Skip to main content
Applied Biological Chemistry
Download PDF

Isolation, identification, and apoptosis activity of the photosensitizer methyl pheophorbide A from Perilla frutescens leaves

Applied Biological Chemistry volume 65, Article number: 52 (2022) Cite this article

A Correction to this article was published on 26 September 2022

This article has been updated

Abstract

Photodynamic therapy (PDT) is a promising cancer treatment with fewer side effects, and it eliminates tumors in target tissues with reactive oxygen species produced by photosensitizers (PS) and light. In this study, we isolated methyl pheophorbide A, which induces photodynamic cell death in the U937 and SK-HEP-1 cells, from perilla leaves. Its potential as a material for the development of a new PS was also evaluated. The methyl pheophorbide A is a dark green porphyrin compound isolated from methanol extract of perilla leaves. Apoptosis occurred was methyl pheophorbide A treated and irradiated with light, and there was no significant change where light not treated. In both U937 and SK-HEP-1 cells, apoptotic body, vesicle formation, and DNA ladder were confirmed in the light-irradiated. The caspase-3/7 activity an important factor in apoptosis, was 101.50 ± 14.24% when treated with 0.25 μg/ml methyl pheophorbide A in U937, and 91.32 ± 16.23% when treated with 1.00 μg/ml in SK-HEP-1 cells. The apoptotic phenomenon appeared more strongly when the methyl pheophorbide A concentration was increased with irradiating light. This study demonstrates the isolation, identification, and phototoxic activity of methyl pheophorbide A in perilla leaves. We expect that this study will be useful in the search for PS candidates using natural products.

Introduction

Photodynamic therapy (PDT) is being attended as a new cancer treatment strategy, since it has less severe side effects compared to conventional cancer treatments. PDT is a medical treatment that uses reactive oxygen species (ROS) production of a photosensitizer (PS). It is based on the fact that PS reacts with oxygen when it reaches to an excited state by light in a specific wavelength, and produces oxidants in tissues to induce cell death [1]. PDT has advantages compared to traditional treatments, such as high tumor selectivity, low side effect, non-invasive nature, and high possibility of repeated therapy without cumulative toxic effects [2].

Cell death mechanisms by PDT are as follows: induction of cytotoxicity due to reactive oxygen species, cell death from activation of an immune response, and local depletion of oxygen and nutrients according to vascular shutdown [3]. The PS currently in use are the Photofrin® and Photogem® based on hematoporphyrin derivatives (HpD). These PS have disadvantages that the available ranges of light wavelength are limited, and the light must be blocked during the PS metabolism except for the light treatment to cancer cells [4]. Therefore, it is necessary to search for various candidate compounds that can solve these problems and to develop a new PS [5].

Perilla frutescens is an annual herb belonging to the Lamiaceae family, and is originated from East Asia countries. The leaves, stems, and seeds of these family plants are widely used in folk medicine. Various phytochemicals exist in seeds, leaves, and stems of P. frutescens, and 271 natural substrates are reported [6]. It is classified into hydrophilic (anthocyanins, phenolic acids, and flavonoids) and hydrophobic (phytosterols, policosanols, and tocopherols) according to its chemical properties. It has been reported various biological activities such as anti-cancer, anti-inflammatory, anti-allergic, antioxidant, and neuroprotective effects [7,8,9,10]. Also, there are functional studies with perilla leaves extracts on cell death due to apoptosis in various cancer cells such as hepatoma HepG2 cells, leukemia HL-60 cells, and colon adenocarcinoma DLD-1 cells [10,11,12].

We, therefore, tested availability of perilla leaves as a source of an improved PS. A solvent extract was obtained from the perilla leaves and isolated a compound which have photodynamic activity. Here we report the identification of methyl pheophorbide A from series of chromatography analyses, which have not been reported previously.

Materials and methods

Solvent extraction

Dried, powdered perilla leaves were extracted with 10 volumes of methanol in a glass container at 150 rpm for 3 h. The extraction was repeated twice and the extract filtered with filter paper (5C. 110 mm, Advantec, Tokyo RoshiKaisha, Ltd.,Tokyo, Japan). The filtrate was concentrated with a rotary vacuum evaporator (EYELA N-1000, Japan).

Isolation of photosensitizer

Open column chromatography with normal phase silica gel (Merck, Darmstadt, Germany) and thin layer chromatography (TLC) with TLC Silica gel 60 RP-18 F254s (Merck, Darmstadt, Germany) were used to separate PS. Chromatography separation techniques were applied to separate in PS from methanol extract of perilla leaves. The extract was applied to open column chromatography, and eluted with hexane: ethyl acetate (from 1:0, 5:1, 5:2, 5:3 to 5:4) gradients. The primary 105 fractions were identified by the TLC and the similar Rf (retention factor) values were divided into nine groups (Additional file 1: Fig. S1B). The factions with similar Rf values were combined and tested PDT-induced apoptosis for U937 and SK-HEP-1 cells. Successive two open column chromatography and a TLC were performed, and a single compound was separated (Additional file 1: Fig. S1C). Liquid chromatography analysis was performed to confirm the purity of the separated compound using Waters 600 HPLC system and photodiode array detector (Waters, USA). The LC grade methanol and ethyl acetate (Fisher, USA) were used as mobile phase. The column using LiChrospher silica-60 (5 μm × 250 mm × 4.6 mm, Merck, Germany). The gradient elution program of methanol (solvent A) and ethyl acetate (solvent B) was as follows: 0–5 min, 20% B; 5–20 min, 20–80% B; 20–23 min 80–100% B; 23–25 min 100% B. The flow rate was 0.5 ml/min and the detection wavelength was 390 nm. NMR and FAB-Mass analyses were performed to identify the structure of the separated compound.

Structure identification

1D-NMR (1H-NMR, 13C-NMR) and 2D-NMR (Homo-Cosy, HSQC, HMBC) were conducted using Agilent 600 MHz NMR system (Santa Clara, CA, USA) by dissolving the compound in Deuterated chloroform (CDCl3, Aldrich, USA) for NMR to identify the structure of the compound. Chemical shifts are reported in δ (ppm). FAB-mass spectroscopy (JMS 700, Jeol, Japan) was conducted to determine the molecular weight of the compound.

Cell line and cell culture

U937 and SK-HEP-1 cells were received from the Korean Cell Line Bank (KCLB, KOREA). U937 and SK-HEP-1 cells were cultured in RPMI and DMEM medium, respectively. Cell culture was conducted with DMEM medium (Dulbecco’s modified eagle) and RPMI 1640 medium (Roswell Park Memorial Institute). FBS (Fetal Bovin Serum) and trypsin–EDTA were purchased from Hyclone (Logan, USA), and penicillin–streptomycin was purchased from Sigma Aldrich (ST. Louis, USA). Medium consisted of 10% (v:v) FBS and 1% (v:v) penicillin–streptomycin. Cells were incubated at 37 °C with 5% CO2 in a humidified atmosphere. Cells were passaged every 36 h to maintain an appropriate number.

Change in the cell morphology

Changes of cell morphology were observed in the light and dark condition. ADCL was used as a mixture of actinomycin D (Sigma-Aldrich, USA) and colcemid (Sigma-Aldrich, USA). The cells were seeded in 48 well plate at 2 × 105 cells/ml in 0.2 ml and incubated for 12 h. The methyl pheophorbide A dissolved in DMSO (with and without light), 0.1 mM ADCL (the positive control), and DMSO (the negative control) were treated to observe the PDT-induced apoptosis. After 4 h incubation, plate of light condition was irradiated at 3250 Lux for 10 min with daylight fluorescent lamps (wavelengths: 440, 550, and 620 nm). The plate of dark condition was incubated under light blocked condition at all time. The changes in cell morphology were observed with an IX51 inverted microscope (Olympus, Japan) at 200 × magnification. The pictures were captured by the iSolution Image analysis software.

Cell viability assay

The cell viability was determined by a Cell viability Glo 2.0 assay kit (Promega, USA). Briefly, cells were seeded at a density of 2 × 105 cells/ml in 96 well white plate. After 4 h of incubation, separated compound were treated with different concentrations in each well. After inducing photodynamic activity, cells were mixed with an equal amount of CellTiter-Glo 2.0 reagent for 10 min. Luminescence was measured with a GloMax™ Multi microplate multimode reader.

Assay of caspase-3/7 activity

Caspase activity was measured using a Caspase-Glo 3/7 assay kit (Promega, USA). Cells were seeded at a density of 2 × 105 cells/ml in 96 well white plate and incubated for 4 h. The methyl pheophorbide A was added at different doses to each well. After inducing photodynamic activity, an equal volume of Caspase-Glo solution reagent was added, plates were shaken for 1 h, and luminescence measured with a GloMax™ Multi microplate multimode reader (Promega, USA).

DNA fragmentation assay

To observe DNA fragmentation, which is the biochemical hallmark of apoptosis, cells were seeded at a density of 1 × 106 cells/ml in 6 well plate and incubated for 4 h. The methyl pheophorbide A, ADCL, and DMSO were then added. After inducing photodynamic activity, cells were harvested and washed with ice-cold PBS. Cell pellets were suspended in cell lysis solution (Promega, USA) at 4 °C for 10 min and centrifuged at 4 ℃, 14,000 rpm for 20 min. After collection, the supernatant was incubated with 2 μl of RNase A (10 mg/ml, Ambion) and 10 μl of proteinase K (20 mg/ml, Roche) at 37 °C for 1 h, separately. DNA was precipitated with nine volumes of isopropyl alcohol at 4 °C overnight and centrifuged at 14,000 rpm for 20 min at 4 °C. Pellets were dissolved with 0.5% TE buffer. Electrophoresis was performed on 1.6% agarose containing ethidium bromide.

Statistical analysis

All experiments were conducted with three replicates. Statistical analysis was performed using IBM SPSS Statistics 26. The different groups between light and dark conditions were evaluated by two-way ANOVA and Duncan’s multiple range test (p < 0.05). Data are expressed as mean ± SD (standard deviation).

Results

Separation and identification of methyl pheophorbide A from P. frutescens leaves

We obtained 288.13 g of methanolic extract from 1.2 kg of dried perilla leaves powder. The PS from perilla leaves was successfully separated with series of chromatography techniques (Additional file 1: Fig. S1A). The primary 105 fractions were obtained by the TLC and the similar Rf values were divided into nine groups (Additional file 1: Fig. S1B). Successive two open column chromatography and a TLC were performed to obtain a single compound (Additional file 1: Fig. S1C). As a result, a single peak was confirmed with HPLC at Rt 6.079 min (Additional file 1: Fig. S1D). The separated compound showed a similar UV spectrum with chlorophyll, a representative compound having a phytyl group (Additional file 1: Fig. S2). The single peak compound was further identified with NMR. The 1H-NMR spectrum is shown in Table 1. The four methine group signals appeared at 8.559 (20-H, s), 9.386 (5-H, s), 9.522 (10-H, s), and 7.98 (31-H, dd) ppm, respectively, showing structural similarity with chlorophyll having porphyrin rings [13, 14]. In contrast, the compound did not have a phytyl group compared to chlorophyll which shows multiple signals at around 0.5–1 ppm.

Table 1 1H-NMR chemical shift of methyl pheophorbide A (600 MHz, CDCl3)
Full size table

Magnesium ions are present in the center of the porphyrin ring of chlorophyll and since these ions are weak in structural bonding, they are easily eliminated, forming NH bond. In the 1H-NMR spectrum of the separated compound, NH signal at -1.65 ppm was detected, showing an absence of magnesium ions. The hydrogen bindings to 31 and 32-vinyl were detected in 31-H (7.98, dd), 32-H (H trans, 6.29, d), and 32-H (H cis, 6.18, d). The 7C-CH3 signal appeared as a double let at 3.22 ppm, indicating that it is a porphyrin compound with the basic structure of chlorophyll a, whereas chlorophyll b has 7C-CHO.

In the 13C-NMR chemical shift (Table 2) of the separated material, the keto carbon [δ189.603(C-131)], the carbonyl carbon [δ169.56(C-133), δ173.324(C-173)], the methine bridge carbon [δ 93.118(C-20), δ97.560(C-5), δ104.454(C-10)], the vinyl carbon [δ128.890(C-31), δ122.800(C-32)] signal was similar to chlorophyll a and the previously reported compounds [5]. The carbon signal of the phytyl group at 30 ~ 40 ppm did not appear in the spectrum of the separated compound, and it was consistent with the 1H-NMR result.

Table 2 13C-NMR chemical shift of methyl pheophorbide A (600 MHz, CDCl3)
Full size table

In addition, FAB-MS analysis was performed to measure the molecular weight, and a fragment signal of [M-H]+ 605.8 was confirmed (Additional file 1: Fig. S8), resulting in a molecular weight of 605.8. The compound isolated from perilla leaves by combining NMR and MS data was identified as a methyl pheophorbide A, a dark green porphyrin compound with a molecular weight of 605.8 (Fig. 1).

Fig. 1
figure 1

Structure of PDT-induced compound isolated from perilla leave. The compound was determined as a methyl pheophorbide A with a molecular weight 605.8 in C36H38N4O5

Full size image

Changes in the cell morphology due to photodynamic activity

To test photodynamic activity of the separated methyl pheophorbide A, apoptosis was observed with two cell lines. The U937 and SK-HEP-1 are originated from histiocytic lymphoma and hepatic adenocarcinoma, respectively and showing different cell growth habitats. U937 cell shows suspension monocyte morphology whereas, SK-HEP-1 cells are epithelial on a culture plate in normal condition.

The methyl pheophorbide A treatment without light did not change cell morphology. However, methyl pheophorbide A with light treatment induced apoptosis with the apoptotic body and vesicle formation in both cell lines similar to 0.1 mM ADCL treatment, the positive control (Fig. 2).

Fig. 2
figure 2

Morphological changes in U937 and SK-HEP-1 cells by treating with methyl pheophorbide A and light. The cells were seeded with 2 × 105 cells/ml in 48 well plate and incubated for 12 h. After 4 h of treatment with the methyl pheophorbide A, DMSO (the negative control), and 0.1 mM ADCL (the positive control), only the plate of light condition was irradiated at 3250 Lux for 10 min with daylight fluorescent lamps (wavelengths: 440, 550, and 620 nm), PDT-induced cell death was observed. The plate of dark condition was incubated under light blocked condition at all time. The morphology of the cells was observed at 200 × magnification with an IX51 inverted microscope (Olympus, Japan)

Full size image

Cell viability by methyl pheophorbide A with light irradiation

The degree of apoptosis was quantified with luminescent cell viability assay for methyl pheophorbide A and light treatment. In U937 cells, ADCL, which induces apoptosis, showed cell viability of 9.79 ± 0.09%, 10.80 ± 0.16% in light and dark conditions, respectively (Fig. 3A). When methyl pheophorbide A was treated at various concentrations and irradiated with light, cell viability rates of 10.34 ± 0.79% (0.25 μg/ml), 14.28 ± 1.73% (0.50 μg/ml), 0.35 ± 0.03% (1.00 μg/ml), and 0.26 ± 0.05% (2.00 μg/ml) were shown, and most of the cells were observed to die. The cell viability under dark condition was 74.36 ± 1.90% (0.25 μg/ml), 92.54 ± 1.24% (0.50 μg/ml), 82.01 ± 2.66% (1.00 μg/ml), 72.11 ± 4.27% (2.00 μg/ml) similar to the cell viability 84.47 ± 8.70% (the light condition), 92.13 ± 3.57% (the dark condition) of DMSO (the negative control).

Fig. 3
figure 3

Cell viability in light and dark conditions according to methyl pheophorbide A treatment for U937 cells (A) and SK-HEP-1 cells (B). The cells were seeded at a density of 2 × 105 cells/ml in 96 well white plate. After 4 h of incubation, the methyl pheophorbide A was treated at a concentration of 0.25, 0.50, 1.00, and 2.00 μg/ml. After inducing photodynamic activity, cells were mixed with an equal amount of CellTiter-Glo 2.0 reagent for 10 min. Luminescence was measured with a GloMax™ multi microplate multimode reader. All experiments were conducted with three replicates, and the results were expressed as mean ± SD (standard deviation). Values were significantly different both by treatment and light with two-way ANOVA (p < 0.05). Different letters represent p-values between light and dark conditions by Post hoc Duncan’s multiple range test

Full size image

The cell viability concentration-dependently decreased by methyl pheophorbide A and light treatment of SK-HEP-1 cells 85.68 ± 1.02% (0.25 μg/ml), 93.16 ± 9.07% (0.50 μg/ml), 16.24 ± 5.16% (1.00 μg/ml), 1.37 ± 0.43% (2.00 μg/ml) (Fig. 3B). The cell viability in dark conditions with methyl pheophorbide A was 78.54 ± 1.89% ~ 89.41 ± 1.39%, and there was no significance with DMSO.

Caspase-3/7 activity in PDT-induced apoptosis

The activity of caspase-3/7, effector caspases, was measured which induces apoptosis by activating nuclease. Caspase is classified into an initiator caspase (caspase-2, -8, -9, and -10) and effector caspase (caspase-3, -6, -7). Effector caspases are known to the end causatives of apoptosis. The RLU value was calculated as the relative ratio (%) of ADCL (the positive control). When U937 cells were treated with methyl pheophorbide A and irradiated with light, caspase activity was measured as 101.50 ± 14.24% (0.25 μg/ml), 71.31 ± 6.41% (0.50 μg/ml), 22.28 ± 4.29% (1.00 μg/ml), and 4.27 ± 0.26% (2.00 μg/ml). In dark condition, caspase activity was measured by 18.89 ± 0.65% (0.25 μg/ml), 6.48 ± 0.43% (0.50 μg/ml), 10.19 ± 2.81% (1.00 μg/ml), and 9.61 ± 2.77% (2.00 μg/ml) (Fig. 4A). In dark conditions, there was no significant difference according to the concentration of methyl pheophorbide A. However, the light condition was concentration-dependently decreased caspase activity. The decrease in concentration-dependent activity is presumed to be due to the rapid inactivation of enzymes due to rapid cell death. Cell viability after treatment with 1.00 μg/ml, 2.00 μg/ml of methyl pheophorbide A was 0.35 ± 0.03%, 0.26 ± 0.05%, and most of the cells died.

Fig. 4
figure 4

Caspase-3/7 activity by PDT-induced apoptosis following methyl pheophorbide A treatment in U937 (A) and SK-HEP-1 cells (B). Cells were seeded at a density of 2 × 105 cells/ml in 96 well white plate and incubated for 4 h. After 4 h of incubation, the methyl pheophorbide A was treated at a concentration of 0.25, 0.50, 1.00, and 2.00 μg/ml in each well. After inducing photodynamic activity, an equal volume of Caspase-Glo solution reagent was added, plates were shaken for 1 h, and luminescence measured with a GloMax™ multi microplate multimode reader. All experiments were conducted with three replicates, and the results were expressed as mean ± SD (standard deviation). Values were significantly different both by treatment and light with two-way ANOVA (p < 0.05). Different letters represent p-values between light and dark conditions by Post hoc Duncan’s multiple range test

Full size image

In SK-HEP-1 cells light condition, caspase activity was 29.22 ± 1.03% (0.25 μg/ml), 30.95 ± 2.79% (0.50 μg/ml), 91.32 ± 16.23% (1.00 μg/ml), and 12.41 ± 1.12% (2.00 μg/ml). In dark condition, caspase activity was measured by 18.48 ± 0.61% (0.25 μg/ml), 19.21 ± 3.46% (0.50 μg/ml), 20.39 ± 4.23% (1.00 μg/ml), and 24.73 ± 1.73% (2.00 μg/ml) (Fig. 4B). The highest caspase activity was shown at 91.32 ± 16.23% in methyl pheophorbide A 1.00 μg/ml treated light irradiated group, but no significant difference was found in other concentration treatments.

DNA fragmentation by PDT-induced apoptosis

DNA fragmentation is one of the major features of cell death caused by apoptosis. When apoptosis is induced, DNA fragmentation occurs and DNA ladder can be observed. The ADCL (the positive control) treatment lead DNA fragmentation independent to irradiation, whereas DMSO did not.

In the methyl pheophorbide A and light irradiation, the DNA ladder was observed in both cell lines. In dark conditions, the DNA ladder was not observed like DMSO (the negative control) treatment (Fig. 5). Therefore, cell death induced by methyl pheophorbide A shows characteristics such as DNA ladders, apoptotic bodies, and vesicles, suggesting that cell death is results of apoptosis.

Fig. 5
figure 5

DNA fragmentation by PDT-induced apoptosis of methyl pheophorbide A for U937 cells (A) and SK-HEP-1 cells (B). The cells were seeded at a density of 1 × 106 cells/ml in 6 well plate. After incubation for 4 h, the methyl pheophorbide A, ADCL, and DMSO were added to each well. After inducing photodynamic activity, cells were harvested. DNA fragmentation was confirmed by extracting DNA from harvested cells and electrophoresis on 1.6% agarose gel containing ethidium bromide (L: Light, D: Dark)

Full size image

Discussion

The PDT research aims to find a PS with minimal cytotoxicity in the dark conditions of tissues, a higher concentration in diseased tissues than in healthy tissues, and a higher efficiency generating singlet oxygen and ROS [15]. Photofrin®, a PS widely used in clinics, is effective in various types of cancer, but induces a longstanding cutaneous photosensitivity, and has the longest absorption wavelength of 630 nm, which is limited in treating deep or larger tumors. Longer wavelengths (> 650 nm) of light are more transmissive and can be used to treat deep or larger tumors. Therefore, the new PS development is focused on finding materials with a long wavelength ranges between 650 and 850 nm [16].

The separated methyl pheophorbide A from perilla leaves in this study have a typical porphyrin structure similar to chlorophyll a. The previously reported porphyrin compounds with photocytotoxicity have a lactone structure between 131-C and 132-C, with or without OH group on 132-C [5]. The spectrums of the lactone containing porphyrin have 131-C signal at 189.603 ppm and 132-C signal at 64.723 ppm, however, the methyl pheophorbide A in this study did not show such signals indicating that it has not a lactone structure.

Pheophorbides are plant metabolites related to chlorophyll and are used as major photoactive compounds in PDT [17]. Anti-cancer activities and photocytotoxicities of pheophorbide a, pyropheophorbide a, and pheophytin-a have been isolated from various plant species including Piper penangense, Phaeanthus ophthalmicus, and Zanthoxylum ailanthoides Sieb. & Zucc has been reported [18,19,20]. This the first study on the photocytotoxicity of methyl pheophorbide A isolated from P. frutescens leaves to out best knowledge. Cytotoxic activity, anticancer activity, antimetastasis effects, and antioxidant response element dependent transcriptional regulation of methyl pheophorbide A isolated from various natural products have been reported [21,22,23].

PDT-induced cell death is mainly resulted by apoptosis among the three major reasons of cell death; apoptosis, autophagy, and necrosis [3]. Two major pathways of PDT-induced apoptosis are the death receptor-mediator extrinsic pathway and the mitochondria-mediated or intrinsic pathway. Cell surface receptors of tumor necrosis factor genes are activated by initiator caspase-8 via adaptor and scaffolding protein in death receptor-mediated extrinsic pathway. Mitochondria-mediated or intrinsic pathway started with a mitochondrial functional disturbance which induces a release of cytochrome c. Cytosolic cytochrome c binds to Apaf-1, which induces Apaf-1 oligomerization in the presence of dATP. This complex, called an apoptosome, activates the initiator caspase-9. In both pathways, activation of the initiator caspase (caspase-8 or caspase-9) leads to the activation of the effector caspase (caspase-3, -6, -7) showing caspase-3/7 is a good indicator for observing apoptosis [24].

In the methyl pheophorbide A treated with the light condition, cell death was induced in U937 and SK-HEP-1 cells with apoptotic body and vesicle formation, which are characteristics of apoptosis. According to light irradiation, caspase-3/7 activity was increased to a similar level as the positive control ADCL. The DNA fragmentation, one of the characteristics of apoptosis, was observed in methyl pheophorbide A treatment with light. In U937 and SK-HEP-1 cells, cell viability and caspase-3/7 activity were found to be dependent on the methyl pheophorbide A concentration. The cell death pathway is due to various mechanisms [25, 26]. The difference in amount and efficiency of PS localization in diseased tissues with types of PS compound and cells should be further investigated.

In this study, methyl pheophorbide A has been isolated from perilla leaves with series of chromatography. This compound is a dark green porphyrin compound, C36H38N4O5, and a molecular weight of 605.8 [M-H]+. The methyl pheophorbide A induces cell death depending on light, and exhibits a characteristic of apoptosis. This compound has the potential to be utilized as a PS which would have apoptosis activity and availability of broad light wavelength. It is important to discover various candidate compounds with physiological activity from natural products. We expect that this study will be useful in the search for PS candidates using natural products.

Availability of data and materials

Not applicable.

Change history

References

  1. Dos Santos AF, De Almeida DRQ, Terra LF, Baptista MS, Labriola L (2019) Photodynamic therapy in cancer treatment—an update review. J Cancer Metastasis Treat 5:25–44

    Google Scholar 

  2. Lan M, Zhao S, Liu W, Lee CS, Zhang W, Wang P (2019) Photosensitizers for photodynamic therapy. Adv Healthc Mater 8:1–37

    Article  Google Scholar 

  3. Mroz P, Yaroslavsky A, Kharkwal GB, Hamblin MR (2011) Cell death pathways in photodynamic therapy of cancer. Cancers 3:2516–2539

    Article  CAS  Google Scholar 

  4. Stender IM, Borgbjerg FM, Villumsen J, Lock-Andersen J, Wulf HC (2006) Pain induced by photodynamic therapy of warts. Photodermatol Photoimmunol Photomed 22:304–309

    Article  CAS  Google Scholar 

  5. Kim KK, Kawano Y, Yamazaki Y (2003) A novel porphyrin photosensitizer from bamboo leaves that induces apoptosis in cancer cell lines. Anticancer Res 23:2355–2361

    CAS  PubMed  Google Scholar 

  6. Ahmed HM (2019) Ethnomedicinal, phytochemical and pharmacological investigations of Perilla frutescens (L.) Britt. Molecules 24:102

    Article  Google Scholar 

  7. Chen ML, Wu CH, Hung LS, Lin BF (2015) Ethanol extract of Perilla frutescens suppresses allergen-specific Th2 responses and alleviates airway inflammation and hyperreactivity in ovalbumin-sensitized murine model of asthma. Evid Based Comple Alternat Med 324265

  8. Huang B, Lin C, Chen Y, Kao S (2014) Anti-inflammatory effects of Perilla frutescens leaf extract on lipopolysaccharide-stimulated RAW264. 7 cells. Mol Med Rep 10:1077–1083

    Article  CAS  Google Scholar 

  9. Wang XF, Li H, Jiang K, Wang QQ, Zheng YH, Tang W, Tan CH (2018) Anti-inflammatory constituents from Perilla frutescens on lipopolysaccharide-stimulated RAW264. 7 cells. Fitoterapia 130:61–65

    Article  CAS  Google Scholar 

  10. Cho BO, Jin CH, Park YD, Ryu HW, Byun MW, Seo KI, Jeong IY (2011) Isoegomaketone induces apoptosis through caspase-dependent and caspase-independent pathways in human DLD1 cells. Biosci Biotechnol Biochem 75:1306–1311

    Article  CAS  Google Scholar 

  11. Lin CS, Kuo CL, Wang JP, Cheng JS, Huang ZW, Chen CF (2007) Growth inhibitory and apoptosis inducing effect of Perilla frutescens extract on human hepatoma HepG2 cells. J Ethnopharmacol 112:557–567

    Article  Google Scholar 

  12. Kwak CS, Yeo EJ, Moon SC, Kim YW, Ahn HJ, Park SC (2009) Perilla leaf, Perilla frutescens, induces apoptosis and G1 phase arrest in human leukemia HL-60 cells through the combinations of death receptor-mediated, mitochondrial, and endoplasmic reticulum stress-induced pathways. J Med Food 12:508–517

    Article  CAS  Google Scholar 

  13. Cheng HH, Wang HK, Ito J, Bastow KF, Tachibana Y, Nakanishi Y, Xu Z, Luo TY, Lee KH (2001) Cytotoxic pheophorbide-related compounds from Clerodendrum calamitosum and C. cyrtophyllum. J Nat Prod 64:915–919

    Article  CAS  Google Scholar 

  14. Kim SK, Byun HG, Park PJ, Adachi K (2002) Purification and structure of antioxidative substance derived from Tetraselmis suecica. J Korean Fish Soc 35:155–161

    Google Scholar 

  15. Josefsen LB, Boyle RW (2008) Photodynamic therapy and the development of metal-based photosensitisers. Met Based Drugs 276109

  16. Comuzzi C, Cogoi S, Overhand M, Van Der Marel GA, Overkleeft HS, Xodo LE (2006) Synthesis and biological evaluation of new pentaphyrin macrocycles for photodynamic therapy. J Med Chem 49:196–204

    Article  CAS  Google Scholar 

  17. Rapozzi V, Miculan M, Xodo LE (2009) Evidence that photoactivated pheophorbide a causes in human cancer cells a photodynamic effect involving lipid peroxidation. Cancer Biol Ther 8:1318–1327

    Article  CAS  Google Scholar 

  18. Tan PJ, Ong CY, Danial A, Yusof HM, Neoh BK, Lee HB (2011) Cyclic tetrapyrrolic photosensitisers from the leaves of Phaeanthus ophthalmicus. Chem Cent J 5:1–7

    Article  Google Scholar 

  19. Kamarulzaman FA, Shaari K, Ho ASH, Lajis NH, Teo SH, Lee HB (2011) Derivatives of pheophorbide-a and pheophorbide-b from photocytotoxic Piper penangense extract. Chem Biodivers 8:494–502

    Article  CAS  Google Scholar 

  20. Chou ST, Chan HH, Peng HY, Liou MJ, Wu TS (2011) Isolation of substances with antiproliferative and apoptosis-inducing activities against leukemia cells from the leaves of Zanthoxylum ailanthoides Sieb. & Zucc. Phytomedicine 18:344–348

    Article  CAS  Google Scholar 

  21. Mahibalan S, Rao PC, Khan R, Basha A, Siddareddy R, Masubuti H, Fujimoto Y, Begum AS (2016) Cytotoxic constituents of Oldenlandia umbellata and isolation of a new symmetrical coumarin dimer. Med Chem Res 25:466–472

    Article  CAS  Google Scholar 

  22. She LC, Liu CM, Chen CT, Li HT, Li WJ, Chen CY (2017) The anti-cancer and anti-metastasis effects of phytochemical constituents from Leucaena leucocephala. Biomed Res 28:2893–2897

    CAS  Google Scholar 

  23. Kandagatla SK, Uhl RT, Graf TN, Oberlies NH, Raner GM (2019) Pheophorbide derivatives isolated from Açaí berries (Euterpea oleracea) activate an antioxidant response element in vitro. Nat Prod Commun. 14

  24. Buytaert E, Dewaele M, Agostinis P (2007) Molecular effectors of multiple cell death pathways initiated by photodynamic therapy. Biochim Biophys Acta 1776:86–107

    CAS  PubMed  Google Scholar 

  25. Lin CW, Shulok JR, Kirley SD, Cincotta L, Foley JW, Lin CW (1991) Lysosomal localization and mechanism of uptake of nile blue photosensitizers in tumor cells. Cancer Res 51:2710–2719

    CAS  PubMed  Google Scholar 

  26. Wood SR, Holroyd JA, Brown SB (1997) The subcellular localization of Zn (II) phthalocyanines and their redistribution on exposure to light. Photochem Photobiol 65:397–402

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a 2-Year Research Grant of Pusan National University.

Funding

This work was supported by a 2-Year Research Grant of Pusan National University.

Author information

Authors and Affiliations

  1. Department of Life Science and Environmental Biochemistry, Pusan National University, Miryang, 50463, Republic of Korea

    Jun Young Ha, Yu Jin Kim, Kwang Min Lee, Chang Oh Hong & Keun Ki Kim

  2. Department of Central Area Crop Science, National Institute of Crop Science, RDA, Suwon, 16429, Republic of Korea

    Jun Young Ha, Hwan Hee Bae & Young Sam Go

  3. Department of Bio-Environmental Chemistry, Chungnam National University, Daejeon, 34134, Republic of Korea

    Gibum Yi

Authors
  1. Jun Young Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gibum Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hwan Hee Bae
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Young Sam Go
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yu Jin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kwang Min Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chang Oh Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Keun Ki Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KKK: Experimental guidance and overall management. 1st author JYH: Separation of photosensitizer. 2nd author GY: Pellia cultivation. 3rd author HHB: Pellia cultivation and statistical processing. 4th author YSG: Pellia cultivation and statistical processing. 5th author YJK: Apoptosis assay. 6th author KML: DNA ladder. 7th author COH: Structure identification. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Keun Ki Kim.

Ethics declarations

Competing interests

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The original version of the article was revised: the sequence of author has been corrected. The first author has been placed as last author.

Supplementary Information

Additional file 1

: Fig. S1. Procedure of the methyl pheophorbide A isolation from Perilla frutescens leaves. (A) Strategy of chromatographies for the methyl pheophorbide A isolation. (B) Rf patterns of the fractions from primary column chromatography with thin layer chromatography. TLC was visualized with visible light (top), UV short wave (middle), UV long wave (bottom). (C) Final purification of methyl pheophorbide A with TLC which was visualized with visible light (left), UV short wave (middle), UV long wave (right). (D) HPLC analysis for the purified methyl pheophorbide A. Fig. S2. Scanning of absorbance (y-axis) of continuous light. Fig. S3. 1H NMR spectrum (CDCl3, 600 MHz) of methyl pheophorbide A. Fig. S4. 13C NMR spectrum (CDCl3, 600 MHz) of methyl pheophorbide A. Fig. S5. COSY NMR spectrum (CDCl3, 600 MHz) of methyl pheophorbide A.  Fig. S6. HMBC NMR spectrum (CDCl3, 600 MHz) of methyl pheophorbide A. Fig. S7. HSQC NMR spectrum (CDCl3, 600 MHz) of methyl pheophorbide A.  Fig. S8. FAB-MS spectrum of methyl pheophorbide A.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ha, J.Y., Yi, G., Bae, H.H. et al. Isolation, identification, and apoptosis activity of the photosensitizer methyl pheophorbide A from Perilla frutescens leaves. Appl Biol Chem 65, 52 (2022). https://doi.org/10.1186/s13765-022-00719-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13765-022-00719-5

Keywords