Comparative Study of Camellia oleifera Seed Oil on Chemical Constituents, Antioxidant Activities and Physicochemical Characteristics from Southern China.

Eleven kinds of Camellia oleifera seed oils (CSOs) were evaluated in terms of chemical constituents, antioxidant activities, acid value (AV) as well as peroxide value (POV). These CSOs contained abundant ß-sitosterol, squalene, α-tocopherol and phenolics, in which the squalene was the distinct constituent with the content between 45.8±0.8 and 184.1±5.5 mg/kg. The ß-sitosterol ranging from 143.7±4.8 to 1704.6±72.0 mg/kg contributed a considerable content to total accompaniments. Palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid were present in these CSOs, in which the dominant fatty acid was oleic acid with the content between 59.66±0.72 and 82.89±2.16 g/100 g. The AV ranged from 0.1±0.0 to 1.3±0.0 mg KOH/g, and the POV was between 0.1±0.0 and 1.0±0.0 g/100 g. These CSOs showed antioxidant activity based on DPPH and ABTS radical scavenging assay. Both α-tocopherol and ß-sitosterol contents showed a positive correlation with DPPH and ABTS values, respectively, while the α-tocopherol content showed a negative correlation with AV. These results suggested that CSO can be categorized into high oleic acid vegetable oil with abundant active constituents, of which the quality presented variation among different origins. These accompaniments may contribute to the delay of its quality deterioration.

Pseudocereal Oils, Authenticated by Fourier Transform Infrared Spectroscopy, and their Chemopreventive Properties.

Amaranth, quinoa, and buckwheat are the representatives of pseudocereals, different parts and by-products of which are used in daily nutrition and food processing industry. However, only scarce information exists on the bioactivity of their oils. Thus, oils obtained from amaranth, buckwheat, and red, yellow, and white quinoa seeds were evaluated in terms of their nutritional (fatty acid profile, squalene), cytotoxic (against normal and neoplastic gastrointestinal, prostate, and skin cells), anti-inflammatory and antiradical (interleukin 6, TNF-alpha, nitric oxide, DPPH, Total phenolics, and superoxide dismutase) potential in the in vitro model. Linoleic (42.9-52.5%) and oleic (22.5-31.1%) acids were the two main unsaturated, while palmitic acid (4.9-18.6%) was the major saturated fatty acid in all evaluated oils. Squalene was identified in all evaluated oils with the highest content in amaranth oil (7.6 g/100 g), and the lowest in buckwheat oil (2.1 g/100 g). The evaluated oils exerted a high direct cytotoxic impact on cancer cells of different origins, but also revealed anti-inflammatory and antiradical potentials. Yellow quinoa oil was the most active, especially toward skin (A375; IC50 6.3 µg/mL), gastrointestinal (HT29 IC50 4.9 µg/mL), and prostate cancer cells (LNCaP IC50 7.6 µg/mL). The observed differences in the activity between the oils from the tested quinoa varieties deserve further studies. High selectivity of the oils was noted, which indicates their safety to normal cells. The obtained results indicate that the oils are good candidates for functional foods with perspective chemopreventive potential.

Functional Characterization of Four Olive Squalene Synthases with Respect to the Squalene Content of the Virgin Olive Oil.

The release of new olive cultivars with an increased squalene content in their virgin olive oil is considered an important target in olive breeding programs. In this work, the variability of the squalene content in a core collection of 36 olive cultivars was first studied, revealing two olive cultivars, 'Dokkar' and 'Klon-14', with extremely low and high squalene contents in their oils, respectively. Next, four cDNA sequences encoding squalene synthases (SQS) were cloned from olive. Sequence analysis and functional expression in bacteria confirmed that they encode squalene synthases. Transcriptional analysis in distinct olive tissues and cultivars indicated that expression levels of these four SQS genes are spatially and temporally regulated in a cultivar-dependent manner and pointed to OeSQS2 as the gene mainly involved in squalene biosynthesis in olive mesocarp and, therefore, in the olive oil. In addition, the biosynthesis of squalene appears to be transcriptionally regulated in water-stressed olive mesocarp.

Characterization of a novel squalene-rich oil: Pachira macrocarpa seed oil.

Pachira macrocarpa is a woody oil crop with high economic and ornamental value. Although P. macrocarpa seeds are rich in oil, little information has been reported about its characterization. In this study, the fatty acids, minor components (tocopherols, squalene, phytosterols, and total phenols), antioxidant activity, cytotoxicity, thermal, and rheological behavior of the P. macrocarpa seed oil (PSO) were investigated for the first time. The results showed that the seeds contained 43.34% lipid, which was mainly composed of palmitic acid (49.96%), linoleic acid (31.22%), and oleic acid (13.48%). The contents of tocopherols, squalene, phytosterols, and total phenols in PSO were 42.01 mg/100 g, 96.78 mg/100 g, 119.67 mg/100 g, and 3.79 mg GAE/100 g, respectively. PSO showed relatively strong DPPH radical scavenging capacity (93.47 µmol TE/100 g) and high melting point (20.8°C). In addition, the oil exhibited Newtonian flow behavior and was not toxic to normal L929 cells at concentrations of 500-8000 µg/ml. As a whole, PSO may be considered as a valuable source for new multipurpose products for industrial utilization. PRACTICAL APPLICATION: Pachira macrocarpa is a woody oil crop and its seeds are rich in oil. Our study has investigated the physicochemical properties and chemical composition of the P. macrocarpa seed oil (PSO). The present study revealed PSO had potential as an edible oil, and it may be considered as a valuable source for new multipurpose products for food industrial utilization.

On the Squalene Content of CV Chondrolia Chalkidikis and Chalkidiki (Greece) Virgin Olive Oil.

This work is a continuation of efforts to establish the nutritional profile of virgin olive oil (VOO) from cv. Chondrolia Chalkidikis and Chalkidiki and to strengthen its positioning in the global VOO landscape. VOOs produced at an industrial scale in different olive mills of the Chalkidiki (Greece) regional unit as well as VOOs obtained at the laboratory scale from drupes of different maturity stages for four consecutive harvesting years were examined for their squalene (SQ) content using both HPLC and GC procedures. The mean values of SQ were found to be 4228 (HPLC) and 4865 (GC) mg/kg oil (n = 15) and were of the same magnitude as that in VOOs from cv Koroneiki (4134 mg/kg, n = 23). Storage of VOOs in the dark at room temperature for 18 months indicated an insignificant mean SQ content loss (~2%) in comparison to a mean loss of 26% for alpha-tocopherol content. This finding strengthens our view that SQ does not act as a radical scavenger that donates hydrogen atoms to the latter. The four consecutive harvest years studied indicated a clear declining trend in VOO SQ concentration upon olive ripening. To our knowledge, this is the first systematic work concerning the SQ content of Chondrolia Chalkidikis and Chalkidiki VOOs.

Chemical Characterization of Chinese Perilla Seed Oil.

Physicochemical properties and chemical composition of Chinese perilla seed oil has been characterized in this study. The result showed that both the cold press oil and the solvent extracted oil possessed low acid value and peroxide value. The fatty acid composition result showed that the oil has high content of linolenic acid (C18:3) up to 66.4 g/100 g, followed by linoleic acid (C18:2) of 15.3 g/100 g. The total triacylglycerol (TAG) profiles results showed that the oil contained 20 TAGs including 17 regioisomers, including LnLnLn (35.8 g/100 g), LLnLn (20.2 g/100 g), LLLn (17.7 g/100 g) and PLnLn (14.9 g/100 g) (Ln, linolenic acid; L, linoleic acid; P, palmitic acid). With content of only 0.57 g/100 g oil, the unsaponifiable matters were mainly composed of phytosterols, squalene, tocopherol, alcohols and hydrocarbons. The total phytosterols content was 0.39 g/100 g oil, in which ß-sitosterol has high content of 0.31 g/100 g oil.

Comparative Study on Functional Components, Physicochemical Properties and Antioxidant Activity of Amaranthus Caudatus L. Oils Obtained by Different Solvents Extraction.

Functional compositions, physicochemical properties and antioxidant activities of Amaranthus caudatus L. oils (ACO) obtained by different solvents were comparatively investigated. All the resulted ACO were enrich in 75% unsaturated fatty acid and in squalene of about 4 g/100 g. Different solvents showed varying in oil extraction, where acetone results a highest yield of 6.80 g/100 g. ACO extracted by ethanol showed a highest tocopherol (1351.26 mg/kg), polyphenols (211.28 mg/kg) and squalene (42519.13 mg/kg). However, phytosterols in ACO extracted by hexane (27571.20 mg/kg) was higher than that by acetone (19789.91 mg/kg), ethanol (22015.73 mg/kg) and petroleum ether (24763.30 mg/kg). Furthermore, antioxidant activity of ACO was also measured by DPPH, ABTS and FRAP assay. According to principal component and correlation analysis, squalene was correlated with the DPPH scavenging ability, but phytosterols and tocopherols was correlated with the ABTS and ferric reducing ability of the oils, respectively. This study provides a promising excellent source of functional oil for food industries.

Green microsaponification-based method for gas chromatography determination of sterol and squalene in cyanobacterial biomass.

Sterol analysis of complex matrices can be very laborious. To minimize the existing drawbacks, a new micro-method of sterols and squalene determination in cyanobacteria was developed and applied to monitor their production of Phormidium autumnale cultured heterotrophically. Sample extraction/saponification and GC analysis of the target compounds were optimized separately using Plackett-Burman design (PB) followed by a central composite rotational design (CCRD). The most influential variables were identified to maximize compound recovery. Chloroform presented the highest capability to extract all target compounds with a horizontal shaker table (HST) for homogenization in the saponification step. For the pretreatment, a small amount of chloroform was used for 90 min at 50 °C and 6 min for the saponification time. The sample introduction in the GC injector was studied by evaluating pressure and injector temperature. High response for sterols and squalene were obtained between 19 and 23 psi and at 310 °C of injection temperature. The new method was able to determine different sterol concentrations: 0.2-0.6 mg kg-1 of squalene, 5-18 mg kg-1 of stigmasterol, 6 mg kg-1 of cholesterol, and 3 mg kg-1 of ß-sitosterol, showing high analytical performance and fulfilling all steps, thus proving to be a promising technique.

Evaluation of Chemical Properties of Commercial Extra Virgin Olive Oil in China.

The aim of the study was to investigate the chemical properties of the most popular commercial extra virgin olive oils (EVOOs) in China. A total of 14 EVOO samples were collected and evaluated, and significant differences were observed with respect to physicochemical properties, fatty acid composition, minor components, and the oxidation stability index (OSI). The results showed that the chemical properties of EVOOs were significantly affected by different producing areas. The oleic acid (C18:1) content (average value: 77.80%), squalene content (average value: 6052.28 mg/kg), and OSI (average value: 9.90 h) of the Spanish olive oil were higher than those of the other oils investigated, while the total phenolic content (average value: 308.34 mg/kg) was the lowest. Greek EVOOs had the lowest total sterol content (average value: 1023.48 mg/kg) and OSI (average value: 4.22 h). The C18:1 content (66.42%) and squalene content (3173.42 mg/kg) of the EVOO from China were lower than those of the other oils, while the palmitic acid (C16:0, 16.82%), linoleic acid (C18:2, 12.18%), total phenolic (553.17 mg/kg), and total sterol content (1904.77 mg/kg) were higher than those of the other olive oils. The EVOOs of the various countries could be distinguished by hierarchical cluster analysis (HCA). In addition, multiple linear regression (MLR) analyses between the OSI and chemical properties revealed that squalene (R = 0.729) and the unsaturation determined by the specific UV adsorption at 232 nm (K232, R = -0.300) were the main factors to affecting the EVOO oxidation stability.

A Simple Screening Method for Extra Virgin Olive Oil Adulteration by Determining Squalene and Tyrosol.

A simple screening method for discrimination between commercial extra virgin olive oils and their blends with other vegetable oils was developed. Squalene, which was contained relatively high amounts in virgin olive oil, was determined by HPLC after a simple pretreatment that was carried out by dilution of oil samples with 2-propanol. Tyrosol, which was contained at relatively high concentration in virgin olive oil among phenolic compounds, was determined by HPLC after a simple liquid-liquid extraction. When using squalene and tyrosol contents as axes, extra virgin olive oils could be discriminated from pure olive oils, blended oils (extra virgin olive oils with sunflower oil or grapeseed oil) and other vegetable oils. These results suggest that determining squalene and tyrosol in seed oil samples could be useful in distinguishing between extra virgin olive oil and blended oils as a screening method.

Isolation and functional analysis of squalene synthase gene in tea plant Camellia sinensis.

Tea contains high quantities and diverse types of triterpenoids, particularly in the form of saponins. However, little is yet known about the molecular basis of triterpenoid biosynthesis in tea plant. Here we report on isolation and functional analysis of squalene synthase (SQS) gene from tea plant (Camellia sinensis var. sinensis), which controls the biosynthesis of triterpenoids precursor. First, a full-length cDNA of squalene synthase, designated CsSQS, was isolated from tea plant. The protein is highly homologous to SQSs from other plants. Using CsSQS-reporter assays, CsSQS was demonstrated to be endoplasmic reticulum membrane-bound. The coding region of CsSQS excluding transmemberane sequence was expressed in Escherichia coli. Recombinant CsSQS catalyzed the formation of squalene using farnesyl-pyrophosphate (FPP) as substrate with NADPH and Mg2+. In tea plant leaves, CsSQS expression was significantly induced by both herbivore and mechanical damages. Consistent with the stronger induction of CsSQS expression by mechanical damage than herbivory, tea plants injured mechanically released squalene as a volatile compound, which however was not detected from herbivore-damaged tea plants. Furthermore, it was found that the flowers of another tea plant cultivar Camellia sinensis var. assamica contain higher concentrations of squalene than the cultivar sinensis, indicating variations among tea plant varieties. With the identification and molecular characterization of squalene synthase in tea plant, next, we can ask the questions about the roles of squalene as a volatile product as well as a precursor for triterpenoids, which may promote product development from diverse tea materials and mining of excellent tea germplasm resources.

Lipid Characteristics of Camellia Seed Oil.

Camellia oleifera, C. japonica and C. sinensis are three representative crops of the genus Camellia. In this work, we systematically investigated the lipid characteristics of these seed oils collected from different regions. The results indicated significant differences in acid value (AV), peroxide value (PV), iodine value (IV), saponification value (SV) and relative density of the above-mentioned camellia seed oils (p < 0.05). The C. japonica seed oils showed the highest AV (1.7 mg/g), and the C. sinensis seed oils showed the highest PV (17.4 meq/kg). The C. japonica seed oils showed the lowest IV (79.9 g/100 g), SV (192.7 mg/g) and refractive index (1.4633) of all the oils, while the C. sinensis seed oils showed the lowest relative density (0.911 g/cm3). The major fatty acids in the camellia seed oils were palmitic acid (16:0), oleic acid (18:1) and linoleic acid (18:2); the oleic acid in C. oleifera and C. japonica seed oils accounted for more than 80% of the total fatty acids. The oleic acid levels in the C. oleifera and C. japonica oils were higher than those in the C. sinensis seed oils, while the linoleic acid levels in the former were lower than those in the latter one. Differences also exist in the triacylglycerol (TAG) composition, although the most abundant TAG molecular species in the camellia seed oils was trioleoylglycerol (OOO). Seven sterol species, squalene and α-tocopherol were detected in the camellia seed oils, however, the contents of tocopherol and unsaponifiable molecules in the C. oleifera and C. japonica seed oils were significantly lower than those in the C. sinensis seed oil. These results demonstrated that the varieties of Camellia affected the seed oil lipid characteristics.

Calibration of Squalene, p-Cymene, and Sunflower Oil as Standard Oxidizable Substrates for Quantitative Antioxidant Testing.

The autoxidation kinetics of stripped sunflower oil (SSO), squalene (SQ), and p-cymene ( p-C) initiated by 2,2'-azobis(isobutyronitrile) at 303 K were investigated under controlled conditions by differential oximetry in order to build reference model systems that are representative of the natural variability of oxidizable materials, for quantitative antioxidant testing. Rate constants for oxidative chain propagation ( kp) and chain termination (2 kt) and the oxidizability ( kp/√2 kt) were measured using 2,6-di- tert-butyl-4-methoxyphenol, 2,2,5,7,8-pentamethyl-6-chromanol, BHT, and 4-methoxyphenol as reference antioxidants. Measured values of kp (M-1 s-1)/2 kt (M-1 s-1)/oxidizability (M-1/2 s-1/2) at 303 K in chlorobenzene were 66.9/3.45 × 106/3.6 × 10-2, 68.0/7.40 × 106/2.5 × 10-2, and 0.83/2.87 × 106/4.9 × 10-4, respectively, for SSO, SQ, and p-C. Quercetin, magnolol, caffeic acid phenethyl ester, and 2,4,6-trimethylphenol were investigated to validate calibrations. The distinctive usefulness of the three substrates in testing antioxidants is discussed.

Monitoring of minor compounds in corn oil oxidation by direct immersion-solid phase microextraction-gas chromatography/mass spectrometry. New oil oxidation markers.

The aim of this study is to shed light on the evolution of the minor compounds in the corn oil oxidation process, through the information provided by direct immersion-microextraction in solid phase followed by gas chromatography/mass spectrometry (DI-SPME-GC/MS). This methodology enables one, in a single run, to establish the identity and abundance both of original oil minor components, some with antioxidant capacity, and of other compounds coming from both main and minor oil components oxidation. For the first time, some of the compounds formed from oil minor components degradation are proposed as new markers of oil incipient oxidation. Although the study refers to corn oil, the methodology can be applied to any other edible oil and constitutes a new approach to characterizing the oxidation state of edible oils.

Simultaneous determination of tocols, γ-oryzanols, phytosterols, squalene, cholecalciferol and phylloquinone in rice bran and vegetable oil samples.

In this study, a simultaneous analytical method of tocols, γ-oryzanols, phytosterols, squalene, cholecalciferol and phylloquinone were developed using HPLC-DAD-FLD. The developed method allowed the quantification of 18 compounds in 30 min. Method validation showed linearity of calibration curves (α = 0.05). RSD of intra-day, inter-day and inter-laboratory precision were less than 4.88%. The limit of detections (LODs) and limit of quantifications (LOQs) were low (0.009-2.166 µg g-1) with recoveries around 96.0-102.9%. Results derived from the established method demonstrated a wide variation of detected compounds in rice bran and vegetable oil samples (22.4-1774.6 µg g-1 tocols, ND-26484 µg g-1 γ-oryzanols, ND-12655 µg g-1 phytosterols, ND-3189 µg g-1 squalene, ND-105.3 µg g-1 cholecalciferol, and ND-54.4 µg g-1 phylloquinone). Thus, the developed HPLC-DAD-FLD method is a powerful analytical tool for the above mentioned compounds useful in food and pharmaceutical application.

Chemical characterization of a variety of cold-pressed gourmet oils available on the Brazilian market.

Different specialty extra virgin oils, produced by cold-pressing fruits/nuts (olive, pequi, palm, avocado, coconut, macadamia and Brazil nut) and seeds (grapeseed and canola), and retailed in the Brazilian region of Minas Gerais, were chemically characterized. Specifically, for each type of oil, the fatty acid composition was elucidated by GC-FID, the contents of selected polyphenols and squalene were determined respectively by UHPLC-MS and UHPLC-PDA, whereas minerals were explored by means of ICP-MS. Olive oil was confirmed to have the highest MUFA content due to a valuable level of oleic acid, while oils from grapeseed, Brazil nut and canola were marked by nutritionally important PUFA levels. The highest SFA content found in coconut oil was mainly due to the high levels of lauric acid, known for its advantageous HDL-raising effects. As for polyphenols, gourmet oils from palm, coconut and canola showed higher levels of phenolic acids (e.g. p-hydroxybenzoic, ferulic, syringic, acids) than olive oil, which was though characterized by peculiar antioxidants, such as tyrosol and hydroxytyrosol. Also, olive oil had the highest amount of squalene, followed by the oil from Brazil nut. Finally, all the investigated oils had very low levels (order of µg/kg) of pro-oxidant elements, such as Cu, Fe and Mn. Overall, these findings may fill the gaps still present in literature on certain compositional aspects of commercially available gourmet oils.

Direct olive oil analysis by mass spectrometry: A comparison of different ambient ionization methods.

Analytical methods based on ambient ionization mass spectrometry (AIMS) combine the classic outstanding performance of mass spectrometry in terms of sensitivity and selectivity along with convenient features related to the lack of sample workup required. In this work, the performance of different mass spectrometry-based methods has been assessed for the direct analyses of virgin olive oil for quality purposes. Two sets of experiments have been setup: (1) direct analysis of untreated olive oil using AIMS methods such as Low-Temperature Plasma Mass Spectrometry (LTP-MS) or paper spray mass spectrometry (PS-MS); or alternatively (2) the use of atmospheric pressure ionization (API) mass spectrometry by direct infusion of a diluted sample through either atmospheric pressure chemical ionization (APCI) or electrospray (ESI) ionization sources. The second strategy involved a minimum sample work-up consisting of a simple olive oil dilution (from 1:10 to 1:1000) with appropriate solvents, which originated critical carry over effects in ESI, making unreliable its use in routine; thus, ESI required the use of a liquid-liquid extraction to shift the measurement towards a specific part of the composition of the edible oil (i.e. polyphenol rich fraction or lipid/fatty acid profile). On the other hand, LTP-MS enabled direct undiluted mass analysis of olive oil. The use of PS-MS provided additional advantages such as an extended ionization coverage/molecular weight range (compared to LTP-MS) and the possibility to increase the ionization efficiency towards nonpolar compounds such as squalene through the formation of Ag+ adducts with carbon-carbon double bounds, an attractive feature to discriminate between oils with different degree of unsaturation.

Health-promoting effects of red palm oil: evidence from animal and human studies.

The fruit of the oil palm tree (Elaeis guineesis) is the source of antioxidant-rich red palm oil. Red palm oil is a rich source of phytonutrients such as tocotrienols, tocopherols, carotenoids, phytosterols, squalene, and coenzyme Q10, all of which exhibit nutritional properties and oxidative stability. Mutagenic, nutritional, and toxicological studies have shown that red palm oil contains highly bioavailable ß-carotene and vitamin A and is reasonably stable to heat without any adverse effects. This review provides a comprehensive overview of the nutritional properties of red palm oil. The possible antiatherogenic, antihemorrhagic, antihypertensive, anticancer, and anti-infective properties of red palm oil are examined. Moreover, evidence supporting the potential effectiveness of red palm oil to overcome vitamin A deficiency in children and pregnant women, to improve ocular complications of vitamin A deficiency, to protect against ischemic heart disease, to promote normal reproduction in males and females, to aid in the management of diabetes, to ameliorate the adverse effects of chemotherapy, and to aid in managing hypobaric conditions is presented.

Fast UPLC/PDA determination of squalene in Sicilian P.D.O. pistachio from Bronte: Optimization of oil extraction method and analytical characterization.

A fast reversed-phase UPLC method was developed for squalene determination in Sicilian pistachio samples that entry in the European register of the products with P.D.O. In the present study the SPE procedure was optimized for the squalene extraction prior to the UPLC/PDA analysis. The precision of the full analytical procedure was satisfactory and the mean recoveries were 92.8±0.3% and 96.6±0.1% for 25 and 50mgL-1 level of addition, respectively. Selected chromatographic conditions allowed a very fast squalene determination; in fact it was well separated in ∼0.54min with good resolution. Squalene was detected in all the pistachio samples analyzed and the levels ranged from 55.45-226.34mgkg-1. Comparing our results with those of other studies it emerges that squalene contents in P.D.O. Sicilian pistachio samples, generally, were higher than those measured for other samples of different geographic origins.

Gas chromatography-mass spectrometry and Raman imaging measurement of squalene content and distribution in human hair.

A sensitive and specific gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the measurement of the squalene content from root to tip, in both Chinese black virgin and bleached hair. Deuterated squalene was used as the internal standard. For quantification, selective ion monitoring (SIM) at m/z 410.0 and 347.0 were monitored for squalene and deuterated squalene, respectively. Different methods for the extraction of squalene from ex vivo human hair were compared including organic solvent extraction and acid/alkali hydrolysis. The best extraction efficiency was obtained by using a mixed solvent consisting of chloroform:methanol = 2:1 (v:v). The linear range of squalene ran from 1.0 to 50.0 µg mL(-1). The limit of detection (LOD) was 0.10 µg mL(-1) (corresponding to 0.005 mg g(-1) in human hair), which enabled quantification of squalene in human hair at very low level. The recovery of squalene was 96.4 ± 1.46% (n = 3). Using the above-mentioned mixed solvent extraction, squalene content in human hair was successfully quantified from root to tip. Meanwhile, a Raman imaging method was developed to visualize the squalene distribution in Chinese white virgin hair from cuticle to medulla.