INTRODUCTION:

Ayurveda is the oldest surviving complete medical system in the world and its origins go back nearly 5000 years. Derived from its ancient Sanskrit roots - ‘Ayus' (life) and ‘ved' (knowledge) and offering a rich, comprehensive outlook to a healthy life. The art of Ayurveda had spread around in the 6th century BC to Tibet, China, Mongolia, Korea and Sri Lanka, carried over by the Buddhist monks travelling to those lands. Ayurveda the science of life, deals with the holistic view of healthy living. It emphasizes on prevention as well as treatment of various disease conditions through holistic approach. Since ancient times, several diseases have been treated by administration of plant material based on traditional method and approaches. Investigation of traditionally used medicinal plants is thus valuable on two levels, firstly, as a source of potential chemotherapeutic drugs, and secondly, as a measure of safety for the continued use of medicinal plants.

The biological diversity in the Indian Himalayan Region especially in Kumaun Himalaya has been a source of medicine for millions in the country and elsewhere¹. At present, the pharmaceutical sector is using nearly 280 medicinal plant species, out of which 175 are from the Indian Himalayan Region². Various plants being the effective source of both traditional and modern medicines are genuinely useful for primary health care.

Juniperus communis L., a gymnosperm, belonging to the family Cupressaceae, (common name-Juniper) is a high altitude shrub. It has the largest range of any woody plant, throughout the cool temperate Northern Hemisphere from the Arctic south in mountains to around 30°N latitude in North America, Europe and Asia. This species is distributed in the Hindu-Kush Himalayan region across Afghanistan, India and Nepal (West and Central) in an altitude range of 1800-3600 meters within India; it has been recorded in Jammu and Kashmir, Himachal Pradesh and Uttar Pradesh. It is known by various vernacular names Hapusa, Visraganga in Sanskrit, Haubera in Hindi, Abhal in Urdu, Hosha in Marathi, Juniper, Common juniper in English.³

Juniper berries act as a strong urinary tract disinfectant if consumed and were used by American Indians as an herbal remedy for urinary tract infections. Western tribes combined the berries of juniperus communis L. with Berberis root bark in a herbal tea to treat diabetes. Clinical studies have verified the traditional use of this treatment in insulin-dependent diabetes. Dioscorides' De materia medica also lists juniper berries, when crushed and put on the penis or vagina before intercourse, as a contraceptive.⁴ Native Americans also used juniper berries as a female contraceptive.⁵

Indigenous peoples from Eurasia made tonics for kidney and stomach ailments and rheumatism. Juniper was used by Great Basin Indians as a haematenic. Native Americans from the Pacific Northwest used tonics made from the branches to treat colds, flu, arthritis, muscle aches, and kidney problems. Aboriginal peoples drank juniper berry tea to relief stomachaches, arthritis and colds. While they are still taken for these ailments, juniper berries are primarily used for their diuretic action. Experts caution against the use juniper berries during pregnancy as they may stimulate uterine contractions. Because of their diuretic action, extended use (more than six weeks) may cause problems for people with weak or damaged kidneys.⁶

In Ayurvedic system of medicine juniperus communis L. is used as ingredient in major formulations namely Chavikasava, Dadhika ghrita, Erand paka, Hinguvachadi churna, Hingvadi churna, Kumaryasava, Narayana churna, Nityanand rasa, Pradarantak lauha, Saptavimsatika guggulu and Trayodasanga guggulu.⁷ It is also used in the treatment of diarrohea, abdominal disorders, urinary tract infection, menstrual disorders, skin diseases, heart ailmnets, sweat inducing, tumors, piles, bronchitis and indigestion.⁸

In herbal medicine, juniper oil has been used as a carminative, diuretic and as a steam inhalant in the management of bronchitis. It has also been used in arthritis as well as antioxidant.⁹ Berries are also recommended in cough, infantile tuberculosis and diabetes¹⁰, whereas, ash of the bark is used for certain skin diseases¹¹. It is noted by German authorities that the tea is diuretic and urinary antiseptic¹². The diuretic activity is thought to be largely due to the tea's content of terpinen-4-ol, a non irritating terpene. Juniper berry tea is listed in the German Pharmacopoeia as a digestive aid, both stimulating appetite as well as relieving flatulence. The oils also help increase the flow of digestive fluids, improve digestion and eliminate gas and stomach cramping¹³. Juniper has been used for centuries as a steam inhalant for bronchitis, and to control arthritis.

The fruit of Juniperus communis L. contains the main constituents were monoterpenoids, α-pinene, myrcene, sabinene, camphene, camphor, cineole, p-cymene¹⁴, δ, γ-cadinenes, bornyl acetate¹⁵, limonene¹⁴, β-pinene, α-terpinene, terpinen-4-ol, α-phellandrene; diterpenoids: caryophyllene⁴, sandaracopinaric acid, isocupressic acid, isopimaric acid, imbricatolic acid, 15,16-epoxy-12-hydroxy-8(17),13(16),14-labdatrien-19-oic acid¹⁶; flavonoids: luteolin-7-O-β-D-glucoside, kaempferol-3-O-β-D-glucoside, quercitrin, apigenin, luteolin, robustaflavone, apodocarpusflavone A, hinokiflavone¹⁷; catechins: (+)-afzelechin, (-)-epiafzelechin, (+)-catechin, (-)-epicatechin, (+)-gallocatechin, (-)-epigallocatechin¹⁸. The leaf contains essential oils: α-pinene (17%), sabinene (12%), terpinen-4-ol (7.7%), phellandrene (7.3%), widdrene (6.4%), γ- terpinene (5.9%), β- terpinene (4.3%), α- terpinene (3.8%)¹⁹, limonene (4.2%), terpinen-4-ol (2.7%), myrcene (2.6%), β-pinene (2.0%)²⁰; and also contains biflavonoids: cupressuflavone, amentoflavone, hinokiflavone, isocryptomerin, sciadopitysin²¹.

MATERIALS AND METHODS:

Plant material:

The samples of Juniperus communis L. were collected from local market of Patiala, Punjab, India. Specimen were identified and authenticated at the National Institute of Ayurvedic Pharmaceutical Research, CCRAS, Patiala. Powder of the sample was used for chemical analysis. Different physicochemical parameters like Total ash, acid insoluble ash, water soluble ash, ethanol soluble extractive value, water soluble extractive value, loss on drying, pH, TLC were carried out on collected the sample.

Preliminary Phytochemical Analysis:

Preliminary phytochemical results showed the presence or absence of certain phytochemicals in the Juniperus communis L. fruit. The tests performed using n-Hexane, Chloroform, Ethyl acetate, alcoholic and water extracts were subjected to various qualitative tests for the identification of phytoconstituents present. Preliminary phytochemical test revealed the presence of coumarin, flavonoids, glycoside, tannin, sugar terpenoids and absence of alkaloids results are given in Table.1.^22-23.

Table 1. Preliminary phytochemical tests for different solvent extract of Juniperus communis L. (Hapusa) fruit.

S. No	Natural product	Test performed	Results
1.	Coumarin	Alkaline test	+Ve
2.	Flavonoid	Shinoda test	+Ve
3.	Alkaloid	Dragendorff’s test Mayer’s test Wagner’s test	-Ve
4.	Glycosides	Legal’s test Baljet’s test	+Ve
5.	Tannin	Neutral FeCl3	+Ve
6.	Sugar	Molisch’s test Fehling’s test	+Ve
7.	Terpenoid	Noller’s test	+Ve

Physico-chemical analysis of Juniperus communis L. (Hapusa) fruit:

Physico-chemical analysis was done to ascertain the quality of the raw material used in the preparation of Ayurvedic formulations. To check the quality, the parameters as per the WHO/AOAC guidelines,^24,25and methodology were adopted as mentioned below:

Foreign Matter:

The 50 gm Juniperus communis L. fruit sample was spread in a thin layer, and the pieces of foreign matter were sorted out by visual inspection. The powder of foreign matter was sifted through a 250 micron sieve. All portions of the foreign matter were pooled and weighed.

Moisture content:

4 g powder of the Juniperus communis L. fruit sample was taken and heated in an oven at 105°C for 5 hour in a previously weighed 100 ml beaker. It was cooled in desiccators and weighed. The procedure was repeated till constant weight is obtained. The percentage of loss in weight of the sample was calculated.

Deterioration time of the plant material depends upon the amount of water present in plant material. If the water content is high, the plant can be easily deteriorated due to fungal attack. The loss on drying at 105°C of Juniperus communis L. fruit was found to be 7.65 %.

Determination of Total ash value:

2 g powder of the sample was taken accurately in a previously ignited and tarred Silica dish. The material was spread evenly and ignited in a muffle furnace by gradually increasing the temperature to 600^oC until it is white, indicating the absence of carbon. The crucible was cooled in desiccators and allowed to stand for 30 minutes and weighed.

Total ash value of plant material indicated the amount of minerals and earthy materials attached to the plant material. Analytical results showed total ash value of Juniperus communis L. fruit was 3.77 %.

Determination of Acid insoluble ash value:

To the dish containing the total ash, 25 ml of 20 % Hydrochloric acid was added covered with a watch glass and boiled gently for 5 minutes. The watch glass was rinsed with a hot water and added to the crucible. The residue was washed with the hot water till the washings were neutral to the litmus. The insoluble material was collected and again placed in a same crucible and again ignited for 6 hr. to constant weight. The residue was cooled a desiccators for 30 minutes and weighed.

Percentage of acid insoluble as was calculated. The amount of acid-insoluble siliceous matter present in the Juniperus communis L. fruit was 0.62%.

Determination of Water soluble extractive value:

4 g of the sample was taken in a glass stoppered flask 100 ml of distilled water was added. The flasks were shaken occasionally for 6 hours and then allowed to stand for 18 hours. The extract was filtered and 25 ml of the filtrate was pipette out in a pre-weighed 100 ml beaker and evaporated to dryness on a water bath. It was kept in a hot air oven for 5 hr at l05°C, cooled in desiccators for 30 minutes and weighed. The procedure was repeated till constant weight.

The water-soluble extractive value indicated the presence of sugar, acids and inorganic compounds. The water soluble extractive value in the Juniperus communis L. fruit sample was found to be 18.37%.

Determination of Alcohol soluble extractive Value:

Same procedure as for the water soluble extractive value was followed. Instead of water, rectified spirit was taken as a solvent.

The alcohol soluble extractive values indicated the presence of polar constituents like phenols, alkaloids, steroids, glycosides, flavonoids and secondary metabolites present in the plant sample. The alcohol soluble extractive value was found to be 16.3% in the Juniperus communis L. fruit.

Determination of pH Value:

10% aqueous solution of sample was prepared and used for determining the pH value by pH meter. The pH value of Juniperus communis L. fruit was found to be 6.83.

RESULT AND DISCUSSION:

Physico-chemical parameters

Different physicochemical parameters like Total ash, acid insoluble ash, water soluble extractive value, ethanol soluble extractive value, loss on drying at 105ºC and pH etc. were studied. (Table. 2)

Table 2. Physicochemical parameters of Juniperus communis L. (Hapusa)) fruit

S. No.	Name of Parameters	Results
1.	Foreign Matter	Nil
2.	pH (10% aqueous solution (v/w)	6.83
3.	Loss on drying at 105^oC (% w/w)	7.65
4.	Total Ash (% w/w)	3.77
5.	Acid-insoluble ash (% w/w)	0.62
6.	Water-soluble extractive (% w/w)	18.37
7.	Alcohol-soluble extractive (% w/w)	16.3

Thin Layer Chromatography:

Thin layer chromatographic technique was used to separate the chemical compounds present in the drug. Various solvent systems were checked to separate the maximum number of chemical compounds in the drug. Take 4g of the Juniperus communis L. fruit sample was soaked in 40 ml of rectified spirit (90%) with occasional shaking for 18 hrs, boiled for 10 minutes and filtered. The filtrate was evaporated and the soluble portion was filtered, concentrated and made upto 10 ml in standard flask. 10μl of the solution was applied on (E. Merck) Aluminium plate precoated with Silica gel 60 F254 of 0.2 mm thickness using Linomat IV applicator. The plate was developed in Toluene: Ethyl acetate: Formic acid (5: 5: 0.3 v/v). After air drying the plate was not visualized in UV 254 and 366 nm. The plate was then dipped in Anisaldehyde sulphuric acid and heated in air oven at 105°C till the spots appeared (Figure. 1) and the results of R_f values given in Table. 3.

Table 3. TLC profile of Juniperus communis L. Fruit (Toluene: Ethyl acetate: Formic acid :: 5: 5: 0.3, v/v/v).

S. No	UV- λ 254 nm		UV- λ 366nm		After derivatization with anisaldehyde-sulphuric acid reagent
S. No	Colour	R_f.	Colour	R_f.	Colour	R_f.
1.	-	-	0.08	Light red	-	-
2.	0.23	Grey	0.23	Grey	-	-
3.	0.27	Grey	0.29	Red	0.32	Light red
4.	0.37	Grey	0.40	Red	-	-
5.	0.45	Grey	0.44	Red	0.46	Green
6.	0.58	Grey	0.54	Blue	0.52	Light blue
7.	-	-	0.61	Light blue	0.59	Light red
8.	0.65	Grey	0.66	Light blue	0.63	Blue
9.	0.79	Grey	0.77	Light blue	-	-
10.	-	-	0.82	Red	0.81	Green
11.	0.88	Grey	0.85	Fluorescent	0.87	Blue
12.	0.96	Grey	0.96	Red	0.97	Green

Under 254 nm, the samples showed 9 spots towards the baseline indicating the highly polar nature of the compounds. At 366 nm the plate showed 12 spots in between the base line and solvent front indicating the presence of compounds. After dipping the plate in a Anisaldehyde sulphuric acid solution, the plate showed 8 different colored compounds indication the presence of compounds with strong chromophoric groups and multiple bonds. The R_fof all the spots was calculated and mentioned in the table 2.

Figure 1. TLC profile of Juniperus communis L. Fruit (Toluene : Ethyl acetate : Formic acid :: 5 : 5 : 0.3 v/v).

λ 254 nm	λ 366 nm	After Derivatization^*

^*Derivatization with anisaldehyde-sulphuric acid reagent and visualized under visible light

CONCLUSION:

Morphology as well as various phamacognostic aspects of the rhizome sample was studied and described along with physio-chemical parameters and TLC. These parameters will be useful in authentification and identifying the adulterants and quality control of raw drugs. Whole plant of Juniperus communis Linn. fruit exhibited a set of diagnostic characters, which will help to identify the drug in dried condition. It has been concluded from this study that estimation of these parameters is highly essential for raw drugs or plant parts used for the preparation of compound formulation. The periodic assessment is essential for quality assurance and safer use of herbal drugs.

ACKNOWLEDGEMENT:

The authors are very grateful to Programme Officer, CCRAS, New Delhi for providing encouragement and facilities for carrying out this work.

REFERENCES:

1. Tripathi M. Threat to medicinal plants of Kumaon Himalaya. Curr. Sci., 2007; 93(10): 25.

2. Dhar U, Rawal RS Upreti J. Setting priorities for conservation of medicinal plants – A case study in the Indian Himalaya., 2000; 57-65.

3. Judith Ladner, ETH Zurich. Food and agriculture research organization, United States.

4. Riddle J M. Contraception and abortion from the ancient world to the Renaissance, Harvard University Press, 1992; 31.

5. Edible and Medicinal Plants of the West, Gregory L. Tilford, ISBN 08784-359-1.

6. Wikipedia, the free encyclopedia, website.

7. Ayurveda consultants.com website.

8. Kirtikar KR, Basu BD. Indian Medicinal Plants. Periodical experts, Delhi, India. 1935; 3.

9. Takacsova M, Pribela A, Faktorova M, Nahrung. Study of the antioxidative effects of thyme, sage, juniper and oregano. Nah rung. Food, 1995; 39: 241-243.

10. Zaman MB, Khan MS. Hundred Drug Plants of West Pakistan. Pakistan Forest Institute Peshawar, Pakistan,1970.

11. Baquar SR. Medicinal and Poisonous plants of Pakistan. Printas Karachi, Pakistan, 1989; 506.

12. Hagar HH J. Hagers Handbuch des pharmazeutischen Praxis, Springer -Verlag, Berlin, 1979; 5, 333.

13. Uphof J C T. Dictionary of Economic Plants, Verlag von Cramer, Germany, 1968; 290.

14. Butkiene R, Nivinskiene O, Mockute D. Volatile compounds of ripe berries (black) of Juniperus communis L. growing wild in north-east Lithuania. Journal of Essential Oil-Bearing Plants. 2005, 8(2): 140-147

15. Barjaktarovic B, Sovilj M, Knez Z. Chemical composition of Juniperus communis L. fruits supercritical CO2 extracts: dependence on pressure and extraction time. Journal of Agricultural and Food Chemistry. 2005, 53(7): 2630-2636

16. Martin AM, Queiroz EF, Marston A, Hostettmann K. Labdane diterpenes from Juniperus communis L. berries. Phytochemical Analysis. 2006, 1: 32-35

17. Hiermann A, Kompek A, Reiner J, Auer H, Schubert-Zsilavecz M. Investigation of flavonoid pattern in fruits of Juniperus communis. Scientia Pharmaceutica. 1996, 64(3-4): 437-444

18. Friedrich H, Engelshowe R. Monomeric tannin products in Juniperus communis L. Planta Medica. 1978, 33(3): 251-257

19. Mastelic J, Milos M, Kustrak D, Radonic A. Essential oil and glycosidically bound volatile compounds from the needles of common juniper (Juniperus communis L.). Croatica Chemica Acta. 2000, 73(2): 585-593

20. Chatzopoulou PS, Katsiotis ST. Chemical investigation of the leaf oil of Juniperus communis L. Journal of Essential Oil Research. 1993, 5(6): 603-607

21. Ilyas M, Ilyas N. Biflavones from the leave of Juniperus communis and a survey on biflavones of the Juniperus genus. Ghana Journal of Chemistry. 1990, 1(2): 143-147

22. Khandelwal KR, Practical Pharmacognosy, Nirali Prakashan, Pune, 2005, 149-153.

23. Pankaj Pradhan, Lincy Joseph, Mathew George, Neha Kaushik, Rahul Chulet. Pharmacognostic, Phytochemical and Quantitative Investigation of Saraca asoca leaves. Journal of Pharmacy Research 2010, 3(4),776-780.

24. World health Organization, Quality control methods for medicinal plant materials, Switzerland, 53.

25. AOAC Official methods of analysis of AOAC ,International, 16th edition, AOAC International, Suite 400, 2200 Wilson Boulevard, Arlington, Virginia, USA, 1995.

Received on 08.10.2011 Modified on 20.10.2011

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 88-91