# The Effects of _Anthyllis vulneraria_ Hydroalcoholic Leaf Extract as an Adjuvant in Wound Healing
## 2 Materials and Methods
### Plant Source  
_A. vulneraria_ seeds were acquired from an Internet gardening shop in Germany ([[https://www.jelitto.com/Seed/Perennials/ANTHYLLIS+vulneraria+var+cocinea+Portion+s.html](https://www.jelitto.com/Seed/Perennials/ANTHYLLIS+vulneraria+var+cocinea+Portion+s.html)]([https://www.jelitto.com/Seed/Perennials/ANTHYLLIS+vulneraria+var+cocinea+Portion+s.html](https://www.jelitto.com/Seed/Perennials/ANTHYLLIS+vulneraria+var+cocinea+Portion+s.html)), accessed on 24 October 2022).  
Both indoor and outdoor plantings were used to grow the plant material. Seeds were planted in February-March of 2023. Indoor plants were grown in 20 \(\times\) 50 cm planter boxes with drainage, with 25 cm of soil, watered at a regular interval, under natural light. The soil used was peat-based potting soil (black peat, sphagnum peat, composted fertilizer of bovine origin, clay) with 40% minimum organic material with an NPK of 0.3:0.1:0.1. Watering was conducted when the topsoil was dry, approximately once a week during the spring months, and once every three days in the summer months.  
The outdoor plants were cultivated in open-field conditions in the southern region of Romania. They were exposed to natural light and watered at regular intervals to prevent excessive soil dry-out. For the soil, based on the geographic location (approximately 45.14\({}^{\circ}\) latitude, 24.38\({}^{\circ}\) longitude) and soil characteristics, the soil was determined to be eutric Cambisol (palebrown, ochrA) based on the SRCS-extended WRB description [26]. Watering was conducted on a similar schedule to the indoor conditions.  
During the first year of vegetative growth, the plants developed only foliar structures, with no evidence of inflorescence formation. Plant material, namely leaves, was collected at regular intervals (every 3-4 weeks, depending on the growth rate) from both indoor and outdoor-grown plants. The harvested plant material was weighed and air-dried in a cool, dry environment, protected from direct light, until a constant mass was achieved. Once dried, the material was transferred to dry paper bags, labeled according to lot type (indoor or outdoor) and collection date, and stored under appropriate conditions until the beginning of the study.  
All plant material used in this study was certified as _A. vulneraria_ by [PERSON], Assistant Professor PhD, from the Department of Pharmaceutical Botany, University of Medine and Pharmacy \"[PERSON]\" Cluj-Napoca, Romania. A voucher specimen (58.16.1.1.2) was deposited in the Herbarium of the Department of Pharmaceutical Botany, Faculty of Pharmacy at \"[PERSON]\" University of Medicine and Pharmacy.  
It is noteworthy to mention the marked difference in leaf size observed between the _A. vulneraria_ specimens cultivated under controlled indoor and outdoor conditions and wild populations collected from local mountain regions, where the species spontaneously grows. Both the indoor and outdoor plants displayed more well-developed leaves compared to their wild counterparts. As illustrated in Figure 1, these differences can probably be attributed to increased nutrient availability and warmer growing conditions in the cultivation environments than in the natural habitat.  
Figure 1: Size comparison of the leaf specimens of _A. vulneraria_ found in three growing climates: (**A**) wild specimens harvested from spontaneous Romanian flora from the Carpathian Mountains area; (**B**) indoor grown specimens; and (**C**) outdoor grown specimens of _A. vulneraria_, grown in the southern region of Romania.