Oriental Pharmacy and Experimental Medicine

, Volume 11, Issue 4, pp 243–249

Pre clinical studies of Streblus asper Lour in terms of behavioural safety and toxicity

Authors

    • Division of Pharmacology, Department of Pharmaceutical TechnologyJadavpur University
  • A. Puratchikodi
    • Department of Pharmaceutical, Engineering and TechnologyAnna University Trichy
  • Angelene Prasanna
    • Division of Pharmacology, Department of Pharmaceutical TechnologyJadavpur University
  • Narayan Dolai
    • National Institute of Pharmaceutical Educations & Research
  • Piyali Majumder
    • Division of Pharmacology, Department of Pharmaceutical TechnologyJadavpur University
  • U. K. Mazumder
    • Division of Pharmacology, Department of Pharmaceutical TechnologyJadavpur University
  • P. K. Haldar
    • Division of Pharmacology, Department of Pharmaceutical TechnologyJadavpur University
    • Department of PharmacologyHimalayan Pharmacy Institute
Research Article

DOI: 10.1007/s13596-011-0040-4

Cite this article as:
Kumar, R.B.S., Puratchikodi, A., Prasanna, A. et al. Orient Pharm Exp Med (2011) 11: 243. doi:10.1007/s13596-011-0040-4
  • 223 Views

Abstract

Streblus asper Lour (Family: Moraceae) is a medicinal plant wildly grows in most part of Asian countries. It has many traditional uses like leprosy, piles, diarrhoea, dysentery, elephantiasis, cancer etc. This present study was relates in terms of behavioural safety and toxicity studies of methanol and petroleum ether extracts of S. asper. Brine Shrimps lethality bioassay method was established for the present study and cytotoxicity was reported in terms of 50% lethality concentration (LC50). Different concentrations of drug solutions were added to the Brine Shrimps, surviving shrimps were counted after 24 h and 50% lethality concentration (LC50) were assessed. Acute toxicity was studied on Swiss albino mice after single dose. Sub-Chronic toxicity was performed for 28 days and assessed with biochemical and histopathological parameters. On cytotoxicity studies of brine shrimps it was found methanol extract of S. asper (MESA) was weakly toxic, may be due to the presence of cardiac glycosides and bioactive compounds, however petroleum ether extract of S. asper (PESA) was non toxic. But, in case of acute and sub-acute toxicity study both extracts were found to be non-toxic.

Keywords

Streblus asperCytotoxicityBrine shrimpsAcute toxicitySub-chronic toxicity

Introduction

Streblus asper Lour (Moraceae) is a medicinal plant commonly found in most areas of Southern Asia such as India, Bangladesh, Sri Lanka, Malaysia, Philippines and Thailand. It has a reputation for its claim on healing properties and used for specific ailments (Rastogi et al. 2006). Based on India’s indigenous knowledge, the plant is traditionally used for a wide range of specific ailments including leprosy, piles, diarrhoea, dysentery, elephantiasis, cancer and other conditions (Rastogi et al. 2006). The Marma tribe in Bangladesh have used the root juice of S. asper to treat irregular menstruation and to promote delayed menstruation (Rastogi et al. 2006) and the leaves are also known to be chewed with salt as an anthelmintic (Alam 1992). Previous bioactivity studies have revealed its bactericidal activity was found in the 50% ethanol extract of its leaves (Bhandary et al. 1995). A few laboratory studies have shown that S. asper exhibits a wide range of biological and pharmacological activities that may substantiate the therapeutic use of this plant in traditional medicine.

The merits of the traditional use of S. asper have also been supported by the isolation and identification of several possible active chemical constituents, including cytotoxic cardiac glycosides (stebloside and mansonin) (Fiebig et al. 1985). For preclinical assessment of the safety of new potential pharmaceuticals represents a special case of the general practice of toxicology, possessing its own peculiarities and special considerations.

Thus the present study was undertaken to investigate the safety of methanol and petroleum ether extract by determining its behavioural and pharmaco-toxicological effects in Swiss albino mice and its cytotoxicity bioassay in brine shrimp which is based on its ability to kill laboratory cultured brine shrimp (Artemia salina) (Michael et al. 1956).

Materials and methods

Chemicals

The chemicals used were podophyllotoxin, methanol, petroleum ether, diethyl ether (MERCK limited, Mumbai, India) and kits of cholesterol, creatinine, glucose, SGOT, SGPT (SPAN diagnostic, Mumbai, India), all the other chemicals are use of high analytical grade form the authorised supplier.

Plant material

The leaves of S.asper was collected from West Bengal, India, during the month of March 2010 and authenticated at Botanical Survey of India, Botanical garden, Howrah, West Bengal, India. A voucher specimen (No.-CNH/I-I/(29/2010/Tech.II) has been preserved at our laboratory for future reference. Air-dried plant leaves material (500 g) was powdered by mechanical grinder and successively extracted with petroleum ether and methanol by using Soxhlet extraction apparatus (Lapornik et al. 2005), extraction process with petroleum ether was completed in 26 h and with methanol 19 h. Then solvent was completely removed under reduced pressure and stored in a vacuum dessicator (yield 6.33% and 9.80%).The petroleum ether of S. asper (PESA) and methanol extract of S. asper (MESA) were used for present studies.

Cytotoxicity bioassay

Cytotoxicity of both PESA and MESA were evaluated for lethality to brine shrimp (Artemia salina) (Meyer et al. 1982; Solis et al. 1993). Brine shrimp eggs were hatched at identical conditions for 48 h (conical flask containing 600 ml of seawater, oxygen supplied by external air pump, temperature 29–30°C and a bright light source) (Cepleanu et al. 1994). The PESA and MESA were dissolved in purified water to obtain a final concentration of 1 mg/ml, in addition 0.1% tween 80 was added to PESA to make it completely soluble in water and diluted with purified water to get different concentrations (100, 200, 400, 600, 800 & 1,000 μg/ml). An aliquot of each concentration (1 ml) was transferred, in triplicate, into clean sterile universal vials and volume was made upto 10 ml with aerated sea water. Ten shrimp nauplii were transferred to each vial (30 shrimps per concentration) in identical conditions and maintained throughout the experiment period. Drug solutions in seawater were used as negative controls. After 24 h the numbers of survivors were counted and percentage of death was calculated. The concentration that killed 50% of the nauplii (LC50 in μg/ml) was determined by the statistical method of analysis.

Criterion of toxicity for PESA and MESA was established. LC50 values >1,000 μg/ml (non-toxic), ≥500≤1,000 μg/ml ( weak toxicity) and <500 μg/ml(toxic). (Deciga-Campos et al. 2007).

Acute toxicity (OECD GUIDLINE NO. 423)

Healthy Male Swiss albino mice weighing 27–30 g were taken. The mice were grouped and housed in poly acrylic cages (38 cm × 23 cm × 10 cm) with not more than six animals per cage and maintained under standard laboratory conditions (temperature 25 ± 2°C and dark/light cycle 14/10 h) for 7 days before commencement of the experiment. They were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. All procedures described were reviewed and approved by the Jadavpur University (367001/C/CPCSEA), Animal Ethical Committee.

The acute toxicity of the extract was determined according to the OECD guideline No. 423. Male albino mice weighing 27–30 g were used for this study. PESA and MESA were given to respective groups (n = 5) of animals at 2,000 mg/kg b.w. The treated animals were under observation for 72 h to check the mortality and 14 days for general behaviour. No death was observed till the end of the study.

Sub-chronic toxicity study

Swiss albino mice weighing 20–25 g were divided in to three groups (n = 6), the Group I served as saline control (0.9% NaCl 5 ml/kg., b.w.), Group II & III received PESA and MESA (400 mg/kg b.w.) daily through oral route for 28 days (Malek et al. 1997). Food and water intake was observed throughout the experiment. Twenty four hours after the last dose (i.e. 29th day) all the animals are anesthetised with di-ethyl ether and blood was collected by cardiac puncher for the estimation of haematological and serum biochemical parameters. Major organs such as brain, liver and kidney were dissected out, to check organ weight, estimation of tissue biochemical assays and histopathological parameters.

Blood analysis

Collected blood was analysed for determination of haemoglobin (Hb), total erythrocyte count (RBC) and leucocytes count (WBC) by standard protocol (Kostis et al. 1984).

Biochemical assays

Serum biochemical assays like serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), Creatinine, cholesterol and glucose were estimated with commercially available kits (Span Diagnostics Ltd. Surat, India.)

Histopathological parameters

One centimeter diameter of liver, kidney and brain were removed, fixed in 10% formalin and embedded in paraffin. Vertical section (5 μm) were cut, mounted on a glass slide and stained with Hematoxylin stain. The pathological changes were observed microscopically after staining.

Statistical analyses

All data are expressed as mean ± SEM Statistical significance (p) calculated by one-way ANOVA followed by Dunnett’s post hoc test, where p < 0.05 and p < 0.01 considered as significant and highly significant respectively. Statistical software (Origin Pro) used for calculations.

Results

Brine shrimp lethality

By enumeration the number of shrimps surviving after 24 h the percentage of inhibition was evaluated. The lethality concentration 50% (LC50) of the reference standard, podophyllotoxin was found to be 0.8 μg confirmed by the curve plotted against concentration vs. % inhibition (Fig. 1 and Table 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs13596-011-0040-4/MediaObjects/13596_2011_40_Fig1_HTML.gif
Fig. 1

Brine Shrimps lethality plot with reference standard, PESA & MESA. Podophyllotoxin was used as a reference standard. % inhibition for MESA was found to be dose dependent. PESA shows non-toxic

Table 1

Percentage inhibition of reference standard Podophyllotoxin in Brine shrimps. T1, T2 and T3: Test vials with 10 shrimps each

Concentration of extract

No. of shrimps surviving after 24 h

Total no. of shrimps alive

% Inhibition

T1

T2

T3

Control

7

8

8

22

26.6

0.1 μg

6

7

8

21

30

0.5 μg

5

7

5

16

46.6

1.0 μg

3

3

2

7

76.6

2.5 μg

1

1

1

3

90

5.0 μg

1

0

1

2

93.3

n = 10, T1+T2+T3=total shrimps. Percentage of inhibition was calculated using statistical software (Origin Pro)

The result obtained for the bioassay with MESA was tabulated in Table 2 and the 50% lethality concentration was found to be 800 μg confirmed by the curve plotted against dose concentration vs. % inhibition (Fig. 1). There was a gradual increase in the percentage of inhibition with the increase in the concentration of MESA and found to be weak toxicity LC50 ≥ 500 ≤ 1,000 μg/mL (Table 2).
Table 2

Percentage inhibition of MESA. T1, T2 and T3: Test vials with 10 shrimps each

Concentration of extract

No. of shrimps surviving after 24 h

Total no. of shrimps alive

% Inhibition

T1

T2

T3

100 μg

10

9

10

29

3.33

200 μg

9

9

10

28

6.66

400 μg

9

8

8

25

16.6

600 μg

7

8

7

22

26.6

800 μg

4

6

5

15

50

1,000 μg

3

2

1

6

80

n = 10, T1+T2+T3=total shrimps. Percentage of inhibition was calculated using statistical software (Origin Pro)

Results for the bioassay of PESA was tabulated in Table 3, and found to be non toxic (LC50 > 1,000 μg/mL).
Table 3

Percentage inhibition of PESA. T1, T2 and T3: Test vials with 10 shrimps each

Concentration of extract

No. of shrimps surviving after 24 h

Total no. of shrimps alive

% Inhibition

T1

T2

T3

100 μg

9

9

8

26

13.3

200 μg

10

7

8

25

16.6

400 μg

8

8

9

25

16.6

600 μg

10

8

8

26

13.3

800 μg

9

7

8

24

20

1,000 μg

10

9

9

28

6.6

n = 10, T1+T2+T3=total shrimps. Percentage of inhibition was calculated using statistical software (Origin Pro)

Acute toxicity study

PESA and MESA were found to be safe on Swiss albino mice up to the dose of 2,000 mg/kg., b.w. orally that does not produce any mortality and body weight of the animals was found to be normal.

Sub-chronic toxicity

Effect of PESA and MESA on general behaviour

PESA and MESA did not produce any noticeable change in the general behaviour of the animals, no significant changes in body weight and food intake. A summary of growth, mortality and gross symptoms of toxicity observed after sub-chronic oral administration of both extracts is represented in the Table 4 & Fig. 2.
Table 4

Mortality rate of animals. Observation was done throughout the experimental period

Dose

Number of animals

Mortality (D/T) #

Symptoms of toxicity

Control

6

0/6

None

PESA 400 mg/kg.b.w

6

0/6

None

MESA 400 mg/kg.b.w

6

0/6

None

D/T Death/Total. n = 6, # number

https://static-content.springer.com/image/art%3A10.1007%2Fs13596-011-0040-4/MediaObjects/13596_2011_40_Fig2_HTML.gif
Fig. 2

Comparison of PESA and MESA on growth rate with normal control. PESA & MESA treated group shows normal growth rate when compared with the control group

Effect of oral PESA and MESA on haematological and biochemical parameters

The haematological parameters such as, haemoglobin concentration, RBC and WBC count and biochemical assays such as SGOT, SGPT, Glucose, Creatinine and Cholesterol in the treated mice did not exhibit any significant change as compared with control group and all the values remained within the normal limits Table 5 and Fig. 3.
Table 5

Haematological and biochemical parameters

Dosage Group

Haemoglobin (g/dl)

RBC count × 108

WBC count 108

Cholesterol (mg/dl)

Creatinine (mg/dl)

Glucose (mg/dl)

SGOT (IU/dl)

SGPT (IU/dl)

Control

12.13 ± 0.15

6.90 ± 0.15

6.32 ± 0.37

38.5 ± 5.23

0.42 ± 0.05

70.83 ± 3.58

42.59 ± 1.14

35.18 ± 1.70

PESA 400 mg/kg

11.99 ± 0.17

6.86 ± 0.22**

5.85 ± 0.44*

43.3 ± 8.62**

0.37 ± 0.03**

76.16 ± 5.59**

42.16 ± 2.14**

34.93 ± 0.80**

MESA 400 mg/kg.b.w

12.22 ± 0.25

7.18 ± 0.33**

5.82 ± 0.29**

49.8 ± 6.40**

0.49 ± 0.04**

68.33 ± 6.03**

38.68 ± 1.87**

32.51 ± 0.72**

Values are expressed as mean ± SEM (n = 6); **p < 0.001; *p < 0.01 compared to normal control

https://static-content.springer.com/image/art%3A10.1007%2Fs13596-011-0040-4/MediaObjects/13596_2011_40_Fig3_HTML.gif
Fig. 3

Haematological and biochemical parameters. **p < 0.001 compared to normal control, *p < 0.01 compared to normal control

Body weight, organ weight & histopathological study

Body and organ weight of mice treated with PESA and MESA is tabulated in Table 6. There was no statistically significant change in the body and organ weights of the treated mice compared with the control group. In addition, there were no histopathological change (morphological and architecture of cell) findings on liver and kidney that could exhibited with PESA and MESA (Figs. 4 & 5).
Table 6

Body weight, organ weight & histopathological study

Dose group

Body weight

Kidney weight

Liver weight

Brain weight

Initial wt

Final wt

Control

21.24 ± 1.10

26.65 ± 0.17

0.76 ± 0.06

2.73 ± 0.07

1.16 ± 0.03

PESA 400 mg/kg.b.w

20.15 ± 1.15

25.00 ± 1.21

0.73 ± 0.02

2.83 ± 0.05

1.17 ± 0.03

MESA 400 mg/kg.b.w

21.16 ± 0.04

25.41 ± 1.56

0.77 ± 0.05

2.65 ± 0.03

1.18 ± 0.07

Values are expressed as mean ± SEM

https://static-content.springer.com/image/art%3A10.1007%2Fs13596-011-0040-4/MediaObjects/13596_2011_40_Fig4_HTML.gif
Fig. 4

Liver section at 400 x. comparison of MESA & PESA has shown no morphological changes when compared with normal control

https://static-content.springer.com/image/art%3A10.1007%2Fs13596-011-0040-4/MediaObjects/13596_2011_40_Fig5_HTML.gif
Fig. 5

Kidney section at 400 xs. glomeruli of MESA & PESA shown normal when compared with the normal control group

Discussion

According to WHO survey 70–80% of world population depends on medicines which are derived from plant sources. This is especially the case of developing countries where the cost of consulting a doctor and cost of medication is beyond the means of most population (Chan 2003) even though medicines derived from plants may produce several biological activities in humans. Scientifically very little is known about their toxicity in different parameter at different rate of variables and the same applies for S. asper. Hence, safety should be the dominant norm in the selection of plants with medicinal properties for use in health care system (Fennell et al. 2004).

Cytotoxicity study on brine shrimps has been an excellent method for preliminary investigation of toxicity for screening medicinal plant (Gupta et al. 1996). This technique is inexpensive, utilize little amount of material and easy to perform (Meyer et al. 1982). This in-vivo test has been successively engaged in cytotoxic bio-assay of active cytotoxins and antitumour agents (Mongelli et al. 1996). The assay is considered a useful tool for preliminary assessment of toxicity and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, pesticides and cytotoxicity testing of dental materials (Barahona and Sanchez-Fortun 1999; Pelka et al. 2000).

The brine shrimps cytotoxicity of PESA may be non toxic (Deciga-Campos et al. 2007) and MESA may have weak toxicity (Deciga-Campos et al. 2007) at the concentration of 800 μg and the toxicity may increase directly proportional to the increase in concentration of MESA.

From the results of acute toxicity study it was found that the PESA & MESA were safe upto the dose of 2,000 mg/kg orally and did not produces any mortality.

Results from the sub-chronic toxicity study of PESA & MESA showed that there was no significant change in animal growth, haematological and biochemical parameters. Histopathological studies also exhibited that there was no changes in the cellular architecture and morphology.

Previously work done on ethanol extract shown high amount of cardiac glycosides (Fiebig et al. 1985), which naturally possess toxicity and bioactivity. May be due to the geographical distribution of collected plant this study shows the variation in the toxicity level. Overall these studies on cytotoxicity on brine shrimps, acute toxicity and sub-chronic toxicity on Swiss albino mice provides valuable preliminary data on the toxicity profile of S. asper that should be useful for the planning of pre-clinical and clinical studies. Collectively form the findings of the present study showed that MESA may be weak toxic to brine shrimps. This minute amount of toxicity on brine shrimps may be effective for antiangiogenesis and apoptosis study in the future. Studies on Swiss albino mice for acute and sub-chronic toxicity evaluations it was found PESA and MESA were non-toxic.

Acknowledgement

This work was fully funded by University Grants Commission (U.G.C), Grant number P-1\RS\115\2010, New Delhi, India. This funding had no role in the collection, analysis and interpretation of data or in the writing of the manuscript.

Copyright information

© Institute of Oriental Medicine, Kyung Hee University 2011