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A Remedial Approach for Naja naja karachiensis Envenomation: Enzymatic Assay for Alkaline Phosphatase Activity in Extracts of Local Plants of Pakistan.

Byline: Muhammad Hassham Hassan Bin Asad Ghulam Murtaza and Izhar Hussain

Abstract Present study was performed to inhibit alkaline phosphatases (ALPases) present in snake venom Naja naja karachiensis by 28 medicinal plants of Pakistan. 2.87 U/mg 4.75 U/mg 6.70 U/mg and 8.6 U/mg of phosphatase activity were estimated for 0.2 mg 0.4 mg 0.6 mg and 0.8 mg of crude venom respectively in dose dependent manner. All plants extract (625 ng to 2.5g) were found to inhibit (80% to 93%) snake venom (0.4 mg) ALPases. Standard antidote was found to pose 93% hindrance at 2.5 g/ml while EDTA (specific metalloenzyme inhibitor) was observed 91% effective. Extract of Sapindus mukorossi Gaertn was declared the best anti-venom as reference standard however isolation and identification of bioactive constituent(s) is necessary for complete treatment of snake bite envenomation.

Key words: Alkaline phosphatases medicinal plants Pakistan cobra antisera.

INTRODUCTION

Snakes venom is complex mixture of hydrolytic enzymes non enzymatic proteins organic and inorganic molecules (Asad et al. 2013; Dhananjaya and D'Souza 2011; Khan 1999). Among hydrolytic enzymes proteases phospholipases (PLA) were more screened pharmacologically whilst nucleases nucleotidases and phosphatases (acidic as well as alkaline phosphatases) were less characterized (Matsui et al. 2000; Marshall 2005). It was due to toxinologists who claimed previously their non-toxic and digestive role (Dhananjaya and D'Souza 2011). However recent studies revealed that ALPases are deadly poisonous owing to their hydrolytic tendency towards phosphate esters non-specifically. In the victims they act on various substrates like 3'- AMP 5'- AMP FMN 5'- dAMP deoxy-dinucleotide phosphates ribose-3-phosphate 5'- phosphoribose- 1-pyrophosphate and ATP (preferably). ALPases act on ATP along with phosphodiesterases (PDE) caused in generation of adenosine moieties (Wuster 1996;

Feroze et al. 2010). Concurrently other enzymes in snake venom like phospholipases proteases cardiotoxin and cytotoxic peptides assist in cell necrosis lead to liberation of nucleic acids (DNA and RNA) (Alirol et al. 2010; Razi et al. 2011). Nucleic acids cleaved by nucleotideases and ALPases and resulted in large amount of adenosine. Adenosine is a multitoxin and induced miscellaneous hazardous effects like hypotention heart block redness inflammation anti-platelet aggregation renal failure unconsciousness pain analgesia via different adenosine (A1 A2a A2b and A3) receptors (Dhananjaya and D'Souza 2011).

Immunoglobulins are effective for snake- bites; however recovery depends on dose neutralization and way of inoculation (Warrell 2010). Nevertheless their high cost serum sickness reactions and scarcity in rural areas have aroused the quest for new sources of venom inhibitors (Razi et al. 2011). Medicinal plants (natural antidotes) have been documented previously to have various constituents to inhibit snake venom enzymes. Pakistan is a hub of medicinal plants where mostly rural people depend on them for their health-related problems (Asad et al. 2011). To the best of our knowledge mostly medicinal plants of Pakistan have not been assessed for their antivenom potentials to neutralize alkaline phosphatases regardless of their ethnobotanical use as antisnake venom (Asad et al. 2011 2014; Khan et al. 2009). Due to this reason venom from Naja naja karchiensis was evaluated scientifically for the first time to neutralize ALPases by local medicinal plants used in Pakistan.

MATERIALS AND METHODS

Collection of Naja naja karachiensis

Cobra snakes were collected from Cholistan desert situated in Southern Punjab province of Pakistan. They were collected with charmers and further duly identified by zoologist.

Milking of snake venom

Pakistani cobra snakes venom was milked by squeezing their glands below their eyes. Whole experiment was carried out in low light surrounding. Subsequently it was lyophilized and then refrigerated in a light resistant container. Before use lyophilized venom was reconstituted in saline in terms of its dry powder weight (Razi et al. 2011).

Plant collection

Medicinal plants of Pakistan were collected from various areas (Khyber Pakhtoonkhawa and Gilgit Biltistan) of the country. Different parts of the plants were collected on the basis of their ethnobotanical claims as anti-venom. Prof. Dr. Altaf Ahmad Dasti identified them and voucher specimens were deposited in the herbarium of the Institute of Pure and Applied Biology Bahauddin- Zakariya University Multan Pakistan. Complete description about collection of medicinal plants of Pakistan is provided in Table I.

Extraction of plant material

Shade dried plants material were chopped and further subjected to simple maceration process. Dried powder of desired part(s) of plant material (1Kg) was soaked in methanol (5L) in the extraction bottles. Homogenates were kept at ambient temperature for a period of four weeks. Subsequently they were filtered two times initially with an ordinary filter paper and later on by Whatman filter paper # 41. Methanol was evaporated at room temperature and different extracts were weighed and stored for further research (Hussain et al. 2007). Milky exudates of Citrullus colocynthis (L.) Schrad were collected lyophilized and then stored for further use.

Reference standard antidote

Reference standard antiserum (antidote) was purchased from pharmacy store (Raza Pharmacy) at Nishtar Hospital Multan Pakistan. It was utilized to compare different plants extract for neutralization of alkaline phospahtases toxicities. Alkaline phosphatases (with enzyme commission number 3.1.3.1) was supplied by Bharat Serums and Vaccines Limited Ambernath India (Asad et al. 2012).

Enzymatic assay for alkaline phosphatases

Activity of alkaline phosphatses (ALPases) in Naja naja karachiensis venom was assessed with the release of p-nitrophenol from its substrate (p- nitrophenyl phosphate) by their hydrolytic action. Briefly reaction mixture was prepared by mixing 0.5 ml of 0.5 M glycine buffer (pH=8.5) 0.5 ml of 0.01M p-nitrophenyl phosphate and 0.3 ml of 0.01 M MgSO4. Subsequently Naja naja karachiensis venom (0.2-0.8 mg/0.1ml) was added. Reaction mixture was incubated at 37 C for half an hour. At the end of this period 2 ml of NaOH (0.2 M) solution was added and further kept for 20 minutes at room temperature to halt the reaction and to confer stable yellow color to p-nitrophenol which absorbed maximally at 400 nm (Kanagasabapathy and Kumari 2000; Sulkowski et al. 1963; Yap et al. 2011). A standard curve of known concentrations of p-nitrophenol was constructed and ALPase activity was expressed as micromole of product released per minute.

To evaluate anti-venom activity of various antidotes (standard antisera and medicinal plants) they were preincubated with venom (0.4 mg) at different concentration ranges from 312 ng/ml to 05 g/ml for 30 minutes before measuring ALPase activity (Ushanandini et al. 2006; Dhananjaya et al. 2006). Specific metalloenzyme inhibitor (EDTA 2mM) was used to monitor the inhibition of ALPases activity. Additionally venom was also evaluated upon heating (0.4 mg 100C 10 min) and then after cooling (25C 45 min) for ALPases activity (Chakrabarty et al. 2000).

Table I.- Different medicinal plants collected from various locations in Pakistan having ethnobotanical evidences as anti- venom.

Sr.###Family (scientific name of the plant)###Area of collection###Voucher###Reference

No###(Part collected)###number

1.###Amaryllidaceae (Allium cepa L).###Bahawalpur (seeds)###STW.42###Makhija and

###Khamar (2010)

2.###Anacardiaceae (Pistacia integerrima J. L. Stewart).###Murree Hills (galls)###STW.458###Baquar (1989)

3.###Apiaceae (Cuminum cyminum L).###Sargodha (seeds)###STW.516###Baquar (1989)

4.###Apocynaceae (Nerium indicum Mill)###Haripur (roots and leaves)###STW.564###Asad et al.

###(2011)

5.###Apocynaceae (Rhazya stricta Dcne)###Leaves (Lakki Marwat)###STW.565###Asad et al.

###(2011)

6.###Apocynaceae (Calotropis procera (Aiton) W.T. Aiton)###Haripur (milky latex and###STW.566(a)###Asad et al.

###flower)###STW.566(b)###(2011)

7.###Bignoniaceae (Stenolobium stans (L.) Seem).###Haripur (roots)###STW.669###Baquar (1989)

8.###Boraginaceae (Trichodesma indicum (L.) Sm).###Sind province (complete###STW.604###Baquar (1989)

###plant)

9.###Fabaceae (Bauhinia variegate L).###Haripur (roots).###STW.374###Shinwari and

###Shah (2007)

10.###Brassicaceae (Brassica nigra (L.) W. D. J. Koch)###Manshera (seeds)###STW.302###Baquar (1989)

11.###Brassicaceae (Matthiola incana (L.) W. T. Aiton)###Rawalpindi (seeds)###STW.322###Baquar (1989)

12.###Cucurbitaceae (Momordica charantia L).###Abbottabad (fruit)###STW.706###Baquar (1989)

13.###Cucurbitaceae (Citrullus colocynthis (L.) Schrad).###Bahawalpur (fruits)###STW.702###Baquar (1989)

14.###Combretaceae (Terminalia arjuna (Roxb. ex DC.)###Islamabad (bark)###STW.502###Baquar (1989)

###Wight and Arn).###Prajapati et al.

###(2010)

15.###Fabaceae (Psoralea corylifolia L).###Peshawar (seeds)###STW.418###Baquar (1989)

16.###Gentianaceae (Enicostema hyssopifolium (Willd.) I.###Jhelum (fresh plant)###STW.553###Daniel (2006)

###Verd).

17.###Lamiaceae (Leucas capitata Desf)###Rawalpindi (whole plant)###STW.615###Shinwari and

###Shah (2007)

18.###Lamiaceae (Ocimum sanctum L)###Islamabad (whole plant)###STW.626###Prajapati et al.

###(2010)

19.###Amaryllidaceae (Allium sativum L).###Bhakkar (bulb)###STW.46###Ugulu (2011)

20.###Malvaceae (Althaea officinalis L).###Rawalpindi (roots)###STW.411###Asad et al.

###(2011)

21.###Fabaceae (Albizia lebbeck (L.) Benth)###Bahawalpur (seeds).###STW.381###Baquar (1989)

22.###Pinaceae (Cedrus deodara (Roxb. ex D. Don) G. Don)###Nathia Gali (bark)###STW.25###Baquar (1989)

23.###Pinaceae (Pinus roxburghii Sarg)###Murree Hills (oleoresin)###STW.26###Baquar (1989)

24.###Rubiaceae (Rubia cordifolia L)###Murree Hills (stems)###STW.689###Baquar (1989)

25.###Rutaceae (Citrus limon (L). Burm. F)###Haripur (fruit)###STW. xx###Rita et al. (2011)

26.###Sapindaceae (Sapindus mukorossi Gaertn)###Niswari Bazaar Rawalpindi###STW.463###Prajapati et al.

###(fruits)###(2010)

27.###Zingiberaceae (Zingiber officinale Roscoe)###Lahore (rhizome)###STW.66###Duke and

###Ayensu (1985)

28.###Zygophyllaceae (Fagonia cretica L)###Lasbella (leaves and twigs)###STW.433###Razi et al. (2011)

Statistical analysis

All numerical values were obtained as mean SEM by using Microsoft Excel version 2007. Student t-test was applied by the guidelines and instructions published by British medical Journal. Level of significance was set at 0.05.

RESULTS AND DISCUSSION

Standard graph of P-NP was generated with positive correlation coefficient (r) equal to 0.999 and y = 0.009x + 0.001. High doses of cobra venom were found with greater enzymatic activity in a dose dependent manner. Various activities of ALPase i.e. 2.87 4.75 6.70 and 8.60 U/mg were found in 0.2 0.4 0.6 and 0.8 mg of crude venom respectively.

Furthermore heated venom resulted in cessation of phosphatase activity as shown in Table II.

Table II.- Enzymatic activity of alkaline phosphatases at various concentrations of Naja naja karachiensis venom in terms of para- nitrophenol (PNP) released.

Concentration of venom###Absorbance at###Enzyme

used for enzymatic###400 nm###activity

activity###(Mean SEM)###(Unit/mg)

0.2 mg/0.1 ml###0.310.01###2.87

0.4 mg/0.1 ml###0.510.01###4.75

0.6 mg/0.1 ml###0.720.02###6.70

0.8 mg/0.1 ml###0.930.02###8.60

Effect of heat on venom###Same as sample###Nil

###blank###

Cooling effect on heated###Same as sample###Nil

venom###blank

Dose of venom was fixed at 0.4 mg to monitor the neutralizing tendencies of miscellaneous antidotes. In vitro experimentations revealed that different medicinal plants within concentration range of 625 ng to 2.5 g were found maximally (80% to 93%) active to cope with deleterious effects of ALPase. Standard antidote was found to pose maximum hindrance (93% at 2.5 g) against ALPase whilst EDTA (specific metalloenzyme inhibitor) was proved to halt 91% activity. Treatment of cobra venom with standard antidote resulted in maximum neutralization of 4.4 U/mg of phosphatase activities however 0.36 U/mg activities were outstanding. On comparison of mean absorbance of various plants extract (anti-venom) with reference standard 21% medicinal plants (Brassica nigra (L.) W. D. J. Koch Enicostema hyssopifolium (Willd.) I. Verd Pinus roxburghii Sarg Rhazya stricta Decne Sapindus mukorossi Gaertn and Stenolobium stans (L.) Seem) were found anti-venom up to 93% effective at pless than 0.5.

Moreover 46% plant extracts (Albizia lebbeck (L.) Benth Allium cepa L Allium sativum L Bauhinia variegate L Citrullus colocynthis (L.) Schrad Citrus limon (L.) Burm. F. Cuminum cyminum L. Matthiola incana (L.) W. T. Aiton Ocimum sanctum L Pistacia integerrima J. L. Stewart Psoralea corylifolia L Rubia cordifolia L and Zingiber officinale Roscoe) were observed (=91%) supportive having P less than 0.5. Remaining 33% plants extract (Althaea officinalis L Calotropis procera (Aiton) W. T. Aiton (both exudates and flowers) Cedrus deodara (Roxb. ex D. Don) G. Don Fagonia cretica L Leucas capitata Desf Momordica charantia L Nerium indicum Mill

Trichodesma indicum (L.) Sm and Terminalia arjuna (Roxb. ex DC.) Wight and Arn) were found =86% valuable with Pless than 0.05 to neutralize snake venom. Among various plant extracts Sapindus mukorossi Gaertn was found as effective as reference standard (93% 2.5 g) to neutralize ALPase activities.

Complete description about inhibition of ALPase activity by medicinal plants of Pakistan (at the most effective concentration) is summarized in Table III.

ALPases have been documented previously for their presence in different spiders and snakes venom. They are more copious in the venom of crotalids elapids and viperids therefore present study was designed to neutralize ALPases present in cobra venom via medicinal plants extracts as a step towards scientifically validating their efficiencies as an antidote (Rodrigues et al. 2006; Dhananjaya and D'Souza 2011).

ALPases produced their toxic effects in dose depended manner however elevated temperature led to permanently cessation of ALPases activities might be due to degeneration of (locus of) this enzyme. Metalloenzyme inhibitor (EDTA) has shown inhibitory effects by sequestration of metallic ions that are necessary for normal functioning of ALPases (Chakrabarty et al. 2000). Neutralizing phenomenon of antidotes can be best explained by considering snake venom as antigen and antidote (antisera) as antibody. Maximum neutralization was observed when neither antigen nor antibody in excess i.e. at equivalence point (Edwards 1985). Therefore different antidotes were exhibited maximum protection (80-93%) at lower concentration comparable to the quantity of ALPases in the cobra venom. Highest protection offered by various plant extracts at different

Table III. List of maximum protection posed by various plant extracts at the most effective concentration to mask alkaline phosphatase activity in 0.4 mg/0.1ml of snake venom.

###Most###Activity of alkaline phosphatase

###Masked

###effective###Before###After###Maximum

Sr.###enzymatic

###Scientific name of anti-dote###concen-###treatment###treatment###protection

No.###activity

###tration###(U/mg)###(U/mg)###offered (%)

###(U/mg)

###(g/ml)

1.###Albizia lebbeck (L.) Benth###1.25###4.75###0.69###4.06###85

2.###Allium cepa L###2.5###4.75###0.63###4.12###87

3.###Allium sativum L###2.5###4.75###0.62###4.13###87

4.###Althaea officinalis L.###0.625###4.75###0.72###4.03###85

5.###Bauhinia variegate L.###1.25###4.75###0.72###4.03###85

6.###Brassica nigra (L.) W. D. J. Koch###1.25###4.75###0.60###4.15###87

7.###Calotropis procera (Aiton) W. T. Aiton###0.625###4.75###0.67###4.08###86

###(exudates)

8.###Calotropis procera (Aiton) W. T. Aiton###0.625###4.75###0.89###3.86###81

###(flower)

9.###Cedrus deodara (Roxb. ex D. Don) G. Don###1.25###4.75###0.86###3.89###82

10.###Citrullus colocynthis (L.) Schrad###0.625###4.75###0.65###4.10###86

11.###Citrus limon (L.) Burm. f.###1.25###4.75###0.42###4.33###91

12.###Cuminum cyminum L###2.5###4.75###0.64###4.11###86

13.###Enicostema hyssopifolium (Willd.) I. Verd###0.625###4.75###0.40###4.35###91

14.###Fagonia cretica L###2.5###4.75###0.85###3.90###82

15.###Leucas capitata Desf###1.25###4.75###0.82###3.93###83

16.###Matthiola incana (L.) W. T. Aiton###2.5###4.75###0.63###4.12###87

17.###Momordica charantia L###0.625###4.75###0.96###3.79###80

18.###Nerium indicum Mill###0.625###4.75###0.86###3.89###82

19.###Ocimum sanctum L###1.25###4.75###0.65###4.10###86

20.###Pinus roxburghii Sarg###1.25###4.75###0.48###4.27###90

21.###Pistacia integerrima J. L. Stewart###2.5###4.75###0.45###4.30###90

22.###Psoralea corylifolia L###0.625###4.75###0.50###4.25###89

23.###Rhazya stricta Decne###2.5###4.75###0.42###4.33###91

24.###Rubia cordifolia L###0.625###4.75###0.44###4.31###91

25.###Sapindus mukorossi Gaertn###2.5###4.75###0.33###4.42###93

26.###Stenolobium stans (L.) Seem###0.625###4.75###0.64###4.11###86

27.###Terminalia arjuna (Roxb. ex DC.) Wight and###2.5###4.75###0.96###3.79###80

###Arn

28.###Trichodesma indicum (L.) Sm###0.625###4.75###0.74###4.01###84

29.###Zingiber officinale Roscoe###1.25###4.75###0.77###3.98###84

concentrations depicted their relative potencies as shown in Table III. Medicinal plant extracts at high doses (10mg/ml) were unable to show their best neutralizing effects (data is not shown). It is due to the equivalence point which was not attained due to excess of antidotes in addition to their intrinsic colors that have created interference in absorbance pattern led to erroneous results (Asad et al. 2011).

CONCLUSIONS

Among all medicinal plants Sapindus mukorossi Gaertn extract proved equally effective as reference standard to inhibit snake venom ALPases. Presence of secondary metabolites like xanthenes phenols quinonoids terpenoids and various flavonoids have previously been documented to inhibit snake venom enzymes. Secondary metabolites neutralize snake venom and pose hindrance in binding of various enzymes to their potential targets thus producing neutralizing effects. However further study is inevitable to evaluate and characterize bioactive component(s) from potential medicinal plant extracts.

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