Advertisment ACS-IndiaSymposium
Journal of Pharmaceutical Negative Results
  Print this page Email this page Small font sizeDefault font sizeIncrease font size 
Search Article 
Advanced search 
 Home | About us | Editorial board | Search | Ahead of print | Current issue | Archives | Submit article | Instructions | Subscribe | Contacts  

  Table of Contents  
Year : 2013  |  Volume : 4  |  Issue : 1  |  Page : 1-4  

Lack of brine shrimp lethality and hemagglutination activity in Grewia asiatica Linn

1 Department of Pharmacy, Hazara University, Havelian Campus, Abbottabad, Pakistan
2 Department of Genetics, Hazara University, Mansehra, Pakistan
3 Department of Horticulture, Faculty of Agriculture, Gomal University, Dera Ismail Khan, Pakistan

Date of Web Publication21-Aug-2013

Correspondence Address:
Abidah Parveen
Department of Pharmacy, Hazara University, Havelian Campus, Abbottabad 22010
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0976-9234.116754

Rights and Permissions

Objective: To investigate the brine shrimp lethality and hemagglutination activity of the fruit, stem bark and leaves of Grewia asiatica L. (Family: Tiliaceae). Materials and Methods: The crude ethanolic extracts and fractions of the fruit, stem bark, and leaves were subjected to cytotoxic assay using brine shrimps and were also investigated for hemagglutination activity. Results: Brine shrimps survived easily and hemagglutination activity was not observed. Conclusions: The fruits, leaves and stem bark of G. asiatica have insignificant brine shrimp lethality and hemagglutination activity was found to be absent.

Keywords: Brine shrimp, crude ethanolic extracts, Grewia asiatica, hemagglutination, traditional medicine

How to cite this article:
Parveen A, Irfan M, Jilani MS, Wasim K, Kiran M, Muhammad F. Lack of brine shrimp lethality and hemagglutination activity in Grewia asiatica Linn. J Pharm Negative Results 2013;4:1-4

How to cite this URL:
Parveen A, Irfan M, Jilani MS, Wasim K, Kiran M, Muhammad F. Lack of brine shrimp lethality and hemagglutination activity in Grewia asiatica Linn. J Pharm Negative Results [serial online] 2013 [cited 2020 Jun 7];4:1-4. Available from:

   Introduction Top

Grewia species are used in traditional medicine for their anticancer property; few have been investigated. Among them, Grewia villosa has been reported to possess strong anticancer activity. [1] The extracts of Grewia tilliaefolia and lupeol isolated from it have been reported to possess weak anticancer activity in three cell lines Vero, HEp-2, and B 16 F 16 . [2] The aqueous extracts of the fruit and leaves of Grewia asiatica L. (Tiliaceae) have also been reported to show strong in-vitro anticancer activity in five cell lines HEK-293, NCI-H522, HELA, HeP-2, and MCF-7 in a concentration-dependent manner. [3] However, the methanolic extract of the fruit has been reported to be active against NCI-H522 and MCF-7 and inactive against the other three cell lines. [4] Its methanolic extract has also been reported to be active against Ehrlich's ascites carcinoma cell lines. [5] The present investigation is to determine the chronic toxicity level using brine shrimps and hemagglutination activity of the crude ethanolic extracts and the fractions of the fruit, stem bark, and leaves of G. asiatica.

   Materials and Methods Top

Collection and identification of plant material

The fruits, leaves, and stem bark were collected from Grewia orchard in Dera Ismail Khan in June 2009. The plant was identified and authenticated by Mr. Hamidullah, Faculty of Pharmacy, Gomal University, Dera Ismail Khan, Pakistan. Voucher specimens were deposited at the Herbarium of the Department of Pharmacognosy, Gomal University, D.I. Khan.

Preparation of extracts

The plant materials were extracted with ethyl alcohol (distilled) by maceration process. This procedure was repeated three times. The crude ethanolic extract was filtered; the solvent was distilled off and then concentrated under vacuum. The crude ethanolic extracts of the fruit, leaves and stem bark were then fractionated with hexane, carbon tetrachloride, chloroform, ethyl acetate, n-butanol, methanol and distilled water. Fractions obtained in considerable quantities were stored in airtight, amber-colored containers for further studies. All solvents were analytical grade Merck Germany. Etoposide was used as a standard reference.

Cytotoxic assay

20 mg of the test sample was dissolved in 2 ml methanol which served as a stock solution. From each stock solution, 10, 100 and 1000 μg/ml were taken in vials in triplicates separately by means of micro syringe for 5, 50 and 500 ppm. [6],[7] The vials were kept overnight for the evaporation of organic solvent. Traces of organic solvent were removed by placing vials in vacuum desiccator. Using a Pasteur pipette, ten mature larvae were placed per vial and the volume made to 5 ml by sea water. The vials were kept for 24 hours at 27 ° C under illumination. The number of survivors and dead in test samples and control were thereafter counted and recorded. The data was analyzed with Finney computer program to determine LD 50 values with 95% confidence intervals.

Hemagglutination activity

The hemagglutination activity was tested against human erythrocytes blood groups ABO. [8] A 2% suspension of erythrocytes, obtained by centrifugation of the blood, was prepared in phosphate buffer (pH 7). [9] The activity was investigated serially in different dilutions in the ratio of 1:1, 1:2, 1:4, 1:8, and 1:16 of each extract against all the blood groups at the doses of 5, 2.5, 1.25, 0.625, and 0.3125 mg/m1. [10] Smooth button-like formation (due to sedimentation of erythrocytes) at the bottom of the test tube indicates the negative activity of the extract where as a rough granular deposition (due to agglutination of erythrocytes) shows positive action.

   Results Top

The crude ethanolic extract of the fruit of G. asiatica and its fractions were found to be cytotoxically inactive because the shrimps survived easily in this extract [Table 1]. The crude ethanolic extract/fractions of the stem bark [Table 2] and leaves of G. asiatica were also found to be non-active against LD 50 [Table 3]. LD 50 was determined by Finney computer program, which showed no lethality. No hemagglutination activity was observed for either of the crude extracts [Table 4].
Table 1: Cytotoxic activity of the fruit of Grewia asiatica

Click here to view
Table 2: Cytotoxic activity of the stem bark of Grewia asiatica

Click here to view
Table 3: Cytotoxic activity of the leaf of Grewia asiatica

Click here to view
Table 4: Agglutination activity of the crude extracts of fruit, leaf and stem bark of Grewia asiatica

Click here to view

   Conclusion Top

Grewia asiatica is used for the treatment of cancer like symptoms [3] in traditional medicine. On the basis of the present study, it might be suggested that the fruit, stem bark, and leaves of G. asiatica have non-significant cytotoxic activity because of the absence of brine shrimp lethality giving a preliminary indication of safety of the plant extracts. It might also be suggested that G. asiatica has only a selective action against cancer cells by virtue of its antioxidant potential. Dietary and endogenous antioxidants prevent cellular damage by reacting with and eliminating oxidizing free radicals. [11] Higher oxidative stress observed in cancer cells can also result from a decrease or inactivation of antioxidants [12],[13] and "oxidation therapy" is a recent strategy developed for the treatment of cancer by inducing cytotoxic oxidative stress for cancer treatment. [14],[15] The anticancer activity of G. asiatica reported in the literature has also been attributed to the presence of antioxidants such as vitamin C, anthocyanins, and folate carotenoids. [3]

The heamagglutination activity of the plant plays an important role to see the effect of the test drug on blood. It also helps to determine the safety margin of the drug in cases of blood disorders such as hemorrhages or clot formation. However, no such activity was observed in the crude ethanolic extracts of the fruit, stem bark, and leaves of G. asiatica. Due to the absence of these activities, the fractions of the crude extracts were not investigated further.

   Abbreviations Top

Vero (a normal African green monkey kidney cell line), Hep-2(a human larynx epithelial carcinoma cell line), B16F10 (Mouse melanoma cell line), HEK-293 (Epidermal kidney cancer cell line), NCI-H522 (Cell lung cancer cell line), HELA (Cervical cancer cell line) and MCF-7 (Breast cancer cell line).

   Acknowledgment Top

The authors acknowledge Mr. Hamidullah, Chairman, Pharmacognosy Department and Dr. Gul Majid, Dean, Faculty of Pharmacy, Gomal University, Dera Ismail Khan, Pakistan for their guidance to execute this study.


The opinions expressed in this publication are those of the authors and do not necessarily represent those of SCIBIOLMED.ORG. Authors are responsible for their citing of sources and the accuracy of their references and bibliographies. The editors cannot be held responsible for any lacks or possible violations of third parties' rights.

   References Top

1.Dictionary of Indian Medicinal Plants, Lucknow: Institute of Medicinal and Aromatic Plants Publication; 1992.  Back to cited text no. 1
2.Badami S, Vijayan P, Mathew N, Chandrashekhar R, Godavarthi A, Dhanraj SA, et al. In vitro cytotoxic properties of Grewia tiliaefolia bark and lupeol. Indian J Pharm 2003;35:250-1.  Back to cited text no. 2
3.Marya B, Dattani KH, Patel DD, Patel PD, Patel D, Suthar MP et al. In- vitro cytotoxicity evaluation of aqueous fruit and leaf extracts of Grewia asiatica using MTT Assay. Der Pharma Chemica 2011; 3:282-7.  Back to cited text no. 3
4.Dattani KH, Patel DD, Marya B, Patel PD, Patel D, Suthar MP, et al. In-vitro cytotoxicity evaluation of methanolic fruit extract of Grewia asiatica using MTT assay. Inventi Impact: Ethnopharmacology 2011;2:354.  Back to cited text no. 4
5.Bibhuti BK, Viyapuri TS, Lakhmanan M, Gupta M, Mazumder UK, Bhaskar Das. Antitumor and in-vitro cytotoxic activity of Grewia asiatica Lin. against Ehrlich's ascites carcinoma cell lines. Pharmacol online 2011;3:956-60.  Back to cited text no. 5
6.Meyer BN, Ferrigni NR, Putnam JM, Jacobsen LB, Nicholas PE, Mclanghlin JL. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med 1982;45:31-4.  Back to cited text no. 6
7.Dey PM, Harbone JB. Methods in the Plant biochemistry. Vol. 6. London N.Y.: Academic Press; 1991. p. 8-10.  Back to cited text no. 7
8.Naqvi SB, Sheikh D, Usmanghani K, Shameel M, Sheikh M. Screening of marine algae of Karachi for haemagglutinin activity. Pharm. J Pharm Sci 1992;5:129-38.  Back to cited text no. 8
9.Cavlovic P, Mankotia M, Pantazopoulos P, Scott PM. Liquid chromatographic determination of solasonine in frozen green peas as an indicator of the presence of night shade berries. JAOAC Int 2003;86:759-63.  Back to cited text no. 9
10.Alam MT, Usmanghani K. Studies on marine algae for haemagglutinic activity. Pak J Pharm Sci 1994;7:1-5.  Back to cited text no. 10
11.Davis WL, Mathew SB. Antioxidants in cancer therapy; their actions and interactions with oncologic therapies. Altern Med Rev 1999;4:304-29.  Back to cited text no. 11
12.Huang P, Feng L, Oldham EA, Keating MJ, Plunkett W. Superoxide dismutase as a target for the selective killing of cancer cells. Nature 2003;5:390-5.  Back to cited text no. 12
13.Conklin KA. Free radicals: The pros and cons of antioxidants. Cancer chemotherapy and antioxidants. J Nutr 2004;134:3201-4.  Back to cited text no. 13
14.Fang J, Nakamura H, Iyer AK. Tumor-targeted induction of oxystress for cancer therapy. J Drug Target 2007;15:475-86.  Back to cited text no. 14
15.Trachootham D, Alexandre J, Huang P. Target cancer cells by ROS mediated mechanisms; a radical therapeutic approach? Nat Rev 2009;8:579-91.  Back to cited text no. 15


  [Table 1], [Table 2], [Table 3], [Table 4]

This article has been cited by
1 Protective action of Grewia asiatica (phalsa) berries against scopolamine-induced deficit in learning and memory using behavior paradigms in rats
Sangeeta Paul,Swapnil Sharma,Sarvesh Kumar Paliwal,S. B. Kasture
Oriental Pharmacy and Experimental Medicine. 2019;
[Pubmed] | [DOI]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  In this article
    Materials and Me...
    Article Tables

 Article Access Statistics
    PDF Downloaded9667    
    Comments [Add]    
    Cited by others 1    

Recommend this journal