Determination of Free Radical Scavenging,Antioxidative DNA Damage Activities and Phytochemical Components of Active Fractions from Lansium domesticum Corr. Fruit

Lansium domesticum Corr. or “long-kong” is one of the most popular fruits in Thailand. Its peel (skin, SK) and seeds (SD) become waste unless recycled or applied for use. This study was undertaken to determine the bioactivity and phytochemical components of L. domesticum (LD) skin and seed extracts. Following various extraction and fractionation procedures, 12 fractions were obtained. All fractions were tested for antioxidant capacity against O₂^−• and OH^•. It was found that the peel of L. domesticum fruits exhibited higher O₂^−• and OH^• scavenging activity than seeds. High potential antioxidant activity was found in two fractions of 50% ethanol extract of peel followed by ethyl acetate (EA) fractionation (LDSK50-EA) and its aqueous phase (LDSK50-H₂O). Therefore, these two active fractions were selected for further studies on their antioxidative activity against DNA damage by hydrogen peroxide (H₂O₂) in human TK6 cells using comet assay. The comet results revealed DNA-protective activity of both LDSK50-EA and LDSK50-H₂O fractions when TK6 human lymphoblast cells were pre-treated at 25, 50, 100, and 200 μg/mL for 24 h prior to H₂O₂ exposure. The phytochemical analysis illustrated the presence of phenolic substances, mainly scopoletin, rutin, and chlorogenic acid, in these two active fractions. This study generates new information on the biological activity of L. domesticum. It will promote and strengthen the utilization of L. domesticum by-products.     

Pigment identification,antioxidant activity,and nutrient composition of Tinospora cordifolia (willd.) Miers ex Hook. f & Thoms fruit

The stem, leaf, and root of Tinospora cordifolia (willd.) have been highly exploited for medicinal preparations; however, the nutritional and nutraceutical potential of its attractive red berries (fruits) have not so far been studied. Pigments, berberine (107.0 mg/100 g) and lycopene (50.8 mg/100 g), were identified in the deseeded fruit. Total phenol content in the fruit was 3.2 mg gallic acid equivalent/g. The IC₅₀ of DPPH^√ (82, and 468 ppm) and OH^√ (100, and 1,000 ppm) scavenging activity, and the EC₅₀ of reducing power (2,616, and 1,472 ppm) of hexane and methanol extracts, respectively, were observed. Oxygen radical absorbance capacities of the fruit extracts (150 and 300 ppm) were 1,107 and 2,614 μM trolox equivalent/g, respectively. Nutrient composition including carbohydrate (18.4%), proteins (1.5%), lipids (6%) of which ～62% was unsaturated fatty acids, vitamins like ascorbic acid (0.24 μg), niacin (0.7 mg), and tocopherols (2.4 mg) in 100 g fruits were determined. Potassium content was 1.2 g/100 g dry tissue of berries. These findings show that components of T. cordifolia fruit could be bioactive and used in food, pharmaceutical, and cosmetics.     

Cardioprotective actions of grape polyphenols

The aim of this review is to discuss the accumulating evidence that suggests that grape extracts and purified grape polyphenols possess a diverse array of biological actions and may be beneficial in the prevention of some inflammatory-mediated diseases including cardiovascular disease. The active components from grape extracts, which include the grape seed, grape skin, and grape juice, that have been identified thus far include polyphenols such as resveratrol, phenolic acids, anthocyanins, and flavonoids. All possess potent antioxidant properties and have been shown to decrease low-density lipoprotein–cholesterol oxidation and platelet aggregation. These compounds also possess a range of additional cardioprotective and vasoprotective properties including antiatherosclerotic, antiarrhythmic, and vasorelaxation actions. Although not exclusive, antioxidant properties of grape polyphenols are likely to be central to their mechanism(s) of action, which also include cellular signaling mechanisms and interactions at the genomic level. This review discusses some of the evidence favoring the consumption of grape extracts rich in polyphenols in the prevention of cardiovascular disease. Consumption of grape and grape extracts and/or grape products such as red wine may be beneficial in preventing the development of chronic degenerative diseases such as cardiovascular disease     

Effects of Elevated O<Subscript>3</Subscript> and/or Elevated CO<Subscript>2</Subscript> on Lipid Peroxidation and Antioxidant Systems in <Emphasis Type="Italic">Ginkgo biloba</Emphasis> Leaves

Four-year-old seedlings of Ginkgo biloba were exposed to elevated O₃, elevated CO₂ and elevated O₃ plus elevated CO₂ in open-top chambers (OTCs) to study the responses of antioxidant system in Ginkgo biloba leaves. No significant changes in reactive oxygen production and scavenging systems were detected in seedlings exposed to high CO₂. Significant increase in H₂O₂ and MDA content were induced by elevated O₃. The ascorbate content and antioxidative enzymes activity were increased significantly by exposure to high O₃ as well. But the promoted ability in scavenging did not prevent the increase in H₂O₂ content and cell membrane lipid peroxidation. The increase was mitigated by high CO₂ in the combined exposure, but the effect was hardly significant.     

Effects of Elevated CO<Subscript>2</Subscript> and O<Subscript>3</Subscript> on Phenolic Compounds in Spring Wheat and Maize Leaves

C₃ crops are generally considered more sensitive than C₄ crops to the elevated CO₂ and O₃, but it is unclear whether the concentrations of phenolic compounds in them are affected. In this paper, an enrichment experiment with open-top chamber was conducted to examine the effects of elevated CO₂, O₃, and their combination on the contents of total phenolic compounds and flavone in the leaves of spring wheat (C₃ crop) and maize (C₄ crop). The results showed for spring wheat, the total phenolic contents in its leaves at jointing stage was significantly higher under elevated CO₂ and/or O₃, with the sequence of CO₂ plus O₃ > O₃ > CO₂ > ambient, while at grain-filling stage, the total phenolic content was lower under CO₂ plus O₃ than under CO₂, O₃, and ambient. The total phenolic content in maize leaves at jointing stage had the similar variation trend with that for wheat, but at grain-filling stage, the total phenolic content was slightly affected by elevated CO₂ and/or O₃. The flavone content in spring wheat leaves was significantly lower under CO₂ and/or O₃ stress at jointing stage, but had lesser difference at grain-filling stage under the stress. The same variation trend was observed in the flavone content in maize leaves at jointing and grainfilling stages, i.e., CO₂ plus O₃ > CO₂ > ambient > O₃. C₃ plant was more sensitive than C₄ plant to the CO₂ and/or O₃ stress. Foundation item: The National Natural Science Foundation of China (No. 30570348 and 30500069).     

Catechin, epicatechin, quercetin, rutin and resveratrol in red grape: Content, in vitro antioxidant activity and interactions

The extracts obtained from skin and seeds of 10 native Tuscan and international Vitis vinifera varieties were evaluated for their antioxidant activity, total phenolic and anthocyanin content and subjected to HPLC–UV analysis to quantify the content of five phenolic constituents of biological interest: catechin and epicatechin in seeds and quercetin, rutin and resveratrol in skin extracts. The antioxidant activity of the extracts and pure compounds was assessed by means of two different in vitro tests: scavenging of the stable DPPH radical and of authentic peroxynitrite (ONOO⁻). All the extracts showed significant antiradical capacity: Merlot skin was most active towards both radicals. All the five phenols investigated possessed strong antiradical activity. Quercetin, catechin and epicatechin showed maximum activity (respectively, IC_50(DPPH) 5.5, 6.7 and 6.8 μM, IC_50(ONOO^-) 48.8, 55.7 and 56.7 μM). Potential antiradical interactive effects among the five compounds were also investigated and results indicated possible synergy between quercetin, rutin and resveratrol towards ONOO⁻. The effect was additive for catechin and epicatechin.     

Bioactive Compounds and Antioxidant Properties of Goji fruits (Lycium barbarum L.) Cultivated in Serbia

Objective: Goji fruit extracts, methanol (MGE) and hexane (HGE), were subjected to evaluation as potential source of phenolic antioxidants and antiradical activity.

Methods: Some phenolic compounds (gallic, protocatechuic, vanillic, chlorogenic, coumaric, caffeic and ferulic acid and catechin and rutin), vitamin C and carotenoids were identified and quantified by HPLC. Antioxidant activity was tested by measuring ability to scavenge DPPH and hydroxyl radicals. Also, reducing power of goji fruit extracts was determined.

Results: HPLC analysis results showed predominance of gallic acid (40.44 mg/g g.f). Vitamin C content in MGE was 716.91 mg vitC/100 g g.f. IC₅₀^DPPH· varied from 26.64 μmolTEAC/g for HGE to 62.15 μmolTEAC/g for MGE, while RP_0.5 values varied from 952.23 μmolTEAC/g for MGE to 1360.48 mg/mL for HGE. IC₅₀·^OH for MGE was 1844.01 μmolTEAC/g.

Conclusions: Our results support the use of goji fruits as rich sources of phytochemicals for further utilization in the food industry as supplements and functional food ingredients.     

Antioxidant and free radical scavenging capacity of the underutilized legume, Vigna vexillata (L.) A. Rich

The raw and processed seeds of the underutilized legume, Vigna vexillata (L.) A. Rich were extracted with 70% acetone and used for the evaluation of its antioxidant potential. The different heat treatments included dry heating, soaking followed by autoclaving with 0.1% sodium bicarbonate, soaking followed by autoclaving and autoclaving without soaking. The raw seed extract contained higher levels of total phenolics (11.7 g/100 g) and tannins (5.9 g/100 g) than the processed seed extracts. The extracts were further evaluated for their antioxidant activities using FRAP, Fe²⁺ chelation, phosphomolybdenum reduction, DPPH and ABTS⁺ radical scavenging, antihemolytic and β-carotene/linoleic acid emulsion systems. In general, the raw seed extracts were the most potent antioxidant suppliers and free radical scavengers. Interestingly, among the various processing methods, acetone extract of dry heated seed sample registered higher antihemolytic activity (86.2%) at the concentration of 500 μg/ml, whereas, in β-carotene/linoleic acid emulsion system, the extracts of raw seeds and the presoaked seeds followed by autoclaving with 0.1% sodium bicarbonate exhibited higher and comparable activity than other processed seed extracts.     

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H₂O₂-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H₂O₂-induced cell damage in vitro.     

Wild-growing lignicolous mushroom species as sources of novel agents with antioxidative and antibacterial potentials

Crude extracts of two edible and two medicinal lignicolous mushroom species: Meripilus giganteus, Agrocybe aegerita, Fomes fomentarius and Xylaria polymorpha, growing wild in Serbia, were analyzed for their antioxidative and antibacterial potentials. Free radical scavenging capacity (RSC) on DPPH^? and ^?OH was evaluated both by spectrophotometer and by Electron Spin Resonance (ESR) spectroscopy against DPPH^?. The highest antioxidant and antibacterial bioactivity was obtained with F. fomentarius extracts (IC₅₀?≈?10.7?µg/ml in DPPH^? assay; 136.6?mg ascorbic acid equivalents (AAE)/g dry weight (d.w.) for ferric reducing antioxidant power FRAP). It also showed the highest total phenol (TP) (82.54?mg gallic acid equivalents (GAE)/g dry weight (d.w.)) and total flavonoid (TF) content (76.8?µg rutin equivalents (RE)/g dry weight (d.w.)). A. aegerita showed the best antioxidant activity (IC₅₀?=?0.87?mg/ml) against DPPH^? in ESR analysis. Total redox potential of extracts was in direct positive correlation with TP content (r^2?=?0.98) and TF content (r^2?=?0.58). GC/MS analysis detected major constituents of extracts, confirming the presence of the following organic and phenolic acids: fumaric, succinic, mallic, 4-hydroxy benzoic, gentisic, protocatechuic, vanillic, gallic and p-coumaric acid.     

Radical scavenging activity of flavonoids from Trollius chinensis Bunge

Objective

Flos trollii is considered as functional tea, as well as a traditional medicinal herb, in China. In this study, total phenolic and flavonoid contents of Flos trollii were determined by a colorimetric method. The antioxidative potential of the hydroalcoholic extract of Flos trollii (FTE, extracted by alcohol:water, 80:20) was also evaluated by various antioxidant assays.

Methods

Chemiluminescence technique was used to determine the radical scavenging activities of FTE toward different reactive oxygen species, including superoxide anion (O_-2·), hydroxyl radical (OH·), lipid-derived radicals (R·), and singlet oxygen (¹O₂).

Results

FTE could effectively scavenge O_-2·, OH·, R·, and ¹O₂ at an efficient concentration (EC₅₀) of 46, 5.64, 5.19, and 3.97 mg/mL, respectively. Moreover, the radical scavenging activities of FTE were higher than those of ascorbic acid. Further, FTE had higher 1,1 diphenyl-2-picrylhydrazyl radical (DPPH) radical-scavenging activity with EC₅₀ 44 mg/mL, compared with butylated hydroxytoluenesynthetic antioxidant with EC₅₀ 52 mg/mL.

Conclusion

All results indicated that FTE is a powerful antioxidant, deserving of better utilization of the extracted Flos trollii as antioxidants.     

Sulfur (S35) metabolism in rats exposed to 535O2 contained in the air with simultaneous use of the method of (S35O2) binding by ammonia

Summary Sulfur metabolism was traced in experimental investigations on rats, and the importance of the method for neutralizing sulfur dioxide with ammonia was estimated.The animals were exposed to S³⁵O₂ by means of inhalation, and radioactivity in several organs at different periods of exposure was traced.Sulfur incorporation into proteins and mucopolysaccharides, as well as elimination of isotope with urine and feces at different periods of time after exposure, were also determined. The second group of animals was exposed to the products of S³⁵O₂ neutralized by ammonia; after performing identical investigations it was found that the use of ammonia to neutralize sulfur dioxide in the air decreased the amount of sulfur which could pass into the organism through the breathing of air contaminated by SO₂. The use of S³⁵O₂ neutralized by ammonia brings about a faster elimination of sulfur from the organism.This work was supported by the Environmental Protection Agency USA, Project No. 5-533-1.     

Antioxidant activities of some Greek wines and wine phenolic extracts

Antioxidant activities and phenolic composition of eight Greek wines were determined. Red wines scavenged 1,1′-diphenyl-2-picryl-hydrazyl radical (DPPH) to a much greater degree than whites, in proportion to their phenolic contents. Red wines inhibited β-carotene bleaching, while white wines were almost inactive. Red wines were more active than quercetin in DPPH and less active in β-carotene assay. Xinomavro-red must exhibited significant activities in both assays. Roditis-white must scavenged DPPH, and exhibited some activity in β-carotene assay. Young Xinomavro and Roditis wines and their extracts were more active in scavenging DPPH than the respective aged wine and extracts. However, young Xinomavro wine and its extracts were less active than those of aged wine in β-carotene assay. Roditis wine extracts exhibited only limited activity in β-carotene assay. Xinomavro young and aged wine extracts rich in anthocyanins and flavanols were more active than the others in both assays used.     

The antioxidant and cytotoxic activities of Sonchus oleraceus L. extracts

This study investigated in vitro antioxidant activity of Sonchus oleraceus L. by extraction solvent, which were examined by reducing power, hydroxyl radical-scavenging activity(HRSA) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assays. 70% MeOH extract had the greatest reducing power while EtOH extract had the greatest HRSA. The antioxidant activity of S. oleraceus extracts was concentration dependent and its IC₅₀ values ranged from 47.1 to 210.5 µg/ml and IC₅₀ of 70% MeOH, boiling water and 70% EtOH extracts were 47.1, 52.7 and 56.5 µg/ml, respectively. 70% MeOH extract of S. oleraceus contained the greatest amount of both phenolic and flavonoid contents. The extracts tested had greater nitrite scavenging effects at lower pH conditions. The cytotoxic activity showed that EtOH extract had the best activity against the growth of stomach cancer cell. These results suggest that S. oleraceus extract could be used as a potential source of natural antioxidants.     

Antioxidant rich grape pomace extract suppresses postprandial hyperglycemia in diabetic mice by specifically inhibiting alpha-glucosidase

Background

Postprandial hyperglycemia is an early defect of type 2 diabetes and one of primary anti-diabetic targets. Treatment of postprandial hyperglycemia can be achieved by inhibiting intestinal α-glucosidase, the key enzyme for oligosaccharide digestion and further glucose absorption. Grape pomace is winemaking byproduct rich in bioactive food compounds such as phenolic antioxidants. This study evaluated the anti-diabetic potential of two specific grape pomace extracts by determining their antioxidant and anti-postprandial hyperglycemic activities in vitro and in vivo.

Methods

The extracts of red wine grape pomace (Cabernet Franc) and white wine grape pomace (Chardonnay) were prepared in 80% ethanol. An extract of red apple pomace was included as a comparison. The radical scavenging activities and phenolic profiles of the pomace extracts were determined through the measurement of oxygen radical absorbance capacity, DPPH radical scavenging activity, total phenolic content and flavonoids. The inhibitory effects of the pomace extracts on yeast and rat intestinal α-glucosidases were determined. Male 6-week old C57BLKS/6NCr mice were treated with streptozocin to induce diabetes. The diabetic mice were then treated with vehicle or the grape pomace extract to determine whether the oral intake of the extract can suppress postprandial hyperglycemia through the inhibition of intestinal α-glucosidases.

Results

The red grape pomace extract contained significantly higher amounts of flavonoids and phenolic compounds and exerted stronger oxygen radical absorbance capacity than the red apple pomace extract. Both the grape pomace extracts but not the apple pomace extract exerted significant inhibition on intestinal α-glucosidases and the inhibition appears to be specific. In the animal study, the oral intake of the grape pomace extract (400 mg/kg body weight) significantly suppressed the postprandial hyperglycemia by 35% in streptozocin-induced diabetic mice following starch challenge.

Conclusion

This is the first report that the grape pomace extracts selectively and significantly inhibits intestinal α-glucosidase and suppresses postprandial hyperglycemia in diabetic mice. The antioxidant and anti-postprandial hyperglycemic activities demonstrated on the tested grape pomace extract therefore suggest a potential for utilizing grape pomace-derived bioactive compounds in management of diabetes.

     

Influence of Elevated Carbon Dioxide and Ozone on the Foliar Nonvolatile Terpenoids in <Emphasis Type="Italic">Ginkgo Biloba</Emphasis>

The aim of this study was to determine the effects of elevated O₃ and elevated CO_2, singly and in combination, on the contents of nonvolatile terpenoids in leaves of Ginkgo Biloba. The results showed that elevated CO₂, alone and in combination with elevated O₃ increased concentrations of all the determined terpenoids, while elevated O₃ alone only increased concentration of bilobalide. These results demonstrated that the metabolism of terpenoids in ginkgo leaves was more sensitive to elevated CO₂ than elevated O₃.     

Ice Cream with Functional Potential Added Grape Agro-Industrial Waste

The objective of this study was to develop grape ice cream spiked with agro-industrial grape residue flour (GRF). The attributes of color, aroma, flavor, texture and overall quality of ice cream with different GRF concentrations [P (0%), F1 (2%), F2 (6%), F3 (10%)] were evaluated by a panel of tasters, using hedonic scale of 9 points. The formulations with the best sensory characteristics were used to determine the chemical composition, the physical and chemical characteristics, the bioactive compounds content and antioxidant capacity, besides the objective evaluation of color (CIELAB). The F1 ice cream (2% GRF) showed satisfactory sensory attributes that allowed to be appointed by the taste panel as the best formulation. The addition of 2% of GRF in to grape ice cream provided an increase in protein, lipids, ash, dietary fiber, total energy value, reducing sugars, total phenolics, flavonoids, flavonols and anthocyanins. Consequently, the radical scavenging ability of DPPH^? (EC50) e ABTS^?+ was higher than the pattern sample (without GRF) (p <0.05). The GRF incorporated in to grape ice cream formulation made changes in color attributes of the product that, although statistically significant, did not affect the visual perception of characteristic color and desired for this type of product. Thus, the addition of grape residue flour (2%) in to ice cream may be considered a viable and healthy technology application, to obtaining a product with organoleptic and nutritional characteristics satisfactory, higher levels of dietary fibers, bioactive compounds and antioxidant potential, and contribute to the use of this waste.     

Non-polyphenolic compounds of a specific kind of dried grape (Maviz) inhibit memory impairments induced by beta-amyloid peptide

Objectives: Although grape has been recently the topic of many investigations, Maviz (a kind of dried one) has remained neglected. The aim of this study was to assess anti-Alzheimer activity of Maviz.

Methods: To reach this goal, total phenolic content (TPC) of ethanolic (Eth) and aqueous (Aq) extracts were determined and radical scavenging activity was assayed by 2,2-diphenyl-1-picrylhydrazyl. Chemical compositions of each extract were also determined via GC-Mass. Behavioral changes were studied via passive avoidance and Morris water maze in Aβ-induced model of Alzheimer's disease. Catalase (CAT) and superoxide dismutase (SOD) determination were also done on rats’ hippocampus.

Results: The results showed that seed Eth extract has a high level of TPC and radical scavenging activity. However, this extract had surprisingly no effect on memory and CAT and SOD activities. In contrast, fruit Aq and Eth extracts (containing furfurals as major compounds) inhibited memory impairment (P?P?Conclusion: It seems that Maviz non-phenolic compounds-most probably 5-hydroxymethylfurfural and other similar derivatives-are responsible for these actions.     

Antioxidant responses of rice seedling to Ce⁴+ under hydroponic cultures

Since the 1980s, rare earth elements have been commonly used in China because of their enriched fertilizers. To understand the potential benefits or damages of Ce⁴⁺ on rice, the antioxidant responses (superoxide dismutase, ascorbate peroxidase, catalase activities, and ascorbate and glutathione contents) of rice seedling to Ce⁴⁺ under hydroponic cultures were investigated. The results showed that Ce⁴⁺ induced H₂O₂ and O₂⁻ production of rice seedling. The inhibition studies with diphenylene iodonium suggested that the key enzyme responsible for oxidative bursts was primarily NADPH oxidase. Ce⁴⁺ (0.02 mM) increased the antioxidant capacity of reduced ascorbate and glutathione and the levels of superoxide dismutase, ascorbate peroxidase, and catalase. However, antioxidant enzymes and antioxidant capacity of rice seedling were decreased by 0.2 mM Ce⁴⁺ treatment, indicating that higher content of Ce⁴⁺ damaged the mechanism of defense responses and emerged the peroxidation of membrane lipids. These results will help us to understand the mechanism of Ce⁴⁺ on rice and concern about its environmental impact in agricultures.     

Discrimination of five Greek red grape varieties according to the anthocyanin and proanthocyanidin profiles of their skins and seeds

The knowledge of grapes phenolic content is proven to be critical for the vinification process and the improvement of wine quality. This study was undertaken to determine the phenolic composition and to employ the phenolic profile as a varietal discrimination tool in five Greek red grape varieties. Ninety grape samples from two seasons (2017 and 2018) were analyzed after extraction with organic solvents. Their proanthocyanidin profile, expressed as percentages of flavan-3-ols, was determined in both skins and seeds by employing phloroglucinolysis followed by HPLC-UV and MS detection, and anthocyanin profile was identified only in the skin extracts by HPLC-UV. Significant differences were observed in proanthocyanidin and anthocyanin profiles of skin extracts between the samples of different varieties, but not in seeds. (-)-Epicatechin was the main subunit in Mandilaria, Kotsifali, Agiorgitiko and Xinomavro grapes while (-)-epigallocatechin gallate in Mavrotragano. Malvidin-3-O-glucoside was the predominant pigment in all grape samples analyzed with the exception of Kotsifali skin extracts, where peonidin-3-O-glucoside was the most abundant anthocyanin. In addition, Mavrotragano skin extracts were the richest in delphinidin and petunidin-3-O-glucosides, while Agiorgitiko and Xinomavro contained the highest amount of malvidin-3-O-glucoside. The results underline the significance of the skin phenolic composition as a tool for the discrimination of the Greek red grape varieties.