JP7533892B2 - Antioxidant and its manufacturing method - Google Patents

Description

The present invention relates to an antioxidant consisting of a plant extract and a method for producing the same.

In animals, oxygen taken into the body is converted into highly reactive compounds called reactive oxygen species. While reactive oxygen species have strong bactericidal properties and play an important role in immune function, excessive production of these species can damage cells and cause various disorders, including aging, cancer, and cardiovascular disorders.
There are three types of active oxygen: alkoxy radical activity, hydroxyl radical activity, and singlet oxygen activity.
Antioxidants are substances that remove excess active oxygen in the body or suppress its oxidative action, and polyphenols and carotenoids are known to have this effect. Polyphenols and carotenoids are plant-derived components and are found in crops such as green tea, black tea, aloe juice, blackberry juice, cucumber juice, lemon, and tomato juice. The antioxidant effect of polyphenols and carotenoids is measured by their ability to scavenge alkoxy radicals and hydroxyl radicals.

However, due to the diversity of polyphenol structures, it is difficult to measure the degree of radical scavenging activity in a fundamentally reliable manner. In particular, there have been very few examples of measuring the singlet oxygen scavenging activity, partly because a measurement method for this activity had not been established.
In recent years, a method for analyzing the antioxidant capacity (functionality) of various antioxidants (catechins, flavonoids, vitamin E, etc.) contained in foods and the like using the ORAC (Oxygen Radical Absorbance Capacity) method has been developed. Based on this ORAC method, a method for quantifying the antioxidant capacity of carotenoids and the like that could not be measured using the ORAC method has been developed using the SOAC (single oxygen absorption capacity) method. However, the SOAC method is specialized for evaluating the singlet quenching activity of foods that are easily soluble in oil-based solvents, such as carotenoids, and is therefore not suitable for evaluating water-soluble samples.

Singlet oxygen is a reactive oxygen species that is involved in various biological phenomena by directly reacting with biomolecules and regulating intracellular signal transduction pathways involving redox. There are light-dependent and light-independent mechanisms for the generation of singlet oxygen in the body, and both have been shown to lead to disease. Singlet oxygen is known to be particularly reactive on the skin surface, and singlet oxygen generated on the skin surface is involved in skin oxidation, the production of acne bacteria, and genetic metabolic disorders such as porphyria. By making an antioxidant capable of quenching singlet oxygen water-soluble, it can be applied to the skin and can easily penetrate the skin, making it very effective as a lotion or serum to prevent and resolve skin problems (Non-Patent Document 1).

The inventors have been engaged in research into the use of oil palm sap for many years, and have previously proposed a method for producing ethanol using the sugars contained in oil palm (Patent Document 1). However, it is known that some oil palm sap has a low sugar concentration (Patent Document 2), and there has been a demand for technology that adds more value to oil palm and its sap and expands the range of uses, including oil palm sap with low sugar concentration that is unsuitable for ethanol production.

Patent No. 4065960 JP 2009-254311 A

Junji Terao, "Generation and elimination of singlet oxygen in living organisms: considering its relationship with oxidative stress", Vitamin Vol. 90, No. 11 (November 2016) Hiromi Kameya, Toshihiko Shoji, Yuji Odagiri, Akio Obata, Vipavee Trivittayasi, Mizuki Tsuta, Evaluation of the Active Oxygen Scavenging Activity of Tomato Juice by ESR Spin Trapping Method, Journal of the Japanese Society for Food and Nutrition, Vol. 27, pp. 267-272, 2017, DOI: 1609753029132_0.4_267 Hiromi Kameya, Mika Kanazaki, Yuji Odagiri, Akio Obata, Evaluation of the active oxygen scavenging ability of tomato juices with different colors by ESR spin trapping method, Journal of the Japan Society of Home Economics 70(5), 250-258, 2019 Journal of the Japanese Society for Food Science and Technology, 57, 525-531(2010) Akihiko Kosugi, Ryohei Tanaka, Kengo Magara, Yoshinori Murata, Takamitsu Arai, Othman Sulaiman, Rokiah Hashim, Zubaidah Aimi Abdul Hamid, Mohd Khairul Azri Yahya, Mohd Nor Mohd Yusof, Wan Asma Ibrahim, Yutaka Mori, “Ethanol and lactic acid production using sap squeezed from old oil palm trunks felled for replanting”, J Biosci Bioeng, 2010 Sep;110(3):322-5., doi: 10.1016/j.jbiosc.2010.03.001

The inventors focused on the polyphenols contained in oil palm sap and investigated its antioxidant power using the ORAC method and the electron spin resonance (ESR) method. They found that the polyphenols have antioxidant activity different from that of polyphenols in conventionally known products such as green tea, black tea, aloe juice, blackberry juice, cucumber juice, lemon, and tomato juice, which led to the completion of the present invention.

The present invention is a technology for utilizing oil palm sap that was developed in consideration of the above points, and aims to utilize components contained in the sap other than the sugar components. In addition to this, it also aims to provide an antioxidant with stronger antioxidant activity and diverse antioxidant activity compared to polyphenols, which are conventionally known as antioxidants, and a method for producing the same.

The antioxidant of the present invention is composed of an extract obtained from a plant, and has at least one of the free radical scavenging abilities of alkoxy radical scavenging ability, hydroxyl radical scavenging ability, and singlet oxygen scavenging ability. In particular, the free radical scavenging ability of the antioxidant of the present invention is characterized by having a stronger singlet oxygen scavenging ability than the antioxidant effect of polyphenols. Here, the plant is preferably a tropical plant, such as oil palm or sugar cane. More preferably, the extract is either (1) sap from oil palm trunks or sugar cane stems, or (2) petiole juice collected from the petioles of the oil palm or sugar cane. The extract is preferably an extract using water or a solvent.
The method for producing an antioxidant of the present invention is a method in which an extract is collected as a water-soluble liquid from either the trunk, petiole, fiber, or crushed chips of oil palm, or the stem, petiole, fiber, or crushed chips of sugarcane.
The sap of the oil palm trunk is preferably sap obtained from any of the upper, middle or lower parts of the oil palm trunk excluding the bark or petiole, or sap obtained from any of the upper, middle or lower parts of the oil palm trunk including the bark or petiole.
The antioxidant components of the water-soluble liquid are preferably concentrated by heating concentration, freezing, drying, freeze drying, membrane concentration, ultrasonic atomization separation or pH adjustment.
Components may be extracted or fractionated from this water-soluble liquid using a non-polar or polar solvent, and then made water-soluble.
The solvent is preferably any one of organic solvents selected from ethanol, acetone and ether.
The extract obtained by extraction from the aqueous liquid with a solvent is preferably subjected to concentration, freezing, drying, membrane concentration, ultrasonic atomization separation, or pH adjustment to extract the antioxidant components.
The present invention may be a food product containing any of the above-mentioned antioxidants.
The present invention may be a cosmetic product containing any of the antioxidants described above.

The antioxidant of the present invention provides a new utilization of oil palm sap, which has previously only utilized the sugar content of the sap, and a method for producing the same. In particular, the antioxidant of the present invention not only has activity at least equivalent to the scavenging ability of plant-derived polyphenols against alkoxy radicals and hydroxyl radicals, but also has a significantly superior ability to scavenge singlet oxygen activity compared to conventionally known plant-derived polyphenols.
Therefore, the present invention provides an antioxidant with high added value that is different from antioxidants derived from other plants, and further provides a method for producing the same.
The antioxidant of the present invention has the ability to eliminate singlet oxygen and can be used in the form of an aqueous solution, so it can be applied to the skin and is easily absorbed into the skin. Therefore, it is very effective in preventing and eliminating skin troubles as a perfume, lotion or beauty essence. The antioxidant of the present invention can be added to a food composition and used as a biological radical scavenger.

FIG. 1 shows the measurement results of the hydroxyl radical scavenging ability of oil palm sap (upper (T), middle (M), and lower (B) parts of the palm trunk) and standard substances. FIG. 1 shows the measurement results of the alkoxy radical scavenging ability of oil palm sap (upper (T), middle (M), and lower (B) parts of the palm trunk) and standard substances. FIG. 1 shows the measurement results of the singlet oxygen radical scavenging ability of oil palm sap (upper (T), middle (M), and lower (B) parts of the palm trunk) and a standard substance. This figure shows the results of comparing the antioxidant activity of oil palm sap (upper (T), middle (M), and lower (B) parts of the palm trunk) and a standard substance using the ORAC method.

The present invention relates to an antioxidant comprising an extract obtained from a plant, and having at least one free radical scavenging ability selected from the group consisting of an alkoxy radical scavenging ability, a hydroxyl radical scavenging ability and a singlet oxygen scavenging ability.
The present invention will be described below by showing the antioxidant capacity of an extract of a tropical plant, ie, the ability to scavenge alkoxy radicals, the ability to scavenge hydroxyl radicals and the ability to scavenge singlet oxygen.
In the following, oil palm and sugar cane will be used as examples of tropical plants, but the present invention is not limited to these, and is not particularly limited as long as the alkoxy radical scavenging ability, hydroxyl radical scavenging ability, and singlet oxygen activity scavenging ability are equal to or greater than those of blackberries, green tea (Benifuki green tea), black tea (BOH), cucumber, aloe, tomato juice, lycopene, and β-carotene.

The extract is sap obtained from the trunk or stem of a plant, petiole sap obtained from the petiole of a plant, and an extract from crushed matter or chips obtained from a plant body. The trunk or stem of a plant may be chemically treated to make it easier to collect sap or petiole sap. When using sap from an oil palm trunk, it may be sap obtained from any of the upper, middle, or lower parts of the oil palm trunk excluding the outer skin or petiole, or it may be sap obtained from any of the upper, middle, or lower parts of the oil palm trunk including the outer skin or petiole. When extracting, the oil palm trunk may be divided into any of the upper, middle, or lower parts to make the extraction operation easier. For example, it may be divided into the above three parts, or into two parts, upper and lower, and if it is easy to perform the extraction operation, the upper, middle, and lower parts may be finely crushed and mixed and used for the extraction operation. It may also be in a plate shape without being made into chips, or it may be used as it is. Furthermore, in order to obtain a sufficient amount of aqueous solution, the cut surface of the oil palm trunk can be painted with paint or coating material to prevent the trunk from drying out, and then crushed using the above method. It is preferable to collect the oil palm trunk within one month after felling. The trunk of the oil palm has an outer skin, but it is acceptable to extract the outer skin as well.
The antioxidant of the present invention is a water-soluble liquid such as sap or petiole juice, but can also be efficiently extracted with hot water, cold water, or a neutral, acidic or alkaline aqueous solution. The resulting aqueous solution is known to contain sugar (see Non-Patent Document 5). In order to prevent spoilage and to efficiently extract components, the antioxidant components may be extracted or concentrated from the water-soluble liquid by any of the following methods: heating and concentration, drying, reduced pressure concentration, vacuum concentration, freeze-drying, membrane concentration, ultrasonic atomization separation, or pH adjustment to an acidic or alkaline side precipitation and removal of other impurities.
It is preferable to obtain sap from crushed plant material or chips by physically squeezing or grinding them, but if it is possible to collect the sap, any method may be used, such as immersion extraction in water, extraction with added water, or extraction using a solvent.

The extract is a water-soluble liquid, but the components may be further fractionated and extracted with a non-polar or polar solvent. Examples of these solvents include organic solvents such as ethanol, methanol, ethyl acetate, acetone, hexane, and cyclohexane. Even if the solvent is an organic solvent, it may be a solvent miscible with water. For example, ethanol or methanol containing 50% water may be used. The solvent is preferably any of ethanol, methanol, acetone, ether, and hexane, and more preferably ethanol, methanol, or acetone. The extract extracted from the water-soluble liquid with the solvent may be concentrated, frozen, dried, membrane concentrated, or pH adjusted to extract antioxidant components.

The antioxidant of the present invention has the ability to eliminate singlet oxygen and can be used in the form of an aqueous solution, so it can be applied to the skin and easily penetrates the skin. Therefore, it is very effective in preventing and resolving skin troubles as a perfume, lotion or beauty serum. The antioxidant of the present invention can be added to a food composition and used as a biological radical scavenger. Furthermore, the antioxidant of the present invention can be used alone or in appropriate mixtures as a biological radical scavenger, as an antioxidant that eliminates oxidation and oxidative stress caused by detergents such as shampoos and soaps or pharmaceuticals. In addition to pharmaceuticals, it can also be mixed in appropriate amounts as an oxidation protection component in foods, cosmetics, and detergents that have other antioxidant components or medicinal properties.

The hydroxyl radical scavenging ability (Example 1) and alkoxy radical scavenging ability (Example 2) of oil palm sap, sugar cane sap, and comparison samples of blackberry, green tea, black tea, cucumber, and aloe were investigated using the ESR method.

Example 1
(Hydroxy radical scavenging ability)
(Preparation of measurement sample)
Sap was prepared from oil palm trunks procured from the outskirts of Kuala Lumpur, Selangor, or Penang, Malaysia, as follows. Oil palm trunks aged 10 to 25 years or more were randomly selected, and 30 to 40 trunks were felled and immediately moved to a covered area to avoid direct sunlight and rain. The felled palm trunks were divided into three parts at intervals of about 4 to 5 m from the top, the upper part (T), the middle part (M), and the lower part (B), immediately after felling, in order to compare the activity of each part. The trunks were cut into appropriate sizes over time and pressure was applied using a small juicer to extract sap. The petioles were cut from the top, cut, and then pressure was applied using a small juicer to extract petiole juice, which was then used as the upper part.
The collected sap and petiole sap were frozen at −20° C. and stored until measurement. After thawing, the sap was serially diluted with distilled water to prepare measurement samples for the measurement of radical scavenging activity.
To compare the strength of the radical scavenging ability of oil palm sap, extracts of blackberry, green tea (Benifuki green tea), black tea (BOH), cucumber, and aloe were used as samples with radical scavenging ability. All standard samples were procured from a supermarket in Tsukuba City, Ibaraki Prefecture.
100 g of aloe, cucumber, and blackberry were weighed out, crushed in a mortar, and filtered through Whatman No. 4 filter paper to prepare squeezed juice, which was then frozen and stored at -20°C.
For green tea and black tea, 2 g each was steeped in 200 ml of hot water at 90° C. to 95° C. and extracted for 5 minutes.
The extract was then filtered through Whatman No. 4 filter paper to obtain an extract. The extract was similarly frozen and stored at -20°C until use. When measuring, the extract was appropriately diluted with distilled water if necessary and used for the measurement.
In addition, for lycopene and β-carotene from tomato juice (produced in Gunma Prefecture), which has been proven to have radical scavenging ability, the literature values reported by Kameya et al. were used as a reference (Non-Patent Document 2).
In addition, sugarcane juice, which is said to have similar components and sugar concentration to oil palm trunk sap, was also used for comparison. Frozen sugarcane (from Ishigaki Island) was squeezed under the same conditions using a small juicer described in Non-Patent Document 2, and the juice was directly used for measurement.

(Method for measuring hydroxyl radical scavenging ability)
The sample or standard reagent (mannitol) _was exposed to UV light for 5 seconds in a mixture of 0.25% _H2O2 as an active oxygen generating reagent and 5 mM CYPMPO (5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide) and DTPA (Diethylenetriaminepentaacetic Acid) as spin trapping agents, and measurements were performed.
Mannitol was used as the standard reagent for the calibration curve.
The ESR measurement was performed using EMX-plus from Bruker Japan.
The radical scavenging ability was evaluated by measuring the radical scavenging ability by the MULTIS method using ESR spin trapping (see Non-Patent Document 3).

The measurement results of the hydroxyl radical scavenging ability are shown in Table 1.
The unit of hydroxyl radical scavenging activity is expressed in "mmol mannitol equivalent/mL."

The hydroxyl radical scavenging activity of the sap prepared from the upper part (T) of the oil palm trunk was 4,984.6 mmol mannitol equivalent/mL on average, that of the sap prepared from the middle part (M) was 5,454.6 mmol mannitol equivalent/mL on average, and that of the sap prepared from the lower part (B) was 2,122.5 mmol mannitol equivalent/mL on average.
The sap from the upper (T) and middle (M) parts of the oil palm trunk showed relatively high hydroxyl radical scavenging activity, while the sap from the lower part (B) was found to have the same scavenging activity as tomato juice.
The hydroxyl radical scavenging capacity of sugarcane juice was (5,587.5 mmol mannitol equivalent/mL).

On the other hand, the hydroxyl radical scavenging ability of the comparison samples was blackberry (5,299.2 mmol mannitol equivalents/mL), green tea (1,962.5 mmol mannitol equivalents/mL), black tea (306.7 mmol mannitol equivalents/mL), cucumber (3,578.8 mmol mannitol equivalents/mL), aloe (6,213.1 mmol mannitol equivalents/mL), tomato juice (1,844 mmol mannitol equivalents/mL), lycopene (111 mmol mannitol equivalents/mL), and beta-carotene (436 mmol mannitol equivalents/mL).

The results of the hydroxyl radical scavenging ability of oil palm sap and the standard substance are shown in Figure 1.
The unit of hydroxyl radical scavenging activity is expressed in "mmol mannitol equivalent/mL."

These results clearly show that sugarcane juice, along with oil palm sap, has a strong ability to scavenge hydroxyl radicals, and it is assumed that juice or sap obtained from tropical plants has a relatively strong ability to scavenge hydroxyl radicals.

Example 2
(Alkoxy radical scavenging ability)
(Method for measuring alkoxy radical scavenging ability)
The sample or standard reagent (Trolox) was irradiated with UV light for 5 seconds in a mixture of 1 mM AAPH (2,2'-azobis[2-aminodipropane] dihydrochloride) as an active oxygen generating reagent and 5 mM CYPMPO (5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide) as a spin trapping agent, and then the measurement was performed. Trolox was used as the standard reagent for the calibration curve. The measurement results are shown in Table 2.
To demonstrate the universality of the results, the distribution of radical scavenging activity for each part of the trunk of the oil palm, approximately 30 samples each, is shown as a box plot in Figure 2. The activity unit is expressed in mmol trolox equivalent/mL.

Based on these results, the alkoxy radical scavenging ability of oil palm sap was compared with that of blackberry (16.4 mmol trolox equivalents/mL), green tea (11.4 mmol trolox equivalents/mL), black tea (0.40 mmol trolox equivalents/mL), cucumber (5.3 mmol trolox equivalents/mL), aloe (14.9 mmol trolox equivalents/mL), tomato juice (12.0 trolox equivalents/mL), lycopene (0.5 trolox equivalents/mL), β-carotene (7.1 trolox equivalents/mL), and sugarcane juice (4.7 trolox equivalents/mL), all of which have known antioxidant effects. The alkoxy radical scavenging ability of oil palm sap was 11.8 mmol trolox equivalent/mL on average for sap prepared from the upper part (T), 10.6 mmol trolox equivalent/mL for sap prepared from the middle part (M), and 9.9 mmol trolox equivalent/mL for sap prepared from the lower part (B), showing that they have the same or relatively high alkoxy radical scavenging activity.

Example 3
(Ability to quench singlet oxygen radicals)
(Measurement of singlet oxygen radical scavenging ability)
The measurement of singlet oxygen radical scavenging was performed using the ORAC method according to the method of Jun Watanabe et al. (see Non-Patent Document 3).

The process of decomposition of the labeling substance fluorescein by peroxy radicals generated by the radical generator 2,2'-Azobis(2-methylpropionamidine)Dihydrochloride (AAPH) was evaluated by measuring the fluorescence intensity of fluorescein over time. Trolox was used as the standard substance for the calibration curve.

The sample or standard reagent (glutathione (GSH)) was irradiated with visible light for 30 seconds in a mixture of 0.1 mM pterine as an active oxygen generating reagent, and 10 mM N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD) and 1 mM DTPA as spin trapping agents, and then measured. Glutathione was used as the standard reagent for the calibration curve.

The results are shown in Table 3 and FIG.
The unit of scavenging activity is expressed as "GSH equivalent/mL."

The singlet oxygen radical scavenging capacity of oil palm sap was compared with that of blackberry (15.8 mmol GSH equivalents/mL), green tea (20.4 mmol GSH equivalents/mL), black tea (25.1 mmol GSH equivalents/mL), cucumber (34.6 mmol GSH equivalents/mL), aloe (43.8 mmol GSH equivalents/mL), tomato juice (63.0 GSH equivalents/mL), lycopene (1.7 GSH equivalents/mL), β-carotene (5.8 GSH equivalents/mL), and sugarcane juice (63.0 GSH equivalents/mL). The singlet oxygen radical scavenging capacity of oil palm sap was 387.7 mmol GSH equivalents/mL on average for sap prepared from the top (T), 239.6 mmol GSH equivalents/mL for sap prepared from the middle (M), and 343.6 mmol GSH equivalents/mL for sap prepared from the bottom (B).

These results show that the singlet oxygen radical scavenging activity of oil palm sap is significantly higher than that of blackberries, green tea, black tea, cucumber, aloe, tomato juice, beta-carotene, lycopene, and sugarcane juice, all of which are known to have antioxidant effects.

(Reference example)
Here, we compared the differences in antioxidant activity between oil palm sap and standard reagents (mannitol and trolox).
Figure 4 shows the results of comparing the antioxidant activity of oil palm sap (upper (T), middle (M), and lower (B) parts of the palm trunk) and a standard substance using the ORAC method. The horizontal axis of Figure 4 is the activity unit, expressed in mmol trolox equivalents/mL.
As shown in Figure 4, the sap from oil palm trunks showed almost the same or slightly inferior antioxidant capacity.
This result indicates that there is a discrepancy between the results measured by the ESR method (Examples 1 and 2) and the results for known antioxidants (catechin, flavonoids, vitamin E, etc.) assumed by the conventional ORAC method.
However, the results of the ESR method showed that oil palm sap contains substances with antioxidant properties that are difficult to detect using the ORAC method, indicating that oil palm sap has a strong radical scavenging effect that is different from existing antioxidants.
Considering the results of Example 3, it is considered that the radical scavenging ability possessed by oil palm sap, which is different from that of known antioxidants, is the ability to scavenge singlet oxygen radicals.

Although the present invention has been described above, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention.

Claims (9)

An antioxidant consisting of at least one of the juice squeezed from oil palm trunks and the juice squeezed from oil palm petioles, and having the ability to quench singlet oxygen. A method for producing an antioxidant having singlet oxygen quenching ability, comprising the steps of:
The method for producing the antioxidant includes at least one of a step of squeezing oil palm trunks to obtain sap and a step of squeezing oil palm petioles to obtain petiole sap,
The method for producing an antioxidant, wherein the sap and the petiole juice are water-soluble liquids. The method for producing an antioxidant according to claim 2, wherein the sap of the oil palm trunk is sap obtained from any of the upper, middle, or lower parts of the oil palm trunk excluding the outer bark or the petiole, or sap obtained from any of the upper, middle, or lower parts of the oil palm trunk including the outer bark or the petiole. 3. The method for producing an antioxidant according to claim 2, further comprising a step of concentrating the antioxidant component in the water-soluble liquid by subjecting the water-soluble liquid to heating concentration, freezing, drying, lyophilization, membrane concentration, ultrasonic atomization separation or pH adjustment. The method for producing an antioxidant according to claim 2, further comprising the step of extracting the antioxidant from the aqueous liquid with a solvent. The method for producing an antioxidant according to claim 5, wherein the solvent is an organic solvent selected from ethanol, acetone, and ether. 7. The method for producing an antioxidant according to claim 5 or 6, further comprising a step of extracting an antioxidant component from the extract by concentrating, freezing, drying, membrane concentrating, ultrasonic atomization separation, or pH adjusting the extract obtained by extracting the aqueous liquid with the solvent. A food product comprising the antioxidant according to claim 1. A cosmetic product comprising the antioxidant described in claim 1.