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Antitumor activity of Albizia lebbeck L. against Ehrlich ascites carcinoma in vivo and HeLa and A549 cell lines in vitro


1 Department of Pharamcology, Gitam Institute of Pharmacy, GITAM (Deemed to be University), Rushikonda, Visakhapatnam, Andhra Pradesh, India
2 Department of Pharmacology, Nirmala College of Pharmacy, Atmakur, Mangalagiri, Guntur, Andhra Pradesh, India

Date of Submission27-Jun-2019
Date of Acceptance21-Jan-2020
Date of Web Publication09-Oct-2020

Correspondence Address:
Chilaka Naga Kavitha,
Department of Pharmacology, Gitam Institute of Pharmacy, GITAM (Deemed to be University), Rushikonda Beach Road, Visakhapatnam - 530 045, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_454_19

 > Abstract 


Aim of the Study: The aim of the present study was to explore the antitumor activity of the ethanolic extract of Albizia lebbeck L. pods against Ehrlich ascites carcinoma (EAC) in Swiss albino mice and its cytotoxic effect against HeLa and A549 cell lines in vitro.
Materials and Methods: Antitumor activity of ethanolic extract of A. lebbeck L. (ALEE) pods was evaluated in Swiss albino mice against EAC cell lines at the doses of 200 and 400 mg/kg body weight which were given by intraperitoneal route of administration and was compared with 5-fluorouracil (5-FU), the reference standard. The extract and 5-FU were administered for 14 consecutive days. After 24 h of the last dose and 18 h of fasting, the mice were sacrificed and the antitumor effect of ALEE was assessed by evaluating tumor volume, viable and nonviable tumor cell count, increase in life span, and hematological parameters of EAC-bearing hosts.In vitro cytotoxicity has been assessed using (2,3-bis[2-Methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt assay method and was compared with cisplatin, the reference standard.
Results: ALEE showed direct cytotoxicity on EAC cells in a dose-dependent manner. ALEE exhibited a significant (P < 0.001) decrease in the body weight, tumor volume, viable cell count, tumor weight, and elevated the life span of EAC tumor-bearing mice. Hematological profile such as red blood cell, hemoglobin, white blood cell, and platelet count was reverted to the normal level in ALEE-treated mice.
Conclusion: The results showed that the ethanolic extract of A. lebbeck L. has a powerful antitumor activity because it was effective in significantly inhibiting the tumor growth in both in vivo and in vitro cancer cell lines.

Keywords: 5-fluorouracil, A549 cell lines, Albizia lebbeck L., cytotoxicity, Ehrlich ascites carcinoma cell lines, HeLa cell lines



How to cite this URL:
Kavitha CN, Raja K D, Rao S K. Antitumor activity of Albizia lebbeck L. against Ehrlich ascites carcinoma in vivo and HeLa and A549 cell lines in vitro. J Can Res Ther [Epub ahead of print] [cited 2020 Oct 27]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=297623




 > Introduction Top


Over the past decade, herbal medicines have been appreciated and accepted all over the world and they have made an impact on both global health and international trade. Hence, medicinal plants continue to play an important role in the health-care system of a majority of the world's population.[1] A traditional Indian medical practice using plant drugs has been successful from very early times in using these natural drugs and preventing or suppressing various tumors with different lines of treatment.[2] In India, people of different ethnic groups inhabiting various terrains possess their own distinct culture, religious rites, food habit, and a rich knowledge of traditional medicine.[3] They practice herbal medicine to cure a variety of diseases. Natural products, especially plants, have been used in the treatment of various diseases for thousands of years. However, only a few medicinal plants have attracted the interest of scientists to investigate the remedy for neoplasm (tumor or cancer).

Cancer is one of the most common life-threatening diseases, with more than 100 different types occurring due to some molecular changes within the cell. It is the third leading cause of death worldwide following cardiovascular and infectious diseases.[4] Cancer is caused by both external factors (tobacco, chemicals, radiation, and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). Due to lack of effective drugs, cost of chemotherapeutic agents, and the side effects of anticancer drugs, cancer can be a cause of death. Therefore, efforts are still being made to search for effective naturally occurring anticarcinogens that would prevent, slow, or reverse cancer development. Medicinal plants have a special place in the management of cancer. It is estimated that plant-derived compounds in one or the other way constitute more than 50% of anticancer agents.[5],[6] Chemoprevention is recognized as an important approach to control malignancy and recent studies have focused on the search for desirable chemopreventive agents. Natural products, particularly dietary substances, have played an important role in creating new chemopreventive agents.[7] Numerous cancer research studies have been conducted using traditional medicinal plants in an effort to discover new therapeutic agents that lack the toxic side effects associated with the present chemotherapeutic agents.

The plant, Albizia lebbeck L., East Indian walnut in English, belongs to the family Mimosaceae and is used in folk and traditional medical practice in India. It is considered a potent alexipharmic, and every part of it is used for the treatment of bites and stings from venomous animals. Its leaves are used in ophthalmic diseases, night blindness, syphilis and ulcer,[8] cold, cough, and respiratory disorders. The bark is bitter, cooling, alexiteric, and anthelmintic, and it also cures diseases of blood, leukoderma, itching, skin disease, piles, excessive perspiration, inflammation, bronchitis, and toothache and strengthens the gums and teeth; it is used for leprosy, deafness, boils, scabies, syphilis, paralysis, and weakness. Its roots alleviate spasms and stimulate the cardiovascular system, besides having antitumor and spermicidal properties.[9] The flowers are aphrodisiac, emollient, and maturant and also used in the treatment of spermatorrhea. The seeds are aphrodisiac, astringent, and brain tonic and used in treating gonorrhea, piles, diarrhea, and scrofulous swellings, whereas the seed oil is applied topically to cure leukoderma. The pod extract is believed to possess antiprotozoal, hypoglycemic, and antidiabetic activity.[10]A. lebbeck is also psychoactive, astringent. In some cultures, it is used to treat boils, cough, gingivitis, flu, eye and lung problems, as a tonic and it is also used to treat abdominal tumors. A. lebbeck has antihistaminic property.[11]A. lebbeck seems to exhibit potent hepatoprotective activity along with various pharmacological activities such as central nervous system activity, cardiotonic activity, lipid-lowering activity, antioxidant activity, and hypoglycemic activity. It is reported to have antiseptic, antimicrobial, antiovulatory, antifertility, antiprotozoal, antidysentric, antitubercular, and anticancer activities.[12],[13],[14],[15]


 > Materials and Methods Top


Chemicals

5-fluorouracil (5-FU) was obtained from Sigma Chemicals Ltd. Petroleum ether, ethanol, trypan blue dye, and 0.9% NaCl solution were obtained from Loba Chemie Pvt. Ltd., Mumbai.

Plant

The pods of A. lebbeck L. were collected locally and were authenticated by Botany Department, Kakatiya University, Warangal, Telangana, and the specimen sample was deposited in the herbarium with the accession number 1882. The pods were dried, powdered by mechanical grinder, and defatted. The defatted powder (210 g) was extracted with absolute ethanol in a Soxhlet apparatus at 40°C for 24 h. After completion of extraction, the solvent was evaporated using a rotary flash evaporator and concentrated to dry residue (yield: 9 g and the %yield: 4.28%).

Animals

Swiss albino mice (20–25 g) were used for this study and they were procured from Mahaveer Enterprises, Hyderabad. They were maintained at 25°C ± 3°C temperature at 12/12 h light/dark cycle in polypropylene cages. These were fed with standard pellet diet and water ad libitum throughout the experimental period. Animals were acclimatized to laboratory conditions for 10 days before initiation of experiments. All the described procedures were reviewed and approved by the Institutional Animal Ethics Committee (IAEC approval No: 008/IAEC/NCPA/M. Pharm/2012-2013).

Acute toxicity studies and dose calculations

As per the OECD-423 guidelines, acute oral toxicity of ethanolic extract of A. lebbeck L (ALEE) was performed on female Swiss albino mice (20–26 g). The animals were fasted overnight with water ad libitum. 3 h before the administration of ALEE, water was also withheld. The ALEE was dissolved in normal saline and administered by oral gavage to three mice at each step. The starting dose of ALEE was 50 mg/kg body weight. The whole experimental protocol was carried out as per the procedure described in Annexure II (b) of OECD 423 guidelines and dose levels of 200, 300, 500, 1000, and 2000 mg/kg body weight were used in the subsequent steps. Animals were closely observed for 3 h and the mortality was noted during and after 24 h. There were no signs of toxicity, i.e., any gross behavioral changes and mortality up to 2000 mg/kg of ALEE. Based on the acute toxicity studies, doses of 200 mg/kg body weight and 400 mg/kg body weight of ALEE were selected for experimental protocol.

Transplantation of tumor cells

Ehrlich ascites carcinoma (EAC) cells were procured from Amala Cancer Institute, Thrissur, Kerala. The EAC cells were maintained in vivo in Swiss Albino mice by intraperitoneal transplantation of 2 × 106/mouse every 10 days[16] which were used as donor mice, i.e., ascitic fluid containing EAC cells aspirated from the peritoneal cavity of the mice was given intraperitoneally (2 × 106 cells/mouse) to fresh mice to develop ascitic tumor for every 10 days.

EAC cells were then collected from the donor mice and suspended in sterile isotonic saline (0.9% NaCl). The viable EAC cells were counted under a microscope with the aid of trypan blue staining.[17] The cells were then adjusted to 106 cells/10 g body weight of animals and were injected (intraperitoneally) into the experimental animals on day 0.

Treatment protocol

All the animals were divided into five groups with 12 animals in each group (n = 6) and treated as follows: grouping of animals was done based on computerized randomization using Random Allocation Software 2.0. 5-FU (20 mg/kg) was used as reference standard.

  • Group I: Served as normal control and treated with vehicle 2% CMC (5 ml/kg body weight p.o.)
  • Group I: Served as EAC control and transplanted with 1 × 106 cells/10 g body weight i.p.ly
  • Group III: Transplanted with 1 × 106 cells/10 g body weight i.p.ly and treated with 5-FU (20 mg/kg i.p.ly)
  • Group IV: Transplanted with 1 × 106 cells/10 g body weight i.p.ly and treated with ethanolic extract of A. lebbeck (200 mg/kg, p.o.)
  • Group V: Transplanted with 1 × 106 cells/10 g body weight i.p.ly and treated with ethanolic extract of A. lebbeck (400 mg/kg, p.o.).


All the groups of animals were treated with either A. lebbeck or 5-FU except normal control and tumor control (EAC) groups, which received vehicle only. The day after tumor induction to the respective animal groups was taken as day 0. One day of incubation was allowed for multiplication of the EAC cells. Treatment with A. lebbeck (200 mg/kg and 400 mg/kg) and reference drug (5-FU) was continued for subsequent 14 days starting from day 1.

Collection of blood samples and ascetic fluid

On the 15th day, 24 h after administration of the last dose, half of the animals (6 mice) in each group were kept fasting for 18 h and 0.5 mL of blood was withdrawn by retro-orbital sinus[18] method under mild ketamine anesthesia for the estimation of hematological parameters. After blood withdrawal, the mice were sacrificed by cervical dislocation method for the collection of ascitic fluid. The remaining half of the animals were allowed for natural death to assess mean survival time (MST) and increase in life span (ILS).

Assessment of parameters

Tumor volume

Ascitic fluid from peritoneal cavity of tumor-bearing mice was quantitatively isolated by peritoneal lavage. The fluid was carefully collected with a 5-mL sterile syringe in a measuring centrifuge tube and the volume was noted and expressed in milliliter (mL). It was then centrifuged at 1000 rpm for 5 min and the packed cell volume was determined.[19]

Viable/nonviable cell count (trypan blue exclusion assay)

The cells were calculated by Neubauer's hemocytometer. Briefly, 40 μL of EAC-aliquot was added to 4 mL 2% trypan blue (dissolved in 0.9% saline) and the mixture was left for 5 min. One drop from the mixture was taken on a Neubauer's hemocytometer and the numbers of stained cells (dead cells) and nonstained cells (viable or alive cells) were counted under a microscope (×40).

Percentage increase in life span

Percentage increases in life span (%ILS) and MST were calculated on the basis of mortality of the experimental mice.[20]



Hematological parameters

The blood samples were used for the estimation of hemoglobin, red blood cell (RBC), and white blood cell (WBC) count by standard procedures.[21],[22]

In vitro cytotoxicity using HeLa and A549 cell lines

In vitro cytotoxic effect of ALEE was assessed using HeLa and A549 cell lines. The human cervical cell lines (HeLa) and lung cancer cell lines (A549) were obtained from Amala Cancer Institute, Kerala. To measure the proliferation response in above cell lines, the (2,3-bis[2-Methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay was used.[23] The tetrazolium salt, XTT, is especially useful in quantifying viable cells. This assay is designed for the spectrophotometric quantification of cell growth and viability and is based on the cleavage of yellow tetrazolium salt, XTT, to form an orange formazan dye by metabolically active cells. HeLa cells or A549 cells were grown in growth medium plus 10% FBS in 96-well plates and were then treated with either different concentrations of ALEE or cisplatin, reference standard for 24 h. At the end of incubation, 50 mL of XTT labeling mixture solution was added to each well, and the cells were incubated at 37°C for 4 h. The formazan dye formed was solubilized in aqueous solutions and the optical density at 450 nm was compared with that of control wells with a screening multiwell spectrophotometer enzyme-linked immunosorbent assay reader. The reference wavelength is 650 nm. The percentage cell inhibition was determined using the following formula and a graph was plotted between concentration and percentage cell inhibition, and concentration required for 50% inhibition concentration (IC50) was calculated.



Statistical analysis

All the data were expressed as mean ± standard error of mean. Statistical significance was calculated by one-way ANOVA followed by Dunnett's multiple comparison test. P <0.05 and P < 0.01 were considered significant and highly significant, respectively.


 > Results Top


Tumor growth response

The development of tumor was observed on day 5; from that day, a steady increase in body weight was observed up to the end of the study (15th day) [Figure 1]a. The maximum gain of body weight was observed in the EAC control group. In case of ALEE- and 5-FU-treated groups, the tumor weight was significantly (P < 0.001) reduced at all the doses [Figure 1]c, [Figure 1]d, [Figure 1]e, [Figure 1]f. The tumor volume, tumor weight, and viable cell count were found to be significantly (P < 0.001) decreased and nonviable cell count was significantly (P < 0.001) increased in ALEE-treated animals at the doses 200 and 400 mg/kg and 5-FU (20 mg/kg) when compared with EAC control animals [Table 1]. With ALEE treatment, the life span of treated mice significantly (P < 0.001) increased as compared to the EAC-bearing control group [Figure 1]b. In ALEE- (200 and 400 mg/kg) and 5-FU-treated groups, %ILS was found to be 30.50 ± 2.90, 37.90 ± 3.02, and 41.70 ± 3.13, respectively, when compared to the EAC control group [Table 1].
Figure 1: Effect of Albizia lebbeck Linn on different parameters of Ehrlich ascites carcinoma. (a) Body weight, (b) life span, (c) packed cell volume, (d) tumor volume, (e) non-viable count, (f) viable count. All values were represented as mean ± SEM (n=10). *P<0.1, **P<0.01 when compared with EAC control &#P<0.01 when compared with normal control

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Table 1: Effect of ethanolic extract of Albizia lebbeck L on tumor parameters

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Hematological parameters

There was a significant increase in the WBC count and a drastic fall in the RBC count and hemoglobin content in the EAC control group as compared to the normal control group [Figure 2]. Administration of both doses of ALEE and 5-FU in EAC-bearing mice significantly (P < 0.001) reverted the above changes to normal [Table 2]. Furthermore, the ALEE has shown concentration-dependent cytotoxicity on HeLa cells and A549 cells in XTT assay [Figure 3], [Figure 4], [Figure 5], [Figure 6], and the IC50 values of ALEE were found to be 183.54 μg and 175.32 μg, respectively. The effect was comparable with that of standard, cisplatin.
Figure 2: Effect of Albizia lebbeck Linn on different hematological parameters. (a) Platelets cells × 103/μL, (b) red blood cells × 106/μL, (c) white blood cells × 103/μL, (d) hemoglobin g/dL. All values were represented as mean ± SEM (n=10). *P<0.1, **P<0.01when compared with normal control

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Table 2: Effect of ethanolic extract of Albizia lebbeck L on hematological parameters

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Figure 3: Comparison of antitumor activity of reference standard and ethanolic extract of Albizia lebbeck L on HeLa cell lines

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Figure 4: (a) Untreated HeLa cell line, (b) HeLa cell line treated with ethanolic extract of Albizia lebbeck L extract

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Figure 5: Comparison of antitumor activity of reference standard and ethanolic extract of Albizia lebbeck L on A549 cell lines

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Figure 6: (a) Untreated A549 cell line, (b) A549 cell line treated with Albizia lebbeck extract

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 > Discussion Top


Nowadays, the use of plant extracts has attracted a great attention in the alternative medicine because of their less toxicity and more efficiency. Hence, an attempt was made to evaluate antitumor activity of ALEE pods. The EAC (1905) is a transplantable, poorly differentiated, malignant tumor, which was initially described as a spontaneous murine mammary rapidly growing adenocarcinoma with a very aggressive behavior and can proliferate in almost in all strains of mice.[16] In 1932, it was named as EAC due to ascitic liquid together with carcinoma cells. The Ehrlich cell (1948) became popular because it has a similarity with human tumors that are most sensitive to chemotherapy due to its rapid growth rate. EAC is referred to as undifferentiated carcinoma. It is highly transplantable with rapid proliferation and 100% malignancy and do not adhere to synthetic surface in vitro. The Ehrlich ascetic tumor implantation induces per se a local inflammatory reaction, with increasing vascular permeability, which is essential for tumor growth because it constitutes a direct nutritional source for tumor cells.[24] Due to these concerns, it has been widely used from several years to study the antitumor properties of several natural and synthetic compounds.[19],[25]

Reduction in viable cell count and increase in nonviable cell count toward normal in tumor host suggest antitumor effect against EAC cell in mice.[26] In this study, the ALEEextract has significantly reduced the tumor volume, tumor weight, and tumor viable cell count when compared to the EAC tumor control group. This implies either a direct cytotoxic effect or an indirect local effect which may involve macrophage activation and vascular permeability inhibition. Literature reports reveal that prolongation of life span of tumor-bearing animals, reduction in tumor volume and viable cell count, and increased nonviable cell count is a reliable criterion for the depiction of potential of any anticancer agent,[19],[24],[27] based on which it was confirmed that ALEE has strong anticancer activity.

The present study showed that the ALEE-treated group has significant increase in the life span when compared with the EAC tumor control. The reliable criteria for judging the value of any anticancer drug are the prolongation of life span of the animals and the decrease of the leukemic cells in the blood.[28] The capacity (potency) to reduce the cell volume and increase in the life span of mice suggest the delaying impact of ALEE on cell division.[29] Earlier literature reveals that A. lebbeck pods contain higher amounts of proteins, saponins and minerals like potassium, sodium, and copper, amino acids like glutamic acid, aspartic acid in the pods[30] and presence of these compounds might contribute to the observed anti tumor activity of A, lebbeck.

Cancer itself is a cause of Anaemia in which RBC worn out quickly. Cancer also causes increase in abnormal WBC and reduction in healthy WBC. In the present study, reduction in hemoglobin, RBC count, and elevated WBC count was observed in the EAC control group, whereas ALEE treatment replenished the hemoglobin content and maintained the normal values of RBC and WBC. In addition, A. lebbeck did not cause myelotoxicity. Moreover, the results support that A. lebbeck has a strong hematopoietic protecting activity, great advantage over conventional chemotherapeutic agents. ALEE also showed potent cytotoxicity against cancerous cell lines HeLa and A549, using XTT assay in a dose-dependent manner.

Previous studies have shown that most of the plant extracts reduce EAC-induced myelotoxicity due to their immune boosting, antioxidant, and free radical scavenging activity.[19] It has been reported that a plant extract having antioxidant potential can inhibit proliferation of cancer cells.[31]A. lebbeck is known for its antioxidant and anti-inflammatory activity,[32] which might be the reason for the hematopoietic activity of ALEE. Similarly, the observed antitumor activity of ALEEin this EAC model might be due to its antioxidant principles as well as other phytoconstituents such as terpenoids, β-sitosterol, saponins, and flavonoids, which are known to be effective against various types of cancers.


 > Conclusion Top


The present study clearly demonstrates the potent antitumor activity of ethanolic extract of A. lebbeck. Earlier phytochemical studies on A. lebbeck revealed the presence of saponins, terpenoids, phenolic compounds and flavonoids. Several such compounds are known to possess strong antitumor and antioxidant properties. The strong antitumor activity observed in this model might be due to the presence of above principles in the A. lebbeck. In addition, ALEE has a significant hemoprotective effect which is advantageous over conventional anticancer drugs. Further studies on the elucidation of its mechanism of action and isolation of its active constituents may prove rewarding in cancer treatment.

Acknowledgment

We acknowledge the support of Nirmala College of Pharmacy, Atmakur, Mangalagiri, Guntur, Andhra Pradesh, to carry out this research work. We are also thankful to Amla Cancer Institute, Kerala for providing EAC cell lines and Hela and A549 cell lines.

Financial support and sponsorship

Nil.

Conflicts for interest

There are no conflicts for interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

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