Industrial applicability of Lilium candidum flowers

Determination of Biochemical Activities of White Lily (Lilium Candidum) and Investigation of Its Usability for Industrial Purposes

In this study, Lilium candidum flowers grown in rural Muğla were collected in May-June and their biochemical activities were investigated. Proteases catalyze the hydrolysis of proteins into peptides and amino acids. It is one of the most important enzyme groups in both industrial and biochemical applications. To purify protease enzyme from Lilium candidum flowers, (NH4)2SO4 precipitation and CM-Sephadex ion exchange chromatography were used. The enzyme's optimum pH and temperature values and KM and Vmax values for casein, azocasein, gelatin, hemoglobin, and azoalbumin substrates were determined. SDS-PAGE was used to verify the purity of the purified protease enzyme. Using gel filtration chromatography, the molecular weight of the enzyme was calculated to be 29.7 kDa. The effects of cations Hg2+, Mn2+, Ca2+, Fe2+, Fe3+, Ni2+, Mg2+ (10 mM, 1 mM, 0.1 mM) and compounds EDTA, SDS, β-mercaptoethanol and iodoacetamide on enzyme activity were also examined. The amounts of vitamin A, vitamin E and vitamin C contained in Lilium candidum flowers were determined as (64 ± 0.23 μg/100 mg), (36 ± 0.15 μg/100 g), and (16 ± 0.32 mg/100 g), respectively. Phenolic compounds, B-group vitamins and free amino acid content were determined using UPLC-ESI-MS/MS method. Volatile and aroma-giving components were analyzed using Headspace GC/MSD method. Antioxidant activities of three different extracts (hexane, ethyl acetate and ethyl alcohol fractions) obtained from Lilium candidum flowers were also determined. For this purpose, total antioxidant, DPPH free radical scavenging and ABTS cation radical scavenging activities were determined. The results obtained showed that Lilium candidum flowers are rich in phenolic compounds. It was found that kaempferol and ferulic acid are present in significant amounts in Lilium candidum flowers. Per 100 grams of flowers, essential amino acids phenylalanine (568 μg), isoleucine (547 μg), tryptophan (520 μg), valine (275 μg) and leucine (81 μg) were also found. According to the results of this research, Lilium candidum flowers contain protease enzymes, all free amino acids, aroma compounds, vitamins (A, C, E and B-group) and phenolic compounds. Due to this rich content, it was concluded that the flower would be an important source in the pharmaceutical and cosmetic industries. Introduction

History of Cosmetics

Cosmetics began with humanity's origin. Its beginning dates back to Adam and Eve. This expression shows how important beauty and grooming are to people. Humans have long benefited from the healing and beautifying properties of plants, oils and elixirs. They were nourished by plants, healed and survived. The natural way of life, passed down from generation to generation, has reached today and has become a lifestyle in modern times. Consuming natural products, personal care with natural cosmetic materials, taking dietary supplements have become an integral part of urban life. All of these are essential elements for natural and healthy living. Natural cosmetics aim at caring for the human body and beautiful enhancement in a healthy way through natural substances. This can only be achieved with materials that are friendly to skin and environment. Natural cosmetics help and revitalize our skin functions. It provides gentle care and helps keep skin of all ages healthy. Natural cosmetics contribute to achieving harmony between body and spirit. Nature, the source of life, provides us with everything we need to live a healthy and long life. What matters is being aware.

Characteristics of Lilium Plant

The plant from the Liliaceae family is distributed in temperate Asia, Europe and North America. It is known that there are over 100 species in the world. In Turkey, 5 species, 1 subspecies and 4 varieties of this flower have been identified (Smyth et al., 1980). The species of the plant in our country are Lilium candidum (White Lily), Lilium martagon (Turkish Lily), Lilium carniolicum, Lilium ciliatum (Ciliate Lily), Lilium monadelphum. Ten different species of the plant have also been defined in the European flora (Tutin et al., 1980).

General Characteristics of Lilium Candidum (White Lily)

The stem is 50–130 cm in height. The flowers are funnel-shaped, snow-white in color and have 2–12 flowers. The leaves are spiral arrangement, shiny and hairy. Filaments are 45–50, rarely 57 mm, and anthers are 9–11 mm with golden-yellow pollen. Style is 35–50, rarely 60 mm in length. The flowering period is May (Davis, 1984). The plant is distributed in sandy and rocky soils, stony wooded areas and grassy areas, at altitudes between 10–1300 m (Davis, 1984). Lilium candidum is a perennial plant. The bulb of the plant is garlic-white in color, and the flowers have a very pungent fragrance. It is grown as an ornamental plant in gardens and also grows wild in Western Anatolia (Izmir, Karaburun, Söke-Bafra Lake, Marmaris–Bozburun). The bulbs of Lilium candidum are used as diuretic, expectorant and boil-ripening agents. Since ancient times, it has been cultivated in Western Anatolia with the aim of obtaining fragrant substances from its flowers. Today it is especially grown as an ornamental plant and its bulbs are exported to foreign countries. Water obtained from the flowers by steam distillation is used in the treatment of skin spots and acne. The extract obtained by keeping the flower in olive oil is used as a fragrance agent. Other names of the flower are white lily, white bulb lily, lord lily (around Marmaris) and white lily (Baytop, 1984). Through the purification and characterization of protease enzyme, it was concluded that this flower could be used in the cosmetic, food and pharmaceutical industries. The volatile components and richness in vitamins suggest that lily can be used in the cosmetic industry. Materials and Methods

Plant Extraction

Lilium candidum flowers were divided into small pieces and extracted with methanol. The flowers of the plant were kept in methanol for 24 hours each time, for a total of 5 extractions. Methanol was evaporated using a rotary evaporator. Thus, crude extract was obtained. The obtained methanol extract was dissolved in water. Back extraction was performed with hexane and ethyl acetate. Subsequently, after evaporation of solvents, crude extracts for each solvent were obtained. The remaining portion from these procedures was called water fraction. The water in this solution was removed using a lyophilizer. The amounts of this extract and fractions were determined by weighing.

Determination of Phenolic Compounds by UPLC-MS/MS

3 g of lilium candidum flowers were ground with 200 mL liquid nitrogen. 30 mL of acetone-water (80:20) mixture was added and kept for 6 hours (at –86°C). The mixture was removed from the cooler, kept in an ultrasonic bath for 15 minutes, then centrifuged at 4000 rpm for 10 minutes (at 20°C). After filtration, the residue was extracted twice with 30 mL of acetone-water mixture. The extracts were combined and acetone was removed using an evaporator. The aqueous phase was washed with n-hexane and diethyl ether. It was then extracted three times with 30 mL ethyl acetate. The extracts were combined. Ethyl acetate was evaporated and the residue was dissolved in methanol-water (80:20) mixture. The solution was passed through 0.20 μm filters (Macherey-Nagel Chromafil Xtra PTFE-20/25). It was analyzed using UPLC-MS/MS (Waters Acquity Ultra Performance LC, Xevo TQ-S MS-MS) apparatus.

Determination of Free Amino Acid Content by UPLC-MS/MS

After lilium candidum flowers were divided into small pieces, they were dried in a lyophilizer for 12 hours. To the homogeneous sample (1 g), water-methanol-formic acid solution (80–19.9–0.1, v/v/v, 5 mL) was added. The mixture was stirred for 10 minutes, then centrifuged at 14000 rpm for 10 minutes (at 4°C). The upper part was filtered through a 0.20 μm filter (Macherey-Nagel Chromafil Xtra PTFE-20/25) and analyzed using UPLC-MS/MS (Waters Acquity Ultra Performance LC, Xevo TQ-S MS-MS) apparatus. The apparatus parameters are the same as those used in phenolic compounds.

Determination of Volatile Components of Lilium Candidum Flowers by Headspace GC/MSD

Freshly ground lilium candidum flowers were weighed in a 20 mL headspace vial (5 g). Subsequently, anhydrous magnesium sulfate (MgSO4) was added and mixed. The vial was placed in the sampler of the apparatus and extracted at 90°C for 30 minutes. Subsequently, the volatile components at the top of the vial were transferred with helium gas to the GC Split/Splitless inlet.

Determination of B Vitamin Content by UPLC-MS/MS

Approximately 3 g of lilium candidum flowers were ground, then 30 mL of H2O-CH3CN (80:20) mixture was added and kept in a cooler for 6 hours (at +4°C). The mixture was kept in an ultrasonic bath for 15 minutes, then centrifuged at 4000 rpm for 10 minutes (at 20°C). After the residue was filtered, it was extracted two more times with 30 mL H2O-CH3CN mixture. The acetonitrile in the combined solution was evaporated using an evaporator (at 40°C). The solution was passed through a 0.20 μm filter (Macherey-Nagel Chromafil Xtra PTFE-20/25) and analyzed using UPLC-MS/MS (Waters Acquity Ultra Performance LC, Xevo TQ-S MS-MS) apparatus. Antioxidant Activity Determination Methods

Total Antioxidant Activity: β-Carotene Color Fading Method

Total antioxidant activity was determined using the β-carotene-linoleic acid method based on measuring the inhibition of conjugate diene hydroperoxides resulting from linoleic acid oxidation (Miller, 1971). This method is based on the fading of β-carotene color. In a 250 mL ground-neck flask, 0.5 mg β-carotene is dissolved in 1 mL chloroform and 200 mg Tween-40 and 20 μL linoleic acid are added. The solvent is completely removed. 100 mL of water saturated with oxygen is added to the residue and shaken. 4 mL β-carotene mixture is added to 1 mL samples containing 0.5–4 mg sample. When the emulsion is placed in test tubes, initial absorbances are measured using a spectrophotometer (β = 470 nm). Methanol is used as control. The tubes are incubated at 50°C and kept until the β-carotene in the control tube loses its color (approximately 120 minutes). The rate of β-carotene color fading (R) and antioxidant activity (AA) are calculated.

DPPH Free Radical Scavenging Activity Method

This property of Lilium candidum flowers was determined using DPPH (1,1-diphenyl-2-picrylhydrazyl) radical (Blois, 1958). 4 mL of 0.004% DPPH solution dissolved in ethanol was added to 1 mL samples containing 0.5–4 mg extract. Ethanol was used as control. After the samples were incubated at room temperature for 30 minutes, their absorbances were measured at 517 nm. Subsequently, free radical scavenging activities were calculated.

ABTS Cation Radical Scavenging Activity Method

The cation radical scavenging activities of the extracts were determined using ABTS (2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (Re et al., 1999). 7 mM ABTS was activated with 2.45 mM K2SO8 to produce 5 mL ABTS radical. This mixture was kept in the dark for 16 hours for cation radical formation. 1 mL was taken from this radical solution and its absorbance was diluted with ethanol to approximately 0.700. 4 mL of ABTS solution was added to 1 mL sample containing 0.5–4 mg extract. Ethanol was used as control. After waiting for 10 minutes at room temperature, absorbances were measured at 734 nm. ABTS cation radical scavenging activity was calculated. Prof. Dr. Nazan Demir / Muğla Sıtkı Koçman University - Cosmetic Products Application and Research Center Prof. Dr. Yaşar Demir / Muğla Sıtkı Koçman University - Cosmetic Products Application and Research Center Dr. Fatih Uçkaya / Bezmialem University Faculty of Pharmacy Research Assistant Ceyhun IŞIK / Graduate Student Sıla Nezahat Daşdemir - Muğla Sıtkı Koçman University Faculty of Science