INTRODUCTION:

Cough is a protective mechanism which helps to expel unwanted materials like food, dirt, etc. that does not belong to our lungs or narrow passages of trachea^1-3. Cough can be grouped into two categories i.e. productive cough and non productive cough. In case of productive cough there is excessive secretion of mucous but non-productive cough is just opposite i.e. dry. Antitussive like dextromethorphan or codeine relieve us from excessive expectoration of mucous. On the other hand non-narcotic antitussives inhibit the stretch receptor in the narrow passage of the respiratory tract and there by suppress the cough reflex at its source. Narcotic antitussives are the cause of inhibition of cough centre located in medulla oblongata ^4-5.

Formulation containing synthetic drug acts on symptoms of a particular disease but crude drug or extracts generally boost up body natural healing process. Natural medicine acclimatizes with the body biochemical process very easily^6-7.

Considering the present situation we have aimed to formulate a traditional cough preparation containing extracts of five different plants such as Ocimum sanctum, Adhatoda vasica, Cinnamomum verum, Cardamomum subulatum, Ephedra sinica and to evaluate its efficacy scientifically. Traditionally Tulsi is used to alliviate diseases like catarrh, bronchitis, gastric and genitor-urinary disorders, malaria fever, rheumatism, skin diseases etc^8-9. Vasaka is used in the treatment of cold, cough, chronic bronchitis and asthma. Cardamom is used to cure from asthma, teeth and gum infections, cataracts, nausea, diarrhoea, cardiac, digestive and kidney problems. Ephedra is used use in traditional Chinese medicine for asthma, bronchitis, allergies, and cold and flu symptoms^10-11.

MATERIALS AND METHODS:

Collection, authentication and processing:

All the crude drugs were collected from Majhitar, Sikkim in the month of August when the plants bear flower. The plant was identified, confirmed and authenticated by Botanical survey of India, Gangtok. The collected plant materials were shade dried. The dried materials were coarsely powdered individually by means of mechanical grinder. Then they were passed though sieve no.40 (aperture size-425mm) to get moderately coarse powder. The resulting powdered materials were used for further studies.

Standardization of the basic crude drugs:

Morphological studies:

Morphological studies of leaf such as size, shape, colour, odour and taste were examined using different terms^12-13.

Proximate analysis:

Determination of ash values such as total ash, acid insoluble ash, water soluble ash, sulphated ash and extractive values like water soluble extractives, alcohol soluble extractives were carried out as method described by Pharmacopoeia of India¹⁴.

Determination of moisture contentwas carried out as method described by Khandelwal K R¹⁵.Fluorescence analysis was carried out as method described by Nisha Raj RS et al¹⁶.

Formulation of herbal cough syrup:

Preparation of simple syrup IP:

66.67gram of sucrose was weighed and sufficient distilled water was added to it. It was then heated to dissolve all the sucrose and the final volume was adjusted to 100ml by adding hot distilled water^17-18.

Preparation of decoction:

Specific amount (each 100gm) of powdered tulsi, vasaka, ephedra, cardamom and cinnamon were taken on a Whatman filter paper and mixed properly. The mixture was taken in a 5000ml round bottom flask and sufficient distilled water was added to it. It was then boiled till the final volume reduced to ¼^th of the initial volume. The mixture was cooled and filtered through Buchner filtration assembly. At the end the filtrate was taken to mix with the simple syrup.

Preparation of final product:

1 part of the decoction was mixed with five parts of the simple syrup IP. Required amount of methyl paraben was added it as a preservative. The solubility of each ingredient was checked by observing visually in the laboratory vicinity.

Evaluation of the herbal formulation:

Physical evaluation:

After the formulation was ready the different physical parameters like colour, odour, taste, specific gravity, pH, total solid content, viscosity, refractive index and finally stability were tasted following the method described by Kumar et al., 2013¹⁹.

In vivo pharmacological evaluation:

Acute toxicity study:

It was conducted to determine the median lethal dose (LD₅₀) i.e. the dose which can kill 50% of the animal used. The acute toxicity test was done following the OECD guide line 421^20-21.

Protocol of the in vivo experimental activity:

Housing of animals:

Mice of either sex (22-25g) were used for expectorant activity. All the animals were housed at room temperature (22-24⁰C) and constant humidity (50-60%) less than 12 hours of dark and light cycle with standard rodent diet.

Grouping of animals:

All male and female mice were randomly allowed into 4 groups (n=3)

Group 1- control group received distilled water only

Group 2- positive control group received ammonium chloride 1000mg/kg body weight

Group 3- herbal formulation (low dose 300mg/kg) treated group

Group 4- herbal formulation (High dose 600mg/kg) treated group

All groups were treated orally with a single dose daily for 5 days and the last dose was given before the i.p. administration of phenol red solution (5% w/v in saline solution, 0.1ml/10g body weight). water was not provided 30 minutes after the administration of drug. 30 minutes after administration of phenol red solution the mice were anaesthetized with pentobarbital of the dose of 75mg/kg body weight and exsanguinated by cutting the abdominal aorta. After being dissected to free from the adjacent organs, the trachea was removed from the thyroid cartilage to the main stem bronchi and put into 1 ml normal saline immediately. After ultra sonication for 5 minutes 1ml NaHCO₃ solution (5% v/v) was added to the saline and the optical density of the resulted solution was measured at 558n.m. using UV-VIS spectroscope^22-24.

CALCULATION:

The Data was expressed as mean±SD. Statistical analysis was done by one-way analysis of variance (ANOVA) using graph pad K Y Plot version 5.1.

RESULT AND DISCUSSION:

In this section the result of various investigations carried out were complied. An attempt has also been made to discuss these results in order to provide convincing reason for the studies performed.

The macroscopic evaluation was carried out with crude drug materials under simple microscope and the results are tabulated in table 1.

Proximate analysis including moisture content, extractive values and ash values were determined and shown in the following table 2.

Table 1: Macroscopic evaluation of crude drug materials

Plant species

Parameters

Observations

Ocimum sanctum

Shape

Size

Colour

Odour

Taste

Elliptical , oblong

2.5-5.0 cm long and 1.5- 3.2 cm broad

Dark green

Aromatic

Warm, pungent

Adhatoda vasica

Shape

Size

Colour

Odour

Taste

Ovate, lanceolate

10-13 cm long and 2.0-3.0 cm broad

Light green

Aromatic

Bitter

Cinnamomum verum

Shape

Size

Colour

Odour

Taste

Depends on the method of cultivation

Depends upon the plant

Brown to reddish brown

Aromatic

Sweet, pungent

Cardamomum subulatum

Shape

Size

Colour

Odour

Taste

Round

0.5 to 1.0 cm

Reddish brown to brown

Aromatic

Strong aromatic

Ephedra sinica

Shape

Size

Colour

Odour

Taste

Long thin and branched

Different sized

Yellowish brown

Astringent

Sweetish

Table 2: Proximate analysis of crude drug materials

Parameters in %(w/w)			Plant species
			*Ocimum sanctum*		*Adhatoda vasica*	*Cinnamomum verum*	*Cardamomum subulatum*	*Ephedra sinica*
Extractive values		Water soluble	14.4 7.2	7.2 20.0		3.2 9.6	8.0 8.8	12.8 19.2
		Ethanol soluble
Moisture content			8.0	8.0		9.5	6.0	6.0
Ash values	Total ash		8.8 0.4 3.8 0.8	1.4 0.9 3.0 0.5		6.6 0.3 2.8 0.3	8.0 1.2 3.1 0.5	7.9 0.8 7.1 0.7
	Acid insoluble
	Water soluble
	Sulphated

Table 3: Fluorescence analysis of crude powdered materials

Treatment	Plant species
	*Ocimum sanctum*		*Adhatoda vasica*		*Cinnamomum verum*		*Cardamomum subulatum*		*Ephedra sinica*
	Viscible light	UV 254 n.m.	Viscible light	UV 254 n.m.	Viscible light	UV 254 n.m.	Viscible light	UV 254 n.m.	Viscible light	UV 254 n.m.
Distilled water	black	black	brown	black	brown	black	brown	black	brown	green
H₂SO₄	brown	black	green	black	Reddish brown	black	black	black	brown	green
HCl	brown	black	green	green	Reddish brown	black	brown	black	brown	green
1(N) NaOH	brown	black	green	green	black	black	brown	black	brown	black
CH₃OH	black	black	green	black	Reddish brown	green	black	black	brown	green
C₂H₅OH	black	black	green	black	dark brown	black	brown	black	brown	green
Glacial CH₃COOH	brown	black	brown	black	brown	black	brown	black	brown	green

Fluorescence analysis of powder of different plant species were carried out in visible rays as well as in UV 254 nm. Wave length of light and recorder in table 3.

The physical evaluation of the finished product was carried out to standardize it and the observations are given in table 4

Table 4: Different physical parameters to evaluate the prepared formulation

Parameters	Obsevation
pH of 10% v/v solution	04.82
Surface tension	54.32 dynes/cm
Total solid content	38.20 % w/v
Specific gravity	1.324 at 25⁰C
viscosity	04.68 c.p.
Refractive index	01.43

The result of in vivo expectorant activity was shown in the fig. No 1. There were significant increase (P<0.001) of expectorant activity with herbal formulation in different doses as compared with the control animals.

Fig. 1: Effect of herbal formulation on optical density of phenol red obtained from mice in vivo. Values are presented as mean ± SD. Data were compared with control group. The P value was found significant ( p<0.001).

The crude drugs were identified by means of Phamacognostical studies which include macroscopical study and proximate analysis. Macroscopical parameters like colour, odour, taste, size and shape and the parameters of proximate analysis such as ash values, extractive values, fluorescence and moisture content determination gave us an idea about the purity of the drug. The ash content of the crude material is the residue obtained after complete incineration which simply represents the inorganic slot like carbonates, phosphates, silicates and silica. Acid insoluble ash values are usually consists of silica. The result suggested that the crude drugs have higher water soluble extractive value that ethanol soluble extractive value. The moisture content was below 10% which indicates less chances of microbial contamination. Fluorescence analysis gave an idea about the nature of phytoconstituients present. After the formulation was ready different physical parameters were tested. Different parameters like colour, odour, taste, pH, surface tension, total solid content, specific gravity, viscosity and refractive index helped us to standardize the formulation and its specificity to the users. It was found that all the parameters were within the limit. Furthermore its acute toxicity was observed for long term (14 days) and LD₅₀ was found 3000 mg/kg. The 1/5^th and 1/10^th of the LD₅₀ was considered as low dose and high dose. In In-vivo animal study it was found that both the doses and the standard drug ammonium chloride produced positive secretion of phenol red in the air way. There was a significant increase in expectorant activity with herbal formulation and the standard as compared to the control animals. Due to the excessive secretion of mucous in trachea the mucous layer becomes thin and phenol red solution can be easily transported to the trachea. The transported phenol red was later estimated. So more optical density indicated that more phenol red present in the trachea and more mucous secreting ability of the herbal formulation. It implied that our poly herbal formulation had possessed good expectorant activity.

CONCLUSION:

The formulation was considered best as it has appropriate concentration of sugar according to the I.P. and good preservative. The pH, specific gravity, viscosity, solid content refractive index and in vivo expectoration were satisfactory to be a ploy herbal cough syrup. Now a day herbal product has high demand because of lowest side effect. So the present study helped to develop a poly herbal cough syrup which can be used effectively and safely for expectoration of cough sputum from bronchial tract.

CONFLICTS OF INTEREST:

There are no conflicts of interest.

ACKNOWLEDGEMENT:

The authors are also thankful to the authority of University of North Bengal, Himalayan Pharmacy Institute and Sikkim Government Pharmacy College for their relentless cooperation in research works.

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Received on 01.06.2020 Modified on 22.03.2021

Research J. Pharm. and Tech 2022; 15(3):949-953.

DOI: 10.52711/0974-360X.2022.00158