Sphingolipid-Enriched Porcine Placental Extract Promotes the Expression of Structural Genes and Desquamation Enzyme Genes in Cultured Human Keratinocytes.

Porcine placental extract (PPE) is commonly used in various health foods and cosmetics. PPE use in cosmetics predominantly consist of the water-soluble fraction derived from the entire placenta. In this report, we examined the effect of the hydrophobic constituents of the PPE, specifically the sphingolipid-enriched fraction designated as the sphingolipid-enriched porcine placental extract (SLPPE), on the expression of genes associated with skin function in cultured normal human epidermal keratinocytes. Using quantitative RT-PCR (qRT-PCR) analysis, we found that SLPPE concentrations ranging from 25 to 100 µg/mL upregulated the gene expression of key components associated with the cornified envelope structure (filaggrin (FLG), involucrin (IVL) and loricrin (LOR)), cornification enzymes (transglutaminase 1 (TGM1) and TGM5) and the desquamation enzymes (kallikrein 5 (KLK5) and KLK7). Additionally, KLK5p and FLG protein (FLGp) were detected in the culture supernatants of keratinocytes treated with SLPPE at these concentrations. These findings suggest that SLPPE is possible to promote the cornification and desquamation in epidermal keratinocytes, and it may offer potential benefits in cosmetics.

Stress-Relieving Effects of Sesame Oil Aroma and Identification of the Active Components.

(1) Sesame oil aroma has stress-relieving properties, but there is little information on its effective use and active ingredients. (2) Methods: ICR male mice were housed under water-immersion stress for 24 h. Then, the scent of sesame oil or a typical ingredient was inhaled to the stress groups for 30, 60, or 90 min. We investigated the effects of sesame oil aroma on mice behavior and the expression of the dual specificity phosphatase 1 (DUSP1) gene, a candidate stress marker gene in the brain. (3) Results: In an elevated plus-maze test, the rate of entering into the open arm of a maze and the staying time were increased to a maximum after 60 min of inhalation, but these effects decreased 90 min after inhalation. As for the single component, anxiolytic effects were observed in the 2,5-dimethylpyrazine and 2-methoxy phenol group, but the effect was weakened in the furfuryl mercaptan group. The expression levels of DUSP1 in the hippocampus and striatum were significantly decreased in 2,5-dimethylpyrazine and 2-methoxy phenol groups. (4) Conclusions: We clarified the active ingredients and optimal concentrations of sesame oil for its sedative effect. In particular, 2,5-dimethylpyrazine and 2-methoxy phenol significantly suppressed the stress-induced changes in the expression of DUSP1, which are strong anti-stress agents. Our results suggest that these molecules may be powerful anti-stress agents.

Smell and Stress Response in the Brain: Review of the Connection between Chemistry and Neuropharmacology.

The stress response in the brain is not fully understood, although stress is one of the risk factors for developing mental disorders. On the other hand, the stimulation of the olfactory system can influence stress levels, and a certain smell has been empirically known to have a stress-suppressing effect, indeed. In this review, we first outline what stress is and previous studies on stress-responsive biomarkers (stress markers) in the brain. Subsequently, we confirm the olfactory system and review previous studies on the relationship between smell and stress response by species, such as humans, rats, and mice. Numerous studies demonstrated the stress-suppressing effects of aroma. There are also investigations showing the effects of odor that induce stress in experimental animals. In addition, we introduce recent studies on the effects of aroma of coffee beans and essential oils, such as lavender, cypress, α-pinene, and thyme linalool on the behavior and the expression of stress marker candidates in the brain. The transfer of volatile components into the brain is also discussed while using the results of thyme linalool as an example. These studies may provide a good opportunity to connect chemical research at the molecular level with neuropharmacological approaches in the future.

Effects of Sesame Oil Aroma on Mice after Exposure to Water Immersion Stress: Analysis of Behavior and Gene Expression in the Brain.

(1) Background: Sesame has been popular as a healthy food since ancient times, and effects of the aroma component of roasted sesame are also expected. However, little research has been reported on its scent; (2) Methods: Jcl:ICR male mice were housed under water immersion stress for 24 h. Then, the scent of saline or sesame oil was inhaled to stress groups for 90 min. We investigated the effects of sesame oil aroma on the behavior and brains of mice; (3) Results: In an elevated plus maze test, the rate of entering to open arm and the staying time were decreased by the stress. These decrements were significantly enhanced by sesame oil aroma. Stress had a tendency to increase the serum corticosterone concentration, which was slightly decreased by the aroma. Expression of Kruppel-like factor-4 (Klf-4) and Dual-specificity phosphatase-1 (Dusp-1) in the striatum were increased by water immersion stress, and the level of Klf-4 and Dusp-1 in the striatum and hippocampus were significantly attenuated by sesame oil aroma (4) Conclusions: The present results strongly suggest that the odor component of sesame oil may have stress suppressing effects. Moreover, Klf-4 and Dusp-1 may be sensitive stress-responsive biomarkers.

Analgesic Effects of Ephedra Herb Extract, Ephedrine Alkaloids-Free Ephedra Herb Extract, Ephedrine, and Pseudoephedrine on Formalin-Induced Pain.

The analgesic effect of Ephedra Herb (EH) is believed to be derived from the anti-inflammatory action of pseudoephedrine (Pse). We recently reported that ephedrine alkaloids-free EH extract (EFE) attenuates formalin-induced pain to the same level as that achieved by EH extract (EHE), which suggests that the analgesic effect of EH may not be due to ephedrine alkaloids (EAs). To examine the contribution of EAs to the analgesic effect of EH, mice were injected with formalin to induce a biphasic pain reaction (first phase, 0-5 min; second phase, 10-45 min) at various time points after oral administration of the following test drugs: ephedrine (Eph), Pse, "authentic" EHE from Tsumura & Co. (EHE-Ts), EFE, and EHE that was used as the source of EFE (EHE-To). Biphasic pain was suppressed at 30 min after administration of Eph, EHE-Ts, and EHE-To. At 6 h after administration of EFE, EHE-To, and Pse-and at 4 to 6 h after administration of EHE-Ts-only second-phase pain was suppressed; however, the effect of Pse at 6 h was not significant. These results suggested that EHE has a biphasic analgesic effect against biphasic formalin-induced pain: in the first phase of analgesia (30 min after administration), biphasic pain is suppressed by Eph; in the second phase of analgesia (4-6 h after administration), second-phase pain is alleviated by constituents other than EAs, although Pse may partially contribute to the relief of second-phase pain.

Ephedrine Alkaloids-Free Ephedra Herb Extract, EFE, Has No Adverse Effects Such as Excitation, Insomnia, and Arrhythmias.

Ephedrine alkaloids-free Ephedra Herb extract (EFE) has been developed to eliminate the adverse effects caused by ephedrine alkaloid-induced sympathetic hyperactivation. Previously, we reported that EFE possesses analgesic, anti-influenza, and cancer metastatic inhibitory effects at comparable levels to that of Ephedra Herb extract (EHE). However, it has not yet been demonstrated that EFE is free from the known side effects of EHE, such as excitation, insomnia, and arrhythmias. In this study, the incidence of these adverse effects was compared between mice administered EHE and those administered EFE. Increased locomotor activity in an open-field test, reduced immobility times in a forced swim test, and reduced sleep times in a pentobarbital-induced sleep test were observed in EHE-treated mice, when compared to the corresponding values in vehicle-treated mice. In contrast, EFE had no obvious effects in these tests. In electrocardiograms, atrial fibrillation (i.e., irregular heart rhythm, absence of P waves, and appearance of f waves) was observed in the EHE-treated mice. It was suggested that this atrial fibrillation was induced by stimulation of adrenaline ß1 receptors, but not by hypokalemia. However, EFE did not affect cardiac electrophysiology. These results suggest that the abovementioned side effects are caused by ephedrine alkaloids in EHE, and that EFE is free from these adverse effects, such as excitation, insomnia, and arrhythmias. Thus, EFE is a promising new botanical drug with few adverse effects.

Synthesis of Theaflavins and Their Functions.

Numerous epidemiological and interventional clinical studies have consistently reported that black tea is good for human health. The polyphenolic compound, theaflavin, and its galloyl esters (theaflavins) are the primary red pigments in black tea that possess several health benefits, including fat-reducing and glucose-lowering capabilities and lifestyle-related disease prevention related to anti-obesity, anticancer, anti-atherosclerotic, anti-inflammatory, antiviral, antibacterial, anti-osteoporotic, and anti-dental caries properties. These compounds are produced by key enzymes, such as polyphenol oxidase and peroxidase, from parent green tea catechins present in fresh green tea leaves during the production of black tea leaves or the fermentation of green tea. However, theaflavins are only present in low concentrations in black tea; thus, their extraction from black tea leaves at sufficient levels for use in medical studies has been difficult. To circumvent this issue, different procedures for the synthesis of theaflavins using chemical oxidizing reagents or enzymes have been studied; however, low yields have limited their utility. Recently, however, several biosynthetic methods have been developed for the mass production of theaflavins. Using these methods, the physiological functions of theaflavins in lifestyle-related diseases in mice and humans have also been studied. In this review, we present the synthesis of theaflavins and their health benefits.

A Simple, Enzymatic Biotransformation Method Using Fresh Green Tea Leaves Efficiently Generates Theaflavin-Containing Fermentation Water That Has Potent Physiological Functions in Mice and Humans.

The polyphenolic compound theaflavin, the main red pigment in black tea, possesses many beneficial properties, such as fat-reducing and glucose-lowering capabilities. To produce theaflavin-containing fermentation water on a large scale, we have developed a simple, inexpensive, and selective enzymatic biotransformation method to obtain sufficient levels from fresh green tea leaves. Subsequent administration of theaflavin-containing fermentation water to obese mice on a high-fat diet inhibited body weight gain, decreased casual blood glucose and fasting blood glucose levels, and lowered mesenteric and total fat composition. To note, there were no significant differences observed in food consumption between the experimental and control (water without theaflavin) mice groups. Next, we investigated the effect of this water on blood glucose levels in healthy humans and found that it significantly inhibited blood glucose levels. Thus, we showed that theaflavin-containing fermentation water can be efficiently generated from fresh green tea leaves and demonstrated its significantly potent effects in vivo.

Induction of the UDP-Glucuronosyltransferase 1A1 during the Perinatal Period Can Cause Neurodevelopmental Toxicity.

Anticonvulsants can increase the risk of developing neurotoxicity in infants; however, the underlying mechanism has not been elucidated to date. Thyroxine [3,5,3',5'-l-tetraiodothyronine (T4)] plays crucial roles in the development of the central nervous system. In this study, we hypothesized that induction of UDP-glucuronosyltransferase 1A1 (UGT1A1)-an enzyme involved in the metabolism of T4-by anticonvulsants would reduce serum T4 levels and cause neurodevelopmental toxicity. Exposure of mice to phenytoin during both the prenatal and postnatal periods significantly induced UGT1A1 and decreased serum T4 levels on postnatal day 14. In the phenytoin-treated mice, the mRNA levels of synaptophysin and synapsin I in the hippocampus were lower than those in the control mice. The thickness of the external granule cell layer was greater in phenytoin-treated mice, indicating that induction of UGT1A1 during the perinatal period caused neurodevelopmental disorders. Exposure to phenytoin during only the postnatal period also caused these neurodevelopmental disorders. A T4 replacement attenuated the increase in thickness of the external granule cell layer, indicating that the reduced T4 was specifically associated with the phenytoin-induced neurodevelopmental disorder. In addition, these neurodevelopmental disorders were also found in the carbamazepine- and pregnenolone-16-α-carbonitrile-treated mice. Our study is the first to indicate that UGT1A1 can control neurodevelopment by regulating serum T4 levels.

Theaflavin Synthesized in a Selective, Domino-Type, One-Pot Enzymatic Biotransformation Method with Camellia sinensis Cell Culture Inhibits Weight Gain and Fat Accumulation to High-Fat Diet-Induced Obese Mice.

The polyphenolic compound theaflavin, which is the main red pigment present in black tea, is reported to elicit various physiological effects. Because of the extremely low concentration of theaflavin present in black tea, its extraction from black tea leaves in quantities sufficient for use in medical studies has been difficult. We have developed a simple, inexpensive, selective, domino-type, one-pot enzymatic biotransformation method for the synthesis of theaflavin that is suitable for use in medical studies. Subsequent administration of this synthetic theaflavin to high-fat diet-induced obese mice inhibited both body weight gain and visceral fat accumulation, with no significant difference in the amount of faeces between the experimental and control mice.

Inhalation administration of the sesquiterpenoid aristolen-1(10)-en-9-ol from Nardostachys chinensis has a sedative effect via the GABAergic system.

Spikenard, the dried roots of Nardostachys chinensis, contains sesquiterpenoids and is widely used as an herbal tranquilizer. We previously demonstrated that spikenard vapor showed a sedative effect when administered by inhalation, and we identified hydrocarbon sesquiterpenoids as active components. Here we investigated the other components that contribute to the effects of spikenard. Six oxygenated sesquiterpenoids, including aristolane- and guaiane-types, were isolated from an acetone extract of spikenard. We evaluated the sedative activities of these oxygenated compounds using an inhalation administration method in a caffeine-treated excitatory mouse model. We identified aristolen-1(10)-en-9-ol and patchouli alcohol as highly effective sedative components. These compounds inhibited locomotion in mice by approximately 60% at a dose of 300 µg/cage. In addition, aristolen-1(10)-en-9-ol prolonged pentobarbital-induced sleep to the same extent as 1 mg/kg diazepam. This effect completely disappeared with the administration of the GABAA-benzodiazepine receptor antagonist flumazenil (3 mg/kg), suggesting that the sedative effect of aristolen-1(10)-en-9-ol is expressed via the GABAergic system. Furthermore, differently from diazepam, inhalation of aristolen-1(10)-en-9-ol for 1 h did not affect the motor coordination in the rota-rod test. In the present study, we identified active components and provided evidence supporting the traditional sedative use of spikenard. Our research suggests that aristolen-1(10)-en-9-ol may be an effective aromatherapy, providing mild sedation.

Inhalation administration of valerena-4,7(11)-diene from Nardostachys chinensis roots ameliorates restraint stress-induced changes in murine behavior and stress-related factors.

Dried Nardostachys chinensis roots contain sesquiterpenoids that are widely used as herbal tranquilizers. We previously identified the highly sedative sesquiterpenoid valerena-4,7(11)-diene (VLD) from this plant. In the present study, we investigated stress reducing effects of VLD and the associated mechanisms of action. Application of 15-min restraint stresses induced excitatory behaviors in mice. Immobility times in the forced swim test and sleeping times in the pentobarbital sleep test were shortened in the stressed group by 47% and 43%, respectively, compared with the control group. Furthermore, restraint stress increased serum corticosterone levels by 75%, and cerebral serotonin (5-HT) and dopamine (DA) levels. Inhaled VLD (300 µg/cage) suppressed stress-induced excitatory behaviors and significantly reduced stress-induced blood corticosterone, cerebral 5-HT, and DA levels. These results suggest that VLD interacts with the hypothalamic-pituitary-adrenal axis and the sympathetic-adrenomedullary system. These interactions appear to involve GABAergic and D2 antagonist activities. Moreover, tests in anosmic and intravenously treated mice showed that the sedative effect of inhaled VLD was expressed via olfactory stimulation and pulmonary absorption. Although more studies are required to further elucidate the properties of this compound, our studies suggest that VLD may be an effective anti-stress aromatherapy for humans.

Metabolic fingerprinting for discrimination of DNA-authenticated Atractylodes plants using 1H NMR spectroscopy.

Identifying different species of the genus Atractylodes which are commonly used in Chinese and Japanese traditional medicine, using chromatographic approaches can be difficult. 1H NMR metabolic profiling of DNA-authenticated, archived rhizomes of the genus Atractylodes was performed for genetic and chemical evaluation. The ITS region of the nuclear rDNA was sequenced for five species, A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana. Our samples had nucleotide sequences as previously reported, except that part of the A. lancea cultivated in Japan had a type 5, hybrid DNA sequence. Principal component analysis (PCA) using 1H NMR spectra of extracts with two solvent systems (CD3OD, CDCl3) was performed. When CDCl3 extracts were utilized, the chemometric analysis enabled the identification and classification of Atractylodes species according to their composition of major sesquiterpene compounds. The 1H NMR spectra using CD3OD contained confounding sugar peaks. PCA removal of these peaks gave the same result as that obtained using CDCl3 and allowed species distinction. Such chemometric methods with multivariate analysis of NMR spectra will be useful for the discrimination of plant species, without specifying the index components and quantitative analysis on multi-components.

Emotional Impairments and Neuroinflammation are Induced in Male Mice Invulnerable to Repeated Social Defeat Stress.

Prolonged stress triggers neuroinflammation, which plays a significant role in the development of depression; however, stressed people do not always suffer from depression because of individual differences in stress vulnerability. Negative cognitive bias (NCB) toward pessimistic judgment often underlies depressive episodes. However, a relationship between stress vulnerability, neuroinflammation, and NCB remains elusive. In addition, an animal model with all the traits would be a powerful tool for studying the etiology of depression and its therapeutic approaches. Accordingly, this study evaluated the effect of stress vulnerability on neuroinflammation and depression-related behaviors, including NCB in males, using a modified version of repeated social defeat stress (mRSDS) paradigm, a validated animal model of psychosocial stress. Exposure to mRSDS, consisting of 5 min of social defeat by unfamiliar CD-1 aggressor mice for five consecutive days, caused NCB, which co-occurred with depressive- and anxiety-like behaviors, and neuroinflammation in male BALB/c mice. Treatment with minocycline, an antibiotic with anti-inflammatory property, blocked mRSDS-induced depressive-like behaviors and neuroinflammation, but not NCB, indicating the limited effect of an anti-inflammatory intervention. In addition, marked differences were found in neuroinflammatory profiles and hippocampal gene expression patterns between resilient and unstressed mice, as well as between susceptible and resilient mice. Therefore, mice resilient to mRSDS are indeed not intact. Our findings provide insights into the unique features of the mRSDS model in male BALB/c mice, which could be used to investigate the etiological mechanisms underlying depression as well as bridge the gap in the relationship between stress vulnerability, neuroinflammation, and NCB in males.

Neuroinflammaging underlies emotional disturbances and circadian rhythm disruption in young male senescence-accelerated mouse prone 8 mice.

Aging causes psychological dysfunction and neurodegeneration, and can lead to cognitive impairments. Although numerous studies have reported that neurodegeneration and subsequent cognitive impairments are involved in neuroinflammation, relationship between psychological disturbance and neuroinflammation with aging (neuroinflammaging) remains unclear. Here, to clarify the relationship, we examined whether neuroinflammaging affects emotional behaviors in senescence-accelerated mouse prone 8 (SAMP8) mice. Microglial inflammatory responses to a subsequent lipopolysaccharide (LPS) challenge were significantly enhanced in male SAMP8 mice relative to normal aging senescence-accelerated mouse resistant 1 (SAMR1) mice at 17 weeks, but not 8 weeks of age. LPS injection also significantly increased brain and systemic inflammation in SAMP8 mice at 17 weeks. In a battery of behavioral tests, SAMP8 mice at 17 weeks, but not 8 weeks, exhibited anxiety- and depression-like behaviors and circadian rhythm disruption. Taken together, SAMP8 mice at 17 weeks possess a brain microenvironment in which it is easier to trigger neuroinflammatory priming; this may lead to an emergence of anxiety- and depression-like behaviors and circadian rhythm disruption. These findings provide new insights into the temporal relationship between neuroinflammaging and emotion.

Comprehensive evaluation of antioxidant effects of Japanese Kampo medicines led to identification of Tsudosan formulation as a potent antioxidant agent.

Oxidative stress due to the overproduction of reactive oxygen species plays an important role in the pathogenesis of various diseases. In the present study, we comprehensively evaluated the antioxidant activities of 147 oral formulations of Japanese traditional herbal medicines (Kampo medicines), representing the entire panel of oral Kampo medicines listed in the Japanese National Health Insurance Drug List, using in vitro radical scavenging assays, including the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity assay, the superoxide anion scavenging activity assay, and the oxygen radical absorption capacity assay. Three of the formulations tested, namely, Tsudosan, Daisaikoto, and Masiningan, showed the most potent in vitro antioxidant activities and were selected for further investigation of their intracellular and in vivo antioxidant effects. The results of the 2',7'-dichlorodihydrofluorescin diacetate assay demonstrated that all three Kampo medicines significantly inhibited hydrogen peroxide-induced oxidative stress in human hepatocellular liver carcinoma HepG2 cells. In addition, Tsudosan significantly increased the serum biological antioxidant potential values when orally administrated to mice, indicating that it also had in vivo antioxidant activity. The potent antioxidant activity of Tsudosan may be one of the mechanisms closely correlated to its clinical usage against blood stasis.

[The Adverse Effects of Ephedra Herb and the Safety of Ephedrine Alkaloids-free Ephedra Herb Extract (EFE)].

Ephedra Herb is defined in the 17th edition of the Japanese Pharmacopoeia (JP) as the terrestrial stem of Ephedra sinica Stapf., Ephedra intermedia Schrenk et C.A. Meyer, or Ephedra equisetina Bunge (Ephedraceae). The stems of Ephedra Herb contain greater than 0.7% ephedrine alkaloids (ephedrine and pseudoephedrine). Despite its high effectiveness, Ephedra Herb exert several adverse effects, including palpitation, excitation, insomnia, and dysuria. Both the primary and adverse effects of Ephedra Herb have been traditionally believed to be mediated by these ephedrine alkaloids. However, our study found that several pharmacological actions of Ephedra Herb were not associated with ephedrine alkaloids. We prepared an ephedrine alkaloid-free Ephedra Herb extract (EFE) by eliminating ephedrine alkaloids from Ephedra Herb extract (EHE) using ion-exchange column chromatography. EFE exerted analgesic, anti-influenza, and anticancer activities in the same manner as EHE. Moreover, EFE did not induce adverse effects due to ephedrine alkaloids, such as excitation, insomnia, and arrhythmias, and showed no toxicity. Furthermore, we evaluated the safety of EFE in healthy volunteers. The number of adverse event cases was higher in the EHE-treated group than in the EFE-treated group, although the difference was not significant. Our evidence suggested that EFE was safer than EHE.

Relationship between augmentation index and flow-mediated vasodilation in the brachial artery.

Recent studies have shown that the augmentation index (AI) is a predictor of cardiovascular complications. Endothelial dysfunction is the initial step in the pathogenesis of atherosclerosis, which in turn can lead to cardiovascular complications. Endothelial function assessed by flow-mediated dilation (FMD) can serve as an independent predictor of cardiovascular events. However, there is little information on the relationship between AI and FMD in the human vasculature, and we therefore investigated this relationship in the present study. A total of 100 subjects (71 males and 29 females; age range, 22-88 years; mean age, 59 +/- 17 years), including 83 patients with cardiovascular diseases (e.g., atherosclerosis, hypertension, coronary heart disease, stroke and peripheral arterial disease) and 17 healthy subjects were enrolled. High-resolution ultrasonography (automated vessel-diameter measurements; eTRACKING system), a linear array transducer (13 MHz) and an arm holding device were used to measure the arterial diameter response to reactive hyperemia and sublingual nitroglycerine (NTG, 75 micrograms) in all subjects. AI measured using an automated device was significantly correlated with FMD (r = -0.38, p < 0.0001). There was no significant correlation between AI and vascular response to NTG. Multiple regression analysis showed that FMD was a significant independent predictor of AI (p < 0.05). These findings suggest that increase in arterial stiffness may be associated with grade of endothelial dysfunction and that AI may be an index of not only arterial stiffness but also endothelial function.

Two flavone C-glycosides as quality control markers for the manufacturing process of ephedrine alkaloids-free Ephedra Herb extract (EFE) as a crude drug preparation.

As part of our continuing study of ephedrine alkaloids-free Ephedra Herb extract (EFE) in pursuit of its approval as a crude drug preparation, we identified two quantitative markers for the quality control of the manufacturing process of EFE and sought to establish cost-effective and simple methods for quantitative analyses. We analysed Ephedra Herb extracts grown in different habitats and collection years by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and detected two notable peaks common to each extract. These peaks were identified as vicenin-2 (1) and isovitexin 2″-O-rhamnoside (2). Quantitative analyses using the isocratic condition of LC/MS showed that the content percentages of 1 and 2 in EFE were 0.140-0.146% and 0.350-0.411%, respectively. We concluded that 1 and 2 were adequate quality control markers for quantitative analysis of EFE. Furthermore, we quantitatively analysed apigenin (3), an aglycon common to 1 and 2, and found that the conversion factors of 1 to 3 and 2 to 3 were 1.3 and 1.5, respectively. Therefore, we concluded that 3 was a secondary standard for quantifying the contents of 1 and 2 in EFE. A series of results obtained from this study will be valuable for the quality control of EFE.