Potential Emphasis of Formulated Herbal Cream on Wound Healing

Shivhare et al Asian Journal of Pharmaceutical Research and Development. 2020; 8(1): 73-78 Available online on 15.02.2020 at http://ajprd.com Asian Journal of Pharmaceutical Research and Development Open Access to Pharmaceutical and Medical Research © 2013-20, publisher and licensee AJPRD, This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited Open Access Research Article Potential Emphasis of Formulated Herbal Cream on Wound Healing Yogesh Shivhare*, Alok Pal Jain Sarvepalli Radhakrishnan University, Bhopal (M.P.) India ABSTRACT Aim: The present study was to formulate and evaluate the herbal cream comprising extracts of different medicinal plants. Experimental: The aqueous extract of all selected plants (Chenopodium album, Coccinia indica, Momordica dioica, Praecitrullus fistulosus and Trichosanthes dioica) were prepared by successive solvent extraction using soxhlet apparatus. Topical herbal cream was formulate and examined on different parameters like colour, consistency, pH, viscosity, spreadability and stability. Pharmacological evaluation of formulated herbal cream was done with excision and incision wound models. Results and Conclusion: Based on the resultant findings obtained from the different evaluation parameters, it can be concluded that prepared herbal formulation was stable and safe to use in wound healing activity. Keywords: Excision, Incision, Evaluation, Herbal Cream A R T I C L E I N F O: Received 31 Oct. 2019; Review Completed 20 Dec. 2019; Accepted 08 Feb. 2020; Available online 15 Feb. 2020 Cite this article as: Shivhare Y*, Jain A P, Potential Emphasis of Formulated Herbal Cream on Wound Healing, Asian Journal of Pharmaceutical Research and Development. 2020; 8(1):73-78. DOI: http://dx.doi.org/10.22270/ajprd.v8i1.639 *Address for Correspondence: Yogesh Shivhare, Sarvepalli Radhakrishnan University, Bhopal (M.P.) India. INTRODUCTION W ounds are defined as physical, chemical or thermal injuries that outcome in an opening or breaking in the reliability of the skin or the interruption of anatomical and functional integrity of living tissues.1 Wound healing is the process of renovate that follows injury to the skin and other soft tissues.2 The effectual management of wounds will reduce the number of complications and allow speedy return to normal function. Therefore, the aim of treating a wound is to either shorten the time required for healing or to minimize the undesired consequences.3 Attention should be aimed at towards discovering an agent, which will accelerate wound healing. The use of medicinal plants in the management of acute and chronic wounds is common in most conventional medicine practices in the world. Herbal medicines are widely used all over the world. However, there is still a huge gap between best scientific evidence and what people actually use to treat a disease. The lack of acceptance of herbal medicines by medical community is based both on false and true grounds. It is important that study of herbal medicine is offered to all health care professionals.4 The use of natural products brings a challenge to their expansion as drugs. Indeed, important medicines were developed and marketed in the past as complexes because it was not possible to purify the individual components at a sufficient scale.5 Clearly, many technologies required for ISSN: 2320-4850 the successful discovery, development and production of herbal medicines are not yet in place and efforts required for their emergence need to be substantial.6 Nevertheless, while new technology is not in place, ethnopharmacology still is more efficient to discover new drugs from plants. Medicinal plants Chenopodium album, Coccinia indica, Momordica dioica, Precitrullus fistulosus and Trichosanthes dioica were used in the formulation of herbal cream for the assessment of wound healing activity. Hence the present work was designed to formulate and evaluate an herbal cream containing different plant extracts for potent healing activity in various wound models. MATERIAL AND METHODS Collection of plant materials All the selected plants (Chenopodium album, Coccinia indica, Momordica dioica, Praecitrullus fistulosus and Trichosanthes dioica) leaves were collected from available sources. The leaves were cleaned, sorted and air dried under shade at room temperature. The dried leaves were converted into powder and kept in an air tight polyethylene bag until needed for the extraction process. [73] CODEN (USA): AJPRHS Shivhare et al Asian Journal of Pharmaceutical Research and Development. 2020; 8(1): 73-78 Preparation of plant extracts Viscosity The powdered materials of all the plants (Chenopodium album, Coccinia indica, Momordica dioica, Praecitrullus fistulosus and Trichosanthes dioica) were extracted successively with water solvent to obtain aqueous extract of all plants by using soxhlet apparatus. Obtained extracts were dried by removing the solvent under vacuum. The dried extract of each plant was weighed and stored for further use. In the current study, viscosity of formulated herbal cream was determined by book field viscometer at 100 rpm using spindle No 7. Extrudability The formulations were filled in collapsible tube container. The extrudability was determined in terms of weight of formulations required to extrude 0.5cm of ribbon of ointment in 10 seconds. Formulation of herbal cream Spreadability Ingredient of oil phase (A) was melted in a beaker by using water bath on stable stirring. Components of aqueous phase (B) were mixed together and warmed to about same temperature of oil phase (up to 700C). The preservative methyl paraban and propyl paraban were added into aqueous phase and heated. Then oil phase was added to water phase little by little on stable stirring. Aqueous extracts of each plant were mixed with uniform stirring in the formulated cream base. Water was added finally and mixed.7-8 The composition of formulated herbal cream is given in table 1. In the current investigation, sample of the formulated cream was sandwiched between 2 slides. A weight of 100gm was placed on upper slide. The weight was removed and extra formulation was scrapped off. The lower slide was fixed on board of apparatus and upper slide was fixed with non flexible string on which load was applied. Time taken by upper slide to slip off was noted down. Spreadability= m ×l /t m = weight tied to the upper slide, l =length of glass slide, t =time taken in seconds. Table 1: Composition of formulated herbal cream Oil Phase Aqueous Phase Plant Extracts Ingredients Qty.% w/w Stearic acid 1% White Beeswax 5% Cetyl alcohol 3% Propylene glycol 5% Glycerine 4% Methyl Paraban 0.5% Propyl Paraban 0.5% Water Upto 100% Chenopodium album 5% Coccinia indica 5% Momordica dioica 5% Praecitrullus fistulosus 5% Test for thermal stability The formulated cream was inserting into glass bottle with the help of spatula, and taped to settle to the bottom. Filled up to two-third capacity of bottle and insert plug and tighten the cap. Filled bottle was kept erect inside the incubator at 4˚±1° for 48 hrs. The sample passed the test, if on removal from the incubator shows no oil separation or any other phase separation. Test for microbial growth in formulated creams The formulated creams were inoculated on the plates of agar media by streak plate method and a control was prepared by omitting the cream. The plates were placed in to the incubator and are incubated at 370C for 24 hours. After the incubation period, plates were taken out and check the microbial growth by comparing it with the control. Pharmacological evaluation of formulated herbal cream Experimental animals and Treatment protocol Albino wistar rats weighing 100-150 g were used. The animals were maintained in well ventilated animal room for a period of 7 days prior to the experimental period. They were kept on standard pellet diet and water ad libitum. Institutional animal ethical committee permitted the study. Animals of either sex were divided into three groups for each excision and incision wound model. The grouping of animals was shown in table 2. Evaluation of formulated herbal cream The formulated herbal cream was evaluated in terms of various evaluation parameters. 9-11 Organoleptic evaluation The formulated herbal cream was evaluated for its organoleptic properties like color, odor and consistency manually. Table 2: Grouping of Animals Wash ability Group I Control (Cream Base) The product was applied on hand was observed under running water. Group II Standard (0.1% silver sulfadiazine cream) Group III Herbal cream formulated with aqueous extracts of all selected plants pH The pH of formulated herbal cream was determined by using digital pH meter. About 1g of the cream was weighed and dissolved in 100 ml of distilled water and its pH was measured. ISSN: 2320-4850 Excision Wound Model An excision wound was inflicted by cutting away a 300 mm2 full thickness of skin from a predetermined shaved area. Rat’s wounds were left undressed to the open [74] CODEN (USA): AJPRHS Shivhare et al Asian Journal of Pharmaceutical Research and Development. 2020; 8(1): 73-78 environment. The cream base, standard drug cream (0.1% Silver sulfadiazine) and formulated herbal cream were applied topically to the control group, standard group and treated group respectively till the wound was completely healed. In this healing evaluation model, wound contraction and epithelialization period was monitored. Wound contraction was measured as percent contraction in each 4 days after wound formation.12 in control rats as shown in fig 1. The increased wound contraction in the formulated herbal cream treated group may be due to the enhanced activity of fibroblasts. Effect of formulated herbal cream on epithelialization period in excision wound model The epithelialization time was measured from initial day. The epithelialization time was significantly reduced in all the groups. The time taken for complete epithelialization of the excision wound was more than 24 days in control, 10 days in standard (silver sulfadiazine 0.01%) and 11 days in formulated herbal cream treated group. Early dermal and epidermal regeneration in the treated group confirmed that formulated herbal cream had a positive effect toward cellular proliferation and epithelialization. The findings were recorded in table 4. Incision Wound Model All animals were anaesthetized before wound creation and two full thickness paravertebral long incisions were made through the skin at the distance of about 1 cm from midline on each side of the depilated back of rat. The both edges kept together and stitched with black silk surgical thread (no. 000) and a curved needle (no. 11) was used for stitching. The continuous threads on both wound edges were tightened for good closure of the wound. After stitching, wound was left undressed. Cream base, standard cream and formulated herbal cream was applied daily up to 9 days. The sutures were removed when wounds were cured thoroughly. Tensile strength, hydroxyproline content in healed tissues was determined.13-14 Effect of formulated herbal cream on hydroxyproline content in incision wound model Formulated herbal cream groups showed significant increased in hydroxyproline content when compared to control group (P< 0.01). The hydroxyproline content of granulated tissue in different groups were 18.12 ± 0.021 mg/g tissue (control), 70.25 ± 0.014 mg/g tissue (standard), 68.26± 0.002 mg/g tissue (formulated herbal cream of aqueous extract). Increase in hydroxyproline content showed probably the formation of new blood vessels which accelerates the process of healing. The findings were recorded in table 4. Histopathological studies The regenerated tissue from the healing wounds was collected and placed in 10% buffered formalin for histopathological studies. Sections from the regenerated tissue were examined for epithelialization, inflammation, collagen and fibroblasts.15 Effect of formulated herbal cream on tensile strength in incision wound model Statistical analysis Tensile strength for all the treated groups on 10 th, 12th, 14th day was found to be significant (P< 0.01) than control group. The mean tensile strength of granulated tissue in different groups was 385.23 g/mm2 (control), 610.15 g/mm2 (standard) and 442.72 (formulated herbal cream of aqueous extract). Tensile strength reflects the sub dermal organization of the collagen fibres in wound. The findings were recorded in table 4. The values were calculated as mean ± S.E.M. P< 0.01 or above was considered to be significant. RESULTS AND DISCUSSION Formulated herbal cream was evaluated by various standard parameters and found acceptable in all limits. The colour was found greenish brown, semisolid consistency, easily washable with good extrudability. The pH, viscosity and spreadability of formulated herbal cream was found 6.10, 5905 and 7.8 respectively. There is no microbial growth and oil separation in prepared formulation. All the findings were reported in table 3. Histopathological studies in excision and incision wound model In control wister rats excision and incision type of wounds shown incomplete healing as in poor fibroblasts cells, collagen fibres and blood vesicles while increased fibroblasts cells collagenation and angiogenesis occurs in standard (silver sulfadiazine 0.01%) and formulated herbal cream treated group as shown in figure 2. Effect of formulated herbal cream on % wound contraction in excision wound model A better healing pattern with complete wound closure was observed in 16 days by standard and formulated herbal cream treated groups while it was about more than 24 days Table 3: Evaluation findings of formulated herbal cream ISSN: 2320-4850 S.N. Parameter Findings 1 Colour Greenish brown 2 Consistency Semisolid 3 Wash ability Easily 4 Extrudability Good 5 pH 6.10 6 Viscosity 5905 7 Spreadability 7.8 8 Stability Stable No Oil Separation 9 Microbial Growth Absent [75] CODEN (USA): AJPRHS Shivhare et al Asian Journal of Pharmaceutical Research and Development. 2020; 8(1): 73-78 Table 4: Findings of pharmacological evaluation of formulated herbal cream Groups Epithelialization Period Hydroxyproline Content (In Days) (mg/g tissue) G-I (Control) 22 18.12 ± 0.021* G-II (Standard) 10 70.25 ± 0.014* G-III (Herbal Cream) 11 68.26 ± 0.002* n= 6 albino rats per group, values are represents mean± SEM *P< 0.01(comparison of I & II with III) A B Tensile Strength (g/mm2) 385.023 ± 2.167* 610.15 ± 2.717* 442.72 ± 2.124* C Figure 2: Histopathological characteristics of rat skin by treatment with cream base (A), Standard (B), Formulated herbal cream (C) in Excision wound model 2. Priya KS, Gnanamani A, Radhakrishnan N, Babu M. Healing potential of Datura alba on burn wounds in albino rats. Journal of Ethnopharmacology. 2002; 83:193-199. 3. Myers KA, Marshal RD, Friedin J. Principles of Pathology in Surgery, 1st edition. Blackwell Scientific Publications London. 1980; 58-82. 4. Fabio Carmona, Ana Maria, Soares Pereira. Herbal medicines: old and new concepts, truths and misunderstandings. Brazilian Journal of Pharmacognosy. 2013; 23(2):379-385. 5. Koehn FE, Carter GT. The evolving role of natural products in drug discovery. Nature Review Drug Discovery. 2005; 4 (3):206-220. 6. Raskin I, Ripoll C. Can an apple a day keep the doctor away. Current Pharmaceutical Design. 2004; 10:3419-3429. 7. Das Trailokya, Debnath Jiban, Nath Bipul, Dash Suvakanta. Formulation and evaluation of an herbal cream for wound healing activity. International Journal of Pharmacy and Pharmaceutical Sciences. 2014; 6(2):693-697. 8. Nair Sujith S, Mathew Molly, Sreena K. Formulation and Evaluation of Herbal Cream containing Curcuma longa. International Journal of Pharmaceutical and Chemical Sciences. 2012; 1(4):1362-1368. 9. Matangi Surya Prabha, Mamidi Santhosh Aruna, Gulshan MD, Raghavamma STV, Nadendla Rama Rao. Formulation and Evaluation of Anti-Aging Poly Herbal Cream. International Journal of Pharmaceutical Sciences Review and Research. 2014; 24(2):133-136. 10. Negi A, Sharma M, Singh M. Formulation and Evaluation of an Herbal Anti-Inflammatory Gel Containing Eupatorium Leaves Extract. Journal of Pharmacognosy and Phytochemistry. 2012; 1:112117. CONCLUSION Plants or chemical entities derived from plants need to be identified and formulated for treatment and management of wounds. In this direction, a number of herbal products are being investigated at present. Obtained findings collectively demonstrated that formulated herbal cream possesses wound healing activity and this justifies its use for treatment of wounds. 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