaL.,Allium sativum L.,Althaea officinalis L.,Bauhinia variegataL.,Brassica nigra(L.) W. D. J. Koch, Calotropis procera(Aiton) W. T. Aiton,Cedrus deodara(Roxb. ex D. Don) G. Don,Citrullus colocynthis(L.) Schrad, Citrus limon(L.) Burm. f., Cuminum cyminumL.,Enicostema hyssopifolium(Willd.) I. Verd,Fagonia creticaL.,Leucas capitataDesf.,Matthiola incana(L.) W. T. Aiton,Momordica charantiaL.,Nerium indicumMill,Ocimum sanctum L., Pinus roxburghiiSarg,Pistacia integerrimaJ. L. Stewart, Psoralea corylifoliaL.,Rhazya strictaDecne,Rubia cordifoliaL., Sapindus mukorossiGaertn,Solanum xanthocarpum Schard and Wendle,Stenolobium stans(L.) Seem,Terminalia arjuna(Roxb. ex DC.) Wight and Arn,Trichodesma indicum(L.) Sm, andZingiber officinaleRoscoe) were collected to test their potentials against toxicities induced byNaja naja karachiensis envenomation (Table 1).Table 1
Detailed description for evaluated medicinal plants having folklore evidences as antivenom.
Sr. number
Botanical name of medicinal plants
Family
Part used
Phytochemicals reported
Reference
1
Albizia lebbeck   (L.) Benth.
Fabaceae
Seed
Carbohydrates, proteins, alkaloids, flavanoids, tannins, echinocystic acid, and different amino acids.
[6]
2
Allium cepaL.
Amaryllidaceae
Bulb
11 g carbohydrates, 1.2 g  proteins, 0.6 g  fibers, and H2O content  (86.8 g)/100 g  of material.
[6]
3
Allium sativum L.
Amaryllidaceae
Bulb
Quercetin, scordinines A and B allicin, thiosulfinates, 2 mercapto-L-cysteines, anthocyanins, alliinase, polysaccharides, sativin I and sativin II, and glycosides of kaempferol.
[6]
4
Althaea  officinalisL.
Malvaceae
Root
37% starch, 11% mucilage, fatty oil, pectin, flavonoids, phenolic acids, asparagines, phytosterol, sucrose, and butyric acid.
[6]
5
Bauhinia variegata L.
Fabaceae
Root
Tannins, fatty oil, lupeol, kaempferol-3-glucoside, gums,β-sitosterol, 5,7-dehydroxy and 5,7-dimethoxy-flavanone-4-0-a-L-rhamnopyranosyl-β-D glucopyranosides.
[6]
6
Brassica nigra (L.) W. D. J. Koch
Brassicaceae
Seed
Essential oil, sinigrin and glucoside.
[6]
7
Calotropis procera (Aiton) W. T. Aiton
Apocynaceae
Exudates and flower
Resins, tannins, calotropin, sterol, uscharin, calotropagenin, calotoxin, and calactin.
[6]
8
Cedrus deodara (Roxb. ex D. Don) G. Don
Pinaceae
Bark
Taxifolin,  dewardiol, dewarene, gum, ascorbic acid, himadarol, cantdarol, cholesterin, allohimachalol, cedrinoside, himachalol, dewarol, cedrin, dihydrodehydrodiconiferyl alcohol, essential oil, isocentdarol, and dihydromyricetin.
[6]
9
Citrus limon (L.) Burm. f.
Rutaceae
Fruit
4-β-glucopyranoside, d-x-pinene camphene, d-limonene, linalool ichangin 4-β-glucopyranoside, nomilinic acid, and essential oil.
[6]
10
Citrullus colocynthis (L.) Schrad
Cucurbitaceae
Fruit
Many alkaloids, glycosides, tannins, citrulluin, citrulluic acid, dihydric alcohol, phydroxybenzyl, citrulluene, hentriacontane, elaterin, citrullol, methyl ether, bitter oil, citbittol, and saponins.
[6]
11
Cuminum cyminum L.
Apiaceae
Seed
Essential oil and cumin oil contain sminaldehyde, 1,3-p-menthadien-7-al, and 1,4-p-menthadien-7-al.
[6]
12
Enicostema hyssopifolium (Willd.) I. Verd.
Gentianaceae
Whole plant
Saponarin, isovetexin, sylyswertin, betulin, swertiamarin, apigenin, genkwanin, erythrocentaurine, swertioside, isoswertisin, enicoflavine, and swertisin.
[6]
13
Fagonia cretica L.
Zygophyllaceae
Leaves
Ursolic acid, pinitol, nahagenin, saponin glycosides, saponin-I and saponin-II, docosyl docosanoate from n-hexane extract, and different proteins from aqueous extract.
[6]
14
Leucas capitataDesf.
Lamiaceae
Whole plant
Alkaloid as well as essential oil.
[6]
15
Matthiola incana (L.) W. T. Aiton
Brassicaceae
Seeds
Oil rich in chlorophylls a, b, N, P, K, and Na,γ-linolenic acid, and carotenoids. Sulforaphene is an important component.
[6]
16
Momordica charantia L.
Cucurbitaceae
Fruit
Carotenoids, momorcharasides A  and  B, glucosides, stigmastadien-3-beta-ol, squalene, mycoses, steroidal glucoside, momordicines I and II, stigmasterol, vicine, cucurbitane triterpenoids, momordicosides, cycloeucalenol, taraxerol, spinasterollophenol, diosgenin, thiocyanogen, 24-methylencycloartenol, and phenyl propanoids.
[6]
17
Nerium indicum Mill
Apocynaceae
Root  and  leaves
Nerioderin, neriodorin, karabin, and odorin.
[6]
18
Ocimum sanctum L.
Lamiaceae
Whole plant
Essential oils are found rich in 3.2% carvacrol, 71.3% eugenol, 1.7% caryophyllene, 20.4% methyl eugenol, eugenol methyl ether, linalool, and methyl chavicol cineole.
[6]
19
Pinus roxburghii Sarg
Pinaceae
Oleoresin
Careen,α-pinene, β-pinene, β-carene, β-longifolene, longifolene, and longicyclene.
[6]
20
Pistacia integerrimaJ. L. Stewart
Anacardiaceae
Galls
1.3% essential oil rich in camphene, cineole, A-terpineol, A-pinene, aromadendrene, caprylic acid, and d-limonene abundant in galls.
[6]
21
Psoralea corylifolia L.
Fabaceae
Seeds
Limonene, linalool, psoralene, angelicin, neobavachalcone, bakuchiol, 4-terpineol, corylidin, neobavaisoflavone,  bavachinin, isobavachin,α-elemene, geranylacetate, stigmasterol, bakuchioltraincontane, isopsoralidin, isopsoralen, bakuchalcone,  isoneobavachalcone, psoralen, psoralidin, raffinose, corylifolinin, corylifolean, and corylifolin.
[6]
22
Rhazya stricta Decne
Apocynaceae
Leaves
Enzymes  (NADPH dependent), glycosides  (roblnin, 3-7-rhamnoside and isorhamnetin-3-7-rhamnoside), alkaloid  (sewarine), and triterpenes  (ursolic acid, Mg quinate, andβ-sitosterol) along with flavonoids rhazianosides A and B.
[6]
23
Rubia cordifolia L.
Rubiaceae
Stem
Pseudopurpurin, xanthopurpurin, purpurin, munjistin, alizarin, and munjistin are found with their glycosides.
[6]
24
Sapindus mukorossi Gaertn
Sapindaceae
Fruit
β-Sitosterol, sapindoside A, sapindoside B, kaempferol, quercetin, saponin, and emarginatoside.
[6]
25
Stenolobium stans (L.) Seem
Bignoniaceae
Root
β-Carotene, β-sitosterol, α-amarine, zeaxanthin, indole metabolizing enzymes, phenolic acids, oleanolic acid, triterpenoids, ursolic acid, indole-oxygenase, and luteinzeaxanthin.
[6]
26
Terminalia arjuna  (Roxb. ex DC.) Wight and Arn
Combretaceae
Bark
β-Sitosterol, arjunolic acid, tomentosic acid,  ellagic acid, essential oil, arjunetin, arjunine, calcium salts, aluminium and magnesium salts, coloring agents, reducing sugars, tannin  (pyrocatechol), and a lactone.
[6]
27
Trichodesma indicum (L.) Sm
Boraginaceae
Whole plant
Seeds oil is rich in linoleic, oleic, stearic, palmitic, and linolenic acids. Apart of it hexacosane, ethyl hexacosanoate, and 21,24-hexacos-adienoic acid ethyl esters are found.
[6]
28
Zingiber officinale Roscoe
Zingiberaceae
Rhizome
D-Curcumene, cineol, potassium oxalate, camphene, citral borneol, singiberine, shogaol,α-curcumene, α-bergamotene, β-gamma-bisabolene, gingerol, and β-phellandrene.
23


## 2. Material and Methods

### 2.1. Collection and Milking of Snake Venom

Black Pakistani cobra snakes (Naja naja karachiensis) were collected from Cholistan desert located in southern Punjab province of Pakistan. After collection, they were properly identified by a zoologist. Venom fromNaja naja karachiensis was collected by pressing their glands below their eyes in low light environment. After collection, it was lyophilized and stored in light resistant container at 2°C to 8°C. For further experiments, venom was used in terms of its dry weight [6].


### 2.2. Collection and Preparation of Plant Extracts

Medicinal plants were collected from different areas in Pakistan. After collection, they were duly identified by expert botanist (Professor Dr. Altaf Ahmad Dasti) and voucher specimens were deposited in the herbarium of the Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan. Complete description about evaluated medicinal plants is summarized in Table1. One kilogram chopped plant material was soaked in 5 L of methanol as solvent in extraction bottles. The homogenates were kept for a period of a month at optimum temperature. After filtration, filtrate was evaporated by using water bath and extracts were weighed and stored for further experimentation [24].


### 2.3. Experimental Animals

Seventy-one healthy growing male rabbits (1
±
0.5 kg) were selected for this study after getting permission from local ethical committee. Animals were acclimatized for a week in the laboratory by maintaining their standards for chow, water, and light. Subsequently, they were divided into various groups in different cages such that experimental rabbits in group I were used for baseline measurements of various biochemical parameters. Animals belonging to groups II and III were evaluated for different doses of cobra venom while group IV was served as control. Group V was redivided into twenty-nine groups such that each subgroup (V/1–V/29) was assigned to a single medicinal plant extract [25].


### 2.4.In Vivo Antivenom Activity of Medicinal Plant Extracts

Venom fromNaja naja karachiensis (400 μg/kg and 800 μg/kg) was injected subcutaneously to evaluate its toxicity to the heart, liver, and kidneys. Before injection of venom, experimental animals were anaesthetized by administration of ketamine (50 mg/kg). To determine antivenom activity of various plants’ extract, they (100 mg/kg) were incubated (at 37°C for 30 minutes) with fixed amount of venom (800 μg/kg) before injection while saline was used as control [26–28].


### 2.5. Biochemical Assays

For serum analysis of various biochemical parameters, blood was collected from marginal ear artery by the use of hypodermic syringe needle after 3 hours of envenomation [25]. Separated serum was tested for alanine aminotransferase (GPT/ALT), aspartate aminotransferase (GOT/AST), urea, creatinine, creatine kinase isoenzyme MB (CK-MB), and lactate dehydrogenase (LDH) by the use of kits (manufactured by Merck) according to the DGKC and IFCC method on the Selectra Junior (Vital Scientific B.V, The Netherlands) [26].


### 2.6. Statistical Analysis

All numerical values were expressed as mean (3 replicates) ± standard error of mean (SEM). They were calculated by the use of Microsoft Excel 2007 and Student’st-test was applied to compare the efficacy of evaluated samples with standard antidote (reference standard).


## 2.1. Collection and Milking of Snake Venom

Black Pakistani cobra snakes (Naja naja karachiensis) were collected from Cholistan desert located in southern Punjab province of Pakistan. After collection, they were properly identified by a zoologist. Venom fromNaja naja karachiensis was collected by pressing their glands below their eyes in low light environment. After collection, it was lyophilized and stored in light resistant container at 2°C to 8°C. For further experiments, venom was used in terms of its dry weight [6].


## 2.2. Collection and Preparation of Plant Extracts

Medicinal plants were collected from different areas in Pakistan. After collection, they were duly identified by expert botanist (Professor Dr. Altaf Ahmad Dasti) and voucher specimens were deposited in the herbarium of the Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan. Complete description about evaluated medicinal plants is summarized in Table1. One kilogram chopped plant material was soaked in 5 L of methanol as solvent in extraction bottles. The homogenates were kept for a period of a month at optimum temperature. After filtration, filtrate was evaporated by using water bath and extracts were weighed and stored for further experimentation [24].


## 2.3. Experimental Animals

Seventy-one healthy growing male rabbits (1
±
0.5 kg) were selected for this study after getting permission from local ethical committee. Animals were acclimatized for a week in the laboratory by maintaining their standards for chow, water, and light. Subsequently, they were divided into various groups in different cages such that experimental rabbits in group I were used for baseline measurements of various biochemical parameters. Animals belonging to groups II and III were evaluated for different doses of cobra venom while group IV was served as control. Group V was redivided into twenty-nine groups such that each subgroup (V/1–V/29) was assigned to a single medicinal plant extract [25].


## 2.4.In Vivo Antivenom Activity of Medicinal Plant Extracts

Venom fromNaja naja karachiensis (400 μg/kg and 800 μg/kg) was injected subcutaneously to evaluate its toxicity to the heart, liver, and kidneys. Before injection of venom, experimental animals were anaesthetized by administration of ketamine (50 mg/kg). To determine antivenom activity of various plants’ extract, they (100 mg/kg) were incubated (at 37°C for 30 minutes) with fixed amount of venom (800 μg/kg) before injection while saline was used as control [26–28].


## 2.5. Biochemical Assays

For serum analysis of various biochemical parameters, blood was collected from marginal ear artery by the use of hypodermic syringe needle after 3 hours of envenomation [25]. Separated serum was tested for alanine aminotransferase (GPT/ALT), aspartate aminotransferase (GOT/AST), urea, creatinine, creatine kinase isoenzyme MB (CK-MB), and lactate dehydrogenase (LDH) by the use of kits (manufactured by Merck) according to the DGKC and IFCC method on the Selectra Junior (Vital Scientific B.V, The Netherlands) [26].


## 2.6. Statistical Analysis

All numerical values were expressed as mean (3 replicates) ± standard error of mean (SEM). They were calculated by the use of Microsoft Excel 2007 and Student’st-test was applied to compare the efficacy of evaluated samples with standard antidote (reference standard).


## 3. Results

Venom fromNaja naja karachiensis was proved to cause severe complications in dose-dependent manner. Venom at the dose of 400 μg/kg led to release of moderate serum levels of ALT (72
±
4.70 U/L, 0.1
>
P
>
0.05), AST (157
±
24.24 U/L, 0.1
>
P
>
0.05), urea (42
±
3.08 mg/dL, 0.05
>
P
>
0.02), creatinine (1.74
±
0.03 mg/dL, 0.01
>
P
>
0.001), CK-MB (21
±
1.5 U/L, 0.05
>
P
>
0.02), and LDH (2064
±
15.98 U/L, P
<
0.001) and thus indicated toxicities to the heart, liver, and kidneys. At 800 μg/kg of cobra venom, severe tissue damage was observed in terms of significant release of ALT (86
±
5.0 U/L, 0.05
>
P
>
0.02), AST (251
±
18.2 U/L, 0.01
>
P
>
0.001), urea (57.6
±
3.84 mg/dL, 0.02
>
P
>
0.01), creatinine (2.1
±
0.10 mg/dL, 0.02
>
P
>
0.01), CK-MB (77
±
11.22 U/L, 0.05
>
P
>
0.02), and LDH (2562
±
25.14 U/L, P
≪
0.001). Complete detail about various biochemical parameters for baseline measurements different doses of cobra venom, and saline (as negative control) is summarized in Table 2.Table 2
Biochemical parameters before and after snake venom administration in different experimental groups of rabbits.
Toxicity determined
Markers of snake venom toxicity
Group I baseline  measurements    (mean ± SEM)
Group II and III  venom injection
Group IV negative control (mean ± SEM)
Normal reference values reported
References
0.4 mg/kg (mean ± SEM)
0.8 mg/kg (mean ± SEM)
Liver
GPT/ALT
(52 ± 3.46) U/L
(72 ± 4.70) U/L
0.1
>
P
>
0.05
(86 ± 5.0) U/L
0.05
>
P
>
0.02
(52 ± 3.48) U/L
(48–80) U/L
[19]
GOT/AST
(65 ± 6.57) U/L
(157 ± 24.24) U/L
0.1
>
P
>
0.05
(251 ± 18.2) U/L
0.01
>
P
>
0.001
(67 ± 3.21) U/L
(14–113) U/L
[19]
Kidneys
Urea
( 28 ± 1.73) mg/dL
(42 ± 3.08) mg/dL
0.05
>
P
>
0.02
(57.6 ± 3.84) mg/dL
0.02
>
P
>
0.01
(28 ± 0.33) mg/dL
(10–28) mg/dL
[26]
Creatinine
(1.0 ± 0.313) mg/dL
(1.74 ± 0.03) mg/dL
0.01
>
P
>
0.001
(2.1 ± 0.10) mg/dL
0.02
>
P
>
0.01
(1 ± 0.06) mg/dL
(0.5–2.5) mg/dL
[26]
Heart
CK-MB
(13.2 ± 2.0) U/L
(21 ± 1.5) U/L
0.05
>
P
>
0.02
(77 ± 11.22) U/L
0.05
>
P
>
0.02
(13 ± 0.56) U/L
(<25) U/L*
[20, 21]
LDH
(714 ± 3.18) U/L
(2064 ± 15.98) U/L
P
<
0.001
(2562 ± 25.14) U/L
P
≪
0.001
(720 ± 4.70) U/L
(559–2077) U/L
[46]
*CK-MB activity is less than 2% in healthy rabbits; however, it is usually 10%–30% of total CK activity.To neutralizein vivo 800 μg/kg of cobra venom, twenty-eight medicinal plant extracts were evaluated. It was noticed that extract ofStenolobium stans (L.) Seem was the best antidote (P
>
0.5) compared to reference standard.Allium sativum L.,Althaea officinalisL.,Citrullus colocynthis(L.) Schrad,Leucas capitataDesf.,Pinus roxburghiiSarg,Psoralea corylifolia L.,Rubia cordifolia L., andSapindus mukorossiGaertn were found to be valuable plants to protect liver damage (P
>
0.5) as standard antidote (ALT = 52.5
±
3.51 U/L and AST = 69.5
±
18.55 U/L); however, required ALT and AST levels were not observed with remaining plant extracts (0.5
>
P
>
0.05) as shown in Table 3 and comparison is shown in Figure 1. Extracts ofLeucas capitataDesf. andAlthaea officinalisL. were observed to be helpful (P
>
0.5) as reference standard (urea = 31.5
±
0.50 mg/dL and creatinine = 1.08
±
0.02 mg/dL); nevertheless, remaining plants were found less valuable (0.5
>
P
>
0.01) to minimize urea and creatinine levels to maintain kidney functions (Table 4 and Figure 2). When compared with standard antidote (CK-MB = 9.0
±
0.85 U/L) eight medicinal plants (Allium cepaL.,Althaea officinalisL.,Bauhinia variegataL.,Cedrus deodara(Roxb. ex D. Don) G. Don,Fagonia cretica L.,Leucas capitataDesf., Momordica charantia L., andOcimum sanctum L.) were found equally capable (P
>
0.5) of normalizing high values of CK-MB; however, rest of all were proved less beneficial (0.5
>
P
>
0.1). To combat highly raised values of LDH,Althaea officinalisL.,Leucas capitataDesf., andTerminalia arjuna (Roxb. ex DC.) Wight and Arn were proved useful to some extent (0.5
>
P
>
0.1) although remaining plant extracts could not be shortlisted (0.1
>
P
>
0.001) as standard antisera (LDH = 763
±
6.01 U/L). Overall detail about LDH is discussed in Table 5 and comparison is shown in Figure 3.Table 3
Hepatoprotective activity of various medicinal plant extracts on ALT and AST levels to neutralize snake bite envenomation in experimental rabbits.
Sr. number
Group V(subgroup)
Name of evaluated sample
GPT/ALT
GOT/AST
U/L(mean  ±  SEM)
P value/comment
U/L (mean  ±  SEM)
P value/comment
1
(V/1)
Albizia lebbeck(L.) Benth.
62 ± 7.02
0.5
>
P
>
0.1
99 ± 18.55
0.5
>
P
>
0.1
2
(V/2)
Allium cepaL.
68 ± 12.03
0.5
>
P
>
0.1
166.5 ± 0.50
0.5
>
P
>
0.1
3
(V/3)
Allium sativumL.
49 ± 5.01
P
>
0.5
82.5 ± 18.55
P
>
0.5
4
(V/4)
Althaea officinalisL.
70 ± 20.05
P
>
0.5
74.5 ± 14.54
P
>
0.5
5
(V/5)
Bauhinia variegataL.
78 ± 4.04
0.5
>
P
>
0.1
149.5 ± 19.9
0.5
>
P
>
0.1
6
(V/6)
Brassica nigra(L.) W. D. J. Koch
45 ± 0.00
0.5
>
P
>
0.1
55 ± 1.00
P
>
0.5
7
(V/7a)
Calotropis procera(Aiton) W. T. Aiton (exudates)
31.5 ± 0.5
0.5
>
P
>
0.1
141 ± 1.01
0.5
>
P
>
0.1
8
(V/7b)
Calotropis procera(Aiton) W. T. Aiton (flowers)
62.5 ± 2.52
0.5
>
P
>
0.1
194 ± 12.12
0.5
>
P
>
0.1
9
(V/8)
Cedrus deodara (Roxb. ex D. Don) G. Don
63.3 ± 4.14
0.5
>
P
>
0.1
72 ± 2.02
P
>
0.5
10
(V/9)
Citrus limon(L.) Burm. f.
30.5 ± 13.63
0.5
>
P
>
0.1
49 ± 24.24
P
>
0.5
11
(V/10)
Citrullus colocynthis(L.) Schrad
47.5 ± 5.55
P
>
0.5
74 ± 9.09
P
>
0.5
12
(V/11)
Cuminum cyminumL.
67 ± 16.16
P
>
0.5
157 ± 14.14
0.5
>
P
>
0.1
13
(V/12)
Enicostemma hyssopifolium(Willd.) I. Verd.
85.25 ± 1.26
0.1
>
P
>
0.05
168.5 ± 7.57
0.5
>
P
>
0.1
14
(V/13)
Fagonia creticaL.
80 ± 7.82
0.5
>
P
>
0.1
170 ± 13.13
0.5
>
P
>
0.1
15
(V/14)
Leucas capitataDesf.
45.5 ± 7.52
P
>
0.5
53.5 ± 0.50
P
>
0.5
16
(V/15)
Matthiola incana(L.) W. T. Aiton
62 ± 8.08
0.5
>
P
>
0.1
121 ± 5.05
0.5
>
P
>
0.1
17
(V/16)
Momordica charantiaL.
62.5 ± 7.52
0.5
>
P
>
0.1
247 ± 46.13
0.5
>
P
>
0.1
18
(V/17)
Nerium indicumMill
88 ± 19.55
0.5
>
P
>
0.1
235 ± 18.05
0.1
>
P
>
0.05
19
(V/18)
Ocimum sanctumL.
61.5 ± 7.52
0.5
>
P
>
0.1
158 ± 10.02
0.5
>
P
>
0.1
20
(V/19)
Pinus roxburghiiSarg
53 ± 9.02
P
>
0.5
57 ± 13.03
P
>
0.5
21
(V/20)
Pistacia integerrimaJ. L. Stewart
41.5 ± 1.5
0.5
>
P
>
0.1
54.5 ± 0.50
P
>
0.5
22
(V/21)
Psoralea corylifoliaL.
54 ± 1.00
P
>
0.5
66.5 ± 11.53
P
>
0.5
23
(V/22)
Rhazya strictaDecne
66 ± 24.24
P
>
0.5
132.5 ± 12.62
0.5
>
P
>
0.1
24
(V/23)
Rubia cordifoliaL.
65.5 ± 17.67
P
>
0.5
129 ± 66.41
P
>
0.5
25
(V/24)
Sapindus mukorossiGaertn
56 ± 6.01
P
>
0.5
78.5 ± 13.54
P
>
0.5
26
(V/25)
Stenolobium stans(L.) Seem
46.5 ± 6.56
P
>
0.5
70.5 ± 3.53
P
>
0.5
27
(V/26)
Terminalia arjuna(Roxb. ex DC.) Wight and Arn
47.5 ± 1.51
0.5
>
P
>
0.1
78 ± 6.06
P
>
0.5
28
(V/27)
Trichodesma indicum(L.) Sm
52 ± 0.00
P
>
0.5
125 ± 4.01
0.5
>
P
>
0.1
29
(V/28)
Zingiber officinaleRoscoe
66.5 ± 32.59
P
>
0.5
117.5 ± 9.52
0.5
>
P
>
0.1
30
(V/29)
Reference standard antidote(standard antisera /immunoglobulin’s)
52.5 ± 3.51
Select to compare
69.5 ± 18.55
Select to compareTable 4
Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomation in experimental rabbits.
Sr. number
Group V (subgroup)
Name of evaluated sample
Urea
Creatinine
mg/dL(mean  ±  SEM)
P value/comment
mg/dL(mean  ±  SEM)
P value/comment
1
(V/1)
Albizia lebbeck(L.) Benth.
57 ± 0.0
0.02
>
P
>
0.01
1.6 ± 0.20
0.5
>
P
>
0.1
2
(V/2)
Allium cepaL.
51 ± 11.03
0.5
>
P
>
0.1
1.50 ± 0.17
0.5
>
P
>
0.1
3
(V/3)
Allium sativumL.
50 ± 4.01
0.5
>
P
>
0.1
1.30 ± 0.06
0.5
>
P
>
0.1
4
(V/4)
Althaea officinalisL.
26 ± 5.05
P
>
0.5
1.18 ± 0.24
P
>
0.5
5
(V/5)
Bauhinia variegataL.
50 ± 12.6
0.5
>
P
>
0.1
1.36 ± 0.24
0.5
>
P
>
0.1
6
(V/6)
Brassica nigra(L.) W. D. J. Koch
22.5 ± 4.51
0.5
>
P
>
0.1
1.07 ± 0.11
P
>
0.5
7
(V/7a)
Calotropis procera(Aiton) W. T. Aiton (exudates)
42.5 ± 2.52
0.5
>
P
>
0.1
1.30 ± 0.01
0.1
>
P
>
0.05
8
(V/7b)
Calotropis procera(Aiton) W. T. Aiton (flowers)
39 ± 2.02
0.5
>
P
>
0.1
1.25 ± 0.03
0.5
>
P
>
0.1
9
(V/8)
Cedrus deodara(Roxb. ex D. Don) G. Don
44 ± 4.04
0.5
>
P
>
0.1
1.44 ± 0.07
0.5
>
P
>
0.1
10
(V/9)
Citrus limon(L.) Burm. f.
39.5 ± 2.52
0.5
>
P
>
0.1
1.40 ± 0.03
0.1
>
P
>
0.05
11
(V/10)
Citrullus colocynthis(L.) Schrad
49.5 ± 3.53
0.5
>
P
>
0.1
1.52 ± 0.15
0.5
>
P
>
0.1
12
(V/11)
Cuminum cyminumL.
54 ± 1.01
0.05
>
P
>
0.02
1.48 ± 0.01
0.05
>
P
>
0.02
13
(V/12)
Enicostemma hyssopifolium(Willd.) I. Verd.
39.5 ± 4.54
0.5
>
P
>
0.1
1.35 ± 0.05
0.5
>
P
>
0.1
14
(V/13)
Fagonia creticaL.
43 ± 2.27
0.5
>
P
>
0.1
1.23 ± 0.13
P
<
0.5
15
(V/14)
Leucas capitataDesf.
31.5 ± 0.50
P
>
0.5
1.07 ± 0.05
P
>
0.5
16
(V/15)
Matthiola incana(L.) W. T. Aiton
43.5 ± 3.53
0.5
>
P
>
0.1
1.44 ± 0.14
0.5
>
P
>
0.1
17
(V/16)
Momordica charantiaL.
57 ± 5.01
0.5
>
P
>
0.1
1.5 ± 0.005
0.05
>
P
>
0.02
18
(V/17)
Nerium indicumMill
47 ± 0.00
P
<
0.05
1.24 ± 0.10
0.5
>
P
>
0.1
19
(V/18)
Ocimum sanctumL.
49.5 ± 7.52
0.5
>
P
>
0.1
1.5 ± 0.005
0.05
>
P
>
0.02
20
(V/19)
Pinus roxburghiiSarg
45 ± 1.00
P
>
0.05
1.4 ± 0.05
0.5
>
P
>
0.1
21
(V/20)
Pistacia integerrimaJ. L. Stewart
66.5 ± 0.50
0.02
>
P
>
0.01
1.68 ± 0.10
0.5
>
P
>
0.1
22
(V/21)
Psoralea corylifoliaL.
42.5 ± 2.50
0.5
>
P
>
0.1
1.27 ± 0.15
0.5
>
P
>
0.1
23
(V/22)
Rhazya strictaDecne
53.5 ± 3.53
0.5
>
P
>
0.1
1.67 ± 0.005
0.05
>
P
>
0.02
24
(V/23)
Rubia cordifoliaL.
36 ± 1.01
0.5
>
P
>
0.1
0.93 ± 0.48
P
>
0.5
25
(V/24)
Sapindus mukorossiGaertn
44 ± 1.00
0.1
>
P
>
0.05
1.43 ± 0.12
0.5
>
P
>
0.1
26
(V/25)
Stenolobium stans(L.) Seem
25.5 ± 6.51
P
>
0.5
1.1 ± 0.06
P
>
0.5
27
(V/26)
Terminalia arjuna(Roxb. ex DC.) Wight and Arn
38.5 ± 1.51
0.5
>
P
>
0.1
1.27 ± 0.23
P
>
0.5
28
(V/27)
Trichodesma indicum(L.) Sm
37 ± 1.00
0.5
>
P
>
0.1
0.46 ± 0.01
0.05
>
P
>
0.02
29
(V/28)
Zingiber officinaleRoscoe
47 ± 1.00
0.05
>
P
>
0.02
1.30 ± 0.01
0.5
>
P
>
0.1
30
(V/29)
Reference standard antidote(standard antisera /immunoglobulin’s)
31.5 ± 0.50
Select to compare
1.08 ± 0.02
Select to compareTable 5
Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation in experimental rabbits.
Sr. number
Group V (subgroup)
Name of evaluated sample
LDH
CK-MB
U/L (mean  ±  SEM)
P value/comment
U/L (mean  ±  SEM)
P value/comment
1
(V/1)
Albizia lebbeck(L.) Benth.
1357 ± 1.00
0.01
>
P
>
0.001
4.1 ± 0.85
0.5
>
P
>
0.1
2
(V/2)
Allium cepaL.
934 ± 13.03
0.1
>
P
>
0.05
14.8 ± 1.65
P
>
0.5
3
(V/3)
Allium sativumL.
1177 ± 20.56
0.05
>
P
>
0.02
6.6 ± 3.30
0.5
>
P
>
0.1
4
(V/4)
Althaea officinalisL.
975.5 ± 33.60
0.5
>
P
>
0.1
14.8 ± 3.30
P
>
0.5
5
(V/5)
Bauhinia variegataL.
1972 ± 3.00
P
>
0.001
8.3 ± 6.76
P
>
0.5
6
(V/6)
Brassica nigra(L.) W. D. J. Koch
855.5 ± 0.50
0.05
>
P
>
0.02
9.0 ± 0.85
0.5
>
P
>
0.1
7
(V/7a)
Calotropis procera(Aiton) W. T. Aiton (exudates)
1022 ± 5.01
0.02
>
P
>
0.01
6.6 ± 3.30
0.5
>
P
>
0.1
8
(V/7b)
Calotropis procera(Aiton) W. T. Aiton (flowers)
1114 ± 1.00
0.02
>
P
>
0.01
61.8 ± 10.9
0.5
>
P
>
0.1
9
(V/8)
Cedrus deodara(Roxb. ex D. Don) G. Don
1230 ± 23.57
0.05
>
P
>
0.02
41.2 ± 31.66
P
>
0.5
10
(V/9)
Citrus limon(L.) Burm. f.
1831 ± 65.69
0.05
>
P
>
0.02
73 ± 14.1
0.5
>
P
>
0.1
11
(V/10)
Citrullus colocynthis(L.) Schrad
827 ± 6.51
0.1
>
P
>
0.05
05 ± 1.66
0.5
>
P
>
0.1
12
(V/11)
Cuminum cyminumL.
1589 ± 22.56
0.02
>
P
>
0.01
5.8 ± 0.80
0.5
>
P
>
0.1
13
(V/12)
Enicostemma hyssopifolium(Willd.) I. Verd.
1615 ± 1.51
P
>
0.001
9.85 ± 1.66
0.5
>
P
>
0.1
14
(V/13)
Fagonia creticaL.
1418 ± 13.03
0.02
>
P
>
0.01
11 ± 2.52
P
>
0.5
15
(V/14)
Leucas capitataDesf.
783 ± 10.02
0.5
>
P
>
0.1
14 ± 0.80
P
>
0.5
16
(V/15)
Matthiola incana(L.) W. T. Aiton
1428 ± 6.51
P
>
0.001
08 ± 1.66
0.5
>
P
>
0.1
17
(V/16)
Momordica charantiaL.
1475.5 ± 3.51
P
>
0.001
15.6 ± 2.45
P
>
0.5
18
(V/17)
Nerium indicumMill
1268 ± 12.03
0.02
>
P
>
0.01
05 ± 1.65
0.5
>
P
>
0.1
19
(V/18)
Ocimum sanctumL.
1335 ± 12.03
0.02
>
P
>
0.01
12.3 ± 2.45
P
>
0.5
20
(V/19)
Pinus roxburghiiSarg
1050 ± 1.00
0.02
>
P
>
0.01
08 ± 1.65
0.5
>
P
>
0.1
21
(V/20)
Pistacia integerrimaJ. L. Stewart
1135.5 ± 0.5
0.02
>
P
>
0.01
13.1 ± 1.65
0.5
>
P
>
0.1
22
(V/21)
Psoralea corylifoliaL.
1153.5 ± 0.5
P
>
0.001
17.3 ± 2.50
0.5
>
P
>
0.1
23
(V/22)
Rhazya strictaDecne
1538 ± 20.05
0.02
>
P
>
0.01
4.1 ± 0.85
0.5
>
P
>
0.1
24
(V/23)
Rubia cordifoliaL.
1078 ± 16.04
0.05
>
P
>
0.02
6.6 ± 0.00
0.5
>
P
>
0.1
25
(V/24)
Sapindus mukorossiGaertn
1460.5 ± 5.51
0.01
>
P
>
0.001
4.1 ± 0.80
0.5
>
P
>
0.1
26
(V/25)
Stenolobium stans(L.) Seem
787 ± 28.08
P
>
0.5
13 ± 1.76
P
>
0.5
27
(V/26)
Terminalia arjuna(Roxb. ex DC.) Wight and Arn
798.5 ± 14.54
0.5
>
P
>
0.1
6.6 ± 0.00
0.5
>
P
>
0.1
28
(V/27)
Trichodesma indicum(L.) Sm
978.5 ± 4.51
0.05
>
P
>
0.02
6.6 ± 3.00
0.5
>
P
>
0.1
29
(V/28)
Zingiber officinaleRoscoe
888 ± 2.00
0.05
>
P
>
0.02
17.3 ± 0.80
0.5
>
P
>
0.1
30
(V/29)
Reference standard antidote(standard antisera /immunoglobulin’s)
763 ± 6.01
Select to compare
09 ± 0.85
Select to compareFigure 1
Comparison of various medicinal plant extracts with reference standard antidote in neutralization of increased GPT and GOT serum level posed byNaja naja karachiensis venom in rabbits.Figure 2
Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea and creatinine level posed byNaja naja karachiensis venom in rabbits.Figure 3
Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levels elevated byNaja naja karachiensis venom in rabbits.


## 4. Discussion

Snake bite has been responsible for tens of thousands of deaths worldwide and numerous physical handicaps [29]. Generally, snake venom is an intricate mixture of various proteins (>90%) and most of them are enzymes, particularly (40%) phospholipases A2 [30]. Among different complications, hepatic injury is one of the deadly venomous effects produced by cobra bite [31, 32].Naja naja karachiensis venom caused significant increase in ALT and AST levels (dose dependently) that are surrogate markers for liver toxicity either by direct action or immunological (hypersensitivity) reaction. The allergic reactions reactions are not dose dependent which clarify lethal effects to the cytoplasm and mitochondrial membranes of the hepatic cells [25, 33]. Phospholipases enzymes, abundant inNaja naja karachiensis venom, are responsible for breakdown of membranous phospholipids and therefore resulted in cellular injury along with inflammation [24]. PLA2 is attributed to decrease in Na+/K+ ATPase activities and led to greater influx of sodium ions and water molecules into the cell. Subsequently, plasma membrane lipid bilayer disorganized and eventually resulted in hepatic cells death [33, 34]. In addition, PLA2 is also found to cause anticoagulation in victims [6]. All pharmacological effects are due to phospholipid hydrolysis or phospholipid competing biding mechanism with other coagulation factors, particularly FXa, or by both ways; however, it is very hard to pinpoint exact one [35]. Anticoagulant response posed by cobra PLA2 (anticoagulant enzyme) may aggravate hepatic injury as many anticoagulant agents cause liver damage that are idiosyncratic in nature [36].Cardiac injury, particularly systolic heart arrest, is one of the well-known toxicities related toNaja naja subspecies [30]. In present study, Pakistani cobra venom was found to release two cystolic enzymes (LDH and CK-MB) that are sensitive indicators of myocyte injury [37]. It is due to the presence of myotoxic PLA2 and other cardiotoxin(s) that are salient features of cobra venom that are responsible for cellular necrosis and cytotoxicity [38, 39]. Cytotoxic effect ofNaja naja karachiensis venom may not be overlooked even when antisera administration is late cause of toxic components to the microvasculature and thrombus produced which poses hindrance in access of immunoglobulins to the site of snake bite [26, 29, 39].Snake venom has been responsible for detrimental effects to the renal tissues [40]. Likewise,Naja naja krachiensisvenom was found to cause severe renal damage by significant rise in serum urea and creatinine levels. Indeed, phospholipases enzymes are responsible for the increase in vascular permeability with hemorrhagic effects to the vital tissues in the victims. Subsequently, numerous lesions are produced related to the glomerular membrane and renal tubules either interstitial or vascular [29, 41, 42]. Presence of lymphocytes (white blood cells) and oedema in the cortical as well as medullary regions of renal tubules further confirmed the idea of renal damage (data has not shown) as reported previously with different snake venoms likeHemiscorpius lepturus [26, 43]. Apart of it, pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope Tc
99
m confirmed that kidneys and urinary bladder are the most saturated organs (>70%) after intravenous injection in experimental rabbits (unpublished data by our group).Medicinal plants of Pakistan are used to inhibit snake venom (PLA2) enzymes [6]. Due to this effect, present study was designed to select twenty-eight medicinal plants of Pakistan to test their potentials as an antidote against toxicities produced byNaja naja karachiensis venom. It was fascinating that extract ofStenolobium stans (L.) Seem showed significant neutralization compared to that of reference standard (antisera) as reported previously by Asad et al., for inhibition of phospholipases A2 anticoagulant activity [6, 44]. Present study indicated that extract ofStenolobium stans (L.) Seem possesses an endogenous inhibitor(s) to nullify venom (PLA2) induced toxicities. Extracts of these plants material are routinely used in Pakistan by simple application in the form of paste to the affected area [45]. This practice provides effective first aid treatment as tiny molecules of an antidote diffuse favorably at the site of snake bite before hospitalization and abrogated the spreading of toxins [39]. It is owing to the various secondary metabolites like phenols, flavonoids, terpenoids, xanthenes, quinonoids, and so forth, as reported earlier to mask various enzymatic actions of cobra venom [6, 23]. Indeed, secondary metabolites pose hindrance in binding of different snake venom enzymes to their potential targets; therefore, antidotal effect evoked. Other plants extract were not proved significantly effective to neutralize cobra venom, therefore cannot be declared useful plants in venom therapy. It is the need of the time to isolate lethal component(s) of cobra venom attributed to its major toxicities. Furthermore, characterization of antivenom compound(s) from medicinal plant extracts would be worth full for complete and effective treatment of snake bite in the future.



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*Source: 970540-2014-04-27.xml*