Wound Healing Activity of Latex of Euphorbia nivulia Buch.-Ham. in Mice

 

SB Badgujar1*, RT Mahajan2 and MZ Chopda2

1Department of Biotechnology, SSBT’s, College of Engineering and Technology, Bambhori, Post Box No. 94, NH – 6, Jalgaon: 425 001, Maharashtra, India.

2Department of Zoology, Moolji Jaitha College, Jalgaon: 425 002, Maharashtra, India.

ABSTRACT:

Wound healing activity of Euphorbia nivulia Buch.-Ham. (Euphorbiaceae) was evaluated using excision wound model in albino mice by applying latex topically on the area of excised wound once a day for 23 days. Plant latex significantly enhanced the rate of wound contraction and period of epithelialization, which is comparable to Soframycin. Latex of this plant significantly reduces the bleeding and whole blood clotting time. Phytochemical analysis revealed the presence of alkaloids, cynogenic glycosides, phenolics, tannins and saponins. The latex of Euphorbia nivulia possesses natural ingredients capable of arresting wound bleeding and accelerating wound healing properties which suggests healing potential to cure wound.

 

Key words: Bleeding time, Euphorbia nivulia, Excision wound, Wound healing.  

 

 

INTRODUCTION:

Wounds are visible results of individual cell death or damage. It is a disruption of tissue integrity that is typically associated with a loss of substance. Wound healing is the process of repair that follows injury to the skin and other soft tissues. It is fundamentally a connective tissue response. Initial stages of wound healing and tissue repair involve a series of biochemical and cellular reactions, beginning with inflammation and followed by the repair and remodeling of the injured tissue. A review on potential wound healers from 81 plant origin has been recorded along with wound healing activity of some herbal formulations.1

 

A comprehensive and an extensive appraisal on 283 wound healing plants of Indian origin was compiled and out of these, about ten members of Euphorbiaceae  family was recorded for wound healing activity.2 Euphorbia nivulia Buch.-Ham., a member of Euphorbiaceae family is a tall shrub with cylindrical stem and branches covered with thorn and commonly known as “Patrasnuhi", able to stop bleeding from wounds.3  Leaves 8.5 – 20 X 3.5 – 6.5 cm, crowded at the end of branches, obovate-oblong or spathulate, glabrous. Cymes – 3 – flowered, born from above the leaf scars on the tubercles. Capsules glabrous, trigonous, seeds globose, dorsally lined, smooth. New leaves appear in rainy season. Generally it is planted as a hedge plant around the agricultural boundaries. It is claimed in folk medicine as latex applied on fresh cut wounds for very good healing.4

 

No reports appeared in literature regarding biological activities in general wound healing activity except antimicrobial5 and cytotoxic activity6 of this plant. Therefore, present investigation was undertaken to evaluate wound healing effect and to search a phytoingredient responsible for such effect of E. nivulia latex.

 

 

 

MATERIALS AND METHODS:

Plant material:

Latex of Euphorbia nivulia Buch.-Ham. (Specimen number: LAT 87) was collected early in the morning (during June 2007 to February 2008) by nipping leaves near the stem or by incision of trunk and branches of plant and allowing the milk to drain in clean glass tube separately, brought to the laboratory and kept in refrigerator (till the experiment starts) and the corresponding voucher specimen was deposited in the Department of Zoology, Moolji Jaitha College, Jalgaon 425 001, Maharashtra, India.

 

Animals and drugs:

Swiss albino mice of either sex (50-100g) were used for the study. The animals were put into separate cages at 28 ± 0.50C and fed with standard diet and water ad libitum. The animals were maintained under standard laboratory conditions in animal house of Moolji Jaitha College, Jalgaon (M.S.), approved by committee for the purpose of control and supervision on experiments on animals (CPCSEA). The experimental protocol was approved by Institutional Animal Ethics Committee. Soframycin skin cream (Aventis Pharma Ltd., Ponda, Goa, India) serve as a positive control.

 

Wound healing activity (Excision wound model):

Albino mice were divided into three groups of six animals in each group. Circular wounds of approximately 300 to 350 mm2 in diameter were inflicted on the shaved skin under mild ether anesthesia.7 Group II and III were treated with plant latex and 1% w/w framycetine sulphate I.P (Soframycin) respectively. Group I was untreated consider as control.

 

Measurement of wound area:

The progressive changes in wound area were recorded in mm2 by tracing the wound boundaries around it on a transparent paper on every day. Wound contraction was expressed as percentage reduction of original wound size .7

 

Pharmacological tests:

Coagulation time of whole blood:

Twelve Khan tubes were arranged in a water bath at 370C, into six of these tubes (test), 0.1ml each of the latex was added and nothing was added to the remaining six tubes (control). 1ml of blood was collected separately from mice by clean venepuncture and 1ml was added into each of the tubes, immediately the blood started flowing into the syringe, the tubes were observed for clot and the clotting time taken using a stop watch. The average of the clotting time of the six tubes with latex (test) and the six tubes without latex (control) were taken as the clotting time respectively .8

 

Bleeding/clotting time test:

The effect of the latex on bleeding from fresh experimentally induced wounds was evaluated using the bleeding/clotting time test in mice.9 After sterilizing the skin with 70% alcohol; a puncture was made on the tail with a sterile sharp blade. Immediately, a drop of the latex (1000 μg/ml) was placed on the cut portion and at the same time a stopwatch was switched on. Sterilized filter paper was used to absorb blood coming out and time taken for ceasing bleeding was recorded, the average was taken as bleeding time (test). The procedure was repeated on the second group of mice but here, after puncturing the tail, a drop of latex was not applied, serves it as a control group of animal.

Preliminary phytochemical screening:

Plant latex was analyzed for their phytochemical composition by qualitative method. Dragendorff’s reagent and Mayer’s reagent were used to know the presence of alkaloids and cynogenic glycosides using cold concentrated sulphuric acid test. Latex was tested for phenolics using Folin Ciocalteu reagent. Flavonoids were detected by appearance of effervescences with pink color by dissolving latex in 10 % HCL and Zinc powder. Latex was also tested for terpenoids and saponins. Tannins were detected using gelatin salt block test. 10, 11

Statistical analysis:

The mean and standard deviation and the level of significance for the difference between means were determined by Tukey's Multiple Comparison Test12 computed by GraphPad Prism 4.

 

RESULTS AND DISCUSSION:

Table 1 summarizes the results obtained on phytochemical investigation of latex. Plant latex gave positive test for alkaloids, cynogenic glycosides, phenolics, tannins and saponins. Figure 1 illustrates significant reduction in bleeding/clotting time in mice. Also, the latex significantly decreases the coagulation time of whole blood in mice (P < 0.001).

 

Table 1. Phytochemical investigation of E. nivulia latex.

 

Sr. No

Qualitative test

Result

1

Alkaloids

+

2

Cynogenic Glycosides

+

3

Phenolics

+

4

Flavonoids

-

5

Terpenoids

-

6

Tannins

+

7

Saponins

+

Phytochemical tests:   - negative and + positive

 

A significantly improved wound healing activity has been observed in mice treated by latex as compared to that of reference standard (Soframycin) and control groups of animal (Table 2). The study reveals that in all three groups of animal, wound area was reduced progressively. However, on 16th post wounding day Group I animals have  92.02 % wound contraction (which may be due to self immunity of the animals) whereas in Group II and III animals exhibit 94.50 and 93.29% effect by Soframycin and plant latex respectively. A latex treated group of animals showed significant reduction in wound contraction area (P < 0.001). A similar type of wound healing activity was reported in the latex of Euphorbiaceae member i.e. Jatropha curcas13 and Euphorbla neriifolia14 and a member of Asclepiadaceae family i.e. Calotropis procera.15 Our results are agreed with the findings of earlier researchers. Present result fully justifies the folkloric use of E. nivulia for healing wound.3, 4

Evaluation of the potentials of E. nivulia in wound management showed that the plant latex has


Table 2. Effect of E. nivulia latex on excision wound parameter in mice

 

Post wounding days

Wound Area and percentage of wound contraction is in parentheses

Group I: Control

Group II: Soframycin

Group III: Latex

0

344.67 ± 1.75

316.22± 3.12

335.83 ± 1.33

4

240.53 ± 1.38 (30.22)

276.09± 3.01 (14.2)

222.75 ± 1.97 (33.7)*

8

162.32 ± 2.25 (52.9)

187.03± 1.38 (41.90)

170.89 ± 1.33 (49.7) *

12

47.67 ± 1.03 (86.17)

53.17± 2.04 (83.45)

38.32 ± 1.63 (88.6) *

16

27.50 ± 1.87 (92.02)

17.77± 1.63 (94.5)

22.67 ± 1.21 (93.29) *

20

8.33 ± 1.03 (97.58)

5.54± 1.87 (98.29)

4.08 ± 1.02 (98.78) *

PE

23.68 ± 1.03

24.17 ± 1.17

22.67 ± 1.37

Values are expressed as mean ± S.D., n=6 animals in each group, PE: Period of epithelialization (days),

* P < 0.001 as compared to control

 

Figure 1. Effect of latex on bleeding and whole blood clotting time Values are expressed as mean ± S.D., n=6 animals in each group, * P < 0.001 as compared to control,

 

haemostatic and wound healing properties. The latex arrested bleeding from fresh wounds by reducing bleeding/clotting and whole blood coagulation time which are important indices of haemostatic activity. Our results are good in agreement with the earlier observations of haemostatic activity of stem latex of another member of Euphorbiaceae family i.e. Jatropha gossypifolia.16 The reduction in coagulation time of whole blood by the latex indicates that the latex may also interfere with the blood coagulation pathways. Thus, this shrub is a promising haemostatic agent and wound healing promoter and it is worth to evaluate it further in detail.

 

ACKNOWLEDGEMENTS:

We are grateful to the Principal of Moolji Jaitha College, Jalgaon, Maharashtra for providing necessary laboratory facilities to carry out the present research work.

 

REFERENCES:

1.       Habbu PV, Joshi H and Patil BS. Potential wound healers from plant origin. Pharmacognosy Review. 2007; 1(2): 271-282.

2.       Chopda MZ, Mahajan RT. Wound healing plants of Jalgaon District, Maharashatra state, India. Ethanobotanical leaflets 2009; 13:1-32.

3.       Kirtikar KR and Basu BD. Indian Medicinal Plants. Vol. I-IV, International Book Distributors, Dehradun. 1995.

4.       Mahajan RT and Badgujar SB. Ethnomedicinal values of Laticiferous plants used by tribal people of North Maharashtra, India. Research Link. 2008; 55, VII (8): 20-23.

5.       Annapurna J, Chowdary IP, Lalitha G, Ramakrishna SV and Iyengar DS. Antimicrobial Activity of Euphorbia nivulia Leaf Extract. Pharmaceutical Biology. 2004; 42 (2): 91 – 93.

6.       Veluri R, Reddy VLN, Reddy AV, Kodela R, Tadikamalla PR, Tejomoorty SR, Kondapi AK, Diwan PV and Yenamandra V. Three New Ingol Diterpenes from Euphorbia nivulia: Evaluation of Cytotoxic Activity, Chem. Pharm. Bull. 2003; 51(4): 431-434.

7.       Sadaf F, Rubeena S, Muhammad A, Syed IA and Navaid-ul-Zafar. Healing potential of cream containing extract of Sphaeranthus indicus on dermal wounds in Guinea pigs. Journal of Ethnopharmacology. 2006; 107: 161–163.

8.       Dacie JV and Lewis SM. Practical Haematology, 4th edition. J and H Churchill, London. 1970.

9.       Okoli CO, Akah PA and Okoli AS. Potentials of use of Aspilia Africana (Compositae) in wound care: an experimental evaluation. BMC Complimentary and Alternative Medicine. 2007; 7: 24.

10.     Wagner H, Bladt S and Zgainski EM. Plant Drug Analysis, 13th edition, Springer-Verlag, 1984.

11.     Harborne JB. Phytochemical methods, A guide to modern techniques of plant analysis. Chapman and Hill, London. 1984.

12.     Daniel WW. Biostatistics, A foundation for the analysis in the health sciences. 7th Edn., John Wiley and sons (Asia) Pvt Ltd, Singapore. 2004.

13.     Nath LK and Dutta SK. Wound healing response of the proteolytic enzyme curcain. Indian Journal of Pharmacology. 1992; 24: 114-115.

14.     Rasik AM, Shukla A, Patnaik GK, Dhawan BN, Kulshrestha DK and Srivastava S. Wound Healing Activity Of Latex Of Euphorbla Neriifolia Llnn. Indian Journal of Pharmacology. 1996; 28: 107-109.

15.     Rasik AM, Ranghubir R, Gupta A and Jain HK. Wound healing potential of Calotropis procera on dermal wounds in guinea pigs. Journal of Ethnopharmacology. 1999; 68: 261-266.

16.     Oduela T, Popoola1 GB, Avwioro OG, Oduola TA, Ademosun1 AA and Lawal MO. Use of Jatropha gossypifolia stem latex as a haemostatic agent: how safe is it? Journal of Medicinal Plants Research. 2007; 1 (1): 14 – 17.

 

Received on 02.04.2009

Accepted on 10.08.2009     

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Research J. Pharmacology and Pharmacodynamics  2009; 1(2): 90-92