The leaves were reported to contain alkaloid mudarine, glycosides calotropin,uscharin and calotoxin, besides yellow bitter acid and resin. The milky latex contains a powerful bacteriolytic enzyme, a very toxic glycoside calactin,calotropin D-I, calotropin D-II,calotropin F-I, calotropin F-II and a non toxic powerful proteolytic enzyme calotopin. The whole plant contains α- amyrin, β-amyrin, teraxasterol,β- sitosterol and a wax^(10,11).

Literature survey revealed that the latex of Calotropis procera¹², Tabernaemontana coronaria¹³ and Calotropis gigantea¹⁴ exhibited significant local anesthetic activity in frog (nerve block anesthesia and muscle twitch method) and in Guinea pig. Moreover, their local anaesthetic activity was potentiated by epinephrine(Vasoconstrictor) and pH(7.2) ^(15,16). No researcher has yet reported potentiation of local anaesthetic activity exhibited by Calotropis gigantea latex. Therefore, it is worth conducting an investigation on local anesthetic activity of Calotropis gigantea latex by infiltration method and to study the effect of vasoconstrictor and pH on local anesthetic activity.

Local anesthetics are the agents which prevent or relieve pain by interrupting nerve conduction. They effectively block and relieve pain, but with a relatively short duration of action, limiting their analgesic effectiveness. Therefore, a long acting local anesthetic would improve the management of pain, but no such agent is yet available for clinical use¹⁷. As early as 1903, Braun discovered that the addition of epinephrine to solutions of local anesthetics increased and prolonged their action. Vasoconstrictors reduce the rate of absorption of a drug by allowing the metabolic rate of the local anesthetic to keep pace with the rate at which it is absorbed into blood stream. This also reduces systemic toxicity. However, prolonged use of a vasoconstrictor on major arteries may cause irreversible tissue damage and can lead to gangrene¹⁸. Therefore, the present work was undertaken to evaluate the potentiation of the local anesthetic activity of milky latex, obtained from Calotropis gigantea, with epinephrine and pH in Guinea pig.

MATERIALS AND METHODS:

Chemicals and equipments: Milky latex of Calotropis gigantea, centrifuge (REMI), Distilled water, Xylocaine (0.25w/v), Epinephrine (Vasocon 1mg/ml), Sodium hydroxide solution (0.2M), Normal saline solution (0.65%Nacl), Hair depleting agent and sharp pins.

Animals: Guinea pig (Male).

Method: Infiltration anaesthesia method.

Collection of milky latex:

The milky latex was collected usually between 8 am and 10 am by cutting the tips of young branches of C. gigantea. Then 1 ml of latex was centrifuged at 5,000 r. p. m after being diluted 10 times its volume with distilled water. The gummy pellet was discarded and the supernatant liquid was used for evaluation of local anesthetic activity.

Dilution of Epinephrine:

Vasocon injection (Epinephrine 1mg /1ml) was purchased from the local market and diluted with distilled water to get a solution containing 10µg/1ml. 0.5 ml of diluted solution was used in the present study.

Table No: 1 Infiltration Anesthesia method:

Group

Time in minutes

Squeak Response

Before administration of the drug

Standard

(Xylocaine alone)

- +

Test

(Latex 1:10 alone)

- +

Standard-I

(Xylocaine + Epinephrine)

Test-I

(Latex1:10+Epinephrine)

- +

Standard-II

(Xylocaine+0.2M Sodium hydroxide)

Test-II

(Latex1:10+0.2M sodium hydroxide)

- +

Preparation of 0.2M Sodium hydroxide solution¹⁹:

0.2 M of sodium hydroxide solution was prepared by dissolving 8.0 gm of sodium hydroxide in 1000 ml of distilled water. 34.7 ml of 0.2M sodium hydroxide solution was used in the present study.

Preparation of standard solution and test solution:

Xylocaine solution (0.25w/v) was purchased from the local market. 0.5 ml of this solution was mixed with 0.5 ml of diluted epinephrine solution (Standard solution-I). Similarly, 0.5 ml of latex (1:10) was mixed with 0.5 ml of diluted epinephrine solution (Test solution-I). Xylocaine solution was mixed with 34.7 ml of 0.2M sodium hydroxide solution (Standard-II) and 50 ml of latex (1:10) was mixed with 34.7 ml of 0.2M sodium hydroxide solution(Test-II).

Infiltration Anaesthesia method:

Six healthy adult Guinea pigs (CPSCEA regd no 1016-a-06-CPSCEA-0112009) were taken and fur on their back was removed by using depleting agents. The depleted area was washed with normal saline and allowed to dry. Then 1 ml each of xylocaine solution, latex (1:10) Standard-I, II and Test-I, II were injected intradermally to six different guinea pigs separately. Squeak or twitch response of animal upon touching the injection site with sharp pin was marked as (+) and (-) if the animal does not show any response to pin prick²⁰.

RESULTS AND DISCUSSION:

In the Infiltration method, before administration of the standard and test there was a sharp response for pin prick. After administration of the standard (Xylocaine alone) there was response for pin prick up to 1 minute. Then the response reproduced at 45^th minute. Test(latex1:10 alone) , Standard –I(Xylocaine+ Epinephrine),Test-I (Latex1:10 + Epinephrine), Standard-II (Xylocaine + 0.2M Sodium hydroxide), Test-II(Latex1:10+0.2M Sodium hydroxide) reproduced response at 32^nd minute,65^th minute,50^th minute,60^th minute and 38^th minute respectively. The results are shown in Table No:1.

The duration of infiltration anesthesia can be prolonged by the addition of epinephrine (5µg/ml) to the injection solution. Epinephrine also decreases peak concentrations of local anesthetics in blood.

Local anesthetics tend to be only slightly soluble as unprotonated amines. Therefore, they are generally marketed as water soluble salts, usually hydrochlorides. Many local anesthetics are weaker bases with typical pK_a values ranging from 8 to 9, but their hydrochloride salts are mildly acidic. This property increases the stability of the local anesthetic esters and any accompanying vasoconstrictor substance.

Although the unprotonated species of the local anesthetics is necessary for diffusion across cellular membranes, it is the cationic species that interacts preferentially with sodium channels. This conclusion has been supported by the results of experiments on anesthetized mammalian non myelinated fibers. In these experiments, conduction could be blocked or unblocked merely by adjusting the pH of the bathing medium to7.2 or 9.6, respectively, without altering the amount of anesthetic present.

CONCLUSION:

The duration of the effect produced by combination of Calotropis gigantea latex and epinephrine was longer than the combination of latex and pH as well as by latex alone.From the above observation, we conclude that, epinephrine and pH 7.2 potentiate the local anesthetic effect of Calotropis gigantea latex in Guinea pig when co-injected intradermally. The present results may probably support the development of a long acting local anesthetic.

ACKNOWLEDGEMENT:

The authors are thankful to Smt. P. Sulochana, Chairperson, Dr. D. Ranganayakulu, Principal, SPSP, Tiruchanoor, Tirupati for providing us facilities to carry out this research work.

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Received on 18.02.2010

Accepted on 14.03.2010

Research J. Pharmacology and Pharmacodynamics 2(2): March –April 2010: 198-200