Antioxidant,Antimicrobial and Wound Healing Potential of Helicteres isora Linn.Leaf Extracts

RENUKA Mahajan,PRAKASH Itankar

Department of Pharmaceutical Sciences,Rashtrasant Tukadoji Maharaj Nagpur University,Nagpur,Maharashtra 440033,India

Keywords

Helicteres isora Linn.

Phytochemical screening

Antioxidant

Antimicrobial

Incision wound

Excision wound

Period of epithelization

ABSTRACT

Objective To investigate the antioxidant,antimicrobial and wound healing potential of Helicteres isora Linn.leaf extracts.

Methods The petroleum ether,chloroform,acetone,ethanol and hydroalcoholic extracts of leaves were screened for phytochemicals.The 1,1-diphenyl-2-picrylhydrazyl(DPPH),nitric oxide(NO)radical scavenging tests and reducing power assays were performed to measure antioxidant activity; disc diffusion methods were used to evaluate antimicrobial potential.Wound healing activity was evaluated by incision and excision wound rat models.

Results The extracts contained mainly sterols and flavonoids.The hydroalcoholic extract showed remarkable antioxidant and antimicrobial potential and significant(P＜0.05)wound healing activity.

Conclusions The identified activities of the hydroalcoholic extract may be attributable to its constituent phytochemicals.

1 Introduction

A wound may be defined as loss or breakage of the cellular,anatomical or functional continuity of living tissues[1].Wounds are characterized by the development of fibrous tissue,new blood vessels and epithelial reconstruction,which involves the migration and proliferation of various cell types,including endothelial cells,epithelial cells,and fibroblasts,deposition of connective tissue,and wound contraction.These processes are controlled by cytokines,growth factors and matrix molecules[2].There are various causes of chronic wounds,including diabetes,autoimmune disorders and ischemia[3].Wound healing,which is necessary for the restoration of skin,starts from the trauma and terminates in scar formation.Acute and chronic wounds heal through a series of biochemical and cellular reactions starting with hemostasis,followed by inflammation,proliferation or granulation,and remodeling or maturation[4].

In traditional medicine systems,plants and other natural products have been used to promote healing.Although healing is a natural process,infection and oxidative damage can increase inflammation and delay the process[5].Therefore,we investigatedHelicteres isora(H.isora)Linn.(the East Indian screw tree),of the family Sterculiaceae,for its antioxidant,antimicrobial and wound healing activity.The plant,known as Muradsheng,Avartaphala,Avartani and Murvai in other languages,is a large herb or small tree with ovate,hairy leaves with a serrate margin.The plant has orange-red flowers,and compound,twisted,screw-like fruit.H.isorais found throughout India and used in many regions for medicinal purposes.The fruits are traditional remedies for gastrointestinal problems,such as internal colic,flatulence,diarrhea,constipation in neonates,and other conditions,such as snake bites or sore ears.They are also effective as a demulcent,an expectorant,astringent and an emollient[6,7].In the Bihar region,the fruits of this plant are used in the treatment of malnutrition-associated diseases.As an ethnomedicine,the paste of the roots has been used for cuts and wounds.Furthermore,in Uttar Pradesh,the decoction of roots is used as a treatment for diarrhea and the aqueous extract is used for the treatment of dog bites.The fresh paste of leaves has been used for the treatment of scabies,skin infections and snake bites,whereas the bark has been used for the treatment of diabetes and diarrhea,and as an anthelmintic[8].

The pharmacological activities ofH.isorahave been successfully evaluated,demonstrating its antidiarrheal,hypolipidemic,wormicidal,antioxidant,antibacterial,cardiac antioxidant,antiperoxidative,antiplasmid,anticancer,brain antioxidant,hepatoprotective and antinociceptive activity.New flavones,i.e.,5,8-dihydroxy-7,4 flavones,herbacetin-8-O-glucoronide(hibifolin)and kaempferol-3-O-galactoside(trifolin)have been isolated from the leaves ofH.isora[9].H.isorapossesses a wide range of nutritional and medicinal properties.However,no scientific study has been performed on its wound healing potential; thus,we considered this a worthwhile investigation.

2 Materials and Methods

H.isoraleaves were collected in the month of August and authenticated by Dr.N.Dongarwar,Professor,Department of Botany,R.T.M.Nagpur University,Nagpur.The voucher specimen number was 9437.

2.1 Extraction

H.isoraleaves were washed,dried in the shade for two weeks,and crushed till formation of coarse powder.The powdered drug(500 g)was transferred into a Soxhlet apparatus and successive extractions were performed using petroleum ether(60 - 80 °C),chloroform,acetone and ethanol.Each cycle for an individual solvent was 24 - 36 h.Finally,the marc was macerated with hydroalcoholic solvent(70% ethanol :30% water,v/v)for 48 h.The obtained extracts were filtered through Whatman filter paper No.42 and concentrated using a rotary vacuum evaporator to obtain the semisolid residue[10].

2.2 Preliminary phytochemical screening

The screening of secondary metabolites was conducted by subjecting extracts to a battery of standard chemical tests[11].

2.3 Antioxidant activity

Various assays were performed to evaluate the antioxidant property of different extracts ofH.isora in vitro.The 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical-scavenging activity,nitric oxide(NO)radicalscavenging activity and reducing power were assayed[12-14].L-ascorbic acid was used as the standard.

2.3.1 DPPH methodA solution of approximately 0.1 mM DPPH in ethanol was prepared and 1.0 mL of this solution was added to 3.0 mL of each of the extract solutions in water; solutions with different concentrations(20,40,60,80 and 100 μg/mL)of the extract were prepared using the same method.After 30 min,the absorbance of the samples at 517 nm was measured.A lower absorbance indicates that high amounts of free radicals have been scavenged.The percentage activity was calculated using the following equation:DPPH scavenging activity(%)=(Acont - Atest)/Acont×100,where Acont was the absorbance of the control reaction and Atest was the absorbance in the presence of the extracts.

2.3.2 NO radical-scavenging activityApproximately 1 mL of sodium nitroprusside(5 mM in 0.5 M phosphate buffer)was mixed with 3.0 mL of different concentrations(20,40,60,80 and 100 μg/mL)of the extracts dissolved in the suitable solvent and the mixtures were incubated at 25 °C for 150 min.These samples were reacted with Griess reagent(1%sulfanilamide in 2% H3PO4and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in water).The absorbance of the samples at 546 nm was measured.The same reaction mixture without the extract was used as the control.The capability to scavenge the NO radicals was calculated from the following equation:NO scavenging(%)=(Acont - Atest)/Acont×100,where Acont was the absorbance of the control reaction and Atest was the absorbance in the presence of the extracts.

2.3.3 Determination of reducing powerThe reducing power of the different extracts was determined in accordance with the method of Oyaizu[15].Various concentrations of the extracts(20,40,60,80 and 100 μg/mL)in 1.0 mL of deionized water were mixed with phosphate buffer(2.5 mL,0.2 M,pH 6.6)and 1% potassium ferricyanide(2.5 mL).The mixtures were incubated at 50 °C for 20 min.Aliquots of trichloroacetic acid(2.5 mL,10%)were added to the mixture,which was then centrifuged for 10 min.The upper layer of solution(2.5 mL)was mixed with distilled water(2.5 mL)and added to freshly prepared FeCl3solution(0.5 mL,0.1%).The absorbance of the samples at 700 nm was measured.

2.4 Antibacterial and antifungal activity

Nutrient agar and Sabouraud dextrose agar were used as the growth media.Freshly subculturedEscherichia coli(NCIM 5 649),Staphylococcus aureus(NCIM 5 842),Pseudomonas aeruginosa(NCIM 5 002)andMicrococcus luteus(NCIM 5 203)were cultured on a nutrient agar slant and incubated for 24 h at 37 °C.The colonies on the slants were washed with 5 mL of sterile saline solution and used as inocula[16].Candida albicans(NCIM 6 430)was cultured on Sabouraud dextrose agar and incubated for 5 d at 22 -27 °C.The growth was washed with 5 mL of sterile saline solution.The residue of each extractive was suspended in a suitable quantity of sterile water to obtain a 5.0% solution(with 1.0% previously sterilized CMC solution added as a suspending agent).The disc diffusion method was used to estimate the antibacterial and antifungal activity of all extracts[17].Each extract concentration(10,5 and 2.5 mg/mL)was tested in triplicate,and the inhibition zone was measured in millimeters.Streptomycin and fluconazole were used as the standards.The cultures were obtained from NCL(National Collection of Industrial Micro-organisms),Pune,India.Based on the antioxidant and antimicrobial potential of extracts,the ethanolic and hydroalcoholic extracts were selected for further studies.

2.5 Preparation of formulation and evaluation

Simple ointments(2% and 5% w/w)were prepared by dissolving methylparaben,propylparaben,sodium lauryl sulphate(SLS)and propylene glycol in an aqueous vehicle.Stearyl alcohol and white petrolatum were collected in a separate beaker,and heated in a steam bath to approximately 75 °C.The previously dissolved ingredients were added,followed by heating again to 75 °C,and stirring until the mixture was congealed.The ointment was evaluated for stability and skin irritation.Extract ointments(0.5 g)were applied once daily and tested using different groups of animals[18].

2.5.1 Physical stability testThe ointments were tested for pH,viscosity and spreadability.Readings were taken in triplicate and the average was recorded.Visual appearance,color and odor were recorded.A pH meter,Brookfield viscometer and parallel plate was used to determine pH,viscosity and spreadability,respectively.Spreadability was calculated from the equationS=M×L/T,whereS=spreadability of ointment,M=weight tied on the upper plate in gram,L=length of the glass plate in centimeter,andT=time taken to slide complete length[19].

2.5.2 Skin irritation testThe acute skin irritation test was performed to check for any signs of inflammation,edema,erythema,or eschar formation from 4 - 48 h after application on rats.The formulated ointments(2% and 5% w/w)were applied dorsally on a shaved area of approximately 500 mm2to two groups of animals,and the third group was considered the normal group[20].

2.6 Experimental animals

Sprague-Dawley rats of either sex(150 - 200 g)were used forin vivostudies.The animals were housed under standard temperature conditions,and exposed to a 10 - 12 h light/dark cycle.The rats were fed a standard pellet diet and water.The animals were acclimatized to laboratory conditions for at least 24 h before experiments were performed[20].The experimental protocol was approved by the Central Animal Ethical Committee of Rashtrasant Tukadoji Maharaj Nagpur University(dated 15/08/2018)(Reg.No.92/1999/CPCSEA,dated 28/04/1999)(Reg.No.IAEC/UDPS/2018/46).The animals were divided into six groups:Group I(standard:1% w/w framycetin sulfate ointment),Group II(simple ointment base:control),Group III and IV(2% and 5% w/w of ethanol extract ointments),Group V and VI(2% and 5% w/w of hydroalcoholic extract ointments).

2.7 Incision wound model

The animals were anesthetized and paravertebral incisions(6 cm)were made through the skin 1.5 cm from the midline back of the rats.After the incision was made,the parted skin was kept together and stitched at 0.5 cm intervals using surgical thread(No.000)and a curved needle(No.11).The thread on both wound edges was tightened to achieve good closure of the wound.The extract ointment,simple ointment base and standard drug(1% w/w framycetin sulfate ointment)were applied once daily.The sutures were removed on the 7thday postwounding.On the 10thday,the wound breaking strength was measured.Histopathological studies were performed on the tissue sections[21-23].

2.8 Excision wound model

A circular piece(300 mm2)of full-thickness skin was excised from the dorsal interscapular region after anesthesia.The group treatments were the same as those for the incision wound model.Wound closure time and wound contraction were the parameters monitored until complete healing.Epithelization time was recorded as the number of days after wounding that were required for the scar to fall off leaving no raw wound behind.The progressive changes were monitored in the wound area by tracing the wound margin on a graph paper[21-23].The percentage wound area was calculated from the following equation:Wound contraction(%)=Healed area/Total wound area×100.

Two-way ANOVA was used to determine the statistical significance between different groups,with six animals in each group; the experimental results were expressed as the mean±SEM.Statistical analysis was performed using GraphPad Prism version 5 software,withP＜0.05 considered to indicate statistical significance.

3 Results

3.1 Extraction

The percentage yields for the petroleum ether,chloroform,acetone,ethanol and the hydroalcoholic extract were 1.29%,1.76%,1.41%,2.18% and 3.23% w/w,respectively.

3.2 Qualitative phytochemical analysis

Preliminary phytochemical analysis of the extracts revealed the presence of sterols,fats and oils in the petroleum ether extract,whereas proteins and amino acids were present in the chloroform and acetone extracts.Tannins,alkaloids,flavonoids,saponins and sugars were present in the ethanol extracts,whereas sterols and higher concentrations of flavonoids were present in the hydroalcoholic extract.The ethanolic and hydroalcoholic extract contained a higher number of phytoconstituents(Table 1).

Table 1 Preliminary phytochemical screening of crude extracts

3.3 Antioxidant effects

The reactivity of the test compound with free radicals was determined by the antioxidant activity of different extracts ofH.isoraLinn.The concentrations of the ethanolic and hydroalcoholic extracts,and Lascorbic acid needed for 50% inhibition free radicals were found to be 86.54 μg/mL,78.05 μg/mL and 54.96 μg/mL,respectively.The inhibition of NO radicals generated from sodium nitroprusside at physiological pH by the ethanolic and hydroalcoholic extracts and L-ascorbic acid was observed at concentrations of 83.13 μg/mL,76.59 μg/mL and 51.69 μg/mL,respectively.The reducing power of the different extracts was found to be directly proportional to the amounts of samples used for treatment(Figure 1,2 and 3).

3.4 Antimicrobial extracts

The hydroalcoholic extract showed concentration dependent broad-spectrum activity against human pathogens,includingEscherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Micrococcus luteusandCandida albicans,which was comparable with that of standard streptomycin(for bacteria)and fluconazole(for fungi),indicating the notable antibacterial and antifungal activity of the extract(Table 2).

3.5 Formulations

The 2% and 5% w/w ointments prepared in simple ointment base were observed for phase separation,and the color and odor were not found to be objectionable.The pH,viscosity and spreadability were found to be within acceptable ranges(Table 3)and the formulation was found to be resistant to centrifugation.

The acute dermal toxicity test showed that the formulated ointment containing extracts ofH.isoraleaves did not induce notable inflammation,swelling,edema,erythema,eschar formation,or any other abnormality.

3.6 Incision wounds

The incision wound study revealed that the topical application of 5% w/w hydroalcoholic extractcontaining ointment resulted in a marked increase in wound breaking strength.Greater contraction was recorded after treatment with the ointment containing the higher concentration of the extract.The histopathological study of the excised skin was performed for a detailed understanding of the stages of wound healing.Histological examination revealed accelerated cellular proliferation,increased collagen synthesis and the formation of a well-keratinized epidermal layer,with the union of dermal fibrous connective tissue in the incision model animals(Table 4,Figure 4 and 5).

3.7 Excision wounds

The untreated rats(control group)displayed complete healing on the 36thday post-wounding.A faster healing rate was observed in the rats treated with 5% w/w hydroalcoholic extract ointment:complete wound contraction occurred on the 24thday post-wounding with 100% wound closure.The period of epithelization and the scar showed the significant healing effect of the topical treatment with the extract-based ointment(Table 5 and Figure 6).

Table 2 Antimicrobial activity of Helicteres isora leaf extracts(zone of inhibition in mm)

Table 3 Measurement of pH,viscosity and spreadability of simple ointment

Table 4 Wound breaking strength

4 Discussion

Herbal drugs are being explored nowadays as potential medicines.In developing countries,a large proportion of the population relies on traditional practices to meet their primary healthcare needs.The present attempt was made to correlate traditional knowledge with modern therapeutics.The investigation was performed to achieve scientific validation of the wound healing potential of different extracts ofH.isoraLinn.leaves.Notable healing activity was observed in the incision and excision wound models,and confirmed by various evaluations,including those of antioxidant and antimicrobial activity.Wound causes bleeding,contraction of tissue,coagulation and inflammation.The healing process was followed by a series of constructive events involving leukocytes and inflammatory mediators,such as histamine,kinins,serotonin,complementary factors,fibrins,fibrinopeptides,platelets and growth factors[24].The pH,viscosity and spreadability of the fabricated ointments assured prolonged contact and increased the absorption of the constituents responsible for the healing effects through the topical biological membrane[25].The incision wound model examined in the present study showed an increase in tensile strength,which may be attributable to the formation and stabilization of collagen fibers.Collagen is a key component in homeostasis,and provides strength and integrity to the wound matrix[26,27].The excision wound model predominantly demonstrated the contraction of the wound.The rate of contraction(centripetal movement of edges to facilitate wound closure)determines the period of epithelization and is directly proportional to the amount of collagen deposited[24].The topical treatment explains the faster rate of wound contraction.The treatment of hydroalcoholic extract may have contributed to the enhancement of collagen formation and epithelization[28].The wound healing effects may be a result of the additive or synergistic effect of the phytoconstituents,which aided the formation of a well-keratinized epidermal layer in a shorter period,compared with the standard treatment(1% w/w framycetin sulfate ointment)[27].

The formation of reactive oxygen species during oxidative stress may delay the wound healing process.The tests for the DPPH radical and NO radicalscavenging activities and reducing power revealed a significant antioxidant effect,which in turn,reduced the formation of free radicals,resulting in no oxidative damage and early healing of the wound[15].The hydroalcoholic extract contained the maximum content of phytoconstituents.The antioxidant effect of the extract,compared with L-ascorbic acid,may be due to the presence of these phytoconstituents,which may effectively reduce the formation of reactive oxygen species.The constituents in extracts that have higher antibacterial and antifungal activity represent non-polar residues[17].Our experiments determined the antimicrobial potential of the extracts againstEscherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Micrococcus luteusandCandida albicans.Thus,it can be concluded that the hydroalcoholic leaf extracts may be a potent natural wound healing compound.The wound healing effect was promoted by the decrease in free radical generation,an antibacterial effect and faster collagen deposition.In further studies,the isolation and characterization of the constituent compounds of such extracts,the elucidation of the molecular mechanisms underlying their action,and acute toxicity testing of the extracts should be performed.

5 Conclusions

The present study provided scientific justification of the traditional claim of the wound healing potential ofH.isoraLinn.leaves.These effects may arise from enhanced cellular proliferation,the formation of well-keratinized epidermal cells,increased collagen synthesis,free radical scavenging,and inhibition of bacterial and fungal growth.The antioxidant,antimicrobial and wound healing potentials of the leaves are likely due to the presence of a combination of phytoconstituents,mainly flavonoids.The study also revealed that the simple ointment base formulation accelerates wound healing.

Table 5 Mean wound area(% wound closure)of different groups in the excision wound model

Acknowledgements

The authors would like to acknowledge Department of Pharmaceutical Sciences,Rashtrasant Tukadoji Maharaj Nagpur University Nagpur for the provision of the necessary facilities to perform the study.

Competing Interests

The authors declare no conflict of interest.