ARTICLE   Open Access    

Carotenoid cleavage dioxygenase catalyzes carotenoid degradation and regulates carotenoid accumulation and petal coloration in Zinnia elegans

  • # Authors contributed equally: Hongsheng Qing, Xinyue Liu

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  • Carotenoids are one of the important coloring substances in the ray florets of Zinnia elegans. Carotenoid cleavage dioxygenase (CCD) directly affects carotenoid contents by cleaving the double bond of carotenoids, thereby affecting the flower color. However, few reports have elucidated its function in Zinnia elegans. In the present study, through transcriptome homology searches, three ZeCCDs, ZeCCD1, ZeCCD4-1, and ZeCCD4-2 were screened from Zinnia elegans. The function of ZeCCDs was verified by bacterial pigment complementation system, and it revealed that all of the three ZeCCDs had the ability to cleave β-carotene and other carotenoids (ε-carotene, zeaxanthin and lycopene). Meanwhile, the expression levels of ZeCCD1 and ZeCCD4-2 in the petals of different cultivars were significantly negatively correlated with the carotenoid content. Furthermore, compared with the cytoplasmic localization of ZeCCD1, ZeCCD4-2 is localized in the plastid. Our results indicate that ZeCCD4-2 is the key gene responsible for the differential accumulation of carotenoids in the petals of different Zinnia elegans cultivars. This study lays a foundation for further research on the molecular mechanism of petal coloration and carotenoid metabolism of Zinnia elegans.
  • Salvia rosmarinus L. (old name Rosmarinus officinalis), common name Rosemary thrives well in dry regions, hills and low mountains, calcareous, shale, clay, and rocky substrates[1]. Salvia rosmarinus used since ancient times in traditional medicine is justified by its antiseptic, antimicrobial, anti-inflammatory, antioxidant, and antitumorigenic activity[1,2]. The main objective of the study is to evaluate the antimicrobial activity of different extracts of Salvia rosmarinus in vitro, and its compounds related to in silico targeting of enzymes involved in cervical cancer. Since the start of the 20th century, some studies have shown that microbial infections can cause cervical cancers worldwide, infections are linked to about 15% to 20% of cancers[3]. More recently, infections with certain viruses like Human papillomaviruses (HPV) and Human immunodeficiency virus (HIV), bacteria like Chlamydia trachomatis, and parasites like schistosomiasis have been recognized as risk factors for cancer in humans[3]. Then again, cancer cells are a group of diseases characterized by uncontrolled growth and spread of abnormal cells. Many things are known to increase the risk of cancer, including dietary factors, certain infections, lack of physical activity, obesity, and environmental pollutants[4]. Some studies have found that unbalanced common flora Lactobacillus bacteria around the reproductive organ of females increases the growth of yeast species (like Candida albicans) and some studies have found that women whose blood tests showed past or current Chlamydia trachomatis infection may be at greater risk of cervical cancer. It could therefore be that human papillomavirus (HPV) promotes cervical cancer growth[3]. Salvia rosmarinus is traditionally a healer chosen as a muscle relaxant and treatment for cutaneous allergy, tumors, increases digestion, and the ability to treat depressive behavior; mothers wash their bodies to remove bacterial and fungal infections, promote hair growth, and fight bad smells[5] .

    The study of plant-based chemicals, known as phytochemicals, in medicinal plants is gaining popularity due to their numerous pharmacological effects[6] against drug resistance pathogens and cancers. The causes of drug resistance to bacteria, fungi, and cancer are diverse, complex, and only partially understood. The factors may act together to initiate or promote infections and carcinogenesis in the human body is the leading cause of death[7]. Antimicrobial medicines are the cornerstone of modern medicine. The emergence and spread of drug-resistant pathogens like bacteria and fungi threaten our ability to treat common infections and to perform life-saving procedures including cancer chemotherapy and cesarean sections, hip replacements, organ transplantation, and other surgeries[7]. On the other hand, information about the current magnitude of the burden of bacterial and fungal drug resistance, trends in different parts of the world, and the leading pathogen–drug combinations contributing to the microbial burden is crucial. If left unchecked, the spread of drug resistance could make many microbial pathogens much more lethal in the future than they are today. In addition to these, cancers can affect almost any part of the body and have many anatomies and molecular subtypes that each require specific management strategies to avoid or inhibit them. There are more than 200 different types of cancer that have been detected. The world's most common cancers affecting men are lung, prostate, colorectal, stomach, and liver cancers[8]. While breast, cervix, colorectal, lung, and stomach cancers are the most commonly diagnosed among women[8]. Although some cancers said to be preventable they seem to still be one of the causes of death to humans, for example cervical cancer. The need to fill the gap to overcome the problem of searching for antimicrobials and anticancers from one source of Salvia rosmarinus is of importance.

    Cervical cancer is a common cancer in women and a prominent cause of death[9]. In Ethiopia, cervical cancer is a big deal for women aged 15 to 44, coming in as the second most common cancer[9]. Globally, it's the fourth most common prevalent disease for women[10]. Aberrant methylation of tumor-suppressor genes' promoters can shut down their important functions and play a big role in causing cervical tumors[10]. There are various cervical cancer repressor genes (proteins turn off or reduce gene expression from the affected gene), such as CCNA1, CHF, HIT, PAX1, PTEN, SFRP4, and TSC1. The genes play a crucial role in causing cervical cancer by regulating transcription and expression through promoter hypermethylation, leading to precursor lesions during cervical development and malignant transformation[11]. The process of DNA methylation is primarily carried out by a group of enzymes known as DNA methyltransferases (DNMT1). It has been reported that DNMT1 (PDB ID: 4WXX), a protein responsible for DNA methylation can contribute to the development of cervical cancer. DNMT1 inhibits the transcription of tumor suppressor genes, facilitating tumorigenesis, which finally develops into cervical cancer. Tumor suppressor gene transcription is inhibited by DNMT1, which helps cancer grow and eventually leads to cervical cancer. Repressive genes' hypermethylation may be decreased, their expression can be increased, and the phenotype of malignant tumors can be reversed by inhibiting the DNMT1 enzyme.

    On the other hand, infection by the human papilloma virus (HPV) phenotype 16, enzyme 6 (PDB ID: 4XR8) has been correlated with a greatly increased risk of cervical cancer worldwide[12]. Based on variations in the nucleotide sequences of the virus genome, over 100 distinct varieties of the human papilloma virus (HPV) have been identified (e.g. type 1, 2 etc.). Genital warts can result from certain types 6 and 11 of sexually transmitted HPVs. Other HPV strains, still, that can infect the genitalia, do not show any symptoms of infection[8]. Persistent infection with a subset of approximately 13 so-called 'high-risk' sexually transmitted HPVs, including such as types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 different from the ones that cause warts may lead to the development of cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). These are precancerous lesions and can progress to invasive cancer. Almost all occurrences of cervical cancer have HPV infection as a required component[13]. Superfluous infection by HPV type 16 E6 (PDB ID: 4XR8) has been correlated with a greatly increased genital risk of precursor cervical cancer worldwide[11]. Scholars more defined in major biochemical and biological activities of HPV type 16 E6 (PDB ID: 4XR8) in high-risk HPV oncogenes and how they may work together in the development of cervical disease and cancer[13].

    One potential approach to treat cervical cancer is to inhibit the activity of the DNMT1 and HPV type 16 E6 enzymes specifically[1316]. Over 50% of clinical drug forms worldwide originate from plant compounds[17]. In the past, developing new drugs was a lengthy and costly process. However, with the emergence of bioinformatics, the use of computer-based tools and methods have become increasingly important in drug discovery. One such method is molecular docking and ADMET profiling which involves using the structure of a drug to screen for potential candidates. This approach is known as structure-based drug design and can save both time and resources during the research process[15]. Structural-based drug designing addresses ligand binding sites with a known protein structure[15]. Using free binding energies, a computational method known as docking examines a large number of molecules and suggests structural theories for impeding the target molecule[17]. Nowadays, due to increasing antibiotic resistance like bacteria, fungi, and cancer cells, natural products remain an important source for discovering antimicrobial compounds and novel drugs for anti-cancers like cervical cancers. Therefore, the purpose of this research is to assess the antimicrobial activity of extracts, molecular docking, ADMET profiling in anticancer properties of compounds isolated from Salvia rosmarinus, on a targeting DNMT1 and HPV type 16 E6 in human cervical cancer. In the present study, various solvent crude extracts obtained from Salvia rosmarinus were used for antimicrobial activity and the isolated compounds 1 and 2 were submitted for in silico study to target the DNMT1 and HPV type 16 E6 enzymes to inhibit the growth of human cervical cancer cells.

    Healthy Salvia rosmarinus leaves were collected in Bacho district, Southwest Showa, Oromia, Ethiopia, during the dry season of November 2022. The plant materials were authenticated by Melaku Wondafrish, Natural Science Department, Addis Ababa University and deposited with a voucher number 3/2-2/MD003-80/8060/15 in Addis Ababa University's National Herbarium.

    The most common organic solvent used in extractions of medicinal plants is 2.5 L of petroleum ether, chloroform/methanol (1:1), and methanol. The test culture medium for microbes was used and performed in sterile Petri dishes (100 mm diameter) containing sterile Muller–Hinton Agar medium (25 mL, pH 7) and Sabouraud Dextrose Agar (SDA) for bacteria and fungi, respectively. A sterile Whatman filter paper (No. 1) disc of 6 mm diameter was used to determine which antibiotics an infective organism is sensitive to prescribed by a minimum zone of inhibition (MZI). Ciprofloxacin antibiotic reference (manufactured by Wellona Pharma Ciprofloxacin tablet made in India) and Ketoconazole 2% (made in Bangladesh) were used as a positive controls for antibacterial and antifungal, respectively and Dimethyl sulfoxide (DMSO) 98.9% was used as a negative control for antimicrobial tests. In the present study, the height of the column was 650 mm and the width was 80 mm. Several studies by previous researchers showed the acceptable efficiency of column chromatography (up to 43.0% w/w recovery) in the fractionation and separation of phenolic compounds from plant samples[18]. In column chromatography, the ideal stationary phase used silica gel 60 (0.200 mm) particles. The 1H-NMR spectrums of the compounds were analyzed using a 600 MHz NMR machine and 150 MHz for 13C NMR. The compounds were dissolved in MeOD for compound 1 and in DMSO for compound 2 for NMR analysis. On the other hand, UV spectroscopy (made in China) used 570 nm ultraviolet light to determine the absorbency of flavonoids (mg·g−1) phytochemicals.

    The samples (extracts) were analyzed to detect the presence of certain chemical compounds such as alkaloids (tested using Wagner's reagents), saponins (tested using the froth test), steroids (tested with Liebermann Burchard's tests), terpenoids (tested with Lidaebermann Burchard's tests), quinones, and flavonoids (tested using Shinoda tests)[19].

    The leaves of Salvia rosmarinus (500 g) were successively extracted using maceration using petroleum ether, chloroform/methanol (1:1), and methanol, every one 2.5 L for 72 h to afford 3.6, 6, and 53 g crude extracts, respectively. The methanol/chloroform (1:1) extract (6 g) was loaded to silica gel (150 g) column chromatography using the increasing polarity of petroleum ether, methanol/chloroform (1:1) solvent system to afford 80 fractions (100 mL each). The fraction obtained from chloroform/methanol 1:1 (3:2) after repeated column chromatography yielded compound 1 (18 mg). Fractions 56-65, eluted with chloroform/methanol (1:1) were combined and purified with column chromatography to give compound 2 (10 mg).

    The microorganisms were obtained from the Ethiopia Biodiversity Institution (EBI). Two gram-positive bacteria namely Staphylococcus aureus serotype (ATCC 25923) and Streptococcus epidermidis (ATCC14990); and three gram-negative bacteria, namely Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 5702), and Klebsiella pneumonia (ATCC e13883) were inoculated overnight at 37 °C in Muller–Hinton Agar/MHA culture medium and two fungus strains of Candida albicans (ATCC 16404) and Aspergillus niger (ATCC 11414) were inoculated overnight at 27−30 °C in Sabouraud Dextrose Agar/SDA culture medium[20].

    The antibacterial and antifungal activities of different crude extracts obtained from Salvia rosmarinus plant leaves were evaluated by the disk diffusion method (in accordance with the 13th edition of the CLSI M02 document on hardydiagnostics.com/disk-diffusion). Briefly, the test was performed in sterile Petri dishes (100 mm diameter) containing solid and sterile Muller–Hinton Agar medium (25 mL, pH 7) and Sabouraud Dextrose Agar (SDA) for bacteria and fungi, respectively. The extracts were placed on the surface of the media that had previously been injected with a sterile microbial suspension (one microbe per petri dish) after being adsorbed on sterile paper discs (5 μL per Whatman disc of 6 mm diameter). To prevent test samples from eventually evaporating, all Petri dishes were sealed with sterile laboratory films. They were then incubated at 37 °C for 24 h, and the zone diameter of the inhibition was measured and represented in millimeters. Ciprofloxacin antibiotic reference (manufactured by Wellona Pharma Ciprofloxacin tablet, India) was used as a positive control and DMSO was used as a negative control for antibacterial activity test while Ketoconazole 2% (Bangladesh) was used as a positive control and 10 μL of 0.2% agar as a negative control for antifungal activity tests[20]. The term 'inhibitory concentration' refers to the minimum sample concentration required to kill 99.9% of the microorganisms present[21]. Three repetitions of the crude extract sample were used to precisely measure the inhibitory halo diameter (in mm), which was then expressed as mean ± standard deviation to assess the anti-microbial activity.

    Cervical cancer-causing protein was identified through relevant literature. The protein molecule structure of DNA (cytosine-5)-methyltransferase 1 (DNMT1) (PDB ID: 4WXX)[21] and HPV type 16 E6 (PDB ID: 4XR8)[21] - a protein known to cause cervical cancer - were downloaded from the Protein Data Bank[22]. The stability of the protein molecule was assessed using Rampage[23].

    Phytochemical constituents of Salvia rosmarinus plant leaves were used to select a source of secondary metabolites (ligands). Ligand molecules were obtained through plant extraction, and isolation, and realized with PubChem (https://pubchem.ncbi.nlm.nih.gov/). The ligands were downloaded in Silver diamine fluoride format (SDF) and then converted to PDB format using an online SMILES translator (https://cactus.nci.nih.gov/translate/). The downloaded files were in PDB format, which was utilized for running various tools and software[24].

    The Biovia Discovery Studio Visualizer software was used to analyze the protein molecule. The protein molecule was converted into PDB format and its hierarchy was analyzed by selecting ligands and water molecules. Both the protein molecule and the water molecules lost their attached ligands during the analysis. Finally, the protein's crystal structure was saved in a PDB file[25].

    PyRx software was utilized to screen secondary metabolites and identify those ligands with the lowest binding energy to the protein target. The ligands with the lowest binding energy were further screened for their drug-likeliness property through analysis. It is worth noting that PyRx runs on PDBQT format. To begin using PyRx, it needs to load a protein molecule. This molecule should be converted from PDB to the protein data bank, partial charge (Q), and Atom Type (PDBQT) format. Once the protein molecule is loaded, it can import ligands from a specific folder in Silver diamine fluoride format. The ligand energy was minimized and changed to PDBQT format. The protein was docked with the ligand and screened based on minimum binding energy (https://cactus.nci.nih.gov/translate/).

    The optimal ligand was selected for final docking using AutoDock Vina and Biovia by modifying the reference of Discovery Studio Client 2021 (https://cactus.nci.nih.gov/translate/).

    The protein target from the Protein Data Bank (PDB) was loaded onto the graphical interface of AutoDock Vina. To prepare the protein for docking, water molecules were removed, hydrogen polar atoms were added, and Kollman charges were assigned to the protein molecule. Ultimately, PDBQT format was used to store the protein. After being imported in PDB format, the Ligand molecule was transformed to PDBQT format. Next, a grid box was chosen to represent the docked region. The command prompt was used to run AutoDock Vina and the outcomes were examined (https://cactus.nci.nih.gov/translate/).

    Docking the ligand with the protein target DNMT1(PDB ID: 4WXX)[22] and HPV type 16 E6 (PDB ID: 4XR8)[21] enzymes were performed using Biovia Discovery Studio Client 2021 by loading the protein target first followed by the ligand in PDB format. The charges were attached to the protein molecule, and the energy was minimized for the ligands. Both the protein and ligand molecules were prepared for docking. Once the docking process was complete, the results were analyzed based on several parameters, including absolute energy, clean energy, conf number, mol number, relative energy, and pose number. The interaction between the protein and ligand was analyzed using structure visualization tools, such as Biovia Discovery Studio Visualizer and PyMol (https://cactus.nci.nih.gov/translate/).

    The process of visualizing the structure was carried out using the PyMol tool. PyMol is a freely available software. Firstly, the protein molecule in PDBQT form was loaded on the PyMol graphical screen. Then, the output PDBQT file was added. The docked structure was visualized and the 'molecule' option was changed to 'molecular surface' under the 'shown as' menu (https://cactus.nci.nih.gov/translate/).

    Drug likeliness properties of the screened ligands were evaluated using the SwissADME online server. SMILE notations were obtained from PubChem and submitted to the SwissADME web server for analysis. The drugs were subjected to Lipinski's rule of five[20] for analysis. Lipinski's rules of five were selected for final docking through AutoDock Vina and Biovia Discovery Studio Client 2021. Ligands 1 and 2 were analyzed using Lipinski's rule of five for docking with AutoDock Vina and Biovia Discovery Studio Client 2021.

    The antimicrobial analysis data generated by triplicate measurements reported as mean ± standard deviation, and a bar graph also generated by GraphPad Prism version 8.0.1 (244) for Windows were used to perform the analysis. GraphPad Prism was used and combined with scientific graphing, comprehensive bar graph fitting (nonlinear regression), understandable statistics, and data organization. Prism allows the performance and modification of basic statistical tests commonly used and determined through the statistical applications in microbiology labs (https://graphpad-prism.software.informer.com/8.0/).

    Phytochemical screening of the different extracts for the presence (+) and absence (−) of alkaloids, steroids, glycosides, coumarins, terpenoids, flavonoids, carbohydrates, tannins, and saponins were done. The present study showed that alkaloids, terpenoids, flavonoids, and tannins tests in S. rosmarinus leaves of petroleum ether, chloroform/methanol (1:1), and methanol extracts were high whereas glycoside, coumarins, and carbohydrates had a moderate presence. The extract of S. rosmarinus leaves contain commonly bioactive constituents such as alkaloids, steroids, terpenoids, flavonoids, tannins, and saponins. These bioactive chemicals have active medicinal properties. Phytochemical compounds found in S. rosmarinus leaves have the potential to treat cancer cells and pathogens. The study also found that these flavonoids are related to natural phenolic compounds with anticancer and antimicrobial properties in the human diet (Table 1).

    Table 1.  Phytochemical screening tests result of petroleum ether, chloroform/methanol (1:1) and methanol extracts of Salvia rosmarinus leaves.
    Botanical name Phytochemicals Phytochemical screening tests Different extracts
    Petroleum ether Chloroform/methanol (1:1) Mehanol
    Salvia rosmarinus Alkaloids Wagner's test ++ ++ ++
    Steroids Libermann Burchard test ++ + ++
    Glycoside Keller-Killiani test +
    Coumarins Appirade test + +
    Terpenoids Libermann Burchard test ++ ++ ++
    Flavonoids Shinoda test ++ ++ ++
    Carbohydrate Fehling's test ++ ++
    Tannins Lead acetate test ++ ++ ++
    Saponins Foam test + + +
    + indicates moderate presence, ++ indicates highly present, − indicates absence.
     | Show Table
    DownLoad: CSV

    Two compounds were isolated and characterized using NMR spectroscopic methods (Fig. 1 & Supplementary Fig. S1ac). Compound 1 (10 mg) was isolated as yellow crystals from the methanol/chloroform (1:1) leaf extract of Salvia rosmarinus. The TLC profile showed a spot at Rf 0.42 with methanol/chloroform (3:2) as a mobile phase. The 1H-NMR spectrum (600 MHz, MeOD, Table 2, Supplementary Fig. S1a) of compound 1 showed the presence of one olefinic proton signal at δ 5.3 (t, J = 3.7 Hz, 1H), two deshielded protons at δ 4.7 (m, 1H), and 4.1 (m, 1H) associated with the C-30 exocyclic methylene group, and one O-bearing methine proton at δH 3.2 (m, 1H), and six methyl protons at δ 1.14 (s, 3H), 1.03 (d, J = 6.3 Hz, 3H), 1.00 (s, 3H), 0.98 (s, 3H), 0.87 (s, 3H), and 0.80 (s, 3H). A proton signal at δ 2.22 (d, J = 13.5 Hz, 1H) was attributed to methine proton for H-18. Other proton signals integrate for 20 protons were observed in the range δ 2.2 to 1.2. The proton decoupled 13C-NMR and DEPT-135 spectra (151 MHz, MeOD, Supplementary Fig. S1b & c) of compound 1 revealed the presence of 30 well-resolved carbon signals, suggesting a triterpene skeleton. The analysis of the 13C NMR spectrum displayed signals corresponding to six methyl, nine methylene, seven methine, and eight quaternary carbons. Among them, the signal observed at δ 125.5 (C-12) belongs to olefinic carbons. The methylene carbon showed signals at δC 39.9, 28.5, 18.1, 36.7, 23.9, 30.4, 26.5, 32.9, and 38.6. The quaternary carbons showed a signal at δC 39.4, 41.9, 38.4, 138.2, 41.8, and 47.8. The signals of exocyclic methylene carbon signals appeared at δ 153.1 and 103.9. The spectrum also showed sp3 oxygenated methine carbon at δ 78.3 and carboxyl carbon at δ 180.2. The spectrum revealed signals due to methyl groups at δC 27.4, 16.3, 15.0, 20.2, 22.7, and 16.4. The remaining carbon signals for aliphatic methines were shown at δC 55.3, 55.2, 53.0, and 37.1. The NMR spectral data of compound 1 is in good agreement with data reported for micromeric acid, previously reported from the same species by Abdel-Monem et al.[26]. (Fig. 1, Table 2).

    Figure 1.  Structure of isolated compounds from the leaves of Salvia rosmarinus.
    Table 2.  Comparison of the 13C-NMR spectral data of compound 1 and micromeric acid (MeOD, δ in ppm).
    Position NMR data of compound 1 Abdel-Monem
    et al.[26]
    1H-NMR 13C-NMR 13C-NMR
    1 38.60 39.9
    2 27.8 28.5
    3 3.2 (m, 1H) 78.3 80.3
    4 39.4 39.9
    5 55.3 56.7
    6 18.1 18.3
    7 36.7 34.2
    8 41.9 40.7
    9 53 48.8
    10 38.4 38.2
    11 23.9 24.6
    12 5.3 (t, J = 3.7 Hz, 1H) 125.5 127.7
    13 138.2 138
    14 41.8 43.3
    15 30.4 29.1
    16 26.5 25.6
    17 47.8 48
    18 δ 2.22 (d, J = 13.5 Hz, 1H) 55.2 56.1
    19 37.1 38.7
    20 153.1 152.8
    21 32.9 33.5
    22 39.0 40.1
    23 27.4 29.4
    24 16.3 16.9
    25 15.0 16.6
    26 20.2 18.3
    27 22.7 24.6
    28 180.2 177.8
    29 16.4 17.3
    30 4.7 (m, 1H), and 4.1 (m, 1H) 103.9 106.5
     | Show Table
    DownLoad: CSV

    Compound 2 (18 mg) was obtained as a white amorphous isolated from 40% methanol/chloroform (1:1) in petroleum ether fraction with an Rf value of 0.49. The 1H NMR (600 MHz, DMSO, Supplementary Fig. S2a) spectral-data showed two doublets at 7.79 (d, J = 8.7 Hz, 2H), and 6.90 (d, J = 8.7 Hz, 2H) which are evident for the presence of 1,4-disubstituted aromatic group. The oxygenated methylene and terminal methyl protons were shown at δ 4.25 (q, J = 7.1 Hz, 2H) and 1.29 (t, J = 7.1 Hz, 3H), respectively. The13C-NMR spectrum, with the aid of DEPT-135 (151 MHz, DMSO, Table 3, Supplementary Fig. S2b & c) spectra of compound 2 confirmed the presence of well-resolved seven carbon peaks corresponding to nine carbons including threee quaternary carbons, one oxygenated methylene carbon, one terminal methyl carbon, and two symmetrical aromatic methine carbons. The presence of quaternary carbon signals was shown at δ 120.9 (C-1), 148.2 (C-4), and ester carbonyl at δ 166.0 (C-7). The symmetry aromatic carbons signal was observed at δ 131.4 (C-2, 6), and 116.8 (C-3, 5). The oxygenated methylene and terminal methyl carbons appeared at δC 60.4 (C-8) and 14.7 (C-9), respectively. The spectral results provided above were in good agreement with those for benzocaine in the study by Alotaibi et al.[27]. Accordingly, compound 2 was elucidated to be benzocaine (4-Aminobenzoic acid-ethyl ester) (Table 3, Fig. 1, Supplementary Fig. S2ac), this compound has never been reported before from the leaves of Salvia rosmarinus.

    Table 3.  Comparison of the 1H-NMR, and 13C-NMR spectral data of compound 2 and benzocaine (DMSO, δ in ppm).
    Position NMR data of compound 2 Alotaibi et al.[27]
    1H-NMR 13C-NMR 1H-NMR 13C-NMR
    1 120.9 119
    2 7.79 (d, J = 8.7 Hz, 2H) 131.4 7.86 (d, J = 7.6 Hz) 132
    3 6.90 (d, J = 8.7 Hz, 2H) 116.8 6.83 (d, J = 7.6 Hz) 114
    4 148.2 151
    5 6.90 (d, J = 8.7 Hz, 2H) 116.8 6.83 (d, J = 7.6 Hz) 114
    6 7.79 (d, J = 8.7 Hz, 2H) 131.4 7.86 (d, J = 7.6 Hz) 132
    7 166.0 169
    8 4.3 (q, J = 7.1 Hz, 2H) 60.4 4.3 (q, J = 7.0 Hz) 61
    9 1.3 (t, J = 7.1 Hz, 3H) 14.7 1.36 (t, J = 7.0 Hz) 15
     | Show Table
    DownLoad: CSV

    The extracts and isolated compounds from Salvia rosmarinus were evaluated in vitro against microbes from gram-positive bacteria (S. aureus and S. epidermidis), gram-negative bacteria (E. coli, P. aeruginosa, and K. pneumoniae) and fungi (C. albicans and A. Niger) (Table 4). The petroleum ether extracts exhibited significant activity against all the present study-tested microbes at 100 μg·mL−1, resulting in an inhibition zone ranging from 7 to 21 mm. Chloroform/methanol (1:1) and methanol extracts demonstrated significant activity against all the present study-tested microbes at 100 μg·mL−1 exhibiting inhibition zones from 6 to 14 mm and 6 to 13 mm, respectively (Table 4). The chloroform/methanol (1:1) extracts were significantly active against bacteria of E. coli and K. pneumonia, and A. Niger fungi at 100 μg·mL−1. On the other hand, chloroform/methanol (1:1) extracts were significantly inactive against the S. rosmarinus and P. aeruginosa of bacteria and C. albicans of fungi, and again chloroform/methanol (1:1) extracts overall significantly active produced an inhibition zone of 12 to 14 mm (Table 4). Methanol extracts exhibited significant activity against S. aureus, E. coli bacteria, and A. Niger fungi at 100 μg·mL−1. The inhibition zone was recorded to be 11 to 13 mm. However, methanol extracts exhibited significant inactivity against K. pneumoniae (Table 4). The overall result of our studies shows that Salvia rosmarinus was extracted and evaluated in vitro, exhibiting significant antibacterial and antifungal activity, with inhibition zones recorded between 6 to 21 mm for bacteria and 5 to 21 mm for fungi. In our study, the positive control for ciprofloxacin exhibited antibacterial activity measured at 21.33 ± 1.15 mm, 15.00 ± 0.00 mm, and 14.20 ± 0.50 mm for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. Similarly, the positive control for ketoconazole demonstrated antifungal activity of 22.00 ± 1.00 mm, 13.67 ± 0.58 mm, and 15.00 ± 0.58 mm for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. Additionally, our findings indicated that the mean values of flavonoids (mg/g) tested were 92.2%, 90.4%, and 94.0% for petroleum ether, chloroform/methanol (1:1), and methanol extracts, respectively. This suggests that the groups of phenolic compounds evaluated play a significant role in antimicrobial activities, particularly against antibiotic-resistant strains.

    Table 4.  Comparison of mean zone of inhibition (MZI) leaf extracts of Salvia rosmarinus.
    Type of specimen, and standard antibiotics for
    each sample
    Concentration (μg·mL−1) of extract
    in 99.8% DMSO
    Average values of the zone of inhibition (mm)
    Gram-positive (+) bacteria Gram-negative (−) bacteria Fungai
    S. aurous S. epidermidis E. coli P. aeruginosa K. pneumoniae C. albicans A. niger
    Petroleum ether extracts
    S. rosmarinus 50 18.50 ± 0.50 15.33 ± 0.58 0.00 ± 0.00 0.00 ± 0.00 10.00 ± 0.00 15.93 ± 0.12 4.47 ± 0.50
    75 19.87 ± 0.06 17.00 ± 0.00 9.33 ± 0.29 10.53 ± 0.50 10.93 ± 0.12 18.87 ± 0.23 5.47 ± 0.50
    100 21.37 ± 0.78 17.50 ± 0.50 11.47 ± 0.50 13.17 ± 0.29 12.43 ± 0.51 20.83 ± 0.76 6.70 ± 0.10
    Standard antibiotics Cipro. 21.33 ± 1.15 18.33 ± 0.58 9.33 ± 0.58 12.30 ± 0.52 15.00 ± 0.00
    Ketocon. 22.00 ± 1.00 10.67 ± 0.58
    Chloroform/methanol (1:1) extracts
    50 5.47 ± 0.42 0.00 ± 0.00 10.33 ± 0.00 0.00 ± 0.00 9.70 ± 0.00 0.00 ± 0.12 8.47 ± 0.50
    S. rosmarinus
    75 5.93 ± 0.06 0.00 ± 0.00 11.33 ± 0.29 0.00 ± 0.50 12.50 ± 0.12 0.00 ± 0.23 10.67 ± 0.50
    100 6.47 ± 0.06 0.00 ± 0.00 14.17 ± 0.50 7.33 ± 0.29 14.17 ± 0.51 0.00 ± 0.76 12.67 ± 0.10
    Standard antibiotics Cipro. 15.00 ± 0.00 11.00 ± 1.00 11.33 ± 0.58 10.00 ± 0.52 12.67 ± 0.00
    Ketocon. 7.00 ± 1.00 13.67 ± 0.58
    Methanol extracts
    50 9.17 ± 0.29 5.50 ± 0.50 0.00 ± 0.00 7.50 ± 0.00 0.00 ± 0.00 6.57 ± 0.12 0.00 ± 0.50
    S. rosmarinus
    75 9.90 ± 0.10 6.93 ± 0.12 9.33 ± 0.29 8.50 ± 0.50 0.00 ± 0.00 8.70 ± 0.23 0.00 ± 0.50
    100 11.63 ± 0.55 7.97 ± 0.06 11.47 ± 0.50 9.90 ± 0.10 0.00 ± 0.00 10.83 ± 0.76 13.13 ± 0.10
    Standard antibiotics Cipro. 13.00 ± 0.00 11.50 ± 0.50 14.20 ± 0.58 13.33 ± 0.29 10.00 ± 0.00
    Ketocon. 12.00 ± 1.00 15.00 ± 0.58
    Mean values of flavonoids (mg·g−1) by 570 nm
    S. rosmarinus
    Petroleum ether extracts Chloroform/methanol (1:1) extracts Methanol extracts
    50 0.736 0.797 0.862
    75 0.902 0.881 0.890
    100 0.922 0.904 0.940
    Samples: Antibiotics: Cipro., Ciprofloxacin; Ketocon., ketoconazole (Nizoral); DMSO 99.8%, Dimethyl sulfoxide.
     | Show Table
    DownLoad: CSV

    Determining the three solvent extracts in S. rosmarinus plants resulted in relatively high comparable with positive (+) control. Especially, the S. rosmarinus petroleum ether leaf extracts against drug resistance human pathogenic bacteria S. aureus, S. epidermidis, E. coli, P. aeruginosa, and K. pneumoniae were minimum zone of inhibition (MZI) recorded that 21.37 ± 0.78, 17.50 ± 0.50, 11.47 ± 0.50, 13.17 ± 0.29, and 12.43 ± 0.51 mm, respectively and against human pathogenic fungi C. albicans and A. niger were minimum zone of inhibition (MZI) recorded that 20.83 ± 0.76 and 6.70 ± 0.10 mm, respectively which was used from bacteria against S. aureus MZI recorded that 21.37 ± 0.78 mm higher than the positive control (21.33 ± 1.15 mm). The S. rosmarinus of chloroform/methanol (1:1) extracts were found to be against E. coli (14.17 ± 0.50 mm) and K. pneumoniae (14.17 ± 0.51 mm) higher than the positive control 11.33 ± 0.58 and 12.67 ± 0.00 mm, respectively. The methanol extracts of leaves in the present study plants were found to have overall MZI recorded less than the positive control. The Salvia rosmarinus crude extracts showed better antifungal activities than the gram-negative (−) bacteria (Table 4, Fig 2, Supplementary Fig. S3). Therefore, the three extracts, using various solvents of different polarity indexes, have been attributed to specific biological activities. For example, the antimicrobial activities of Salvia rosmarinus extracts may be due to the presence of alkaloids, terpenoids, flavonoids, tannins, and saponins in natural products (Table 1).

    Figure 2.  Microbes' resistance with drugs relative to standard antibiotics in extracts of Salvia rosmarinus. The figures represent understudy of three extracts derived from Salvia rosmarinus. (a) Petroleum ether, (b) chloroform/methanol (1:1), and (c) methanol extracts tested in Salvia rosmarinus.

    Compounds 1 and 2 were isolated from chloroform/methanol (1:1) extract of Salvia rosmarinus (Fig. 1, Tables 2 & 3). The plant extract exhibited highest antibacterial results recorded a mean inhibition with diameters of 21 and 14 mm at a concentration of 100 mg·mL−1 against S. aureus and E. coli/K. pneumoniae, respectively. After testing, overall it was found that the highly active petroleum ether extract of Salvia rosmarinus was able to inhibit the growth of S. aureus and C. albicans, with inhibition zones of 21 and 20 mm, respectively. The petroleum ether extracts showed good efficacy against all tested microbes, particularly gram-positive bacteria and fungi (Table 4). This is noteworthy because gram-negative bacteria generally exhibit greater resistance to antimicrobial agents. Petroleum ether and chloroform/methanol (1:1) extracts of the leaves were used at a concentration of 100 mg·mL−1, resulting in impressive inhibition zone diameters of 11 and 14 mm for E. coli, 13 and 7 mm for P. aeruginosa, and 12 and 14 mm for K. pneumoniae, respectively.

    The present study found that at a concentration of 50 μg·mL−1, petroleum ether, chloroform/methanol (1:1), and MeOH extracts did not display any significant inhibition zone effects against the tested microbes. This implies that the samples have a dose-dependent inhibitory effect on the pathogens. The leaves of Salvia rosmarinus have been found to possess remarkable antimicrobial properties against gram-negative bacteria in different extracts such as E. coli, P. aeruginosa, and K. pneumoniae with 14.17 ± 0.50 in chloroform/methanol (1:1), 13.17 ± 0.29 in petroleum ether and 14.17 ± 0.51 in chloroform/methanol (1:1), respectively. However, in the present study, Salvia rosmarinus was found to possess remarkable high zones of inhibition with diameters of 21.37 ± 0.78 and 17.50 ± 0.50 mm antimicrobial properties against S. aureus, and S. epidermidis of gram-positive bacteria, respectively (Supplementary Fig. S3). The results are summarized in Fig. 2ac.

    The crystal structure of human DNMT1 (351-1600), classification transferase, resolution: 2.62 Å, PDB ID: 4WXX. Active site dimensions were set as grid size of center X = −12.800500 Å, center Y = 34.654981 Å, center Z = −24.870231 Å (XYZ axis) and radius 59.081291. A study was conducted to investigate the binding interaction of the isolated compounds 1 and 2 of the leaves of Salvia rosmarinus with the binding sites of the DNMT1 enzyme in human cervical cancer (PDB ID: 4WXX), using molecular docking analysis.

    The study also compared the results with those of standard anti-cancer agents Jaceosidin (Table 5 & Fig. 3). The compounds isolated had a final fixing energy extending from −5.3 to −8.4 kcal·mol−1, as shown in Table 4. It was compared to jaceosidin (–7.8 kcal·mol−1). The results of the molecular docking analysis showed that, compound 1 (−8.4 kcal·mol−1) showed the highest binding energy values compared with the standard drugs jaceosidin (–7.8 kcal·mol−1). Compound 2 has shown lower docking affinity (–5.3 kcal·mol−1) but good matching amino acid residue interactions compared to jaceosidin. After analyzing the results, it was found that the isolated compounds had similar residual interactions and docking scores with jaceosidin.

    Table 5.  Molecular docking results of ligand compounds 1 and 2 against DNMT1 enzyme (PDB ID: 4WXX).
    Ligands Binding affinity

    ( kcal·mol−1)
    H-bond Residual interactions
    Hydrophobic/electrostatic Van der Waals
    1 −8.4 ARG778 (2.85249), ARG778 (2.97417), VAL894 (2.42832) Lys-889, Pro-879, Tyr-865, His-795, Cys-893, Gly-760, Val-759, Phe-892, Phe-890, Pro-884, Lys-749
    2 −5.3 ARG596 (2.73996), ALA597 (1.84126), ILE422 (2.99493), THR424 (2.1965), ILE422 (2.93653) Electrostatic Pi-Cation-ARG595 (3.56619), Hydrophobic Alkyl-ARG595 (4.15839), Hydrophobic Pi-Alkyl-ARG595 (5.14967) Asp-423, Glu-428, Gly-425, Ile-427, Trp-464, Phe-556, Gln-560, Gln-594, Glu-559, Gln-598, Ser-563
    Jaceosidin −7.8 ASP571 (2.93566), GLN573 (2.02126), GLU562 (2.42376), GLN573 (3.49555), GLU562 (3.46629) Hydrophobic Alkyl-PRO574 (4.59409), Hydrophobic Alkyl-ARG690 (5.09748), Hydrophobic Pi-Alkyl-PHE576 (5.1314), Hydrophobic Pi-Alkyl-PRO574 (4.97072), Hydrophobic Pi-Alkyl-ARG690 (5.07356) Glu-698, Cys-691, Ala-695, Pro-692, Val-658, Glu-566, Asp-565
     | Show Table
    DownLoad: CSV
    Figure 3.  The 2D and 3D binding interactions of compounds against DNMT1 enzyme (PDB ID: 4WXX). The 2D and 3D binding interactions of compound 1 and 2 represent against DNMT1 enzyme, and jaceosidin (standard) against DNMT1 enzyme.

    Hence, compound 1 might have potential anti-cancer agents. However, anti-cancer in vitro analysis has not yet been performed. Promising in silico results indicate that further research could be beneficial. The 2D and 3D binding interactions of compounds 1 and 2 against human cervical cancer of DNMT1 enzyme (PDB ID: 4WXX) are presented in Fig. 3. The binding interactions between the DNMT1 enzyme (PDB ID: 4WXX), and compound 1 (Fig. 3) and compound 2 (Fig. 3) were displayed in 3D. Compounds and amino acids are connected by hydrogen bonds (green dash lines) and hydrophobic interactions (non-green lines).

    Crystal structure of the HPV16 E6/E6AP/p53 ternary complex at 2.25 Å resolution, classification viral protein, PDB ID: 4XR8. Active site dimensions were set as grid size of center X = −43.202782 Å, center Y = −39.085513 Å, center Z = −29.194115 Å (XYZ axis), R-value observed 0.196, and Radius 65.584122. A study was conducted to investigate the binding interaction of the isolated compounds 1 and 2 of the leaves of Salvia rosmarinus with the binding sites of the enzyme of human papilloma virus (HPV) type 16 E6 (PDB ID: 4XR8), using molecular docking analysis software. The study also compared the results with those of standard anti-cancer agents jaceosidin (Table 6 & Fig. 4). The compounds isolated had a bottom most fixing energy extending from −6.3 to −10.1 kcal·mol−1, as shown in Table 6. It was compared to jaceosidin (–8.8 kcal·mol−1). The results of the molecular docking analysis showed that, compound 1 (−10.1 kcal·mol−1) showed the highest binding energy values compared with the standard drugs jaceosidin (–8.8 kcal·mol−1). Compound 2 has shown lower docking affinity (–6.3 kcal·mol−1) but good matching amino acid residue interactions compared to jaceosidin. After analyzing the results, it was found that the isolated compounds had similar residual interactions and docking scores with jaceosidin.

    Table 6.  Molecular docking results of ligand compounds 1 and 2 against HPV type 16 E6 (PDB ID: 4XR8).
    Ligands Binding affinity
    (kcal·mol−1)
    H-bond Residual interactions
    Hydrophobic/electrostatic Van der Waals
    1 −10.1 ASN101 (2.25622), ASP228 (2.88341) Asp-148, Lys-176, Lys-180, Asp-178, Ile-179, Tyr-177, Ile-334, Glu-382, Gln-336, Pro-335, Gln-73, Arg-383, Tyr-100
    2 −6.5 TRP63 (1.90011), ARG67 (2.16075), ARG67 (2.8181) Hydrophobic Pi-Sigma-TRP341 (3.76182), Hydrophobic Pi-Pi Stacked-TYR156 (4.36581), Hydrophobic Pi-Pi T-shaped-TRP63 (5.16561), Hydrophobic Pi-Pi T-shaped-TRP63 (5.44632), Hydrophobic Alkyl-PRO155 (4.34691), Hydrophobic Pi-Alkyl-TRP341 (4.11391), Hydrophobic Pi-Alkyl-ALA64 (4.61525) Glu-154, Arg-345, Asp-66, Met-331, Glu-112, Lys-16, Trp-231
    Jaceosidin −8.8 ARG146 (2.06941), GLY70 (3.49991), GLN73 (3.38801) Electrostatic Pi-Cation-ARG67 (3.93442), Hydrophobic Pi-Alkyl-PRO49 (5.40012) Tyr-342, Tyr-79, Ser-338, Arg-129, Pro-335, Leu-76, Tyr-81, Ser-74, Tyr-71, Ser-80, Glu-46
     | Show Table
    DownLoad: CSV
    Figure 4.  The 2D and 3D binding interactions of compounds against HPV type 16 E6 (PDB ID: 4XR8). The 2D and 3D binding interactions of compound 1 and 2 represent against HPV type 16 E6 enzyme, and jaceosidin (standard) against HPV type 16 E6 enzyme.

    Hence, compounds 1 and 2 might have potential anti-cancer agents of HPV as good inhibitors. However, anti-cancer in vitro analysis has not been performed yet on HPV that causes cervical cancer agents. Promising in silico results indicate that further research could be beneficial. The 2D and 3D binding interactions of compounds 1 and 2 against human papilloma virus (HPV) type 16 E6 enzyme (PDB ID: 4XR8) are presented in Fig. 4. The binding interactions between the HPV type 16 E6 enzyme (PDB ID: 4XR8) and compound 1 (Fig. 4) and compound 2 (Fig. 4) were displayed in 3D. Compounds and amino acids are connected by hydrogen bonds (magenta lines) and hydrophobic interactions (non-green lines).

    In silico bioactivities of a drug, including drug-likeness and toxicity, predict its oral activity based on the document of Lipinski's Rule[25] was stated and the results of the current study showed that the compounds displayed conform to Lipinski's rule of five (Table 7). Therefore, both compounds 1 and 2 should undergo further investigation as potential anti-cancer agents. Table 8 shows the acute toxicity predictions, such as LD50 values and toxicity class classification (ranging from 1 for toxic, to 6 for non-toxic), for each ligand, revealing that none of them were acutely toxic. Furthermore, they were found to be similar to standard drugs. Isolated compound 1 has shown toxicity class classification 4 (harmful if swallowed), while 2 showed even better toxicity prediction giving results of endpoints such as hepatotoxicity, mutagenicity, cytotoxicity, and irritant (Table 8). All the isolated compounds were predicted to be non-hepatotoxic, non-irritant, and non-cytotoxic. However, compound 1 has shown carcinogenicity and immunotoxicity (Table 9). Hence, based on ADMET prediction analysis, none of the compounds have shown acute toxicity, so they might be proven as good drug candidates.

    Table 7.  Drug-likeness predictions of compounds computed by Swiss ADME.
    Ligands Formula Mol. Wt. (g·mol−1) NRB NHA NHD TPSA (A°2) Log P (iLOGP) Log S (ESOL) Lipinski's rule of five
    1 C30H46O3 454.68 1 3 2 57.53 3.56 −6.21 1
    2 C 9H11NO2 165.19 3 2 1 52.32 1.89 −2.21 0
    Jaceosidin C17H14O7 330.3 3 7 3 105 1.7 1 0
    NHD, number of hydrogen donors; NHA, number of hydrogen acceptors; NRB, number of rotatable bonds; TPSA, total polar surface area; and log P, octanol-water partition coefficients; Log S, turbid metric of solubility.
     | Show Table
    DownLoad: CSV
    Table 8.  Pre ADMET predictions of compounds, computed by Swiss ADME.
    Ligands Formula Skin permeation value
    (logKp - cm·s−1)
    GI
    absorption
    Inhibitor interaction
    BBB permeability Pgp substrate CYP1A2 inhibitor CYP2C19 inhibitor CYP2C9 inhibitor CYP2D6 inhibitor
    1 C30H46O3 −4.44 Low No No No No No No
    2 C 9H11NO2 −5.99 High Yes No No No No No
    Jaceosidin C17H14O7 −6.13 High No No Yes No Yes Yes
    GI, gastrointestinal; BBB, blood brain barrier; Pgp, P-glycoprotein; and CYP, cytochrome-P.
     | Show Table
    DownLoad: CSV
    Table 9.  Toxicity prediction of compounds, computed by ProTox-II and OSIRIS property explorer.
    Ligands Formula LD50
    (mg·kg−1)
    Toxicity
    class
    Organ toxicity
    Hepatotoxicity Carcinogenicity Immunotoxicity Mutagenicity Cytotoxicity Irritant
    1 C30H46O3 2,000 4 Inactive Active Active Inactive Inactive Inactive
    2 C 9H11NO2 NA NA Inactive Inactive Inactive Inactive Inactive Inactive
    Jaceosidin C17H14O7 69 3 Inactive Inactive Inactive Inactive Inactive Inactive
    NA, not available.
     | Show Table
    DownLoad: CSV

    Rosemary is an evergreen perennial plant that belongs to the family Lamiaceae, previously known as Rosmarinus officinalis. Recently, the genus Rosmarinus was combined with the genus Salvia in a phylogenetic study and became known as Salvia rosmarinus[28,29] and it has been used since ancient times for various medicinal, culinary, and ornamental purposes. In the field of food science, rosemary is well known as its essential oil is used as a food preservative, thanks to its antimicrobial and antioxidant properties, rosemary has many other food applications such as cooking, medicinal, and pharmacology uses[30]. According to the study, certain phytochemical compounds found in Salvia rosmarinus leaves have the potential to halt the growth of cancer cells, and pathogens or even kill them[31]. In literature, alkaloids are found mostly in fungi and are known for their strong antimicrobial properties, which make them valuable in traditional medicine[32,33]. However, in the present study, S. rosmarinus species have been shown to possess alkaloids. Most alkaloids have a bitter taste and are used to protect against antimalarial, antiasthma, anticancer, antiarrhythmic, analgesic, and antibacterial[33] also some alkaloids containing nitrogen such as vincristine, are used to treat cancer.

    Steroids occur naturally in the human body. They are hormones that help regulate our body's reaction to infection or injury, the speed of metabolism, and more. On the other hand, steroids are reported to have various biological activities such as chronic obstructive pulmonary disease (COPD), multiple sclerosis, and imitate male sex hormones[34]. It is a natural steroid compound occurring both in plants and animals[35]. Thus, were found in the present study. Terpenoids are derived from mevalonic acid (MVA) which is composed of a plurality of isoprene (C5) structural units. Terpenoids, like mono-terpenes and sesquiterpenes, are widely found in nature and more than 50,000 have been found in plants that reduce tumors and cancers. Many volatile terpenoids, such as menthol and perillyl alcohol, are used as raw materials for spices, flavorings, and cosmetics[36]. In the present study, high levels of these compounds were found in Salvia rosmarinus leaves.

    Flavonoids are a class of phenolic compounds commonly found in fruits and vegetables and are considered excellent antioxidants[37]. Similarly, the results of this study revealed that S. rosmarinus contain flavonoids. According to the literature, these flavonoids, terpenoids, and steroid activities include anti-diabetic, anti-inflammatory, anti-cancer, anti-bacterial, hepatic-protective, and antioxidant effects[36]. Tannins are commonly found in most terrestrial plants[38] and have the potential to treat cancer, and HIV/AIDS as well as to treat inflamed or ulcerated tissues. Similarly, in the present study, tannins were highly found in the presented plant. On the other hand, due to a sudden rise in the number of contagious diseases and the development of antimicrobial resistance against current drugs, drug development studies are vital to discovering novel medicinal compounds[30] and add to these cancer is a complex multi-gene disease[39] as in various cervical cancer repressor genes[11] that by proteins turn off or reduce gene expression from the affected gene to cause cervical cancer by regulating transcription and expression through promoter hypermethylation (DNMT1), leading to precursor lesions during cervical development and malignant transformation.

    In a previous study[40], a good antibacterial result was recorded at a median concentration (65 μg·mL−1). Methanol extract showed a maximum and minimum zone antibacterial result against negative bacteria E. coli 14 + 0.71 and most of the petroleum ether tests show null zone of inhibition. However, in the present study at a concentration of 100 μg·mL−1, the methanol extract demonstrated both maximum and minimum antibacterial zones against E. coli 11.47 ± 0.50. Conversely, the test conducted with petroleum ether exhibited a good zone of inhibition by increasing concentration. Further research may be necessary to determine the optimal concentration for this extract to maximize its efficacy. The results obtained in gram-negative bacteria such as E. coli, P. aeruginosa, and K. pneumoniae are consistent with previous research findings[41]. However, in the present study, Salvia rosmarinus has been found to possess high zones of inhibition with diameters of 21.37 ± 0.78 and 17.50 ± 0.50 mm antimicrobial properties against S. aureus, and S.epidermidis of gram-positive bacteria, respectively (Table 4 & Fig. 2, Supplementary Fig. S3). According to a previous study[42], the ethanolic leaf extract of Salvia rosmarinus did exhibit activity against C. albicans strains. In the present study, the antifungal activity of petroleum ether extracts from Salvia rosmarinus were evaluated against two human pathogenic fungi, namely C. albicans and A. niger. The findings showed that at a concentration of 100 μg·mL−1, the extracts were able to inhibit the growth of C. albicans 20.83 ± 0.76 resulting in a minimum zone of inhibition.

    Antimicrobial agents can be divided into groups based on the mechanism of antimicrobial activity. The main groups are: agents that inhibit cell wall synthesis, depolarize the cell membrane, inhibit protein synthesis, inhibit nucleic acid synthesis, and inhibit metabolic pathways in bacteria. On the other hand, antimicrobial resistance mechanisms fall into four main categories: limiting the uptake of a drug; modifying a drug target; inactivating a drug; and active drug efflux. Because of differences in structure, etc., there is a variation in the types of mechanisms used by gram-negative bacteria vs gram-positive bacteria. Gram-negative bacteria make use of all four main mechanisms, whereas gram-positive bacteria less commonly use limiting the uptake of a drug[43]. The present findings showed similar activity in chloroform/methanol (1:1) and methanol extracts of leaves of Salvia rosmarinus than gram-negative bacteria like P. aeruginosa and Klebsiella pneumoniae. However, Staphylococcus epidermidis of gram-positive bacteria under chloroform/methanol (1:1) extracts have similarly shown antimicrobial résistance. This occurred due to intrinsic resistance that may make use of limiting uptake, drug inactivation, and drug efflux that need further study. The structure of the cell wall thickness and thinners of gram-negative and gram-positive bacteria cells, respectively when exposed to an antimicrobial agent, there happen two main scenarios may occur regarding resistance and persistence. In the first scenario, resistant cells survive after non-resistant ones are killed. When these resistant cells regrow, the culture consists entirely of resistant bacteria. In the second scenario, dormant persistent cells survive. While the non-persistent cells are killed, the persistent cells remain. When regrown, any active cells from this group will still be susceptible to the antimicrobial agent.

    Ferreira et al.[44] explained that with molecular docking, the interaction energy of small molecular weight compounds with macromolecules such as target protein (enzymes), and hydrophobic interactions and hydrogen bonds at the atomic level can be calculated as energy. Several studies have been conducted showing natural products such as epigallocatechin-3-gallate-3-gallate (EGCG), curcumin, and genistein can be used as an inhibitor of DNMT1[4547] . In the literature micromenic (1) is used for antimicrobial activities and for antibiotic-resistance like methicillin-resistant Staphylococcus aureus (MRSA)[48], and benzocaine (2) is used to relieve pain and itching caused by conditions such as sunburn or other minor burns, insect bites or stings, poison ivy, poison oak, poison sumac, minor cuts, or scratches[49]. However, in the present study, Salvia rosmarinus was used as a source of secondary metabolites (ligands) by using chloroform/methanol (1:1) extract of the plant leaves yielded to isolate micromeric (1) and benzocaine (2) in design structure as a candidate for drugs as inhibitors of the DNMT1 enzyme by inhibiting the activity of DNMT1 that prevent the formation of cervical cancer cells.

    Cervical cancer is one of the most dangerous and deadly cancers in women caused by Human papillomaviruses (HPV). Some sexually transmitted HPVs (type 6 owner of E6) may cause genital warts. There are several options for the treatment of early-stage cervical cancer such as surgery, nonspecific chemotherapy, radiation therapy, laser therapy, hormonal therapy, targeted therapy, and immunotherapy, but there is no effective cure for an ongoing HPV infection. In the present study, Salvia rosmarinus leaves extracted and isolated compounds 1 and 2 are one of the therapeutic drugs design structure as a candidate drug for inhibiting HPV type 16 E6 enzyme. Similarly, numerous researchers have conducted studies on the impact of plant metabolites on the treatment of cervical cancer. Their research has demonstrated that several compounds such as jaceosidin, resveratrol, berberin, gingerol, and silymarin may be active in treating the growth of cells[47].

    Small-molecule drugs are still most commonly used in the treatment of cancer[50]. Molecular docking in in silico looks for novel small-molecule (ligands) interacting with genes or DNA or protein structure agents which are still in demand, newly designed compounds are required to have a specific even multi-targeted mechanism of action to anticancer and good selectivity over normal cells. In addition to these, in the literature, anti-cancer drugs are not easily classified into different groups[51]. Thus, drugs have been grouped according to their chemical structure, presumed mechanism of action, and cytotoxic activity related to cell cycle arrest, transcription regulation, modulating autophagy, inhibition of signaling pathways, suppression of metabolic enzymes, and membrane disruption[52]. Another problem for grouping anticancers often encountered is the resistance that may emerge after a brief period of a positive reaction to the therapy or may even occur in drug-naïve patients[50]. In recent years, many studies have investigated the molecular mechanism of compounds affecting cancer cells and results suggest that compounds exert their anticancer effects by providing free electron charge inhibiting some of the signaling pathways that are effective in the progression of cancer cells[53] and numerous studies have shown that plant-based compounds such as phenolic acids and sesquiterpene act as anticancer agents by affecting a wide range of molecular mechanisms related to cancer[53]. The present investigations may similarly support molecular mechanisms provided for the suppression of metabolic enzymes of cervical cancer.

    The main aim of the study was to evaluate the antimicrobial activity of different extracts of Salvia rosmarinus in vitro, and its compounds related to in silico targeting of enzymes involved in cervical cancer. The phytochemical screening tests indicated the presence of phytochemicals such as alkaloids, terpenoids, flavonoids, and tannins in its extracts. The plant also exhibited high antimicrobial activity, with varying efficacy in inhibiting pathogens in a dose-dependent manner (50−100 μg·mL−1). However, this extract exhibited a comparatively high inhibition zone in gram-positive and gram-negative bacteria had lower inhibition zones against E. coli, P. aeruginosa, and K. pneumoniae, respectively, and stronger antifungal activity 20.83 ± 0.76 mm inhibition zone against C. albicans fungi. Molecular docking is a promising approach to developing effective drugs through a structure-based drug design process. Based on the docking results, the in silico study predicts the best interaction between the ligand molecule and the protein target DNMT1 and HPV type 16 E6. Compound 1 (–8.3 kcal·mol−1) and 2 (–5.3 kcal·mol−1) interacted with DNMT1 (PDB ID: 4WXX) and the same compound 1 (–10.1 kcal·mol−1) and 2 (–6.5 kcal·mol−1) interacted with HPV type 16 E6 (PDB ID: 4XR8). Compounds 1 and 2 may have potential as a medicine for treating agents of cancer by inhibiting enzymes DNMT1 and HPV type 16 E6 sites, as well as for antimicrobial activities. None of the compounds exhibited acute toxicity in ADMET prediction analysis, indicating their potential as drug candidates. Further studies are required using the in silico approach to generate a potential drug through a structure-based drug-designing approach.

  • The authors confirm contribution to the paper as follows: all authors designed and comprehended the research work; plant materials collection, experiments performing, data evaluation and manuscript draft: Dejene M; research supervision and manuscript revision: Dekebo A, Jemal K; NMR results generation: Tufa LT; NMR data analysis: Dekebo A, Tegegn G; molecular docking analysis: Aliye M. All authors reviewed the results and approved the final version of the manuscript.

  • All data generated or analyzed during this study are included in this published article.

  • This work was partially supported by Adama Science and Technology University under Grant (ASTU/SP-R/171/2022). We are grateful for the fellowship support from Adama Science and Technology University (ASTU), the identification of plants by Mr. Melaku Wendafrash, and pathogenic strain support from the Ethiopian Biodiversity Institute (EBI). We also thank the technical assistants of the Applied Biology and Chemistry departments of Haramaya University (HU) for their help.

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 Primer sequences used in this research.
    Supplemental Table S2 The GenBank accession of carotenoid cleavage dioxygenases sequences from other species.
    Supplemental Table S3 The primer sequences for qPCR used in this study.
    Supplemental Fig. S1 Protein sequence alignment analysis of CCD1, CCD4-1 and CCD4-2 in Zinnia elegans of different cultivars. The black arrow refers to the histidine residue that binds to the Fe2+ cofactor.
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  • Cite this article

    Qing H, Liu X, Chen J, Li L, Qian J, et al. 2024. Carotenoid cleavage dioxygenase catalyzes carotenoid degradation and regulates carotenoid accumulation and petal coloration in Zinnia elegans. Ornamental Plant Research 4: e005 doi: 10.48130/opr-0024-0003
    Qing H, Liu X, Chen J, Li L, Qian J, et al. 2024. Carotenoid cleavage dioxygenase catalyzes carotenoid degradation and regulates carotenoid accumulation and petal coloration in Zinnia elegans. Ornamental Plant Research 4: e005 doi: 10.48130/opr-0024-0003

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Carotenoid cleavage dioxygenase catalyzes carotenoid degradation and regulates carotenoid accumulation and petal coloration in Zinnia elegans

Ornamental Plant Research  4 Article number: e005  (2024)  |  Cite this article

Abstract: Carotenoids are one of the important coloring substances in the ray florets of Zinnia elegans. Carotenoid cleavage dioxygenase (CCD) directly affects carotenoid contents by cleaving the double bond of carotenoids, thereby affecting the flower color. However, few reports have elucidated its function in Zinnia elegans. In the present study, through transcriptome homology searches, three ZeCCDs, ZeCCD1, ZeCCD4-1, and ZeCCD4-2 were screened from Zinnia elegans. The function of ZeCCDs was verified by bacterial pigment complementation system, and it revealed that all of the three ZeCCDs had the ability to cleave β-carotene and other carotenoids (ε-carotene, zeaxanthin and lycopene). Meanwhile, the expression levels of ZeCCD1 and ZeCCD4-2 in the petals of different cultivars were significantly negatively correlated with the carotenoid content. Furthermore, compared with the cytoplasmic localization of ZeCCD1, ZeCCD4-2 is localized in the plastid. Our results indicate that ZeCCD4-2 is the key gene responsible for the differential accumulation of carotenoids in the petals of different Zinnia elegans cultivars. This study lays a foundation for further research on the molecular mechanism of petal coloration and carotenoid metabolism of Zinnia elegans.

    • Carotenoids, a group of widely distributed natural pigments, influence the color development of petals in many plants[1,2]. The final accumulation of carotenoids, determined by a series of biosynthetic genes and degradation genes, has a great influence on petal coloration[3]. Since the C-C double bond on the carotenoid skeleton is vulnerable to oxygen attack, non-enzymatic or enzymatic oxidative cleavage occurs, resulting in various forms of carotenoid derivatives produced by the enzymatic cleavage of the carotenoid cleavage oxygenases (CCO) family. CCO family composed of multiple members in plants can be divided into two main categories: carotenoid cleavage dioxygenase (CCD) and 9-cis-epoxy carotenoid dioxygenase (NCED)[4].

      Among different CCD gene members, CCD1 and CCD4 participating in the degradation of carotenoids, are regarded as the key factor to regulate carotenoid accumulation in several plant species[57]. In plants, the CCD1 enzyme only exists in the cytoplasm due to the lack of plastid localization peptides. Since carotenoids are mainly produced in the plastids, CCD1 cannot directly act on the C40 carotenoid substrates in the plastids[8,9]. Only C27 apocarotenoids, the product of C40 carotenoids cleaved by CCD4, can be further cleaved by CCD1 into C13 and/or C14 small-molecule volatiles, such as α-ionone and β-ionone[1012]. But in the bacterial pigment complementation system, when the CCD1 prokaryotic expression vector and the engineering plasmid capable of producing carotenoids are expressed in Escherichia coli, CCD1 can contact and cleave several C40 carotenoids, including lycopene, α-carotene, β-carotene, ε-carotene and zeaxanthin[13,14]. Based on these results, it is presumed that CCD1 can directly act on both apocarotenoids produced by other CCD proteins and C40 carotenoids under certain conditions.

      In contrast, the plastid localization of CCD4 allows access to carotenoid substrates that were synthesized and stored in plastids[15]. CCD4 cleaves the same position (9, 10 and 9', 10' double bond) of different C40 carotenoids to produce C13 apocarotenoids, but CCD4 exhibited different cleavage activity for diversiform C40 carotenoids, among which β-carotene was considered to be the preferred substrate for CCD4[16]. It has been reported that the transcriptional level of CCD4 is a key factor affecting the total carotenoid content, which leads to color differences such as white, pale yellow, and dark orange in plant petals[1719]. For example, the transcription level of IbCCD4 was negatively correlated with the total carotenoid content during flower coloration changes from yellowish-cream to white in Lilium brownie var. colchesteri[20]. More importantly, in chrysanthemum (Chrysanthemum morifolium) petals, there was a significant negative correlation between CmCCD4a expression level and total carotenoid content of petals, and white petals turned yellow due to carotenoid accumulation after the RNAi construct of CmCCD4a was introduced[21]. It is suggested that some carotenoid cleavage dioxygenase genes, rather than carotenoid biosynthesis genes, are largely responsible for carotenoid accumulation in many plant species.

      Zinnia elegans is an annual herbaceous ornamental flower, which is widely used as summer and autumn flower in domestic and foreign gardens due to its diversity in flower types, color, and versatility of landscaping. Petal color of Zinnia elegans ranges widely from red to orange and yellow, which is mainly caused by different content of carotenoids and anthocyanins[22,23]. β-carotene is the main carotenoid in the petals, accounting for 65.89% and 82.25% of the total identified carotenoids in red and yellow petals, respectively[24]. What’s more, the cyclizing function of lycopene cyclase genes intimately associated with β-carotene synthesis, has been verified from Zinnia elegans[24] by bacterial pigment complementation system[25]. However, how the carotenoid cleavage dioxygenase genes, which are closely related to β-carotene degradation, participate in the carotenoid metabolism in the ray florets of Zinnia elegans and regulate the molecular mechanism of petal coloration in different color cultivars of Zinnia elegans is still unclear and requires further study.

      In this study, the red, orange and yellow flower cultivars of Zinnia elegans, with different carotenoid content in the petals, were used as study materials. Three CCD genes (one CCD1 and two CCD4) from Zinnia elegans were cloned, and further characterized the cleavage ability of these genes on four kinds of C40 carotenoids using the bacterial pigment complementation system[25]. In addition, the carotenoid content and the expression patterns of three CCD genes were also analyzed among the red, orange and yellow cultivars. Finally, the subcellular localization of ZeCCDs was analyzed. This study will help to further explore the molecular mechanism of carotenoid metabolism in Zinnia elegans.

    • The experimental materials were three cultivars from Zinnia elegans 'Dreamland' series: Zinnia elegans 'Dreamland Red' (DRE), Zinnia elegans 'Dreamland Coral' (DC) and Zinnia elegans 'Dreamland Yellow' (DY). These plants were cultured in the climate room (photoperiod: 13 h light and 11 h dark, temperature cycle: 23 °C day and 18 °C night) of the College of Landscape Architecture, Zhejiang Agriculture and Forestry University (China). In accordance with previous studies[2224], the developmental stages of petals were divided into four stages: the elongation stage (S1), the coloring stage (S2), the extension stage (S3) and the blooming stage (S4). The roots, stems, leaves, phyllary and petals of DY at S4 stage were collected for gene expression pattern analysis, and the outermost ray florets at four different developmental stages of DRE, DC and DY were collected to determine total carotenoid content and gene expression patterns. All samples were rapidly collected, frozen in liquid nitrogen, and subsequently stored at −80 °C until analysis.

    • Total carotenoid content in petal was determined by spectrophotometric according to Lichtenthaler[26]. Fresh petals samples (0.2 g) stored at −80 °C were fully ground, mixed with 95% (v/v) ethanol solution, vortexed, and extracted for 15 min away from light, and centrifuged at 2,500× g for 10 min. The absorbance at 470 nm was measured with the supernatant, and the total carotenoid content of the petals at different developmental stages of different cultivars were calculated using Lichtenthaler's formula[26].

    • The petal samples of four developmental stages of DY were mixed, and the total RNA in the samples was extracted using UltraClean Polysaccharide and phenol Plant RNA Purification Kit (DNA free) (Novogene, Beijing, China). One μg of high-quality RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix (Perfect Real Time) Kit (TaKaRa, Dalian, China) for cDNA library construction and sequenced with DNBSEQ platform (Beijing Genomics Institute, Shenzhen, China). CCD1 and CCD4 proteins from Helianthus annuus (HaCCD1 XP_022023037.1; HaCCD4 XP_021977754.1) was used as bait, and the single gene sequence containing BLASTx (E value < 10−5) was searched in the NCBI non-redundant (Nr) protein database (www.ncbi.nlm.nih.gov). Finally, three CCDs (one CCD1 and two CCD4) were obtained, named ZeCCD1, ZeCCD4-1 and ZeCCD4-2, respectively.

      The full-length open reading frames (ORFs) of the three ZeCCDs amplified from the petals of DRE, DC, and DY were cloned into the vector pMAL-c5X (New England BioLabs, NEB) using homologous cloning technique (ClonExpress® II One Step Cloning Kit, Vazyme, Nanjing, China) with cloning primers (Supplemental Table S1) to identify gene sequence of each ZeCCD from different cultivars. The recombinant plasmids pMAL-ZeCCDs were transformed into E. coli Top10 competent cells and coated on Luria-Bertani (LB) solid medium containing 100 µg/mL ampicillin for screening. Single bacteria were selected for Polymerase Chain Reaction (PCR) positive identification and cultured overnight in LB liquid medium containing 100 µg/mL ampicillin. The plasmids were extracted and sequenced by Tsingke Biotechnology Co., Ltd (Hangzhou, China).

    • Using the ProtParam tool (http://web.expasy.org/protparam/), we analyzed the biochemical properties of the proteins encoded by ZeCCDs from DY, such as protein length, molecular weight (MW), isoelectric point (pI), instability index, aliphatic index and average hydrophilicity (GRAVY). The amino acid sequences of CCDs from Arabidopsis thaliana, Vitis vinifera, Triticum aestivum, Nicotiana tomentosiformis, Zea mays, Crocus sativus, Crocus angustifolius, Crocus chrysanthus, Rosa rugosa, Osmanthus fragrans, Lycium chinense, Helianthus annuus, Nicotiana tabacum and Solanum lycopersicum were downloaded from GenBank. The GenBank accession numbers of the downloaded sequences are shown in Supplemental Table S2. The sequence alignment was performed with ZeCCDs using MEGA X software[27], and a maximum likelihood phylogenetic tree was constructed based on the JTT matrix-based model (JTT) with 500 bootstrap replicates.

    • In order to characterize the cleavage function of ZeCCDs, the above pMAL-ZeCCD1, pMAL-ZeCCD4-1, pMAL-ZeCCD4-2, and the empty vector pMAL-c5X, were respectively co-transformed into E. coli Top10 competent cells[2830] with four engineering plasmids (including pAC-β, β-carotene; pAC-ε, ε-carotene; pAC-ZEA, zeaxanthin; pAC-LYC, lycopene). The transformed E. coli cells were coated on LB solid medium containing 34 μg/mL chloramphenicol and 100 μg/mL ampicillin, and cultured overnight at 37 °C. Positive single colonies were inoculated in 2 mL LB liquid medium containing corresponding antibiotics. After 12 h of shaking culture at 37 °C, 1 mL of bacterial culture was expanded to 100 mL of medium containing the same antibiotics, and cultured again at 37 °C for 12 h. The bacterial cells were collected by centrifugation, and carotenoids were extracted with 1 mL of acetone, and shaken until the sediment was colorless. The supernatant was carefully aspirated after centrifugation at 2,500× g for 5 min, filtered through an organic filter membrane with a pore size of 0.22 μm, and stored in a brown glass sample bottle for subsequent carotenoid analysis.

    • In order to quantitatively analyze the expression of ZeCCDs genes, AceQ® qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) was used, and the primers for quantitative real-time PCR (qRT-PCR) are available in Supplemental Table S3. The reaction system and conditions were set according to our previous studies[24]. The qRT-PCR analyzes of the target genes were performed on the LightCycler® 480II (Roche Diagnostics, Germany) instrument. With ZeACT as the internal reference gene[2224], Cq values were automatically calculated by LightCycler® 480II software and the relative expression of target genes was quantified by the 2−ΔCᴛ[31]. All qPCR reactions were set up with three biological replicates.

      The total carotenoid content and the expression levels of ZeCCDs in the petals of different cultivars at different developmental stages were subjected to Person correlation analysis in SPSS 20.0 software (SPSS Inc., Chicago, IL, USA).

    • The coding sequences of ZeCCDs from DY without stop codon were inserted into pCNHP-eYFP vector, so that each of the three ZeCCDs was fused with the eYFP protein to produce the 35S: ZeCCDs-eYFP fusion construct (primers used are shown in Supplemental Table S1). The recombinant plasmids verified by sequencing were transferred into Agrobacterium tumefaciens GV3101. The Agrobacterium tumefaciens GV3101 containing the target plasmid was respectively injected into 4-week-old tobacco (Nicotiana benthamiana) leaves. After 48 h of infection in the dark, the fluorescence signal was observed by confocal fluorescence microscope (ZEISS, Germany) with chloroplast autofluorescence as the marker.

    • Total carotenoids were extracted from fresh petals of three Zinnia elegans cultivars DRE, DC and DY (Fig. 1a) at four developmental stages (Fig. 1b) and quantified by spectrophotometry. The results showed that the total carotenoid content of DY petals was consistently higher than that of DRE and DC at different developmental stages, while that of DRE was always the lowest. Especially at S4, the total carotenoid content in DC petals was 2-fold higher than that of DRE, and the DY content was almost 10-fold higher than that of DRE (Fig. 1c).

      Figure 1. 

      The total carotenoid content in the petals of Zinnia elegans. (a) Picture of flowers from DRE, DC and DY, and at (b) different developmental stages. (c) Total carotenoid content in petals from three cultivars and different developmental stages. These are mean ± standard errors, three biological replicates. Different letters indicate significant differences in the one-way ANOVA Duncan test, p < 0.05.

    • Carotenoid cleavage dioxygenase can breakdown the different sites of different carotenoids, cleave carotenoids, decrease the content of carotenoids in plants, and thus change the plant coloration. Therefore, the isolation, identification and characterization of ZeCCDs are essential for understanding carotenoid metabolism in the petals of Zinnia elegans. In the current study, the petal transcriptome was used for homology-based search to identify the carotenoid cleavage dioxygenase of Zinnia elegans[24]. Using HaCCD1 and HaCCD4 from Helianthus annuus as bait, three homologous genes with the highest sequence identity were identified in Zinnia elegans, which were named ZeCCD1, ZeCCD4-1 and ZeCCD4-2 respectively. Sequence analysis of ZeCCDs in DRE, DC and DY showed that sequence identity of ZeCCD1, ZeCCD4-1 and ZeCCD4-2 in three different cultivars were 100%, 98.21% and 99.78%, respectively. More importantly, we found four histidine residues binding to Fe2+ cofactors conserved in the amino acid sequences of ZeCCDs from three cultivars, which are essential for carotenoid cleavage dioxygenase activity, and their sites in the three cultivars are consistent (Supplemental Fig. S1). Subsequent analysis was carried out with ZeCCDs from DY, and coding regions of ZeCCD1, ZeCCD4-1 and ZeCCD4-2 were 1629 bp, 1734 bp and 1791 bp, respectively (Table 1).

      Table 1.  Biochemical properties of ZeCCDs.

      ProteinORF length
      (bp)
      Protein length
      (aa)
      Molecular weight
      (Da)
      Isoelectric
      point
      Aliphatic
      index
      Grand average of
      hydropathy
      Instability index
      ZeCCD11,62954261,102.985.8578.54−0.26733.71
      ZeCCD4-11,73457764,307.796.9988.01−0.10931.13
      ZeCCD4-21,79159665,784.956.0579.13−0.19645.14

      The amino acid sequences of the ZeCCDs were compared with protein sequences of Helianthus annuus, Osmanthus fragrans, Lycium chinense, Arabidopsis thaliana and other species. It was found that four histidine residues binding to Fe2+ cofactors, which are essential for carotenoid cleavage dioxygenase activity, were well conserved in the amino acid sequences of CCDs in the above different specie (Fig. 2a).

      Figure 2. 

      Sequence alignment and phylogenetic analysis of CCDs proteins. (a) Alignment of the deduced amino acid sequences of CCDs homologs. Black represents the 100% homology level of the sequence, dark gray represents the ≥ 75% homology level, and light gray represents the ≥ 50% homology level. The black arrow refers to the histidine residue that binds to the Fe2+ cofactor. Sequences of functionally characterized HaCCD1 (XP_022018126.2) and HaCCD4 (CTP93722.1) from Helianthus annuus, OfCCD1 (AXQ60417.1) and OfCCD4 (ABY60887.1) from Osmanthus fragrans, LcCCD1 (AIY62758.1) and LcCCD4 (AIY62809.1) from Lycium chinense, AtCCD1 (NP_191911.1) from Arabidopsis thaliana. (b) Phylogenetic analysis of CCDs homologs from Zinnia elegans and other species (see Supplemental Table S2 for their GenBank accession numbers). The phylogenetic tree was built using MEGA X software with 500 bootstrap replicates based on the JTT matrix-based model (JTT). The scale bar represents the phylogenetic distance calculated based on the number of differences.

      Phylogenetic analysis showed that CCDs were mainly divided into two major branches, one of which consisted of CCD1, CCD2 and CCD4. In this branch, CCD1 and CCD4 were clustered into a small branch (Fig. 2b).

    • The pAC plasmids, which can provide the genes essential for efficient production of C40 carotenoids in E. coli, were used to establish a system based on the bacterial pigment complementation system[25] to verify the cleavage activity of three CCDs on four C40 carotenoid substrates. Due to the accumulation of carotenoids, cells transformed with different pAC plasmids showed diversiform coloration, which is a convenient system for characterizing enzymes that cleave carotenoids.

      The results showed that all three ZeCCDs could make E. coli cells, which could produce β-carotene and other carotenoids, show different degrees of discoloration. Compared with pMAL-c5X, the cells co-transformed with four pAC plasmids (pAC-β, pAC-ε, pAC-ZEA, pAC-LYC) and pMAL-ZeCCD1 were the lightest in coloration and close to white, while co-transfected with pAC plasmids and pMAL-ZeCCD4 were lighter in coloration (Fig. 3). Carotenoids extracted from E. coli cells were examined by spectrophotometer. The results showed that the content of total carotenoids in E. coli, which transformed plasmids expressing ZeCCD1 with pAC, was significantly reduced compared to the control, and the content in cells transformed plasmids expressing ZeCCD4-1 and ZeCCD4-2 was also reduced and not significantly different between each other. The above results indicated that these three ZeCCDs in E. coli cells showed different cleavage activity for four carotenoids (β-carotene, ε-carotene, zeaxanthin, lycopene), among which ZeCCD1 had the strongest cleavage activity (Fig. 3).

      Figure 3. 

      Functional characterization of ZeCCDs proteins. The pMAL-ZeCCD1, pMAL-ZeCCD4-1, pMAL-ZeCCD4-2 and pMAL-c5X empty vectors were co-transformed into the engineered E. coli strains that were modified to accumulate (a) β-carotene, (b) ε-carotene, (c) zeaxanthin and (d) lycopene. These are mean ± standard errors, three biological replicates. Different letters indicate significant differences in the one-way ANOVA Duncan test, p < 0.05.

    • The tissue specificity and expression patterns during flower development of ZeCCDs were analyzed by qPCR in order to further understand the possible biological functions of ZeCCDs. In DY, ZeCCD1 had the highest expression level in leaves and the lowest expression level in petals, while ZeCCD4-1 had the highest expression level in petals and stems. The expression level of ZeCCD4-2 was the highest in leaves and phyllaries (Fig. 4a). Significantly, the expression levels of ZeCCD1 and ZeCCD4-2 in petals were much lower than that of ZeCCD4-1.

      Figure 4. 

      The expression patterns of ZeCCDs. (a) Expression patterns of three genes in different tissues of DY. The expression patterns of (b) ZeCCD1, (c) ZeCCD4-1 and (d) ZeCCD4-2 in different cultivars and developmental stages. These are mean ± standard errors, three biological replicates. Different letters indicate significant differences between cultivars at the same developmental stage in the one-way ANOVA Duncan test, p < 0.05.

      The results shows that ZeCCD1 has the highest expression level in DRE, followed by DC, and the lowest in DY at all four developmental stages (Fig. 4b). Compared with ZeCCD1, ZeCCD4-1 had the highest expression level in S2, S3 and S4 stages of DY. At the S1 stage, the expression level of ZeCCD4-1 in DC was higher than that in other cultivars (Fig. 4c). The expression pattern of ZeCCD4-2 was similar to that of ZeCCD1. During petal development, both ZeCCD4-2 and ZeCCD1 had the highest expression level in DRE and the lowest expression level in DY (Fig. 4d).

      To further confirm the relationship between the expression levels of ZeCCDs and total carotenoid content, person correlation analysis showed that the expression levels of ZeCCD1 and ZeCCD4-2 were significantly negatively correlated with the total carotenoid content (Table 2).

      Table 2.  Correlation analysis of total carotenoid content and expression levels of ZeCCDs in petals from Zinnia elegans.

      ZeCCD1ZeCCD4-1ZeCCD4-2
      Total carotenoid content−0.915**0.543−0.757*
      '*' and '**' indicates significant negative correlation at p < 0.05 and p < 0.01 level between ZeCCDs expression levels and total carotenoid content respectively.
    • To determine the subcellular localization of ZeCCDs, we constructed a Pro35S::ZeCCDs-eYFP fusion structure and expressed it in epidermal cells of tobacco leaves, which was then visualized by using fluorescence microscopy. The results of transient expression in tobacco leaves demonstrated that the 35S::ZeCCD1-eYFP fusion protein was localized in the cytosol, while control protein (35S::eYFP) was spread throughout the cell. It is worth noting that the 35S::ZeCCD4-1-eYFP and 35S::ZeCCD4-2-eYFP fusion protein was co-localized with chloroplasts (Fig. 5).

      Figure 5. 

      The subcellular localization of ZeCCDs. The fusion constructs (Pro35S::ZeCCDs-eYFP) were transiently transformed into Nicotiana benthamiana leaf epidermal cells, and observed by confocal fluorescence microscope (ZEISS, Germany) with chloroplast autofluorescence as the maker. For each construct, yellow (eYFP), red (chloroplasts), gray (bright-field), and merged are shown. Bars = 50 μm.

    • CCDs can change the type and content of carotenoids in plants by acting on different sites of carotenoids, especially CCD1 and CCD4 which are closely related to plant flower color. In order to explore the metabolic mechanism of carotenoids in the petals of Zinnia elegans, three CCDs, including one CCD1 and two CCD4, were identified and isolated from Zinnia elegans by using homology-based search on petal transcriptome, and named ZeCCD1, ZeCCD4-1, and ZeCCD4-2 (Table 1). This result was similar to that of Crocus sativus, which had two isoforms of CCD4[32]. Noteworthy, four histidine residues essential for carotenoid cleavage activity were conserved in these proteins. Four histidine residues together with a divalent iron center form a 4-His-ligated iron center, which is required for substrate recognition and correct positioning of substrates in the active site[33], suggesting that four histidine residues are necessary for CCD to perform its cleavage function. The specific structure of the 4-His-ligated iron center is conserved in the CCD family, which requires the amino acid sequences of CCDs to be evolutionarily conserved[34].

    • The pigment complementation system in E. coli showed that ZeCCD1, ZeCCD4-1 and ZeCCD4-2 cleaved four carotenoids, including β-carotene (Fig. 3), which is the most abundant carotenoids in the petals of Zinnia elegans[24]. This is similar to the cleavage activity of CCD1 from other plants with extensive affinity for C40 carotenoids. For example, LeCCD1 from tomato can catalyze the cleavage of several carotenoids such as lycopene, β-carotene, ε-carotene and zeaxanthin[10]. DoCCD1 from Dendrobium officinale and RdCCD1 from Rosa damascene also catalyze the cleavage of lycopene, β-carotene and zeaxanthin[35,36]. Similarly, two ZeCCD4 have catalytic activity for all four carotenoids, consistent with the reported cleavage activity of CCD4 from Camellia sinensis in E. coli[37]. In contrast, GmCCD4 from soybeans (Glycine max) can only specifically catalyze the cleavage of β-carotene and has affinity or cleavage activity for lycopene[38]. Although CCD4 enzymes from different plant species exhibit differences in substrate specificity and regioselectivity, it is generally accepted that β-carotene is recognized as the most favorable substrate[5]. Moreover, ZeCCD1 seemed to have higher cleavage activity for diversiform C40 carotenoids, and only a small quantity of carotenoids could be extracted from E. coli co-transfected with different pAC plasmids, compared with ZeCCD4-1 and ZeCCD4-2 (Fig. 3). This could be partly attributed to the differences of cleavage site and substrate specificity. The CCD1 enzymes are characterized by their extensive substrate and double-bond specificity, and they cleave different linear and cyclic C40 carotenoids and apocarotenoid carotenoids at 9, 10 and 9′, 10′ positions[36]. Accordingly, we surmise that ZeCCD1 catalyzes the cleavage of both ends of carotenoids in two steps, from C40 carotenoids via C27 apocarotenoids to C13/C14 apocarotenoid end-products in E. coli, with the second reaction proceeding in a way that may promote the first step. This hypothesis is supported by CCD1 breakdown both C40 carotenoids and C27 apocarotenoids[11]. In contrast, ZeCCD4-1 and ZeCCD4-2 may catalyze single cleavage of C40 carotenoids at 9, 10 (9′, 10′), which is similar to research on CCD4 from potato (Solanum tuberosum)[39], sweet orange (Citrus sinensis)[40] and Satsuma mandarin (Citrus unshiu)[41]. In Citrus species, this cleavage led to the production of C30 apocarotenoid pigments, such as β-citraurinene and β-citraurin, which are essential for peel pigmentation in citrus fruits[42]. Although, it has been reported that CCD4 catalyzes double cleavage of C40 carotenoids at 7, 8 (7′, 8′), they are well correlated with the biosynthesis of some specific substances in plants, such as crocins and bixin[43,44], which contain the conjugated polyene system of carotenoids. Therefore, it is necessary to study CCD, which will provide a better understanding of the original biological functions of these enzymes and further determine their hypothetical roles in material metabolism.

    • We analyzed the expression patterns of ZeCCDs at four developmental stages in the petals of different Zinnia elegans cultivars (Fig. 4). The expression levels of ZeCCD1 and ZeCCD4-2 were significantly negatively correlated with total carotenoid content in petals of Zinnia elegans (Table 2). In general, the expression levels of CCDs are negatively correlated with carotenoid content due to the cleavage function of CCDs on carotenoids. The higher expression level of SiCCD1 at the grain maturity stage was considered to be the primary reason for the lower carotenoid content in white cultivars of Setaria italica[45]. What’s more, the expression level of CmCCD4a in chrysanthemum, which belongs to Asteraceae family, also showed a significant negative correlation with the total carotenoid content. After the introduction of an RNAi construct of CmCCD4a in the white cultivar, the petals turned yellow[8]. The results of the subcellular localization assay showed that ZeCCD1 was localized in the cytosol while ZeCCD4-1 and ZeCCD4-2 were localized in the chloroplasts (Fig. 5). This is consistent with previous studies in Prunus mume that PmCCD1 and PmCCD1-like-c were localized in the cytoplasm, and PmCCD4 was localized in the plastid[46]. In plants, the enzymes involved in carotenoid biosynthesis are located in the plastids[35]. At the same time, carotenoids, as the hypothetical substrate of CCDs, are mostly stored in plastids due to their hydrophobicity, while CCD1 enzyme, due to the lack of plastid localization peptides, is the only member of the CCDs family located in the cytoplasm. It cannot directly obtain the C40 carotenoid substrate in the plastid, but acts on the C27 apocarotenoid, the cleavage product of the C40 carotenoid that is transported to the cytoplasm[47]. Unlike the CCD1 enzymes located in the cytoplasm, most of the CCD4 enzymes are located in the plastids[13], suggesting that the CCD4 enzymes can contact its carotenoid substrate. For example, in Satsuma mandarin (Citrus unshiu), CitCCD4 is located in the plastids and can cleave the carotenoid substrates β-cryptoxanthin and zeaxanthin stored in the plastids to produce β-citraurin[45]. Therefore, it was presumed that ZeCCD4-2 might be a critical gene affecting carotenoid metabolism in the petals of Zinnia elegans. Our work preliminarily revealed the function of carotenoid cleavage dioxygenase genes in carotenoid accumulation in Zinnia elegans, and laid a foundation for further exploring the molecular mechanism of carotenoid metabolism in Zinnia elegans.

      Carotenoid accumulation in plants is not only affected by carotenoid metabolic genes, transcription factors can also be involved in the regulation of carotenoid accumulation. For example, the promoter region of CCD2 in Crocus sativus contained a MYB-binding region, and the expression level of CstCCD4b was significantly increased in transgenic lines for the MYB transcription factors CstMYB1 and CstMYB1R2[48]. Another study showed that in Chrysanthemum morifolium, two MADS box-containing transcription factors, APETALA3 (AP3) and ULT1 Interacting Factor 1 (UIF1), could be connected by PISTILLATA (PI) to form the CmAP3-CmPI-CmUIF1 protein complex, regulated carotenoid metabolism by directly activating the expression of CmCCD4a-2[49]. At the same time, there were many GATA binding sites in the promoter region of CmCCD4a-5, and the GATA family transcription factor CmGATA4 of Chrysanthemum morifolium could bind directly to the promoter of CmCCD4a-5 and inhibit its activity[50]. The WRKY transcription factor OfWRKY3 and the ethylene responsive factor (ERF) transcription factor OfERF61 of Osmanthus fragrans were found to bind directly to the promoter of OfCCD4 and significantly promote the expression of OfCCD4[51]. Transcription factors related to the regulation of CCDs in Zinnia elegans have not been determined, so more studies should be conducted to identify and reveal the function of up-stream potential transcription factors, which could directly regulate the expression of CCDs in Zinnia elegans.

    • Three CCDs were isolated from Zinnia elegans. These three ZeCCDs could cleave β-carotene, ε-carotene, zeaxanthin and lycopene. The expression levels of ZeCCD1 and ZeCCD4-2 were significantly negatively correlated with the total carotenoid content in petals from different cultivars. Subcellular localization analysis showed that ZeCCD1 was localized in the cytosol, while ZeCCD4-1 and ZeCCD4-2 were localized in the chloroplasts. Based on the above results, it is speculated that ZeCCD4-2 plays an more important role in carotenoid cleavage in Zinnia elegans and affects the petal coloration of ray florets.

    • The authors confirm contribution to the paper as follows: literature collation: Qing H; experimental design and implementation: Qing H, Fu J, Zhang C; data analysis and visualization: Qing H, Liu X, Chen J, Li L, Qian J; writing and polishing: Qing H, Liu X; resources and project administration: Fu J, Zhang C; review and editing: Fu J, Zhang C. All authors reviewed the results and approved the final version of the manuscript.

    • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

      • We are grateful to Drs E. Gantt and F.X. Cunningham for the E. coli functional complementation system and Dr. Xing-Qi Huang for the gift of plasmids pAC-LYC, pAC-β, pAC-ε, pAC-Zea and pMAL-C5X. This research was supported by Zhejiang Provincial Natural Science Foundation of China (Grant No. LY24C150001) and College Students Research Training Program of Zhejiang Agriculture and Forestry University (No. 2023KX044).

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Hongsheng Qing, Xinyue Liu

      • Supplemental Table S1 Primer sequences used in this research.
      • Supplemental Table S2 The GenBank accession of carotenoid cleavage dioxygenases sequences from other species.
      • Supplemental Table S3 The primer sequences for qPCR used in this study.
      • Supplemental Fig. S1 Protein sequence alignment analysis of CCD1, CCD4-1 and CCD4-2 in Zinnia elegans of different cultivars. The black arrow refers to the histidine residue that binds to the Fe2+ cofactor.
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (5)  Table (2) References (51)
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    Qing H, Liu X, Chen J, Li L, Qian J, et al. 2024. Carotenoid cleavage dioxygenase catalyzes carotenoid degradation and regulates carotenoid accumulation and petal coloration in Zinnia elegans. Ornamental Plant Research 4: e005 doi: 10.48130/opr-0024-0003
    Qing H, Liu X, Chen J, Li L, Qian J, et al. 2024. Carotenoid cleavage dioxygenase catalyzes carotenoid degradation and regulates carotenoid accumulation and petal coloration in Zinnia elegans. Ornamental Plant Research 4: e005 doi: 10.48130/opr-0024-0003

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