Wound healing activity of Cynodon dactylon (L.) Pers. in albino wistar rats

Wound healing activity of Cynodon dactylon (L.) Pers. in albino wistar rats

By Anand Kumar*1 and Pranita Kashyap2

1Head, Department of Pharmacy, University Teaching Department, Sarguja University, Ambikapur – 497001, (C.G.), India.

2Principal, Shri Rawatpura Sarkar Institute of Pharmacy, Behind Holiday Resort, Kumhari – 490042, (C.G.), India.

1.Introduction

Cynodon dactylon, also known as Vilfa stellata, Bermuda grass, Dhoob, dūrvā grass, dubo, dog’s tooth grass, Bahama grass, devil’s grass, couch grass, Indian doab, arugampul, grama, wiregrass and scutch grass, is a grass that originated in Africa. Although it is not native to Bermuda, it is an abundant invasive species there. It is presumed to have arrived in North America from Bermuda, resulting in its common name. In Bermuda it has been known as crab grass (also a name for Digitaria sanguinalis).

More than 80% of the world’s population depends upon traditional medicines for various skin diseases 1. Recently, the traditional use of plants for wound healing has received attention by the scientific community2. Approximately one-third  of all  traditional  medicines  in  use are  for the treatment  of wounds  and  skin  disorders, compared to only 1-3 % of modern drugs3.

Wound healing activity of Cynodon dactylon (L.) Pers. in albino wistar rats
Cynodon dactylon (bermuda grass)

The processes of wound healing involve a variety of biological responses, such as an acute inflammation, cellular proliferation and a contraction of the collagen lattice formed 4-5. Wounds caused by environmental insults such as mechanical and chemical injuries, may extend from the epidermis deep into the muscles depending on the severity of damage. Wound thus caused can be healed by a spontaneous process in the organism through a cascade of events, which starts by switching on various chemical signals in the body; this facilitates the restoration of anatomical continuity and function.

While partial thickness wound heals by mere epithelialization, the healing of full thickness wound which extends through the entire dermis involves more complex well-regulated biological events resulting in the formation of hypertrophic scars 6-7. The healing process begins with the clotting of blood and is completed with re-modelling of the cellular layers of the skin. However, the wound healing process may be prevented by the presence of ROS (Reactive oxygen species) or microbial infection, since the type of cells to be first recruited to the site of injury is the neutrophil which is produced in response to cutaneous injury8 and which has a role in antimicrobial defense and may cause cellular damage  by  peroxidation  of  membrane  lipids 9-10.

Appropriate  method  for  healing  of  wounds  is  essential  for  the restoration of damaged tissue anatomical continuity and disturbed functional status of the skin 11. Research on wound healing agents is one of the developing areas in modern biomedical sciences and many traditional practitioners across the world particularly in countries like India and China have valuable information of many plants for treating wounds and burns12.

For this reason traditional plant based remedies are back and find increasing application as source of direct therapeutic agents. Considerable scientific data have been generated worldwide in this direction and there are spurt of study on natural product study. Based on this rationale, the study has been compiled and provides data on potential medicinal properties, action and uses of plants in order to contribute material leading to the discovery of new drugs of natural origin13. Among numerous species of plants growing wild in India, doob ghas, or Durva or taxonomically the Cynodon dactylon (L.) Pers. family Poaceae occupies its unique place and key position in ethnomedicinal practices and traditional medical (Ayurvedic, Unani, Nepalese, and Chinese) knowledge systems. The herbal preparations of this grass are being based on folklore and traditional wisdom14.

According to Ayurveda, India’s traditional pharmacopoeia, Cynodon plant is pungent, bitter, fragrant, heating, appetizer, vulnerary, anthelmintic, antipyretic, alexiteric. It destroys foulness of breath, useful in leucoderma, bronchitis, piles, asthma, tumors, and enlargement of the spleen. In Homoeopathic systems of medicine, it is used to treat all types of bleeding and skin troubles15.

It contains essential oil triticin 12.4%. The other chemical constituents are glycosides, saponins, tannins, flavonoids, & carbohydrates16. It also contains agropyrene, arunodin, furfural, furfural alcohol,β-ionine, 2-(4’hydroxy phenyl) propionic acid, 2-(3’methoxy-4’hydroxy-phenyl) propionic acid, 3-methoxy-4-hydroxy benzoic acid, phytol, β- sitosterol-D-glucoside,   stigmasterol   acetate,   phagostimulant   phytone   (6,10-14-trimethyl   pentadecane-2-one) 17. Cuticular wax contains triacontane, docosanol. tetracosanol, hexacosanol, octacosanol, eicosanic acid & docosanoic acid (spencer & chapmann)18.

Materials and Methods

Collection of plant material

The whole plant of Cynodon dactylon (L.) Pers. were collected from the Botanical garden of Shri Rawatpura Sarkar Institute of Pharmacy, Kumhari, Dist.- Durg, Chhattisgarh, India in September 2010, cleaned and dried at room temperature in shade and away from direct sunlight. The plant authenticated by Dr. Mrs. Ranjana Shrivastava, HOD, Dept. of Botany, Govt. V.Y.T. P.G. Autonomous College, Durg (C.G.), by comparing morphological features and a sample voucher of specimen of plant was deposited for future reference sample voucher specimen no. is GPG/SRIP/PH/10/21.

Preparation of extract

The whole plant of Cynodon dactylon (L.) Pers. were collected and dried in the shade and then pulverized in a grinder. The powdered drug was utilized for extraction. Material was passed through 120 meshes to remove fine powders and coarse powder was used for extraction. A method was used for extraction of powdered material 18. Extraction was done by using Hydroalcoholic solvent (70:30% ratios). The yield of the extract was found to be 11.5%w/w.

Preliminary phytochemical screening

Phytochemical screening was carried out according to standard methods. Extracts shows the presence of carbohydrates,  glycosides,  flavonoids,  saponins,  alkaloids,  phenolic compounds,  tannins,  fixed  oil,  &  mucilage19. Inorganic constituents present in selected plant C. dactylon are calcium, magnesium, chloride, iron & sulphur20. Thin layer  chromatography of various extracts  of  C.  dactylon  has  confirmed  the presence of Secondary metabolites like: alkaloids, glycosides, saponins, tannins & flavonoids as the colour of spots & their Rf value of standard21 matches to that of sample (Khandelwal 2005)22.

Experimental design

Healthy male Albino Wistar rats (180-200 g) of 2-3 months of age were used, and the animal experiments were performed in accordance with legislation on welfare (CPCSEA). The animals were acclimatized to standard laboratory conditions for a week prior to the initiation of the experiment. Environmental conditions of temperature (22 ± 3OC), humidity (60 ± 5%) and a 12-h light/dark cycle. The animals were fed with a standard laboratory diet (Godrej Agro Food Industries, Bangalore, India) and were provided with clean drinking water ad libitum. Twelve hours before the start of the experiment, rats were deprived of food, but given free access to water. Excisions of wounds were made as described by Morton and Malone23.

Excised wound healing activity

Wound induction and evaluation extracts for properties wound healing before the beginning of the wound healing experiments. Animals were anesthetized with 1.5 mg/kg, i.p. of Ketamin and Xylazine and placed in operation table in its natural position the dorsal skin of the Wistar rats were cleaned by using a solution of hair remover. A full thickness of the excision wound (circular area about 150 mm2 and 2 mm depth) was created along the markings using toothed forceps, a surgical blade and pointed scissors24 was made on depilated ethanol-sterilized dorsal thoracic region of male Albino Wistar rats weighing 180-200g.

Excision Wound Model

The animals were divided randomly into nine groups of six rats each. Group 1 was left untreated served as (Blank);  group  2  was  treated  with  Cipladine  (Povidone-iodine)  ointment  and  served  as  a  reference  (Allopathic Standard); groups 3 was treated with Durva Ghrita ointment served as (Ayurvedic Standard); group 4 was treated with the Ghrita Base served as (Control); group 5 was treated with Hydrophilic Base served also as (Control); group 6 was treated with FM- 1: 7.5%, group 7 was treated with FM- 2: 10%; group 8 was treated with Oleaginous Base also served as (Control); group 9 was treated with FM-3: 10%, all these group {6,7, & 9} were served as (Test), and treated topically with the respective ointments & ointment bases, till the epithelialization was complete. The percentage of wound closure and period of epithelialization were recorded

During the wound healing period, the wound contraction was studied by tracing the raw wound area manually subsequently on day 1, 4, 8, 12, 16, 18 and 21 on graph paper. Scar residue, area and time for complete epithelialization were also measured and photographed. The wound area at days 7th, 14th and 21st the experiment was terminated and the wound area was removed from the surviving animals for histological examination25. The excision skin biopsies were fixed in 4% formaldehyde solution 48h during the experimentation period. The percentage of wound closure was calculated as follows using the initial and final area drawn on glass slides during the experiments 26: % of wound closure = (wound area on day 0 wound area on day n)/wound area on day 0 × 100 where n is a number of days (7 th, 14th, and 21stday).

Statistical Analysis

All the data were expressed as Mean ± S.D. Tukey-Kramer multiple comparison test and one way analyses of variance (ANOVA) were performed. A value of P<0.05 was considered significant.

Results

Many studies indicate that plant products are potential agents for wound healing and largely preferred because of the absence of unwanted side effects and their effectiveness. In this report, we show for the first time that the topical application of hydroalcholic extracts of C. dactylon (L.) Pers. promoted wound healing activity in excision model in rats.

 

Treatment

 

Inflammation cells

 

Collagen fibres

Organization of the collagen

 

Necrosis

 

Fibrins

 

Day

7

Day

14

Day

21

Day

7

Day

14

Day

21

Day

7

Day

14

Day

21

Day

7

Day

14

Day

21

Day

7

Day

14

Day

21

Blank

+++

++

+

+

+

+++

++

+

+++

++

+

Povidone-Iodine

++

+

+

++

+++

+

++

++

+

++

+

Durva Ghrita

++

+

+

++

+++

+

++

++

+

++

+

Ghrita Base

+++

++

+

+

+

++

+

+

+++

++

++

+

+

Hydrophilic Base

++

+

+

+

+

++

+

+

+++

++

++

+

+

FM-1: 7.5%

++

+

+

++

+++

+

+

++

++

+

++

+

FM-2: 10%

++

+

+

++

+++

+

+

++

++

+

++

+

Oleaginous Base

++

+

+

+

++

+

++

+++

++

++

+

+

FM-3: 10%

+

+

++

+++

+++

++

+++

+++

+

+

Effect of the treatments on the evolution of wounds in rats after 7, 14 and 21 days of topical application.

Note: + : Slight, ++ : Moderate, +++ : Extensive,  – : Absent.

Wound healing is a process by which damaged tissue is restored as closely as possible to its normal state and wound contraction is the process of shrinkage of the area of the wound. It is mainly dependent upon the type and extent of damage, the general state of health and the ability of the tissue to repair. In this study the HAE of C. dactylon (L.) Pers. topical application of formulation-III which contains 10% HAE formulated in oleaginous base improved wound contraction and closure, and the effects were distinctly visible starting from 7th post-wounding day. Also formulation-II which contains 10% HAE formulated in hydrophilic base also significantly improved the rate wound contraction and significantly shortened the period of epithelization, and collagen turnover. Collagen, the major component which strengthens and supports extracellular tissue, is composed of the amino acid, hydroxyproline, which has been used as a biochemical marker for tissue collagen26  which was more or less similar to the result of Cipladine (Povidone-iodine) treated group and it was less in control animals.

The preliminary phytochemical analysis of the flower extract of C. dactylon (L.) Pers. named Durva, doob showed the presence of polyphenol compounds such as tannins, flavonoids, & carbohydrates17. It also contains agropyrene,  arunodin,  furfural,  furfural  alcohol,β-ionine,  2-(4’hydroxy  phenyl)  propionic  acid,  2-(3’methoxy- 4’hydroxy-phenyl) propionic acid, 3-methoxy-4-hydroxy benzoic acid, phytol, β-sitosterol-D-glucoside, stigmasterol acetate, phagostimulant phytone (6,10-14-trimethyl pentadecane-2-one). Polyphenol compound may be responsible for antimicrobial activity. It may be either due to the individual or additive effect of the phytoconstituents that hasten the process of wound healing. The exact component of the extract that is responsible for this effect, however, was not investigated. Further phytochemical studies are needed to isolate the active compound(s) responsible for these pharmacological activities.

Table: Comparison of means of the wound areas at different days of evolution by Tukey-Kramer test. The means with different letters are significantly different at 0.05 probability level according to Tukey-Kramer Multiple Range test

Conclusion

The present study demonstrated that hydroalcholic extract of C. dactylon (L.) Pers. was capable of promoting wound healing activity. Enhanced wound contraction and histological observations suggest that C. dactylon (L.) Pers. has potential in the management of wound healing and suggests further study.

Acknowledgement

Author sincerely thanks to Dr. Mrs. Pranita Kashyap (Phd, MBA) for her professional expertise guidance, and also to Mr. H. A. Sawarkar & Mr. V. S. Deshmukh for their prompt suggestions and technical support.

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Wound healing activity of Cynodon dactylon (L.) Pers. in albino wistar rats

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