A Novel Derivative of Curcumol, HCL-23, Inhibits the Malignant Phenotype of Triple-Negative Breast Cancer and Induces Apoptosis and HO-1-Dependent Ferroptosis.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype of breast cancer. Curcumol, as a natural small molecule compound, has potential anti-breast cancer activity. In this study, we chemically synthesized a derivative of curcumol, named HCL-23, by structural modification and explored its effect on and underlying mechanism regarding TNBC progression. MTT and colony formation assays demonstrated that HCL-23 significantly inhibited TNBC cells proliferation. HCL-23 induced G2/M phase cell cycle arrest and repressed the capability of migration, invasion, and adhesion in MDA-MB-231 cells. RNA-seq results identified 990 differentially expressed genes including 366 upregulated and 624 downregulated genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) revealed that these differentially expressed genes were obviously enriched in adhesion, cell migration, apoptosis, and ferroptosis. Furthermore, HCL-23 induced apoptosis via the loss of mitochondrial membrane potential and the activation of the caspase family in TNBC cells. In addition, HCL-23 was verified to trigger ferroptosis through increasing cellular reactive oxygen species (ROS), labile iron pool (LIP), and lipid peroxidation levels. Mechanistically, HCL-23 markedly upregulated the expression of heme oxygenase 1 (HO-1), and the knockdown of HO-1 could attenuate ferroptosis induced by HCL-23. In animal experiments, we found that HCL-23 inhibited tumor growth and weight. Consistently, the upregulation of Cleaved Caspase-3, Cleaved PARP, and HO-1 expression was also observed in tumor tissues treated with HCL-23. In summary, the above results suggest that HCL-23 can promote cell death through activating caspases-mediated apoptosis and HO-1-dependent ferroptosis in TNBC. Therefore, our findings provide a new potential agent against TNBC.

Potential roles and molecular mechanisms of bioactive ingredients in Curcumae Rhizoma against breast cancer.

BACKGROUND: Breast cancer is the most prevalent cancer worldwide, with high morbidity and mortality. Despite great advances in the therapeutic strategies, the survival rate in the past decades of patients with breast cancer remains unsatisfactory. Growing evidence has demonstrated that Curcumae Rhizoma, called Ezhu in Chinese, showed various pharmacological properties, including anti-bacterial, anti-oxidant, anti-inflammatory and anti-tumor activities. It has been widely used in Chinese medicine to treat many types of human cancer. PURPOSE: To comprehensively summarize and analyze the effects of active substances in Curcumae Rhizoma on breast cancer malignant phenotypes and the underlying mechanisms, as well as discuss its medicinal value and future perspectives. METHOD: We used "Curcumae Rhizoma" or the name of crude extracts and bioactive components in Curcumae Rhizoma in combination with "breast cancer" as key words. Studies focusing on their anti-breast cancer activities and mechanisms of action were extracted from Pubmed, Web of Science and CNKI databases up to October 2022. The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 guideline was followed. RESULTS: Crude extracts and 7 main bioactive phytochemicals (curcumol, ß-elemene, furanodiene, furanodienone, germacrone, curdione and curcumin) isolated from Curcumae Rhizoma have shown many anti-breast cancer pharmacological properties, including inhibiting cell proliferation, migration, invasion and stemness, reversing chemoresistance, and inducing cell apoptosis, cycle arrest and ferroptosis. The mechanisms of action were involved in regulating MAPK, PI3K/AKT and NF-κB signaling pathways. In vivo and clinical studies demonstrated that these compounds exhibited high anti-tumor efficacy and safety against breast cancer. CONCLUSION: These findings provide strong evidence that Curcumae Rhizoma acts as a rich source of phytochemicals and has robust anti-breast cancer properties.

Spatial Distribution and Estimation Model of Soil pH in Coastal Eastern China.

Soil pH is an essential indicator for assessing soil quality and soil health. In this study, based on the Chinese farmland soil survey dataset and meteorological dataset, the spatial distribution characteristics of soil pH in coastal eastern China were analyzed using kriging interpolation. The relationships between hydrothermal conditions and soil pH were explored using regression analysis with mean annual precipitation (MAP), mean annual temperature (MAT), the ratio of precipitation to temperature (P/T), and the product of precipitation and temperature (P*T) as the main explanatory variables. Based on this, a model that can rapidly estimate soil pH was established. The results showed that: (a) The spatial heterogeneity of soil pH in coastal eastern China was obvious, with the values gradually decreasing from north to south, ranging from 4.5 to 8.5; (b) soil pH was significantly correlated with all explanatory variables at the 0.01 level. In general, MAP was the main factor affecting soil pH (r = -0.7244), followed by P/T (r = -0.6007). In the regions with MAP < 800 mm, soil pH was negatively correlated with MAP (r = -0.4631) and P/T (r = -0.7041), respectively, and positively correlated with MAT (r = 0.6093) and P*T (r = 0.3951), respectively. In the regions with MAP > 800 mm, soil pH was negatively correlated with MAP (r = -0.6651), MAT (r = -0.5047), P/T (r = -0.3268), and P*T (r = -0.5808), respectively. (c) The estimation model of soil pH was: y = 23.4572 - 6.3930 × lgMAP + 0.1312 × MAT. It has been verified to have a high accuracy (r = 0.7743, p < 0.01). The mean error, the mean absolute error, and the root mean square error were 0.0450, 0.5300, and 0.7193, respectively. It provides a new path for rapid estimation of the regional soil pH, which is important for improving the management of agricultural production and slowing down soil degradation.

Garmultin-A Incites Apoptosis in CB3 Cells Through miR-17-5p by Attenuating Poly (ADP-Ribose) Polymerase-1.

Background: Leukemia accounts for a large number of deaths, worldwide, every year. Treating this ailment is always a challenging job. Recently, oncogenic miRNA leading to apoptosis are highly promising targets of many natural products. In this study, Garmultin-A (GA), isolated from the bark of Garcinia multiflora, was elucidated for its anti-leukemic effect in CB3 cells. Methods: The effect of the compound on CB3 cell viability was detected by MTT assay and apoptosis by FITC Annexin V/PI and Hochest 33258 staining. The western blot analysis assessed the BAX, BCL2, cMYC, pERK, and PARP-1 protein levels. Autodock analysis predicted the ligand-protein interactions. q-RT-PCR quantified the miR-17-5p expression. Luciferase assay confirmed the interaction between PARP-1 and miR-17-5p. Results: We uncover that GA leads to apoptosis by inducing overexpression of miR-17-5p and significantly downregulate PARP-1 protein levels in CB3 cells. The overexpression of miR-17-5p promotes apoptosis, and the miR-17-5p antagomirs restore GA-triggered apoptosis. Notably, we disclose that PARP-1 is a direct target of miR-17-5p. Increased pro-apoptotic and reduced anti-apoptosis protein levels were also observed in GA-treated CB3 cells. Conclusion: These results provide critical insights that GA could induce apoptosis in CB3 cells through targeting miR-17-5p by attenuating PARP-1. Thus, GA could act as a novel therapeutic agent for erythroleukemia.

Integrating UPLC-HR-MS/MS, Network Pharmacology, and Experimental Validation to Uncover the Mechanisms of Jin'gan Capsules against Breast Cancer.

In the theory of traditional Chinese medicine (TCM), "liver-qi" stagnation and heat-induced toxicity represent the main etiologies of breast cancer. Recently, several TCMs with heat-clearing and detoxification efficacy have shown inhibitory effects on breast cancer. Jin'gan capsules (JGCs), initially approved to treat colds in China, are a heat-clearing and detoxification TCM formula. However, the anticancer activity of JGCs against breast cancer and its underlying mechanisms remain unclear. First, we assessed the antiproliferative activity of JGCs in breast cancer cell lines and evaluated their effects on cell apoptosis and the cell cycle by flow cytometry. Furthermore, we identified the potential bioactive components of JGCs and their corresponding target genes and constructed a bioactive compound-target interaction network by ultra-performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-HR-MS/MS) and network pharmacology analysis. Finally, the underlying mechanism was investigated through gene function enrichment analysis and experimental validation. We found that JGCs significantly inhibited breast cancer cell growth with IC50 values of 0.56 ± 0.03, 0.16 ± 0.03, and 0.94 ± 0.09 mg/mL for MDA-MB-231, MDA-MB-468, and MCF-7, respectively. In addition, JGC treatment dramatically induced apoptosis and S phase cell cycle arrest in breast cancer cells. Western blot analysis confirmed that JGCs could regulate the protein levels of apoptosis- and cell cycle-related genes. Utilizing UPLC-HR-MS/MS analysis and network pharmacology, we identified 7 potential bioactive ingredients in JGCs and 116 antibreast cancer targets. Functional enrichment analysis indicated that the antitumor effects of JGCs were strongly associated with apoptosis and the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. Western blot analysis validated that JGC treatment markedly decreased the expression levels of p-JAK2, p-STAT3, and STAT3. Our findings suggest that JGCs suppress breast cancer cell proliferation and induce cell cycle arrest and apoptosis partly by inhibiting the JAK2/STAT3 signaling pathway, highlighting JGCs as a potential therapeutic candidate against breast cancer.

Design, Synthesis, and Antileukemic Evaluation of a Novel Mikanolide Derivative Through the Ras/Raf/MEK/ERK Pathway.

Chronic myeloid leukemia (CML) accounts for a major cause of death in adult leukemia patients due to mutations or other reasons for dysfunction in the ABL proto-oncogene. The ubiquitous BCR-ABL expression stimulates CML by activating CDK1 and cyclin B1, promoting pro-apoptotic, and inhibiting antiapoptotic marker expression along with regulations in RAS pathway activation. Thus, inhibitors of cyclins and the RAS pathway by ERK are of great interest in antileukemic treatments. Mikanolide is a sesquiterpene dilactone isolated from several Asteraceae family Mikania sp. plants. Sesquiterpene dilactone is a traditional medicine for treating ailments, such as flu, cardiovascular diseases, bacterial infections, and other blood disorders. It is used as a cytotoxic agent as well. The need of the hour is potent chemotherapeutic agents with cytotoxic effects inhibition of proliferation and activation of apoptotic machinery. Recently, ERK inhibitors are used in clinics as anticancer agents. Thus, in this study, we synthesized 22-mikanolide derivatives that elucidated to be potent antileukemic agents in vitro. However, a bioactive mikanolide derivative, 3g, was found with potent antileukemic activity, through the Ras/Raf/MEK/ERK pathway. It can arrest the cell cycle by inhibiting phosphorylation of CDC25C, triggering apoptosis, and promoting DNA and mitochondrial damage, thus suggesting it as a potential chemotherapeutic agent for leukemia patients.

Germacranolide sesquiterpenes from Carpesium cernuum and their anti-leukemia activity.

In this study, three new germacranolide sesquiterpenes (1-3), together with six related known analogues (4-9) were isolated from the whole plant of Carpesium cernuum. Their structures were established by a combination of extensive NMR spectroscopic analysis, HR-ESIMS data, and ECD calculations. The anti-leukemia activities of all compounds towards three cell lines (HEL, KG-1a, and K562) were evaluated in vitro. Compounds 1-3 exhibited moderate cytotoxicity with IC50 values ranging from 1.59 to 5.47 µmol·L-1. Mechanistic studies indicated that 2 induced apoptosis by decreasing anti-apoptotic protein Bcl-2 and activating the caspase family in K562 cells. These results suggest that compound 2 is a potential anti-leukemia agent.

L20, a Calothrixin B analog, induces intrinsic apoptosis on HEL cells through ROS/γ-H2AX/p38 MAPK pathway.

Erythroleukemia is a malignant disease in the blood system. Quinones consists of a class of antitumor agents. Calothrixin B is a carbazole-1,4-quinone alkaloid isolated from Calothrix cyanobacteria with a unique indolo[3,2-j] phenanthridine framework. This study aimed to investigate the anti-leukemic effect of the new Calothrixin B derivative, L20, and to dig up the underlying mechanisms. Cytotoxicity analysis of L20 has revealed that it shows significant IC50 concentrations in HEL cells at low doses (1.10 ± 0.05 µM) than in K562, and KG-1a (5.46 ± 3.09, and 1.82 ± 1.08 µM respectively). The study even revealed that the L20 could induce a dose and time-dependent cellular death in HEL cells. The L20 increased expression of phospho-γ-H2A.X and phospho-p38 in HEL cells, causing DNA damage and nuclear alterations due to the G2/M phase cell cycle arrest. The HEL cells even lost the mitochondrial membrane potential (MMP) and resulted in the release of reactive oxygen species (ROS). Additionally, L20 inhibited the proliferation of HEL cells by inducing apoptosis through the mitochondrial pathway, depending on the caspase family. The study even established this may be due to the upregulation of the p-P38MAPK and downregulation of p-ERK. Pretreatment with P38/ERK inhibitors, SB203580, and U0126, decreased L20-induced apoptosis. These findings indicated that L20 induced mitochondrial mediated-apoptosis and G2/M arrest through DNA damage and modulation of p38 MAPK pathways. Thus, the study suggests L20, a chemical analog of Calothrixin B, as a novel chemotherapeutic agent against erythroleukemia.

Drying methods and structure-activity relationships of hydroxycinnamic acid derivatives in Idesia polycarpa Maxim. Leaves.

Idesia polycarpa Maxim. leaves are an excellent source of hydroxycinnamic acid derivatives and have drawn special attention due to their various biological activities. However, the effects of post-harvest treatment on the structure-activity relationships of hydroxycinnamic acid derivatives in leaves of I. polycarpa are still unknown. In the current study, we compared the contents of unstable compounds in leaves with four drying methods, namely sun-drying, freeze-drying, shade-drying, and oven-drying. We found that the four hydroxycinnamic acid derivative isomers of leaves were significantly affected after drying processing with four different drying methods. Consequently, the underlying mechanisms responsible for the variation of these compounds during the drying processes have been well elucidated: UV lighting induced the isomerization of 1-[(6'-O-(E)-p-coumaroyl)-ß-d-glucopyranosyl]-oxy-2-phenol (1) and 1-[(4'-O-(E)-p-coumaroyl)-ß-d-glucopyranosyl]-oxy-2-phenol (3) into 1-[(6'-O-(Z)-p-coumaroyl)-ß-d-glucopyranosyl]-oxy-2-phenol (2) and 1-[(4'-O-(Z)-p-coumaroyl)-ß-d-glucopyranosyl]-oxy-2-phenol (4). Also, heat (exceeding 20 °C) led to the rearrangement of the (E/Z)-p-coumaric acid moiety of compounds 3 and 4, of which the 4-O-acylglucoses changed into the 6-O-acylglucoses to generate compounds 1 and 2, respectively. Interestingly, the hepatocyte-free fatty acid accumulation in OA-induced steatosis-conditioned HepG2 cells decreased by 65.00%, 10.69%, and 47.00%, respectively, following treatment with compounds 2, 3 and 4, and compound 1 presented no lipid-lowering activity. In addition, the bioactivities of compounds 2 and 4 were substantially enhanced by 58.42% and 25.33% with the sun-drying method compared to the freeze-dying methods. Our study suggests that sun-drying processing is the best method among the four drying processing methods of I. polycarpa Maxim. leaves.

Paris Saponin VII Induces Apoptosis and Cell Cycle Arrest in Erythroleukemia Cells by a Mitochondrial Membrane Signaling Pathway.

BACKGROUND AND PURPOSE: Leukemia is considered a top-listed ailment, according to WHO, which contributes to the death of a major population of the world every year. Paris Saponin VII (PS), a saponin which was isolated from the roots of Trillium kamtschaticum, from our group, was reported to provide hemostatic, cytotoxic and antimicrobial activities. However, its molecular mechanism underlying the anti-proliferative effects remains unclear. Thus, this study hypothesized to assess that mechanism in PS treated HEL cells. METHODS: The MTT assay was used to analyze the PS inhibited cell viability in the HEL cells. We further found that PS could induce S phase cell cycle arrest through flow cytometry as well as the western blot analysis of intrinsic and extrinsic apoptotic molecules. RESULTS: The MTT assay showed the IC50 concentration of PS as 0.667µM. The study revealed that PS treatment inhibits cell proliferation dose-dependently. It further caused mitochondrial membrane potential changes by PS treatment. Mechanistic protein expression revealed a dose-dependent upsurge for Bid and Bim molecules, while Bcl2 and PARP expression levels were significantly (P<0.05) down-regulated in PS treated HEL cells resulting in caspase -3 release and increased the Bim levels upon 24h of incubation. CONCLUSION: These findings indicate that PS possesses an excellent anti-leukemic activity via the regulation of the mitochondrial pathway, leading to S phase cell cycle arrest and caspase-dependent apoptosis, suggesting it as a potential alternative chemotherapeutic agent for leukemia patients.

A natural small molecule induces megakaryocytic differentiation and suppresses leukemogenesis through activation of PKCδ/ERK1/2 signaling pathway in erythroleukemia cells.

Kaempferol-3-O-rhamnoside (KOR) has multiple potency involved in anti-cancer, anti-inflammatory and antibacterial actions. However, the potential roles of KOR and the analogues isolated from the leaves of Cyclocarya paliurus in anti-erythroleukemia remain unclear. In the present study, KOR and the two analogues (Kaempferol-3-O-(4″-O-acetyl-a-L-rhamnopyranoside) (KLR) and (kaempferol-3-O-α-L-(4″-E-p-coumaroyl) rhamnoside) (KCR) were isolated from leaves of Cyclocarya paliurus. Cell viability assay showed that KCR exerted an excellent anti-erythroleukemia activity. We observed that KCR not only significantly increased the percentage of G2 phase and apoptotic cells compared with control group, but also induced megakaryocytic differentiation in HEL and K562 cells by flow cytometry, indicating that KCR might inhibit cell proliferation through inducing differentiation-mediated apoptosis and cell cycle arrest. Mechanism investigation revealed that KCR treatment obviously increased phosphorylation levels of PKCδ and ERK1/2 as well as GATA1 expression. Taken together, these findings demonstrate that KCR induces megakaryocytic differentiation and suppresses leukemogenesis at least partly through activation of PKCδ/ERK1/2 signaling pathway in erythroleukemia cells. KCR may also serve as a promising natural compound for human erythroleukemia treatment.

BW18, a C-21 steroidal glycoside, exerts an excellent anti-leukemia activity through inducing S phase cell cycle arrest and apoptosis via MAPK pathway in K562 cells.

C-21 steroids displayed the activities of immunosuppressive, anti-inflammatory and anti-virus effects by the reports. However, its antitumor effects and molecular mechanism remain unclear. We previously isolated and identified a C-21 steroidal glycoside (BW18) from the root of Cynanchum atratum Bunge. This study was aimed to assess anti-leukemia activity and its underlying mechanism in K562 cells. MTT assay results showed that BW18 inhibited cell viability and proliferation of K562 cells. We also found that BW18 could induce S phase cell cycle arrest and apoptosis. Furthermore, our results demonstrated that BW18 regulated the expression of apoptosis and cell cycle related proteins. Mechanism investigation revealed that the anti-leukemia activity of BW18 may be mediated through MAPK pathway. These findings indicate that BW18 possesses an excellent anti-leukemia activity via regulating MAPK pathway leading to S phase cell cycle arrest and apoptosis, which suggested BW18 could be as a potential alternative therapeutic agent for CML patients.

UPLC-MS-MS Determination of Dihydrocodeine in Human Plasma and Its Application to a Pharmacokinetic Study.

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) method was developed to determine dihydrocodeine (DHC) in human plasma using diazepam as the internal standard (IS). Sample preparation was accomplished through a liquid-liquid extraction procedure with ethyl acetate. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by multiple reaction monitoring (MRM) of the transitions at m/z 302.3 → 199.2 for DHC and m/z 285.1 → 193.1 for IS. The linearity of this method was found to be within the concentration range of 0.5-100 ng/mL with a lower limit of quantification of 0.5 ng/mL. The overall run time was 4.0 min. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of DHC in healthy Chinese volunteers after oral administration.

Determination of kurarinone in rat plasma by UPLC-MS/MS.

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine kurarinone in rat plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid-liquid extraction procedure with ethyl acetate to 0.2 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 437.0→301.2 for kurarinone and m/z 168.1→132.1 for IS. The linearity of this method was found to be within the concentration range of 20-2000 ng/mL with a lower limit of quantification of 20 ng/mL. Only 3.0 min was needed for an analytical run. The matrix effect was 94.7-107.2% for kurarinone. The intra- and inter-day precision (RSD%) were less than 8.2% and accuracy (RE%) was within ±9.0%. The recovery ranged from 77.3% to 85.6%. Kurarinone was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of kurarinone in rats.

UPLC-MS/MS method for determination of avicularin in rat plasma and its application to a pharmacokinetic study.

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of avicularin in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile-methanol (9:1, v/v) to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 1.60 min and the elution of avicularin was at 1.20 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 434.1→301.3 for avicularin and m/z 237.2→194.3 for carbamazepine (IS), respectively. The calibration curve was linear over the range of 10-3000 ng/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. Mean recovery of avicularin in plasma was in the range of 84.2-89.5%. Intra-day and inter-day precision were both <12%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0mg/kg avicularin in rats.

[Chlorophyll content and net photosynthetic rate of Machilus pauhoi and M. leptophylla].

By using SPAD-502 chlorophyll meter, LI-6400 portable photosynthesis system, and spectrophotometer, the leaf SPAD value, net photosynthetic rate (Pn), and chlorophyll (a + b) content (Ct) of 3-year-old Machilus pauhoi and M. leptophylla seedlings were measured, and the relationships of SPAD value with Pn and Ct were analyzed. The M. pauhoi seedlings were grown from the seeds originated from Suichuan County of Jiangxi Province and Jian'ou County of Fujian Province, named as MPS and MPJ, respectively; while the M. leptophylla seedlings were grown from the seeds originated from Shangyou County of Jiangxi Province, named as MLG. There were significant differences in the mean chlorophyll content of MPS, MPJ, and MLG. The SPAD value and the contents of chlorophyll (a + b) (Ct), chlorophyll a (Ca) and chlorophyll b (Cb) were in the order of MPS < MLG < MPJ, with the mean SPAD value being 43.80, 45.12, and 50.67 and the Ct value being 1.944, 2.831, and 3.447 mg c g(-1), respectively. The chlorophyll content was influenced by the maturing degree of mesophyll tissues of M. pauhoi and M. leptophylla, being lower in current-year leaves than in 2-year-old leaves. The Ct of same age leaves at different crown layers of MPS and MPJ and of MLG was in the order of upper layer < middle layer < lower layer and of upper layer < lower layer < middle layer, respectively, and the SPAD value of the same lamina at different positions was in the order of apex < middle < base. SPAD value had a significant positive linear correlation with Ct, and a statistically not significant positive correlation with Pn.