Toxicology Laboratory, Faculty of Biotechnology (Biomaterials), College of Applied Life Science, SARI, Jeju National University, Jeju, Republic of Korea
Received: 13 Jul 2017 Revised and Accepted: 31 Aug 2017
Objective: The objective of this study was undertaken to examine pancreatic lipase and tyrosinase inhibitory activities of Camellia mistletoe (Korthalsella japonica) extracts and conduct their antiproliferative activity on several human cancer (Hela, A375, HCT 116, HepG2 and A549) and normal cells (TK6) lines.
Methods: The lipase inhibitory activity of acts of Camellia mistletoe extracts were determined from the hydrolytic reaction of p-nitrophenyl butyrate with pancreatic lipase and cytotoxicity against human cancer and normal cell lines were also evaluated using the MTT assay. The in vitrotyrosinase inhibitor potency of Camellia mistletoe extracts was estimated using mushroom tyrosinase.
Results: Both methanol and ethanol extracts of Camellia mistletoe exhibited aninhibitory effect on lipase inhibition activity with IC50 values of 0.39 and 0.49 mg/ml, respectively. Moreover, Camellia mistletoe extracts were judged to be strongly active against the cancer cell lines, while they had no antiproliferative activity against the normal human cell lines. However, they had little inhibitory effects on tyrosinase activity.
Conclusion: Camellia mistletoe extracts could be the good sources of natural lipase inhibition and anticancer agents with a great potential to be used in food and therapeutic fields.
Keywords: Camellia mistletoe, Pancreatic lipase inhibitory activity, Tyrosinase inhibitory activity, Antiproliferative activity
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Medicinal plants are an importanttherapeutic aid for various ailments. In recent times, focus on plant research has increased all over the world and a large body of evidence has collected to show immense potential of medicinal plants used in various traditional systems . Plants are a rich source of medicines because they produce awide array of bioactive molecules, most of which probably evolved as achemicaldefence against predation or infection. The increasing use of plant extracts in the food, cosmetic and pharmaceutical industries suggests that in order to find active compounds, a systematic study of medicinal plants is very important. Compounds from medicinal plants in the food and cosmetics industry have a central role and have a wide range of applications.
Mistletoes are native to Asia, Africa, Europe and Australia, and 70-100 species have been recorded . Mistletoe extracts are used for thetreatment of many diseases such as cancer, hypertension, and diabetes [3-5]. Camellia mistletoe, Korthalsella japonica (family Santalaceae), is a semiparasitic plant which is distributed in Jeju Island, Korea. Recently there has been considerable interest in the antioxidant potential and antimicrobial activities of Camellia mistletoe extracts [6, 7]. Although a lot of work was carried out with regard to the phytochemical and pharmacological evaluation of European mistletoe, Viscum album L. , but little is known of actual physiological benefits of Camellia mistletoe, K. japonica. Therefore, the present study was aimed to evaluate inhibitory activities of lipase, tyrosinase and cancer cell proliferation by the methanol and ethanol extracts from Camellia mistletoe.
Chemicals and reagents
Porcine pancreatic lipase, p-nitrophenyl butyrate (p-NPB), mushroom tyrosinase, tyrosine, ascorbic acid were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Cell culture reagents were obtained from Lonza (Walkersville, MD, USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell proli-feration kit was purchased from Boehringer Mannheim (Indianapolis, IN, USA). All ingredients used in this study were of analytical grade.
Plant material and extraction procedure
Camellia mistletoe (K. japonica) samples were collected during May 2015fromCamellia Hill located in Andeok-myeon, Seogwipo-si, Jeju-do Province, South Korea and the voucher specimens (KHUP-0803) have been deposited in the herbarium of College of Applied Life Science, Jeju National University.Samples were prepared using a previously described protocol. The dried and powdered plant material (20 g) wasextracted with 100 ml of 100% methanol or 70% ethanol for 72 h and filtered, concentrated using a rotary evaporator (BuchiRotavapor R-200, New Castle, DE, US), freeze-dried and stored at-20 USyin storage vials for experimental use. The average extraction yields (freshsample/methanol or ethanol) were7.1 and 10.4%, respectively.Dried extracts were reconstituted in dimethyl sulfoxide (DMSO, Amresco, Solon, Ohio, USA).DMSO acts as a solvent without changing any property of the methanol and ethanol extracts.
Pancreatic lipase inhibitory activity
The lipase inhibitory activity of Camellia mistletoe extracts was estimated from the ability to inhibit the in vitro porcine pancreatic lipase activity as previously described with slight modifications. The reaction mixture consisted of 6 μLof 10 mg/ml of porcine pancreatic lipaseand170 μLof Tris buffer. Then 20 μL of Camellia mistletoe extracts was added and incubated at 37°7for 15 min.Four microliters of 10 mMp-NPB was then added and incubated at 37°t for 60 min.
Lipase activity was determined by measuringthe hydrolysis of p-NPB to p-nitrophenol at 400 nm using a Spectra MR microplate reader (Dynex Technologies, Inc., Chantilly, VA, US). IC50, the concentrationof a tested compound giving 50% inhibitionof the enzyme activity, was evaluated from the leastsquareregression line of the plot of the logarithm ofconcentration vs. the inhibitory activity. Hesperidin was used as the positive control.
Tyrosinase inhibitory activity
Tyrosinase inhibitory activity of Camellia mistletoe extracts was carried out by a previouslydescribed method with some modifications. Briefly, mushroom tyrosinase (40 𝜇l, 110 unit/ml) was mixed with 100 𝜇l of phosphate buffer (0.175 M, pH 6.8) and 40 𝜇l of 10 mmoltyrosine and 20𝜇l of differentconcentrated Camellia mistletoe extracts (0.125, 0.25 and0.5 mg/ml). The mixturewas then incubated for 15min at room temperature. Following incubation,absorbance of the mixture was determined at 475 nm byusing a Spectra MR microplate reader. The concentration of the extract which caused a halfmaximal inhibition of the lipase activity (IC50) wasobtained from a semilog plot of Camellia mistletoe extract concentrationsagainst the percentage of enzyme inhibition.Ascorbic acid (500 𝜇g/ml) as positive control was used for assay.
In vitro cytotoxic activity
The in vitro cytotoxic activity of Camellia mistletoe extractswasdetermined from the mitochondrial activity of cell lineswhich represent the number of viable cells after thetreatment, by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cytotoxic assay on six different human cell lines intissue culture. The human lymphoblastoid cells (TK6), cervical cancer (Hela), malignant melanoma (A375), colon cancer (HCT 116), liver cancer (HepG2) and non-small cell lung adenocarcinoma(A549) cell lines werekindly provided byProfessor Wogan (Massachusetts Institute of Technology, MA, US). Hela and A375 cells werecultured in Dulbecco’s modified Eagle’s Medium(DMEM) supplemented with10% heat-inactivated fetal bovine serum, 100units/ml penicillin, 100 μg/ml streptomycin and 2 mmol L-glutamine at 37°C with 5% CO2 in ahumidified atmosphere. TK6, HCT 116, HepG2 and A549 cell lines weremaintained in RPMI 1640, McCoy’s 5A, MEM and Ham’s F-12 media supplemented asabove with L-glutamine, antibiotics and FBS, respectively.
The inhibitory effect of Camellia mistletoe extracts on theproliferation of human normal and cancer cells wasdetermined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly,cell suspensions (5×103 per well) wereloaded into 96-well culture plates and treated withfresh medium containing various concentrations (0-200μM) of each extract for 72 h. Cells werewashed once with phosphatebuffered saline andreacted with the MTT solutionat 37°C for 4 h to produce theformazan salt. Finally, the formazan salt formed ineach cultured cells was dissolved in DMSO, and theoptical density (OD) value of each solution wasmeasured at 540 nm using a Spectra MR microplate reader. The OD value detected for thecontrol (cells without treatment with any samples)from the treated cells was plotted on the x-axis,designated as proliferation (% of control), todemonstrate the effect of each sample on the viabilityof the related cells.The concentration of the extract which caused a half-maximal inhibition of cell proliferation (IC50), as determinedby the MTT assay, was obtained from a semi-logplot of Camellia mistletoe extract concentrations against the percentageof cell survival.
Data were analyzed using SPSS statistical software (SPSS, ver. 12.0; SPSS Inc., Chicago, IL, US). Comparisons of all results between methanol and ethanol extracts of Camellia mistletoe were made by using a non-parametric test (Mann-Whitney U test) with p<0.05.
Lipase inhibitory activity
Obesity is now recognized as a major risk factor in the metabolic syndrome, diabetes mellitus, hypertension and some cancers . Obesity is considered an extremely costly health problem which in developed nations accounts for 2-6% of total health care costs. Currently Orlistat, a potent competitive inhibitor of gastric and pancreatic lipase, has been approved by FDA and is available as an antiobesity drug . However, this medication is associated with incidence of side-effect including intestinal flatulence, borborygmi and abdominal cramps . Therefore, new approaches for the treatment of obesity tried to reduce energy intake through gastrointestinal mechanisms, without altering any vital mechanisms . Inour current study, methanol and ethanol extracts of Camellia mistletoeinhibited thelipase activity in a dose-dependent manner in theassay system with p-NPBas a substrate (table 1). With respect to the lipase inhibition, the methanolextract showed more effective activity than ethanol extract (p<0.05). Nevertheless, this was weak(IC50 value of 0.39 mg/ml), compared to that forthe positive control of hesperidin (IC50 value of 0.032 mg/ml). However, the pancreatic lipase inhibitory effect of Camellia mistletoe was superior to those when various dietary plants were screened by Kim et al.  and Confortiet al. .
Tyrosinase inhibitory activity
Tyrosinase inhibitor has been used frequently in cosmetics and depigmenting agents for hyperpigmentation. It catalyzes two different reactions usingmolecular oxygen the ortho-hydroxylation of tyrosine (mono-phenols) named monophenolase activity and the oxidation of 3,4-Dihydroxyphenylalanine (DOPA) to dopaquinine (o-quinones) named diphenolase activity . A large number of moderate to potent tyrosinaseinhibitors from natural and synthetic resources have been reportedduring the last decade[20, 21]. However, some tyrosinase inhibitors are considered to be cytotoxic to melanocytes and potentially mutagenicto mammalian cells[21, 22]. Therefore, it remains necessary tosearch for new tyrosinase inhibitors without side effects. In this study, we found that Camellia mistletoe extractsfraction had asmall inhibitory effect at the highest dose tested (table 1).However theinhibitory effect is small and it is not economically feasible to bedeveloped further.Ascorbic acid, used as a positive control, reduced mushroom tyrosinasedrastically even at the lowest dose used. Although the results of this work are inconsistent with those reported by Lee et al. in that high tyrosinase inhibitory activity was detected from hot water (64%) and ethanol (63%) extracts of mistletoe (Viscumalbum) at the concentration of 100 mg/ml, our study was conducted with different extraction methods, different species (K. japonica) and relatively very low test concentrations (0.125, 0.25 and 0.5 mg/ml).
Table 1: Effects of the methanol and ethanol extracts from Camellia mistletoe (K. japonica) on lipase and tyrosinase inhibitory activities
|100% MeOH||0.125||40.1 ± 0.90||0.39 ± 0.0136*|
|0.25||41.9 ± 3.93|
|Lipase inhibitory activity||0.5||53.3 ± 3.60|
|70% EtOH||0.125||14.9 ± 5.39||0.49 ± 0.0654|
|0.25||25.9 ± 2.85|
|0.5||50.5 ± 7.71|
|100% MeOH||0.125||10.8 ± 1.85||1.40 ± 0.159*|
|0.25||16.6 ± 0.61|
|Tyrosinase inhibitory activity||0.5||17.8 ± 1.67|
|70% EtOH||0.125||7.3 ± 2.98||1.56 ± 0.107|
|0.25||12.1 ± 1.70|
|0.5||16.0 ± 1.40|
|Each values is expressed as mean ± standard deviation (n = 3). *p < 0.05 compared to ethanol extract of Camellia mistletoe.|
Fig.1: The percentage of viable Hela and A375 cells afterCamelliamistletoe(K. japonica)treatment,Cell extracts for 72 h. Each point is the mean±SD of three experiments, *p<0.05 compared to ethanol extract of Camelliamistletoe, Cell viability was determined by MTT assay aftertreatment with 0, 50, 100 and 200 μg/ml of methanol and ethanol
In vitro cytotoxic activity
Because of serious side effects and toxicity of chemotherapeutic anticancer agents, many cancer patients seek alternative and/or complementary methods of treatment. Plants have been used for treating various diseases of human beings and animals since time immemorial. They maintain the health and vitality of individuals and also cure diseases, including cancer without causing toxicity. More than 50% of all modern drugs in clinical use are of natural products, many of which have the ability to control cancer cells . Medicinal plants are therefore being investigated for their anticancer properties, and the demand for natural anticancer agents increasing.
Fig.2: The percentage of viable HCT116 and HepG2 cells afterCamelliamistletoe(K. japonica)treatment,Cell viability was determined by MTT assay aftertreatment with 0, 50, 100 and 200 μg/ml of methanol and ethanol extracts for 72h, Each point is the mean±SD of three experiments, *p<0.05 compared toethanol extract of Camelliamistletoe
Fig.3: The percentage of viable A549 and TK6 cells afterCamelliamistletoe(K. japonica)treatment, Cell viability was determined by MTT assay aftertreatment with 0, 50, 100 and 200 μg/ml of methanol and ethanol extracts for 72 h, Each point is the mean±SD of three experiments, *p<0.05 compared to ethanol extract of Camelliamistletoe
In the present study, human cervical cancer (Hela), malignant melanoma (A375), colon cancer (HCT 116), liver cancer (HepG2) and non-small cell lung adenocarcinoma (A549) cells were treated with various concentrations (0-200 μ0/ml) of Camellia mistletoe extracts for 72 h, and then analyzed by MTT assay for the cell viability (% control), as shown in fig. 1-3. Overall, both methanol and ethanol extracts of Camellia mistletoe were found to exhibits significantly growth inhibitory effect in all human cancer cell lines, and methanol extract was more active than ethanol extract (p<0.05) (fig. 1-3). The highest cytotoxic activity (IC50 of 64.6 μo/ml) was obtained against the Hela cell line by the methanol extract of Camellia mistletoe (fig. 1), and the rank order of potency of the methanol and ethanol extracts of Camellia mistletoe for cell line was A375 (IC50 of 135.6 and 156.3 μo/ml), HCT116 (IC50 of 154.0 and 171.3 μg/ml), HepG2 (IC50 of 149.4 and 193.3 μo/ml), A549 (IC50 of 217.2 and 226.5 μo/ml), respectively (fig. 1-3). Based on the results obtained, Camellia mistletoe extracts were effective in inhibiting all tested cancer cell proliferation. For the development of anticancer agents from natural products, it is important not only to screen the selectivity (or specificity) of the natural products among several cancer cells but also to assay if natural products exhibited any cytotoxicity in non-cancer cells. Thus, the effect of Camellia mistletoe extracts on non-cancer cells (human fibroblast TK6 cells) was tested. The TK6 cells did not exhibit cytotoxic effect with any extracts (fig.3B). One drawback of cytotoxic drug therapy for treatment of malignant diseases is serious toxicity. The data described above suggested that these Camellia mistletoe extracts are expected to be candidates for a non-toxic antitumor agent
Our study proposed Camellia mistletoe as acandidate for natural lipase inhibitor and anticancer agent. Thesein vitroantilipase and antiproliferativeactivities of Camellia mistletoe extractsseems to be closely correlated with its lectin contents or phenolic components. Further study is also needed to investigate the mechanism of antilipase and antiproliferative activities of Camellia mistletoe extracts.
Design, experimental part of the work and writing of the manuscript was done by the corresponding author Dr. M. Y. Kim.
We declare that there is no conflict of interest
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How to cite this article
Min Young Kim. Inhibitory activities of Camellia mistletoe (K. japonica) extracts on pancreatic lipase, tyrosinase and cancer cell proliferation.Int J Pharm Pharm Sci 2017;9(10):187-190.