Medicinal Properties of Lilium candidum L. and Its Phytochemicals

Lilium candidum L., known as Madonna, meadow, or white lily, is a bulbous plant from the Liliaceae family, originating in the Middle East. L. candidum has been abundantly used in folk medicine since ancient times to relieve a variety of ailments, including age-related diseases, burns, ulcers, and coughs. The aim of this article is to investigate the anti-inflammatory and anti-diabetic activities of L. candidum extracts and its active phytochemicals. Some active volatile phytochemicals were identified using gas chromatography–mass spectrometry (GC-MS) analysis. Significant (p < 0.001) anti-diabetic properties of the extracts kaempferol, linalool, citronellal, and humulene were demonstrated by an elevation in glucose uptake by adipocytes. The significant (p < 0.01) effect of the plant extracts kaempferol, citronellal, and humulene on the secretion of pro-inflammatory cytokines interleukin 6 (IL-6) and interleukin 8 (IL-8) was demonstrated using enzyme-linked immunosorbent assay. Altogether, L. candidum and its rich collection of phytochemicals hold promising medicinal potential, and further investigations of its therapeutic prospects are encouraged.


Introduction
The prevalence of age-related diseases (ARDs) including cardiovascular diseases, cancer, type 2 diabetes, neurodegenerative diseases, and obesity is rapidly increasing worldwide [1]. Recent studies provide strong evidence suggesting an essential role of chronic inflammation in the pathogenesis of the above-mentioned ARDs [2][3][4]. In addition, genotoxic stress, which is a component of a wide variety of pathological conditions, not only causes extensive DNA damage but also activates pathways leading to chronic inflammation (the ERK, JNK, and p38 MAPK pathways) and transcription of pro-inflammatory cytokines (TNF-α, IL-1β, interleukin 6 (IL-6)), chemokines (interleukin 8 (IL-8)), adhesion molecules (VCAM-1, ICAM-1, P-, E-selectin), and other pro-inflammatory enzymes including iNOS and COX-2 [5]. This in turn leads to an elevated pro-inflammatory status that is likely to set the stage for increased vulnerability to many ARDs [6].
Unfortunately, current therapeutic agents have inadequate efficacy and many serious adverse effects in treating all kinds of ARDs [7]. All possible options should be considered in order to develop new drugs that are more effective. In fact, many medicinal plants are able to cope with inducers and/or consequences of stress such as thermal or oxidative insults, ionizing radiation, DNA damage,

Results
We identified the presence of many novel volatile compounds using gas chromatography-mass spectrometry (GC-MS) analysis, which, to the best of our knowledge, were not mentioned in the literature. Among those, we found linalool, citronellal, caryophyllene, humulene (Figure 1), and neridiol (not represented), which were isolated and identified not only from L. candidum but from other plants as well.  Table 1 provides a list of these phytochemicals and their medicinal properties.
In the case of kaempferol, a significant number of interacting protein targets were found to be associated with three large groups: UGT (uridine 5'-diphospho-glucuronosyltransferase), AHR (aryl hydrocarbon receptor), and CYP1B1 (cytochrome P450 family 1 subfamily B member 1) ( Figure 2). The majority of the targets are connected to the UGT enzymes, which participate in cellular detoxification in different tissues of the digestive system [66]. Their involvement in various cancerassociated processes is also well-known [66,67]. AHR is a ligand-activated transcription factor, which may interact with different pathways regulating cellular homeostasis including cellular regeneration
In the case of kaempferol, a significant number of interacting protein targets were found to be associated with three large groups: UGT (uridine 5'-diphospho-glucuronosyltransferase), AHR (aryl hydrocarbon receptor), and CYP1B1 (cytochrome P450 family 1 subfamily B member 1) ( Figure 2). The majority of the targets are connected to the UGT enzymes, which participate in cellular detoxification in different tissues of the digestive system [66]. Their involvement in various cancer-associated processes is also well-known [66,67]. AHR is a ligand-activated transcription factor, which may interact with different pathways regulating cellular homeostasis including cellular regeneration in the context of aging and diseases [68]. In addition, AHR regulates CYP1B1 [69], which belongs to the cytochrome P450 superfamily of enzymes. Cytochrome P450 plays an important role in cellular detoxification and in the formation of reactive intermediates of thousands of chemicals [70]. Thus, the medicinal effects of kaempferol are apparently mediated by its direct involvement in many pathological processes.
Plants 2020, 9, x FOR PEER REVIEW 4 of 12 in the context of aging and diseases [68]. In addition, AHR regulates CYP1B1 [69], which belongs to the cytochrome P450 superfamily of enzymes. Cytochrome P450 plays an important role in cellular detoxification and in the formation of reactive intermediates of thousands of chemicals [70]. Thus, the medicinal effects of kaempferol are apparently mediated by its direct involvement in many pathological processes. It is well acknowledged that diabetes mellitus (DM) pathogenesis is linked to oxidative stress [71]. Although many phytochemicals from L. candidum have anti-oxidant properties (Table 1), to the best of our knowledge, no research on its anti-diabetic properties has been performed so far. Thus, we investigated the anti-diabetic activity of bulbs and leaves of L. candidum. For this purpose, we treated adipocytes with an ethanolic extract from L. candidum bulbs and leaves, while the glucose uptake of the adipocytes was estimated after these treatments, as previously. L. candidum extracts increased glucose uptake in 3T3-L1A cells better than insulin. Importantly, the anti-diabetic activity of leaf extracts was higher than those from bulb extracts and of insulin, which was used as a positive control (Figure 3). The difference between negative control (untreated adipocytes) and cells that were treated with extracts was highly significant (p < 0.001). All known phytochemicals of L. candidum were tested in this experiment. As seen in Figure 3, kaempferol, linalool, citronellal, and humulene significantly increased glucose uptake in 3T3-L1 adipocytes (p < 0.001). It is well acknowledged that diabetes mellitus (DM) pathogenesis is linked to oxidative stress [71]. Although many phytochemicals from L. candidum have anti-oxidant properties (Table 1), to the best of our knowledge, no research on its anti-diabetic properties has been performed so far. Thus, we investigated the anti-diabetic activity of bulbs and leaves of L. candidum. For this purpose, we treated adipocytes with an ethanolic extract from L. candidum bulbs and leaves, while the glucose uptake of the adipocytes was estimated after these treatments, as previously. L. candidum extracts increased glucose uptake in 3T3-L1A cells better than insulin. Importantly, the anti-diabetic activity of leaf extracts was higher than those from bulb extracts and of insulin, which was used as a positive control (Figure 3). The difference between negative control (untreated adipocytes) and cells that were treated with extracts was highly significant (p < 0.001). All known phytochemicals of L. candidum were tested in this experiment. As seen in Figure 3, kaempferol, linalool, citronellal, and humulene significantly increased glucose uptake in 3T3-L1 adipocytes (p < 0.001).
Elevated levels of circulating inflammatory mediators including cytokines and chemokines are hallmarks of chronic inflammation and progression of metabolic diseases [2]. The extracts and their phytochemicals (identified by us and known) were investigated from the perspective of chronic inflammation. As seen in Figure 4, plant extracts, kaempferol, citronellal, and humulene significantly decreased secretion of IL-6 and IL-8 cytokines by senescent human pulmonary fibroblasts (HPFs) and human dermal fibroblasts (HDFs) as measured by enzyme-linked immunosorbent assay (ELISA) (p < 0.01). Elevated levels of circulating inflammatory mediators including cytokines and chemokines are hallmarks of chronic inflammation and progression of metabolic diseases [2]. The extracts and their phytochemicals (identified by us and known) were investigated from the perspective of chronic inflammation. As seen in Figure 4, plant extracts, kaempferol, citronellal, and humulene significantly decreased secretion of IL-6 and IL-8 cytokines by senescent human pulmonary fibroblasts (HPFs) and human dermal fibroblasts (HDFs) as measured by enzyme-linked immunosorbent assay (ELISA) (p < 0.01).

Discussion
Taking into account the therapeutic properties of L. candidum known from folk tradition, we expected to discover anti-inflammatory and anti-diabetic activities in L. candidum extracts. It is important to emphasize here that senescent cells (HPFs and HDFs) are one of the most widely used

Discussion
Taking into account the therapeutic properties of L. candidum known from folk tradition, we expected to discover anti-inflammatory and anti-diabetic activities in L. candidum extracts. It is important to emphasize here that senescent cells (HPFs and HDFs) are one of the most widely used models for studying inflammatory processes. Accumulation of senescent cells in an organism leads to disruption of tissues and cellular structure and function [72]. The phenomenon of cellular senescence has been demonstrated to play a causal role in driving ageing [72] and chronic diseases [7,9,72]. Namely, the above-mentioned cells are accepted models [7,9] used to investigate the effect of plant extracts and pure compounds on inflammation.
Our hypothesis was confirmed, as the plant extracts indeed possess anti-inflammatory and anti-diabetic properties (Figures 3 and 4). We also identified selected phytochemicals behind these properties: anti-diabetic properties were associated with kaempferol, linalool, citronellal, and humulene ( Figure 3). In turn, kaempferol, citronellal, and humulene had a significant impact on the secretion of pro-inflammatory cytokines IL-6 and IL-8 (p < 0.01), (Figure 4). It is known that the above-mentioned cytokines are directly connected to delay in the wound healing process and are known to be secreted by HPFs [9,73]. Thus, our results might provide an explanation of the effect of L. candidum extract on wound healing, as described in ethnopharmacological publications [13,14]. Further investigation is important in order to establish which phytochemicals are connected with wound healing directly, and to elucidate their modes of action.
Although the therapeutic activity of L. candidum extract and its phytochemicals was demonstrated [74][75][76], their mechanism of action is still unknown.
With its rich collection of phytochemicals, L. candidum has promising therapeutic potential. Our results demonstrate a bright future for this plant and its compounds as prophylactic and therapeutic agents. However, additional clinical studies are warranted in order to establish the effectiveness of compounds from L. candidum in the treatment of chronic diseases.

Bioinformatic Assessment
For bioinformatical assessment, we selected the STITCH database found at http://stitch.embl.de/. This database contains most of the available information regarding proteins' interaction with different chemicals. It can be searched according to the name of the chemical or its PubChem ID [63][64][65].

Preparation of Plant Material
Aerial parts of L. candidum were collected from the greenhouse at Ben-Gurion University of the Negev, Beer-Sheva, Israel. Leaves, flowers, and bulbs of L. candidum were dried by lyophilization and grounded for GC-MS analysis.
Ethanolic extracts were prepared from leaves and bulbs of L. candidum as described previously [27]. Plant tissues were homogenized, incubated at room temperature for 48 h in ethanol, centrifuged at 2000 rpm for 10 min, and the supernatant was evaporated by lyophilization. The pellet was dissolved in a minimal amount of 95% ethanol (0.5 mL) and diluted with water to a final concentration of 10 mg/mL.

GC-MS Analysis
Identification of volatile compounds is described in our previous publication [7].
Human pulmonary fibroblasts (HPFs), human dermal fibroblasts (HDFs), and 3T3-L1 adipocytes were propagated in DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% antibiotic mixture, which included a combination of penicillin, streptomycin, and nystatin. The cells were grown in an incubator at 37 • C. The relative humidity was set at 95%, and the CO 2 content was 5%.

Measurement of Anti-Diabetic Activity
Ethanolic bulb and leaf extracts of L. candidum were tested for their anti-diabetic properties. The glucose uptake of the 3T3 adipocytes was determined as described previously [7,71]. Briefly, before the measurement, adipocytes were transferred to low-glucose serum-free media. After an overnight incubation, the cells were treated with bulb or leaf ethanol extracts of L. candidum for 1 h. For negative control, adipocytes were treated with the vehicle only. Insulin at a concentration of 100 nM was used as a positive control. After the 1-h incubation, the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) was added for 30 min. The cells were then rinsed with PBS, and the fluorescence of intracellular 2-NBDG with excitation at 467 nm and emission at 538 nm was measured using a fluorescence microplate reader (POLARstar Omega, BMG LABTECH GmbH, Ortenberg, Germany).

Measurement of Interleukin Release
Senescent HPFs or HDFs were treated with specified concentrations of the extracts or compounds. After 3 days of exposure, we collected the medium and measured the concentrations of interleukin 6 and 8. The measurement was performed with R&D Systems ELISA kits according to the manufacturer's instructions. For each experiment, standard curves were built, and the concentrations of interleukins were calculated.

Statistical Analysis
Experiments were repeated at least three times. All data were analyzed using Statistica, version 7, for Windows software (StatSoft, Inc., Tulsa, Oklahoma), and p < 0.05 was chosen as the minimal acceptable level of significance. Simple regression models were subsequently used to eliminate non-significant effects. Values are presented as means ± SD.