Perilla seed oil improves bone health by inhibiting bone resorption in healthy Japanese adults: A 12-month, randomized, double-blind, placebo-controlled trial.

Accumulating evidence suggests the beneficial effect of omega-3 polyunsaturated fatty acids (PUFAs) on bone mineral density (BMD). However, the effects of perilla (Perilla frutescens) seed oil (PO), a rich source of α-linoleic acid (LNA), on human bone have not yet been elucidated. This randomized, double-blind, placebo-controlled trial investigated the effects of long-term PO intake on bone health in Japanese adults. After screening for eligibility, 52 participants (mean age 54.2 ± 6.4 years) were randomly assigned to placebo (n = 25) and PO (n = 27) groups, which received 7.0 ml of olive oil and PO daily, respectively. At baseline and 12-month, quantitative ultrasound of the right calcaneus was measured with an ultrasound bone densitometer and percentage of the Young Adult Mean (%YAM) was calculated. Serum levels of tartrate-resistant acid phosphatase 5b (TRACP-5b), and bone alkaline phosphatase (BALP) were evaluated. In addition, PUFA levels in the erythrocyte plasma membrane (RBC-PM), serum biological antioxidant potential (BAP), and diacron reactive oxygen metabolites (d-ROM) were evaluated. Compared with the placebo group, %YAM levels increased and serum TRACP-5b levels decreased significantly in the PO group at 12-month, while serum BALP levels remained unchanged. Moreover, RBC-PM LNA levels and BAP/d-ROM ratios increased significantly in the PO compared with the placebo group. These results suggest that long-term PO intake may improve age-related BMD decline by suppressing bone resorption and increasing LNA levels.

Genetic variation of seed oil characteristics in native Korean germplasm of Perilla crop (Perilla frutescens L.) using SSR markers.

BACKGROUND: In order to maximize the use of valuable native Perilla germplasm in South Korea, knowledge of the Perilla seed oil content and genetic variation among native Perilla germplasm resources is very important for the conservation and development of new Perilla seed oil varieties using the native Perilla germplasm accessions preserved from the Rural Development Administration Genebank (RDA-Genebank) collection from South Korea. OBJECTIVES: In this study, we studied population structure and association mapping to identify Perilla SSR markers (PSMs) associated with the five fatty acid contents and two seed characteristics of the native Korean Perilla germplasm accessions of cultivated var. frutescens of the RDA-Genebank collected in South Korea. METHODS: For an association mapping analysis to find PSMs associated with the five fatty acid contents and two seed characteristics of the Perilla germplasm accessions of cultivated var. frutescens, we evaluated the content of five fatty acids of 280 native Korean Perilla germplasm accessions and used 29 Perilla SSR primer sets to measure the genetic diversity and relationships, population structure, and association mapping of the native Korean Perilla germplasm accessions of the RDA-Genebank collected in South Korea. RESULTS: Five fatty acids of 280 native Korean Perilla accessions were identified as follows: palmitic acid (PA) (5.30-8.66%), stearic acid (SA) (1.60-4.19%), oleic acid (OA) (9.60-22.5%), linoleic acid (LA) (8.38-25.4%), and linolenic acid (LNA) (52.7-76.4%). In a correlation analysis among the five fatty acids and two seed characteristics of the 280 Perilla accessions, the combinations of PA and SA (0.794**) and SA and OA (0.724**) showed a particularly high positive correlation coefficients compare to other combinations. By using an association analysis of the 29 PSMs and the five fatty acids in the 280 Perilla accessions, we found 17 PSMs (KNUPF1, KNUPF2, KNUPF4, KNUPF10, KNUPF16, KNUPF25, KNUPF26, KNUPF28, KNUPF37, KNUPF55, KNUPF62, KNUPF71, KNUPF74, KNUPF77, KNUPF85, KNUPF89, and KNUPF118) associated with the content of the five fatty acid components and two seed characteristics. CONCLUSIONS: These PSMs are considered to be useful molecular markers related to five fatty acid components and two seed characteristics for selecting accessions from the germplasm accessions of the Perilla crop and their related weedy types through association mapping analysis and marker-assisted selection (MAS) breeding programs.

Enhancement of Oxidative Stability of Perilla Seed Oil by Blending It with Other Vegetable Oils.

Perilla seed oil is mainly composed of omega-3 fatty acid (α-linolenic acid, ALA). Despite being nutritionally favorable and rich in unsaturated fatty acids, its low oxidative stability limits its application in food. Thus, the present study aimed to formulate a stable oil blend using perilla seed oil with selected vegetable oil of higher stability characteristics and balance the ratio of the fatty acids. Hence, improving the nutritional and functional value of the blended oil. Perilla seed oil was blended with different edible oil (palm olein, coconut oil, and groundnut oil) in ratios of 20:80 and 30:70. All the blended oils were studied for their fatty acid composition, physicochemical properties, oxidative stability, and nutritional quality index. It was found that perilla seed oil blended with saturated oil like palm olein had improved physicochemical properties and oxidative stability (0.5 h to 6.5 h). The fatty acids ratio of perilla and palm olein blends was close to the recommended value given by the World health organization (WHO). The nutritional quality indices (atherogenic index, the thrombogenic index, and hypocholesterolemic: hypercholesterolemic ratio) of blended oil were also improved compared to the individual oils.

Effect of microwave pretreatment of perilla seeds on minor bioactive components content and oxidative stability of oil.

This study investigated the effects of different microwave power (380 W, 540 W, 700 W) and time (0-10 min) on the minor bioactive components content and oxidative stability of perilla oil. The results indicated that fatty acids in perilla oil were slightly affected by microwave treatment. The oxidative stability of perilla oil increased with increasing microwave treatment intensity and the oil from perilla seeds treated at 700 W for 10 min had the highest oxidative stability. Compared with other microwave treatments, treatment with 700 W for 10 min resulted in significant increases in the total phytosterols content, Maillard reaction products and DPPH radical scavenging activity of perilla oil, while showed dramatic reductions in the total tocopherol content, phenolic compounds content and lipase activity. These results proved that microwave treatment of perilla seeds was an effective way to improve the quality of perilla oil.

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.

Presence of Cholesterol in Non-Animal Organisms: Identification and Quantification of Cholesterol in Crude Seed Oil from Perilla frutescens and Dehydrated Pyropia tenera.

Studies have reported that cholesterol, a molecule found mainly in animals, is also present in some plants and algae. This study aimed to determine whether cholesterol exists in three dehydrated algae species, namely, Pyropia tenera, Saccharina japonica, and Undaria pinnatifida, and in one plant species, namely, Perilla frutescens (four perilla seed oil samples were analyzed). These species were chosen for investigation because they are common ingredients in East Asian cuisine. Gas chromatography-flame ionization detection (GC-FID) analysis found that cholesterol was present in P. tenera (14.6 mg/100 g) and in all four perilla seed oil samples (0.3-0.5 mg/100 g). High-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) also demonstrated that cholesterol was present in P. tenera (14.2 mg/100 g) and allowed the separation of cholesterol from its isomer lathosterol. However, cholesterol could not be detected by HPLC-ELSD in the perilla seed oil samples, most likely because it is only present in trace amounts. Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the presence of cholesterol in both P. tenera and perilla seed oil. MRM results further suggested that lathosterol (a precursor of cholesterol) was present in P. tenera.

The anti-tussive, anti-inflammatory effects and sub-chronic toxicological evaluation of perilla seed oil.

BACKGROUND: Perilla seed oil (PSO) is the main constituent of perilla seeds currently being used in the food industry, however it also has great clinical potential in the regulation of lung function as a nutrition supplement because of the high content of α-linolenic acid (ALA). In this study, the pharmacological activities including anti-tussive, expectorant and anti-inflammatory effect of PSO were performed. Furthermore, the 90-day sub-chronic oral toxicity with a 30 day recovery period was evaluated in Wistar rats. RESULTS: The pharmacological studies demonstrated that PSO inhibited cough frequency induced by capsaicine in mice. PSO also inhibited the leukotriene B4 (LTB4) release from the calcium ionophore A23187-induced polymorphonuclear neutrophils (PMNs) to some extent. In this sub-chronic toxicity study, mortality, clinical signs, body weight, food consumption, hematology, serum biochemistry, urinalysis, organ weight, necropsy, and histopathology were used to evaluate the toxicity of PSO. Lower body weight and various negative impacts on liver related parameters without histopathological lesion were observed in the 16 g kg-1 groups. No clinically significant changes were discovered in the 4 g kg-1 group during the test period. CONCLUSION: In summary, PSO exhibited anti-tussive and anti-inflammatory activities in vivo and in vitro. These sub-chronic toxicity studies inferred that the 'no-observed adverse effect level' (NOAEL) of PSO in Wistar rats was determined to be 4 g kg-1 . These results may provide a safety profile and a valuable reference for the use of PSO. © 2020 Society of Chemical Industry.

Influence of refining processes on the bioactive composition, in vitro antioxidant capacity, and their correlation of perilla seed oil.

The effect of chemical refining process on the bioactive composition, in vitro antioxidant capacity, and their correlation of perilla seed oil (PSO) were investigated. In this paper, seven samples corresponding to each step of the refining process (degumming, neutralization, bleaching, deodorization, winterization, crude, and refined oils) were studied. The results showed that phenolic compounds and tocopherols were removed from PSO to a degree of 19.4% and 5.4%, respectively. In addition, the carotenoid content of PSO decreased during the refining process. The main carotenoid of PSO was found to be lutein, and the compound was lost completely during the bleaching step of the refining process. In this paper, we analyzed the variation of carotenoid content in PSO during the refining process for the first time. Neutralization affected the contents of phytosterols the most, followed by the effects of degumming and bleaching. The demonstrated results of Pearson product-moment correlation indicated that total tocopherols were significantly correlated with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorption capacity (ORAC) values, whereas carotenoids were significantly correlated with the DPPH value. However, phenolic compounds and phytosterols have no significant difference with DPPH, 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, ORAC, and ferric reducing antioxidant power values. The collected information can be applied to seeking out optimum factors needed to suffice the fundamental requirements for PSO production and minimize micronutrient losses to enhance its market value. PRACTICAL APPLICATION: The present study aimed to determine influence of chemical refining in the bioactive composition of perilla seed oil (PSO) as well as its antioxidant capacity in vitro. Moreover, we also intend to find the correlation between them. Results indicated that this study supplies a good reference for the industrial parameters of the refining process to minimize micronutrient losses and further obtain high-quality PSO products for consumers.

Effects of dietary perilla seed oil supplementation on lipid metabolism, meat quality, and fatty acid profiles in Yellow-feathered chickens.

This study evaluated the effect of the dietary replacement of 1% lard (CT) with 1% perilla oil (PO), 0.9% perilla oil + 0.1% anise oil (PA), or 0.9% perilla oil + 0.1% ginger oil (PG) on indices of lipid metabolism, antioxidant capacity, meat quality, and fatty acid profiles from Yellow-feathered chickens at day 63. Compared with the CT chickens, those given perilla oil had decreased (P < 0.05) plasma lipid levels including triglycerides (TG), total cholesterol (TCH), and low-density lipoprotein cholesterol (LDL-C). Hepatic TG, TCH levels, and fatty acid synthase activity were also decreased (P < 0.05) in chickens fed diets containing perilla oil. Abdominal fat percentage was significantly decreased in birds fed the PG compared to CT diets. Birds fed the PA or PG diets had increased (P < 0.05) hepatic total SOD, glutathione peroxidase, and glutathione-S-transferase than in chickens given PO alone. In addition, the content of reduced glutathione (GSH) in breast muscle was lower (P < 0.05) in birds fed PO compared with those given PG, and the reverse was true for content of malondialdehyde. Compared with the CT diet, the PO diet decreased breast muscle shear values and increased yellowness (b*) of breast muscle (P < 0.05). Birds fed the PA or PG diets had meat with better overall acceptability than those fed the CT diet. Chickens fed perilla oil diets exhibited higher contents of α-linolenic acid (C18:3n-3), DHA (22:6n-3), polyunsaturated fatty acids, and n-3 fatty acids, together with a lower content of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), total saturated fatty acids, and n-6/n-3 ratio compared to controls (P < 0.05). These findings indicate that perilla oil has the potential to decrease lipid-related indices and improve fatty acid profiles of breast meat in chickens without adverse effect on antioxidant status or meat quality; this was even better when perilla oil was given together with anise oil or ginger oil.

Preparative and scaled-up separation of high-purity α-linolenic acid from perilla seed oil by conventional and pH-zone refining counter current chromatography.

α-Linolenic acid is an essential omega-3 fatty acid needed for human health. However, the isolation of high-purity α-linolenic acid from plant resources is challenging. The preparative separation methods of α-linolenic acid by both conventional and pH-zone refining counter current chromatography were firstly established in this work. The successful separation of α-linolenic acid by conventional counter current chromatography was achieved by the optimized solvent system n-heptane/methanol/ water/acetic acid (10:9:1:0.04, v/v), producing 466 mg of 98.98% α-linolenic acid from 900 mg free fatty acid sample prepared from perilla seed oil with linoleic acid and oleic acid as by-products. The scaled-up separation in 45× is efficient without loss of resolution and extension of separation time. The separation of α-linolenic acid by pH-zone refining counter current chromatography was also satisfactory by the solvent system n-hexane/methanol/water (10:5:5, v/v) and the optimized concentration of trifluoroacetic acid 30 mM and NH4 OH 10 mM. The separation can be scaled up in 180× producing 9676.7 mg of 92.79% α-linolenic acid from 18 000 mg free fatty acid sample. pH-zone refining counter current chromatography exhibits a great advantage over conventional counter current chromatography with 20× sample loading capacity on the same column.

Development of Perilla seed oil and extra virgin olive oil blends for nutritional, oxidative stability and consumer acceptance improvements.

This study reports the blending at different levels (25, 30, 35, 40 and 45%) of Perilla seed oil (PO) with extra virgin olive oil (EVOO). Pure oils and blends were evaluated in terms of free acidity, peroxide value, fatty acid composition, sterols, tocopherols and biophenols content, oxidation stability, sensory acceptability and food pairing. Blends with high content of ω - 3 and ω - 6 fatty acids, biophenols, tocopherols, sterols and satisfying oxidation stability were obtained, representing products with improved nutritional properties. All blends resulted acceptable by consumers. Two groups of consumers with opposite preferences for samples with low (25-35%) and high (40-45%) levels of PO were identified. Blends containing 40-45% of PO were mainly paired to strong-flavour and cooked foods, while blends with less PO were preferably matched with raw meat and vegetables. Consequently, PO and EVOO blends showed promising potential as innovative vegetable oils with improved nutritional properties and versatile gastronomic use.

Acute and sub-chronic 90-day oral toxicity study of Perilla seed oil in rodents and Beagle dogs.

Perilla seeds are used as food and traditional medicine in China. This study aimed to investigate the toxicity profile of Perilla seed oil (PSO), which is the main constituent of Perilla seeds in rodents and Beagle dogs. No significant treatment-associated toxicity or mortality was observed at PSO dosages of up to 50 g/kg and 20 g/kg in KM mice and Wistar rats, respectively, suggesting that PSO was well tolerated by the experimental rodents. Sub-chronic oral toxicity of PSO was studied in dogs at doses of 3, 6 and 12 g/kg/d for 90 days followed by a 30 day recovery period. The results indicated that the body weight increased in all-dose groups more than control group, typical of animals on diets rich in fatty acids. Treatment-related side effects, including changes in hematology and serum biochemistry parameters, histopathology of liver and lymph glands, were observed in the high and moderate-dose dogs. However, these changes disappeared after the doses were withdrawn during the recovery period, except for alteration of liver in the high-dose group. In conclusion, the "no observed adverse effect level" (NOAEL) of oral administration of PSO for 90 days in Beagle dogs was considered to be 3 g/kg/d.

Outcomes of Perilla Seed Oil as an Additional Neuroprotective Therapy in Patients with Mild to Moderate Dementia: A Randomized Control Trial.

BACKGROUND: Dementia is a common medical disorder in the elderly. Oxidative stress plays a major role in the process of cognitive decline in dementia. Perilla seed oil demonstrates its neuroprotective effects via anti-oxidative mechanisms against dementia. We investigate neuroprotective effects of perilla seed oil as an additional treatment in patients with mild to moderate dementia. METHOD: A double-blind, randomized-control trial (perilla seed oil versus placebo) in patients with mild to moderate dementia was conducted. Perilla seed oil or placebo was added on with standard treatment for six months. Cognitive function was compared at nine months after enrollment. RESULT: 182 patients, with 94 in the experimental group and 88 in the placebo group, were able to complete the study. Cognitive function is not significantly different compared between groups. However, the total cholesterol and LDL cholesterol were significantly lower in the experimental group. Perilla seed oil had no adverse effect to kidney, liver, blood components or glucose metabolism. CONCLUSION: Perilla seed oil as additional neuroprotective therapy in patients with mild to moderate dementia does not improve cognitive function. Perilla seed oil significantly reduced total cholesterol and LDL cholesterol. A clinical trial is needed to prove the benefit of cholesterol-lowering effects with perilla seed oil in human.

Evaluating the safety of phytosterols removed perilla seed oil-based lipid emulsion.

OBJECTIVES: The aim of this study was to ascertain the potential toxicity of perilla seed oil-based lipid emulsion (POLE) caused by phytosterols and confirm the efficacy of the technique for removing phytosterols from perilla seed oil, and evaluate the safety of a low phytosterol POLE in a long-term tolerance study in dogs. METHODS: A comparison between a soybean oil lipid emulsion (Intralipid group A) and POLE with high (group B) versus low (group C) levels of phytosterols was made with regard to their effects on the general condition, hematological and biochemical parameters, urinalysis and histopathological changes in nine dogs receiving daily infusions for four weeks at dosage levels of 6, 6, 9 g fat /kg. RESULTS: Dogs in group A and group C remained in good condition and gained weight during the infusion period and no diarrhea or gastrointestinal bleeding occurred. Only a moderate degree of anemia was observed, the biochemical parameters changed only slightly and returned to normal after treatment had ceased. However, the dogs in group B exhibited significant symptoms of 'fat overload syndrome'. Vomiting, diarrhoea and blood in the faeces were observed. Moreover, triglyceridemia, cholesteremia, and dark urine as well as microscopic signs of liver and gastrointestinal tract damage and generalized jaundice were clearly seen. CONCLUSIONS: Phytosterols promote 'fat overload syndrome' in long-term tolerance studies of POLE in dogs by producing cholestatic liver injury and interfering with fat metabolism. And the toxicity of POLE was reduced by removing phytosterols.

The Effect of Spa Therapy Combined with Dietary Supplementation with n-3 Fatty Acids on Serum Eosinophil Cationic Protein in Asthmatic Subjects / 日本温泉気候物理医学会雑誌

Recently, we have also reported that spa therapy combined with dietary supplementation of perilla seed oil would be effective for patients, and would suppress the generation of leukotriene C4 (LTC4) by peripheral leucocytes. However, it is still unclear how the combination spa therapy and dietary supplementation of perilla seed oil influences on the pathophysiology of bronchial asthma. In the present study, the effects of spa therapy combined with dietary supplementation of perilla seed oil were examined in patients with asthma in relation to the serum eosinophil cationic protein (ECP) levels to investigate the effect on bronchial asthma. Ten adult asthmatic patients with moderate type asthma in terms of severity were taken to have a complex spa therapy and consume perilla seed oil-rich diet for 4 weeks. The generation of LTC4 by peripheral leucocytes, serum ECP level and pulmonary function were measured. Significant decreases were observed for LTC4 and ECP for 4 weeks. Forced vital capacity (FVC), which was one of the pulmonary function tests, improved significantly at 4 weeks (p<0.05). The number of eosinophils decreased for 4 weeks, but the differences were not significant. The results obtained here suggest that spa therapy combined with dietary supplementation of perilla seed oil leads to decrease in LTC4 and ECP and improves pulmonary function and asthma control.

Effects of Spa Therapy Combined with Dietary Supplementation with n-3 Fatty Acids on Bronchial Asthma / 日本温泉気候物理医学会雑誌

N-3 fatty acids are reportedly effective for asthma. In addition, spa therapy has been reported to be effective for patients with asthma. In the present study, the effects of spa therapy combined with perilla seed oil-rich diet (rich in n-3 fatty acid) were examined on asthma. A total of 14 asthmatic patients had a complex spa therapy and consumed a perilla seed oil-rich diet-rich in α-linolenic acid (α-LNA) for 8 weeks. Generation of leukotriene (LT) C4 by leucocytes, respiratory function were analyzed. The generation of LTC4 by leucocytes decreased significantly for 2, 4 and 8 weeks (P<0.05). Peak expiratory flow (PEF) values increased significantly for 2, 4, 6 and 8 weeks (P<0.05). The values of ventilatory parameters [forced vital capacity (FVC), forced expiratory volume in one second (FEV₁), forced expiratory flow after 25% of expired FVC (FEF₂₅), forced expiratory flow after 75% of expired FVC (FEF₇₅), mean expiratory flow during the middle half of the FVC (FEF_25-75)] revealed a significant increase after 4 and 8 weeks of the modified diet (P<0.05). The results suggest that spa therapy combined with a perilla seed oil-rich diet are effective in the treatment of asthma in terms of its ability to suppress LTC4 generation by leucocytes, and in inducing an improvement of pulmonary function.