Journal of Pharmaceutical Research International 20(6): 1-13, 2017; Article no.JPRI.39090 ISSN: 2456-9119 (Past name: British Journal of Pharmaceutical Research, Past ISSN: 2231-2919, NLM ID: 101631759) In vitro Proliferation and Wound Healing Effects of Narcissus tazetta L. Bulb on Primary Human Dermal Fibroblasts Maryam Rameshk1,2, Fariba Sharififar1*, Mitra Mehrabani1, Abbas Pardakhty3 and Alireza Farsinejad4 1Herbal and Traditional Medicines Research Center, Department of Pharmacognosy, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. 2Department of Traditional Pharmacy, Faculty of Persian Medicine, Kerman University of Medical Sciences, Kerman, Iran. 3Pharmaceutics Research Center, Neuropharmacology Institute, Kerman University of Medical Sciences, Kerman, Iran. 4Department of Hematology and Blood Banking, Faculty of Allied Medical Sciences, Kerman University of Medical Sciences, Kerman, Iran. Authors’ contributions This work was carried out in collaboration between all authors. Author FS designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors MM and MR managed the analyses of the study. Authors AP and AF managed the literature searches. All authors read and approved the final manuscript. Article Information DOI: 10.9734/JPRI/2017/39090 Editor(s): (1) Barkat Ali Khan, Department of Pharmaceutics, Gomal University, Dera Ismail Khan, Pakistan. Reviewers: (1) Naveed Muhammad, Hazara University, Pakistan. (2) Katarzyna Sosnowska, Medical University of Białystok, Poland. (3) Nyoman Kertia, Gadjah Mada University, Dr. Sardjito Hospital, Indonesia. Complete Peer review History: http://www.sciencedomain.org/review-history/23057 Received 9th November 2017 th Original Research Article Accepted 30 January 2018 Published 6th February 2018 ABSTRACT Aims: Narcissus tazetta L. bulbs have been used in Traditional Persian medicine (TPM), as "Zaroor" for wound healing. Toward quality assurance of the plant, pharmacognostic and phytochemical studies, physicochemical characterization, and healing effects of "Zaroor" were studied. Study Design: Original Research Article. _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected]; Rameshk et al.; JPRI, 20(6): 1-13, 2017; Article no.JPRI.39090 Place and Duration of Study: The study took place in herbal and traditional medicines research center at Faculty of Pharmacy and stem cell research center at Faculty of Allied Medical Sciences of Kerman, Iran from March 2016 to September 2017. Methodology: Total flavonoid content (TFC) of the plant was measured and high-performance thin-layer chromatography (HPTLC) fingerprint profiling was planned for qualitative assessment of the plant with reference to rutin as the marker compound. Antioxidant activity was studied using DPPH and FRAP assay. Proliferation and wound healing effect of the plant was evaluated on the primary human dermal fibroblast by neutral red and scratch assay. Results: The pharmacognostic studies and physicochemical characteristics indicated characters, which are of diagnostic value for plant standardization and quality control. HPTLC chromatogram of the plant extract confirmed the presence of rutin in comparison to Rf value of the standard. Maximum inhibition of DPPH radical and IC50 value was estimated at 10000 µg/ml (99.89%) and 2379.82±37.59 µg/ml respectively. In FRAP test, the antioxidant value was estimated 0.29±0.02 mM/mg SO4Fe. Plant extract exhibited no significant effects on cell proliferation in HDF cells after 48 hr treatment using neutral red assay. The greatest reduction in gap width was considered after 48 hr at 1.562- 6.25 µg/ml. This activity was significantly different from untreated cells as a control (p<0.01). Conclusion: N. tazetta would be effective for wound healing through different mechanisms such as anti-inflammatory and antioxidant effect, which needs to be studied in more details. Keywords: Narcissus tazetta; wound healing; scratch assay; human dermal fibroblast; antioxidant; HPTLC. ABBREVIATIONS wound healing. Narcissus tazetta L. bulb from Amaryllidaceae family with the common name of HDFs : Primary human dermal fibroblasts “Narjes” has been confirmed in several sources NR : Neutral red of TPM [4-8]. This ornamental plant has a white PEN : Percolated extract of N. tazetta and yellow flower that appear from January to TPM : Traditional Persian medicine April and have desired odor [9]. Amaryllidaceae SC : Stratum corneum plants are predominantly tropical or subtropical TFC : Total flavonoid content plants, while N. tazetta occurs primarily in Southwestern Europe and Mediterranean region, 1. INTRODUCTION with a center of diversity in the Iberian Peninsula Skin is one of the most important organs, which (Spain and Portugal) as well as in Iran and can protect the body against a variety of external Kashmir [10]. and internal harmful agents such as microbes, toxins, temperature, UV radiation, and especially Presence of alkaloids such as galantamine [11], water retention. Stratum corneum (SC), the outer lycorine [12], flavonoids like rutin, quercetin and layer of skin, is composed of different parts such kaempferol, phenolic acids [13] and a as cholesterol, proteins, fatty acids, and glycoprotein called lectin [14] has been reported ceramides [1]. Alteration in SC integrity causes in the plant bulbs. major skin dysfunction. External factors such as abrasion, inflammation, wound, and burn can According to TPM: "the bulb of Narcissus is hot damage skin and severely distress human life and dry in the third and has been proposed as a and health [2]. There are different types of the purifier, absorbent, and anthelmintic". The plant wounds like pressure ulcer, bedsore wounds and purifies the uterus and improves the internal and diabetic foot ulcer that affect importantly lifestyle external ulcers. In TPM sources, a preparation of of patients. Such wounds cause induction of N. tazetta bulb powder known as "Zaroor" is pain, deformity, disability, and dependence on recommended for wound healing. "Zaroor" has others and even osteomyelitis and death an astringent effect and can stop bleeding in [3]. Considering the limitations and topical uses. In TPM, the term "Zaroor" includes unresponsiveness of current drugs, the use of the fine powder of dried plants that sprinkled on traditional treatments and medicinal plants is organ for treatment aims [4,5,7]. A combination desired. of "Zaroor" with honey and /or vinegar has been recommended for burns, nerve injury, and In TPM (Traditional Persian Medicine), different internal ulcers. Moreover, "Zaroor" can be used natural treatments have been suggested for to remove skin stain [6,8]. 2 Rameshk et al.; JPRI, 20(6): 1-13, 2017; Article no.JPRI.39090 The aim of the present study is the 2.4.1 Maceration pharmacognostic study, preparation, standardization and quality control of "Zaroor" The plant materials (30 g) were macerated in and studying its healing effects on primary methanol 80% for 72 hr. The extract was human dermal fibroblast (HDF) migration and replaced with fresh solvent every 24 hr [16]. proliferation by scratch wound assay. Finally, it was filtered, pooled, concentrated under vacuum, dried, and weighed. 2. MATERIALS AND METHODS 2.4.2 Percolation 2.1 Chemicals and Reagents Plant materials (85 g), were mixed with 80% Rutin, ethyl acetate, formic acid, acid acetic methanol, transferred to a percolator, and glacial, and Dimethyl sulfoxide (DMSO) were allowed to stand for 24 hr, then to which the purchased from Merck (Germany). 1, 1-Diphenyl- same solvent was added (totally 9 liters). 2-picrylhydrazyl (DPPH); butylated Percolation was set to a flow rate of 10 drops/min hydroxytoluene (BHT); 2, 4, 6-tri (2-pyridyl)-s- [16]. Extraction was continued until the positive triazine (TPTZ) and phosphate buffered saline test of an eluted extract of alkaloid reagent. (PBS) were supplied by Sigma–Aldrich. Dulbecco’s modified eagle’s medium (DMEM) 2.4.3 Sonication and fetal bovine serum (FBS) were used of grade Gibco (Germany). Penicillin-G, streptomycin and In this step, 10 g of dried powder was mixed with amphotericin B were purchased from Biostar 150 ml of methanol 80% and was put in bath (Argentina). Primary human dermal fibroblasts ultrasonic for half an hour, three times. The (HDF) were prepared from Afzalipour School of extract was filtered through Whatman filter paper Medicine in Kerman. The other chemicals were and dried in an oven at 50 °C after concentrating of analytical grade. the extract under vacuum. 2.2 Plant Materials 2.4.4 Warm extraction Fresh bulbs were purchased from a local market This method was carried out for 1, 2 and 4 hours in Kerman (Iran) in December of 2015. The bulbs extraction. An amount of 5 gr of dried powder were skinned, chopped, and dried in the shade at was mixed with 50 ml methanol 80% separately room temperature, milled, and passed through a and was put in Mary Bath respectively for 1, 2 sieve (mesh 60). and 4 hours. The extracts were filtered through Whatman filter paper, dried in oven 50 ̊C after 2.3 Authentication concentrating the extract by rotary and weighed. For authentication of N. tazetta, the bulbs were 2.5 Total Flavonoid Content planted in December 2015 in the Botanic garden of the school of Pharmacy, Kerman University of Total flavonoid content was determined by Medical Sciences, Iran. After flowering in March colorimetric assay [17].