DESC:DESCRIPTION:
Field of the invention:
[0001] The present disclosure pertains to the technical field of extracting chemical substances, specifically focusing on the extraction and isolation of a bioactive compound derived from the leaves of Vitex negundo, thereby demonstrating significant anti-cancer activity, particularly against A549 cell lines, a human lung carcinoma model and providing potential therapeutic applications in oncology.
Background of the invention:
[0002] Cancer is becoming prevalent not only in undeveloped countries but also in developing countries. The cancer death rate is alarming around the world. Several research groups are working on prevention, treatment, and finding natural drugs to avoid chemotherapeutic side effects in patients. Reasons to prevent cancer occurrence have not even been reached by the researchers, besides suggesting a systematic approach to life, which may not be assured again. However, there are many drugs identified against various cancers that are not effective in treating the cancer without any side effects. Further, research organizations are working on various natural anti-cancer products, including plant products that are abundantly available from natural resources. A number of studies represents the anti-cancer compounds against various cancers from different plant species.

[0003] Vitex negundo, commonly known as the five-leaved chaste tree or the Chinese chaste tree, is a medicinal plant native to Southeast Asia and commonly found in tropical and subtropical regions 1, 2. It belongs to the family Verbenaceae and is a small tree or shrub that can reach up to 8 metres in height. The plant has been traditionally used in Ayurveda, Siddha, and Unani medicine to treat a variety of ailments, including respiratory infections, fever, cough, and rheumatism. The leaves, flowers, and roots of Vitex negundo contain a range of phytochemicals, including flavonoids, terpenoids, and alkaloids, that have been shown to possess anti-inflammatory, analgesic, antimicrobial, and antitumor properties. Vitex negundo is considered safe for use in recommended doses and has been approved by the World Health Organization for use in traditional medicine.

[0004] Out of all cancers, lung cancer is the most dangerous and may not be detected in its early stages. It is difficult to treat lung cancer patients with a diagnosis of metastasis. There are identified drugs used for treating these cases, with a very low survival rate. The two ways that lung cancer patients can survive are by identifying the markers to detect in the early stages of cancer or finding an effective drug even at the metastasis stage. Since there is no indication from the cancer tissue in the lungs, finding early-stage markers is difficult. Nature has a treasure trove of plants holding various types of compounds with anti-cancer activity. Screening these natural compounds can help find effective lung cancer drugs. In this series, our research group hypothesized to isolate and characterize the compounds that are present in Vitex negundo for their lung cancer preventive activity.

[0005] By addressing all the above-mentioned problems, there is a need for a bioactive compound for anti-cancer activity against human lung cancer cells. There is also a need for a bioactive compound for an anti-cancer activity, which have no side effects on other human organs. There is also a need for a bioactive compound for anti-cancer activity, which is less expensive and can be easily made a formulated drug by medicinal chemistry for early-stage intervention.
Objectives of the invention:
[0006] The primary objective of the present invention is to provide a bioactive compound that shows anti-cancer activity on lung cancer A549 cell lines.

[0007] Another objective of the present invention is to provide a bioactive compound that exhibits significant anti-cancer activity.

[0008] Another objective of the present invention is to provide a bioactive compound for an anti-cancer activity, which have no side effects on normal cells in the human body.

[0009] Further objective of the present invention is to provide a bioactive compound for the treatment of cancer by natural extraction of leaves of Vitex negundo, and the particular bioactive compound can be made and will be provided through medicinal chemistry for future generations.
Summary of the invention:
[0010] The present disclosure proposes a bioactive compound for an anti-cancer activity. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0011] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a bioactive compound from leaves extracts of Vitex negundo for anti-cancer activity on A549 human lung cancer cell.

[0012] According to one aspect, the invention provides a bioactive compound for an anti-cancer activity. Initially, one or more solvent extracts of Vitex negundo leaves are extracted using an ultra-assisted extraction procedure in order to obtain one or more bioactive compounds. The one or more solvent extracts are isolated and purified using PREP-HPLC followed by a lyophilization process for obtaining the bioactive compound Isoorientin, which exhibits significant anti-cancer activity on specific lung hypotriploid alveolar basal epithelial cells (A549) moreover, It’s also demonstrated for first time the cytotoxic activity of obtained Isoorientin from the Vitex negundo against HEK293 cells. The results demonstrate the nontoxic to human organs, suggesting that Isoorientin may act as a potential beneficial molecule in A549.

[0013] The one or more solvent extracts are subjected to preparative high-performance liquid chromatography (PREP-HPLC) for isolated based on their IC50 values. The solvents were labelled as VN-Hexane-extract, VN-Chloroform- extract, VN-Ethyl acetate- extract, and VN-ETOH+H2O- extract. The VN-ETOH+H2O- extract have IC50 value of (31.88±0.362) against lung hypotriploid alveolar basal epithelial cells (human lung adenocarcinoma cell line) A549 among the other solvent extracts. The VN-ETOH+H2O-extract is purified by reverse phase PREP-HPLC to get pure bioactive compounds. The reverse phase PREP-HPLC with 0.1% Formic acid and 100% Acetonitrile, C18 column (Kromasil C18 (150 X 25mm, 7µm) is used to obtain the bioactive compounds. The obtained bioactive compounds labelled as VN-EtOH+H2O-P1, VN-EtOH+H2O-P2, VN-EtOH+H2O-P3, VN-EtOH+H2O-P4.

[0014] The obtained four pure bioactive compounds are elucidated and identified their molecular structure through spectral (NMR, LC-MS, and FTIR) analysis, the four pure bioactive compounds are 4-OH Benzoic acid (VN-ETOH+H2O-P1), Negundoside (VN-ETOH+H2O-P2), Isoorientin (VN-ETOH+H2O-P3), Agnuside (VN-ETOH+H2O-P4). The VN-ETOH+H2O-P3-Isoorientin exhibits anti-tumor activity against the lung epithelial cell line (A549) and it has the significant anti-cancer property.

[0015] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0016] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0017] FIGs. 1A-1D illustrate cell viability graphs for a crude extract for VN-Hexane, VN-Chloroform VN-Ethyl acetate, VN-EtOH+H2O microscopic images of its action on A549 cell lines (5 to 300µg/ml), in accordance to an exemplary embodiment of the invention.

[0018] FIG. 2 illustrates microscopic images of VN-EtOH+H2O (1:1) Extract action on A549 cell lines (5 to 300µg/ml), in accordance to an exemplary embodiment of the invention.

[0019] FIG. 3 illustrates a graph representing LC-MS data for VN-EtOH +H2O (1:1) extract, in accordance with an exemplary embodiment of the invention.

[0020] FIG. 4 illustrates a graph representing the purification of VN-EtOH +H2O (1:1) extract, in accordance to an exemplary embodiment of the invention.

[0021] FIGs. 5A-5D illustrate molecular structures of obtained bioactive molecules of VN-EtOH +H2O-P1 (4-OH Benzoic acid). VN-EtOH +H2O-P2 (Negundoside), VN-EtOH +H2O-P3 (Isoorientin), and VN-EtOH +H2O-P4 (Agnuside), in accordance to an exemplary embodiment of the invention.

[0022] FIGs. 6A-6D illustrate cell viability graphs of hydro-alcoholic (1:1) fractionates VN-Ethanol+H2O-P1 (4-OH Benzoic acid), VN-Ethanol+H2O-P2 (Negundoside), VN-Ethanol+H2O-P3 (Isoorientin), VN-Ethanol+H2O-P4 (Agnuside), in accordance to an exemplary embodiment of the invention.

[0023] FIG. 7 illustrates microscopic images of Isoorientin VN-EtOH+H2O-P3 action on A549 cell lines (5 (11.1µM) µg/mL to 300 (669µM) µg/mL), in accordance to an exemplary embodiment of the invention.

[0024] FIGs. 8A-8B illustrate graphs representing cell line HDF cells treated post 48 hr treatment, the viability percentage graphs for VN-EtOH+H2O-P3- Isoorientin, Doxorubicin used as standard control, in accordance to an exemplary embodiment of the invention.

[0025] FIGs. 9A-9B illustrate the microscopic images for VN-EtOH+H2O-P3- Isoorientin treated HDF cells post 48 hr treatment and the microscopic images for Standard control Doxorubicin treated HDF cells post 48 hr treatment, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0026] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0027] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a bioactive compound from leaves extracts of Vitex negundo for anti-cancer activity on A549 cell lines. The bioactive compound from Vitex negundo leaves extraction exhibits significant anti-cancer property, it showed anti-cancer activity against lung epithelial cells (A549 cell line) and the bioactive compound has no side effects on human organs.

[0028] According to one exemplary embodiment of the invention, FIGs. 1A-1D refer to cell viability graphs (100, 102, 104, 106) for a crude extract for VN-Hexane, VN-Chloroform VN-Ethyl acetate, VN-EtOH+H2O microscopic images of its action on A549 cell lines (5 to 300µg/ml). Generally, Vitex negundo belongs to the family of verbenaceae and is popularly known as sambhaloo or Chinese chaste tree. It is a widely used Indian medicinal shrub to prevent a variety of ailments including cancer. Initially, four solvent extracts of Vitex negundo leaves are extracted using an ultra-assisted extraction procedure. The VN-EtOH+H2O-extract purified through PREP-HPLC followed by a lyophilization process and obtained a bioactive compound Isoorientin, which exhibits significant anti-cancer activity on lung epithelial A549 cell line and cytotoxicity of Isoorientin has been evaluated with HDF. The results demonstrate the non-toxic to human organs, it is suggested that Isoorientin may act as a beneficial molecule and be used therapeutically.

[0029] The method of extracting bioactive compound from one or more solvent extracts of Vitex negundo comprises, initially 1 lit of hexane mixed in 400g of Vitex negundo leaf powder and mixed for 30 min by a stirrer to form a consolidated solution. The obtained solution is subjected to UAE procedure, by ultrasonic waves penetrate leaf and are scattered and absorbed within the tissue to get phytochemicals.

[0030] Next, the solution is filtered through vacuum using a Buchner funnel to obtain filtrate solution and solid residue were separated. Next, the filtrate solution is concentrated by a buchi rotavapor at a temperature of 30°C and 100 ppm vacuum pressure. The process is repeated for the other solvent extracts like ethyl acetate, chloroform, and ethanol-water (1:1) respectively to obtain extracts of them.

[0031] Cytotoxicity of the one or more solvent extracts of Vitex negundo leaf powder is evaluated with A549 human lung epithelial cell line. The anticancer activity of the crude extracts is tested. The concentrations (5µg/ml to 300µg/ml) by using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The MTT colorimetric assay is an established method of determining viable cell number in proliferation and cytotoxicity studies for assessing cell metabolic activity.

[0032] Based on the IC50 values, the extract that have anticancer activity is subjected to further purification procedures to obtain purified bioactive compounds through a reverse phase PREP-HPLC followed by lyophilization process. Lyophilization is a process of making a powder material from liquid fraction, and subjecting the powder to evaporation of water at -80°C under vacuum (sublimation). IC50 represents the concentration at which a substance exerts half of its maximal inhibitory effect. This value is typically used to characterize an antagonist of a biological process.

[0033] The purification steps comprised of subjecting the crude extract to a preparative-scale high performance liquid chromatography (PREP-HPLC) based on IC50 values, thereby obtaining isolated compounds through a lyophilization process. The structure of the isolated bioactive compounds are characterized using 1H nuclear magnetic resonance (1H-NMR), Carbon-13 nuclear magnetic resonance (13C-NMR), homonuclear correlation spectroscopy (COSY), Heteronuclear Multiple Bond Correlation (HMBC), heteronuclear single quantum coherence (HSQC), Nuclear overhauser Effect Spectroscopy (NOESY), Fourier transform infrared spectroscopy (FTIR), and liquid chromatography-mass spectrometer (LCMS) methods. In one embodiment herein, the four solvents were characterized as a VN-hexane-extract, a VN-chloroform- extract, a VN-ethyl acetate- extract, a VN-ETOH+H2O- extract, respectively. In one embodiment herein, the cytotoxic effects of four solvent extracts (hexane, chloroform, ethyl acetate, and water-ethanol) derived from VN (Vitex negundo) are evaluated against a specific cell line to determine their half-maximal inhibitory concentration (IC50) and maximum inhibition percentages.

[0034] In one embodiment herein, the hexane extract of VN demonstrated an IC50 value of 63.03 µg/mL with a maximum inhibition of 77.4%. The dose-response curve indicates a gradual decline in cell viability with increasing concentrations of the extract, reflecting the cytotoxic potential of the hexane extract. In one embodiment herein, the chloroform extract showed an IC50 value of 79.12 µg/mL, achieving a maximum inhibition of 82.83%. The dose-response relationship, as observed in the graph, illustrates that cell viability decreases as the concentration of the chloroform extract increases, suggesting its effectiveness in reducing cell viability at higher concentrations.

[0035] In one embodiment herein, the ethyl acetate extract, the IC50 value was found to be 65.70 µg/mL, with a maximum inhibition of 82.83%. The dose-response curve reflects a significant cytotoxic effect, demonstrating the extract’s ability to inhibit cell viability effectively at concentrations near its IC50. In one embodiment herein, the water-ethanol extract exhibited the highest cytotoxicity among the tested extracts, with an IC50 value of 44.31 µg/mL and a maximum inhibition rate of 80.52%. The dose-response curve for this extract reveals a steep decline in cell viability as concentration increases, indicating a potent cytotoxic response.

[0036] Each dose-response graphs (100, 102, 104, 106) illustrate the relationship between the log concentration of each extract and the percentage of cell viability, thereby allowing for a comparison of their inhibitory effects on cell growth. The IC50 values suggest varying levels of potency across the different extracts, with the water-ethanol extract being the most effective, followed by the hexane, ethyl acetate, and chloroform extracts. The maximum inhibition percentages reflect the peak cytotoxic effects achieved by each extract. Cell line A549 cells treated with solvent extracts are shown in Table 1.

[0037] Table 1: Cell line A549 cells treated with solvent extracts showing the IC50 values as follow in the table provided (5 to 300µg/mL).
S. No. Compounds
(Extracts) IC50(µM) Max Inhibition
1 VN-Hexane 63.03 82.83
2 VL-Chloroform 79.12 77.4
3 VN-Ethyl acetate 65.70 82.83
4 VN-H20+Ethanol 44.31 80.52

[0038] According to another exemplary embodiment of the invention, FIG. 2 refers to microscopic images 200 of VN-EtOH+H2O (1:1) extract action on A549 cell lines (5 to 300µg/ml). The microscopic images 200 illustrate the morphological changes in cells treated with various concentrations of a compound (VN-EtOH+H2O-P1) over a range of 5 to 300 µg. At 5 µg low concentration, the cells still maintain a relatively normal appearance. There are no significant changes in cell morphology compared to the control. At 10 µg, some minor changes in cell morphology may be observed. Some cells may appear slightly rounded or less elongated compared to the control. However, the overall cell morphology remains largely intact. At 25 µg, as the concentration increases to 25 µg, the cells start to exhibit more pronounced changes. Some cells may appear rounded or shrunken, and there may be evidence of cell detachment from the surface. However, many cells still retain a relatively normal morphology.

[0039] At 50 µg, the cytotoxic effects of the compound become more evident. The cells exhibit significant morphological alterations, such as cell shrinkage, rounding, and membrane blebbing. There is also evidence of cell death, as indicated by the presence of cellular debris. At 100 µg, the cells are severely affected by the compound. The majority of the cells are rounded and shrunken, and there is extensive cell death. The remaining cells may exhibit abnormal morphologies, such as membrane blebbing and cytoplasmic vacuolation. At 150 µg, the cells are almost completely destroyed. The majority of the cells are dead or dying, and the remaining cells exhibit severe morphological alterations. The overall image is characterized by cellular debris and a lack of intact cells.

[0040] At 200 µg, 250 µg, and 300 µg, as the concentration increases, the cytotoxic effects become even more pronounced. At these concentrations, the cells are almost completely destroyed, with only a few remaining cells exhibiting severe morphological alterations. The microscopic images 200 are dominated by cellular debris and a lack of intact cells. The microscopic images 200 demonstrate a concentration-dependent cytotoxic effect of VN-EtOH+H2O-P1 on the cells. At low concentrations, the compound has minimal effects on cell morphology, while at higher concentrations, it induces significant cell death and morphological alterations.

[0041] In one embodiment herein, the crude Ethanol-Water (1:1) extract (VN-ETOH+H2O) effectively inhibited the proliferation of A549 cells as compared to other solvent extracts. Therefore, for further analysis Ethanol-Water (1:1) extracts are used for purification studies. Accordingly, four bioactive compounds were isolated from the VN-ETOH+H2O extract using PREP-HPLC. The RT’S (min) 1.28, 1.68, 1.75 and 1.85 respective peaks obtained via LCMS. The bioactive compounds are subjected to structure confirmation using NMR, LCMS and FTIR analytical technics. The VN-ETOH+H2O-extract is purified by reverse phase PREP-HPLC to obtain pure compounds. The Reverse phase PREP-HPLC with 0.1% Formic acid and 100% Acetonitrile; C18 column (Kromasil C18 (150 X 25mm, 7µm) is used to get the bioactive compounds. The obtained bioactive compounds labelled as VN-EtOH+H2O-P1, VN-EtOH+H2O-P2, VN-EtOH+H2O-P3, VN-EtOH+H2O-P4.

[0042] According to another exemplary embodiment of the invention, FIG. 3 refers to a graph 300 representing LC-MS data for VN-EtOH +H2O (1:1) extract. The graph 300 illustrate the mass spectra of four different compounds, labeled as VN-EtOH+H2O (P1), VN-EtOH+H2O (P2), VN-EtOH+H2O (P3), and VN-EtOH+H2O (P4). These spectra were obtained using a mass spectrometer, which ionizes molecules and measures their mass-to-charge ratio (m/z). The peaks in the spectra correspond to different ions generated from the compounds. The m/z value of each peak represents the mass-to-charge ratio of the ion. The intensity of a peak is proportional to the abundance of the corresponding ion in the sample. The peak with the highest m/z value often corresponds to the molecular ion, which represents the intact molecule with an added charge.

[0043] VN-EtOH+H2O (P1) spectrum shows a prominent peak at m/z 136.99, which likely corresponds to the molecular ion [M-H]-. The peak intensity suggests that this ion is relatively abundant in the sample. VN-EtOH+H2O (P2) spectrum shows a peak at m/z 495.36, which likely corresponds to the molecular ion [M-H]-. The peak intensity suggests that this ion is relatively abundant in the sample. VN-EtOH+H2O (P3) spectrum shows a peak at m/z 447.30, which likely corresponds to the molecular ion [M-H]-. The peak intensity suggests that this ion is relatively abundant in the sample. VN-EtOH+H2O (P4) spectrum shows a peak at m/z 465.32, which likely corresponds to the molecular ion [M-H]-. The peak intensity suggests that this ion is relatively abundant in the sample. The mass spectra provide information about the molecular weights of the four compounds. The m/z values of the molecular ion peaks can be used to calculate the molecular weight of each compound. The relative intensities of the peaks can provide information about the abundance of different ions in the sample.

[0044] In one embodiment herein, the formic acid is easily volatile on evaporation process either by lyophilization (or) Rotavapors. The formic acid may not form the salt formation with structure. Formic acid may not desecrate the original structure of extracted components. Formic acid can ionize the molecules during the MS-Directed auto purification to collect or to get the component fractions by their respective mass i.e., m/z value. In one embodiment herein, the obtained pure bioactive compounds from the VN-ETOH+H2O-extract are characterized through spectral analysis and named as VN-ETOH+H2O-P1 (4-OH Benzoic acid), VN-ETOH+H2O-P2 (Negundoside), VN-ETOH+H2O-P3 (Isoorientin), VN-ETOH+H2O-P4 (Agnuside). The obtained pure bioactive compounds are identified by LCMS and NMR (With 2D-NMR) analysis as shown in below Table 2.

[0045] Table 2: The identification of VN-EtOH+ H2O fractionates and its obtained weights from PREP-HPLC.
S. No Obtained compound name After identification by NMR and LCMS compound name Molecular weight Obtained quantity by RP-PREP-HPLC
[M-H]- (mg)
1 VN-EtOH+ H2O (P1) 4-OH Benzoic acid 136.99 140
2 VN-EtOH+ H2O (P2) Negundoside 495.36 50
3 VN-EtOH+ H2O (P3) Isoorientin* 447.3 80
4 VN-EtOH+ H2O (P4) Agnuside 465.32 100

[0046] According to another exemplary embodiment of the invention, FIG. 4 refers to a graph 400 representing the purification of VN-EtOH +H2O (1:1) extract. In one embodiment herein, the chromatographic analysis of the water-ethanol extract of VN (Vitex negundo) is performed using high-performance liquid chromatography (HPLC) to identify major peaks representing specific compounds. The analysis is conducted at a wavelength of 254 nm, with the absorbance measured in milliampere units (mA) over an elapsed time period.

[0047] In one embodiment herein, at peak at 136.99 m/z ([M-H]?) is observed at 9.88 minutes with an m/z value of 136.99, indicating the presence of a compound in the water-ethanol extract. This peak was labeled as P1 (VN EtOH + H2O P1) and likely corresponds to a low molecular weight compound due to the smaller m/z value. The moderate absorbance reflects a noticeable concentration of this compound within the extract.

[0048] In one embodiment herein, the peak at 495.36 m/z ([M-H]?) is detected at 13.67 minutes with an m/z value of 495.36, marked as P2 (VN EtOH + H2O P2). This peak represents a compound with a higher molecular weight, suggesting the presence of a potentially complex molecule within the extract. The absorbance is higher than that of P1, indicating a greater abundance of this compound in the sample. In one embodiment herein, the peak at 447.30 m/z ([M-H]?) is observed at 14.20 minutes with an m/z value of 447.30, labeled as P3 (VN EtOH + H2O P3). The appearance of this peak at a later retention time and its molecular weight suggests a compound with moderate polarity. The peak’s absorbance is similar to that of P2, indicating a comparable concentration within the extract.

[0049] In one embodiment herein, the peak at 465.32 m/z ([M-H]?) is detected at 15.82 minutes with an m/z value of 465.32, marked as P4 (VN EtOH + H2O P4). The retention time suggests this compound is less polar than the earlier compounds, and the relatively high absorbance indicates it is present in a significant amount within the sample. Each identified peak represents a distinct compound in the water-ethanol extract, with the differences in retention time and m/z values reflecting variations in polarity and molecular weight. The chromatogram provides a detailed profile of the chemical constituents in the VN extract, aiding in the identification and potential isolation of bioactive compounds. The observed peak patterns and intensity at 254 nm provide insights into the abundance and composition of these compounds in the VN water-ethanol extract.

[0050] According to another exemplary embodiment of the invention, FIGs. 5A-5D refer to molecular structures (500, 502, 504, 506) of obtained bioactive molecules of VN-EtOH +H2O-P1 (4-OH Benzoic acid) 500, VN-EtOH +H2O-P2 (Negundoside) 502, VN-EtOH +H2O-P3 (Isoorientin) 504, and VN-EtOH +H2O-P4 (Agnuside) 506. The VN-EtOH+H2O-P1 identified as 4-OH Benzoic acid it was a crystalline powder, MP 188 - 193°C. Exact Mass: 138.12g/mol HRMS: 137.0445[M-H]-, C7H5O3 Mass-137.05 [M-H]-, IR Spectrum bands showed the stretching of -OH group (3394.72cm-1), -C=O group (1681.93cm-1), -C-O group (1238.30cm-1). 1H and 13C NMR data. In one embodiment herein, the VN-EtOH+H2O-P2 identified as Negundoside it was a crystalline powder, MP 148 - 152°C. Exact Mass: 496.5g/mol; HRMS: 495.1518[M-H], C23H27O12 Mass-495.15 [M-H]-, IR absorption bands showed the stretching of -OH group (3257.77cm-1), -C=O group (1720.50cm-1), -C-O group (1269.16cm-1) and Carboxylic-OH (2872.01). 1H and 13C NMR data.

[0051] In one embodiment herein, the VN-EtOH+H2O-P3 identified as Isoorientin it was an off-white crystalline powder, MP 148 - 152°C. Exact Mass: 448.38g/mol; HRMS: 447.2279[M-H], C23H27O12 Mass-447.23 [M-H]-, IR absorption bands showed the stretching of -OH group (3379.29cm-1), aryl -C-C (1614.42cm-1 and 1490.97cm-1) -C-O-C- group (1080.10cm-1), 1H and 13C NMR data. In one embodiment herein, the VN-EtOH+H2O-P4 identified as Agnuside it was an off-white crystalline powder, MP 145 - 149°C. Exact Mass: 466.44g/mol, HRMS: 465.2046[M-H]-, C22H26O1 Mass-447.23 [M-H]-, IR absorption bands showed the stretching of -OH group (3379.29cm-1), C=O group (1701.22cm-1), aryl -C-C (1602.85cm-1 and 1450.47cm-1) -C-O-C- group (1130.29cm-1), 1H and 13C NMR data.

[0052] In one embodiment herein, 1 H and 13C NMR spectral data for the compounds VN-EtOH+H2O-P1 and EtOH+H2O-P2 is shown in Table 3.

[0053] Table 3: 1 H and 13C NMR spectral data for the compounds VN-EtOH+H2O-P1 and VN-EtOH+H2O-P2.
4-hydroxy Benzoic acid (VN-EtOH+H2O-P1) Negundoside (VN-EtOH+H2O-P2)
Atom No Type of Atom 1H Chemical Shift (PPM) Coupling Const(J) 13C Chemical Shift (PPM) Atom No Type of Atom 1H Chemical Shift (PPM) Coupling Const(J) 13C Chemical Shift (PPM)
1, 5 CH 7.79(d, 8.5Hz, 2H) 131.4 1 CH 3.2(t, 8.5Hz, 1H) 70.12
2, 4 CH 6.82(d, 8.5Hz, 2H) 114.99 2 CH 3.29(m, 1H) 77.43
3 C - 161.42 3 O - -
6 C - 122.17 4 CH 4.84(d, 8.0Hz, 1H) 95.94
7 C - 167.54 5 CH 4.70(t, 8.5Hz, 1H) 73.39
8 O - - 6 CH 3.49(t, 8.5Hz, 1H) 74.14
9 OH 12.0(hump, 1H) - 7 OH 5.28(hump, 1H) -
8 O - -
9 OH 5.2(hump, 1H) -
10 CH2 3.74(d, 11.3Hz, 1H) 3.50(dd, 11.3, 6.5Hz, 1H) 60.83
11 OH 4.63(hump, 1H) -
12 O - -
13 CH 5.29(d, 2.0Hz, 1H) 93.46
14 O - -
15 CH 7.03(s, 1H) 148.72
16 C - 112.29
17 CH 2.73(m, 1H) 29.84
18 CH 1.97(m, 1H) 50.54
19 C - 167.27
20 O - -
21 OH 11.0(hump, 1H) -
22 CH2 2.03(m, 1H) 1.25(m, 1H) 28.98
23 CH2 1.50(m, 2H) 39.78
24 C - 77.75
25 CH3 1.13(s, 3H) 24.23
26 OH 4.62(s, 1H) -
27 C - 164.68
28 C - 120.65
29 O - -
30, 34 CH 7.72(d, 8.0Hz, 2H) 131.26
31, 33 CH 6.80(d, 8.0Hz, 2H) 115.08
32 C - 161.54
35 OH 10.22(hump, 1H) -

[0054] In one embodiment herein, 1 H and 13C NMR spectral data for the compounds VN-EtOH+H2O-P3 and VN-EtOH+H2O-P4 is shown in Table 4.

[0055] Table 4: 1 H and 13C NMR spectral data for the compounds VN-EtOH+H2O-P3 and VN-EtOH+H2O-P4.

Atom No Type of Atom 1H Chemical Shift (PPM) Coupling Constant (J) 13C Chemical Shift (PPM) Atom No Type of Atom 1H Chemical Shift (PPM) Coupling Constant (J) 13C Chemical Shift (PPM)
Isoorientin (VN-EtOH+H2O-P3) Agnuside (VN-EtOH+H2O-P4)
1 CH 3.12 (t, 9.0Hz, 1H) 70.59 1 CH 3.04 (td, 9.0, 5.0Hz, 1H) 70.03
2 CH 3.20 (t, 8.5Hz, 1H) 78.92 2 CH 3.10 (dd, 9.5, 5.6Hz, 1H) 77.17
3 CH 4.04 (d, 9.0Hz, 1H) 70.16 3 O - -
4 CH 4.59 (d, 9.8Hz, 1H) 73.01 4 CH 4.53 (d, 7.8Hz, 1H) 98.29
5 O - - 5 CH 2.99 (td, 8.6, 5.0Hz, 1H) 73.33
6 CH 3.17 (m, 1H) 81.55 6 CH 3.16 (td, 9.0, 4.6Hz, 1H) 76.61
7 C - 108.85 7 CH2 3.39 (dt, 11.0, 5.6Hz, 1H); 3.65 (dd, 11.0, 5.6Hz, 1H) 61.11
8 OH 4.67 (hump, 1H) - 8 OH 4.38 (t, 5.6Hz, 1H) -
9 CH2 3.70 (d, 11.0Hz, 1H); 3.41 (dd, 11.0, 5.7Hz, 1H) 61.46 9 OH 4.94 (d, 5.0Hz, 1H) -
10 OH 4.67 (hump, 1H) - 10 OH 4.96 (d, 5.0Hz, 1H) -
11 OH 4.67 (hump, 1H) - 11 OH 4.98 (d, 5.0Hz, 1H) -
12 OH 4.67 (hump, 1H) - 12 O - -
13 C - 160.65 13 CH 4.86 (d, 7.0Hz, 1H) 95.76
14 C - 103.36 14 O - -
15 C - 156.16 15 CH 6.37 (d, 6.0Hz, 1H) 140.34
16 CH 6.47 (s, 1H) 93.46 16 CH 5.07 (dd, 5.5, 4.2Hz, 1H) 104.65
17 C - 163.6 17 CH 2.56 (m, 1H) 44.81
18 OH 13.56 (s, 1H) - 18 CH 2.83 (t, 7.5Hz, 1H) 46.69
19 OH 9.81 (hump, 1H) - 19 CH 4.35 (t, 5.0Hz, 1H) 80.58
20 C - 181.83 20 CH 5.80 (s, 1H) 132.28
21 CH 6.67 (s, 1H) 102.76 21 C - 139.8
22 C - 163.6 22 OH 5.16 (d, 5.6Hz, 1H) -
23 O - - 23 CH2 4.88 (d, 14.5Hz, 1H); 4.90 (d, 14.5Hz, 1H) 62.04
24 O - - 24 O - -
25 C - 121.38 25 C - 165.13
26 CH 7.40 (d, 2.0Hz, 1H) 113.27 26 C - 120.18
27 C - 145.72 27 O - -
28 C - 149.69 28, 32 CH 7.87 (d, 8.5Hz, 2H) 131.54
29 CH 6.90 (d, 8.2, 1H) 116.02 29, 31 CH 6.87 (d, 8.5Hz, 2H) 115.37
30 CH 7.42 (dd, 8.2, 2.0Hz, 1H) 118.94 30 C - 162.05
31 OH 9.81 (hump, 1H) - 33 OH 10.29 (broad hump, 1H) -
32 OH 9.81 (hump, 1H) -

[0056] According to another exemplary embodiment of the invention, FIGs. 6A-6D refer to cell viability graphs (600, 602, 604, 606) of hydro-alcoholic (1:1) fractionates VN-Ethanol+H2O-P1 (4-OH Benzoic acid), VN-Ethanol+H2O-P2 (Negundoside), VN-Ethanol+H2O-P3 (Isoorientin), VN-Ethanol+H2O-P4 (Agnuside). The viability dose of the bioactive compound VN-EtOH+H2O-P3- Isoorientin on A549 cell lines at 5 (11.1µM) µg/mL to 300 (669µM) µg/mL) concentrations. Among the obtained bioactive compounds of Ethanol-Water- extract of Vitex negundo the VN-Ethanol-Water-P3 (VN-EtOH+H2O-P3- Isoorientin) purified compound showed significant anticancer activity against the A549 cells lines. Cell lines are found to have IC50 value of 18.50µM as compared to other bioactive compounds tested (as shown in Table 5). The VN-ETOH+H2O-P3-Isoorientin shows anti-tumor activity against the lung epithelial cell line (A549) and exhibits significant anti-cancer properties. The viability graph representing IC50 value of the bioactive compound VN-EtOH+H2O-P3- Isoorientin at 5 (11.1µM) µg/mL to 300 (669µM) µg/mL).

[0057] According to another exemplary embodiment of the invention, FIG. 7 refers to microscopic images 700 of Isoorientin VN-EtOH+H2O-P3 action on A549 cell lines (5 (11.1µM) µg/mL to 300 (669µM) µg/mL). In one embodiment herein, the microscopic images 700 illustrate the morphological changes in cells treated with various concentrations of a compound (VN-EtOH+H2O-P1) over a range of 5 to 300 µg/mL.

[0058] In one embodiment herein, at 5 µg/mL (11.1 µM), the cells appear to have a normal morphology with intact cell membranes and distinct cell boundaries. There is no visible evidence of cell death or significant morphological alterations. At 10 µg/mL (22.3 µM), the cells still maintain a relatively normal appearance, although some minor changes may be observed, such as slight cell shrinkage or rounding. However, the overall cell morphology remains largely intact.

[0059] At 25 µg/mL (55.7 µM), as the concentration increases to 25 µg/mL, the cells start to exhibit more pronounced changes. Some cells may appear rounded or shrunken, and there may be evidence of cell detachment from the surface. However, many cells still retain a relatively normal morphology. At 50 µg/mL (111.5 µM), the cytotoxic effects of the compound become more evident. The cells exhibit significant morphological alterations, such as cell shrinkage, rounding, and membrane blebbing. There is also evidence of cell death, as indicated by the presence of cellular debris.

[0060] At 100 µg/mL (223 µM), the cells are severely affected by the compound. The majority of the cells are rounded and shrunken, and there is extensive cell death. The remaining cells may exhibit abnormal morphologies, such as membrane blebbing and cytoplasmic vacuolation. At 150 µg/mL (334.5 µM) (high concentration), the cells are almost completely destroyed. The majority of the cells are dead or dying, and the remaining cells exhibit severe morphological alterations. The overall image is characterized by cellular debris and a lack of intact cells.

[0061] At 200 µg/mL (446 µM), 250 µg/mL (557.5 µM), and 300 µg/mL (669 µM), as the concentration increases further, the cytotoxic effects become even more pronounced. At these concentrations, the cells are almost destroyed, with only a few remaining cells exhibiting severe morphological alterations. The microscopic images 700 are dominated by cellular debris and a lack of intact cells. The microscopic images 700 demonstrate a concentration-dependent cytotoxic effect of VN-EtOH+H2O-P1 on the cells. At low concentrations, the compound has minimal effects on cell morphology, while at higher concentrations, it induces significant cell death and morphological alterations. The Cell line A549 cells treated Hydro alcoholic fractionates of (5 (11.1µM) µg/mL to 300 (669µM) µg/mL) are shown in Table 5.

[0062] Table 5: Cell line A549 cells treated Hydro alcoholic fractionates showing the IC50 values as follows in the table provided (5 (11.1µM) µg/mL to 300 (669µM) µg/mL)).
Compounds IC50(µM/mL) Max Inhibition
VN-EtOH+H2O-P1 (4-OH Benzoic acid) 51.4 77.33
VN-EtOH+H2O-P2 (Negundoside) 21.32 68.91
VN-EtOH+H2O-P3 (Isoorientin) 18.50 71.84
VN-EtOH+H2O-P4 (Agnuside) 83.06 76.43

[0063] According to another exemplary embodiment of the invention, FIGs. 8A-8B refer to graphs (800, 802) representing cell line HDF cells treated post 48 hr treatment, the viability percentage graphs for VN-EtOH+H2O-P3- Isoorientin, Doxorubicin used as standard control. The graphs (800, 802) refer to the viability graph representing IC50 value (416.57±0.01) of the bioactive compound VN-EtOH+H2O-P3- Isoorientin on HDF cell lines at 5 (11.1µM) µg/mL to 300 (669µM) µg/mL) concentrations and Doxorubicinn standard used as a standard control (IC50 value (19.8±0.01)) The graphs (800, 802) present the results of an MTT assay conducted on HDF cells treated with a test compound and Doxorubicin (a standard chemotherapeutic drug) at various concentrations. The assay measures cell viability by assessing the ability of cells to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a colored formazan product.

[0064] The graph 800 represents test compound-treated HDF cells, the graph 800 shows a dose-dependent decrease in cell viability with increasing concentrations of the test compound. The curve is linear, indicating a consistent cytotoxic effect of the compound. The IC50 value (concentration required to inhibit cell growth by 50%) can be estimated from the graph. It represents the potency of the compound in inducing cell death. The maximum inhibition achievable by the compound can also be inferred from the graph. It represents the highest level of cell killing that can be induced by the compound.

[0065] The graph 802 represents doxorubicin treated HDF cells. Similar to the test compound, Doxorubicin also exhibits a dose-dependent decrease in cell viability. However, the curve is slightly less steep, suggesting a slightly lower potency compared to the test compound. The IC50 value for Doxorubicin can be estimated from the graph. It is likely to be higher than that of the test compound, indicating a lower potency. The maximum inhibition achievable by Doxorubicin can also be inferred from the graph. It may be slightly lower than that of the test compound. Based on the IC50 values, the test compound appears to be more potent than Doxorubicin in inducing cell death in HDF cells. The test compound may also exhibit a higher maximum inhibition compared to Doxorubicin. Overall, the results suggest that the test compound has a significant cytotoxic effect on HDF cells and may be a promising candidate for further investigation as a potential anticancer agent.

[0066] In one embodiment herein, the bioactive compounds VN-EtOH+H2O-P1, VN-EtOH+H2O-P2, VN-EtOH+H2O-P3, and VN-EtOH+H2O-P4 isolated from Vitex negundo have great potential to act against the lung cancer cell lines. Out of four tested VN-EtOH+H2O-P3-isoorientin is more powerful to show apoptotic activity than the other three compounds. Releasing drugs of this kind in the market will help lung cancer patients to survive and allow them to lead a normal life after treatment with fewer side effects. The compound VN-EtOH+H2O-P3 of Vitex negundo has great potential in the treatment of lung cancer in the future. The results indicate that the purified product VN-EtOH+H2O-P3 of EtOH+H2O-leaf extract of V. negundo shows significant anticancer activity against the lung cell lines, A549 based on marked inhibition of cancer cell line. Further, the toxicity of the compound shows a low level of cytotoxicity even at the highest concentration against the HDF cells (as shown in Table 6).

[0067] Table 6. Cell line HDF cells treated post 48 hr treatment, the IC50 values as follow in the table provided (a) VN-EtOH+H2O-P3- Isoorientin (b) Doxorubicin used as standard control.

[0068] (a):
Test Compound Treated HDF Cells Post 48 Hr Treatment
Concentration (µM) Cell Viability % IC50 (µM)
Control 100 416.57 ± 0.01
11.1 88.48
22.3 88.73
55.7 83.02
111.5 83.02
223 55.51
334.5 53.60
446 44.41
557.5 41.84
669 30.24
[0069] (b):
Doxorubicin treated HDF cells-standard control post 48 Hr treatment
Concentration (µM) Cell Viability % IC50 (µM)
Control 100 19.8 ± 0.01
5 53.36
10 50.87
25 47.38
50 46.63

[0070] Isoorientin is treated with HDF (human dermal fibroblast) cell lines at different concentrations (Concentrations were 5 (11.1µM) µg/mL to 300 (669µM) µg/mL). The IC50 value of 416.57 ± 0.01 µM (as shown in Table 6) for the VN-EtOH-H2O-P3 compound with HDF cells over a 48-hour treatment and Doxorubicin was used for standard control for the analysis the IC50 value of 19.8 ± 0.01 µM (as shown in Table 6), indicating its cytotoxicity threshold. This value suggests that at higher concentrations (closer to the IC50), the compound does begin to have a notable cytotoxic effect on HDF cells, but below this threshold, it appears to be non-toxic. Additionally, the presence of Isoorientin in VN-EtOH-H2O-P3, proven to be non-toxic, strengthens the safety profile, at least in vitro.

[0071] According to another exemplary embodiment of the invention, FIGs. 9A-9B refer to microscopic images (900, 902) for VN-EtOH+H2O-P3- Isoorientin treated HDF cells post 48 hr treatment and the microscopic images for Standard control Doxorubicin treated HDF cells post 48 hr treatment. In one embodiment herein, the microscopic images 900 illustrate the morphological changes in cells treated with various concentrations of a compound (VN-EtOH+H2O-P1) over a range of 11.1 to 669.0 µM.

[0072] In one embodiment herein, control (10X), the control image shows a monolayer of cells with a normal morphology. The cells have a typical elongated shape with well-defined cell boundaries. At 11.1 µM (10X) the cells still maintain a relatively normal appearance. There are no significant changes in cell morphology compared to the control. At 22.3 µM (10X) some minor changes in cell morphology may be observed. Some cells may appear slightly rounded or less elongated compared to the control. However, the overall cell morphology remains largely intact. At 55.7 µM (10X), as the concentration increases to 55.7 µM, the cells start to exhibit more pronounced changes. Some cells may appear rounded or shrunken, and there may be evidence of cell detachment from the surface. However, many cells still retain a relatively normal morphology.

[0073] At 111.5 µM (10X), the cytotoxic effects of the compound become more evident. The cells exhibit significant morphological alterations, such as cell shrinkage, rounding, and membrane blebbing. There is also evidence of cell death, as indicated by the presence of cellular debris. At 223 µM (10X), the cells are severely affected by the compound. The majority of the cells are rounded and shrunken, and there is extensive cell death. The remaining cells may exhibit abnormal morphologies, such as membrane blebbing and cytoplasmic vacuolation. At 334.5 µM (10X), the cells are almost completely destroyed. The majority of the cells are dead or dying, and the remaining cells exhibit severe morphological alterations. The overall image is characterized by cellular debris and a lack of intact cells. At 446.0 µM (10X), 557.5 µM (10X), and 669.0 µM (10X), as the concentration increases further, the cytotoxic effects become even more pronounced. At these concentrations, the cells are almost completely destroyed, with only a few remaining cells exhibiting severe morphological alterations. The images are dominated by cellular debris and a lack of intact cells.

[0074] In one embodiment herein, the microscopic images 902 illustrate the morphological changes in cells treated with various concentrations of a compound (VN-EtOH+H2O-P1) over a range of 5 to 100 µM. At control (10X), the control image shows a monolayer of cells with a normal morphology. The cells have a typical elongated shape with well-defined cell boundaries. At 5 µM (10X), the cells still maintain a relatively normal appearance. There are no significant changes in cell morphology compared to the control.

[0075] At 10 µM (10X), some minor changes in cell morphology may be observed. Some cells may appear slightly rounded or less elongated compared to the control. However, the overall cell morphology remains largely intact. At 25 µM (10X), as the concentration increases to 25 µM, the cells start to exhibit more pronounced changes. Some cells may appear rounded or shrunken, and there may be evidence of cell detachment from the surface. However, many cells still retain a relatively normal morphology. At 50 µM (10X), the cytotoxic effects of the compound become more evident. The cells exhibit significant morphological alterations, such as cell shrinkage, rounding, and membrane blebbing. There is also evidence of cell death, as indicated by the presence of cellular debris. At 100 µM (10X), the cells are severely affected by the compound. The majority of the cells are rounded and shrunken, and there is extensive cell death. The remaining cells may exhibit abnormal morphologies, such as membrane blebbing and cytoplasmic vacuolation.

[0076] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure a bioactive compound from Vitex Negundo leaves, is disclosed. The proposed invention provides a bioactive compound from leaves extract of Vitex negundo for anti-cancer activity on A549 cells. The proposed invention provides a bioactive compound from leaves extract of Vitex negundo exhibits significant anti-cancer property. The bioactive compound from leaves extract of Vitex negundo have no side effects on normal cells. Isoorientin is a bioactive compound that has anti-cancer properties that can be formulated into a drug through medicinal chemistry.

[0077] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
,CLAIMS:CLAIMS:
I/We Claim:
1. A bioactive compound for an anti-cancer activity, comprising:
one or more solvent extracts extracted from the leaves of Vitex negundo using an ultra-assisted extraction procedure for obtaining one or more bioactive compounds, wherein. the solvent extracts are isolated using a reverse phase PREP-HPLC followed by lyophilization process for obtaining at least one bioactive compound Isoorientin, which exhibits significant anti-cancer activity on specific lung hypotriploid alveolar basal epithelial cells (human lung adenocarcinoma cell line) A549 and cytotoxic activity of obtained isoorientin is evaluated by toxicity analysis on HDF cells to prove that the Isoorientin is not toxic to human organs.
2. The bioactive compound as claimed in claim 1, wherein the solvent extracts of Vitex negundo leaves includes chloroform, hexane, ethyl acetate, and ethanol-water (1:1).
3. The bioactive compound as claimed in claim 1, wherein the solvents are characterized as a VN-hexane-extract, a VN-chloroform- extract, a VN-ethyl acetate- extract, a VN-ETOH+H2O- extract, respectively.
4. The bioactive compound as claimed in claim 3, wherein the VN-ETOH+H2O- extract have IC50 value of 44.31µg against human lung adenocarcinoma cell line (A549) among the other solvent extracts.
5. The bioactive compound as claimed in claim 3, wherein the VN-ETOH+H2O-extract is purified by reverse phase PREP-HPLC to obtain one or more bioactive compounds, wherein the obtained pure bioactive compounds are tested and labelled as VN-EtOH+H2O-P1, VN-EtOH+H2O-P2, VN-EtOH+H2O-P3, VN-EtOH+H2O-P4.
6. The bioactive compound as claimed in claim 1, wherein the solvent extracts are subjected to preparative high performance liquid chromatography (PREP-HPLC) for isolation and purification based on IC50 values.
7. The bioactive compound as claimed in claim 1, wherein the obtained bioactive compounds are characterized through spectral analysis and labelled as VN-ETOH+H2O-P1 (4-OH Benzoic acid), VN-ETOH+H2O-P2b(Negundoside), VN-ETOH+H2O-P3 (Isoorientin), VN-ETOH+H2O-P4 (Agnuside).
8. The bioactive compound as claimed in claim 1, wherein the anti-cancer activity of solvent extracts are tested in different concentrations using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.
9. The bioactive compound as claimed in claim 1, wherein the obtained Isoorientin exhibits anti-tumor activity against human lung adenocarcinoma cell line A549 and exhibits significant anti-cancer property.
10. The bioactive compound as claimed in claim 1, wherein the reverse phase PREP-HPLC with 0.1% Formic acid and 100% Acetonitrile, C18 column (Kromasil C18 (150 X 25mm, 7µm) is used for purification of extracts to get one or more bioactive compounds.