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. 2024 Nov-Dec;38(6):2629-2638.
doi: 10.21873/invivo.13739.

Potential Medicinal Efficacy of Alkaline Extract of Pine Seed Shell: Anti-UVC Activity and Macrophage Activation

Alejandro Mena Acra #  1   2 , Shin Uota #  1   3 , Masaaki Yoshihara  3 , Yukio Murakami  4 , Hiroshi Sakagami  5
Affiliations

Potential Medicinal Efficacy of Alkaline Extract of Pine Seed Shell: Anti-UVC Activity and Macrophage Activation

Alejandro Mena Acra et al. In Vivo. 2024 Nov-Dec.

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.

Keywords: UVC protection; alkaline extract; macrophage activation; pine seed shell.

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Conflict of interest statement

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.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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.
Figure 3
Figure 3
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.
Figure 4
Figure 4
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.
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