Abstract
Background/Aim: Alkaline extracts of several plants which contain lignin degradation products have several unique biological activities. In order to search for new biological activities of alkaline extracts of pine seed shell (APs), their anti-ultraviolet C (UVC) and macrophage stimulation activity were investigated. Materials and Methods: Anti-UVC activity was determined by the ratio of the 50% cytotoxic concentration against human melanoma cell line COLO679 to the 50% UVC-protective concentration. Extracellularly secreted nitrite (NO2−) by unstimulated and lipopolysaccharide (LPS)-stimulated mouse macrophage-like cells RAW264.7 was determined by Griess method. Results: APs showed significantly higher anti-UVC activity than previously reported hot-water extracts of medical herbs. Anti-UVC activity of AP and vanillic acid was maintained for much longer than that of sodium ascorbate and vanillin. APs enhanced the production of NO2− to the level induced by LPS. Simultaneous addition of AP and LPS did not further increase NO2− production, suggesting their mechanisms of action overlap. Conclusion: The present study suggests the possible application of APs as UVC protectors and immunopotentiators via macrophage activation.
There are many useful medicinal plants in nature. In folklore, home-made concoctions of medicinal plants have been used to treat diseases in daily life. In fact, most Japanese traditional herbal (‘Kampo’) medicines and their constituent plant extracts are prepared by hot-water extraction (1, 2). The medicinal efficacy of alkaline extracts of plants have been reported much less, since alkali extraction and neutralization cannot be reproduced in ordinary households. The unique biological activities of alkaline extracts are generated by the presence of lignin and degradation products in the alkaline extract.
Lignin is formed by dehydrogenative polymerization of three phenylpropanoids: p-coumaryl, p-coniferyl and sinapyl alcohols (3, 4). Lignin can be isolated by several solvents, such as sulfuric acid (5), thioglycolic acid (6), phosphoric acid (7), or sodium hydroxide (8). The size and reactivity of lignin fragments considerably varies depending on which extraction solvents are used (9). Lignin in plant cell walls is linked to polysaccharides to form lignin–carbohydrate complexes (LCCs), with a broad range of molecular weights from 1.5-200 kDa (10-13). The polysaccharide moiety of LCC of pine cone of Pinus parviflora Sieb et Zucc. contains heterologous sugars: glucose, arabinose, mannose, galactose, fucose, and uronic acid (13).
The anti-HIV activity of lignin from cone and seed shell of pine tree, husk and mass of cacao bean, and bark of catuaba is higher than that of hot-water extract such as Kampo medicines and constituent plant extracts (14). The antiviral activity of lignin is produced by the rapid inactivation of virus by tight association with lignin (15). Digestion of lignin moiety by sodium chlorite considerably diminished its anti-influenza virus activity, suggesting the importance of the lignin moiety for anti-viral action (16). This was supported by the finding that dehydrogenation polymer of phenylpropanoid (that does not contain polysaccharides) showed prominent anti-human immunodeficiency virus (HIV) activity (17). In an alkaline solution, lignin is removed and ester bonds between lignin and hemicellulose are cleaved, thus producing an accumulation of lignin degradation products such as phenylpropanoids (18) that have higher anti-ultraviolet C (UVC) activity (19) but lower anti-HIV activity (17) than the parent LCC. This suggests the following rule: As the alkaline strength increases, anti-UVC activity increases, but anti-HIV activity decreases. It was therefore a great surprise that vanillic acid (4-hydroxy-3-methoxybenzoic acid), which has more simple structure than phenylpropanoids, was found to have higher anti-UVC activity than sodium ascorbate, due to its higher stability during prolonged incubation in culture medium, suggesting its possible application in skin care products (20). Since the coronavirus disease 2019 (COVID-19) pandemic is not over yet, UVC sterilizers are still being used in many public facilities. Therefore, it is necessary to search for non-toxic and stable anti-UVC substances to protect ourselves from the health hazards caused by UVC radiation.
The present study aimed to investigate the anti-UVC activity of alkaline extracts of pine seed shell (AP), and the optimal conditions of alkaline extraction for future manufacture of anti-UVC protective substances or skin care products. Sakagami et al. found that alkaline extract of the leaves of Sasa sp., which contains p-coumaric acid (a phenylpropanoid), induced anti-inflammatory activity in interleukin-1β-stimulated human gingival fibroblast via various metabolic pathways for cell survival, apoptosis, and leukocyte recruitment (21). Therefore, the present study also investigated whether AP inhibits NO2− production by macrophages activated by the endotoxin lipopolysaccharide (LPS).
Materials and Methods
Materials. Pine seed shells of Pinus koraiensis. Sieb. et Zucc. (grown in China) and Pinus sylvestris L. (grown in Mongolia) were imported from these two countries and provided by Mr. Masaaki Yoshihara, Nippon Sunshow Medicine Manufacture Co. Ltd. Fetal bovine serum (FBS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA); dimethyl sulfoxide, LPS from Escherichia coli, Dulbecco’s modified Eagle’s medium (DMEM), phenol red-free DMEM, glutamine, pyruvic acid, penicillin-streptomycin solution (×100), 0.25% trypsin-1 mM EDTA-4Na were from Fujifilm Wako Pure Chemical Ind. (Osaka, Japan); sodium ascorbate, vanillic acid and vanillin (4-hydroxy-3-methoxybenzaldehyde) were from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); and 96-microwell plates were from Techno Plastic Products AG (Trasadingen, Switzerland).
Preparation of AP. Pine cones [Pinus koraiensis. Sieb. et Zucc. (lot #1, 2, 3, 4, 5, 6, 8, 9, 11; #3 was mixed with Syzygium aromaticum), Pinus sylvestris L. (lot #7, 10, 12; #10 was mixed with Syzygium aromaticum)] were extensively washed with distilled water to remove the contaminating soil, and then extracted with alkaline solution as described previously (22) with minor modifications. In brief, 10 g of pine seed shells were extracted with 100 ml of 0.1 M KOH for 1 h at 55°C (#1, 3, 4, 5, 6, 9, 10, 12), 60°C (#11), or 65°C (#2, 7, 8). For future large-scale manufacturing of bioproducts, 10 kg of #5 and 100 kg of #9 were extracted with 100 or 1,000 l of 0.1 M KOH, respectively (Table I). Hydrochloric acid was added to the extract to adjust the pH to 7.0±0.2. Neutralized extracts were then passed through an 18-8 stainless steel tea strainer (size: 68 mm, depth: 68 mm, mesh size: 40), purchased from Eve-mode, Amazon.co.jp) to remove the pine seed shell debris and then through filter paper (thickness: 0.22 mm, pore size: 7 μm; Advantec®, Tokyo, Japan) under reduced pressure to remove the insoluble materials. After lyophilization, the powder was dissolved in pure water at 100 mg/ml.
Conditions of extraction of seed shells of Pinus koraiensis. Sieb. et Zucc. and Pinus koraiensis. Sieb. et Zucc.
Cell culture. Human melanoma cells (COLO679, catalog number R21-0267; Riken Cell Bank, Tsukuba, Japan) (19, 20) and RAW264.7 mouse macrophage-like cells (23) were cultured at 37°C in DMEM supplemented with 10% heat-inactivated (56°C for 30 min) FBS, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate in a humidified atmosphere with 5% CO2. For subculture and experiments, cells were detached by treatment with 0.25% trypsin-EDTA. Viable cell numbers were measured using the MTT method. From the dose–response curve, the 50% cytotoxic concentration (CC50) was determined.
UVC protection assay. COLO679 cells (3×104/ml, 0.1 ml) were inoculated in the inner 60 wells of a 96-microwell plate, surrounded with 150 μl of sterile distilled water to minimize the evaporation of water during culture, and incubated for 48 h to achieve complete attachment to the plate. After replacing the medium with fresh culture medium containing different concentrations [0 (control), 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 or 1,000 μg/ml] of AP, cells were irradiated for 3 min with UVC (1.193 W/m2, 254 nm using a GL15 germicidal lamp; Toshiba Co. Ltd., Tokyo, Japan) (Figure 1A) or not irradiated. Cell cultures were then split into two. The culture medium of half of each cell culture was then changed with fresh culture medium without test AP and the viable cell number was determined by MTT method after 48 h (Method I in Figure 1B). The other half of the cell culture was incubated for 48 h without medium change, and then the viable cell number was determined similarly (Method II in Figure 1B). From the dose–response curve, the CC50 and the concentration that restored UVC-induced loss of viability by 50% (EC50) were determined in triplicate. The selectivity index (SI) was then determined using the following equation: SI=CC50/EC50 (20).
Experimental procedures of measurement of anti-ultraviolet C (UVC) activity. (A) The plates with cells were placed at 555 mm from the center of a UVC lamp set within a safety cabinet. (B) Near-confluent COLO679 cells were irradiated for 3 min and then cultured for 48 h with (Method I) or without medium change (Method II). (C) From the dose–response curve, the 50% cytotoxic concentration (CC50) and concentration that restored UVC-induced loss of viability by 50% (EC50) were determined. Anti-UVC activity was expressed as the selectivity index (SI=CC50/EC50). Each value represents the mean±standard deviation of triplicate determinations. Note that concentration axes are not linear. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay.
Assay for NO2− production. RAW264.7 cells were inoculated at 3×104/ml (100 μl) in a 96-microwell plate, surrounded with 150 μl of sterile distilled water to minimize the evaporation of water during culture, and incubated for 24 h to achieve complete adherence to the plate. Initially, we expected that APs may inhibit NO2− production by LPS-activated macrophages, like other anti-inflammatory natural products (23). Therefore, the effects of APs on RAW264.7 cells were investigated without and with LPS. Since the order of addition of LPS and AP may affect the experimental results, two methods for addition were adopted (Figure 2A). In Method III, cells were first treated with AP and 30 min later with LPS or not. In Method IV, cells were treated in the reverse order. Near-confluent cells were treated for 24 h without or with 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500 or 1,000 μg/ml of AP in phenol red-free DMEM (reconstructed with glutamine and pyruvic acid) supplemented with 10% FBS in the presence or absence of 0.1 μg/ml LPS. The NO2− production by RAW264.7 cells was quantified by Griess reagent (23) using a standard curve of NaNO2 (Figure 2B).
Experimental procedures for measurement of macrophage activation. Near-confluent RAW264.7 cells were treated first with alkaline extracts of pine seed shell (AP) and 30 min later then lipopolysaccharide (LPS) (Method I) or first with LPS and 30 min later AP (Method II) and extracellular nitrite (NO2−) was quantified by Griess method (A). Range of linearity for NO2− determination (B) and dose effect of LPS on NO2− production (C) are shown. Macrophage activation (D) and inhibition (E) were quantified. Each value represents the mean±standard deviation of triplicate determinations. Note that concentration axes are not linear.
The LPS concentration (100 ng/ml) used throughout experiments was based on Figure 2C. When samples did not inhibit the NO2− production by LPS-stimulated RAW264.7 cells, the possibility of macrophage activation was investigated. The macrophage stimulation index (MSI) was determined by dividing the 50% cytotoxic concentration (CC50) of samples by the EC50, i.e. that which stimulated NO2− production to the 50% level attained by LPS (0.1 μg/ml; Figure 2D). When samples inhibited te NO2− production by LPS-stimulated RAW264.7 cells, the macrophage inhibition index (MII) was determined by dividing the CC50 by the 50% inhibitory concentration (IC50) of NO2− production by LPS-treated cells (Figure 2E).
Statistical analysis. Experimental data are expressed as the mean±standard deviation of triplicate determinations. Student’s t-test was performed for statistical analysis between two groups (unpaired). The significance level was set at p<0.05.
Results
APs exhibited stronger and longer-lasting anti-UVC activity than hot-water extracts. Firstly, the direct anti-UVC activity of AP was measured using Method I. Since AP was present only during the 3-min irradiation, not during the following 48 h, AP against COLO679 cells was practically not cytotoxic (Figure 3A: CC50 values >1,000 μg/ml, indicated in orange). UVC irradiation reduced the cell viability to approximately 10% of unirradiated cells. By adding increasing concentrations of AP, cell viability gradually returned to the control level. Repeated experiments gave reproducible results (Figure 3A, Table II).
Anti-ultraviolet C (UVC) activity of 12 alkaline extracts of pine cone (APs). Near-confluent COLO679 cells were irradiated (blue) or not (orange) for 3 min and then cultured for 48 h with (Method I) or without medium change (Method II). From the dose–response curve, the 50% cytotoxic concentration (CC50) (blue) and concentration that restored UVC-induced loss of viability by 50% (EC50) (orange) were determined and are indicated in each figure. Anti-UVC activity was expressed as the selectivity index (SI=CC50/EC50). Residual anti-UVC activity after 48 h of contact with sample (RSI) was determined as the ratio of the SI of Method I to the SI of Method II, multiplied by 100 and listed in Table II. Each value represents the mean of triplicate determinations. Note that concentration axes are not linear.
Anti-ultraviolet C (UVC) activity measured after 48 h incubation in the absence (Method I) or presence (Method II) of alkaline extract of pine seed shells (AP). Data of Figure 3 were used.
Secondly, how long exposure to AP affects anti-UVC activity was investigated (Method II) (Figure 3B). The anti-UVC activity remaining after 48 h in contact with AP was determined by the following equation: Remaining SI (RSI)=[anti-UVC activity (i.e. SI by Method II)/anti-UVC activity (i.e. SI by Method I)]×100. Since most APs and vanillic acid showed very weak cytotoxicity against COLO679 cells during 48 h (most with CC50>1,000 μg/ml) (Figure 2), they had reproducibly higher remaining anti-UVC activity (RSI=72.0-76.8%) than vanillin (RSI=30.6%) and sodium ascorbate (RSI=0.21%) (Table II). These data demonstrate that APs had much longer-lasting anti-UVC activity than vanillin and ascorbic acid.
APs potently stimulated macrophages. LPS is known to bind to and activate Toll-like receptor 4 and promote the production of various inflammatory cytokines (such as interleukin-6 and tumor necrosis factor-α) and NO in mouse macrophage-like RAW264.7 cells (24-27). For investigating the anti-inflammatory effects, many authors have added the test substance 30-60 min before adding LPS in order to produce more clear-cut, positive effects. Therefore, in Method III, AP was first added to RAW264.7 cells and 30 min later LPS was added. LPS induced the production of 50-60 μM NO2− (Figure 2C) and morphological spreading (characteristic of activated macrophages, data not shown) (28). In contrast to our expectation, AP did not inhibit LPS-induced NO2− production, but in fact stimulated it to a level comparable with that attained by LPS alone. On the contrary, common antioxidants sodium ascorbate and vanillic acid did not induce NO2− production. Sodium ascorbate, but not vanillic acid, inhibited LPS-stimulated NO2− production (IC50=55.7 μg/ml), mostly due to higher cytotoxicity.
A similar experiment was performed using the reverse order of addition of LPS and AP (Method IV). We obtained similar results (Figure 4B, Table III).
Macrophage activation by alkaline extracts of pine seed shell (AP). Near-confluent RAW264.7 cells were treated first with AP and 30 min later then lipopolysaccharide (LPS) (Method III) (A) or first with LPS and 30 min later with AP (Method IV) (B), and extracellular nitrite (NO2−) was then quantified by Griess method. Macrophage activation and inhibition were then calculated. It should be noted that all APs enhanced NO2− production by RAW264.7 cells, whereas sodium ascorbate (SA) inhibited NO2− production. Vanillic acid (VA) neither enhanced nor inhibited NO2− production. Each value represents the mean±standard deviation of triplicate determinations. Note that concentration axes are not linear.
Macrophage activation by alkaline extract of pine seed shells (AP). Data of Figure 4 were used for the calculations.
Discussion
For the development of skin care substances, it is necessary to consider not only their instantaneous UVC-blocking activity (hydroxyl radical scavenging) but also damage to skin, since the administered substances remain in/on the skin for a long time. In the present study, two methods were devised to quantify the UVC-protective activity of APs. Method I is useful for detecting the hydroxyl radical-scavenging activity (26), while Method II predicts the stability of anti-UVC activity. The present study demonstrated that although the direct anti-UVC activity of AP [that reflects hydroxyl radical-scavenging activity (29)] was considerably lower than that of vanillic acid, vanillin (Table II) and sodium ascorbate (30), but significantly (p<0.0001) higher than that of hot-water soluble medicinal herb extracts (n=108), hot-water extracts of Japanese traditional medicines (n=10) and their constituent plant extracts (n=25) (31). APs had stronger anti-UVC activity (hydroxyl radical-scavenging activity) than the hot-water extract. This finding is consistent with our previous report that the anti-UVC activity of alkaline extracts of green tea and Oolong tea leaves was stronger than that of the corresponding hot-water extracts (32). From the experiments using Method II, it was found that an average of 72% of the anti-UVC activity of AP remained even after 48 h-incubation in culture medium. Similarly, 76.8% of the anti-UVC activity of vanillic acid remained after 48-h incubation (Figure 3, Table II), confirming the reproducibility of the previous report (20), suggesting that vanillic acid can be used as a standard compound for the measurement of anti-UVC activity. On the other hand, only 30.6% of the anti-UVC activity of vanillin remained after 48 h incubation, indicating that replacement of the aldehyde group in vanillin with carboxylic acid potentiates the anti-UVC activity. We previously reported that 99.8% of the anti-UVC activity of sodium ascorbate was lost during the 48-h incubation period, demonstrating rapid decay of its anti-UVC activity (20) (Table II).
The present study also demonstrated that APs stimulated NO2− production by mouse macrophage-like RAW264.7 cells to an extent comparable to LPS (0.1 μg/ml, Figure 4). The combination of both AP and LPS did not further increase NO2− production, regardless of the order of addition. The macrophage-stimulating activity of APs from lots #5 and #9 was observed at 15.6 and 250 μg/ml, respectively, approximately 1/156 and 1/2,500 that of LPS. Further purification of APs is necessary to identify the active component(s). We have reported that the lignin–carbohydrate complex Fr. VII of pine cone of Pinus parviflora Sieb. et Zucc. contained only 0.003% (w/w) endotoxin contamination (19). Since the ester bond of LPS is easily cleaved during alkaline extraction, the possibility that contaminating LPS is responsible for the macrophage activation induced by AP is very low.
In nature, high-molecular-weight substances such as lignin–carbohydrate complexes from lignified plants have an extremely high anti-HIV activity whereas phenylpropanoids (p-coumaric acid, ferulic acid, caffeic acid) were inactive (14). On the other hand, phenylpropanoids (p-coumaric acid, trans-ferulic acid, isoferulic acid, caffeic acid) and vanillic acid have greater anti-UVC activity than lignins (alkaline lignin, ligonosulfonate) (19). We reported previously that the major components responsible for the anti-UVC activity of the alkaline extract of the leaves of Sasa sp. are p-coumaric acid derivatives (33). However, it is still unclear whether macrophage activators are small molecules or large molecules. Therefore, we compared the macrophage activity of 12 APs (Table I), based on their MSI (Figure 2D). We found that lots #5 and #9, which were extracted under the mildest conditions of 55°C for 1 h, using much larger amounts of APs (either 10 or 100 kg, as compared with 10 g for other samples) produced the highest MSI [357 and 145 (Method III); and 132 and 167 (Method IV), respectively], whereas MSI of other lots of APs were one order lower (MSI=1-29) (Table III). This suggests the possibility that substances with relatively high molecular weight may have higher macrophage-stimulatory activity. This was supported by our previous finding that lignified materials stimulated macrophage activation, whereas monomeric phenolic compounds structurally related to lignin components had little or no macrophage-stimulating activity (28), and the present finding that vanillic acid did not produce detectable amounts of NO2− (MSI=1) (Table III). However, further purification of the active component(s) is needed to confirm this point.
The extraction efficiency seems to be better with pine seed shell from China than with pine seed shell from Mongolia (Table I). Since Syzygium aromaticum (clove) is a traditional aromatic spice that has been used for food preservation and possesses various pharmacological activities (34), we tested whether the addition of this spice would affect the macrophage-stimulating activity of APs. We found that it did not clearly modify the macrophage-stimulating activity.
This study demonstrated that sodium ascorbate, a well-known antioxidant, did not activate macrophages and rather inhibited LPS-stimulated macrophage activation. However, its MII was only 3.3-3.6 (Table III). This is possibly due to the cytotoxic action of hydrogen peroxide and other oxidized products generated from sodium ascorbate under an oxygen-rich environment (CC50=91-146 μg/ml) (CC50=91-146 μg/ml) (35, 36).
We reported that one of seven lignin–carbohydrate fractions isolated from Lentinus edodes mycelial extract enhanced the expression of dectin-2 and toll-like receptor-2 prominently in mouse macrophage-like J774.1 cells, while LPS did not affect their expression (37). Further identification of active components in APs will clarify the mechanism of UVC-protective and macrophage stimulatory actions.
In conclusion, the present study demonstrated that APs had (ii) higher anti-UVC activity than hot-water extracts, (ii) higher stability of anti-UVC activity than sodium ascorbate and vanillin, and (iii) potent macrophage-stimulating activity. These data suggest their possible application as UVC protectors, and for immunopotentiation through macrophage activation.
Acknowledgements
This work was supported in part by Miyata Research Fund B.
Footnotes
Authors’ Contributions
AMA, SU, and HS performed the experiments. AMA and HS wrote the article. AMA, HS, SU, MY and YM reviewed the article. AMA, HS, SU and MY designed and interpreted the experimental results. HS provided interpretation of experimental results and edited the article. All Authors read and approved the final version of the article.
Conflicts of Interest
MY is a representative director of Nippon Sunshow Medicine Manufacture Co. Ltd. and provided all pine seed shells used in this study. SU is a visiting researcher from Nippon Sunshow Medicine Manufacture Co. Ltd. AMA is a visiting researcher from the Autonomous University of the State of Mexico (UAEMex), Toluca, Mexico. However, it was confirmed that such support did not influence the outcome of the experimental study. The Authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
- Received June 9, 2024.
- Revision received July 15, 2024.
- Accepted July 17, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
