18 Journal of Food, Agriculture & Environment, Vol.14 (3&4), July- October 2016

www.world-food.net Journal of Food, Agriculture & Environment Vol.14 (3&4): 18-22. 2016

WFL PublisherScience and Technology

Meri-Rastilantie 3 B, FI-00980Helsinki, Finlande-mail: info@world-food.net

Received 15 May 2016, accepted 12 September 2016.

Wound healing potential of methanolic extracts of some plants native to Al-QassimRegion, Saudi Arabia

Khaled B. Al-Harbi*, E.A. El-Tigani-Asil, Ahmed F. Ahmed, Ibrahim M. El-Ashmawy and Naser A. Al-WabelDepartment of Veterinary Medicine, Faculty of Agriculture and Veterinary Medicine, Qassim University, Buraidah, 51452 P.O. 6622, Saudi Arabia. *e-mail: kbhrby@qu.edu.sa, aboabdelelah17@hotmail.com, dr.abd.el.kader@hotmail.com

AbstractFolklore phytotherapy is a worldwide practice extensively used for the treatment of various conditions like wounds which are rising as one of thehealth care obstacles. This study was undertaken to evaluate wound healing effect of methanolic extracts of some plants native to Al-Qassim Region,Saudi Arabia. Wistar rats were used as an animal model for the experiment. Qualitative and quantitative screening of the extracted phytoconstituentswas done using standard procedures. A formula of soft paraffin-based ointment (10% conc.) of each crude extract was prepared and used to testwound healing effect in the incision and excision wounded rat models. Wound healing response assessment using inflammation, granulation, regeneration,wound contraction, clossuring rate and reepithelialization parameters were adopted. Phytochemical analysis showed flavonoids, tannins and saponinswith various amounts. Healing response signs; viz., inflammation, granulation, regeneration, wound contraction and re-epithelialization were observedin the skin sections of sacrified animals. Extracts of Heliotropium bacciferum, Rhanterium epapposum, Achillea fragrantissima, Artemisia herba-alba, Haloxylon salicornicum, Deverra triradiata, Echinops hussoni, Prosopis farcta, Cyperus conglomeratus and Astragalus spinosus were themost potent wound healers that showed complete wound contraction surface (P< 0.05) by the end of the experiment and this may possible be dueto high contents of the phytoconstituents. This study concluded that some plant extracts have potential wound healing effect and further investigationsare needed to obtain complete profile and its various actions.

Key words: Wound healing, methanolic plant extracts, phytoconstituents.

IntroductionFolklore traditional therapy is an affordable primary health carepractice widely distributed across the globe especially in thedeveloping countries where modern medical care is not availableor highly expensive. WHO reports showed about 70-80% of theworld’s populations depend on medicinal plants for treating severaldiseases1. Phytochemical plant extracts proved to possess manyactive ingredients such as alkaloids, carotenoids, flavonoids,tannins, polysaccharides, minerals and vitamins; these chemicalcompounds showed different therapeutic activities against severaldisorders 2-5. On the aspect of wound healing, herbal extractsthought to have an immense potential as blood coagulators,antimicrobial, antioxidants or antidiabetic agents which mayimprove or accelerate healing of the wounds 6,7. However, someplants have certain factors including keratinocyte growth factor-1 and type I collagen growth factor which have a significant role inregeneration and repair of wound respectively 4. Tannins aresuggested to chelate free radical, promote wound contraction,increase angiogenesis and proliferate the fibroblasts 8,9. Whileflavonoids exhibit antioxidant, astringent and antibacterial effectsresponsible for defense, contraction and re-epithelization10-12.Prosopis farcta, one among the plants native to Saudi Arabia,was well investigated elsewhere and proved to have a woundhealing effect due to its high content of flavonoids, tryptaminesand alkaloids 13. Wounds are a disruption of skin due to physical, chemical,mechanical, microbial or immunological agents leading to the

breakdown of tissue surfaces or basement membranes14. Woundsare classified as open or closed wounds on the basis ofdiscontinuity, as acute or chronic on the basis of healing timeand to surgical or contaminated wound on the basis of sterility.Recent reports illustrate that six million people across the globesuffer from chronic wounds 15. Impaired wound healing can resultinto a severe morbidity leading to long hospitalization of patients.The aim of treating wounds is to shorten the time taken for healingand to reduce risks of undesired complications 16 . Wound healing is a complex and intricate biological process torestore the normal tissue anatomic structure and function 17. Thehealing process consists of three overlapped phases includinginflammatory, proliferative and regenerative phases 18,19. Thehealing events are consisting of integrated cellular andbiochemical interactions mainly platelets aggregation and bloodclotting, formation of fibrin, inflammatory response andinflammatory crust formation, fibrous matrix, angiogenesis, re-epithelialization and finally the disrupted skin surfaces are firmlyscared by collagen 4. Wound healing is affected by several factorsincluding contamination, tissue oxignization, drugs, age,radiations, foreign bodies, starvation and diseases (diabetes) 13,

20. Experimental rats are the most preferable models used inevaluating the effects of various phytochemical agents in theprocess of wound healing. Incisional (38.2%), excisional (38.2%)and burns (18.8%) are wounds that were widely adopted for suchinvestigations during 2000-2003.

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The presence of diverse bioactive phytoconstituents in theplants has encouraged researchers to study qualitative andquantitative analysis and subsequently determining potentialproperties associated with wound healing. Hence the objectiveof this research was drowned to evaluate the effect of methanolicextracts of some plants native to Al-Qassim Region, Saudi Arabiain wound healing in rats.

MethodologyMethanol extracts: The plants (n = 21) belonging to 13 familieswere selected from Al Qassim Region, cleaned under running tapewater, air dried, pulverized, extracted, harvested and subjected toqualitative and quantitative phytoconstitent analysis 21, 22. Theextracts were kept at – 4°C until used for the phytochemicalanalysis and wound healing activity.

Preparation of the extract ointments: A formula of soft paraffinbased plant extract ointments (10% conc.) was prepared for thetopical application as prescribed elsewhere. Control positiveantibiotic ointment (Gentamicin, Garamycin®, Schering Plough,Co. USA) and pure soft paraffin control ointment (negative control)was used for the comparative analysis 23, 24.

Animal models: Experimental male Wistar rats 200-250 g. b. wt.were obtained from Laboratory Animal Unit, Faculty of Agricultureand Veterinary Medicine, Qassim University. Feeding, watersupply and adaptation period were standarized 24.

Incision wound models: Expermental animals were anesthetizedwith ether, their dorsum skin was shaved, cleaned and disinfectedwith alcohol. Then aseptically a full-thickness skin surgical incisionwounds of 20 mm in length were made in the dorsal skin of the ratmodels. Observation and assessment of wound contraction andclosuring was adopted to evaluate healing response in createdincision wounds 25.

Excision wound models: Expermental animals were anesthetizedwith ether, their dorsal skin was shaved, cleaned and disinfectedwith alcohol. Then aseptically a full-thickness skin surgical woundsarea of about 400 mm2 and 2 mm in depth were created for excisionwounds. Observation and assessment of the wounds, mainly,granulation tissue formation, re-epithelization and scaring wereadopted to evaluate healing response in the created excisionwounds 25.

Treatment of the wounded models: Both incision and excisionanimals were divided into three groups for testing the potential ofextract ointments in wound healing. The wounded rats weretopically treated with tested and control ointments once daily for10 days as follows: Group 1 was extracts treated group, Group 2was gentamicin treated group and Group 3 was sole paraffin waxtreated group. Animals were individually caged throughout theexperment. Tracking, observation and assessment of woundscontraction, closuring and re-epithelization were adopted toevaluate healing response in created wounds each five days upto 20th day post medication 16.

Histopathological examination: The animals were sacrificed onthe 10th day post treatment and tissue sections were obtained

from both incision and excision wounds. Fixated in 10% formalsaline, routine paraffin wax processed, sectioned and stained withHeamatoxilin&Eosin (H&E) for histopathological analysis 26.

Statistical analysis: Results were expressed as mean ± SE.Difference between means in different groups were tested forsignificance using one-way analysis of variance (ANOVA)followed by Duncan’s test and P value of 0.05 or less wasconsidered as significant.

ResultsPhytoconstituent of methanolic plant extracts: The qualitativeanalysis of plant extracts revealed various phytoconstituentagents mainly alkaloides, saponines, tannins, and flavonoids asactive ingredients. The quantitative analysis of thesephytoconstituents were determined as detailed in Table 1.

Gross wound observation in the treated versus control rats:Observation of wound surface diameter (at day 5, 10, 15 and 20th)of treated rats with tested ointments induced various degrees ofhealing response when compared to controls as shown in Table 2and Fig. 1. Wound surface contraction value less than P< 0.05was considered as signifcant.

Histopathological findings: Paraffin wax H&E stained skinsections illustrated various degrees of wound healing. Concerningincision wound healing response a fibrotic contraction and closureof the wound edges was observed with hyperplasia of epithelialcells and hyperkeratosis (Fig. 2). While excision wounds healing response was seen in theinflammatory healing phase a formation of protective inflammatorycrust covered the excised ulcerative wounds. Proliferation offibroblasts, fibrocytes and collagenous fiber materials with newcapillaries penetrating the matrix were obviously seen beneaththe inflammatory crust as a response of granulation andregeneration phases. Re-epithelization of epidermis with mitosisand forming of keratinocyte epithelial cells were noticed (Figs 3and 4). In our study the healing response of both incised and excisedtreated wounds was the response at 10th day post treatmentaccording to the degree of healing. Failure of contraction orclosuring the incised wound, dead space minimizing of the excisedwound and adhesion of the inflammatory crust are the mostcharacteristics of the weak healing response. Prominentdesquamated inflammatory crust with deposition of granulationtissue, fibrosis, revascularization and re-epithelization were thesigns of moderate response. In contrast to a complete woundclosuring, well desquamated inflammatory crust, maturecollagenous fibrotic matrix, angiogenesis, hyperplasia, mitosis,re-epithelization and formation of keratinized layer were theprominent signs of excellent healing response in both incised andexcised wounds (Figs 3 and 4).

DiscussionRecently the use of traditional plant extracts and alternative therapyhas promoted awareness across the globe especially in developingcountries for their availability, effectiveness and less side effectsacross the globe especially in developing countries for theiravailability effectiveness. On the aspect of wound healing several

20 Journal of Food, Agriculture & Environment, Vol.14 (3&4), July- October 2016

plants have shown a reliable therapeutic effect when topicallyapplied and referred to high content of active phytochemicalingredients; flavonoids, tryptamines, alkaloids and tannins whichproved to promote healing of the wounds 8-11, 13 . Qualitative andquantitative phytoconstituent analysis of the tested plant extractsproved to pose various active ingredients having a potential effectin wound healing response 3, 4, 6. In correlation to qualitative andquantitative phytoconstituents, macroscopic pathologicalobservation of both incision and excision wound surface diameterindicated excellent healing response where P value of the woundsurface area contraction was less than P< 0.05 at day 10 post

Table 1. Quantitative analysis of phytochemical constituent of the plant extracts.

No. Plant Total phenolics Tannin phenolics

Non-tannin phenolics

Total flavonoids

1 Rhazya stricta 52.59 26.55 26.04 18.6 2 Heliotropium bacciferum 60.52 13.85 46.67 58.1 3 Tribulus longipetalus 30.93 19.16 11.77 24.0

4 Rhanterium epapposum 124.26 _ 124.26 30.2 5 Achillea fragrantissima 55.57 36.62 18.95 80.4

6 Artemisia herba-alba 94.19 71.60 22.59 99.7 7 Haloxylon salicornicum 45.83 30.63 15.20 36.1

8 Plantago coronopus 64.46 29.43 35.03 38.5

9 Moltkiopsis ciliata 59.48 11.4 48.08 31.9 10 Lasiurus scindicus 47.42 22.96 24.46 51.2

11 Lasiurus hirsutus 29.28 14.15 15.13 39.6 12 Hordeum distichon 37.71 15.17 23.54 54.1

13 Eragrostis poaeoides 52.94 28.87 24.07 59.5 14 Deverra triradiata 41.3 15.92 25.38 110.3

15 Lycium shawii 101.7 47.66 54.04 59.8 16 Echinops spinosus L. 66.54 20.28 46.26 44.6

17 Echinops hussoni 46.86 22.90 23.96 23.8 18 Prosopis farcta 271.92 81.24 190.68 135.0

19 Cyperus conglomeratus 164.29 _ 164.3 123.5 20 Astragalus spinosus 43.46 _ 43.4 86.6

21 Lepidium sativum seeds 45.1 26.7 18.4 19.8

treatment and the wound surface areas were completely closedby the end of the experment. Methanolic plant extracts ofHeliotropium bacciferum, Rhanterium epapposum, Achilleafragrantissima, Artemisia herba-alba, Haloxylon salicornicum,Deverra triradiata, Echinops hussoni, Prosopis farcta, Cyperusconglomeratus and Astragalus spinosus were found to enhancethe wound healing activity. This may be due to high content offlavonoids and, phenolic compounds, mainly tannins, which werereported to have wound healing properities that promote theinflammatory wound healing response in contrast to those plantswhich have low content of flavonoids and phenolic tannins. These

No. Plant name Day 5(cm2) Day 10(cm2) Day 15(cm2) Day 20(cm2) 1 Rhazya stricta 3.56 ± 0.05 2.10 ± 0.16 1.02 ± 0.05* 0.10 ± 0.05* 2 Heliotropium bacciferum 3.48 ± 0.04 1.94 ± 0.20* 0.98 ± 0.07* 0 3 Tribulus longipetalus 3.50 ± 0.04 1.88 ± 0.07* 1.18 ± 0.08 0.13 ± 0.05* 4 Rhanterium epapposum 3.60 ± 0.04 1.82 ± 0.09* 0.74 ± 0.08* 0 5 Achillea fragrantissima 3.60 ± 0.05 1.76 ± 0.12* 0.84 ± 0.06* 0 6 Artemisia herba-alba 3.44 ± 0.05 1.76 ± 0.05* 0.80 ± 0.05* 0 7 Haloxylon salicornicum 3.54 ± 0.08 1.72 ± 0.07* 0.80 ± 0.11* 0 8 Plantago coronopus 3.50 ± 0.04 1.76 ± 0.09* 1.18 ± 0.08 0.10 ± 0.02* 9 Moltkiopsis ciliata 3.48 ± 0.03 2.24 ± 0.08 1.06 ± 0.08* 0.19 ± 0.05* 10 Lasiurus scindicus 3.54 ± 0.05 2.60 ± 0.08 1.24 ± 0.09 0.43 ± 0.09 11 Lasiurus hirsutus 3.48 ± 0.06 2.66 ± 0.07 1.16 ± 0.11 0.42 ± 0.04 12 Hordeum distichon 3.50 ± 0.07 1.68 ± 0.11* 1.04 ± 0.05* 0.20 ± 0.03* 13 Eragrostis poaeoides 3.48 ± 0.04 1.84 ± 0.19* 1.10 ± 0.08 0.24 ± 0.05* 14 Deverra triradiata 3.44 ± 0.06 1.82 ± 0.14* 0.84 ± 0.10* 0 15 Lycium shawii 3.52 ± 0.03 2.20 ± 0.14 1.12 ± 0.10 0.18 ± 0.03* 16 Echinops spinosus 3.56 ± 0.05 2.66 ± 0.10 1.44 ± 0.16 0.21 ± 0.06* 17 Echinops hussoni 3.52 ± 0.05 1.88 ± 0.09* 0.74 ± 0.08* 0 18 Prosopis farcta 3.46 ± 0.06 1.68 ± 0.10* 0.72 ± 0.08* 0 19 Cyperus conglomeratus 3.52 ± 0.03 1.70 ± 0.14* 0.64 ± 0.09* 0 20 Astragalus spinosus 3.48 ± 0.03 1.76 ± 0.09* 0.80 ± 0.09* 0 21 Lepidium sativum seeds 3.50 ± 0.05 1.66 ± 0.13* 0.86 ± 0.07* 0.10 ± 0.01* 22 Gentamicin oint. 3.48 ± 0.03 1.74 ± 0.14* 0.74 ± 0.11* 0 23 Control 3.60 ± 0.03 2.58 ± 0.15 1.40 ± 0.17 0.41± 0.01

Table 2. Mean ± S.E. value of wound contraction surface area (cm2) in tested rat groups.

* Significantly different compared to control (p< 0.05).Values are expressed as means ± S.E. (n = 5).

Journal of Food, Agriculture & Environment, Vol.14 (3&4), July- October 2016 21

Figure 1. Macroscopic observation that represents the contractionrate of wound surface area (cm2) in treated rat groups.

Figure 2. Incision wounds treated with Tribulus longipetalu (A)and Prosopis farcta(B) extract ointments showing moderate (A)versus excellent (B) healing response at 10th day of necropsy. A:Clot formation (1), Deposition of tissue granules (2), Fibrosis (3)and re-vascularization (4). B: Complete closuring of the wound (2)with re-epithelization (5) and formation of keratinized layer (1).

findings were in line with previous studies 5, 7-12. Most ofresearchers have proved that flavonoids and tannins haveantimicrobial and astringents effects which promote woundcontraction, increase angiogenesis and proliferate fibroblasts andin turn accelerate wound healing. The rest plant extracts whichcontained low amounts of flavonoids, phenolics and saponinsexert a moderate healing response when the P value of woundsurface contraction rate was P>0.05 and histopathological sectionsfrom tested rats showed not completed healing stage despite ofthe evidence and observation of inflammation, deposting ofgranulation tissue and re-epithelization.

ConclusionsHeliotropium bacciferum, Rhanterium epapposum, Achilleafragrantissima, Artemisia herba-alba, Haloxylon salicornicum,Deverra triradiata, Echinops hussoni, Prosopis farcta, Cyperusconglomeratus and Astragalus spinosus have potential woundhealing effect in rat models and further investigation of theseplant extracts native to Al-Qassim Region, Saudi Arabia, are neededfor more exploration.

Figure 3. Excision wounds treated with Achillea fragrantissima (C) andHaloxylon salicornicum (D) Extract ointments showing excellentdefinitive healing response phases were characterized by welldevelopment of the inflammatory crust (1) Deposition of tissue granules,(2) Fibrosis and re-vascularization (3,4) and re-epithelization (5) wereseen at the 10th day of necropsy.

22 Journal of Food, Agriculture & Environment, Vol.14 (3&4), July- October 2016

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Figure 4. Excision wounds treated with Lasiurus scindicus (E) andAstragalus spinosus (F) ointments showing moderate (E) versus excellent(F) healing response at the 10th day of post treatment.E: Moderatehealing response showed well developed and adherent inflammatorycrust (1), deposition of tissue granules (2) with fibrosis and re-vascularization (3,4) and re-epithelization (5). F: Excellent healingresponse showed desquamated inflammatory crust and fibrosis (3),angiogenesis (4), hyperplasia and mitosis and re-epithelization(5).