Antipyretic Activity of Piper nigrum and Nyctanthes arbor-tristis in Different Dosage Forms

 

NB Ghiware1* and TM Nesari2

1Department of Pharmacology, Nanded Pharmacy College, Nanded-431605, India

2Department of Dravyaguna, Tilak Ayurved Mahavidyalaya, Pune-411011, India

*Corresponding Author E-mail: ashish_roge@rediffmail.com

 

ABSTRACT:

Acute oral toxicity studies of the Piper nigrum Linn (Family: Piperaceae), and Nyctanthes arbor-tristis Linn (Family: Oleaceae), were carried out in rats. All external morphological changes, change in body weight, general systemic toxicity and necropsy were recorded. During acute toxicity study there was normal weight gain in the treatment group was observed. There were no significant signs of systemic toxicity and death was noted.  No abnormalities were noted at necropsy. Tablet, liquid oral and suspension dosage forms of dried unripe fruits of Piper nigrum and dried leaves of Nyctanthes arbor-tristis in combination, were tested in brewer’s yeast-induced pyrexia in rabbits to assess their antipyretic activity. The percent inhibition rate against increasing rectal temperature in rabbits was assessed for all test dosage forms and was found significant compared to that of control. The results indicate that liquid oral and tablet dosage forms possess good antipyretic property compared to suspension dosage form.

 

KEYWORDS: Piper nigrum, Nyctanthes arbortristis, Safety and Efficacy, Antipyretic activity. 

 


 

INTRODUCTION:

Pyrexia or fever is caused as a secondary impact of infections like malaria, typhoid; tissue damage, inflammation, graft rejection, malignancy or other diseased states. It is the body's natural defense to create an environment where infectious agent or damaged tissue cannot survive1.

 

Piper nigrum (Family: Piperaceae)2, 3 and Nyctanthes arbor-tristis (Family: Oleaceae)4, 5 used traditionally in India, for the treatment of many diseases characterized by rise in body temperature 6, 7, 8. Piper nigrum is commonly known as ‘Pepper’ and it is a unique spice through out world and marketed in four versions after processing9. Nyctanthes arbor-tristis is commonly known as ‘Harsinghar’ or as ‘Night Jasmine10.  Even though a large number of studies have been done on the benefits of these plants11, 12, so far no systematic investigation of toxicity and anti-pyretic activity was carried out with reference to different possible dosage forms acceptable and palatable to all age group of patients. So the aim of this study was to determine the acute toxicity of Piper nigrum and Nyctanthes arbor-tristis and to study antipyretic activity of different dosage forms of Piper nigrum and Nyctanthes arbortristis in combination form.

 

MATERIAL AND METHODS:

I) Acute oral toxicity:

Acute oral toxicity of the Piper nigrum and Nyctanthes arbor-tristis following a single oral administration was carried out in albino rats according to Organization for Economic Co-operation and Development (OECD) guidelines, No.423“Acute Oral Toxicity- Acute Toxic Class Method” 13- 15.

 

Test material:

Dried unripe fruits of Piper nigrum were collected from known sources from Nanded District, Maharashtra, India and fresh leaves of Nyctanthes arbor-tristis were collected early in the morning from widely grown trees from medicinal gardens at Nanded District, Maharashtra, India. Collected samples authenticated through Department of Pharmacognosy, Nanded Pharmacy College, Nanded. Samples are authenticated as Piper nigrum Linn (Family: Piperaceae) and Nyctanthes arbor-tristis Linn (Family: Oleaceae) respectively. Specimen of both samples has been retained in the Department

 

For the purpose of study, the test material was suspension of dried unripe fruits of Piper nigrum and suspension of shed dried leaves of Nyctanthes arbor-tristis, freshly prepared separately at the appropriate concentration. Homogeneity and stability was carried out prior to the test.

 

Animal and animal husbandry:

Male and female Wistar albino rats (200-250g) were selected for the study and were kept randomly in to the cages. Separate groups are made for the testing of suspension of Piper nigrum and Nyctanthes arbor-tristis. The animals are housed in groups of three by sex in cages. With the exception of an overnight fast before dosing and for 3-4 hrs, after dosing, free access for drinking water and food was allowed throughout the study. Institutional ethical committee approved protocol of study.

 

Table-1: Acute Oral Toxicity: Dosage pattern for Piper nigrum test suspension

Group No.

Dose level

(mg/kg)

Concentration

(mg/ml)

Dose volume

(ml/kg)

1-a

2000

200

10

1-b

2000

200

10

2-a

200

20

10

2-b

200

20

10

3-a

25

2.5

10

3-b

25

2.5

10

Group 1, 2 and 3 indicates dose level 2000mg/kg, 200mg/kg and 25mg/kg respectively. a and b  indicates male and female rats group respectively at that dose level.

 

Experimental:

For acute oral toxicity of Piper nigrum and Nyctanthes arbor-tristis, dose selection, dose volume, number of animals per sex for different groups was as per guidelines and is given in Table 1 and Table 2 respectively.

 

All animals were dosed accordingly once by metal cannula attached to a graduated syringe. Then animals are observed for any death or overt signs of toxicity at ½, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days.

 

Individual body weights are recorded prior to dosing and on 7th and 14th day after treatment. At the end of the initial observations, some of the animals from each group are sacrificed and are subjected to gross pathological examination.

 

Evaluation of data:

Data evaluations include the relationship between the exposure of the animal to the test suspension of Piper nigrum and Nyctanthes arbor-tristis with the incidence and severity of all abnormalities including behavioral, clinical observations, gross lesions, body weight changes, mortality and any other toxicological effects. Toxicological observations include evaluation of skin and fur, eyes and mucous membrane, circulatory and respiratory effects in term of any signs of cyanosis, change in breathing rate etc. Observations also include autonomic effects such as salivation, urination, CNS effects such as tremors, convulsions, gait, posture etc.

 

Using the mortality data obtained, estimate of the acute oral median lethal dose (LD50) of the Piper nigrum and Nyctanthes arbor-tristis was made.

 

II) Anti-pyretic Activity:

Study was carried out to assess antipyretic activity of formulated three dosage forms of dried unripe fruits of Piper nigrum and dried leaves of Nyctanthes arbor-tristis, in combination form, in pyrexia induced animal model 16- 18.

 

 

Table-2: Acute Oral Toxicity: Dosage pattern for Nyctanthes arbor-tristis test suspension

Group No.

Dose level

(mg/kg)

Concentration

(mg/ml)

Dose volume

(ml/kg)

1-x

2000

200

10

1-y

2000

200

10

2-x

200

20

10

2-y

200

20

10

3-x

25

2.5

10

3-y

25

2.5

10

Group 1, 2 and 3 indicates dose level 2000mg/kg, 200mg/kg and 25mg/kg respectively. a and b  indicates male and female rats group respectively at that dose level.

 

 

Test material:

For the purpose of present study, powdered form of dried unripe fruits of Piper nigrum and dried leaves of Nyctanthes arbor-tristis was formulated in combination, in three different dosage forms, viz. Tablet, liquid oral and suspension 19, 20, 21. Tablets were prepared by wet granulation method which contains equal quantity of powdered form of dried unripe fruits of Piper nigrum and dried leaves of Nyctanthes arbor-tristis. Liquid oral was prepared using 1 part of decoction of both plants in equal quantities and mixed with 5 parts of concentrated syrup. High solid content deflocculated suspension of powdered form of dried unripe fruits of Piper nigrum and dried leaves of Nyctanthes arbor-tristis was prepared. All the three formulations contain same quantity of powdered form of dried unripe fruits of Piper nigrum and dried leaves of Nyctanthes arbor-tristis. All the formulations were standardized as per WHO guidelines prior to their use for experimentation.

 

All the glassware, syringes and needles washed with water for injection and sterilized in oven at 2500c for 30 min. All the formulations and solutions of formulations were prepared or diluted with pyrogen free saline solution. Yeast, which was selected to induce pyrexia in anti-pyretic model, was prepared as suspension in pyrogen free solution.

 

Selection of animals and animal grouping:

12 healthy adult rabbits of either sex, weighing not less than 1.5kg were selected for the study and they were kept individually in an area of uniform temperature and uniform humidity. Rabbits with rectal temperature more than 39.80c and less than 380c were not selected for the test. Selected 12 rabbits were equally divided in to four groups.

 

 

Experimental:

3mg/kg of 10% yeast suspension was subcutaneously injected onto the back of individual rabbits. Rectal temperature (0c) was recorded (Telethermometer-Aerotrone) of individual rabbits. Rabbits with rising body temperature, then divided into four equal groups as Control, Gr.-A, Gr.-B and Gr.-C. For present study, type of formulation, route of administration, dose selection and dose volume of test dosage forms was as given in Table 3.

 


Table-3: Type and dosage pattern of formulations for anti-pyretic testing

Group

Type of dosage form

Route of administration

Dose level (mg/kg)

Conc. (mg/ml)

Dose volume (ml/kg)

No. of rabbits

Control

Placebo

Oral

--

--

10

3

Gr.-A

Tablet

Oral

8mg/kg

0.8

10

3

Gr.-B

Liquid Oral

Oral

8mg/kg

0.8

10

3

Gr.-C

Suspension

Oral

8mg/kg

0.8

10

3

 

Table-4: Mean values of increasing rectal temperature in different groups of rabbits before and after dosing

Group

Formulation

Temp. before drug dosing

Increased temperature (0C) after dosing

1hr

2hr

3hr

5hr

Control

Placebo

38.64±0.27

1.40±0.32

1.52±0.22

1.49±0.21

0.51±0.36

Gr.-A

Tablet

38.72±0.28

0.36±0.22

0.34±0.20

0.72±0.23

0.34±0.26

Gr.-B

Liquid Oral

38.78±0.30

0.32±0.23

0.24±0.19

0.25±0.13

0.18±0.16

Gr.-C

Suspension

38.74±0.21

0.50±0.25

0.42±0.25

0.30±0.20

0.37±0.22

Values are mean ±S.D., n(number of observations)= 03/group

 

Table-5: Percent inhibition rate against increasing rectal temperature of rabbits by different formulations

Group

Formulation

Percent inhibition rate against increasing temperature

1hr

2hr

3hr

5hr

Control

Placebo

00

00

00

00

Gr.-A

Tablet

74.28*

77.63

51.67

33.33

Gr.-B

Liquid Oral

77.14*

84.21

83.22

64.70

Gr.-C

Suspension

64.28**

72.36

79.86

27.45

*P<0.001, **P<0.05

 


 

Formulated dosage forms of drugs were given orally in a single dose. Tablet dosage form of drugs was crushed and solution was prepared to get final concentration. Control group receives only vehicle by oral route. All the rabbits accordingly dosed by metal cannula attached to a graduated syringe.

 

Rectal temperature (0c) was recorded of individual rabbits from all four groups before dosing and following dosing at 1, 2, 3 and 5 h. Increase or decrease in temperature value was the temperature difference before and after dosing of test solution.

 

Evaluation of Data:

Data evaluation includes change in rectal temperature (0c) in rabbits due to administered solution of dosage forms of Piper nigrum and Nyctanthes arbor-tristis. Separate proforma was prepared to record rectal temperature before dosing and to record increased temperature (0c) after dosing in rabbits of different groups.

 

Mean value of rectal temperature difference within group at varying time interval of four groups was considered for comparison.

 

Finally effectiveness of formulated dosage forms of Piper nigrum and Nyctanthes arbor-tristis was judged as percent inhibition rate against increasing rectal temperature in rabbits of test group in comparison to control group at varying time intervals. Data was subjected to statistical analysis like student’s t-test and ANOVA for level of significance in treatment group.

 

Results:

Acute oral toxicity:

In the acute oral toxicity, no mortality was observed at 2000mg/kg dose of Piper nigrum and Nyctanthes arbor-tristis. There were no significant signs of systemic toxicity noted. However, some   animals during the day of dosing of 2000mg/kg p.o., dose of both the drugs, shows lethargy. All the animals appeared normal one day after dosing. Another two groups with next decreasing order dose 200mg/kg p.o., and 25mg/kg p.o was made. No lethargy and any other systemic toxicity were observed at these two doses of individual drugs. All animals showed normal expected gain in body weight during the study period. On necropsy no abnormalities was observed.

 

Anti-pyretic activity:

Mean values of increasing rectal temperature before and after dosing and percent inhibition rate against increasing rectal temperature in different groups of rabbits due to three formulations of Piper nigrum and Nyctanthes arbor-tristis, in combination form, were given in Table 4 and Table 5 respectively.

 

Protection against increasing temperature was more after 1 h, of dosing with Liquid Oral (77.14%, P<0.001) and Tablet (74.28%, P<0.001) dosage form than Suspension dosage form (P<0.05) of drugs.

 

Discussion and conclusion:

Herbal medicines are very popular in developing and underdeveloped countries. Reports indicate that the ideal herbal drugs are very safe and free from side effect is somewhat misleading. Therefore, clear understanding of potential adverse effect of herbs used by human population is necessary for implementing safety measures to the public 22. In the case of Piper nigrum and Nyctanthes arbor-tristis, available reports are more or less common on the efficacy of the plant but no systematic safety study had been done so far and hence, in present work, we have studied the acute toxicity both the plants in rats.

 

The results of acute study showed the absence of lethality or toxic side effect on oral administration of dried unripe fruits of Piper nigrum and dried leaves of Nyctanthes arbor-tristis test suspension even at a dose of 2000mg/kg body wt p.o. This clearly indicates the non-toxic nature of the plants. The results of general systemic toxicity and necropsy further confirm non-toxic nature of the plants. The acute oral median lethal dose (LD50) of Piper nigrum and Nyctanthes arbor-tristis was found to be not less than 2000 mg/kg body weight.

 

Search for herbal remedies with potent antipyretic activity received momentum recently as the available antipyretics, such as paracetamol, nimesulide etc have toxic effect to the various organs of the body. Present study identifies antipyretic activity of different formulations of Piper nigrum and Nyctanthes arbor-tristis in yeast induced pyrexia in rabbits and effects were recorded as percent inhibition rate against increasing rectal temperature in animals. Three different formulations were selected to consider their likely effectiveness in all age groups of patients.

 

Maximum protection against increasing rectal temperature in animals was observed in Gr.-B (P<0.001) received liquid oral dosage form of Piper nigrum and Nyctanthes arbor-tristis. Significant values for protection against increasing rectal temperature in animals were observed in Gr.-A and Gr.-B after 1 and 2 h., (P<0.05) of treatment.

 

It was evident from ANOVA that all the three formulations of Piper nigrum and Nyctanthes arbor-tristis showed antipyretic activity (P<0.01), although they were differing in magnitude and time course of antipyretic activity.

 

ACKNOWLEDGEMENT:

The authors gratefully acknowledge Dr. R.N. Gangal, Principal, Tilak Ayurved Mahavidyalaya, Pune; Dr. S.M. Vadvalkar, Principal and Dr. N Y Gond, Head, Dept. of Pharmacognosy, Nanded Pharmacy College, Nanded for their help and support during study.

 

REFERENCES:

1.        Chattopadhyay D, Arunachalam G and Ghosh L, Antipyretic activity of A. macrophylla wall exa. DC: An ethnomedicine of Andaman Island, JPPS, 2005, 8(3), 558-564.

2.        Chaudhari RD, Herbal Drug Industry- A practical approach to Industrial Pharmacognosy, Piper nigrum Linn, 1st edition ( Reprint), Eastern Publishers, Delhi, 1998, pp. 150-52, 205, 239-44, 367, 370.

3.        Rastogi RP and Mehrotra BN,  Piper nigrum Linn. In: Compendium of Indian Medicinal Plants, Central Drug Research Institute, Lucknow and Publications and Information Directorate, New Delhi, 1995, pp. 317,539, 503-04, 570.

4.        Chaudhari RD, Herbal Drug Industry- A practical approach to Industrial Pharmacognosy, Nyctanthes arbor-tristis Linn. 1st edition ( Reprint), Eastern Publishers, Delhi, 1998, pp. 54, 88, 243, 260.

5.        Rastogi RP and Mehrotra BN,  Nyctanthes arbor-tristis Linn. In: Compendium of Indian Medicinal Plants, Central Drug Research Institute, Lucknow and Publications and Information Directorate, New Delhi, 1995, pp. 494-95, 453, 509.

6.        Daniel M and Sabnis S D, A numerical evaluation of the taxonomical status of Nyctanthes, Indian Bot Rep, 1984, 52-57.

7.        Khajuria A and Zutshi U, Piper nigrum Linn, Indian Drugs, 1997, 34, 557.

8.        Sharma PV and Chaturvedi C,  Preliminary observations on the effects of Piper nigrum Linn, IJMS, 1964, 5(1), 51-57.

9.        Khory RN, Principles and Practice of Ayurvedic Medicines, Biotech Books, Delhi, 2004, pp. 211-219.

10.     Chopra IC, Handa KL and Kapur LD, Chopras Indigenous Drugs of India, 2nd edition, Academic publisher, 1994, pp. 250, 278, 520.

11.     Ravishankar B and Sasikala CK, Pharmacological evaluation of compound Ayurvedic preparation containing Piper nigrum, Ancient Science of Life, 1983, 3(1), 11-18.

12.     Tripathi G, Tripathi GS and Tripathi YB, Thyrogenic response of Piper nigrum, Fitoterapia, 1989, 60(6), 539-542.

13.     Public Draft, 1996,  Acute Oral Toxicity, United State environmental Protection Agency Prevention, Pesticides and Toxic Substances.

14.     Public Draft, 1996, Acute Oral Toxicity- Acute Toxic Class Method, OECD Guidelines for the Testing of Chemicals No.423.

15.     Stitzel K and Carr G, Statistical basis for estimating acute oral toxicity       comparison of OECD Guidelines-401, 420, 423 and 425, Up-and-Down   procedure, Peer panel report, 1999

16.     Lu WL, Zhang Q and Wang H, Antipyretic, analgesic and anti-inflammatory activities of ketoprofen in animals, Biol Pharmaceut Bull, 2004, 27(10), 1515-20.

17. Saxena et al, Analgesics, antipyretic and ulcerogenic activity of Nyctanthes arbor- tris leaf extract. JEP,1987, 19(2), 193-200.

18.     Majumdar AM et al, Anti-inflammatory activity of piperine, Jpn J Med Sci Biol, 1990, 43(3), 95-100.

19.     Ansel HC, Pharmaceutical dosage forms and drug delivery systems, 7th   edition. Lippincott, 2000, pp. 347-56.

20.     Aulton ME, Pharmaceutics: The science of Dosage form. Churchill Livingston, 1996, pp. 304.

21.     Lachman L, Liberman HA and Kanig JL, The theory and Practice of Industrial Pharmacy, 3rd edition. Vargees Publication House, 1987, pp. 293-329.

22.     Sheeja SS and Vabhav J, Toxicology of Herbs, Pharma Times, 2005, 37(6), 27-30.

 

 

 

 

 

Received on 18.08.2009          Modified on 16.10.2009

Accepted on 19.11.2009         © RJPT All right reserved

Research J. Pharm. and Tech. 3(1): Jan. - Mar. 2010; Page 157-160