Patent Application
20220175728
This non-provisional application claims priority of Taiwan Invention Patent Application No. 109143031, filed on Dec. 7, 2020, the contents thereof are incorporated by reference herein.
The present invention is directed to pharmaceutical use of a chlorophyll derivative for anti-viral infection, and more particularly to pharmaceutical use of chlorophyllide for anti-viral infection.
Plants are the foundation of traditional medicines. A number of plant extracts possess anti-cancer properties, including Annona muricata L., Carica papaya, Colocasia gigantea, Annona squamosa Linn, Murraya koenigii L., Olea europaea L., Pandanus amaryllifolius Roxb., Chenopodium quinoa, Toona sinensis, Myristica fragrans, Thermopsis rhombifolia, and Cannabis sativa. The potential anti-cancer activities of these plants are associated with various bioactive compounds, including chlorophyll, pheophorbide, alkaloid, terpenoid, polysaccharide, lactone, flavonoid, carotenoid, glycoside, and cannabidiol. Beside the possibility of anti-cancer functions, compounds in plant extract demonstrate to exert function of anti-oxidation, anti-inflammation and attenuate side effects induced by chemotherapeutics. Additionally, those bioactive factors, especially chlorophyll and its derivatives, demonstrate potential for the treatment of cancer.
Chlorophyll, the most abundant pigment on earth, is present at high levels in green leafy plants, algae, and cyanobacteria. The catabolic derivatives of chlorophyll are chlorophyllide (chlide), pheophytin, pheophorbide, and phytol. Studies demonstrate that chlorophyll can reduce the growth and proliferation of MCF-7 breast carcinoma cells. Chlorophyll is also reported to promote cell differentiation, and to induce cell cycle arrest and apoptosis in HCT116 colon cancer cells. Chlorophyllide a/b and pheophorbide a/b are reported to reduce hydrogen peroxide-induced strand breaks and oxidative damage, and aflatoxin B1-DNA adduct formation in hepatoma cells. Chlorophyllide is shown to decrease the levels of hepatitis B virus without affecting cell viability and viral gene products in tetracycline-inducible HBV-expressing HepDE19 cells. In human lymphoid leukemia molt 4B cells, pheophorbide a and phytol are able to induce programmed cell death. Phytol can also reduce inflammation by inhibiting neutrophil migration, reducing the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and oxidative stress. Pheophorbide a, in photodynamic therapy, is found to increase the levels of cytosolic cytochrome c, and is also tested against human pancreatic cancer cells (Panc-1, Capan-1, and HA-hpc2), hepatocellular carcinoma cells (Hep 3B), uterine sarcoma cells, human uterine carcinoma cells, and Jurkat leukemia cells. Pheophorbide is also shown to decrease the levels of procaspase-3 and -9 in Hep3B, Hep G2, and human uterine sarcoma MES-SA cells.
Extensive studies are performed with chlorophyllin (chllin, Cu-chl). Chlorophyllin, a semisynthetic, Cu-coupled, and water-soluble derivative of chlorophyll, is shown to significantly decrease the growth of mutagen-induced cancer cells. In vitro and in vivo studies are suggested that chlorophyllin possesses anti-genotoxic functions against compounds present in cooked meat, including N-nitroso compound and fungal toxin, aflatoxin B1 (AFB1), and dibenzo[d,e,f,p]chrysene (DBC). The regulation of cancer growth by chlorophyllin seems to involve the deactivation of key signal transduction pathways, including the nuclear factor kappa B, Wnt/b-catenin, phosphatidylinositol-3-kinase/Akt, and expressed E-cadherin and alkaline phosphatase pathways.
The amount of chlorophyll degraded globally each year is estimated to exceed 1000 million tons, and this is mostly derived from agriculture and food processing waste. Except for the edible parts of vegetables and fruits, most chlorophyll from low-value agricultural waste can only be degraded naturally. By using low-value agricultural waste as sources to collect chlorophyll, the cost of extraction can be reduced and maximum value of agriculture waste can be reached. Therefore, agricultural waste is potentially useful in the biomedical industry as a high-value nutraceutical and pharmaceutical material.
The present invention is made based on the discovery that a product obtained by treating a plant leaf extract with chlorophyllase exhibits anti-viral properties, wherein its active ingredient at least includes chlorophyllide.
Therefore, an embodiment of the present invention provides a method for treating or preventing virus infection, the method including the step of: administering a therapeutically effective concentration of chlorophyllide to a subject in need thereof.
Preferably, the therapeutically effective concentration is 1.5625 μg/mL to 100 μg/mL.
Preferably, the therapeutically effective concentration is 6.25 μg/mL to 100 μg/mL.
Preferably, the therapeutically effective concentration is 25 μg/mL to 100 μg/mL.
Preferably, the virus is flavivirus.
Preferably, the flavivirus is yellow fever virus, Japanese encephalitis virus, dengue virus, West Nile virus, or Zika virus.
Preferably, the virus is alphavirus.
Preferably, the alphavirus is chikungunya virus.
Preferably, the virus is severe acute respiratory syndrome coronavirus 2.
Another embodiment of the present invention provides a method for treating or preventing virus infection, the method including the step of: administering a product obtained by treating a plant leaf extract with chlorophyllase to a subject in need thereof, wherein the product comprises a therapeutically effective concentration of chlorophyllide.
Preferably, the product is produced by the following steps of: providing plant leaves; performing extraction on the plant leaves with a solvent to obtain a crude extract; and treating the crude extract with chlorophyllase to obtain the product.
Preferably, the therapeutically effective concentration is 1.5625 μg/mL to 100 μg/mL.
Preferably, the therapeutically effective concentration is 6.25 μg/mL to 100 μg/mL.
Preferably, the therapeutically effective concentration is 25 μg/mL to 100 μg/mL.
Preferably, the solvent is ethanol or hexane.
Preferably, the virus is flavivirus.
Preferably, the flavivirus is yellow fever virus, Japanese encephalitis virus, dengue virus, West Nile virus, or Zika virus.
Preferably, the virus is alphavirus.
Preferably, the alphavirus is chikungunya virus.
Preferably, the virus is severe acute respiratory syndrome coronavirus 2.
The detailed description and preferred embodiments of the invention will be set forth in the following content, and provided for people skilled in the art to understand the characteristics of the invention.
The compound chlorophyllide used in the content is represented by a general formula (I)
in which Me is a Mg atom, and R is a CH3 group or a CHO group. Generally, while R is a CH3 group, the compound is called “chlorophyllide a”; while R is a CHO group, the compound is called “chlorophyllide b.”
The leaves of guava, sweet potato, banana, Chinese toona, logan, wax apple, mango, caimito, and cocoa were used to extract chlorophyll. 10 g (wet weight) of leaves were washed, dried, and ground into powder with a pestle and mortar. Leaf mixtures were then frozen in liquid nitrogen and stored at −80° C. in a deep freezer. Chlorophyll was extracted by immersing leaves in ethanol solvent (or hexane solvent) for 48 h. Ethanol crude extracts (or hexane crude extracts) from leaves were centrifuged at 1500 g for 5 min and keep at −20° C. for further experiments. To measure the concentrations of chlorophyll a/b, the crude extracts were passed through a 0.22-μm filter and the absorbance was measured at 649 and 665 nm, which were the major absorption peaks of chlorophyll a and b, respectively. The estimated concentrations of chlorophyll a and b in crude extracts were calculated according to the following equation:
chlorophyll a concentration(μg/mL)=13.7×A665−5.76×A649;
chlorophyll b concentration(μg/mL)=25.8×A665−7.6×A649.
The chlorophyll a/b concentrations in crude extracts calculated with the empirical equation were multiplied by the volume of the solvent that resulted in the relative chlorophyll mass values in the given samples. When the dry and wet weights of the plant species are known, the content of chlorophyll a/b and the mass of crude extracts relative to the mass of the dry plant can be calculated and expressed as mg/gDW.
Chlamydomonas reinhardtii chlorophyllase was produced as described previously (Molecules. 2015 Feb. 24; 20(3):3744-57; Biotechnol Appl Biochem. 2016 May; 63(3):371-7). Recombinant Chlamydomonas reinhardtii chlorophyllase was expressed, purified, and then lyophilized. The reaction mixture contained 0.5 mg of recombinant chlorophyllase, 650 μL of the reaction buffer (100 mM sodium phosphate, pH 7.4, and 0.24% Triton X-100), and 0.1 ml of crude extracts from leaves (100 mM chlorophyll). The reaction mixture was incubated at 37° C. for 30 min in a shaking water bath. The enzymatic reaction was stopped by adding 4 mL of ethanol, 6 mL of hexane, and 1 mL of 10 mM KOH, respectively. The reaction mixture was vortexed vigorously and centrifuged at 4000 rpm for 10 min to separate the two phases. The upper layer contained the untreated chlorophyll a/b; the bottom layer was chlorophyllase-treated crude extracts comprising chlorophyllide a/b. The chlorophyllase-treated crude extracts containing chlorophyllide a/b mixtures were then concentrated and the solvent was removed by evaporation under reduced pressure at 40° C. on a rotary evaporator. The concentrated crude extracts were processed by lyophilization, weighed, and stored at −80° C. for further experiments.
Chlorophyll was extracted from leaves of 9 plant species, including guava, sweet potato, banana, Chinese toona, logan, wax apple, mango, caimito, and cocoa. Ethanol crude extracts were treated with chlorophyllase to generate chlorophyllide, and then lyophilized in order to measure the weight. The results are listed in Table 1. Significantly, the most chlorophyll a level was observed in Chinese toona (9.8 mg/gDW), followed by mango (8.407 mg/gDW). The lowest chlorophyll a levels were present in banana (2.921 mg/gDW) and sweet potato (3.481 mg/gDW). For chlorophyll b, Chinese toona possessed the highest content (5.419 mg/gDW), followed by cocoa (4.485 mg/gDW) and mango (2.599 mg/gDW). The lowest levels of chlorophyll b were found in sweet potato (0.996 mg/gDW), banana (1.031 mg/gDW), and caimito (1.493 mg/gDW). Of the species analyzed, leaves of cocoa and caimito contained the highest level of ethanol crude extracts, at 412.65 and 397.62 mg/gDW, respectively. The lowest weight of ethanol crude extracts was obtained from sweet potato (43.175 mg/gDW), banana (47.76 mg/gDW), and wax apple (94.29 mg/gDW).
Ipomoea batatas
Syzygium
samarangense
Psidium guajava
Musa paradisiaca
Toona sinensis
Dimocarpus longan
Mangifera indica
Pouteria Caimito
Theobroma cacao
To analyze chlorophyll and chlorophyllide, the mixtures containing chlorophyllase-treated crude extracts were analyzed by using HPLC as described previously. Chlorophyllide was detected at a wavelength of 667 nm and identified by absorption spectra, peak ratios, and co-migration with authentic standards.
The HPLC separation system was applied to determine the amount of chlorophyll a/b and chlorophyllide a/b in crude extracts. Since the provision of commercial standards was limited, it was not possible to identify all peaks in all crude extracts by HPLC. Herein, the standards used in this study, including chlorophyll a, chlorophyll b, chlorophyllide a, and chlorophyllide b were selected based on our previous studies. HPLC results were obtained using mobile phases consisting of ethyl acetate/methanol/H2O2=44:50:6. Samples were quantified using photodiode array detection in the region 200-400 nm based on the retention times and UV spectra compared with the standards.
African green monkey kidney Vero cells were infected with chikungunya virus at a multiplicity of infection (MOI) of 0.1 or 0.01 in the presence of chlorophyllide at indicated dosages, and then incubated for 24 hr. Total RNA in the infected Vero cells was isolated with NucleoZOL (Macherey-Nagel) and quantified by RT-qPCR using Mic qPCR (Bio Molecular Systems). The QuantiTech SYBR Green RT-qPCR kit was used to prepare mixture samples, followed by a program of 50° C. for 30 min, 95° C. for 15 min, and then 45 cycles at 95° C. for 15 sec, 57° C. for 25 sec, and 72° C. for 10 sec. Meanwhile, after incubation with chlorophyllide for 24 hr, the culture supernatant was collected to determine virus spread by using the TCID50 assay.
African green monkey kidney Vero cells were infected with chikungunya virus at an MOI of 0.001 in the presence of chlorophyllide at indicated dosages, and then incubated for 3 days, followed by the micro-neutralization test.
The gene expression profiles of the African green monkey kidney Vero cells infected with chikungunya and those infected with chikungunya in combination with chlorophyllide treatment were analyzed by using next generation sequencing (NGS). Table 2 shows both test samples are good samples according to quality evaluation in NGS data.
The Gene Ontology analysis was performed to classify the foregoing 2 differentially expressed gene groups. Based on sequence homology,
The KEGG pathway enrichment analysis was also performed to classify the foregoing 2 differentially expressed gene group. In the KEGG pathway enrichment analysis, all of the foregoing differentially expressed genes were classified according to different KEGG pathways including “metabolism”, “genetic information processing”, “environmental information processing”, “cellular processes”, “organismal systems”, and “human diseases.”
African green monkey kidney Vero cells were infected with Zika virus at an MOI of 1 or 0.1 in the presence of chlorophyllide at indicated dosages, and then incubated for 2 days. Total RNA in the infected Vero cells was isolated with NucleoZOL (Macherey-Nagel) and quantified by RT-qPCR using Mic qPCR (Bio Molecular Systems). The QuantiTech SYBR Green RT-qPCR kit was used to prepare mixture samples, followed by a program of 50° C. for 30 min, 95° C. for 15 min, and then 45 cycles at 95° C. for 5 sec, and 60° C. for 34 sec. Meanwhile, after incubation with chlorophyllide for 2 days, the culture supernatant was collected to determine virus spread by using the TCID50 assay.
African green monkey kidney Vero cells were infected with Zika virus at an MOI of 0.1 in the presence of chlorophyllide at indicated dosages, and then incubated for 4 to 5 days, followed by the micro-neutralization test.
African green monkey kidney Vero cells were infected with dengue-2 virus at an MOI of 0.1 in the presence of chlorophyllide at indicated dosages, and then incubated for 3 days. Total RNA in the infected Vero cells was isolated with NucleoZOL (Macherey-Nagel) and quantified by RT-qPCR using Mic qPCR (Bio Molecular Systems). The QuantiTech SYBR Green RT-qPCR kit was used to prepare mixture samples, followed by a program of 50° C. for 30 min, 95° C. for 15 min, and then 45 cycles at 95° C. for 15 sec, 55° C. for 30 sec, and 72° C. for 30 sec. Meanwhile, after incubation with chlorophyllide for 3 days, the culture supernatant was collected to determine virus spread by using the TCID50 assay.
The gene expression profiles of the African green monkey kidney Vero cells infected with dengue and those infected with dengue in combination with chlorophyllide treatment were analyzed by using next generation sequencing (NGS). Table 3 shows both test samples are good samples according to quality evaluation in NGS data.
The Gene Ontology analysis was performed to classify the foregoing 2 differentially expressed gene groups.
The KEGG pathway enrichment analysis was also performed to classify the foregoing 2 differentially expressed gene groups. In the KEGG pathway enrichment analysis, all of the foregoing differentially expressed genes were classified according to different KEGG pathways including “metabolism”, “genetic information processing”, “environmental information processing”, “cellular processes”, “organismal systems”, and “human diseases.”
While the invention has been described in connection with what is considered the most practical and preferred embodiments, it is understood that this invention is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.
| Number | Date | Country | Kind |
|---|---|---|---|
| 109143031 | Dec 2020 | TW | national |
