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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                                                                                                    Research Article 

Chemo protective activity of Phyllanthus niruri and Opuntia ficus indica plant extract

Krati Sharma*, Rajeev Malviya, Vishal Gupta

School of Pharmacy, Mansarovar Global University, Kolar road, Bhopal 462042, Madhya Pradesh, India

INTRODUCTION   

Cancer is one of the most dreaded diseases threatening the health conditions of mankind. It is among the killer diseases in Nigeria, and a name associated with a groupof diseases in which the body cells divide, multiply and spread uncontrollably ¹. They may also beregarded as a large family of diseases which show features suggestive of malignancy. They form a subsep of neoplasm. The disease causes about 13% of all world death ². Worldwide, approximately 18% of cancer deaths are related to infections, such as hepatitis B, hepatitis C, and human papilloma virus. This proportion varies in different regions of the world from a high of 25% in Africa to less than 10% in the developed world. Deaths from cancer worldwide are projected to continue rising with an estimated 12 million deaths in 2030; case reports from 2007 indicated that about 7.6 million people died from cancer in the world ³. In 2012, about 14.1 million new cases of cancer occurred globally. It caused about 8.2 million deaths or 14.6% of all human deaths ⁵.

MATERIAL AND METHODS 

Plant Extract Collection

Detail of Plant Extract

Animal:

The animal experimental protocol was approved by the Department of Pharmacy, Mansarovar University Bhopal Institutional Animals Ethical Committee (IAEC), Deshpande Laboratories Pvt. Ltd. Bhopal (M.P.)

CPCSEA Registration no: 1582/PO/Re/11/CPCSEA

In Vitro Study: Treatment groups for the individual study, in vitro: Activity of Opuntia ficus indica fruits extract and Phyllanthus niruri herb on NSCLC cells, A549 and H52 A549 and H522 cells were treated with different concentrations of Opuntia ficus indica fruits extract and Phyllanthus niruri herb. Representative groups which were maintained for this study are as follows:

Group 1: Negative control (untreated) This group received no drug or alcohol. A549 and H522 cells were cultured in DMEM and RPMI-1640, respectively, supplemented with 10% heat inactivated FBS and 1% antibiotic antimicotic solution, maintained at 37^oC with 5% CO2 in a humidified incubator.

Group 2 & 3: Opuntia ficus indica fruits extract and Phyllanthus niruri herb –treated A549 and H522 cells were cultured in DMEM and RPMI-1640, respectively. Cells were treated with different concentrations of Opuntia ficus indica fruits extract and Phyllanthus niruri herb for 24h and 48h. For Opuntia ficus indica fruits extract and Phyllanthus niruri herb on NSCLC cell line, H460 H460 & HCT15cells were treated with different concentrations of CGS. Representative groups maintained for this study are as follows:

Group 1: Negative control (untreated) This group received no drug or alcohol. H460 & HCT15cells were cultured in DMEM and RPMI-1640, respectively, supplemented with 10% heat inactivated FBS and 1% antibiotic antimicotic solution, maintained at 37^oC with 5% CO2 in a humidified incubator.

Group 2 & 3: Opuntia ficus indica fruits extract and Phyllanthus niruri –treated H460 & HCT15cells were cultured in DMEM and RPMI-1640, respectively. Cells were treated with different concentrations of Opuntia ficus indica fruits extract and Phyllanthus niruri herb for 24h and 48h.

Determination of cell viability by MTT assay Principle

This is a colorimetric assay that measures the reduction of yellow coloured MTT by mitochondrial succinate dehydrogenase. The MTT enters into the cells and passes to the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent (isopropanol) and the released, solubilised formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells. Cell viability was assessed using MTT followed by the method described by ⁷. NSCLC cells (1x10⁵cells/ml) were cultured for 24h on 96-well micro plates. The cells were incubated for different time-points for different modes of studies with and without drugs.

Materials for MTT

Freshly prepared MTT (5mg/ml in PBS) was filtered and kept in dark, at 4ºC. Acidic isopropanol (0.1N HCl in absolute isopropanol) 96 flat bottom well plate. Cell viability was assessed using MTT followed by the method described by ⁸.

DPPH and Hydroxyl (OH) radical scavenging assay Activity Opuntia ficus indica fruits extract and

Phyllanthus niruri in Lung Cancer cell lines Principle

DPPH and Hydroxyl (OH) catalyses the oxidation of lactate to pyruvate accompanied by the simultaneous reduction of NAD to NADH. DPPH AND HYDROXYL (OH) activity in any protein content is proportional to the increase in absorbance due to the reduction of NAD. Working reagent 1.1ml of DPPH and hydroxyl (OH) (buffered substrate) was added to the vial labelled with 1DPPH and hydroxyl (OH) (co-enzyme), mixed well to dissolve and kept for 5min prior to use.

Observation of cell morphology by light microscopy (LM)

The changes in cell morphology were checked by phase contrast light microscope (Axiscope plus 2, Zeiss, Germany) following the method ⁹.

By scanning electron microscopy (SEM) Principle

A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that can be detected which contain the information about the sample's surface topography and composition. The electron beam is generally scanned in a raster scan pattern, and the beam's position is combined with the detected signal to produce an image. SEM can achieve resolution better than 1nm. Specimens can be observed in high vacuum, in low vacuum, and (in environmental SEM) in wet conditions. The most common mode of detection is by secondary electrons emitted by atoms excited by the electron beam. By scanning the sample and detecting the secondary electrons, an image displaying the tilt of the surface is created. To study the surface exposure and/or any deformity superficially of any unsectioned specimen, scanning electron microscopy is used.

RESULTS AND DISCUSSION:

In Vitro Antitumor Evaluation by MTT assay                                   

Antitumor evaluation of extracts

Opuntia ficus indica and Phyllanthus niruri ethanol extracts were subjected to MTT assay. Different cell lines used were cultured in A459, H522, H460 cells. The results are depicted in the TABLE 1: It is very clear from the results that Opuntia ficus indica and Phyllanthus niruriare showing comparative activity comparative to control treated group.

TABLE 1: The IC50 value of extracts by MTT assay

Antitumor evaluation of fractions: By first fractionation of the ethanolic extract of Opuntia ficus indica (OFI) and Phyllanthus niruri (PN) five different fractions namely OFI1 to OFI5 and PN1 to PN5. All the fractions were subjected to in vitro antitumor evaluation against the cancer cell lines A459, H522, H460. From the TABLE 2: Clear that PN3 and OFI4 showed better activity at 100µg/ml on different cell lines. Table 3 and Fig.1, Fig.2, show the result of Opuntia ficus indica and Phyllanthus nirurifractions.

Antitumor evaluation of isolated components: The compounds isolated from Opuntia ficus indica and Phyllanthus niruri respectively. They showed the better activity was exhibited by OFI4 of Opuntia ficus indica and PN3 of Phyllanthus niruri. It is shown in the TABLE 2: and graphically shown in the Fig.1.

TABLE 2: The percentage inhibition by MTT assay of the fractions (Opuntia ficus indica & Phyllanthus niruri) at 100µg/ml

TABLE 3: The percentage inhibition by MTT assay of the fractions from Opuntia ficus indica and Phyllanthus niruriat 200 and 100µg/ml

Figure 1: The percentage inhibition by MTT assay of the fraction Opuntia ficus indica at 200µg/ ml

A549 H522                             H460            HPN15             HeLa

Cell lines used

Figure 2: The percentage inhibition by MTT assay of the fraction Phyllanthus niruri at 100µg/ ml

A549                  H522           H460         HPN15

Cell lines used

The following figure represents the pathophysiology of cell lines. 

Figure 3: (a) indicates A549 control Figure 3(b): indicates A549 cell lines treated with OFI4 and Figure 3(c): indicates A549 cells treated with PN3.

Figure 4: (a) Indicates H522 control, Figure 4: (b) Indicates H522 cell lines treated with OFI4 and Figure 4: (c) Indicates H522cells treated with PN3. Figure 5: (a) Indicates H460 control Figure 5: (b) Indicates H460 cell lines treated with OFI4 Figure 5: (c) Indicates H460 treated with PN3.

Figure 3: A549 control and the cells treated with the isolated compounds (MTT assay)

Figure 4: H522 control and the cells treated with the isolated compounds (MTT assay)

Figure 5: The H460 control and the cells treated with the isolated compounds (MTT assay)

1. H460 Control                             (b) H460OFI4                 (c) H460PN3

In vitro antitumor evaluation by Lactate Dehydrogenase Assay of isolated compounds:The isolated components from Phyllanthus niruri PN1, PN2, PN3, PN4, and from Opuntia ficus indica, OFI1, OFI2, OFI3 and OFI4 were subjected to Lactate Dehydrogenase Assay. The cell lines used were A459, H522, and H460 at 100µg/ml. The results are showed in the TABLE 4: It is represented graphically in the figure6.18. In Lactate Dehydrogenase Assay, nuclear condensation is taking place and the dead cells become shrinked and the percentage inhibition can be calculated by counting the number of nor OFIl cells and shrinked cells. The following figure represents the pathophysiology of cell lines. Fig.6: (a) Indicates A549 control, Fig.6: (b) indicates A549 cells treated with OFI4 Figure Fig.5: (c) indicates A549 cells treated with PN3. Fig.6: (a) indicates H522 control, Fig.6: (b) indicates H522 cells treated with OFI4 and Fig.6: (c) indicates H522 cells treated with PN3. Fig.7: (a) indicates H460 control, Fig.7: (b) indicates H460 cells treated with OFI4 and Fig.7: (c) indicates H460 cells treated with PN3. The results were comparable with the results obtained by MTT assay.

TABLE 4: The percentage inhibition by Lactate Dehydrogenase Assayof the isolated compounds at 100µg/ml

Figure 6: A549 control and the cells treated with the isolated compounds (Lactate Dehydrogenase Assay)

(a) H522 Control                   (b) H522OFI4                       (c) H522PN3

Figure 7: H522 control and the cells treated with the isolated compounds (Lactate Dehydrogenase Assay)

a) H460 Control                       (b) H460OFI4                        (c) H460PN3

Figure 8: The HPN15 control and the cells treated with the isolated compounds (MTT assay)

In Vivo Study

In vivo antitumor evaluation of the fractions OFI4 and PN3

The in vivo evaluation was done using PN3 fraction obtained from Phyllanthus niruri and OFI4 fraction obtained from Opuntia ficus indica . Two different models, solid tumour study and ascites tumour analysis were done. Dalton’s lympho OFI ascites (DLA) cell lines were used for the study. Both the fractions showed marked activity against cancer since there is marked reduction in tumour volume in case of solid tumour study. It is represented in the TABLE 6 Also there is increase in life span of mice in case of ascites tumour study. It is shown in the TABLE 5.

Ascites tumour model

Effect on tumour growth response

Body weight: Tumour development was noticed from day 6 and there was a gradual rise in body weight due to accumulation of ascites fluid in the peritoneum of EAC mice, especially in the positive control group (45.54±.66), whereas a significant reduction of gain in body weight was found in the standard (cisplatin) treated groups (7.12±.09). On comparing the extract treated groups, all groups exhibited significant differences in a dose-dependent OF Inner and OFI 200 mg/kg showed a maximum decrease in body-weight (19.38±.19). Both extract groups showed significant differences of gain in body weight when compared with the positive control group indicating the effectiveness of the test extracts. Cisplatin (Standard) treated group caused 91% decrease in body weight, while group IV and V OFI (200mg/kg) and PN (100mg/kg) registered only 60.95 and 54.24% respectively TABLE 6. A regular sudden rise in ascites tumour volume was seen in EAC bearing hosts. The direct nutritional source of tumour cells is the ascites fluid and perhaps the sudden rise in ascites fluid with tumour growth could be a way to meet the nutritional needs of tumour cells. ¹⁰

Effect on mean survival time (MST) and percentage increase in life span (% ILS)

Mean survival time and percentage increase in life span are two important parameters used for screening of any anticancer agent. A compound can be considered as cytotoxic if it exhibits more than 25% increase in percentage life span. A significant drop in MST was observed in EAC induced mice (14 days) when compared with standard mice (33 days). In the control group, on day 15 the first death was reported andall mice were dead by day 18. However, cisplatin caused a significant increase in the MST (33 days) and % ILS (115) when compared to the vehicle group. Both petroleum and ethanol extracts significantly improved the MST and all groups exhibited a dose-dependent increase in the life span (Figure 5.1f). Among the six treatments, the petroleum ether extract of OFI200 mg/kg was found to be the most effective percentage inhibition of 81.8% (TABLE 5).

TABLE 5: Effect of different concentrations of petroleum ether and ethanol extracts of Opuntia ficus indica and Phyllanthus niruri gain in body weight, MST and % ILS.

n=6, values are expressed as mean ± SEM. Statistica l analyses were done by one-way ANOVA followed by Tukey’s multiple comparison tests. ^xp <0.001, ^yp<0.01and ^zp<0.05 as compared to control, ^a p<0.001, ^b p<0.01 and ^c p<0.05 as compared to standard, ns – non significant.

Tumour volume and packed cell volume:

At the doses of 200 mg/kg OFI4treated groups significantly (p<0.001 and p<0.001) reduced tumour volume and packed cell volume in a dose dependent Manner compared with that of the EAC control. All groups showed significant differences in tumour volume and packed cell volume, when compared with the positive control group except low dose of the ethanol extract of OFI4 (TABLE 6)

TABLE 6: Effect of petroleum ether and ethanol extracts of Opuntia ficus indica and Phyllanthus niruri on tumour growth response of EAC inoculated Swiss albino mice.

n=6, values are expressed as mean ± SEM. Statistica l analyses were done by one-way ANOVA followed by Tukey’s multiple comparison tests. ^x p <0.001, ^yp<0.01 and ^zp<0.05 as compared to control, ^a p<0.001 ^b p<0.01 and ^c p<0.05 as compared to standard, ns- non significant.

The reliability criterion for judging the value of any anticancer drug is the prolongation of life span of the ani OFI (Clarkson D. & Burchneal JH. 1965) and reduction of WBC (Obeling et.al., 1954). The OFI4 and PN3 have decreased the ascites fluid volume. In all OFI4 treated groups viable cell count was greatly reduced in a dose dependent OFI. In contrast, in the treated groups compared with EAC control hosts the percentage of trypan blue positive dead tumour cells (non-viable) also increased. It OFI y be concluded that OFI4 caused a decrease in the fluid volume by arresting the tumour growth and thereby increased the life span of EAC bearing mice.

Solid tumour model

Effect of ethanol extracts of Opuntia ficus indica and Phyllanthus niruri on solid tumour development in DLA inoculated mice.

Tumour volumes of mice treated with different doses of extracts were found significantly lower than that of control group. The average tumour volume of the vehicle control group on 30^th day was 3.21±0.02 mm³. Animals treated with extracts of Opuntia ficus indica 200 mg/kg and Phyllanthus niruri 100 mg/kg for a period of 30 consecutive days showed the reduction in tumour volume to 1.88±0.03 mm³, 1.26±0.04mm³ respectively. The standard drug (cisplatin 3.5 mg/kg) was found to be efficient (1.26±0.04 mm³) in preventing the development of DLA induced tumour as shown in Figure 5.7. Treatment with Opuntia ficus indica and Phyllanthus niruri also revealed a decrease in the tumour volume as compare to Positive control respectively. These observations revealed that the Opuntia ficus indica and Phyllanthus niruri had the ability to inhibit the tumour progression.

Percentage inhibition of tumour weight and tumour volume

To confirm these findings, the tumour weight after excision was also recorded. A significant reduction in average tumour weight was found in the treated Animals compared to the control Animals (TABLE 7). There was a reduction in tumour weight of Opuntia ficus indica 200 mg/kg and Phyllanthus niruri 100 mg/kg treated Animals (77.54% and 75.42%) and was comparable with that of standard cisplatin (95%).

TABLE 7: Effect of Opuntia ficus indica and Phyllanthus niruri on percentage inhibition of tumour weight and tumour volume of DLA induced Swiss albino mice.

N=6, values expressed as mean ± SEM

Effect on mean survival time (MST) and percentage increase in life span (% ILS)

Mean survival time and percentage increase in life span are two important parameters used for screening of any anticancer agent. A compound can be considered as cytotoxic if it exhibits more than 25% increase in percentage life span. A significant drop in MST was observed in EAC induced mice (15.6 days) when compared with standard mice (20 days). In the control group, on day 15 the first death was reported and all mice were dead by day 19. However, cisplatin caused a significant increase in the MST (22 days) and % ILS (58) when compared to the vehicle group. Both petroleum and ethanol extracts significantly improved the MST and all groups exhibited a dose-dependent increase in the life span. Among the six treatments, the petroleum ether extract of OFI 200 mg/kg was found to be the most Effective showing a percentage inhibition of 48.8% and survival time is (21 days)

TABLE 8: Effect on mean survival time (MST) and percentage increase in life span (% ILS) on solid tumour study of Opuntia ficus indica and Phyllanthus niruri

CONCLUSION

The in vitro anticancer activity of the ethanolic extracts of the four plants was carried out using three different cell lines, A549, H522, and H460 cells. Based on the results two plants, Phyllanthus Niruri and, Opuntia ficus-indica were selected for further study.IC50 value of the most active extract, the ethanol extract of Phyllanthus Niruri and, Opuntia ficus-indica was found to be 32.88, 38.80, 42.77 µg/mL A459, H522, H460, and, Opuntia ficus-indica was found to be 40.00, 42.77, 45.88 µg/mL also increased the mean life span and exhibited 71% of tumour inhibition. The active component isolated (Phyllanthus Niruri and, Opuntia ficus-indica) from the same could bring down the IC50 values to 10 and 15 µ g/mL.

Under the phytochemical investigation, the plant extracts were subjected to fractionation by column chromatography. five different fractions PN1 to 5 from Phyllanthus Niruriand OFI1 – OFI5 from Opuntia ficus-indica were obtained. Among the Phyllanthus Niruri fractions, PN4 and among the Opuntia ficus-indica OFI3 fractions, were found to be the more active fractions based on the in vitro anticancer evaluation and were selected for further column chromatographic fractionation.Again they were subjected to in vitro anticancer evaluation and it was found that the PN4 and OFI3 were comparatively the more active fractions. From the gas chromatography of these fractions it was clear that they contain four components each and by preparative thin layer chromatography all the eight components were separated. They were again subjected to the in vitro anticancer evaluation by MTT assay. PN4 and OFI3 were found to be more active. These selected components were subjected to further characterization to identify the structure of the compounds.

The in vivo study was also carried out using the PN4 and OFI3 fractions by solid tumour and ascites tumour study using the DLA cell lines. The plant fractions were also exhibited better anticancer activity. Effect of different doses of Phyllanthus Niruri and, Opuntia ficus-indica on solid tumour development, percentage inhibition of tumour growth and histopathological studies were also assessed.Phyllanthus Niruri (100mg/kg) and, Opuntia ficus-indica (200 mg/kg) exhibited 77% and 55% inhibition in tumour growth, whereas in the standard drug (cisplatin) caused an inhibition of 95%. Histopathological studies revealed the presence of fibrous cells. Absence of necrosis, inflammatory changes and blood vessels in the high dose treated group proved its efficacy as an anticancer agent. Hence we selected petroleum ether extract of Phyllanthus Niruri and Opuntia ficus-indica for the isolation of the active compounds.

AKNOWLEDGEMENT:

All authors are thankful to the School of Pharmacy, Mansarovar Global University, Bhopal, Madhya Pradesh for providing the needs of experiment like laboratory and library facilities.

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