INTRODUCTION:
Wound is defined as a loss or breaking of cellular and anatomic or functional continuity of living tissues [1]. Proper healing of wounds is essential for the restoration of disrupted anatomical continuity and disturbed functional status of the skin. Healing of wound is a biological process that is initiated by trauma and often terminated by scar formation [2, 3]. Wound healing is a process of reconstruction of injured skin, coordinated by interaction of various epithelial and mesenchymal cells with cytokines, chemokines and growth factors [4].
According to a WHO report, about 70%–80% of the world’s populations rely on nonconventional medicine mainly from herbal sources in their primary health care and in spite of phenomenal development of the synthetic drug industry and antibiotics, medicinal plants still constitute an important part of pharmacopoeias in both the developed and developing countries [5].
Many medicinal plants are claimed to be useful for wound healing in the traditional system of medicine. These plant remedies (both single plant and multi-herbal reparations) are used since ancient times even if the mechanism of action and efficacy of very few of them have been evaluated scientifically [6]. Current estimates indicate that nearly six million people suffer from chronic wound [7] causing great physiological and mental trauma. Wound healing is the interaction of a complex cascade of cellular and biochemical actions leading to the restoration of structural and functional integrity with regain of strength of injured tissue. It involves continuous cell–cell interaction and cell–matrix interactions that allow the process to proceed in different overlapping phases and processes including inflammation, wound contraction, re-epithelialization, tissue remodeling and formation of granulation tissue with angiogenesis [8].
Amaranthus spinosus (Amaranthaceae) have been used as medicinal plants in many countries. Modern pharmacological studies reported several biological activities of A. spinosussuch as analgesic and anti-inflammatory, anti-malarial, anti-fertility, antidepressant, anti-diabetic and antioxidant [9]. A. spinosus is very rich in proteins (12.6 to 18.0%), fat (5 to 8%), saccharides (60 to 65%), and crude fiber (3 to 5%). Concerning the chemical characterization of this plant, phenolic acids such as ferulic, vanillic, syringic and sinapic acids have been isolated from stem bark [10]. By considering the possible antagonistic effects of plant-derivedproducts on human health, this study aimed to evaluate the possible wound healing, antimicrobial and antioxidant effects of A. spinosusin rats.
Polyherbal formulations are better than the pure chemical wound healing formulations because the crude poly herbal formulation have various phyto-constituents which have antimicrobial, anti-inflammatory properties and antioxidant potential. Hence phytoherbal formulation used in the treatment of wound owes assured safety and high efficacy. Hence scientific investigation is needed to explore the pharmacological activities of medicinal plants and to elucidate the claims made about them in folklore medicines.Polyherbal formulation in terms of mobilization of fibroblasts and keratinocytes to the site of injury and their angiogenic potential as well as its in vivo efficacy.
MATERIAL AND METHODS:
Drugs and reagents:
Tween 80 (Lobachem, India), Soframycin (MicroLab, India), Diclofenac (GlaxoSmithKline) carrageenan (Sigma), were used in the study.
Acquisition and authentication of plant:
The whole plant of the Amaranthus spinonouswas collected from the Andhra Pradesh and authenticated by K. Madhava Chetty, Venkateswara University, Tirupati, India and the Voucher number is 1671.
Preparation of ethanolic extract:
Selected and Collected the Fresh whole plant Amaranthus spinosuswere properly washed and cleaned from normal tap water and after distilled water for removing extraneous materials and shade-dried, and pulverized to a coarse powder and passed through a 40 mesh sieve. About 500g of powdered material was soaked 65% ethanol for 72 h in beaker and mixture was stirred every 15 h using a sterile glass rod. Filtrate was obtained three times after passing through Whatman filter paper No. 1 and the solvent was removed by rotary evaporator at (Buchi R-200 USA) 40℃ and stored at 4℃ for further use.
Management of Experimental Animals:
Sprague Dawley rats of either sex were taken from the animal house of the National Laboratory Animal Centre, Lucknow, India. They were kept under controlled conditions of temperature 27±2℃ and relative humidity 44-56%, light/dark cycles of 12 h respectively for one week before and during the experiments. Animals were provided with a standard rodent pellet diet (Amrut, India) and the food was withdrawn 18-24h before the experiment with supply of water ad libitum. All experiments were implemented in the morning accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals approved by the Institutional Committee for Ethical use of Animals and Review Board (Approval No. 1732/GO/RE/CPCSEA).
Acute dermal toxicity tests:
Selection of dosage:
In acute dermal toxicity studies tested 65% ethonolic whole plant extract of Amaranthus spinosus. Selection of dosage was guided by procedures stipulated in Organisation for Economic Co-operation and Development draft guideline no 434. Sighting study was conducted to all extracts tested. The limit dose of 2000 mg/kg was selected for the main study based on the fact that the 1000mg/kg as a start dose in the sighting study could not show any sign of toxicity.
Animals and preparation:
The test in the albino rats were performed according to the OECD draft guideline number 434 [11]. A total of twelve animals (all females) were divided into two groups of six animals each for a treatment and a control groups. Approximately 24hr before the study, fur was removed from the dorsal area of the trunk of the animals by clipping to obtain at least 10% of the body surface area while taking care to avoid abrading the skin. Depending on the type of extract; applied uniformly over a shaved area using a small spatula. The test substances were held in contact with the skin with a porous gauze dressing and non-irritating tape throughout a 24- hour exposure period. At the end of the exposure period, residual test substance was removed using water or sunflower oil or both. Cage side observation was made daily, but weight measurement was taken weekly for 15 days. Observation included evaluation of skin and fur, eyes, respiratory effects, salivation, diarrhoea, urination, and central nervous system effects (tremors and convulsion, gait and posture, reactivity to handling or sensory stimuli and altered strength). By the 15th day rats were humanely sacrificed and organs were carefully taken out for gross and histopathological examinations.
Incision wound healing activity:
Surgical procedure:
The incision wound model used according to Udupaet al., 1995; Govindarajanet al., 2004; Perez Gutierrez et al., 2006 [12-15]. The animals were divided into four groups of six rats each and kept in separate cages. Rats were anesthetized and two paravertebral longincisions made through the skin and cutaneous muscles at a distance of about 1.5 cm from the midline on each side of their depilated back. Aseptic techniques were not applied and no local or systemic antimicrobial was used throughout the experiment. Each of the four groups of animals was treated in the same manner as for theexcision wound model. The parted skin was kept together by stitching with a black silk surgical thread (No.000) and curved needle (No.11) and continuous threads on both wound edges were tightened for good wound closure. Group I animals (control) were treated topically with simple ointment base, group II and III animals received ethanolic extract of Amaranthus spinosus twice a day for 9 days. The tensile strength of a wound represented the degree of wound healing, so wound healing agents usually provide a gain in tensile strength. The tensile strength was calculated from the following equation: wound healing agents usually provide a gain in tensile strength. The tensile strength was calculated from the following equation:
Tensile strength= Total breaking load
 |
Cross sectional area
Anti-inflammatory activity:
The anti-inflammatory activity of the extracts was determined according to the method of [16-17]. The rats were divided into six groups of six rats each. The control group received 1% (v/v) Tween 80 in water, p.o. The STD group was treated standard drug Diclofenac (10mg/kg) p.o. Different extracts were administered to the other groups in doses of 50,100,200 and 400 mg/kg as shown in graph 1. All the different type of concentration of drug were administered 30 min before the induction of oedema by administering 0.1 ml of 1% w/v carrageenan in saline [18-19]. The degree of paw oedema of all the groups was measured using a Plethysmometer at 0hr, 1hr, 2hr, 3hr, 4hr and 5hrafter the administration of carrageenan to each group.
Analgesic activity (Hot plate method & Tail Flick method):
The method was first described by (Eddy and Leimbach).Female Swiss mice weighing 20-25 g were used and divided into six groups with six animals per group. The animals were kept at 18 hour fasting prior to the experiment with water ad libitum.After administration of test and standard drug, the test for analgesia was carried out by placing the mice on electrically heated plate at 55o C ±0.5o C and noted the signs of discomfort like fore paw licking or jumping out of the plate. The time was noted in seconds. Test was carried out similarly for animals of control group. The observation made at 0, 30, 60 and 90 min after oral administration of the samples [20].
Table 1. Grouping for Hot plate and Tail flick Methods
|
Group |
Treatment |
No of animals |
|
Group I |
Normal saline |
6 |
|
Group-II |
ASE(50mg/kg)p.o. |
6 |
|
Group-III |
ASE(100mg/kg)p.o. |
6 |
|
Group-IV |
ASE(200mg/kg)p.o. |
6 |
|
Group-V |
ASE(400mg/kg)p.o. |
6 |
|
Group-VI |
Diclofenac(10mg/kg) |
6 |
Statistical analysis:
The statistical significance of the results was analysed by one way analysis of variance (ANOVA) followed by Student–Newman–Keul's procedure. Experimental results concerning this study were mean ± SEM of six parallel observations and p<0.05, p<0.01 and p<0.001 was considered as significant.
RESULTS:
Phytochemical screening:
Phytochemical evaluation were carried out on the 65% ethanolic extract of A. spinosus and it revealed the presence of tannins, phenolics, saponins, flavonoids, terpenoids and carbohydrates. The percentage yield of the plant extract was found to be 10.8% w/w.
Acute dermal toxicity:
No toxic effect was observed on the behavioural response of rats treated with a dose of 2000 mg/kg of Amaranthus spinonous. All rats were dosed once and observed for 14 days. Moreover, there were no signs of changes in the behaviour patterns, skin, eyes, salivation, and diarrhoea of the rats. Neither mortality nor significant weight loss was also observed.
Cage–side observations for In-vivo acute dermal toxicity:
Table 1.1 Key: - ASE: - Amaranthus spinonous extract; N: - Normal; (-):- Not detected
|
Sr. No |
Toxicity Parameters evaluated |
OBSERVATION |
|
|
Ctrl |
ASE (2000mg) (w/v) |
||
|
1 |
Alertness |
N |
N |
|
2 |
Irritability |
- |
- |
|
3 |
Fearfulness |
- |
- |
|
4 |
Touch Response |
N |
N |
|
5 |
Restlessness |
- |
- |
|
6 |
Abdominal Tone |
N |
N |
|
7 |
Tremors |
- |
- |
|
8 |
Writhing |
- |
- |
|
9 |
Corneal reflexes |
N |
N |
|
10 |
Defection |
N |
N |
|
11 |
Diarrhea |
- |
- |
|
12 |
Urination |
N |
N |
|
13 |
Food and water intake |
N |
N |
|
14 |
Respiration rate |
N |
N |
|
15 |
Pupil size color |
N |
N |
|
16 |
Pupil reaction to light |
N |
N |
|
17 |
Skin color & texture |
N |
N |
|
18 |
Fur color |
N |
N |
|
19 |
Spontaneous activity |
N |
N |
|
20 |
Heart rate |
N |
N |
|
21 |
Convulsion |
- |
- |
|
22 |
Aggressiveness |
- |
- |
|
23 |
Vomiting |
- |
- |
Incision wound healing activity:
The effect of wound healing activity by incision model was evaluated by tensile strength of the incision wound of different group viz control group treated (Figure 1) with simple ointment base, standard group treated with 10%w/w soframycin ointment and the test group treated with the ethonolic whole plant extract of Amaranthus spinonous (5 and 10%).
Figure 1. Effect of ASE on wound healing activity
n=6 Data is represented as mean ±SEM, *p<0.05, **p<0.01, ***p<0.001 vs. control; (One way ANOVA and t-test). From the above data we conclude that ASE 10% show significantly higher activity when compared to the control group (blank ointment).
Anti-inflammatory activity:
The anti-inflammatory effects were observed in carrageenan induced paw edema in rats. The extract and reference ointment were applied topically to rat paw and percent inhibition was observed significantly increase in 5% w/w ASE ointment group and comparable to reference group of animals on 3 to 5th h after induction (Figure 2). Standard drug showed a 61.9% of inhibitory effect at 3 h and inhibition of edema of 73.9% at time 5 h after induction of inflammation.
Figure 2. Effect of ASE on inflammation activity
n=6 Data is represented as mean ±SEM, *p<0.05, **p<0.01, ***p<0.001 vs. control; (One way ANOVA and t-test). From the above data we conclude that, ASEpossesses significant anti-inflammatory activity in comparison to control. Where ASE =50, ASE=100, ASE=200, ASE=400 mg/kg, (Diclofenac)STD= 10mg/kg.
Analgesic activity of hot plate method for Amaranthus spinosus:
Normal mice react to this stimulus by licking their paws in an average time, which were recorded before drug treatment. After 0, 30, 60 and 90 mins, each mouse was tested on the hot plate and time from its reaching the hot plate until it first licks a paw was recorded (Figure 3). The average reaction time was calculated for each mouse. Increase in the reaction time was considered to be indicative of analgesic activity.
Figure 3. Effect of ASE on analgesic activity by tail flick method
n=6 Data is represented as mean ±SEM, *p<0.05, **p<0.01, ***p<0.001 vs. control; (One way ANOVA and t-test). From the above data we conclude that, NBRI-AS (Tail flick method) possess significant analgesic activity in comparison to control. ASE=50, ASE=100, ASE=200, ASE=400 mg/kg, STD= 10mg/kg.
Analgesic activity of tail flick method for Amaranthus spinosus:
Result of analgesic activity (Tail flick method) for 65% ethonolic extract of Amaranthus spinosus are presented in graph no .3the extract found to exhibited a does dependent simultaneously increase in latency time when compared with control group1 (Figure 4).. At 90 minutes, the reaction time of different doses (50,100,200,400mg/kg) were found to be statistically significant (p<0.001).
Figure 4. Effect of ASE on analgesic activity by hot plate method
n=6 Data is represented as mean ±SEM, *p<0.05, **p<0.01, ***p<0.001 vs. control; (One way ANOVA and t-test). From the above data we conclude that, NBRI-AS possess significant analgesic activity (Hot plate method) in comparison to control. Where ASE =50, ASE=100, ASE=200, ASE400 mg/kg, STD= 10mg/kg.
DISCUSSION:
Preliminary qualitative phytochemical screening reveals the presence of alkaloids, carbohydrates, glycosides, tannins, gums, flavonoids & alkaloids in Amaranthus spinosus. Therefore, it is assumed that these compounds may be responsible for the observed anti-inflammatory and analgesic activity. Flavonoids were reported to have a role in analgesic activity primarily by targeting prostaglandins (PG) [21-22]. There are also reports on the role of tannins in anti-nociceptive activity [23]. Besides alkaloids are well known for their ability to inhibit pain perception.
At the end of the observation period in dermal toxicity it was noted that all the treated test animals were normal. During the whole experimental period, no other abnormal behaviours were observed and no deaths occurred. No hazardous signs were recorded in the test animals during 14 days of observation after the topical application of two different doses of the 65% ethanolic whole plantextract of Amaranthus spinosus. Table.1 records the overall findings of various cage-side observations noted for the acute dermal toxicity study.The observations and findings of the dermal toxicity study showed no signs of dermal toxicity, nor any signs of skin corrosion, skin abrasion, etc. and hence it was concluded that the doses of 1000 mg/kg and 2000mg/kg that were evaluated in the present study caused any mortality nor brought any changes in the cellular structure of the skin tissue in response to the treatment of the test animals.
In incision wound model, significant increase in skin breaking strength was observed. Groups treated with 10% and 5% (w/w) extracts and standard ointments showed statistically significant increase in tensile strength as compared to simple ointment base treated group. However, the difference in tensile strength was not statistically significant among standard drug and 10% and 5% (w/w) ointment treated groups. The increase in tensile strength in the incision model may be due to the antioxidant activity of the extract, increase in collagen synthesis and maturation, formation of stable intra- and intermolecular cross-link, matrix deposition, and cell migration [5, 24-26].
Another possible reason for enhanced wound healing effect could be due to the whole plant extract of Amaranthus spinosus which may possess antioxidant, free radical scavenging properties and promote cell proliferating properties.
The development of carrageenan induced edema is biphasic, the 1st phase is attributed to release of histamine, 5-HT, Kinin, while 2nd phase is related to the release of Prostaglandin.Therefore, it is proposed that the anti-inflammatory property of extracts is due to inhibition ofone of the pain mediators like histamine, 5-HT, Kinin or Prostaglandin. The ASE showed varying degrees of anti-inflammatory activities with statistical significance at all tested dose levels 50-400mg/kg. The percentage inhibition of inflammation of ASE are dose dependent, as increase in doeslevel of extracts increases percentage of inhibition accordingly.
65% of ethonolic extracts of the Amaranthus spinosus and standard drug diclofenac sodium (10mg/kg) also presented a longer latency time than the control group in the hot plate test and tail flick method in a dose related manner. At 90 minutes & 400 mg/kg, p.o. administration of the plant extracts the percent inhibition was found 62.22% & 56.56% for Amaranthus spinosus respectively.
CONCLUSION:
On the basis of the results finding in the present investigation, it is concluded that the 65% ethanolic extract Amaranthus spinosus highest wound healing activity as compared to other control. The wound healing activity of the ethanolic extract may be due to the combined effect of the above phytochemicals. Comprehensive evaluation on the plants with wound healing activity on the basis of traditional medicine may possibly give new compounds that could be used as prominent drugs in wound healing therapy. Further investigations are needed for identification of active principles responsible for the wound healing activity.
CONFLICT OF INTEREST:
None.
ACKNOWLEDGMENT:
The authors are thankful to our Honorable Director, CSIR-NBRI, Lucknow, for their assistance and providing laboratory facilities. I amso much thankful to my colleague Dr. Pritt Verma (SRF) and Parag Jain for their constant support during the experiment and Animal work.Further, we extend our gratitude to the University Grant Commission-RGNSRF, New Delhi, India, for providing a grant to the perform thestudy (Grant Number F1-17.1/ 2014-15-RGNF-2014-15-SC-UTT-60684).
REFERENCES:
1. Ayello, EA, Ads. Skin Wound Care. 2005; 18: 98-109.
2. Rubin E, Fabrex JL. Repair, regeneration and fibrosis. In: Pathology, 2nd ed. J.B. Lippincott, Philadelphia, 1996; 7-15.
3. Mantle D, Gok MA, Lennard TWJ. Adverse Drug Reactions and Toxicological Reviews. 2001; 20: 89-103.
4. Martin P, Leibovich SJ. Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol. 2005;15: 599-607.
5. Chan K. Some aspects of toxic contaminants in herbal medicines. Chemosphere. 2003; 52:1361–1371.
6. Nagappa AN, Binu C. Wound healing activity of the aqueous extract of Thespesiapopulnea fruit. Fitotherapia. 2001; 72: 503-506.
7. Kumar K, Singh KK, Asthana AK, Nath V. Ethno therapeutics of Bryophyte Plagiochasmaappendiculatum among the Gaddi Tribes of Kangra Valley, Hima-chal Pradesh, India. Pharm Biol. 2000; 38: 353–356.
8. Martin P. Wound healing aiming for perfect skin regeneration. Science 1997; 276: 75–81.
9. Ilhem R, Anouar BS, Najla H. Oxidative damage and hepatotoxicity associated with deltamethrin in rats: The protective effects of Amaranthus spinosus seed extract Biomedicine & Pharmacotherapy. 2016; 84: 853–860.
10. Hilou OG. Nacoulma TR. Guiguemde. In vivo antimalarial activities of extracts from Amaranthus spinosus L. and Boerhaaviaerecta L. in mice, J. Ethnopharmacol. 2006; 103: 236–240.
11. OECD. Acute dermal toxicity. In: OECD Draft Guidelines for Testing of Chemicals, No. 434, fixed procedures. Organization for Economic Cooperation and Development. Paris, France; 2004.
12. Udupa AL, Rao SG, Kulkarni DR. Hound healing profile of septilin. Indian J Physiol Pharmacol. 1989; 33: 39-42.
13. Govindarajan R, Vijayakumar M, Rao CV, Shirwaikar A, Mehrotra S, Puspangadan P. Healing potential of Anogeissuslatifolia for dermal wound in rats. Acta Pharm. 2004; 54: 331-338.
14. Perez Gutierrez RM, Vargas SR. Evaluation of the wound healing properties of Acalyphalangiana in diabetic rats. Fitoterapia. 2006; 77: 286-289.
15. Gbedema SY, Emelia K, Francis A, Kofi A, Eric W. Wound healing properties and kill kinetics of Clerodendronsplendens G. Don, A Ghanaian wound healing plant. Pharmacog. Res. 2010; 2: 63-68.
16. Borgi W, Ghedira K, Chouchane N. Antiinflammatory and analgesic activities of Zizyphus lotus root barks. Fitoterapia. 2007; 78: 16-19.
17. Vogel HG, Vogel WH. Drug Discovery and Evaluation. Verlag Berlin Heidelberg, New York, Springer, 1997; 390-417.
18. Akindele AJ, Adeyemi OO. Anti inflammatoryactivity of the aqueous leaf extract of Byrsocarpuscoccineus. Fitoterapia. 2007; 78: 25-25.
19. Dimo T, Fotio AL, Nguelefack TB, Asongalem EA, Kamtchouing P. Antiinflammatory activity of leaf extracts of Kalanchoecrenata Andr. Indian J Pharmacol. 2006; 38: 115-119.
20. Raquibul Hasan SM, Hossain MM, Akter R, Jamila M, Mazumder MEH, Alam MA, Faruque A, Rana S, Rahman S. Analgesic Activity of the Different Fractions of the Aerial Parts of Commelinabenghalensis Linn. International Journal of Pharmacology. 2010; 6: 63-67.
21. Rajnarayana K, Reddy MS, Chaluvadi MR, Krishna DR. Biflavonoids classification, pharmacological, biochemical effects and therapeutic potential. Indian Journal of Pharmacology. 2001; 33: 2-16.
22. Rao MR, Rao YM, Rao AV, Prabhkar MC, Rao CS, Muralidhar N. Antinociceptive and antiinflammatory activity of a flavonoid isolated from Caralluma attenuate. Journal of Ethnopharmacology. 1998; 62: 63-66.
23. Vanu MR, Palanivelu S, Panchanatham S. Immunomodulatory and anti-inflammatory effects of Semecarpusanacardium Linn. Nut milk extract in experimental inflammatory conditions. Biological and Pharmaceutical Bulletin. 2006; 29: 693-700.
24. Assefa E, Alemayhu I, Endale M, Mammo F. Iridoid glycosides from the root of Acanthus sennii. Journal of Pharmacy and Pharmacognosy Research. 2016; 4: 231–237.
25. Getie M, Gebre-Mariam T, Rietz R, et al. Evaluation of the anti-microbial and anti-inflammatory activities of the medicinal plants Dodonaeaviscosa, Rumexnervosus and Rumexabyssinicus. Fitoterapia. 2003; 74: 139-143.
26. Arun M, Satish S, Anima P. Evaluation of wound healing, antioxidant and antimicrobial efficacy of Jasminumauriculatum Vahl. Leaves. Avicenna Journal of Phytomedicine. 2016; 6: 295.
Received on 11.12.2019 Modified on 10.01.2020
Accepted on 06.02.2020 © RJPT All right reserved
Research J. Pharm. and Tech 2020; 13(5): 2439-2444.
DOI: 10.5958/0974-360X.2020.00437.0