Comparative Physicochemical Evaluation of Fruits and Anti depressant Potential of volatile oils of fruits of Local Piper Species

Introduction

Piper Linn., belonging to family Piperaceae is a very large genus of shrub,rarely herbs and trees, distributed throughout the tropical and sub-tropical regions of the world. About 30 species of the genus in India and 700 species in the world have been reported, of which, P. nigrum, the Black Pepper and P. betle Linn., the  Pan or Betel are widely cultivated². Five species are used as herbal ingredients of Asian medicines and they are  P. betle , P. cubeba Linn. f. (Cubebs), P. retrofractum Vahl syn. P. chaba Hunter non Blume (Java Long Pepper), P. longum Linn.  (Indian Long Pepper) and P. nigrum Linn³. The leaf juice of P. betle is used as eye drop⁴.  P. cubeba is used as antibacterial⁵, expectorant⁶ and as gastroprotective⁷.  P. longum is used as bioavailability enhancer⁸, digestive and in the treatment of bronchitis⁹ and also as hepatoprotective agent¹⁰. Scientists have received US patent on obtaining a diabetes mellitus therapeutic agent from P. longum¹¹. P. nigrum is used as nervine tonic, and in the treatment of constipation, itching and flatulence¹². Some of the Piper species contain a piperidine type alkaloid, piperine, which is a central nervous system depressant¹³. Most of the piper fruits contain volatile oils¹⁴.

Figure 1  Click here to View figure

 

Materials and methods

Collection of chemicals and plant material:

For the present study fruits of all the five selected species of Piper (P. betle Linn, P. cubeba Linn. f., Piper chaba (retrofractum) Vahl, P. longum Linn & P. nigrum Linn ), were collected from APMC Market, Vashi, Navi Mumbai, India. The fruits were identified, confirmed and authenticated by Prof. H.M. Pandit, Botany Dept., Khalsa College, Mumbai. Fruits of five Piper species were shade dried & ground to coarse powder. All reagents used in quantitative analysis and chemical investigation were of analytical grade and manufactured by E-Merck, Ranbaxy, Loba chemicals, S.D. fine chemicals and Yucca Enterprizes, Mumbai.

Physicochemical parameter

Determination of ash value¹⁴

Total Ash/ Water Soluble Ash/ Acid Insoluble Ash Value

For its detection, 2g of powdered material of each formulation and the individual ingredients of the powers were placed separately in a suitable tarred crucible of silica previously ignited and weighed. The powdered drugs were spread into an even layer and weighed accurately. The materials were incinerated by gradually increasing the heat, not exceeding 450°C until free from carbon, cooled in a desiccator, weighed and percentage ash was calculated by taking in account the difference of empty weight of crucible & that of crucible with total ash. The water soluble and acid insoluble ash value were then determined as per standard procedure.

Master Table

Determination of Solvent Extractive Value¹⁴

Water/Alcohol/Ether Soluble Extractive Value

About 5g of coarsely powdered air-dried drug was macerated with 100 ml of chloroform water/ alcohol/ ether respectively in three different closed flask for twenty-four hours, shaking frequently during six hours and allowed to stand for eighteen hours. These were then filtered rapidly; taking precautions against loss of solvent. 25 ml of the filtrate was evaporated to dryness in a tarred flat-bottomed shallow dish at 105°C to constant weight and weighed. The percentage of water / alcohol / ether soluble extractive were calculated with reference to the air dried drug and is represented as% value.

Master Table 1

Determination of Moisture Content¹⁴

About 2g of drug samples of each fruit was accurately weighed in a dried and tarred flat weighing bottle and dried at 105 ⁰C for 5 hrs. Percentage was calculated with reference to initial weight.

Master Table 1

Determination of Mucilage Content¹⁵

About 1 gm of drug samples of each fruit/powder was accurately weighed and taken in a test tube. 10 ml of distilled water was added to it kept for 24 hours for maceration. On the next day fruit/powder solutions were filtered. The filtrate then treated with 5 ml of ethanol. The mucilage appeared in the ethanol which was then filtered through an already weighed watmann filter paper and difference in the weight was then calculated.

Master Table 1

Determination of Crude Fibre Content¹⁶

About 2 gm of drug sample extracted with 50 ml of methylene chloride. Filtered and to the residue added 50 ml of dilute sulphuric acid, boiled for 30 minutes, filtered. The residue then washed with water.  Ignited already weighed crucible and contents/residue in an electric muffle furnace at 600°C. Then difference in weight was calculated.

Master Table 1

Determination of Volatile Oil Content¹⁷

About 100 gms of drug samples of each fruit was accurately weighed and transferred to a 500ml round bottom flask. Sufficient amount water was added and a Clevenger Apparatus then attached to the RBF with condenser and mixture was then heated for six hour for so as to isolate the volatile oil. The volatile oil of individual drug was then collected in graduated tube of Clevenger Apparatus and separated and stored in vials.

Master Table 1

Determination of Piperine Content¹⁸

Instrument

UV Spectro-photometer: UV-1800-Shimatzu

Preparation of standard solution of Piperine

Accurately weighed Piperine (10 mg) was transferred in 100 ml volumetric flask and dissolved in & diluted to 100 ml with ethanol. The final solution contained 100 mg of the Piperine per ml of the solution.

Preparation of Piperine extract of different Piper Extracts

Accurately weighed 1 gm of Piper fruit powder reflux with 40 ml of ethanol for 1 hour. Filtered the extract and re-reflux the marc I left with 30 ml of ethanol for another 1 hours. Filtered and combined the previous filtrate. Further reflux the mark II left with 20 ml of ethanol. Again filtered and combined filtrate with previous filtrates, finally make up the volume up to 100 ml with ethanol in a volumetric flask.

Preparation of calibration curve for Piperine

Standard solutions of Piperine (0.1, 0.2, 0.3, 0.4 and 0.5 ml) were pipetted in a series of five 10 ml volumetric flask so as to give concentration range 1-5 ug/ml The absorbance of the Piperine was measured at 342.6 nm against ethanol. The concentration and then the percentage of Piperine in different Piper fruits were calculated.

Master Table 1

Master Table 1: Physicochemical Evaluation of Fruits of Local Piper Species

Sr. No.

Name of the Drug

Ash Value

Extractive Value

Moisture Content

Fruit Mucilage Content

Crude Fibre Content

Volatile Oil Content

Piperine Content (gm percent)

T.A.V.

W.S.A.V.

A.I.A.V.

W.S.E.V.

A.S.E.V.

E.S.E.V.

1

Piper nigrum

10.8±0.4

8.8±0.2

1.0±0.1

2.5±0.1

6.2±0.2

5.8±0.2

1.80

3.8

0.35

1.6

1.70 gm %

2

Piper longum

5.7±0.2

3.2±0.1

1.5±0.1

18.5±0.4

6.8±0.2

9.8±0.2

1.35

7.9

0.45

0.2

0.30 gm %

3

Piper chaba

6.85±0.8

3.6±0.1

3.0±0.1

6.0±0.2

8.6±0.2

7.2±0.2

1.25

8.6

0.15

0.8

0.95 gm %

4

Piper cubeba

5.9±0.55

2.8±0.1

3.0±0.1

7.5±0.2

26.0±0.4

23.8±0.3

1.75

4.4

0.45

4.4

0.15 gm %

5

Piper betle

7.65±0.5

4.6±0.2

2.0±0.1

11.5±0..3

16.0±0.3

5.6±0.2

1.65

9.1

0.35

0.2

0.90 gm %

T.A.V.-Total Ash Value, W.S.A.V.-Water Soluble Ash Value, A.I.A.V.-Acid Insoluble Ash Value

W.S.E.V.Water Soluble Extractive Value, A.S.E.V. Alcohol Soluble Extractive Value & E.S.E.V.Ether Soluble Extractive Value

Antidepressant Activity^19-22

Forced Swimming Method

The forced swimming test is adopted here is a modification of the method described by Porsolt et al. (1977). Mice were individually forced to swim for 15 min in glass cylinders (height: 20 cm, diameter: 14 cm), containing 10 cm of water at 25 °C, which is a pre-test, and then mice were removed and dried before being returned to cages. Then standard Fluoxetine and essential oil under tests were administered. Four hours later, mice were placed in the cylinders again for a 6-min test in the same system depicted above. The duration of immobility was recorded during the last 4 min of the 6-min testing period. Groups of 6 mice were treated with vehicle (10 mg/kg, p.o.), drug-treated groups (10 ml/kg, p.o.), Fluoxetine (1 mg/kg,i.p.). Table 2

Table 2: Antidepressant Activity of Essential Oils of Piper Fruits: Forced Swimming Method

	Normal control	Standard	T-1	T-2	T-3	T-4	T-5
Body weight (g)	49.33 ± 2.06#	60.67 ± 3.53*	69.50 ± 1.78**	64.17 ± 2.17**	55.33 ± 2.40	53.17 ± 4.31	56.67 ± 2.95
Mobility (second)	239.00 ± 17.03##	293.50 ± 11.83**,	338.83 ± 6.75**, ##	334.17 ± 4.02**, #	332.17 ± 4.92**, #	348.83 ± 3.02**, ##	350.5 ± 0.76**, ##
Immobility (second)	121.67 ± 17.36##	66.50 ± 11.83**	21.16 ± 6.745**, ##	25.83 ± 4.02**, #	27.83 ± 4.92**, #	11.16 ± 3.02**, ##	9.50 ± 0.76**, ##

Values are represented as Mean ± SEM, one way ANOVA followed by Dunnett’s test for multiple comparisons

*P<0.05, **P<0.01 vs. normal control.

^#P<0.05, ^##P<0.01 vs. standard.

Result and Discussion

In the exhaustive study of Piper fruits, the conclusions drawn are; (a) Piperine content in Piper nigrum is maximum followed by Piper chaba and minimum in Piper cubeba. Here an easy UV Spectroscopic method is used for Piperine determination (b) Volatile oil content is in maximum amount in Piper cubeba followed by Piper nigrum and Piper chaba and least in remaining two species of Piper. Here hydrodistillation method is used for isolation of Volatile oils. (c) Crude fibres are fat free organic substances which are insoluble in acid and alkaline media. The crude fibre content estimation showed that Piper longum and Piper cubeba have equal and maximum crude fibre content followed by Piper nigrum and Piper betle in equal amount. (d) The mucilage content in Piper betle was in maximum amount followed by Piper chaba and then Piper longum. It is revealed that circular or rounded shaped fruit do not contain mucilage in large amount. (e) It is found that moisture content in Piper nigrum is in maximum amount while in Piper chaba it in least amount.

All volatile oils of Piper fruits have shown more activity than the standard compound. Fluoxetine, which is a Selective Serotonin Reuptake inhibitor was taken as standard, therefore for all these oils which have shown comparable and more activity to that of Fluoxetine should be further studied with different models of Antidepressant Activity.

Acknowledgement

The author is thankful to Mr.Waseem Khan, Managing Director, Oriental Education Society for a suitable financial assistance and permission to carry out the work on the Piper species available in local market. The author is also thankful to Mr.Amjad Ali, Mr. Imtiyaz Ansari, Ms.Poonam Sarang, Ms.Sonal Girkar and Nilesh Kharwate, Mr.Altamash, Mr. Amogh and Ms. Jyoti, for their help in the Laboratory.

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